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Apply the latest scientific and clinical advances with Wall & Melzack's Textbook of Pain, 6th Edition. Drs. Stephen McMahon, Martin Koltzenburg, Irene Tracey, and Dennis C. Turk, along with more than 125 other leading authorities, present all of the latest knowledge about the genetics, neurophysiology, psychology, and assessment of every type of pain syndrome. They also provide practical guidance on the full range of today's pharmacologic, interventional, electrostimulative, physiotherapeutic, and psychological management options.

  • Consult this title on your favorite e-reader with intuitive search tools and adjustable font sizes. Elsevier eBooks provide instant portable access to your entire library, no matter what device you're using or where you're located.
  • Benefit from the international, multidisciplinary knowledge and experience of a "who's who" of international authorities in pain medicine, neurology, neurosurgery, neuroscience, psychiatry, psychology, physical medicine and rehabilitation, palliative medicine, and other relevant fields.
  • Translate scientific findings into clinical practice with updates on the genetics of pain, new pharmacologic and treatment information, and much more.
  • Easily visualize important scientific concepts with a high-quality illustration program, now in full color throughout.
  • Choose the safest and most effective management methods with expanded coverage of anesthetic techniques.
  • Stay abreast of the latest global developments regarding opioid induced hyperalgesia, addiction and substance abuse, neuromodulation and pain management, identification of specific targets for molecular pain, and other hot topics.

Sujets

Ebooks
Savoirs
Medecine
Allodynia
Metamizole
Pain scale
Arthropathy
Reward
Behaviour therapy
Family medicine
Neuralgia
Microglia
Neoplasm
Endoscopic thoracic sympathectomy
Toothache
Ileus
Succinate dehydrogenase
Spinal cord injury
Transient receptor potential channel
Postherpetic neuralgia
Hyperalgesia
Gardner's syndrome
Interneuron
Epidural
Urticaria
Polyneuropathy
Opioid dependence
Anti-inflammatory
Random sample
Peripheral neuropathy
Abdominal pain
Cannabinoid receptor
Threshold of pain
Receptor (biochemistry)
Chest pain
Inhibitor
Bradykinin
Variegate porphyria
Diabetic neuropathy
Opioid
Osteoarthritis
Gabapentin
Itch
Pain management
Self-hypnosis
Childcare
Arthralgia
Long-term potentiation
Somatization disorder
Hypersensitivity
Tension headache
Fibromyalgia
Trigeminal neuralgia
Cluster headache
Palliative care
Imaging
Comorbidity
Cannabinoid
Irritable bowel syndrome
Internal medicine
General practitioner
Sexual dimorphism
Knee
Anticonvulsant
Local anesthetic
Ibuprofen
Back pain
Myalgia
Chronic pain
Placebo
Common cold
Paracetamol
Headache
Epidemiology
Glutamic acid
Carpal tunnel syndrome
Complex regional pain syndrome
Ophthalmology
Humanities
Multiple sclerosis
Dementia
Tricyclic antidepressant
Transcription factor
Radiation therapy
Rheumatoid arthritis
Peripheral nervous system
Pelvic inflammatory disease
Pharmacology
Plasticity
Positron emission tomography
Physiology
Non-steroidal anti-inflammatory drug
Neurology
Mechanics
Magnetic resonance imaging
Interstitial cystitis
Ion channel
Médecine
Sumatriptán
Lumbalgia
Vómito
Analgésico
Chronic fatigue syndrome
Brain stimulation
Knee pain
Opiate
Parkinson's disease
Spinal cord
Myocardial infarction
Peripheral nerve injury
Central pain syndrome
Ageing
Bicifadine
Neck pain
Membrane channel
Ascend
LMNA
Cognitive therapy
Radiculopathy
Rhizotomy
Medical procedure
Spinal manipulative therapy
Hyperesthesia
Immunity
Epilepsy
Epinephrine
Major depressive disorder
Dentistry
Conditioning
Chemotherapy
Antidepressant
Analgesic
Allele
Alternative medicine
Arthritis
Anxiety
Classical
Addictions
Human
Phantoms (film)
Amitriptyline
Heroin
Brain
Acupuncture
Gene
PubMed
Substance P
Neuraxis
Sex
Nociception
Insomnia
Release
Histamine
Fatigue
Delta
MU
Cytokine
Thorax
Inflammation
Menstruation
Death
Morphine
London
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Copyright

Informations

Publié par
Date de parution 01 mars 2013
Nombre de lectures 0
EAN13 9780702053740
Langue English
Poids de l'ouvrage 11 Mo

Informations légales : prix de location à la page 0,0744€. Cette information est donnée uniquement à titre indicatif conformément à la législation en vigueur.

Exrait

Wall & Melzack’s Textbook of Pain
Sixth Edition

Stephen B. McMahon, FMedSci, FSB
Sherrington Professor of Physiology, Director, London Pain Consortium, Academic Lead, Europain, Wolfson Centre for Age-Related Diseases, King’s College London, London, UK

Martin Koltzenburg, MD, FRCP
Professor of Clinical Neurophysiology, UCL Institute of Neurology
Co-Director, MRC Centre for Neuromuscular Diseases, University College London
Head of Department, Department of Clinical Neurophysiology, The National Hospital for Neurology and Neurosurgery, UCLH NHS Foundation Trust, Queen Square, London, UK

Irene Tracey, MA (Oxon.), PhD, FRCA
Nuffield Professor of Anaesthetic Science, Director, Oxford Centre for Functional Magnetic Resonance Imaging of the Brain
Head, Nuffield Division Anaesthetics, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK

Dennis C. Turk, PhD
John and Emma Bonica Professor of Anesthesiology and Pain Research, Director, Center for Pain Research on Impact, Measurement, and Effectiveness (C-PRIME), Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, Washington, USA
Table of Contents
Cover
Title page
Copyright
Dedication
Contributors
Foreword
Preface
Abbreviations and Acronyms
Section I: Neurobiology of Pain
Chapter 1: Peripheral Mechanisms of Cutaneous Nociception
Introduction
Properties of Nociceptors in Uninjured Skin
Relationship of Nociceptor Activity to Acute Pain Sensations
Hyperalgesia: Role of Nociceptors and Other Afferent Fibers
Primary Hyperalgesia
Inflammatory Mediators and Nociceptors
Second Messengers and Signal Transduction Pathways
Postoperative Pain and Hyperalgesia
Role of the Sympathetic Nervous System in Inflammation
Secondary Hyperalgesia
Effect of Aging on Nociceptive Properties
Efferent and Trophic Functions of Nociceptors
Nociceptors and Neuropathic Pain
Nociceptors and the Sympathetic Nervous System
Chapter 2: Molecular Biology of Sensory Transduction
Introduction
Chemo-, Thermo-, and Mechanotransducers
Polymodality
Indirect Signaling Pathways
Lessons Learned from Injury-Induced Changes
Conclusion
Chapter 3: Inflammatory Mediators and Modulators of Pain
Introduction
Overview Of Inflammatory Mediator Actions
Specific Pain Mediators
Immune Cells and Pain
Immune Cell and Neurotrophic Factors as Pain Mediators and Modulators
Conclusion
Chapter 4: Microglia: Critical Mediators of Pain Hypersensitivity after Peripheral Nerve Injury
Introduction
Spinal Microglia as Intermediaries in the Pathobiology of Neuropathic Pain
What are the Upstream Regulators of the Spinal Microglia Response to Peripheral Nerve Injury?
Interpreting Findings with so-Called Glial Inhibitors
The Critical Role of Microglial P2X4 Receptors in Peripheral Nerve Injury–Induced Pain Hypersensitivity
Transformation of Lamina I Output May Underlie Symptoms of Neuropathic Pain
Conclusion
Chapter 5: Neuroanatomical Substrates of Spinal Nociception
Introduction
The Laminae of Rexed
Primary Afferent Fibers
Projection Neurons, Substance P, and the Neurokinin 1 Receptor
Spinal Interneurons
Descending Monoaminergic Axons
Conclusion
Chapter 6: Spinal Cord Plasticity and Pain
Some Useful Definitions
Information Flow in Spinal Nociceptive Pathways
Plasticity in the Spinal Dorsal Horn Contributes to Hyperalgesia or Allodynia
Plasticity in Excitatory Nociceptive Pathways of the Spinal Dorsal Horn
Plasticity in Inhibitory Nociceptive Pathways of the Spinal Dorsal Horn
Plasticity in Descending Pathways
Other Proposed Mechanisms of Hyperalgesia and Allodynia
Non-Neuronal Pro-Nociceptive Helper Cells
Mechanisms of Secondary or Widespread Hyperalgesia
Multiplicity of Signaling Pathways
Sex Differences in Spinal Nociception and Antinociception
Reversal of Spinal Plasticity and Pain
Conclusion
Chapter 7: Representation of Pain in the Brain
Historical Perspective
Defining a Pain Network in the Brain
Brain Processing of the Multidimensionality of Pain
How do we Distinguish Location and Quality of Pain?
The Brain’s Role in Modulating Pain
Brain Structure and Function in Clinical Pain
Specific Chronic Pain Conditions
Conclusion
Chapter 8: Central Nervous System Mechanisms of Pain Modulation
Introduction
Descending Modulatory Control
Physiology of Pain-Modulating Neurons in the PAG–RVM System
The PAG–RVM System In Humans
Conclusion
Chapter 9: Development of Pain Pathways and Mechanisms
Introduction
Development of Peripheral Nociceptors
Maturation of Nociceptive Synaptic Transmission
Functional Maturation of Intact Spinal Nociceptive Circuits
Maturation of Supraspinal Descending Control Pathways
Development of Cortical Pain Processing
Development of Persistent Pain Mechanisms
Long-Term Effect of Early Injury on Nociceptive Processing
Chapter 10: Genetics of Pain
Individual Differences in Pain
Pain Genetics in Laboratory Animals
Pain Genetics Studies in Humans
Conclusions
Chapter 11: Animal Models of Pain
Introduction
Common Stimuli and Quantification of Evoked Responses
Models of Arthritis
Models of Neuropathic Pain
How well do the Models Reflect Clinical Conditions?
Behavioral Readouts
Use of Pharmacological Tools
Conclusion
Chapter 12: Ascending Projection Systems
Introduction
Ascending Nociceptive Pathways
Functional Role of Anterolateral Tract Axons
The Thalamus and Pain
An Overview of Spinothalamocortical Systems and Pain
Chapter 13: Autonomic, Endocrine, and Immune Interactions in Acute and Chronic Pain
Introduction
Bidirectional Communication Between the Brain and Body in Tissue Protection
Cytokines Generating Ongoing Pain and Hyperalgesia
Modulation of the Immune System by the Sympathetic Nervous System
Pain and Sympathetic Nervous System–Sympathetic Afferent Coupling
Neuroendocrine Modulation of Hyperalgesia
Synopsis
Chapter 14: Itch
Peculiarity of Itch Sensations
Classification of Itch
Primary Afferent Pruriceptive Neurons
Non-Histaminergic Pruriceptive Neurons
Spinal Pruriceptive Neurons
Supraspinal Itch Processing
Itch Modulation by Painful and Non-Painful Stimuli
Itch Mediators
Sensitization to Itch
Perspectives
Section II: Assessment and Psychology of Pain
Chapter 15: Gender Differences in Pain and Its Relief
Introduction
What are the Sex and Gender Differences in Pain?
Mechanisms Underlying Sex Differences in Pain
Sex and Gender Differences in Analgesia and Their Underlying Mechanisms
Genetic Factors
Future Directions
Chapter 16: Epidemiology of Pain
Introduction
Nature of the Epidemiological Evidence of Pain
Epidemiology of Regional Pain Syndromes and Widespread Body Pain
Conclusion
Chapter 17: Emotion, Motivation, and Pain
Perception of Aversiveness
Motivation and Value
Relief, Reward, and Opponency
Action and Decision Making
Economic Approaches to Pain
Conclusion
Chapter 18: Cognitive and Learning Aspects
Introduction
The Biobehavioral View
Cognitive Factors
Non-Associative Learning
Associative Learning
The Role of Social Learning
Conclusion
Acknowledgment
Chapter 19: Psychiatric Pain-Associated Co-morbidities
Affective Disorders (Depression)
Anxiety Disorders
Post-Traumatic Stress Disorder
Substance Dependence
Smoking/Nicotine Dependence
Pain Disorder
Conversion Disorder
Sleep Disorders
Sexual Disorders
Cognitive Disorders
Personality Disorders
Suicidality
Irritability, Anger, and Violence
Childhood Sexual Abuse
Fatigue
Obesity
Treatment Adherence
Secondary Gain, Litigation, Workers’ Compensation Status, and Malingering
Unexplained Symptoms
Non-Organic Signs/Waddell’s Signs
Myofascial Pain Syndrome and Non-Specific Low Back Pain
Neuropathic Pain
Illness Uncertainty
Somatization
Somatic Symptoms
Chapter 20: Studies of Pain in Human Subjects
Methods of Experimental Pain Stimulation
Properties of Stimulation Methods
Subjective Measures: Pain Psychophysics
Non-Verbal Measures
Relevance of Experimental Methods
Acknowledgment
Chapter 21: Pain Measurement in Adult Patients
Introduction
Dimensions of the Pain Experience
The Language of Pain
Pain Rating Scales
The Mcgill Pain Questionnaire
The Short-Form Mcgill Pain Questionnaire
Multidimensional Pain Experience
The Descriptor Differential Scale
The Pain Quality Assessment Scale
Behavioral Approaches to Pain Measurement
Physiological Approaches to Pain Measurement
Acknowledgment
Chapter 22: Pain in Older Persons
Introduction
Assessment of Pain Across the Adult Life Span
Age Differences in Experimental Pain
Neurobiology of Pain and Aging
Age Differences in Clinical Pain
Conclusion
Acknowledgment
Chapter 23: Measurement and Assessment of Pediatric Pain
Introduction
Assessment and Measurement
Strategies of Pain Measurement
Policy Implementation of Measures
Clinical Significance of Pain Measures
Coping Measures for Pain in Children
Conclusion
Chapter 24: Assessment of Pain Beliefs, Coping, and Function
Introduction
Pain Beliefs and Attitudes
Pain Coping
Function
Conclusion
Chapter 25: Hypnotic Analgesia
Introduction
What is Hypnosis?
Producing Hypnotic Analgesia
Effectiveness of Hypnotic Interventions for Acute and Chronic Pain
Conclusion
Chapter 26: Pain, Opiates, and Addiction
Introduction
Evidence of Tolerance and Dependence with Chronic Opioid Treatment
Evidence of the Analgesic Efficacy of Opiates Without Addiction
The Neurobiology of Addiction
Reward and the Mesolimbic Dopaminergic Pathway
Theories of Addiction
Molecular Changes Following Opiate Administration
Human Imaging Studies
Pain and Opiate Addiction
Pain and Reward can be Dissociated at a Molecular Level
Conclusion
Chapter 27: Placebo Analgesia
Introduction
Terminology
Active Placebo Responses Versus Statistical Artifacts
Evidence for Placebo Analgesia
Ingredients of Placebo Analgesia: What Makes a Placebo Responder?
Mechanisms of Placebo Analgesia
The Placebo Response in Clinical Practice
Acknowledgment
Section III: Pharmacology and Treatment of Pain
Chapter 28: Spinal Pharmacology of Nociceptive Transmission
Introduction
Excitatory Transmitters in the Afferent Components of Nociceptive Processing
Primary Afferents: Transmitters and Receptor Systems
Ascending Afferent Tracts
Modulation of the Encoding of Afferent-Evoked Activity
Spinal Dorsal Horn Receptor Systems
Conclusion
Acknowledgments
Chapter 29: Methods of Therapeutic Trials
Pain Measurement for Trials
Study Design and Validity
Trial Size
Pain “Models”
Checking Validity
Adverse Effects
Chapter 30: Opioids: Basic Mechanisms
Introduction
Opioid Receptors: Molecular Aspects
Opioid Physiology and Pain
Opioids and Neuropathic Pain
Opioids and Inflammation
Opioids in Cancer Pain
Anti-Opioid Systems
Opioid-Induced Hyperalgesia
Morphine Metabolites
Other Opioids
Side Effects of Opioids
Conclusion
Chapter 31: Opioids: Clinical Use
Introduction
Barriers to Clinical use of Opioids
Clinical Aspects of Various Opioid Analgesics
Routes of Administration for Opioids
Adverse Effects of Opioids
Use of Opioids in Specific Clinical Situations
Acknowledgment
Chapter 32: Cyclooxygenase Inhibitors: Basic Aspects
Mode of Action
Properties of NSAIDs and Coxibs in Clinical Use
Non-Acidic Antipyretic Analgesics
Chapter 33: Cyclooxygenase Inhibitors: Clinical Use
Introduction
Clinical Efficacy
Safety
Chapter 34: Antidepressant Analgesics
Introduction
Pharmacodynamics and Pharmacokinetics
Methods
Results
Pharmacogenetics and Antidepressants
Adverse Events (Table 34-7)
Discussion
Clinical Meaningfulness, Comparative Effectiveness Research, External Validity, Negative Randomized Controlled Trials, Combination Therapy
Approach to Therapy
Conclusion
Appendix A
Chapter 35: Mechanism of Action of Anticonvulsants as Analgesic Drugs
Introduction
Anticonvulsants with a Mechanism of Action Against Voltage-Gated Sodium Channels
Anticonvulsants with a Mechanism of Action Against Voltage-Gated Calcium Channels: Gabapentin and Pregabalin
Alternative Mechanisms of Action
Chapter 36: Anticonvulsants: Clinical
Introduction
Anticonvulsants that Act by Blocking Voltage-Gated Sodium Channels
Anticonvulsants that Act by Modulating the α2δ Subunits of VGSCs (Gabapentanoids)
Anticonvulsants Selectively Targeting Alternative Molecular Targets
Anticonvulsants that Act via Multiple Mechanisms of Action
Conclusion and Future Direction
Chapter 37: Local Anesthetic Blocks and Epidurals
Introduction
Peripheral and Sympathetic Blocks
Epidural and Intrathecal Analgesia
Epidural Steroids for Spinal Radicular Pain
Chapter 38: Cannabinoids
The Endocannabinoid System
Plant-Derived and Synthetic Cannabinoids
Mechanisms of Analgesia
Studies in Animal Models of Pathological Pain
Clinical Trials Testing the Efficacy of Cannabinoids
Chapter 39: Analgesic Drugs in Development
Introduction
The Drug Discovery and Development Process
Variations on the Theme of Non-Steroidal Anti-Inflammatory Drugs and Cyclooxygenase-2 Blockers
Opioid Analgesics
Cannabinoids and Adenosine Receptor Ligands
Adrenoceptor Agonists
Serotonin Receptor Ligands and Uptake Blockers
Excitatory Amino Acid Receptor Antagonists
Antagonists of the Actions of Substance P and other Neuropeptides
Voltage-Gated Ion Channel Blockers
Nicotinic Receptor Agonists
Capsaicin (TRPV1) Receptor Activators and Blockers
Prospects for the Future
Chapter 40: Neurosurgical Approaches to the Treatment of Pain
Decompression Procedures
Ablative Procedures
Intracranial Neuroablative Procedures
Conclusion
Acknowledgments
Chapter 41: Spinal Cord and Brain Stimulation
Introduction
Spinal Cord Stimulation
Intracranial Stimulation
Concluding Remarks: Central Stimulation for Pain
Chapter 42: The Cognitive-Behavioral Approach to Pain Management
Introduction
Overview of the Cognitive–Behavioral Perspective
Effectiveness of the Cognitive–Behavioral Approach
Chapter 43: A Critical Appraisal of Complementary and Alternative Medicine
Introduction
Expectations and Attitudes
Definitions of Complementary and Alternative Medicine and Scope of this Chapter
Acupuncture
Chiropractic Treatment
Massage Therapy
Herbal Medicine
Other Complementary and Alternative Medicine Options
Comment
Section IV: Clinical States/Deep Somatic Tissue
Chapter 44: Joint Pain: Basic Mechanisms
Introduction—Pain in Joints
Primary Afferent Neurons Supplying Joints
Spinal Cord Neurons with Input from the Joint
Processing of Joint Input in the Cortex, Thalamus, and Amygdala
Afferent Effects of the Nervous System on Joint Inflammation
Chapter 45: Basic Mechanisms of Muscle Pain
Introduction
General Properties of Muscle Nociceptors
Sensitization of Nociceptors
Occupational Muscle Pain (Chronic Work-Related Myalgia, Repetitive Strain Injury)
Spinal Mechanisms of Muscle Pain
Central Sensitization by Muscle Input
Influence of Muscle Pain on Locomotion
Glial Cells and Central Sensitization
Supraspinal Processing of Nociceptive Input from Muscle
Conclusions
Chapter 46: Postoperative Pain and Its Management
Introduction
Physiology of Postoperative Pain
Pharmacological Treatment of Postoperative Pain
Surgical Stress Response
Non-Pharmacological Treatments
Risk Factors for Chronic Pain
Postoperative Management Practice
Conclusions
Chapter 47: Osteoarthritis and Rheumatoid Arthritis
Introduction
Osteoarthritis
Rheumatoid Arthritis
Future Developments
Chapter 48: Fibromyalgia Syndrome and Myofascial Pain Syndrome
History of Soft Tissue Pain
Classification of Soft Tissue Pain
Diagnosis of Myofascial Pain Syndrome
Diagnosis of Fibromyalgia Syndrome
Assessment of Pain Severity in Fibromyalgia Syndrome
Epidemiology
Co-Morbid Conditions
Differential Diagnosis
Pathogenesis
Management Objectives
Outlook
Chapter 49: Low Back Pain
Introduction
Prevention
Treatment
Economic Evaluation
Guidelines
Conclusion
Chapter 50: Non-specific Arm Pain
Introduction
Key Signs and Symptoms
Risk Factors
Pathophysiology
Treatment
Rehabilitation
Prevention
Similarities with other Conditions
Acknowledgments
Section V: Clinical States/Viscera
Chapter 51: Visceral Pain: Basic Mechanisms
Introduction
Structural Basis of Visceral Nociception
Functional Basis of Visceral Nociception
Excitability of Visceral Afferent Neurons
Visceral Hypersensitivity
Visceral Organ Cross-Sensitization
Chapter 52: Thoracic Pain
Introduction
Innervation
Thoracic Pain—Clinical Findings and Syndromes
Analgesic Strategies
Conclusion
Chapter 53: A Clinical Perspective on Abdominal Pain
Introduction
Differential Diagnosis Of Abdominal Pain
Functional Gastrointestinal Disorders
Treatment Approaches To Patients With Abdominal Pain
Specific Treatments Of Abdominal Pain Associated With Functional Gastrointestinal Disorders
Conclusion
Acknowledgments
Chapter 54: Genitourinary Pain
Background
Clinical Assessment
Acute–Recurrent Urogenital Pain Syndromes
Chronic Urinary Tract Pain Syndromes
Chronic Urogenital Pain Unique to Women
Chronic Urogenital Pain Unique to Men
Acknowledgment
Chapter 55: Obstetric Pain
Introduction
Measurement and Severity of Obstetric Pain
Meaning, Significance, and Impact of Obstetric Pain
Mechanism and Pathways of Pain of Childbirth
Neurobiological Basis of Labor Pain
Physiological Effects of Labor Pain
Methods to Relieve Labor Pain
Side Effects and Complications of Neuraxial Labor Analgesia
Anesthesia-Related Maternal Mortality
Section VI: Clinical States/Headache and Facial Pain
Chapter 56: Trigeminal Mechanisms of Nociception
Introduction
Trigeminal Primary Afferents from Oro- and Craniofacial Tissues That Convey Nociceptive Input
Trigeminal Brain Stem Sensory Nuclei Integrate Nociceptive Input from Oro- and Craniofacial Primary Afferents
Trigeminal Brain Stem Second-Order Neurons: A Widespread Convergence Locus from Oro- and Craniofacial Input
Trigeminal Brain Stem Interneurons: A Substrate for Central Sensitization Phenomena
Spinal Trigeminal Complex Neurons Convey Craniofacial Nociceptive Input To Several Brain Regions
Endogenous, Central Mechanisms of Modulation of Trigeminal Nociception
Conclusion
Acknowledgment
Chapter 57: Acute and Chronic Orofacial and Dental Pain
Introduction
Neurobiology Of Orofacial Pain
Acute Orofacial Pain
Chronic Orofacial Pain
Conclusion
Chapter 58: Migraine and the Trigeminal Autonomic Cephalalgias
Introduction
Anatomy and Pathophysiology of Headache
Migraine
Trigeminal Autonomic Cephalalgias
Cluster Headache
Paroxysmal Hemicrania
Short-Lasting Unilateral Neuralgiform Headache Attacks with Conjunctival Injection and Tearing
Hemicrania Continua
Chapter 59: Tension-Type Headache
Classification and Diagnostic Features
Epidemiology
Pathophysiology
Treatment
Conclusion
Chapter 60: Pain in and around the Eye
Introduction
Ocular Sensory Innervation
Physiology of the Ocular Sensory Innervation
Sensations Evoked by Stimulation of the Ocular Surface
Pain Evoked by Ocular Diseases
Pain Evoked by Ocular Surgery
Trophic Consequences of Nerve Damage
Management of Ocular Pain
Prevention of Surgical Pain
Section VII: Clinical States/Neuropathic Pain
Chapter 61: Neuropathic Pain: Pathophysiological Response of Nerves to Injury
Introduction
Ectopic Impulse Discharge Is A Fundamental Cause of Spontaneous and Evoked Neuropathic Pain
Beyond Ectopia: Other Peripheral Nervous System Mechanisms That Distort Sensory Signals In Neuropathy
Cellular Processes In Nerve Pathophysiology
Therapeutic Efficacy: Why Some Drugs Work and Others
Chapter 62: Animal Models of Experimental Neuropathic Pain
Introduction
Animal Models of Neuropathic Pain
Outcome Measures in the Assessment of Neuropathic Pain in Animals Models
Conclusion
Chapter 63: Central Consequences of Peripheral Nerve Damage
Introduction
Gene Expression in Dorsal Root Ganglia After Nerve Injury
Some Methodological Aspects
Global Gene Expression Studies
Classic Growth Factors
Species Differences
Role of Voltage-Gated Sodium Channels
Mechanisms Underlying Injury-Induced Phenotypic Changes
Role of the DRG Neuron Cell Soma
Functional Consequences of Altered Gene Expression in the DRG
Cancer Pain
The Sprouting Paradigm
Functional Aspects of Spinal Cord Plasticity after Peripheral Nerve Damage
Conclusion
Acknowledgments
Chapter 64: Phantom Limb
Introduction
Clinical Characteristics
Mechanisms of Phantom Pain
Treatment
Prevention
Chapter 65: Painful Peripheral Neuropathies
Introduction
Pain in Peripheral Neuropathy
Clinical Features and Investigations of Peripheral Neuropathies
Polyneuropathies
Mononeuropathies and Multiple Mononeuropathies
Conclusion
Chapter 66: Trigeminal and Glossopharyngeal Neuralgia
Introduction and Aims
Trigeminal Neuralgia
Glossopharyngeal Neuralgia
Chapter 67: Complex Regional Pain Syndromes
Introduction
Definition of Crps
History
Epidemiology
Clinical Characteristics
Pathophysiological Mechanisms
Diagnostic Procedure
Therapy for CRPS
CRPS in Children
Prevention Studies
Prognosis
Acknowledgments
Chapter 68: Pain following Spinal Cord Injury
Introduction
Prevalence of Pain
Factors Related to Pain
Types of Pain after Spinal Cord Injury
Mechanisms
Patient Evaluation
Management
Chapter 69: Central Pain
Introduction
Overview of Central Pain Conditions
Epidemiology of Central Pain
General Symptoms and Signs of Central Pain
Specific Features
Diagnosis of Central Pain
Is Pain Associated with Central Nervous System Disease always Neuropathic?
Mechanisms of Pain
Management
Conclusion
Acknowledgments
Chapter 70: Pharmacological Therapy for Neuropathic Pain
Introduction
Mechanisms Underlying Neuropathic Pain and their Implications for Treatment
Assessment of Neuropathic Pain and its Implications for Treatment
Clinical Trial Evidence and Development of Treament Recommendations for Neuropathic Pain
Treatment of Neuropathic Pain—Pharmacological Classes
The Future of Neuropathic Pain Research and Clinical Practice
Summary and Conclusion
Chapter 71: Surgery for Back and Neck Pain (Including Radiculopathies)
Introduction
Diagnosis of Spinal Pain
Disease States for which Correlation with Pain is Strong
Back and Neck Pain as a Manifestation of Unexpected or Intercurrent Disease
Spondylotic Low Back Pain (Idiopathic)
Back and Neck Pain as a Manifestation of Psychiatric Disease: Implications for Surgical Therapy
Selction of Patients for Surgery
Alternatives to Surgery for Low Back Pain
Surgical Procedures: Lumbar and Cervical
Surgery for Tumors and Infection
Surgery for Spinal Hematoma
Section VIII: Clinical States/Cancer Pain
Chapter 72: Cancer Pain: Causes, Consequences, and Therapeutic Opportunities
The Clinical Problem
Development of a Murine Model of Bone Cancer
The Tumor Environment and Cancer Pain
Tumor-Induced Nerve Injury and Neuropathic Pain
Tumor-Induced Nerve Sprouting and Neuroma Formation
Central SensiTIzation in Cancer Pain
Conclusion
Chapter 73: Cancer Pain Assessment and Syndromes
Approach to Cancer Pain Assessment
Stepwise Approach to the Evaluation of Cancer Pain
Acute Pain Syndromes
Chronic Pain Syndromes
Breakthrough Pain
Conclusion
Chapter 74: Analgesic Therapy and Palliative Care in Children
Cancer in Children: Epidemiology and Prognosis
Pain and Distress at the Time of Initial Diagnosis of Cancer
Pain Secondary to Cancer Treatment
Chronic Pain in Long-Term Survivors of Childhood Cancer
Pain Caused by Tumor Progression
Interventional Approaches to Pain Management in Children with Cancer
Sedation in End-of-Life Care
Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome
Cystic Fibrosis
Neurodegenerative Disorders
Home, Hospice, or Hospital Care
Conclusion
Chapter 75: Cancer Pain: Treatment Overview
Introduction
Aims and Goals in Managing Cancer Pain
Treatment Options
Non-Pharmacological Measures for Relief of Pain
Specific Cancer Treatments
Specific Pain Problems in Cancer Patients
Chapter 76: Pain Control in the Care of the Dying
Introduction
An Overview of the Evidence
Diagnosing Dying
Fear and Impending Death
Pain and Sudden Death
Total Pain
Managing Pain in the Dying Patient
Conclusion
Index
Copyright

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WALL AND MELZACK’S TEXTBOOK OF PAIN  ISBN: 978-0-7020-4059-7
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Library of Congress Cataloging-in-Publication Data
Wall and Melzack’s textbook of pain / edited by Stephen B. McMahon ... [et al.]. -- 6th ed.
  p. ; cm.
 Textbook of pain
 McMahon’s name appears first on the 5th edition.
 Includes bibliographical references and index.
 ISBN 978-0-7020-4059-7 (hardcover : alk. paper)
 I. McMahon, S. B. (Stephen B.) II. Title: Textbook of pain.
 [DNLM: 1. Pain. 2. Pain Management--methods. WL 704]
616’.0472--dc23   2012029999
Executive Content Strategist: Michael Houston
Manager, Content Development: Rebecca Gruliow
Publishing Services Manager: Patricia Tannian
Senior Project Manager: Sharon Corell
Design Direction: Steven Stave
Printed in the United States of America
Last digit is the print number 9 8 7 6 5 4 3 2 1
Dedication



To Patrick Wall teacher, colleague, and friend
Contributors

Zahid Ali, PhD, Senior Director, Clinical Research, Pfizer Neusentis, Cambridge, UK

David A. Andersson, PhD, Lecturer in Physiology, Wolfson Centre for Age-Related Diseases, King’s College London, London, UK

A. Vania Apkarian, PhD, Professor, Department of Physiology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois, USA

Mark L. Baccei, PhD, Associate Professor, Department of Anesthesiology, University of Cincinnati, Cincinnati, Ohio, USA

Miroslav (Misha) Bačkonja, MD
Medical Director of Neuroscience, CRILifeTree Clinical Research, Salt Lake City, Utah
Emeritus Professor, Department of Neurology, University of Wisconsin, Madison, Wisconsin, USA

Ralf Baron, MD, Professor, Chair of the Division of Neurological Pain Research and Therapy, Department of Neurology, University Hospital Schleswig-Holstein, Kiel, Germany

Simon Beggs, PhD, Research Associate, Assistant Professor, Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada

Inna Belfer, MD, PhD, Associate Professor, Departments of Anesthesiology and Human Genetics, Director, Molecular Epidemiology of Pain Program, University of Pittsburgh/UPMC, Pittsburgh, Pennsylvania, USA

Carlos Belmonte, MD, PhD, Professor, Instituto de Neurociencias de Alicante, Universidad Miguel Hernandez-CSIC, Alicante, Spain

David L.H. Bennett, MB, PhD, Reader in Pain Neurosciences, The Nuffield Department of Clinical Neuroscience, The University of Oxford, Oxford, UK

Charles B. Berde, MD, PhD, Chief, Division of Pain Medicine, Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children’s Hospital, Professor of Anesthesia and Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

Odd-Geir Berge, DDS, PhD, Adjunct Professor, Department of Surgical Sciences, Uppsala, Uppsala Berzelii Center, Uppsala, Sweden

Stuart Bevan, PhD, Professor of Pharmacology, Wolfson Centre for Age-Related Diseases, King’s College London, London, UK

Klaus Bielefeldt, MD, PhD, Associate Professor of Medicine, Division of Gastroenterology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA

Andreas Binder, MD, Consultant, Division of Neurological Pain Research and Therapy, Department of Neurology, University Clinic Schleswig-Holstein, Kiel, Germany

Harald Breivik, MD, DMedSci, Professor, University of Oslo, Consultant, Department of Pain Management and Research, Oslo University Hospital, Rikshospitalet, Oslo, Norway

Kay Brune, MD, PhD, Doerenkamp Professor, Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany

M. Catherine Bushnell, PhD, Harold Griffith Professor, Department of Anesthesia, McGill University, Montreal, Quebec, Canada

Asokumar Buvanendran, MD, Director of Orthopedic Anesthesia, Professor of Anesthesiology, Rush University Medical Center, Chicago, Illinois, USA

James N. Campbell, MD, Professor Emeritus, Department of Neurosurgery, The Johns Hopkins University, CEO, Arcion Therapeutics, Baltimore, Maryland, USA

H. Isaac Chen, MD, Resident, Department of Neurosurgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, USA

Nathan I. Cherny, MBBS, FRACP, FRCP, Norman Levan Chair of Humanistic Medicine, Cancer Pain and Palliative Medicine Service, Shaare Zedek Medical Center, Jerusalem, Israel

John J. Collins, MBBS, PhD, FAChPM, FFPMANZCA, FRACP, Head of Department, Pain Medicine and Palliative Care, The Children’s Hospital at Westmead, Clinical Associate Professor, Discipline of Paediatrics and Child Health, University of Sydney, Sydney, New South Wales, Australia

A.D. (Bud) Craig, PhD
Atkinson Research Scientist, Barrow Neurological Institute, Phoenix, Arizona
Research Professor, Cell Biology and Anatomy, University of Arizona College of Medicine, Tucson, Arizona
Research Professor, Department of Psychology, Arizona State University, Tempe, Arizona, USA

John B. Davis, PhD, Biology Head, Convergence Pharmaceuticals Ltd., Cambridge, UK

John M. Dawes, PhD, Wolfson Centre for Age-Related Diseases, King’s College London, London, UK

Marshall Devor, PhD, Department of Cell and Developmental Biology, Institute of Life Sciences and the Center for Research on Pain, The Hebrew University of Jerusalem, Jerusalem, Israel

Anthony Dickenson, BSc, PhD, Professor of Neuropharmacology, Neuroscience, Physiology, and Pharmacology, University College London, London, UK

Andrew Dickman, DPharm, MSc
Consultant Pharmacist, Department of Palliative Care, Blackpool Teaching Hospitals NHS Foundation Trust, Blackpool, UK
Consultant Pharmacist, Marie Curie Palliative Care Institute Liverpool (MCPCIL), University of Liverpool, Liverpool, UK

Andrew Dilley, PhD, Lecturer in Anatomy, Division of Clinical and Laboratory Investigation, Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, UK

Ray J. Dolan, MD, Mary Kinross Professor of Neuropsychiatry, Wellcome Trust Centre for Neuroimaging, University College London, London, UK

Michael J. Dorsi, MD, Neurosurgeon, Ventura County Neurosurgical Associates, Community Memorial Hospital, Ventura, California, USA

Jonathan O. Dostrovsky, BSc, MSc, PhD
Professor Emeritus, Department of Physiology, Faculty of Medicine
Department of Oral Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada

John E. Ellershaw, MBBCh, MA, FRCP, Professor of Palliative Medicine, Marie Curie Palliative Care Institute Liverpool (MCPCIL), University of Liverpool, Liverpool, UK

Edzard Ernst, MD, PhD, FMedSci, FSB, FRCP, FRCPEd, Professor, Department of Complementary Medicine, Peninsula Medical School, University of Exeter, Exeter, Devon, UK

David Felson, MD, MPH, Professor of Medicine and Epidemiology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

Howard L. Fields, MD, PhD, Professor, Department of Neurology, University of California, San Francisco, San Francisco, California, USA

Nanna Brix Finnerup, MD, DrMedSc, Associate Professor, Danish Pain Research Center, Aarhus University, Aarhus, Denmark

David A. Fishbain, BSC (Hon), MSC, MD, Distinguished FAPA
Professor, Department of Psychiatry, Miller School of Medicine, University of Miami, Adjunct Professor, Departments of Neurological Surgery and Anesthesiology, Miller School of Medicine
Professor, Research, Rosomoff Pain Center, Miami, Florida, USA

Maria Fitzgerald, BA, PhD, FMedSci, Professor of Developmental Neurobiology, Department of Neuroscience, Physiology, and Pharmacology, University College London, London, UK

Herta Flor, PhD, Scientific Director, Department of Cognitive and Clinical Neuroscience, Central Institute of Mental Health and University of Heidelberg, Mannheim, Mannheim, Germany

Karen Forbes, MBChB, EdD, Professorial Teaching Fellow, University of Bristol, Honorary Consultant in Palliative Medicine, Department of Palliative Medicine, University Hospitals Bristol NHS Foundation Trust, Bristol, UK

Lucia Gagliese, PhD, Associate Professor, School of Kinesiology and Health Science, York University, Senior Scientist, Ontario Cancer Institute, University Health Network, Scientist, Department of Anesthesia and Pain Management, University Health Network, Toronto, Ontario, Canada

Gerald F. Gebhart, PhD, Director, Center for Pain Research, Department of Anesthesiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Jennifer L. Gibbs, MAS, DDS, PhD, Assistant Professor, Department of Endodontics, New York University, New York, New York, USA

Ian Gilron, MD, MSc, FRCPC, Director of Clinical Pain Research, Department of Anesthesiology & Perioperative Medicine, Queen’s University, Professor, Departments of Anesthesiology & Perioperative Medicine and Biomedical & Molecular Sciences, Queen’s University, Kingston, Ontario, Canada

Peter J. Goadsby, MD, PhD, DSc, Professor, Headache Group–Department of Neurology, University of California, San Francisco, San Francisco, California, USA

Michael S. Gold, PhD, Professor, Department of Anesthesiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Veeraindar Goli, MD, MBA, DFAPA
Vice President, Clinical Disease Area Expert–Pain, Primary Care Clinical Sciences, Pfizer, Inc., Cary, North Carolina
Emeritus Professor, Duke University Medical Center, Durham, North Carolina, USA

Allan Gottschalk, MD, PhD, Associate Professor, Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University, Baltimore, Maryland, USA

Richard H. Gracely, PhD
Professor, Regional Center for Neurosensory Disorders, University of North Carolina, Chapel Hill, North Carolina
Adjunct Professor, Department of Internal Medicine–Rheumatology, University of Michigan, Ann Arbor, Michigan, USA

Jane Greening, PhD, MSc, MCSP, Hon. Senior Research Fellow, Division of Clinical and Laboratory Investigation, Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, UK

Joel D. Greenspan, PhD
Professor and Chair, Department of Neural and Pain Sciences, University of Maryland School of Dentistry
Professor, Program in Neuroscience, University of Maryland, Baltimore, Maryland, USA

Arpana Gupta, PhD, Postdoctoral Research Fellow, Oppenheimer Family Center for Neurobiology of Stress, Semel Institute of Neuroscience and Human Behavior, Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA

Hermann O. Handwerker, MD, Professor, Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander, University Erlangen-Nuremberg, Erlangen, Germany

Kenneth M. Hargreaves, DDS, PhD, Professor, Departments of Endodontics, Pharmacology, Physiology, and Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

Jennifer A. Haythornthwaite, PhD, Professor, Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Mary M. Heinricher, PhD, Professor, Departments of Neurological Surgery and Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon, USA

Raymond G. Hill, BPharm, PhD, DSc (Hon), FMedSci, Visiting Professor of Pharmacology, Department of Medicine, Imperial College London, London, UK

Andrea G. Hohmann, PhD, Linda and Jack Gill Chair of Neuroscience and Professor, Department of Psychological & Brain Sciences, Indiana University, Bloomington, Indiana, USA

Tomas G.M. Hökfelt, PhD, MD, Professor of Histology and Cell Biology, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

Peter Hoskin, MD, FRCP, FRCR, Consultant in Clinical Oncology, Cancer Centre, Mount Vernon Hospital, Northwood, UK, Professor in Clinical Oncology, University College London, London, UK

Stephen P. Hunt, BSc, PhD, Professor of Molecular Neuroscience, Department of Cell and Developmental Biology, University College London, London, UK

Smriti Iyengar, PhD, Senior Research Scientist, Eli Lilly and Company, Indianapolis, Indiana, USA

Wilfrid Jänig, MD, Professor, Department of Physiology, Christian-Albrechts University Kiel, Kiel, Germany

Troels Staehelin Jensen, MD, DMSc, Professor, Department of Neurology and Danish Pain Research Center, Aarhus University Hospital, Aarhus, Denmark

Gareth T. Jones, BSc (Hon), MScEcon, PhD, Senior Lecturer in Epidemiology, Aberdeen Pain Research Collaboration (Epidemiology Group), School of Medicine and Dentistry, University of Aberdeen, Aberdeen, UK

Joel Katz, PhD
Professor and Canada Research Chair in Health Psychology, Department of Psychology, York University
Professor, Department of Anesthesia, University of Toronto, Director, Acute Pain Research Unit, Department of Anesthesia and Pain Management, Toronto General Hospital, Toronto, Ontario, Canada

Brigitte L. Kieffer, PhD, Translational Medicine and Neurogenetics, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France

H. Richard Koerber, PhD, Professor, Department of Neurobiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA

Bart Koes, MSc, PhD, Professor, Department of General Practice, Erasmus MC, Rotterdam, The Netherlands

Martin Koltzenburg, MD, FRCP, Professor of Clinical Neurophysiology, UCL Institute of Neurology, Co-Director, MRC Centre for Neuromuscular Diseases, University College London, Head of Department, Department of Clinical Neurophysiology, The National Hospital for Neurology and Neurosurgery, UCLH NHS Foundation Trust, Queen Square, London, UK

Jeffrey S. Kroin, PhD, Professor, Department of Anesthesiology, Rush Medical College, Chicago, Illinois, USA

Promil Kukreja, MD, PhD, Assistant Professor, Department of Anesthesiology, Medical Director, Regional Anesthesia Pain Service, University of Alabama at Birmingham, Birmingham, Alabama, USA

John Y.K. Lee, MD, Assistant Professor, Department of Neurosurgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Fred A. Lenz, MD, PhD, FRCS(C), A . Earl Walker Professor, Department of Neurosurgery, The Johns Hopkins Hospital, Baltimore, Maryland, USA

Jon D. Levine, MD, PhD, Professor, Department of Medicine, University of California, San Francisco, San Francisco, California, USA

Bengt Linderoth, MD, PhD, Professor, Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska Institutet/Karol University Hospital, Stockholm, Sweden

Arthur G. Lipman, PharmD, FASHP, University Professor, Department of Pharmacotherapy, College of Pharmacy, Adjunct Professor, Department of Anesthesiology, School of Medicine, Director of Clinical Pharmacology, Pain Management Center, University Healthcare, University of Utah Health Sciences Center, Salt Lake City, Utah, USA

Richard Lipton, MD, Edwin S. Lowe Professor, Vice Chair of Neurology, Professor of Epidemiology and Population Health, Professor of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, New York, USA

Donlin M. Long, MD, PhD, Distinguished Professor of Neurosurgery, Johns Hopkins Medical Institute, Baltimore, Maryland, USA

Timothy R. Lubenow, MD, Professor, Department of Anesthesiology, Rush University Medical Center, Chicago, Illinois, USA

Gary J. Macfarlane, BSc (Hon), MBChB, PhD, CStat, MD (Hon), Professor of Epidemiology, Aberdeen Pain Research Collaboration (Epidemiology Group), School of Medicine and Dentistry, University of Aberdeen, Aberdeen, UK

Patrick W. Mantyh, PhD, JD, Professor, Department of Pharmacology, University of Arizona College of Medicine, Tucson, Arizona, USA

Mitchell B. Max, MD † , , Departments of Anesthesiology and Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Emeran A. Mayer, MD
Professor, Department of Medicine, Physiology, and Psychiatry, Division of Digestive Diseases, University of California at Los Angeles
Director, Oppenheimer Family Center for Neurobiology of Stress, University of California at Los Angeles, Los Angeles, California, USA

John McBeth, MA, PhD
Reader, Arthritis Research UK Primary Care Centre, Keele University, Keele, UK
Honorary Reader, Arthritis Research UK Epidemiology Unit, University of Manchester, Manchester, UK

Patrick J. McGrath, OC, PhD, FRSC, FCAHS, Professor, Department of Psychology, Pediatrics, Psychiatry, Dalhousie, Vice President, Research and Innovation, IWK Health Centre and Capital District Health Authority, Halifax, Nova Scotia, Canada

Stephen B. McMahon, FMedSci, FSB, Sherrington Professor of Physiology, Director, London Pain Consortium, Academic Lead, Europain, Wolfson Centre for Age-Related Diseases, King’s College London, London, UK

Henry J. McQuay, DM, FRCA, FRCP, Emeritus Fellow, Balliol College, Oxford, Oxon, UK

Ronald Melzack, PhD, Professor Emeritus, Department of Psychology, McGill University, Montreal, Quebec, Canada

Siegfried Mense, MD, Professor, Department of Neuroanatomy/Neurophysiology, Heidelberg University, Medical Faculty Mannheim, Mannheim, Germany

Richard A. Meyer, BSEE, MS, Professor Emeritus, Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Björn A. Meyerson, MD, PhD, Professor Emeritus, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden

Jeffrey S. Mogil, PhD
E.P. Taylor Professor of Pain Studies, Department of Psychology and Alan Edwards Centre for Research on Pain, McGill University, Montreal, Quebec, Canada
Professor, Department of Anesthesiology and Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Andrew Moore, MA, DPhil, CChem, FRSC, FRCA, DSc, Nuffield Division of Anaesthesia, Nuffield Department of Clinical Neuroscience, University of Oxford, Oxford, UK

Valerie Morisset, PhD, Head of Electrophysiology, Convergence Pharmaceuticals Ltd., Cambridge, UK

Tuhina Neogi, MD, PhD, FRCPC
Associate Professor of Medicine, Clinical Epidemiology Research and Training Unit, and Rheumatology, Boston University School of Medicine
Associate Professor, Department of Epidemiology, Boston University School of Public Health, Boston, Massachusetts, USA

Timothy J. Ness, MD, PhD, Simon Gelman Professor, Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama, USA

Lone Nikolajsen, MD, PhD, DMSc, Clinical Associate Professor, Department of Anesthesiology, Danish Pain Research Center, Aarhus University Hospital, Aarhus, Denmark

Rodrigo Noseda, DVM, PhD, Instructor in Anesthesia, Department of Anesthesia, Critical Care, and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

E. Andrew Ochroch, MD, MSCE, Associate Professor, Department of Anesthesiology, Critical Care, and Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Michael H. Ossipov, PhD, Research Professor, Department of Pharmacology, University of Arizona College of Medicine, Tucson, Arizona, USA

Joanne E. Palmer, BSc (Hon), MSc, PhD, Head of Clinical Operations, Convergence Pharmaceuticals Ltd., Cambridge, UK

Peter H. Pan, MSEE, MD, Professor and Director of Clinical Research, Section of Obstetric and Gynecologic Anesthesia, Department of Anesthesiology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA

Bruce G. Pollock, MD, PhD, FRCPC, Professor and Director, Division of Geriatric Psychiatry, University of Toronto, Vice President, Research, Centre for Addiction and Mental Health, Toronto, Ontario, Canada

Frank Porreca, PhD, Professor, Department of Pharmacology, University of Arizona College of Medicine, Tucson, Arizona, USA

Donald D. Price, PhD, Professor Emeritus, Department of Oral and Maxillofacial Surgery, University of Florida, Gainesville, Florida, USA

Pierre Rainville, PhD
Professor, Department of Stomatology, Université de Montréal
Director, Laboratoire de recherche en neuropsychologie de la douleur, Centre de recherche de l’institut universitaire de gériatrie de Montréal, Montreal, Québec, Canada

Srinivasa N. Raja, MD
Professor, Department of Anesthesiology and Critical Care Medicine, Professor, Department of Neurology
Director, Division of Pain Medicine, The Johns Hopkins University, Baltimore, Maryland, USA

Andrew S.C. Rice, MBBS, MD, FRCA, FFPMRCA, Professor of Pain Research, Department of Surgery and Cancer, Imperial College, Hon. Consultant in Pain Medicine, Chelsea and Westminster Hospital NHS Foundation Trust, London, UK

Matthias Ringkamp, MD, Associate Professor, Department of Neurosurgery, The Johns Hopkins University, Baltimore, Maryland, USA

I. Jon Russell, MS, MD, PhD, ACR Master, Director, Fibromyalgia Research and Consulting, Arthritis and Osteoporosis Center of South Texas, Retired Faculty, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

Michael W. Salter, MD, PhD
Associate Chief, Science Strategy, Neurosciences & Mental Health Program, Hospital for Sick Children
Professor, Department of Physiology, University of Toronto Centre for the Study of Pain, University of Toronto, Toronto, Ontario, Canada

Jürgen Sandkühler, MD, PhD, Director, Center for Brain Research, Medical University of Vienna, Vienna, Austria

Simona Liliana Sava, MD, Headache Research Unit, University Department of Neurology Citadelle Hospital, University of Liège, Liège, Belgium

John W. Scadding, MD, FRCP, Honorary Consultant Neurologist, The National Hospital for Neurology and Neurosurgery, London, UK

Hans-Georg Schaible, MD, Director, Institute of Physiology 1/Neurophysiology, Jena University Hospital–Friedrich Schiller University Jena, Jena, Germany

Martin Schmelz, MD, PhD, Karl Feuerstein Professorship, Department of Anesthesiology, Heidelberg University, Mannheim, Germany

Jean Schoenen, MD, PhD, Professor, Department of Neurology, Headache Research Unit, University of Liège, Liège, Belgium

Stephan A. Schug, MD, FANZCA, FFPMANZCA, Professor and Chair of Anaesthesiology, Pharmacology and Anaesthesiology Unit, University of Western Australia, Director of Pain Medicine, Department of Anaesthesia and Pain Medicine, Royal Perth Hospital, Perth WA, Australia

Petra Schweinhardt, MD, PhD, Assistant Professor, Alan Edwards Center for Research on Pain, McGill University, Montreal, Quebec, Canada

Ben Seymour, MBChB, MRCP, PhD, Principal Investigator, Center for Information and Neural Networks, National Institute of Communications Technology, Japan, Wellcome Clinical Fellow, Computational and Biological Learning Lab, Department of Engineering, University of Cambridge, Consultant Neurologist, Addenbrookes Hospital, Cambridge, UK

Philip J. Siddall, MBBS, MM (Pain Mgt), PhD, FFPMANZCA, Associate Professor, Department of Pain Management, Greenwich Hospital, HammondCare, University of Sydney, Sydney, New South Wales, Australia

Maree T. Smith, BPharm (Hon), PhD, Director, Centre for Integrated Preclinical Drug Development, Professor of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia

Linda S. Sorkin, PhD, Professor, Department of Anesthesiology, University of California, San Diego, La Jolla, California, USA

Simon N. Tate, BSc, Chief Scientific Officer, Convergence Pharmaceuticals Ltd., Cambridge, UK

Timo T. Tervo, MD, PhD, Department of Ophthalmology, University of Helsinki, Chief Physician, Helsinki University Central Hospital, Helsinki, Finland

Mick Thacker, PhD, Lecturer, Biomedical Sciences, Centre of Human and Aerospace Physiological Sciences, King’s College London, London, UK

Andrew J. Todd, MBBS, PhD, Professor, Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, UK

Thomas R. Toelle, MD, PhD, Professor, Department of Neurology, Technische Universität München, München, Germany

Richard J. Traub, PhD, Professor, Department of Neural and Pain Sciences, University of Maryland School of Dentistry, Baltimore, Maryland, USA

Dennis C. Turk, PhD, John and Emma Bonica Professor of Anesthesiology and Pain Research, Director, Center for Pain Research on Impact, Measurement, and Effectiveness (C-PRIME), Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, Washington, USA

Anita M. Unruh, PhD, MSW, OT(c), Reg NS, Associate Dean (Research & Academic), Faculty of Health Professions, Dalhousie University, Halifax, Nova Scotia, Canada

Catherine E. Urch, MRCP, PhD, Palliative Medicine Consultant, Honorary Senior Lecturer, Imperial College Healthcare NHS Trust, Department of Palliative Care, Charing Cross Hospital, London, UK

Maurits van Tulder, PhD, Professor, Department of Health Sciences, Faculty of Earth and Life Sciences, VU University, Amsterdam, The Netherlands

Marcelo Villar, MD, PhD, Professor, Department of Neuroscience, Austral University, Buenos Aires, Argentina

Luis Villanueva, DDS, PhD, Director of Research, CNRS, Head, Pain Group, INSERM, Centre de Psychiatrie et Neurosciences, Paris, France

Tor D. Wager, PhD, Associate Professor, Department of Psychology and Neuroscience, University of Colorado, Boulder, Boulder, Colorado, USA

C. Peter N. Watson, MD, FRCPC, Assistant Professor, Department of Medicine, Division of Neurology, University of Toronto, Toronto, Ontario, Canada

Zsuzsanna Wiesenfeld-Hallin, PhD, Professor and Head of Section of Integrative Pain Research, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

Heng Yu Wong, MD, Director, HY Wong Gastrointestinal and Liver Specialist Clinic, Mount Elizabeth Medical Center, Singapore

Paul J. Wrigley, MBBS, MM, PhD, FANZCA, FFPMANZCA, Senior Lecturer, Pain Management Research Institute and Kolling Institute of Medical Research, University of Sydney, Pain Medicine Senior Staff Specialist, Pain Management Research Centre, Royal North Shore Hospital, Sydney, New South Wales, Australia

Xiao-Jun Xu, PhD, Associate Professor, Department of Physiology and Pharmacology, Section of Integrative Pain Research, Karolinska Institutet, Stockholm, Sweden

Tony L. Yaksh, PhD
Professor and Vice Chair for Research, Department of Anesthesiology
Professor, Department of Pharmacology, University of California, San Diego, La Jolla, California, USA

Joanna Maria Zakrzewska, MD, FDSRCS, FFDRCSI, FFPMRCA, FHEA, Professor, Head of Facial Pain Unit, Division of Diagnostic, Surgical and Medical Sciences, Eastman Dental Hospital, UCLH NHS Foundation Trust, London, UK

Hanns Ulrich Zeilhofer, MD
Professor, Institute of Pharmacology and Toxicology, University of Zurich
Professor, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland

Xu Zhang, PhD, Professor, Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

† Deceased
Foreword
The gate control theory of pain, which Patrick Wall and I published in 1965, led to an explosion of research on pain mechanisms in the spinal cord and brain and provided the rationale for a variety of new approaches to pain therapy. In 1984 we decided to edit a book with the latest information in the rapidly growing field so that clinicians could read about the status of laboratory and clinical research and scientists could learn about major clinical advances in the fight against pain. The first edition of the Textbook of Pain in 1984 was sold out in a year. It was followed by new editions that tracked the remarkable advances in the field of pain research and therapy. Shortly after publication of the fourth edition in 1999, Patrick Wall became ill. Our discussions about the Textbook of Pain now centered on the need to maintain a balance in presenting the two facets of the field of pain—research and therapy. That goal was achieved in 2006 by Stephen McMahon and Martin Koltzenburg in the fifth edition.
The scope of this sixth edition of the Textbook of Pain has been expanded by the addition of two new editors—Dennis Turk and Irene Tracey—who have made outstanding contributions to our understanding of the behavioral and brain mechanisms that underlie acute and chronic pain. We are all very grateful to Michael Houston, Elsevier’s outstanding publishing manager who ensured the timely publication of this up-to-date edition. I am delighted with it and I know that Patrick, who died on August 8, 2001, would be equally pleased.
Wall and I always aimed to achieve the broadest coverage of the field of pain in order to promote the fight against pain and suffering from every possible angle. Stephen McMahon, Dennis Turk, Irene Tracey, and Martin Koltzenburg have maintained this goal by producing this outstanding new edition. It is up to date and comprises a whole, unified body of knowledge that touches on every aspect of pain. The torch has been handed to an exciting new generation of editors and contributors. Pain—particularly chronic pain—continues to destroy the lives of millions of people worldwide. There is no nobler goal than achieving the relief of pain and suffering. This new edition will bring that day closer.


Patrick Wall (left) and Ronald Melzack.


Ronald Melzack (left) and Patrick Wall.

Ronald Melzack, Professor Emeritus, McGill University Montreal, Canada
Preface
The last edition of Wall and Melzack’s Textbook of Pain –the fifth edition–was published in 2006. There has been a considerable increase in our understanding of the nature and mechanisms of pain since that date. This is reflected in the enormous amount of published literature on pain. PubMed finds more than 160,000 publications since the last edition was published, using the search term “pain.” This represents about a 40% increase in publications compared with an equivalent period before publication of the fifth edition. Bibliometric data also shows how some topics within the pain field have become a greater focus of attention than others. For instance, a search for the phrase “neuropathic pain” shows a nearly 90% increase in publication numbers since publication of the last edition of this textbook. “Headache,” by contrast, shows a more modest increase, amounting to less than 30%. Technology has allowed some topics to be explored by greater numbers of researchers. The falling cost of DNA and RNA sequencing and associated technologies is likely to have contributed to some of the 60% increase in publications found with the search terms “genetics” and “pain.” Between the beginning of 2001 and the end of 2006, PubMed finds but a single publication with the search terms “epigenetics” and “pain.” Since then, 19 papers have emerged, and one suspects this will be the beginning of a new flood of interest.
The current edition of Wall and Melzack’s Textbook of Pain, the sixth, tries to capture and report on the most important developments in the field over the last 6 years. Collectively, the 147 authors who contribute to the current edition have probably read a large proportion of those 160,000 new publications. In this new edition we have retained the same general structure that we created for the fifth edition, but we have added some chapters to reflect new developments and merged others. The increasing body of literature also places burdens on the editors. For that reason I am tremendously grateful that Irene Tracey and Dennis Turk have joined the editorial team and applied their distinct expertise to refining this textbook.
Despite advancing knowledge in the field, the burden of pain remains unacceptably high. Epidemiological studies, many reviewed in this book, point to the high prevalence of chronic pain across the world associated with staggering socioeconomic costs. Unfortunately, existing therapies fail to offer good (let alone complete) pain relief to the majority of these sufferers. There have been some modest advances with the approval of some new therapies, such as topical capsaicin patches in some countries. A step chance in analgesic drug efficacy seems possible, too, as evidenced by the dramatic pain relief offered by blockers of NGF in a series of clinical trials–also reviewed in this book. We are still waiting to find out if side effects will limit or block this initiative. But the example serves to illustrate that a good understanding of pain and pain mechanisms can lead to effective therapies.
This is a difficult time for pharmaceutical companies, who have struggled with the many problems associated with translating new knowledge into new therapies in this area and many others. We hope that this new edition of Wall and Melzack’s Textbook of Pain will help all those interested in this field–academic scientists, clinicians, and industry leaders–to do their work more effectively. We sincerely hope they succeed in their efforts to bring about a positive change for another group of stakeholders here–the sufferers of pain.

Stephen B. McMahon, FMedSci, FSB
London
Abbreviations and Acronyms


ABC ATP-binding cassette
AC adenylate cyclase
ACC anterior cingulate cortex
ACG anterior cingulate gyrus
ACh acetylcholine
ACL anterior cruciate ligament
ACOG American College of Obstetricians and Gynecologists
ACPA anti–cyclic citrulated peptide antibody
ACR American College of Rheumatology
ACTH adrenocorticotropic hormone
ADAPT Arthritis Diet and Activity Promotion Trial
ADEPT attitude, diagnosis, education, physical treatment, living
ADP adenosine diphosphate
AEA arachidonyl ethanol amide
AED antiepileptic drug
2-AFC two alternative forced choice (method)
AFP atypical facial pain
2-AG 2-acylglycerol; 2-arachidonoylglycerase
AIA antigen-induced monarthritis
AIM ancestry informative marker
AIP acute inflammatory polyneuropathy
AMH A-fiber mechano-heat–sensitive nociceptor; A fibers responsive to mechanical and heat stimuli
AMI acute myocardial infarction
AMP adenosine monophosphate
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ANS autonomic nervous system
AO atypical odontalgia
AP action potential
APF antiproliferative factor
APM Association for Palliative Medicine of Great Britain and Ireland
APML acute promyelocytic leukemia
APSF Anesthesia Patient Safety Foundation
AS anxiety sensitivity
ASA American Society of Anesthesiologists
ASIC acid-sensing ion channel
ATF3 activated transcription factor 3
ATL aspirin-triggered lipoxin
ATP adenosine triphosphate
AU action unit
AUA American Urological Society
AVM arteriovenous malformation
BBB blood–brain barrier
BCG bacille Calmette-Guérin
BDI Beck Depression Inventory
BDNF brain-derived neurotrophic factor
BH 4 tetrahydrobiopterin
bHLH basic helix–loop–helix
BKN bradykinin
BMD bone mineral density
BMI body mass index
BMS burning mouth syndrome
BOCF baseline observation carried forward
BOLD blood oxygenation level–dependent
BPI Brief Pain Inventory
BPS bladder pain syndrome
BTcP breakthrough cancer pain
CABG coronary artery bypass grafting
CAIEB clinician-administered intermittent bolus
CAM complementary and alternative medicine; constitutively activated mutant
CAMKII calcium–calmodulin–dependent kinase II protein
CASPAR2 contactin-associated protein 2
CAV cyclophosphamide, Adriamycin (doxorubicin), and vincristine
CBF cerebral blood flow
CBT cognitive–behavioral therapy
CCI chronic constriction injury (model)
CCK cholecystokinin
CDH chronic daily headache
CEI continuous epidural infusion
CEP cortical evoked potential
CER control event rate
CES-D Center for Epidemiological Studies–Depression Scale
CFA complete Freund’s adjuvant
CFACS Child Facial Action Coding System
CFS chronic fatigue syndrome
cGMP cyclic guanosine monophosphate
CGRP calcitonin gene–related peptide
CH cluster headache
CHEOPS Children’s Hospital of Eastern Ontario Pain Scale
CHEP contact heat–evoked potential
CI confidence interval
CIA collagen-induced polyarthritis
CIBP cancer-induced bone pain
CIDP chronic inflammatory demyelinating polyneuropathy
CIPA congenital insensitivity to pain with anhidrosis
CISS constructive interference steady state (MRI)
CL centralateral
CLASS Celecoxib Long-term Arthritis Safety Study
CMH C-fiber mechano–heat–sensitive nociceptors
CMM conventional medical management
CMT Charcot–Marie–Tooth (disease)
CNCP chronic non-cancer pain
CNS central nervous system
COMT catechol O -methyltransferase
CONSORT Consolidated Standards of Reporting Trials
Cox, COX cyclooxygenase
CP chronic prostatitis
CPCI Chronic Pain Coping Inventory
CPM conditional pain modulation
CPP chronic pain patient; chronic pelvic pain; conditioned place preference
CPPS chronic pelvic pain syndrome
CPR cardiopulmonary resuscitation
CPSP central poststroke pain
CR conditional response
CREB cyclic AMP response element–binding protein
CRF corticotropin-releasing factor
CRH corticotropin-releasing hormone
CRHCS complexity regarding the health care system
CRPS complex regional pain syndrome
CS conditioned stimulus
CSCI continuous subcutaneous infusion
CSD cortical spreading depression
CSE combined spinal epidural (technique)
CSF cerebrospinal fluid; colony-stimulating factor
CSQ Coping Strategies Questionnaire
CSS CRPS severity score
CT computed tomography
CTB cholera toxin B
CTS carpal tunnel syndrome
CTTH chronic tension-type headache
CWP chronic widespread pain
CXCL1 C-X-C motif ligand 1
DA dopamine
DAG diacylglycerol
DAP depolarizing afterpotential
DAT dopamine transporter
DBS deep brain stimulation
DC dendritic cell
DCN dorsal column nuclei
ddC 2′,3′-dideoxycytidine
ddI 2′,3′-dideoxyinosine
DDS-I Descriptor Differential Scale: intensity dimension
DEG/ENac degenerin/epithelial sodium channel
DGL (DAGL) diacylglycerol lipase
DH dorsal horn
DHE dihydroergotamine
DHPG dihydroxyphenylglycine
DLPFC dorsolateral prefrontal cortex
DMARD disease-modifying antirheumatic drug
DMSO dimethylsulfoxide
DN4 Douleur Neuropathique en 4 questions
DNI distal nerve injury (model)
DNIC diffuse noxious inhibitory control
DOMS delayed-onset muscle soreness
DOR δ-opiate receptor
DPN diabetic painful neuropathy
DREAM downstream regulatory element antagonistic modulator
DREZ dorsal root entry zone
DRG dorsal root ganglion
DRR dorsal root reflex
DSM-IV Diagnostic and Statistical Manual of Mental Disorders, 4th edition
d4t stavudine
DTI diffusion tensor imaging
DVT deep vein thrombosis
DZ dizygotic
EAACI excitatory amino acid carrier 1
EC epidural compression
EDTMP ethylene diamine tetramethylene phosphonate
EEG electroencephalogram
EER experimental event rate
EERW enrolled enrichment with randomized withdrawal
EET epoxyeicosatrienoic acid
EGF epidermal growth factor
eGFR estimated GFR
EII embryonic day II
EM extensive metabolizer
EMDR eye movement desensitization and reprocessing
EMEA European Medicines Evaluation Agency
EMG electromyography
ENaC epithelial Na + channel
ENF epidermal nerve fiber
eNOS endothelial nitric oxide synthase
EP etoposide and cisplatin
EPH episodic paroxysmal hemicrania
EPSC excitatory post-synaptic current
EPSP excitatory post-synaptic potential
EQ European Quality of Life instrument
ERK extracellular signal–regulated kinase
ERP event-related potential
ES1 exteroreceptive suppression (silent) period 1
ESR erythrocyte sedimentation rate
ESSIC European Society for the Study of Interstitial Cystitis
ET-1 endothelin 1
ET a endothelin receptor A
ETTH episodic tension-type headache
EULAR European League Against Rheumatism
FA fractional anisotropy
FAAH fatty acid amide hydrolase
FAI femoral acetabular impingement (syndrome)
FAPS functional abdominal pain syndrome
FBSS failed back surgery syndrome
FCA Freund’s complete adjuvant
FD functional dyspepsia
FDA Food and Drug Administration
FGF fibroblast growth factor
FGID functional gastrointestinal disorder
FHM familial hemiplegic migraine
FIESTA fast imaging employing steady-state acquisition (MRI)
FIQ Fibromyalgia Impact Questionnaire
FISH fluorescence in situ hybridization
FLACC Face, Legs, Activity, Cry, Consolability
FM fibromyalgia
FMH familial hemiplegic migraine
FMPL N -formylmethionyl-leucyl-phenylalanine
fMRI functional magnetic resonance imaging
FMS fibromyalgia syndrome
FPS focal pain scale
FRAP fluoride-resistant acid phosphatase
5-FU 5-fluorouracil
GA gestational age
GABA γ-aminobutyric acid
GAD glutamic acid decarboxylase
GAT-1 GABA transporter type 1
GBS Guillain-Barré syndrome
G-CSF granulocyte colony-stimulating factor
GDNF glial cell line–derived neurotrophic factor
GEn gabapentin enacarbil
GERD gastroesophageal reflux disease
GFAP glial fibrillary acidic protein
GHQ General Hospital Questionnaire
GI gastrointestinal
GIRK G-protein–coupled inward rectifying potassium channel
Gly-IR glycine immunoreactivity
GlyR glycerine receptor
GM-CSF granulocyte–macrophage colony-stimulating factor
GON greater occipital nerve
GP general practitioner
GPCR G protein–coupled receptor
GpER extended-release gabapentin
GPN glossopharyngeal neuralgia
GREP Gender Role Expectations in Pain
GRPR gastrin-releasing peptide receptor
GS gastrocnemius–soleus
GW gestational weeks
GWAS genome-wide association study
HAART highly active antiretroviral therapy
HADS Hospital Anxiety and Depression Scale
HC hemicrania continua
hCG human chorionic gonadotropin
HCN hyperpolarization-activated cyclic nucleotide–gated (ion channel)
HD homeodomain
HETE hydroxyeicosatetraenoic acid
HGF hepatocyte growth factor
HIT Headache Impact Test
HIV human immunodeficiency virus
HLA human leukocyte antigen
HNC healthy normal control
HPC polymodal nociceptive cells
HPETE hydroperoxyeicosatetraenoic (acid)
HPOA hypertrophic pulmonary osteoarthropathy
HR heart rate
HRQoL health-related quality of life
HRT hormone replacement therapy
HSAN hereditary sensory and autonomic neuropathy
HSMN hereditary sensory and motor neuropathy
HSV herpes simplex virus
HT high-threshold (stimuli)
5-HT 5-hydroxytryptamine
HTN high-threshold (mechanoreceptor) mechanosensitive
5-HTP 5-hydroxytryptophan
IA intra-articular
IADR International Association for Dental Research
IASP International Association for the Study of Pain
IB4 isolectin B4
IBD inflammatory bowel disease
IBS irritable bowel syndrome
IBS-C irritable bowel syndrome with constipation
IBS-D irritable bowel syndrome with diarrhea
IBS-M irritable bowel syndrome with mixed bowel habits
IC insular cortex; interstitial cystitis
IC 50 inhibitive concentration of 50%
ICC intraclass correlation coefficient
ICD-9 International Classification of Diseases, ninth revision
ICHD International Classification of Headache Disorders
ICSS intracranial self-stimulation
IENF intraepithelial nerve fiber
IGLE intraganglionic laminar ending
IHS International Headache Society
IL interleukin
IM intermediate metabolizer
iMA intramuscular array
IMMPACT Initiative on Methods, Measurements, and Pain Assessment in Clinical Trials
INCB International Narcotics Control Board
iNOS inducible nitric oxide synthase
INR international normalized ratio
IP 3 inositol triphosphate
IPG implantable pulse generator
IPL inferior parietal lobule
IPSC inhibitory post-synaptic current
IPSP inhibitory post-synaptic potential
ISB interscalene brachial plexus blockade
IT intrathecal
ITS iontophoretic transdermal system
IVRS intravenous regional sympatholysis
JCAHO Joint Commission on Accreditation of Healthcare
JNK c-Jun N-terminal kinase
K/C kaolin and carrageenan
KCC2 potassium–chloride co-transporter-2
K/L Kellegren–Lawrence (OA grading system)
LANSS Leeds Assessment of Neuropathic Symptoms and Signs (pain scale)
LASIK laser in situ keratomileusis
LBP low back pain
LC locus coeruleus; Langerhans cell
LCP Liverpool Care Pathway for the Dying Patient
LEP laser-evoked potential
LFS low-frequency stimulation
LGI1 leucine-rich inactivated 1
LHRF luteinizing hormone–releasing factor
LHRH luteinizing hormone–releasing hormone
LIDSI lack of information about diagnosis or severity of the illness
LIF leukemia inhibitory factor
LIG leucine-rich repeat and immunoglobulin
LLI leg length inequality
LLLT low-level laser therapy
L -NAME N -nitro- L -arginine methyl ester
L -NMA N G -methyl- L -arginine
L -NMNA N G -monomethyl- L -arginine hydrochloride
LOCF last observation carried forward
LOX lipoxygenase
5-Lox 5-lipoxygenase
LP lumbar puncture
LPb lateral parabrachial area
LPS lipopolysaccharide
LS lumbosacral
LT low-threshold (stimuli)
LTB 4 leukotriene B 4
LTD long-term depression
LTM low-threshold mechanoreceptive/mechanosensitive (cell, afferent)
LTP long-term potentiation
M1 primary motor cortex
MA mechanically activated
MAO monoamine oxidase
MAP mitogen-activated protein (kinase)
MAPK mitogen-activated protein kinase
mBSA methylated bovine serum albumin
MCP-1 monocyte chemoattractant protein-1
MCS motor cortex stimulation
MD medial dorsal (nucleus)
MDD major depressive disorder
MDvc medial dorsal (nucleus), ventral caudal portion
MeCP2 methyl CpG binding protein 2
M3G morphine-3-glucuronide
M6G morphine-6-glucuronide
MEG magnetoencephalogram
MEK mitogen-activated protein/ERK kinase
MELAS mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes
MEP magnetic evoked potential
mEPSC miniature excitatory post-synaptic current
MFG medial frontal gyrus
MFPS myofascial pain syndrome
MGL monoacylglycerol lipase
mGlu metabotropic glutamate (receptor)
MGS mouse grimace scale
MGUS monoclonal gammopathy of undetermined significance
MHC major histocompatibility complex
MIA mechanically insensitive afferent (afferent fibers); mono-iodoacetate (model)
MIDAS Migraine Disability Assessment Scale
MINI Mini International Neuropsychiatric Interview
mIPSC miniature inhibitory post-synaptic current
MMPI Minnesota Multiphasic Personality Inventory
MOR μ-opioid receptor
MPEP 2-methyl-6-(phenylethynyl) pyridine
MPI Multidimensional Pain Inventory
MPQ McGill Pain Questionnaire
MPS myofascial pain syndrome
mPP mitochondrial permeability pore
MRA magnetic resonance angiography
mRFF minimum rhythmic firing frequency
Mrgprd Mas-related G protein–coupled
MRI magnetic resonance imaging
MRS magnetic resonance spectroscopy
MS multiple sclerosis
MSA mechanically sensitive afferent
MSG monosodium glutamate
mTOR mammalian target of rapamycin
MVD microvascular decompression
MZ monozygotic
N noradrenergic
NAA N -acetylaspartate
NA c nucleus accumbens
NAC N -acetylcysteine
nAChR nicotinic acetylcholine receptor
NADPH nicotinamide adenine dinucleotide phosphate
NAPE N -arachidonylphosphatidylethanolamine
nBR nociceptive component of the blink reflex
NCCP non-cardiac chest pain
NDPH new daily persistent headache
NDSA non-dermatomal sensory abnormality
NE norepinephrine
NET norepinephrine transporter
NF200 neurofilament 200
NFACS Neonatal Facial Action Coding System
NFCI non-freezing cold injury
NF-κB nuclear factor κB
NGF nerve growth factor
Ngn1 neurogenin 1
NGT nitroglycerin (glyceryl trinitrate)
NHANES National Health and Nutrition Examination Survey
NHP Nottingham Health Profile
NICU neonatal intensive care unit
NK natural killer (cell)
NK1 neurokinin 1
NKA neurokinin A
NMDA N -methyl- D -aspartate
NNH number needed to harm
nNOS neuronal nitric oxide synthase
NNQ number needed to harm
NNT number needed to treat
NO nitric oxide
NOS nitric oxide synthase
NP neuropathic pain
NPQ Neuropathic Pain Questionnaire
NPS Neuropathic Pain Scale
NPFF neuropeptide FF
NPY neuropeptide Y
NRS numerical rating scale
NS nociceptive-specific (cell)
NSAID non-steroidal anti-inflammatory drug
NSAP non-specific arm pain
NSRI serotonin–norepinephrine reuptake inhibitor
NT3 neurotrophin 3; neurotrophic factor 3
NYHA New York Heart Association
OA osteoarthritis
OARSI Osteoarthritis Research Society International
OFC orbitofrontal cortex
OIH opioid-induced hyperalgesia
OMERACT Objective Measures of Randomized Clinical Trials
OMIM Online Mendelian Inheritance in Man (database)
ONJ osteonecrosis of the jaw
OR odds ratio
OSA obstructive sleep apnea
PACAP pituitary adenyl cyclase–activating peptide
PAD primary afferent depolarization
PAF platelet-activating factor
PAG periaqueductal gray
PAOD peripheral arterial occlusive disease
PAP prostatic acid phosphatase
PAR protease-activated receptor
PASS Pain Anxiety Symptoms Scale
PB parabrachial nucleus (of the dorsolateral pons)
PBMC peripheral blood mononuclear cell
PCA patient-controlled analgesia
PCEA patient-controlled epidural analgesia
PCIA patient-controlled intravenous analgesia
PD personality disorder; Parkinson’s disease
PDA personal digital assistant
PDI Pain Disability Index
PDN painful diabetic neuropathy
PDPH post–dural puncture headache
PEA palmitoylethanolamine
PEPD paroxysmal extreme pain disorder
PET positron emission tomography
Pf parafascicular (nucleus)
PFC prefrontal cortex
PFMS primary fibromyalgia syndrome
PG prostaglandin
pgACC perigenual anterior cingulate cortex
PGP protein gene product (e.g., PGP 9.5)
PH paroxysmal hemicrania
PHN post-herpetic neuralgia
PI3K phosphatidyl-3′-kinase
PIEB programmed intermittent epidural bolus
PI-IBS postinfectious IBS
PIP 2 phosphatidylinositol 4,5-bisphosphate
PIPP Premature Infant Pain Profile
PKA protein kinase A
PKC protein kinase C
PLA 2 phospholipase A 2
PLD phospholipase D
PLC phospholipase C
PLP phantom limb pain
PLS phantom limb sensation
PM poor metabolizer
PMN polymorphonuclear
PNB peripheral nerve block
PNI peripheral nerve injury
PNS parasympathetic nervous system; peripheral nervous system
Po posterior complex (nucleus)
POMS Profile of Mood States
PONV postoperative nausea and vomiting
PoT posterior triangular (nucleus)
PPAR-γ peroxisome proliferator–activated receptor γ
PPT pressure pain threshold
PQAS Pain Quality Assessment Scale
Pr5 primary sensory trigeminal nucleus
PREP pain-related electrically evoked potential
PRI pain rating index
PRI-A pain rating index (affective)
PRI-S pain rating index (sensory)
PRI-T pain rating index (total)
PRK photorefractive keratectomy
PROMIS Patient-Reported Outcome Measurement Information System
PROSPECT Procedure-Specific Postoperative Pain Management
PSDC post-synaptic dorsal column pathway
PSNL partial sciatic nerve ligation (model)
PSQI Pittsburgh Sleep Quality Index
PT physical therapy
PTCA percutaneous transluminal coronary angioplasty
PTSD post-traumatic stress disorder
PV partial ventral
PVAS pain visual analog scale
PVB cis -platinum–vinblastine–bleomycin
PVD peripheral vascular disease
PVG periventricular gray
QC quick C (fiber)
QoL quality of life
QSART quantitative sudomotor axon reflex test
QST quantitative sensory test/testing; quantitative somatosensory thermotest
QTL quantitative trait locus
RA rheumatoid arthritis
rACC rostral anterior cingulate cortex
RAIC rostral anterior insular cortex
RANK receptor activator of NF-κB
RANKL RANK ligand
rCBF regional cerebral blood flow
RCT randomized controlled trial
REM rapid eye movement (sleep)
RET receptor tyrosine kinase
RF receptive field
RFT radiofrequency thermorhizotomy
r-HuEPO alfa recombinant human epoetin alfa
RLS restless legs syndrome
RR relative risk
RSD reflex sympathetic dystrophy
RSI repetitive strain injury
rTMS repetitive transcranial magnetic stimulation
RVM rostral ventromedial medulla; rostroventral medulla
S serotonergic
SC slow C (fiber)
SCI spinal cord injury
SCL-90R Symptom Checklist–90 Revised
SCORE Serious Complication Repository
SCR skin conductance response
SCS spinal cord stimulation
SCT spinocervicothalamic
SDH superficial dorsal horn
SDT sensory decision theory
SEP somatosensory evoked potential
SERP somatosensory event-related potential
SERT serotonin transporter
SF-36 36-item short form of the Medical Outcomes Society
SFL spontaneous foot-lifting (behavior)
SF-MPQ short-form McGill Pain Questionnaire
SFMS secondary fibromyalgic syndrome
SG substantia gelatinosa
SHT spinohypothalamic tract
SI, SII primary and secondary somatosensory cortices
sIL-6R soluble IL-6 receptor
SIP Sickness Impact Profile; sympathetically independent pain
siRNA small interfering RNA
sLORETA source analysis method of low-resolution brain electromagnetic tomography; standardized low-resolution brain electromagnetic tomography
Sm submedius (nucleus)
SMA supplementary/supplemental motor area
SMON subacute myelo-optic neuropathy
SMP sympathetically maintained pain
SNI spared nerve injury (model)
SNL spinal nerve ligation (model)
SNP single nucleotide polymorphism
SNRI serotonin–noradrenaline reuptake inhibitor
SNS sympathetic nervous system
SNSR sensory neuron–sensitive receptor
SOPA Survey of Pain Attitude
SP substance P
Sp5 spinal sensory trigeminal nucleus
Sp5C spinal sensory trigeminal nucleus caudalis subnucleus
Sp5I spinal sensory trigeminal nucleus interpolaris subnucleus
Sp5O spinal sensory trigeminal nucleus oralis subnucleus
SPECT single-photon emission computed tomography
SP–SAP substance P–saporin
SQUID superconductivity quantum induction device
SRD subnucleus reticularis dorsalis
SRF serum response factor
SSRI selective serotonin reuptake inhibitor
SSS Somatic Symptoms Score
sst/SST somatostatin
STAI State–Trait Anxiety Inventory
STD short-term depression
StEP Standardized Evaluation of Pain
STh sensory thalamic (nuclei)
STP soft tissue pain (syndrome)
STT spinothalamic tract
SUNA short-lasting unilateral neuralgiform headache attacks
SUNCT short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing
SV2A synaptic vesicle 2A
SVC superior vena cava
TA treatment adherence
TAC trigeminal autonomic cephalgia
TASK TWIK-related acid-sensitive K + channel
TBNS trigeminal brain stem nuclear complex
TCA tricyclic antidepressant
TCM traditional Chinese medicine
TENS transcutaneous electric nerve stimulation
TGF transforming growth factor
TGVS trigeminovascular system
THC tetrahydrocannabinol
TL thoracolumbar
TLR toll-like receptor
Tm transmembrane
TMD temporomandibular disorder
TMJ temporomandibular joint
TN trigeminal neuralgia
TNF-α tumor necrosis factor-α
TRAAK TWIK-related arachidonic acid K + channel
TRAK-1 TWIK-related K + channel 1
TRESK TWIK-related spinal cord potassium channel
trkA tyrosine kinase receptor A; tropomyosin-related kinase A
TRP transient receptor potential
TRPA1 transient receptor potential ankyrin 1
TRPV1 transient receptor potential vanilloid 1
TST sectioning of the tibial and sural nerves while leaving the common peroneal nerve intact (model)
TTH tension-type headache
TTS total tenderness score
TTX tetrodotoxin
TTXr tetrodotoxin-resistant
TUNEL terminal deoxynucleotidyl transfer nick end labeling
TWIK tandem of P domains in a weak inward rectifying K + channel
UK United Kingdom
UM ultrarapid metabolizer
UR unconditioned response
US unconditioned stimulus; United States
UTP uridine triphosphate
UVB ultraviolet B
VAS visual analog scale
Vc ventral caudal (nerve)
VCAM vascular cell adhesion molecule
Vcpc parvicellular part of the ventral caudal (nucleus)
VDS verbal descriptor scale
VGAT vesicular GABA transporter
VGCC voltage-gated calcium channel
VGLUT vesicular glutamate transporter
VGSL voltage-gated sodium channel
VIP vasoactive intestinal polypeptide
VL ventral lateral (nerve)
VLO ventral lateral orbital (cortex)
VMb basal part of the ventral medial (nucleus)
VMl ventromedial (thalamus)
VMpo posterior part of the ventral medial (nucleus)
VP ventral posterior (nucleus)
VPI ventroposterior inferior (nucleus)
VPL ventral posterior lateral (nucleus)
VPM ventral posterior medial (nucleus)
VRP ventral root potential
VRS verbal rating scale
VTA ventral tegmental area
VZV varicella-zoster virus
WDR wide–dynamic range (cell neuron)
WHO World Health Organization
WPI Widespread Pain Index
WC workers’ compensation
WOMAC Western Ontario and McMaster (Universities) Osteoarthritis Index
WS Waddell’s sign
YAG yttrium–aluminum–garnet
Section I
Neurobiology of Pain
Chapter 1
Peripheral Mechanisms of Cutaneous Nociception

Matthias Ringkamp, Srinivasa N. Raja, James N. Campbell and Richard A. Meyer


SUMMARY
Nociceptors are a specialized class of primary afferents that respond to intense, noxious stimuli. Unmyelinated nociceptors signal the burning pain from intense heat stimuli applied to the glabrous skin of the hand, as well as the pain from sustained pressure. Myelinated nociceptors signal the sharp pain from heat stimuli applied to hairy skin and from sharp mechanical stimuli. Both myelinated and unmyelinated nociceptors signal pain from chemical stimuli. Following a cutaneous injury, enhanced pain in response to cutaneous stimuli, called hyperalgesia , develops at the site of injury (primary hyperalgesia) and in the surrounding uninjured skin (secondary hyperalgesia). Tissue injury leads to enhanced responsiveness of nociceptors, called sensitization , which accounts for primary hyperalgesia. This sensitization is due to the local release of inflammatory mediators. Secondary hyperalgesia is due to sensitization of neurons in the central nervous system. When nerves are severed, spontaneous activity and ectopic mechanical, thermal, and chemical sensitivity develop in the injured nociceptors. The properties of nearby, uninjured nociceptors are also changed. In both injured and uninjured nociceptors, responsiveness to adrenergic agents can develop, which may account for involvement of the sympathetic nervous system in certain forms of neuropathic pain.


Introduction
One of the vital functions of the nervous system is to provide information about the occurrence or threat of injury. The sensation of pain, by its inherent aversive nature, contributes to this function. In this chapter we consider the peripheral neural apparatus that responds to noxious (injurious or potentially injurious) stimuli and thus provides a signal to alert the organism to potential injury. Investigators have studied cutaneous sensibility by recording from single nerve fibers in different species, including humans. Stimuli are applied to the receptive field (i.e., area of the tissue responsive to the applied stimulus) of single fibers, and the characteristics of the neural response are noted. We concentrate on the skin for three reasons. First, sensory receptors in the skin have been more thoroughly studied than receptors in any other tissue. Second, the opportunity to perform correlative psychophysical studies in animals and humans allows powerful inferences to be made regarding function. Third, cutaneous pain sensation is of great clinical significance. Diseases such as post-herpetic neuralgia and others associated with small-fiber neuropathies have profound effects on cutaneous sensory function and often lead to severe pain.
Highly specialized sensory fibers, alone or in concert with other specialized fibers, provide information to the central nervous system (CNS) not only about the environment but also about the state of the organism itself. In the case of the sensory capacity of the skin, cutaneous stimuli may evoke a sense of cooling, warmth, or touch. Accordingly, certain sensory fibers are selectively sensitive to these stimuli. Warm fibers, which are predominately unmyelinated, are exquisitely sensitive to gentle warming of their punctate receptive fields. These fibers have been shown to exclusively signal the quality and intensity of the warmth sensation ( Johnson et al 1979 ). Similarly, a subpopulation of the thinly myelinated, Aδ fibers respond selectively to gentle cooling stimuli and encode the sense of cooling ( Darian-Smith et al 1973 ). For the sense of touch, different classes of mechanoreceptive afferent fibers are exquisitely sensitive to deformations of the skin. These low-threshold mechanoreceptors encode such features as texture and shape.
A relatively high threshold for an adequate stimulus distinguishes the remaining class of cutaneous receptors. Because these receptors respond preferentially to noxious stimuli, they are termed nociceptors ( Sherrington 1906 ). Among the many varieties of sensory receptors, nociceptors are distinctive in that they typically respond to the multiple energy forms that produce injury (thermal, mechanical, and chemical stimuli) and provide information to the CNS regarding the location and intensity of noxious stimuli. Nociceptors may be subclassified with respect to four criteria: (1) unmyelinated C-fiber afferents (conduction velocity <2 m/sec) versus myelinated A-fiber afferents (conduction velocity >2 m/sec), (2) modalities of stimulation that evoke a response, (3) response characteristics, and (4) distinctive chemical markers (e.g., receptors expressed on the membrane). We first consider the properties of cutaneous nociceptors and then review how their function is thought to relate to the sensation of pain.
Tissue damage results in a cascade of events that lead to enhanced pain in response to natural stimuli, termed hyperalgesia . A corresponding increase in the responsiveness of nociceptors, called sensitization , occurs. The characteristics of hyperalgesia and its neurophysiological counterpart sensitization are discussed in a later section. Finally, we consider how nociceptors may play a role in accounting for the often severe pain that accompanies nervous system injury and disease.

Properties of Nociceptors in Uninjured Skin
Nature might have designed nociceptors such that each had the capacity to respond to the full complement of stimulus energy forms that pose potential risks to the organism (thermal, mechanical, and chemical). What nature has adopted instead is a mixed strategy whereby many nociceptors respond to multiple stimulus modalities (polymodal) and others have more specialized response properties. These specialized response properties probably at least in part account for different aspects of nociceptive sensory function (e.g., burning, aching, pricking, prickle, itch). As delineated later, nociceptors have distal effector functions as well, and specialization may also play a role here. The end result is that nociceptors have a complex biology and heterogeneous properties.
The receptive field of a nociceptor is often first localized by use of mechanical stimuli. Various other stimulus modalities are then applied to this receptive field. In most early studies of nociceptors, only heat and mechanical stimuli were used to study nociceptors. Therefore, the nomenclature of CMH and AMH is often used to refer to C-fiber mechano-heat–sensitive nociceptors and A-fiber mechano-heat–sensitive nociceptors, respectively. If a fiber responds to heat and mechanical stimuli, the fiber will in most cases respond to chemical stimuli as well ( Davis et al 1993b ). Thus, CMHs and AMHs may also be referred to as polymodal nociceptors.
The issue of whether a given nociceptor responds to a particular stimulus modality is perilous because the presumed lack of response to a given modality may in fact represent failure to apply the stimulus with sufficient intensity. The problem with the application of high-intensity stimuli is that the stimulus may alter the properties of the nociceptor in an enduring manner. A selection bias occurs: nociceptors with lower thresholds are more likely to be studied. The easiest way to find a nociceptor for electrophysiological study is to apply squeezing (mechanical) stimuli to the skin and thus identify the receptive field. This selection process identifies what are termed mechanically sensitive afferents (MSAs). In time it has become apparent that selection bias from this approach has led to oversight of an important class of nociceptors: mechanically insensitive afferents (MIAs). Because these fibers by definition have high mechanical thresholds (or are unresponsive to mechanical stimuli), finding the mechanical receptive field of these fibers is difficult. An alternative technique described by Meyer and colleagues (1991) has been to apply electrical stimuli to the skin to identify the putative receptive field. With this technique it turns out that about half of the Aδ-fiber nociceptors and 30% of the C-fiber nociceptors are MIAs, with MIAs being defined as afferents that have very high mechanical thresholds (>6 bar = 600 kPa = 60 g/mm 2 ) or are unresponsive to mechanical stimuli ( Handwerker et al 1991 , Meyer et al 1991 ). MIAs have also been reported in the knee joint ( Schaible and Schmidt 1985 ), viscera ( Häbler et al 1988 ), and cornea ( Tanelian 1991 ). As will be seen, this MIA–MSA distinction is of significance with regard to distinguishing nociceptor types. From the perspective of nomenclature, it is well to emphasize that MIAs are not defined as fibers that have no response to mechanical stimuli but rather as fibers that have a very high threshold (or no sensitivity at all) such that demonstration of a response to mechanical stimuli in electrophysiological studies is difficult.

C-Fiber Nociceptors
CMHs are commonly encountered cutaneous afferents, and activity of sufficient magnitude in these fibers is thought to evoke a burning pain sensation. The size of the receptive field appears to scale with the size of the animal. Typical values for monkey are between 15 and 20 mm 2 ( LaMotte and Campbell 1978 ), and for human they are near 100 mm 2 ( Schmidt et al 1997 ). There are often discrete areas of mechanical sensitivity (hot spots) within the receptive field, but in many fibers the areas of mechanical responsiveness tend to fuse over the region of the receptive field. Most CMHs respond to chemical stimuli (though not as well as A-fiber nociceptors; Davis et al 1993b ) and can therefore be considered polymodal.
Responses to heat stimuli have been studied in considerable detail. The response of a typical CMH to a random sequence of heat stimuli ranging from 41–49°C is shown in Figure 1-1 A. It can be seen that the response increases monotonically with stimulus intensity over this temperature range, which encompasses the pain threshold in humans. One ion channel involved in the transduction of heat at nerve terminals is thought to be the neuronal transient receptor potential ion channel V1 (TRPV1); activity in this channel increases with increasing temperature ( Caterina et al 1997 ). A detailed description of the neuronal ion channels involved in stimulus transduction is presented in Chapter 2 (for review see Dubin and Papapoutian 2010 ). Signal transduction molecules on keratinocytes may also play a role in heat transduction by inducing the release of adenosine triphosphate (ATP), which activates purinergic receptors (P2X 3 and P2Y 2 ) on the free nerve endings (see Fig. 1-4 ).


Figure 1-1 Responses of a typical C-fiber nociceptor and a warm fiber to heat stimuli. Heat stimuli ranging from 41-49°C and lasting 3 seconds were presented at 25-second interstimulus intervals to the glabrous skin of the monkey hand. Each stimulus occurred with equal frequency and was preceded by every other stimulus an equal number of times. Within these constraints, the order of stimulus presentation was randomized. Base temperature between stimuli was 38°C. A, Monotonic stimulus–response function for a typical nociceptor. B, Non-monotonic stimulus–response function for a typical warm fiber. The solid line represents the total response to a given temperature averaged across all presentations. The dotted lines represent the stimulus–response functions obtained when the preceding temperature was of low (41 and 43°C) or high (47 and 49°C) intensity. (Reproduced with permission from LaMotte RH, Campbell JN 1978 Comparison of responses in warm and nociceptive C-fiber afferents in monkey with human judgements of thermal pain. Journal of Neurophysiology 41:509–528.)
Two types of heat response are observed following a stepped heat stimulus. Quick C (QC) fibers exhibit their peak discharge during the rising phase of the heat stimulus, whereas slow C (SC) fibers exhibit their peak discharge during the plateau phase ( Fig. 1-2 B). The heat thresholds ( Fig. 1-2 C) and mechanical thresholds of QC fibers are significantly lower than those of SC fibers, thus suggesting that they may be located more superficially in the epidermis. QC fibers respond more vigorously to pruritic stimuli than do SC fibers, which suggests that they may be important in itch sensations ( Johanek et al 2008 ).


Figure 1-2 Two types of heat responses are observed in C-fiber nociceptors. A, Stepped heat stimulus (49°C, 3 seconds) used to classify heat response. B, The quick C (QC) fiber (yellow circles) exhibits a high-frequency discharge during the rising phase of the stimulus that adapts quickly (within 1 second). The slow C (SC) fiber (blue circles) exhibits a relatively uniform discharge throughout the stimulus period. Each circle represents the time of occurrence of an action potential. C, A histogram of the heat thresholds reveals that the distributions of QC and SC fibers are almost non-overlapping. (From Johanek LM, Meyer RA, Friedman RM, et al 2008 A role for polymodal C-fiber afferents in nonhistaminergic itch. Journal of Neuroscience 28:7659–7669.)
Thermal modeling studies combined with electrophysiological analysis have indicated that (1) the heat threshold of CMHs depends on the temperature at the depth of the receptor and not the rate of increase in temperature, (2) transduction of heat stimuli (conversion of heat energy to action potentials) occurs at different skin depths for different CMHs ( Tillman et al 1995b ), and (3) suprathreshold responses of CMHs vary directly with the rate of increase in temperature ( Tillman et al 1995a , 1995b ; Yarnitsky et al 1992 ). The depth of the heat-responsive terminals of CMHs varies quite widely (ranging from 20–570 μm; Tillman et al 1995b ). When a stepped temperature stimulus is applied to the skin, the temperature increases in the subsurface levels more slowly because of thermal inertia. The disparity in the surface temperature and the temperature at the level of the receptor varies directly with depth and indirectly with time. Given that the depth of CMH terminals varies widely, true heat thresholds are obtained when the rate of increase in temperature is very gradual or when the duration of the stimulus is very long. Although the literature reflects a wide range of heat thresholds for CMHs, when tested with these types of heat stimuli, the heat threshold of the majority of CMHs is in a remarkably narrow range of 39–41°C ( Tillman et al 1995b ).
The response of CMHs is also strongly influenced by the stimulus history. Both fatigue and sensitization are observed. One example of fatigue is the observation that the response to the second of two identical heat stimuli is substantially less than the response to the first stimulus. This fatigue is dependent on the time between stimuli, with full recovery taking longer than 10 minutes. A similar reduction in the intensity of pain after repeated heat stimuli is observed in human subjects ( LaMotte and Campbell 1978 ). Fatigue is also apparent in Figure 1-1 A, where the response to a given stimulus varied inversely with the intensity of the preceding stimulus. A decrease in the response to heat is also observed following mechanical stimuli applied to the receptive field or electrical stimuli applied to the nerve trunk ( Peng et al 2003 ). This suggests that fatigue in response to a given stimulus modality can be induced by heterologous stimulation, that is, by excitation with a stimulus of a different modality. Interestingly, recovery from cross-modal or heterologous fatigue is faster than recovery from fatigue induced by a stimulus of the same modality. Presumably, this is because these heterologous stimuli do not activate and therefore do not fatigue the stimulus transduction apparatus in the same way. Alternatively, fatigue may arise from independent effects on spike initiation (from antidromic stimulation) and transduction (from natural stimulation at the receptive field). Fatigue in response to heat stimuli is also seen in vitro when small (and presumably nociceptive) dorsal root ganglion (DRG) cells are repetitively tested with heat stimuli ( Greffrath et al 2002 ). The enhanced response, or sensitization, that may occur in CMHs after tissue injury is described below in the section on hyperalgesia.
Responses to mechanical stimuli are covered in more detail later. Suffice it here to indicate that CMHs usually display a slowly adapting response to mechanical stimuli of a given force. As noted later, MSA CMHs have a graded response to punctate stimuli, but their stimulus–response functions become saturated at levels substantially below the threshold for pain.
C-fiber MIAs are heterogeneous with regard to responses to chemical and heat stimuli, and some respond only to mechanical stimuli (but of course with a very high mechanical threshold). The sensitivity to mechanical stimuli has no obvious correlation to the heat threshold ( Davis et al 1993b ). In contrast to CMH afferents, some C-fiber MIAs in humans are vigorously excited when challenged with histamine or capsaicin. In addition, the activity observed in these C-fiber MIAs parallels the duration of the perception of itch (histamine) or burning pain (capsaicin) ( Schmelz et al 1997 , 2000b ). C-fiber MIAs may therefore act as chemosensors. In addition to pronounced chemosensitivity, these fibers have some other interesting properties that could account for pain in response to tonic pressure stimuli or the neurogenic flare response (see below).
Low-threshold C-fiber mechanoreceptors that do not respond to heat have been described in the cat ( Bessou and Perl 1969 ) and rabbit ( Shea and Perl 1985 ). In primates, including humans, these fibers have been found in proximal areas of the body ( Kumazawa and Perl 1977 , Nordin 1990 ) and the hairy skin on the forearm ( Vallbo et al 1999 ). These afferents are strongly activated by innocuous mechanical stimuli moved slowly across the receptive field, but they also respond to pinprick stimuli. The neuronal activity in these fibers is not critical for the perception of touch and, according to one imaging study, leads to the activation of the insular but not the sensory cortex (Olausson et al 2003). Low-threshold C-fiber mechanoreceptors are thought to mediate the sensation of “pleasant” touch and may therefore play an important role in “affiliative” behavior ( Vallbo et al 1999 , Wessberg et al 2003 , Löken et al 2009 ).
Some mechano-insensitive C fibers are reported to be activated by non-noxious and noxious cold and hot stimuli. It has been hypothesized that activity in these afferents may mediate the “hot–burning” sensations caused by such stimuli. These afferents may also be involved in mediating psychophysical phenomena such as “paradoxical heat” or the thermal grill illusion ( Campero et al 2009 ).
C-fiber afferents differ not only in their receptive features but also in their conductive properties. In fact, their conductive and receptive properties appear to correlate. When unmyelinated C-fiber afferents are activated repetitively by electrical stimuli, their conduction latency increases gradually (i.e., the conduction velocity of the afferent decreases). In addition, with increasing stimulation frequency, the amount of this activity-dependent slowing increases. Slowing in C-fiber MIAs is greater than in C-fiber MSAs ( Weidner et al 1999 ), and mechanosensitive nociceptive afferents show more pronounced slowing than do cold-sensitive C fibers, low-threshold C fibers, or sympathetic efferent C fibers ( Gee et al 1996 , Serra et al 1999 , Obreja et al 2010 , Ringkamp et al 2010 ). This difference in slowing properties indicates that the ion channels responsible for conduction may be different and suggests that the ion channels responsible for spike initiation at the receptive terminal may also differ between C-fiber classes.

A-Fiber Nociceptors
A-fiber nociceptors are thought to evoke pricking pain, sharpness, and perhaps aching pain. As a general rule, A-fiber nociceptors do what C-fiber nociceptors do, but do it more robustly. They respond at higher discharge frequencies, and the discriminable information supplied to the CNS is greater (e.g., Slugg et al 2000 ).
Two types of A-fiber nociceptors are apparent ( Dubner et al 1977 , Treede et al 1998 ). A summary of their properties is presented in Table 1-1 . Type I fibers are typically responsive to heat, mechanical, and chemical stimuli and may therefore be referred to as AMHs or polymodal nociceptors. Because the heat thresholds are high with short-duration stimuli (typically >53°C), the responsiveness of these fibers to heat has in some studies been overlooked. Consequently, these fibers have been called high-threshold mechanoreceptors (HTMs) by many investigators (e.g., Burgess and Perl 1967 ). Heat sensitivity in type I fibers is most likely mediated by the vanilloid receptor–like protein 1 (VRL1, renamed TRPV2) since it has a similar high threshold for activation by heat and is expressed in neurons with small myelinated axons ( Caterina et al 1999 ). When heat thresholds are determined with long-duration temperature stimuli, however, thresholds are in the mid-40–50°C range ( Treede et al 1998 ). Type I AMHs are seen in hairy and glabrous skin ( Campbell et al 1979 ) and have also been described in the cat and rabbit ( Fitzgerald and Lynn 1977 , Roberts and Elardo 1985 ). The mean conduction velocity of type I AMHs in the monkey is 25 m/sec and extends as high as 55 m/sec. Thus, by conduction velocity criteria, type I AMHs fall into a category between that of Aδ and Aβ fibers. Nearly all type I AMHs are MSAs. Their receptive field size is similar to that of CMHs, but the presence of “hot spots” in response to mechanical stimuli is much more obvious.

Table 1-1
Comparison of Type I and Type II A-Fiber Nociceptors
CHARACTERISTIC
TYPE I
TYPE II Heat threshold to short stimuli High Low Heat threshold to long stimuli Low Low Response to intense heat Slowly increasing Adapting Response latency to intense heat Long Short Peak latency to intense heat Late Early Mechanical threshold Most are MSAs Most are MIAs Conduction velocity Aδ and Aβ fibers Aδ fibers Sensitization to heat injury Yes No Location Hairy and glabrous skin Hairy skin
MIAs, mechanically insensitive afferents; MSAs, mechanically sensitive afferents.
Type II A-fiber nociceptors were encountered only infrequently in early studies. It turns out that this is because the thresholds to mechanical stimuli place the majority of these fibers in the MIA category. Many have no demonstrable response to mechanical stimuli. When an unbiased electrical search stimulus is used, however, the prevalence of type I and type II A-fiber nociceptors in the hairy skin of the primate is similar. They do not occur in the glabrous skin of the hand (where type I AMHs are prevalent). Their mean conduction velocity, 15 m/sec, is also lower than that of type I AMHs. Their responses to heat resemble those observed in CMHs, and they may also be mediated by the vanilloid receptor 1 (VR1 or TRPV1). Responses to endogenous inflammatory/algesic mediators resemble those seen with type I A-fiber nociceptors ( Davis et al 1993b ).
Examples of the differing responses of the two types of A-fiber nociceptors to a heat stimulus are shown in Figure 1-3 . Type I fibers exhibit a distinctive, gradually increasing response to heat. They sensitize to burn and chemical injury and probably play a role in the development of hyperalgesia. Type II fibers respond to heat in similar fashion to CMHs: early peak frequency and a slowly adapting response ( Treede et al 1995 ). As noted later, type II A-fiber nociceptors are thought to signal first pain sensation in response to heat and may also contribute to pain caused by the application of capsaicin to the skin ( Ringkamp et al 2001 ).


Figure 1-3 A-fiber nociceptors exhibit two types of responses to a heat stimulus. A, Scatter plot of peak discharge latency versus response latency for mechanically insensitive afferents (MIAs; purple symbols) and mechanically sensitive afferents (MSAs; green symbols) in response to a 53°C, 30-second stimulus. Receptors that had a long peak discharge latency were considered to have a type I heat response (squares). Receptors that had a short response latency and a peak discharge near stimulus onset were considered to have a type II heat response (circles). The type II heat response was found more frequently in the MIA group ( p ≤ 0.05, χ²-test). B, Average peristimulus frequency histogram (obtained with a 0.2-second bin width) of the response to the 53°C, 30-second stimulus for A-fiber nociceptors that had a type I heat response. C, Average peristimulus frequency histogram for A-fiber nociceptors that had a type II heat response. (Reproduced with permission from Treede RD, Meyer RA, Campbell JN 1998 Myelinated mechanically insensitive afferents from monkey hairy skin: heat-response properties. Journal of Neurophysiology 80:1082–1093.)
The conduction velocity of small myelinated Aδ fibers is, by definition, faster than that of unmyelinated C fibers. However, the terminal cutaneous branches of nociceptive Aδ fibers may conduct at a velocity characteristic of unmyelinated fibers (i.e., <2 m/sec) ( Peng et al 1999 ). In addition, these unmyelinated terminals may branch off the main axon several centimeters proximal to their cutaneous receptive field.

Nociceptors Can Be Classified by Molecular Markers
The anatomical and biochemical features of nociceptive afferents have been studied extensively to correlate these features with their receptive properties. A wide range of cell markers have been used to classify nociceptive afferents and to study their peripheral and central projections. These markers include molecules expressed on the cell surface (e.g., receptors, glycoconjugates), molecules stored and released from nociceptive afferents (e.g., peptides), and enzymes. Expression of receptors for neurotrophic factors is of interest since these factors may regulate the sensitivity of nociceptive afferents in physiological and pathological states such as inflammation and neuropathy. The size of neuronal populations expressing or co-expressing different markers varies between species ( Zwick et al 2002 ) and changes with the developmental stage ( Molliver et al 1997 , Guo et al 2001 ). Inflammation of the innervated tissue or a peripheral nerve lesion can cause substantial changes in the expression of these molecules. With the ongoing discovery of new marker molecules and the refinement of histological techniques, classification of nociceptive afferents is undergoing constant change and revision. Despite these “challenges,” however, classification of nociceptive afferents based on the expression of biochemical markers is instructive inasmuch as certain different neuronal populations are distinguishable across species. Sophisticated genetic manipulations have allowed the peripheral and central projections of defined neuronal populations to be studied in great detail. In addition, ablation experiments have been used to study the role of defined neuronal populations in animal behavior.
The cell bodies of nociceptive somatic and visceral afferents are located in DRGs. Slowly conducting Aδ and C fibers, including nociceptors, have small cell bodies ( Lawson and Waddell 1991 ). Some of these are labeled with an antibody directed against a neurofilament protein (NF200) and are therefore thought to correspond to the somata of small myelinated Aδ afferents.
Small DRG cells are subdivided into peptidergic neurons (i.e., neurons containing peptides such as substance P [SP], calcitonin gene–related peptide [CGRP], and somatostatin [SST]) and “non-peptidergic” neurons. In the rat, about 40% of DRG cells, 50% of C fibers, and 20% of Aδ fibers are classified as peptidergic ( McCarthy and Lawson 1989 , Lawson et al 1996 ). Non-peptidergic, nociceptive neurons contain fluoride-resistant acid phosphatase (FRAP) ( Silverman and Kruger 1988a ), and their somata and axons bind the plant isolectin B4 (IB4) from Griffonia simplicifolia ( Silverman and Kruger 1988b ). It is common practice to classify neurons as “peptidergic” or “non-peptidergic” based on their binding of IB4. However, considerable co-localization of SP or CGRP and IB4 or FRAP has been reported in rats but less so in mice ( Carr et al 1990 , Wang et al 1994 , Bergman et al 1999 , Price and Flores 2007 ). In vivo intracellular recordings combined with immunohistochemistry have shown that cells containing SP or CGRP or cells binding IB4 are nociceptive and that non-nociceptive cells do not label with these markers ( Lawson et al 1997 , 2002 ; Gerke and Plenderleith 2001 ).
A group of mas-related genes (Mrgs) have been discovered that are selectively expressed in small DRG neurons and encode G protein–coupled receptors (GPCRs) ( Dong et al 2001 ). Independently, sensory neuron–specific GPCRs (so-called sensory neuron–specific receptors [SNSRs]) in which the encoding genes were identical to some of the previously described Mrgs were identified shortly thereafter ( Lembo et al 2002 ). For some Mrgs (MrgA–C) identified in mice, no ortholog genes exist in human or non-human primates, but closely related Mrgs (so-called MrgXs) have been identified. For other Mrgs (MrgD–G), however, ortholog genes exist in humans. Mrgs are expressed mainly in non-peptidergic, IB4-positive neurons, with some Mrgs being expressed in distinct IB4 subpopulations. In in vitro recordings, MrgD + DRG cells showed characteristics typical of nociceptors (e.g., broad action potentials, expression of tetrodotoxin [TTX]-resistant sodium channels) ( Drussor et al 2008 ). Receptors encoded by Mrgs respond to a variety of ligands, including β-alanine, cortistatin, peptides derived from different opioid precursors, and different RFamide peptides ( Dong et al 2001 , Han et al 2002 , Lembo et al 2002 , Robas et al 2003 , Shinohara et al 2004 ), and they probably modulate excitability and sensitivity in this class of nociceptive afferents.
Expression of some markers appears to be related to the peripheral target tissue innervated by the neuron. Thus, almost all visceral afferents are peptidergic, but only about half the afferents projecting to the skin are (e.g., Perry and Lawson 1998 ) and only a small percentage of afferents projecting to muscle are labeled with IB4 ( Plenderleith and Snow 1993 , Ambalavanar et al 2003 ). MrgD-positive fibers exclusively innervate the skin, and they terminate in more superficial skin layers than do their peptidergic counterparts ( Fig. 1-4 ) ( Zylka et al 2005 ). Peptidergic and non-peptidergic afferents project to distinct dorsal horn laminae, with peptidergic fibers projecting mainly to lamina I and lamina II outer and IB4-binding afferents projecting preferentially to lamina II inner (e.g., Hunt and Rossi 1985 , Silverman and Kruger 1988b ; but see also Woodbury et al 2000 ).


Figure 1-4 Schematic illustration of unmyelinated fiber terminations in the epidermis. Non-peptidergic, MrgD + neurons terminate as free nerve endings in the most superficial layers of the epidermis. Peptidergic neurons terminate in deep layers of the epidermis. Some of the signaling receptors found on keratinocytes and free nerve endings are also illustrated. (Artwork by Ian Suk, Johns Hopkins University; adapted from Dussor G, Koerber HR, Oaklander AL, et al 2009 Nucleotide signaling and cutaneous mechanisms of pain transduction. Brain Research Reviews 60:24–35.)
Although all nociceptive neurons depend on nerve growth factor (NGF) during early development, in the adult only peptidergic neurons express its receptor TrkA (tropomyosin-related kinase A) ( Averill et al 1995 ). In contrast, most IB4-positive DRG cells do not express TrkA ( Molliver et al 1995 , but see also Kashiba et al 2001 ) but express one of the glial-derived neurotrophic factor (GDNF) family receptors (GDNFRα1–4) together with receptor tyrosine kinase Ret ( Bennett et al 1998 , Orozco et al 2001 ).
Peptidergic and non-peptidergic neurons express different receptors involved in signal transduction, and they may therefore display different sensitivity to a given stimulus. Thus the P2X 3 receptor, which mediates nociceptor excitation by ATP, is primarily expressed in IB4-positive neurons ( Vulchanova et al 1998 ). In contrast, TRPV1, which mediates responses to heat, capsaicin, and protons, is expressed in only a minority of IB4-positive cells in mice ( Zwick et al 2002 ). In rats, however, this segregation is less obvious since about half of both IB4-positive and -negative cells express TRPV1 ( Caterina et al 1997 ; Michael and Priestley 1999 ; Guo et al 1999 , 2001 ). Species differences also exist in the co-expression of different Mrgs and their co-expression with other markers of nociceptive neurons ( Zylka et al 2003 ).

Coupling between C-Fiber Nociceptors
Activation of one fiber by action potential activity in another is referred to as coupling . Coupling of action potential activity occurs between C fibers in the normal peripheral nerve of the monkey ( Meyer et al 1985a ). Coupling frequently involves conventional CMHs. Coupling is eliminated by injecting small amounts of local anesthetic at the receptive field of the CMH, thus indicating that the site of coupling is near the receptor. Collision studies indicate that the coupling is bidirectional. Sympathetic fibers do not appear to be involved in this coupling as demonstrated by experiments in which the sympathetic chain is stimulated or ablated ( Meyer and Campbell 1987 ). The role of coupling is unknown but it may relate to the flare response or other efferent functions of nociceptors (see below). Coupling between peripheral nerve fibers is also one of the pathological changes associated with nerve injury (e.g., Blumberg and Jänig 1982 , Meyer et al 1985b ). In this case, coupling occurs at the site of axotomy.

Anatomical Studies of Cutaneous Nociceptors
Immunostaining for protein gene product (PGP) 9.5, a carboxy-terminal ubiquitin hydrolase, has proved particularly sensitive in identifying small-diameter afferents in the skin ( Hsieh et al 1996 ). Vertical sections reveal that epidermal axons emerge from the superficial dermal nerve plexuses running beneath the epidermis. Schwann cells encase the axons at the dermal level, but as the axons rise into the epidermis between keratinocytes, the Schwann cell encasements are lost ( Kruger et al 1981 ). Both clear round and large dense-core vesicles are noted at the epidermal penetration site. The vesicles are similar morphologically to vesicles present in other cells involved in hormone and neurotransmitter secretion. It is presumed that these vesicles secrete their contents into tissues on activation (see the efferent role of nociceptors below). Some of these fibers appear to innervate Langerhans cells. In small-fiber neuropathies in which patients have pain and deficits in cutaneous pain sensibility, these axonal terminals stained by PGP 9.5 are markedly decreased ( Holland et al 1998 ).
As illustrated in Figure 1-4 , free nerve endings can be traced far into the epidermal layer. These free nerve endings are probably sensory and serve the sensations of pain, temperature, and itch. The parent axons of these unmyelinated terminals are probably both myelinated and unmyelinated. Some of these free nerve endings are peptidergic and contain SP or CGRP ( Gibbons et al 1987 ). Others are non-peptidergic and reach into the superficial layers of the epidermis.

Relationship of Nociceptor Activity to Acute Pain Sensations

Nociceptors and Pain in Response to Heat Stimuli

CMHs Signal Pain from Heat Stimuli to Glabrous Skin
We now examine the evidence that CMHs signal pain. In glabrous skin of the hand, two types of fibers, CMHs (not AMHs) and warm fibers, respond to short-duration heat stimuli (≤5 seconds) at temperatures near the pain threshold in humans (i.e., around 45°C). It is of interest, therefore, to compare how warm fibers and CMHs encode information about noxious heat stimuli. Warm fibers respond vigorously to gentle warming of the skin and are thought to signal the sensation of warmth ( Johnson et al 1979 ). An example of the response of a warm fiber to stimuli in the noxious heat range is shown in Figure 1-1 B. The response of warm fibers is not monotonic over this temperature range. In the example shown in Figure 1-1 B, the total response evoked at 49°C was less than that at 45°C. Psychophysical studies in humans demonstrate that pain increases monotonically with stimulus intensities between 40 and 50°C. Because the responses of CMHs increase monotonically over this temperature range ( Fig. 1-1 A) and the responses of warm fibers do not ( Fig. 1-1 B), it follows that CMHs probably signal the sensation of heat pain to the glabrous skin of the hand ( LaMotte and Campbell 1978 ).
Other evidence in support of a role for CMHs in pain sensation includes the following: (1) human judgments of pain in response to stimuli over the range of 41–49°C correlate well with the activity of CMH nociceptors over this range ( Fig. 1-5 , Meyer and Campbell 1981b ); (2) selective A-fiber ischemic blocks or C-fiber (local anesthetic) blocks indicate that C-fiber function is necessary for perception of thermal pain near the pain threshold ( Torebjörk and Hallin 1973 ); (3) the stimulus interaction effects observed in psychophysical experiments ( LaMotte and Campbell 1978 ) are also observed in recordings from CMHs ( Fig. 1-1 A); (4) the latency to pain sensation on glabrous skin following stepped changes in temperature is long and consistent with input from CMHs ( Campbell and LaMotte 1983 ); and (5) in patients with congenital insensitivity to pain, microscopic examination of peripheral nerves indicates an absence of C fibers ( Bischoff 1979 ).


Figure 1-5 Correlation of the response of C-fiber nociceptors in the monkey with pain ratings in human subjects. The close match between the curves supports a role for C-fiber nociceptors in heat pain sensation from glabrous skin. The first stimulus of the heat sequence was always 45°C. The remaining nine stimuli ranged from 41–49°C in 1°C increments and were presented in random order. Human judgments of pain were measured with a magnitude estimation technique: subjects assigned an arbitrary number (the modulus) to the magnitude of pain evoked by the first 45°C stimulus and judged the painfulness of all subsequent stimuli as a ratio of this modulus. The response to a given stimulus was normalized by dividing by the modulus for each human subject or by the average response to the first 45°C stimulus for the C-fiber mechano-heat–sensitive nociceptors (CMHs). (Originally published in Meyer RA, Campbell JN 1981 Peripheral neural coding of pain sensation. Johns Hopkins APL Technical Digest 2:164–171. Copyright 1981 AAAS.)

Human Microneurographic Recordings
Microneurography has been used to record from nociceptive afferents in awake humans and allows correlations between the discharge of afferents and the reported sensations of the subject. The technique involves percutaneous insertion of a microelectrode into fascicles of nerves such as the superficial radial nerve at the wrist. These studies have demonstrated that the properties of nociceptors in humans and monkeys are similar. In some experiments the microelectrode is also used to stimulate an identified, single nerve fiber in awake human subjects to evoke specific sensations. Some, however, argue that the size of the stimulating electrode is too large to stimulate individual units ( Wall and McMahon 1985 ). Given this reservation, the following evidence from microneurographic studies in humans points to the capacity of CMH activity to evoke pain: (1) intraneural electrical stimulation of presumed single identified CMHs in humans elicits pain ( Torebjörk and Ochoa 1980 ), (2) the heat threshold for activation of CMHs recorded in awake humans is just below the pain threshold ( Van Hees and Gybels 1981 ), and (3) a linear relationship exists between responses of CMHs recorded in awake humans and ratings of pain over the temperature range 39–51°C ( Torebjörk et al 1984 ).

Correlations between Psychophysical Measures of the Heat Pain Threshold and Neurophysiological Results
We noted above that the heat threshold of CMHs is dependent on temperature at the level of the receptor and is independent of the rate of change in temperature. At the same time when threshold temperature is measured at the surface of skin, CMHs have a lower threshold when the rate of increase in temperature is slow. As discussed earlier, the reason for this relates to thermal inertia.
Human pain thresholds are sometimes measured as the temperature that corresponds to the first report of pain as skin temperature is increased linearly (Marstock technique). Investigators have noted that faster rates of change in temperature lead to lower estimates of the heat pain threshold ( Yarnitsky and Ochoa 1990 , Tillman et al 1995a ). This is the opposite of the situation with the surface temperature threshold of CMHs but fits with the finding that suprathreshold responses of CMHs vary directly with the rate of increase in temperature. Thus it is unlikely that the threshold responses of CMHs are responsible for the heat pain thresholds. Rather, it appears that nociceptors must reach a certain discharge frequency (about 0.5 impulses/sec) for pain to be perceived ( Van Hees 1976 , Yarnitsky et al 1992 , Tillman et al 1995a ).

A-Fiber Nociceptors and Heat Pain
As shown in Figure 1-6 , a long-duration heat stimulus applied to the glabrous skin of the hand in human subjects evokes substantial pain for the duration of the stimulus. CMHs exhibit a prominent discharge during the early phase of the stimulus, but this response adapts within seconds to a low level. In contrast, type I AMHs are initially unresponsive but then discharge vigorously. Therefore, type I AMHs probably contribute to the pain during a sustained, high-intensity heat stimulus ( Meyer and Campbell 1981a ).


Figure 1-6 Ratings of pain by human subjects during a long-duration, intense heat stimulus (53°C, 30 seconds) applied to the glabrous skin of the hand compared with responses of C-fiber mechano-heat–sensitive nociceptors (CMHs) and type I A-fiber mechano-heat–sensitive nociceptors (AMHs). A, Pain was intense throughout the stimulus. B, The brisk response of CMHs at the beginning of the stimulus changed to a low rate of discharge after 5 seconds. C, The response of AMHs increased during the first 5 seconds and remained high throughout the stimulus. (Reprinted with permission from Meyer RA, Campbell JN 1981 Myelinated nociceptive afferents account for the hyperalgesia that follows a burn to the hand. Science 213:1527–1529.)
In hairy skin, stepped heat stimuli evoke a double pain sensation ( Lewis and Pochin 1937 ). The first perception is a sharp pricking sensation, and the second sensation is a burning feeling that occurs after a momentary lull during which little if anything is felt. Myelinated afferent fibers must signal the first pain since the latency of response to the first pain is too small to be carried by C fibers ( Campbell and LaMotte 1983 ). Type II A-fiber nociceptors (see Fig. 1-3 ) are ideally suited to signal this first pain sensation: (1) the thermal threshold is near the threshold temperature for the first pain ( Dubner et al 1977 ), (2) the receptor utilization time (time between onset of the stimulus and activation of the receptor) is short ( Treede et al 1998 ), and (3) the burst of activity at the onset of the heat stimulus is consistent with the perception of a momentary pricking sensation. The absence of a first pain sensation to heat stimuli applied to the glabrous skin of the human hand correlates with the failure to find type II A-fiber nociceptors on the glabrous skin of the hand in the monkey.
The preceding discussion indicates that nociceptors may signal pain in response to heat stimuli. However, two caveats are in order: (1) This does not mean that activity in nociceptors always signals pain. It is clear that low-level discharge rates in nociceptors do not always lead to sensation (e.g., Van Hees and Gybels 1981 , Cervero et al 1993 ). Central mechanisms, including attentional and emotional states, quite obviously play a crucial role in whether and how much nociceptor activity leads to the perception of pain. (2) It is probable that receptors other than nociceptors signal pain in certain circumstances. For example, the pain in response to light touch that occurs after certain nerve injuries or with tissue injury appears to be signaled by activity in low-threshold mechanoreceptors (see below).

Nociceptors and Pain in Response to Controlled Mechanical Stimuli

A-Fiber Nociceptors Signal Sharp Pain
A-fiber and C-fiber MSAs respond well to punctate mechanical stimuli. When a controlled-force stimulus is applied to the receptive field, the response is greatest at the onset of the stimulus and then slowly adapts. Like heat, repeated presentations of a mechanical stimulus lead to pronounced fatigue. A-fiber nociceptors recover faster from fatigue than do C-fiber nociceptors ( Fig. 1-7 ).


Figure 1-7 A-fiber nociceptors recover faster from fatigue than do C-fiber nociceptors. Mechanical stimuli were presented to the receptive field of A-fiber and C-fiber nociceptors at different interstimulus intervals (with 10 minutes between stimulus pairs). The A-fiber response (triangles) recovered within 60 seconds, whereas the C-fiber response (circles) took more than 150 seconds to recover. To normalize the data, the response to the test stimulus was divided by the response to the immediately preceding conditioning stimulus. (Adapted from Slugg RM, Meyer RA, Campbell JN 2000 Response of cutaneous A- and C-fiber nociceptors in the monkey to controlled-force stimuli. Journal of Neurophysiology 83:2179–2191.)
Much has been learned about the features of a mechanical stimulus that determine the response of nociceptors to mechanical stimuli. The discharge of nociceptors increases with increased force and pressure, but these functions vary depending on probe size: the smaller the probe, the greater the response ( Garell et al 1996 ). For cylindrical probes of different diameter, the discharges are comparable if the intensity of the stimulus is calculated according to force per length of the perimeter of the cylindrical probe. This suggests that the stress/strain maximum that occurs at the edge of the cylindrical stimulus is the critical parameter for excitation of nociceptor terminals.
For a given probe size, the response of A-fiber nociceptors increases monotonically with force, whereas the response of C-fiber nociceptors becomes saturated at higher force levels ( Fig. 1-8 A; Slugg et al 2000 ). In general, the discharge in A fibers is greater than that in C fibers.


Figure 1-8 Comparison of responses of nociceptors to mechanical stimuli in the monkey with pain ratings in human subjects. These data provide evidence that A-fiber nociceptors signal the pain reported from sharp probes. A, Average responses of A-fiber nociceptors (triangles) and C-fiber nociceptors (circles) to controlled-force stimuli. The A fibers exhibited a monotonically increasing response, whereas the response of the C fibers reached a plateau at the higher force levels (0.4-mm-diameter cylindrical probes; the total response to a stimulus 3 seconds in duration is plotted). B, Average pain ratings in response to controlled-force stimuli (open circle) increased monotonically in a manner comparable to that observed for the A-fiber nociceptors. Selective block of A-fiber function led to a significant decrease in pain ratings (filled circles). All pain ratings for a given subject were normalized by dividing by that subject’s average rating of the maximum stimulus (0.2-mm-diameter cylindrical probes, stimulus duration of 1 second). ( A, Adapted from Slugg RM, Meyer RA, Campbell JN 2000 Response of cutaneous A- and C-fiber nociceptors in the monkey to controlled-force stimuli. Journal of Neurophysiology 83:2179–2191; B, adapted from Magerl W, Fuchs PN, Meyer RA, et al 2001 Roles of capsaicin-insensitive nociceptors in cutaneous pain and secondary hyperalgesia. Brain 124:1754–1764.)
The area of the receptive field that responds to mechanical stimuli also responds to heat stimuli ( Treede et al 1990 ). However, the transducer elements that account for mechanosensitivity are probably different from those responsible for heat. For example, the heat response of nociceptors is readily sensitized by a heat injury, whereas the mechanical response is not (see below).
A-fiber nociceptors appear to be responsible for the sharp pain reported in response to punctate mechanical stimuli: (1) the reaction time to perception of pain is short, (2) the stimulus–response function of A-fiber nociceptors ( Fig. 1-8 A) is comparable to the pain ratings of human subjects ( Fig. 1-8 B) over a similar force range, and (3) the pain in response to sharp probes is dramatically reduced during selective blockade of A-fiber function ( Fig. 1-8 B; Magerl et al 2001 ).
Pretreatment of the skin with capsaicin abolishes heat pain sensitivity but does not greatly affect mechanical pain ( Magerl et al 2001 ). This suggests that the A-fibers involved in sharp pain are capsaicin insensitive; they could be type I AMHs or HTMs.

C-Fiber MIAs Signal Pain in Response to Tonic Pressure
When long-duration mechanical stimuli are applied to human subjects, the pain increases throughout the stimulus ( Adriaensen et al 1984 ). However, the response of MSAs to long-duration suprathreshold stimuli adapts with time. Although C-fiber MIAs are, by definition, normally insensitive to mechanical stimuli, they develop a response to prolonged mechanical stimulation ( Schmidt et al 2000 ). In addition, the pain associated with a tonic stimulus persists through selective A-fiber blockade ( Andrew and Greenspan 1999b ). Thus it appears that C-fiber MIAs signal the pain associated with tonic pressure.

Nociceptors and Cold Pain Sensation
Cold pain differs from heat pain in a number of important factors: (1) the cold pain threshold (≈14°C on hairy skin; Harrison and Davis 1999 ) is much farther from resting skin temperature (33°C) than the heat pain threshold (about 45°C), (2) the slope of the stimulus–response function is much steeper for heat pain than for cold pain ( Morin and Bushnell 1998 ), and (3) the lag in response between stimulus onset and pain report suggests that cold pain is subserved by deeper receptors whereas heat pain seems to be subserved by superficial receptors. Klement and Arndt (1992) demonstrated that cold pain could be evoked by cold stimuli applied within the veins of human subjects. A local anesthetic applied within the vein, but not in the overlying skin, abolished cold pain sensibility. It is therefore possible that cold pain is served, at least in part, by vascular receptors.
Just as the sensation of warmth is served by a specific set of primary afferents (predominantly C fibers), the sense of cooling is served by a specific set of primary afferents (i.e., cold fibers). Cold fibers are predominantly of the A type. They exhibit ongoing activity at room temperature, and their response increases markedly with gentle cooling. Stimuli that induce cold pain are not encoded well by these cold fibers. Although the majority of nociceptors have some response to ice stimuli applied to the skin, Simone and Kajander (1997) showed that all A-fiber nociceptors respond to cold stimuli below 0°C. C-fiber nociceptors may play a role in signaling cold pain sensation as well ( LaMotte and Thalhammer 1982 ). A non-selective cation channel has been identified (called ANKTM1 or transient receptor potential ankyrin 1 [TRPA1]) that has an activation threshold (17.5°C) comparable to the cold pain threshold ( Story et al 2003 ). This channel is found in a subset of nociceptive sensory neurons that are responsive to intense heat and capsaicin. However, the role of TRPA1 in mediating noxious cold is still debated.

Nociceptors and Chemically Evoked Sensations
Many chemical agents produce pain when applied to the skin. In many cases the pain from these agents probably results from tissue injury and is therefore indirect. (Chemical mediators associated with inflammation are described later.) One exception that has received a lot of attention is capsaicin. Intradermal injection of capsaicin produces intense burning pain that lasts for several minutes. When capsaicin is injected into the receptive field of C-fiber MSAs, the response is weak (relative to the heat response) and of short duration ( Baumann et al 1991 ). In contrast, A-fiber and C-fiber MIAs exhibit a long-lasting, vigorous response to capsaicin ( Schmelz et al 2000b , Ringkamp et al 2001 ), thus suggesting that these fibers are responsible for the pain induced by capsaicin. The pungent effects of capsaicin appear to be mediated by the TRPV1 receptor expressed on nociceptive fibers. This receptor appears to be activated by heat and protons (acid) as well.
Another chemical of interest is histamine, which produces a long-lasting itch when applied to the skin. Injection of histamine into the receptive field of C-fiber MSAs leads to a lasting response ( Johanek et al 2008 ). Iontophoresis of histamine into the receptive field of a subpopulation of C-fiber MIAs also produces a vigorous, long-lasting response ( Schmelz et al 1997 ), which suggests that both CMHs and C-fiber MIAs may play a role in histamine-induced itch. Histamine probably activates nociceptors via the H 1 receptor located on peripheral terminals.
Because cowhage spicules produce an intense itch that is not blocked by topical antihistamines ( Johanek et al 2008 ), and they provide a useful tool to investigate the chronic itch in patients that is resistant to antihistamine treatment. In about half of normal subjects, cowhage-induced itch is greatly attenuated during selective blockade of myelinated fibers. Although C-fiber MIAs do not respond to cowhage, QC fibers and A-fiber nociceptors respond vigorously to cowhage ( Ringkamp et al 2011 ). The active ingredient in cowhage is the cysteine protease mucunain, which activates nociceptive terminals via protease-activated receptor 2 (PAR-2) and PAR-4 ( Reddy et al 2008 ).

Hyperalgesia: Role of Nociceptors and Other Afferent Fibers
To understand the peripheral neural mechanisms of pain induced by noxious stimuli is to understand only one aspect of pain sensibility. There is, in fact, a dynamic plasticity that relates stimulus intensity and sensation. Of great biological importance in this regard is the phenomenon of hyperalgesia. Hyperalgesia is defined as a leftward shift of the stimulus–response function that relates the magnitude of pain to stimulus intensity. An example of this is seen in Figure 1-9 A, which shows human judgments of pain induced by heat stimuli before and after a burn. It is evident that the threshold for pain is lowered and pain in response to suprathreshold stimuli is enhanced.


Figure 1-9 Hyperalgesia and nociceptor sensitization after a cutaneous burn injury. Responses to heat stimuli were obtained 5 minutes before and 10 minutes after a 53°C, 30-second burn on the glabrous skin of the hand. The burn resulted in increases in the magnitude of pain (hyperalgesia) in human subjects that were matched by enhanced responses (sensitization) in type I A-fiber mechano-heat–sensitive nociceptors (AMHs) in the monkey. In contrast, C-fiber mechano-heat–sensitive nociceptors (CMHs) exhibited decreased sensitivity after the burn. A, Human judgments of pain. B, Responses of type I AMHs in the monkey. C, Responses of CMHs in the monkey. The same type of random heat sequence and normalization described in Figure 1-5 was used. Because the AMHs did not respond to the 45°C stimulus before the burn, the AMH data were normalized by dividing by the response to the first 45°C stimulus after the burn. (Reprinted with permission from Meyer RA, Campbell JN 1981 Myelinated nociceptive afferents account for the hyperalgesia that follows a burn to the hand. Science 213:1527–1529.)
Hyperalgesia is a consistent feature of somatic and visceral tissue injury and inflammation. Pharyngitis is associated with hyperalgesia in pharyngeal tissues such that merely swallowing induces pain. Micturition in the presence of a urinary tract infection is painful, again reflecting the presence of hyperalgesia. In arthritis, slight motion of the joint results in pain. A sunburn leads to pain with light touch and gentle heating.
The peripheral neural mechanisms of hyperalgesia have been studied in various tissues, including the joints, cornea, testicle, gastrointestinal tract, and bladder. Much of the theoretical work on hyperalgesia, however, has evolved from studies of the skin, and it is this work that will receive attention here.
Hyperalgesia occurs not only at the site of injury but also in the surrounding uninjured area. Hyperalgesia at the site of injury is termed primary hyperalgesia , whereas hyperalgesia in the uninjured skin surrounding the injury is termed secondary hyperalgesia ( Lewis 1935 ). Hyperalgesia exemplifies the functional plasticity of the nervous system. As we will see, the neural mechanisms for primary and secondary hyperalgesia differ.
In discussing hyperalgesia, it is useful to consider the following variables: (1) energy form of the injury, (2) type of tissue involved, (3) energy form of the test stimulus, and (4) location of the testing relative to the area injured. These variables interact in complex ways. For example, it will be shown that nociceptors will become sensitized to mechanical stimuli (the energy form of the test stimulus), but only after certain forms of injury (i.e., injection of inflammatory mediators).
An experimental design frequently used for study of the neural mechanisms of hyperalgesia is to characterize the response properties of a given fiber, then apply a manipulation that under usual circumstances would produce hyperalgesia, and finally assess whether this manipulation has altered the response properties of the fiber in question. Cutaneous hyperalgesia has been studied after thermal injury (burn or freeze lesions), after local administration of chemicals (e.g., capsaicin, mustard oil, or menthol), after a mechanical injury to the skin (e.g., incision, crushing), and after exposure to ultraviolet radiation. The main features of the hyperalgesia that develops after these various injuries are quite similar.
As shown in Figure 1-10 , the relative locations of the injury site, the test site, and the receptive field of the sensory neuron being studied dictate whether the experiment provides information regarding the mechanisms of primary or secondary hyperalgesia ( Treede et al 1992 ). These three variables may interact in any of six ways. As shown in Figure 1-10 , when the injury and the test site coincide ( Fig. 1-10 A and B), the study has provided a basis by which to consider the mechanism of primary hyperalgesia, whereas when the test site and the injury site diverge ( Fig. 1-10C– F), the study has provided a basis by which to account for secondary hyperalgesia.


Figure 1-10 Experimental configurations for testing the neural mechanisms of primary and secondary hyperalgesia. To study primary hyperalgesia, the site of injury (indicated by filled circles) and the site of testing (indicated by the X’s) must coincide. Alterations in the stimulus–response function from stimuli applied to the original receptive field (RF) (A) and expansion of the RF toward the injury site (B) are substrates for primary hyperalgesia. To study secondary hyperalgesia, the site of injury and the site of testing must not coincide (C and D) . Sensitization of the stimulus–response function as revealed by testing within the original RF may occur following injuries within (C) or outside the RF (D) . Expansion of the RF to include a test site outside the original RF may occur for injuries within (E) or outside (D) the RF. (Reprinted from Treede RD, Meyer RA, Raja SN, et al 1992 Peripheral and central mechanisms of cutaneous hyperalgesia. Progress in Neurobiology 38:397–421. Copyright 1992, with permission from Elsevier.)
When the paradigms shown in Figure 1-10 A and B are used, it is found that under certain circumstances, nociceptors exhibit an increased response to the test stimulus. Thus, peripheral neural mechanisms are likely to account for at least some aspects of primary hyperalgesia. In contrast, primary afferent nociceptors do not develop an enhanced response to the test stimulus when the paradigms shown in Figure 1-10C– F are investigated. By default, therefore, the mechanism for secondary hyperalgesia must reside within the CNS.

Primary Hyperalgesia

Hyperalgesia to Heat Stimuli
We first consider the situation in which a burn injury is applied to the skin and the test stimulus is heat applied to the location of the burn injury. When a burn is applied to the glabrous skin of the hand, marked hyperalgesia to heat develops as shown in Figure 1-9 A ( Meyer and Campbell 1981a ). The hyperalgesia is manifested as a leftward shift of the stimulus–response function that relates the magnitude of pain to stimulus intensity. For example, the 41°C stimulus was not painful before the burn but after the injury was as painful as the 49°C stimulus before the injury.

Peripheral Sensitization as a Mechanism for Primary Hyperalgesia to Heat Stimuli
Substantial evidence favors the concept that the primary hyperalgesia to heat stimuli that develops at the site of a burn injury is mediated by sensitization of nociceptors ( Meyer and Campbell 1981a , LaMotte et al 1982 ). Sensitization is defined as a leftward shift of the stimulus–response function that relates the magnitude of the neural response to stimulus intensity. Sensitization is characterized by a decrease in threshold, an augmented response to suprathreshold stimuli, and ongoing spontaneous activity. These properties correspond to the properties of hyperalgesia ( Table 1-2 ).

Table 1-2
Comparison of Characteristics of Hyperalgesia and Sensitization
HYPERALGESIA (SUBJECT RESPONSE)
SENSITIZATION (FIBER RESPONSE) Decreased pain threshold Decreased threshold for response Increased pain in response to suprathreshold stimuli Increased response to suprathreshold stimuli Spontaneous pain Spontaneous activity
To explain the hyperalgesia that occurs with a burn on the glabrous skin of the hand, a correlative analysis of subjective ratings of pain in humans with responses of nociceptors (CMHs and type I AMHs) in anesthetized monkeys was performed ( Meyer and Campbell 1981a ). Test heat stimuli were applied to the glabrous skin of the hand before and after a 53°C, 30-second burn. The burn led to prominent hyperalgesia in the human subjects ( Fig. 1-9 A). The CMHs showed a decreased response following the burn ( Fig. 1-9 C), whereas the type I AMHs were markedly sensitized ( Fig. 1-9 B). Thus, it is likely that for thermal injuries on the glabrous skin of the hand, AMHs, not CMHs, code for the heat hyperalgesia.
Sensitization is not a uniform property of nociceptors. Tissue type and the nature of the injury are important variables. For example, CMHs that innervate hairy skin become sensitized, whereas as described above, CMHs that innervate the glabrous skin of the hand do not become sensitized to a burn injury ( Campbell and Meyer 1983 ). Thus, CMHs appear to play a role in accounting for hyperalgesia to heat stimuli on hairy skin ( LaMotte et al 1983 ). These data support the conclusion that the hyperalgesia to heat stimuli that occurs at the site of an injury is due to sensitization of primary afferent nociceptors.

Hyperalgesia to Mechanical Stimuli
Distinguishing hyperalgesia to mechanical stimuli in the primary and secondary zones may be incorrect in some respects since the mechanism for hyperalgesia in the two zones may have some common elements. The mechanisms discussed in this section, however, will be limited to those applicable to the primary zone.
Different forms of mechanical hyperalgesia have been characterized. One form is evident when the skin is gently stroked with a cotton swab and is referred to as “stroking hyperalgesia,” “dynamic hyperalgesia,” or “allodynia.” The second form of hyperalgesia is evident when punctate stimuli, such as von Frey probes, are applied and, accordingly, has been termed “punctate hyperalgesia.” Hyperalgesia to tonic stimulation with a blunt probe, called “pressure hyperalgesia,” and impact hyperalgesia to shooting small bullets against the skin at a controlled velocity have also been described in the primary hyperalgesic zone ( Kilo et al 1994 ). As discussed in the later section on secondary hyperalgesia, the mechanism for these different forms of mechanical hyperalgesia is probably different. Stroking hyperalgesia is thought to be signaled by low-threshold mechanoreceptors, whereas punctate hyperalgesia is mediated at least in part by nociceptors. Pressure hyperalgesia and impact hyperalgesia are probably mediated by sensitized C fibers. Another form of mechanical hyperalgesia termed “progressive tactile hypersensitivity,” which may contribute to the allodynia associated with inflammation, has been described ( Ma and Woolf 1997 ).

Nociceptor Sensitization as a Mechanism for Mechanical Hyperalgesia in the Primary Zone
Primary hyperalgesia to mechanical stimuli appears to be due, at least in part, to sensitization of primary afferent nociceptors to mechanical stimuli. This sensitization is manifested in several ways.

Lowered Threshold
Thresholds to mechanical stimulation of either CMHs or AMHs recorded in primates or humans, as measured with von Frey hairs (a punctate stimulus), are not changed by heat and/or mechanical injury (e.g., Thalhammer and LaMotte 1982 , Campbell et al 1988a ). However, MIAs have been shown to develop mechanical sensitivity after inflammation. Figure 1-11 shows the response of an Aδ-fiber MIA to mechanical stimuli before and after exposure to a mixture of algesic inflammatory mediators (bradykinin, histamine, serotonin, and prostaglandin E 1 [PGE 1 ]). This MIA was unresponsive to the 5-bar von Frey probe initially, but a robust response to this probe developed after inflammation.


Figure 1-11 Example of sensitization to mechanical stimuli for an Aδ-fiber nociceptor following a chemical injection. A, The fiber did not respond to the application of a 5-bar stimulus for 15 seconds to the most sensitive area within its receptive field. The initial mechanical threshold for this fiber was 10 bar, and therefore it was a mechanically insensitive afferent (MIA). B, This MIA responded vigorously to a 10-μL intradermal injection of a chemical mixture containing 10 nmol bradykinin, 0.3 nmol prostaglandin E 1 , 30 nmol serotonin, and 30 nmol histamine. (Each asterisk indicates the time of needle insertion; bin size = 5 seconds). C, Sensitization to mechanical stimuli was demonstrated in this fiber 30 minutes after chemical injection. The fiber now responded to application of the 5-bar stimulus. Each vertical tic corresponds to the time of occurrence of an action potential. The von Frey threshold decreased (from 10 to 4 bar), and the receptive field area increased (from 9 to 88 mm 2 ). No response to heat was observed either before or after the injection. (Reproduced with permission from Davis KD, Meyer RA, Campbell JN 1993 Chemosensitivity and sensitization of nociceptive afferents that innervate the hairy skin of monkey. Journal of Neurophysiology 69:1071–1081.)

Increased Response to Suprathreshold Stimuli
Although inflammation does not result in a reduction in the mechanical threshold of AMHs and CMHs, responses to suprathreshold stimuli may be augmented ( Cooper et al 1991 ). Inflammation of the rat paw results in an enhanced response to suprathreshold mechanical stimuli, spontaneous activity, and expanded receptor fields for both A- and C-fiber nociceptors ( Andrew and Greenspan 1999a ).

Expansion of the Receptive Field
The receptive fields of AMH fibers, as well as some CMH fibers, expand modestly into the area of an adjacent heat ( Thalhammer and LaMotte 1982 ) or mechanical ( Reeh et al 1987 ) injury. As a result of this expansion, heat or mechanical stimuli delivered after the injury will activate a greater number of fibers. This spatial summation would be expected to induce more pain.

Loss of Central Inhibition as a Mechanism of Mechanical Hyperalgesia in the Primary Zone
Under usual circumstances, production of pain from activation of nociceptors with mechanical stimuli is inhibited in the CNS by the concurrent activation of low-threshold mechanoreceptors (e.g., Bini et al 1984 ). There is evidence that injury decreases the responsiveness of low-threshold mechanoreceptors. Hyperalgesia to mechanical stimuli in the primary zone could therefore be due to injury to low-threshold mechanoreceptors, which would lead to central disinhibition of nociceptor input and thus result in enhanced pain (i.e., hyperalgesia).

Inflammatory Mediators and Nociceptors
Injury results in the local release of numerous chemicals from non-neuronal cells (e.g., fibroblasts, mast cells, neutrophils, monocytes, and platelets), as well as from the sensory terminals of primary afferent fibers that mediate or facilitate the inflammatory process. Inflammatory mediators include prostaglandins, leukotrienes, bradykinin, serotonin, histamine, SP, thromboxanes, platelet-activating factor, purines such as adenosine and ATP, protons, and free radicals ( Fig. 1-12 , see also Basbaum et al 2009 ). Cytokines, such as interleukins and tumor necrosis factor, and neurotrophins, especially NGF, are also generated during inflammation. NGF not only is necessary for the survival of nociceptors during development but may also play an important role during inflammatory processes in adult animals. Some of these agents can directly activate nociceptors, whereas others act indirectly via inflammatory cells, which in turn release algogenic agents. Other mediators lead to sensitization of the nociceptor response to natural stimuli and therefore play a role in primary hyperalgesia. The variety of chemical mediators released during inflammation can have a synergistic effect in potentiating nociceptor responses.


Figure 1-12 Potential mediators of peripheral sensitization after inflammation. Tissue injury and inflammation lead to the release of numerous chemicals from non-neuronal and neuronal cells, such as mast cells, macrophages, platelets, immune and endothelial cells, Schwann cells, keratinocytes, fibroblasts, and peripheral nociceptor terminals. Mediators released include protons (H + ), purines (adenosine, adenosine triphosphate), nerve growth factor (NGF), cytokines such as tumor necrosis factor (TNF-α) and interleukins (IL-1β, IL-6), leukemia inhibitory factor (LIF), prostaglandin E 2 (PGE 2 ), bradykinin, histamine, serotonin (5-HT), platelet activating factor (PAF), and endothelin. These mediators may act directly to alter the sensitivity of peripheral nociceptors or indirectly via coupling to one or more peripheral membrane-bound receptors, including transient receptor potential (TRP) channels, acid-sensitive ion channels (ASICs), purinergic (P2X) receptors, G protein–coupled receptors (GPCRs), two-pore potassium channels (K 2P ), and receptor tyrosine kinase (RTK). Binding of the ligands to these receptors can initiate a cascade of events that includes activation of second-messenger systems (protein kinase A [PKA] and C [PKC]) and alteration of gene regulation. (Artwork by Ian Suk, Johns Hopkins University; adapted from Woolf CJ, Costigan M 1999 Transcriptional and posttranslational plasticity and the generation of inflammatory pain. Proceedings of the National Academy of Sciences of the United States of America 96:7723–7730.)
A variety of metabotropic and ionotropic receptors, including purinergic and glutamatergic receptors, have been identified on DRG cells and on the peripheral terminals of nociceptive afferent fibers. Activation of these receptors may modulate the sensitivity of peripheral nociceptors to exogenous stimuli ( Carlton and Coggeshall 1998 ).

Arachidonic Acid Metabolites
The prostaglandins, thromboxanes, and leukotrienes are a large family of arachidonic acid metabolites collectively known as eicosanoids. The eicosanoids are generally considered to not activate nociceptors directly but rather enhance the sensation of pain in response to natural stimuli and other endogenous chemicals by increasing the frequency of action potential firing (for reviews see Schaible et al 2002 , Cunha and Ferreira 2003 , Momin and McNaughton 2009 ). A sensitizing and direct excitatory effect of PGE 2 and PGI 2 , however, has been demonstrated in afferents innervating joints. Prostaglandins are synthesized by the constitutive enzyme cyclooxygenase-1 (Cox-1) and by Cox-2, an enzyme induced in peripheral tissues by inflammation ( Ballou et al 2000 ). Several prostaglandins, PGI 2 , PGE 1 , PGE 2 , and PGD 2 , are considered to play a role in inflammatory pain and hyperalgesia. Prostaglandins reduce the threshold for initiation of action potentials and increase the excitability of sensory neurons by decreasing the threshold for activation of a nociceptor-specific voltage-activated Na current, Na v 1.8, and increasing intracellular cyclic adenosine monophosphate (cAMP) levels ( England et al 1996 , Gold et al 1996 ). The prostaglandin-induced increase in firing frequency may also result from an increase in the hyperpolarization-activated current (Ih), which leads to faster depolarization toward the action potential threshold, the consequence of which is a decrease in the time interval between successive action potentials ( Momin and McNaughton 2009 ). Of the leukotrienes (metabolites of the lipoxygenase pathway), LTD 4 and LTB 4 have been suggested to play a role in hyperalgesia ( Levine et al 1984 ) and in sensitization to mechanical stimuli ( Martin et al 1987 ).

Bradykinin
Several lines of evidence suggest that bradykinin may also play a critical role in inflammatory pain and hyperalgesia (see Couture et al 2001 , Meini and Maggi 2008 for reviews). Bradykinin is released on tissue injury (e.g., from plasma), is present in inflammatory exudates, and excites and sensitizes unmyelinated and myelinated nociceptors to natural stimuli ( Beck and Handwerker 1974 , Khan et al 1992 ). Administration of exogenous bradykinin produces pain and transient hyperalgesia to heat in humans ( Manning et al 1991 ). Bradykinin acts on B 1 and B 2 receptors to induce nociceptor sensitization by activation of phospholipase C (PLC) and protein kinase C (PKC), production of arachidonic acids, and modulation of the TRPV1 channel (see the section on the vanilloid receptor below) ( Reeh and Sauer 1997 , Banik et al 2001 ).

Protons
The low pH levels found in inflamed tissue have led to the hypothesis that local acidosis may contribute to the pain and hyperalgesia associated with inflammation. Continuous administration of low-pH solutions in humans causes pain and hyperalgesia to mechanical stimuli ( Steen and Reeh 1993 ). This correlates with the observation that protons selectively activate nociceptors and produce sensitization of nociceptors to mechanical stimuli. Excitation of nociceptors by protons does not undergo tachyphylaxis or adaptation, and a synergistic excitatory effect of protons and a combination of inflammatory mediators has been reported ( Steen et al 1996 ).
A class of acid-sensing ion channels (ASICs), a subgroup of the degenerin/epithelial sodium channel (DEG/ENaC) family of proteins, has emerged as sensors of low pH (see Holzer 2009 , Sluka et al 2009 for review). ASICs signal moderate decreases in extracellular pH, in contrast to TRPV1, which is activated by severe acidosis (pH values below 6). ASIC1A and ASIC3 have been identified in DRG neurons, and their expression is increased by inflammation, nerve injury, and bone cancer, thus suggesting that ASICs may play a role in mediating or modulating pain in these conditions. The observation that a non-selective ASIC inhibitor, amiloride, reduces cutaneous acid-evoked pain in humans suggests that ASICs may be a potential therapeutic target for inflammatory pain ( Ugawa et al 2002 ).

Serotonin
Mast cells, on degranulation, release platelet-activating factor, which in turn leads to the release of serotonin (5-hydroxytryptamine [5-HT]) from platelets. Serotonin causes pain when applied to a human blister base ( Richardson and Engel 1986 ) and can activate nociceptors ( Lang et al 1990 ). Serotonin can also potentiate the pain induced by bradykinin and enhance the response of nociceptors to bradykinin. Additional evidence for a role of 5-HT in nociception stems from observations that 40% of lumbar DRG neurons, mostly small to medium-sized cells, are immunoreactive for the 5-HT 2A receptor and many of these cells also express the TRPV1 receptor ( Van Steenwinckel et al 2009 ).

Histamine
Release of SP from nociceptor terminals can cause the release of histamine from mast cells. Histamine can lead to a variety of responses, including vasodilatation and edema. The role of histamine in pain sensation is less clear since application of exogenous histamine to the skin produces itch and not pain sensations ( Simone et al 1991a ). Histamine excites polymodal visceral nociceptors, especially when applied in high concentrations ( Koda et al 1996 ), and potentiates the responses of nociceptors to bradykinin and heat ( Mizumura et al 1995 ). Mechanosensitive cutaneous nociceptors in rats and humans respond only weakly to histamine ( Lang et al 1990 ), but a subpopulation of mechano-insensitive C fibers was vigorously excited by histamine ( Schmelz et al 1997 ). Activation of histamine H 3 receptors, a ligand-gated ion channel that modulates the influx of Na + , however, leads to decreased release of inflammatory peptides and reduced pain and inflammation ( Cannon et al 2007 ).

Purines
During inflammation and tissue injury, purines such as adenosine and its mono- or polyphosphate derivatives (AMP, ADP, ATP) may be released or leak into the extracellular space and activate nociceptors (for review see Burnstock 2009 ). Platelets are a rich source of ATP, and aggregation of platelets or lysis of cells can lead to release of ATP. Adenosine and its phosphates have been reported to induce pain in a human blister base. Intra-arterial or intradermal injection of adenosine also causes pain, and intravenous/intracoronary infusion of adenosine induces angina-like symptoms ( Sylvén et al 1986 ). In animals, adenosine enhances the response to formalin, presumably via the A 2 receptor. Animals lacking the adenosine A 2a receptor are hypoalgesic to heat stimuli ( Ledent et al 1997 ).
A number of lines of evidence support the potential role of ATP as a peripheral mediator of pain. ATP is found at increased levels at sites of inflammation and can activate nociceptors. Psychophysical studies in humans indicate that iontophoresis of ATP into normal skin results in dose-related pain. ATP-induced pain is dependent on capsaicin-sensitive neurons; repeated topical application of capsaicin reduces the ATP-induced pain to 25% of normal. In addition, the ATP-induced pain is increased two- to three-fold when iontophoresed into skin made hyperalgesic by acute capsaicin treatment or by ultraviolet inflammation. Thus, in inflammatory conditions ATP may activate nociceptors and serve as an endogenous mediator of pain ( Hamilton et al 2000 ). In human microneurographic studies, injection of ATP activated 60% of mechano-responsive and mechano-insensitive C-nociceptive fibers without sensitizing these fibers to mechanical or heat stimuli ( Hilliges et al 2002 ).
Receptors for ATP have been found on primary sensory neurons both in the DRG and in the periphery. Multiple purinergic (P2) receptors have been suggested to be involved in pain signaling and modulation. ATP presumably activates nociceptive neurons in normal skin via the P2X 3 receptor and the heteromeric P2X 2 /P2X 3 receptor ( Chen et al 1995 , Lewis et al 1995 , Cook et al 1997 ). Messenger RNA for most of the P2X receptors (1–6) has been found in DRG neurons. In particular, both mRNA for the P2X 3 receptor and the receptor protein itself have been found in small-diameter neurons in the DRG. Local intradermal injection of agents activating P2X receptors results in dose-related pain behavior in rodents that is mediated by capsaicin-sensitive neurons ( Bland-Ward and Humphrey 1997 ) and enhanced pain behavior in response to formalin ( Sawynok and Reid, 1997 ). The proportion of C-fiber nociceptors responding and the magnitude of their response are increased by P2X agonists in inflamed skin. Activation of homomeric P2X 3 receptors is thought to contribute to acute nociception and inflammatory pain, whereas activation of heteromeric P2X 2/3 receptors appears to modulate the longer-lasting nociceptive sensitivity associated with nerve injury or chronic inflammation ( Burnstock 2009 ).
Recently, it has been suggested that peripheral adenosine receptors may also be involved in the modulation of inflammatory pain. A 1 adenosine receptors are expressed in DRG cells, and peripheral activation of these receptors results in a reduction in inflammatory hyperalgesia via interactions with the nitric oxide/cyclic guanosine monophosphate/protein kinase G intracellular signaling pathways ( Lima et al 2010 ).

Cytokines
During inflammation, cytokines (e.g., interleukin-1β [IL-1β], tumor necrosis factor α [TNF-α], IL-6) are released by a variety of cells (e.g., macrophages, Schwann cells) and regulate the inflammatory response (see Miller et al 2009 , Schaible 2010 ). Clinical studies have shown that TNF-α levels in synovial fluid are increased in painful joints ( Shafer et al 1994 ). Treatment with antibodies against TNF-α has been reported to improve the symptoms accompanying rheumatoid arthritis, including pain ( Elliott et al 1994 ). Studies in animals have demonstrated mechanical and thermal hyperalgesia after systemic or local injection of IL-1, IL-6, and TNF-α. Additionally, treatment with antiserum against TNF-α is able to inhibit or delay the onset of hyperalgesia in experimental models of inflammation ( Woolf et al 1997 ).
Cytokines may excite nociceptors either by rapid alterations in the properties of ion channels expressed in sensory neurons; indirectly by stimulating the release of other mediators such as prostaglandins, neurotrophins, and ATP; and by longer-term changes resulting from new gene transcription. Direct excitation and sensitization of nociceptive afferent fibers to thermal and mechanical stimuli have been shown for IL-1β and TNF-α ( Fukuoka et al 1994 ). When applied along a peripheral nerve, TNF-α induces ectopic activity in nociceptive afferent fibers ( Sorkin et al 1997 ).
IL-6 in combination with its soluble IL-6 receptor can sensitize nociceptors to heat as evidenced by increased heat-evoked intradermal release of CGRP ( Obreja et al 2002 ). Other cytokines, IL-1β and TNF-α, also produce transient sensitization of heat-evoked release of CGRP from nociceptors in rat skin ( Oprée and Kress 2000 ). IL-6–deficient mice show reduced mechanical and thermal hyperalgesia following inflammation ( Xu et al 1997 ). These studies provide evidence for a role of cytokines in inflammation-associated hyperalgesia. The sensitization of nociceptors by cytokines may be mediated by p38-induced phosphorylation of TTX-resistant sodium channels, as well as by up-regulation of TRPV1 expression and function ( Jin and Gereau 2006 ; for review see Ma and Quirion 2007 ).

Excitatory Amino Acids
A number of excitatory amino acids (EAAs) and peptide receptors are present at post-synaptic sites in the dorsal horn. These receptors have been found on DRG cells and the presynaptic terminals of primary afferents and are considered to play a role in the modulation of nociceptive impulses (see Carlton 2001 , Goudet et al 2009 ). The most studied EAA, glutamate, can act either through ligand-gated ion channels (ionotropic glutamate receptors [iGluRs]) or through G protein–coupled metabotropic receptors (mGluRs). Based on sequence homology and physiological and pharmacological properties, the mGluRs have been further divided into three groups—group I (mGluR 1 and 5), group II (mgluR 2 and 3), and group III (mGluR 4, 6, 7, and 8). iGluR, mGluR1, and mGluR5 receptors have been identified on unmyelinated axons in the skin ( Bhave et al 2001 , Zhou et al 2001 ). About 40% of lumbar DRG cells contain mGluR2/3 immunoreactivity, and a majority of these cells are IB4 + small cells.
Several lines of evidence indicate a role of peripheral mGluRs in nociception and inflammatory pain. Peripheral application of glutamate activates nociceptors, and peripheral administration of ligands binding to glutamate receptors induces pain behavior in animals. Involvement of peripheral iGluR, mGluR1, and mGluR5 in formalin-induced pain behavior and glutamate-induced thermal hyperalgesia has been demonstrated ( Davidson et al 1997 ). Intraplantar, but not intrathecal or intracerebroventricular administration of an mGluR5 antagonist reduced inflammatory hyperalgesia. Neurons in the DRG can be double-labeled with antisera for mGluR5 and VR1, thus suggesting that mGluR5 is expressed on the peripheral terminals of nociceptive neurons and contributes to inflammatory hyperalgesia ( Walker et al 2001 ). In particular, mGluR1 activates PLC, which leads to release of Ca 2+ from intracellular stores and activation of PKC.
Endogenous sources of glutamate in the periphery include plasma, macrophages, epithelial and dendritic cells in the epidermis and dermis, and Schwann cells. In addition, peripheral processes of the primary afferents contain glutamate, and nociceptor stimulation can cause peripheral release of glutamate from the terminals of these afferents.
Peripheral mGluRs are also considered to have antinociceptive effects. Peripheral administration of group II mGluR agonists blocks PGE 2 -induced thermal hyperalgesia, and activation of these receptors results in depression of the responses of nociceptors sensitized by exposure to formalin or inflammatory soup ( Yang and Gereau 2002 , Du 2008 ). These observations suggest that selective group II agonists may be a therapeutic target for inflammatory pain states.

Nerve Growth Factor
NGF may contribute to inflammatory pain via direct and indirect mechanisms (for review see Pezet and McMahon 2006 , Watson et al 2008 ). Pro-inflammatory cytokines stimulate the release of NGF from various sources, including fibroblasts, keratinocytes, Schwann cells, and inflammatory cells (lymphocytes, macrophages, and mast cells). NGF stimulates mast cells to release histamine and serotonin. NGF can also induce heat hyperalgesia by acting directly on the peripheral terminals of primary afferent fibers ( Chuang et al 2001 ). Transgenic animals modified to overexpress NGF show hyperalgesic pain behavior ( Davis et al 1993a ). NGF sensitizes nociceptors and may alter the distribution of Aδ fibers such that a greater proportion of fibers have nociceptor properties ( Stucky et al 1999 ). NGF has been implicated in the inflammation-induced changes in nociceptor response properties, such as an increase in the incidence of ongoing activity, increase in the maximum fiber following frequency, and changes in the configuration of the action potential of DRG neurons ( Djourhi et al 2001 ). The inflammation-induced changes in nociceptive neurons are prevented by sequestration of NGF ( Koltzenburg et al 1999 ). Cultured DRG neurons from inflamed animals exhibit spontaneous activity, and cultured DRG neurons from non-inflamed animals exhibit spontaneous activity when cultivated for 1 day with NGF ( Kasai and Mizumura 2001 ). These studies suggest that in inflamed rats NGF may play a role in inducing spontaneous activity in DRG neurons.
NGF modulates the activity of ligand- and voltage-gated ion channels involved in nociception, such as TRPV1, P2X 3 , ASIC3, and Na v 1.8. NGF potentiates responses of the TRPV1 receptor (see the section on the vanilloid receptors), and NGF-induced hyperalgesia is absent in TRPV1 knockout mice ( Chuang et al 2001 ). NGF-induced hyperalgesia may be mediated via its actions on the TTX-resistant sodium channel Na v 1.8. NGF-induced thermal hyperalgesia failed to develop in mice with a mutation in the Na v 1.8 gene ( Kerr at al 2001 ). Binding of NGF to TrkA stimulates the mitogen-activated protein kinase (MAPK), phosphatidyl-3′-kinase (PI3K), and PLC-γ intracellular signal transduction pathways (for details see Cheng and Ji 2008 ). Potential clinical therapeutic approaches being explored include humanized monoclonal antibodies to NGF or its tyrosine kinase receptor TrkA and sequestration of NGF via soluble receptor protein that binds NGF.

Other Receptors
A number of other receptor systems have been reported to play a role in the peripheral modulation of nociceptor responsiveness.

Vanilloid Receptors
The vanilloid receptor TRPV1 (also known as VR1) is present on a subpopulation of primary afferent fibers and is activated by capsaicin, heat, and protons (see Chapter 2 ). Following inflammation, axonal transport of TRPV1 mRNA is induced, the proportion of TRPV1-labeled unmyelinated axons in the periphery is increased by almost 100% ( Carlton and Coggeshall 2001 ), and the sensitivity of DRG neurons and primary afferent fibers to capsaicin increases ( Nicholas et al 1999 , Tohda et al 2001 ). Certain inflammatory mediators, such as bradykinin, lower the threshold of TRPV1-mediated heat-induced currents in DRG neurons and increase the proportion of DRG cells that respond to capsaicin ( Stucky et al 1998 , Sugiura et al 2002 ). NGF also potentiates the responses of TRPV1, and NGF-induced thermal hyperalgesia is absent in TRPV1 knockout mice. These observations, along with other experiments performed in mice lacking TRPV1, indicate that this channel protein plays a critical role in inflammation-induced heat hyperalgesia ( Caterina et al 2000 , Davis et al 2000 ).
Inflammatory mediators activate or sensitize TRPV1 through a diverse array of second-messenger pathways. For example, the thermal hyperalgesia induced by bradykinin and NGF is thought to be mediated, in part, by PLC-dependent phosphorylation of TRPV1 by PKC. Activation of PLC also leads to hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and consequent reversal of TRPV1 disinhibition by that lipid ( Chuang et al 2001 ). A PIP 2 binding site that is critical for the thermal sensitivity of TRPV1 has been identified ( Prescott and Julius 2003 ). Functional coupling between protein kinase A (PKA) and TRPV1 also appears to play an important role in inflammatory hyperalgesia ( Rathee et al 2002 , Distler et al 2003 ). Finally, some inflammatory mediators activate TRPV1 indirectly via the production of fatty acid agonists ( Shin et al 2002 ). For instance, bradykinin, acting at B 2 receptors, excites cutaneous nociceptors via production of the 12-lipoxygenase metabolite of arachidonic acid 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which in turn acts as a TRPV1 agonist.

Endothelin Receptors
Endothelins are vasoactive peptides that are widely distributed in somatic and visceral tissue (for reviews see Hans et al 2009 , Khodorova et al 2009 ). Endothelin-1 (ET-1) is synthesized and released by endothelial cells, as well as by leukocytes and macrophages, and acts via GPCRs—ET A and ET B . ET A receptors are found in a large proportion of small cells in DRGs. ET B receptors are expressed mainly in keratinocytes, DRG satellite cells, and Schwann cells and may induce the synthesis and release of PGE 2 . Peripheral administration of ET-1 results in hyperalgesia that is attenuated by ET A antagonists. ET-1 also potentiates the effects of other algogens such as PGE 2 , capsaicin, and formalin. Activation of ET A receptors on neurons results in enhanced function of TRPV1 and TTX-resistant Na channels and an increase in intracellular Ca 2+ levels, which in turn activates PKC and other second-messenger systems and leads to enhanced excitability of nociceptors. Endothelins have been implicated in the pain and hyperalgesia associated with inflammation, skin incision, cancer, and sickle cell crisis. ET B receptors have been reported to mediate both pro- and antinociceptive effects. Activation of ET B receptors on keratinocytes results in the release of β-endorphins, which inhibit nociceptor activity by binding to opioid receptors on the peripheral terminals of nociceptors ( Khodorova et al 2003 ).

Peripheral Modulators of Nociceptor Activity
GPCRs, present on the plasma membrane and terminals of nociceptive neurons, play an important role in the modulation of pain signaling. GPCRs involved in antinociceptive mechanisms include opioid, cannabinoid, SST, muscarinic acetylcholine, γ-aminobutyric acid (GABA B ), mGlu, and α 2 -adrenergic receptors ( Fig. 1-13 , for review see Pan et al 2008 ). Most GPCR agonists that have antinociceptive action are coupled to G i/o proteins, which modulate voltage-gated Ca 2+ channels and result in a decrease in presynaptic Ca 2+ entry and inhibition of neurotransmitter release. GPCRs also modulate an inwardly rectifying K + channel, the GIRK channel, which plays a critical role in maintaining resting membrane potential and excitability.


Figure 1-13 Potential peripheral modulatory mechanisms of nociceptor activity. Several metabotropic G protein–coupled receptors (GPCRs) may play a role in inhibition of the initiation, transduction, or conduction of pain signals from peripheral nociceptive terminals. These GCPRs include opioid, cannabinoid (CB), somatostatin (SSTR), muscarinic acetylcholine (M 2 ), γ-aminobutyric acid B (GABA B ), metabotropic glutamate (mGluR), adenosine 1 (A 1 ), and α 2 -adrenergic (α 2 ) receptors. Activation of these GCPRs by their endogenous ligands leads to inhibition of voltage-gated Ca 2+ channels (VGCCs), which results in a decrease in presynaptic Ca 2+ entry and inhibition of neurotransmitter release. GPCRs also modulate an inwardly rectifying K + channel, the GIRK channel, which plays an important role in maintenance of the duration and excitability of the resting membrane potential. GPCRs also regulate the function and kinetics of ion channels involved in sensory transduction, such as the transient receptor potential vanilloid (TRPV) channels and sodium channels (Na v ). (Artwork by Ian Suk, Johns Hopkins University.)
The GPCRs on peripheral nociceptors are attractive potential therapeutic targets for the development of new drugs that may have some benefit, in contrast to the more traditional analgesics, which work at the level of the CNS. Drugs acting at the periphery and inhibiting the generation and signaling of nociceptive input toward the spinal cord and the brain may prevent central plastic changes such as wind-up and central sensitization. In addition, these drugs may provide analgesia without the undesirable adverse effects, such as sedation, dizziness, and cognitive dysfunction, associated with drugs acting on the CNS system (see Stein et al 2009 ).

Opioids
Besides their central analgesic action, morphine and other opioids produce analgesia in inflamed tissues by a peripheral mechanism (see Stein et al 2009 ). Opioid receptors have been demonstrated on the peripheral terminals of afferent fibers, and axonal transport of these receptors is enhanced during inflammation. Peripheral analgesia by opioids appears to be part of a physiological antinociceptive system since increased amounts of endogenous opioids have been found in inflamed tissues. Inflammatory cells such as macrophages, monocytes, and lymphocytes contain opioid peptides. Release of endogenous opioids and antinociception can be induced by IL-1β and corticotropin-releasing hormone (CRH) originating from the inflamed tissue.
An alternative mechanism for activation of endogenous opioid analgesia at the site of tissue injury has been described (see Khodorova et al 2009 for review). ET-1, a potent vasoactive peptide, is synthesized and released by epithelia after tissue injury. Although ET-1 can trigger pain by activating ET A receptors on nociceptors, it also has an analgesic effect through its actions on ET B receptors. Activation of ET B receptors on keratinocytes by ET-1 results in the release of β-endorphins and analgesia mediated via peripheral μ- and κ-opioid receptors linked to GIRKs (see Fig. 1-13 ).

Cannabinoids
Cannabinoids have recently emerged as a potential therapy for chronic pain. Clinical use of non-selective cannabinoids is, however, limited by their CNS actions, which lead to psychotropic effects, temporary memory impairment, and dependence. The endocannabinoid system includes the two cloned metabotropic receptors CB1 and CB2, possibly the orphan receptor GPR55, and the endogenous ligands anandamide and 2-arachidonoylglycerol. CB1 and CB2 receptors are GPCRs expressed in neural and non-neural immune cells. They are distributed at many key sites in the pain-signaling pathway, including the peripheral and central terminals of primary afferent fibers, spinal dorsal horn neurons, and the brain stem and brain. CB1 and CB2 mRNA and protein are widely expressed in the majority of DRG nociceptive neurons ( Agarwal et al 2007 ), and their expression has been shown to be up-regulated following inflammation ( Amaya et al 2006 ) and nerve injury ( Beltramo et al 2006 , Mitrirattaanakul et al 2006 ). Multiple lines of evidence suggest that the analgesic effects of CB1 and CB2 agonists may be mediated via their actions on nociceptive primary afferents. Cannabinoids regulate the function and kinetics of ion channels involved in sensory transduction, such as the TRP channels (e.g., TRPV1, TRPA1, TRPM8) and purinergic ion channels (P2X 2 , P2X 2/3 ), as well as channels that directly affect neuronal excitability (various K + and Ca 2+ channels). Studies in animal models suggest that peripheral CB1 and CB2 receptors may be important targets in controlling the pain associated with inflammation, neuropathy, and bone cancer (see Anand et al 2009 , Kress and Kuner 2009 for reviews). CB receptor agonists also enhance the analgesic effects of opioid agonists and non-steroidal anti-inflammatory drugs in experimental pain models.

Somatostatin
SST is a regulatory peptide that is widely distributed in neural and non-neural cells such as immune cells, fibroblasts, and neuroendocrine cells. Found in a subpopulation of capsaicin-sensitive peptidergic DRG neurons, SST binds to G protein–coupled membrane receptors. Activation of SST receptors opens various K + channels and inhibits voltage-gated Ca 2+ channels, which results in its anti-inflammatory and analgesic effects. SST decreases the release of peptides such as SP and CGRP from sensory nerve endings in the periphery and reduces neurogenic inflammation (for review see Pinter et al 2006 ). The analgesic effects of SST are thought to result from inhibition of the TRPV1 ion channel ( Carlton et al 2004 ) and possibly via an interaction with opioid receptors. Intraplantar administration of the SST receptor agonist octreotide reduces the phase II response after formalin injection, decreases the response of CMHs to heat stimuli, and attenuates the thermal responses of nociceptors sensitized by bradykinin. Endogenous release of SST from nociceptive afferents is considered to play a modulatory role in inflammatory and neuropathic pain. Intra-articular injection of SST into the knee resulted in pain relief in patients with osteoarthritis and rheumatoid arthritis. Synthetic SST agonists may have potential as anti-inflammatory and analgesic drugs.

Cholinergic Receptors
Non-neuronally released acetylcholine, acting on peripheral cholinergic receptors, may have a modulatory role on nociception. Nicotine has a weak excitatory effect on C-fiber nociceptors and induces mild sensitization to heat, but no alterations in mechanical responsiveness. In contrast, muscarine desensitizes C nociceptors to mechanical and heat stimuli ( Bernardini et al 2001 ). Thus, nicotinic and muscarinic receptors may have opposing effects on cutaneous nociceptors. Studies in mice with targeted deletions of the M 2 receptor gene suggest that M 2 receptors on cutaneous nerve endings depress the responsiveness of nociceptive fibers to noxious stimuli (see Wess et al 2003 for review). High levels of expression of M 2 mRNA and considerably lower levels of M 3 and M 4 mRNA are detected in medium-sized and small DRG neurons in the rat ( Tata et al 2000 ). M 2 , M 3 , and M 4 muscarinic receptor subtypes may be involved in the modulation of nociceptive transduction.

γ-Aminobutyric Acid Receptors
The inhibitory neurotransmitter GABA activates both ionotropic (GABA A and GABA C ) and metabotropic (GABA B ) receptors. GABA A receptors have been found in DRG cells and on their central terminals in the dorsal horn. GABA A receptors have been reported to be present in 10–14% of the unmyelinated primary afferent axons in the glabrous skin of the cat ( Carlton et al 1999 ). Behavioral studies suggest a bimodal effect of GABA A receptors on the modulation of peripheral nociceptive transmission; a low concentration of GABA A agonists attenuates and a high concentration enhances formalin-induced pain behavior. GABA B receptors are also present in primary afferents, and GABA B mRNA is expressed in DRG cells ( Towers et al 2000 ). Degeneration of primary afferent fibers by administration of capsaicin to neonatal rats decreases GABA B receptor density by 50%, thus indicating that these receptors are localized in TRPV1-expressing nociceptive afferents ( Price et al 1987 ). Activation of the GABA B receptor by agonists such as baclofen inhibits neuronal excitability by inhibition of N-type Ca 2+ currents and potentiation of voltage-dependent K + currents ( Takeda et al 2004 ).

α 2 -Adrenoceptors
Traditionally, the analgesic effects of α 2 -adrenergic agonists, such as clonidine and dexmedetomidine, are thought to be secondary to their actions in the CNS (for review see Pertovaara 2006 ). However, peripheral α 2 -adrenoceptors may also be involved in modulation of nociceptor activity. Studies using selective α 2 -subtype knockout mice have shown that the α 2A -adrenergic receptors are primarily involved in the analgesic effect of α 2 -adrenoceptor agonists ( Stone et al 1997 ). Selective removal of TRPV1-expressing sensory neurons induces a large decrease in α 2A - but not in α 2C -adrenoceptors in the spinal dorsal horn, which suggests that α 2A -adrenoceptors are located on the central terminals of primary afferent neurons whereas the α 2C subtype is located primarily on spinal dorsal horn neurons ( Stone et al 1998 , Chen et al 2007 ). α 2 -Adrenergic agonists may inhibit the depolarization-induced Ca 2+ influx and induce a GIRK current in nociceptors.

Second Messengers and Signal Transduction Pathways
As described above, inflammation is associated with the release of a host of chemical mediators ( Fig. 1-12 ). Although some of these agents may directly activate nociceptors, most of the inflammatory mediators lead to changes in the sensory neuron rather than directly activating it. Such changes in sensory neurons include early post-translational alterations in the peripheral terminals of nociceptors (peripheral sensitization) and a delayed transcription-dependent alteration (see Woolf and Costigan 1999 , Kidd and Urban 2001 ). Peripheral sensitization can be the result of changes in the transducer molecule (e.g., TRPV1 receptor) or in voltage-gated ion channels (e.g., sodium channels) secondary to the phosphorylation of membrane-bound proteins. Inflammation can also induce delayed and longer-lasting transcription-dependent changes in effector genes in DRG cells as a result of electrical activity and retrograde transport of specific signal molecules such as NGF. An increase in intracellular calcium induced by electrical activity activates a host of intracellular transcription factors such as the cAMP-response element–binding protein (CREB; Ji and Rupp 1997 ).
Considerable attention has been focused on the signal transduction mechanisms of primary afferent neurons and their alteration by inflammation. Two principal signaling pathways have been postulated to mediate inflammation-induced hyperalgesia. Inflammatory mediators such as PGE 2 , serotonin, and adenosine activate PKA ( Gold et al 1998 ), whereas NGF, bradykinin, and epinephrine induce hyperalgesia in part by activating PKA but also through an ε isozyme of PKC ( Khasar et al 1999 ). PKA and PKC sensitize nociceptors to heat by modulating the activity of TTX-resistant sodium currents. As described above, these signaling pathways also interact with the heat transducer TRPV1, which results in sensitization of the receptor to heat.
MAPKs are also reported to be involved in the transduction of extracellular stimuli (e.g., signals from extracellular growth factors such as NGF) into diverse intracellular responses and neuronal plasticity. Three subfamilies of MAPKs have been well characterized—the extracellular signal–regulated kinases (ERKs), the c-Jun amino-terminal kinases (JNKs), and the p38 enzymes. ERK is present in primary afferent neurons, is phosphorylated by nociceptive stimuli, and is thought to play a role in inflammatory hyperalgesia ( Dai et al 2002 ). Inflammation also activates p38 in the soma of C-fiber nociceptive cells in the DRG ( Ji et al 2002 ). Inhibiting the activation of p38 in the DRG reduces the inflammation-induced increase in TRPV1 receptors in the DRG and attenuates heat hyperalgesia. Activation of p38 in the DRG is dependent on peripheral production of NGF during inflammation. Thus, MAPKs and NGF play important regulatory roles in TRPV1 receptor expression and maintenance of heat hyperalgesia after inflammation.

Postoperative Pain and Hyperalgesia
The pain resulting from different tissue injuries may differ in its characteristics and mechanisms. Postoperative, incisional pain is a unique but common form of acute pain. Studies in rodents have characterized the primary hyperalgesia to mechanical and thermal stimuli caused by a surgical incision ( Brennan et al 1996 , Pogatzki and Raja 2003 ). Primary hyperalgesia to mechanical stimuli lasts for 2–3 days, whereas hyperalgesia to heat lasts longer—6–7 days after plantar incision. As with other types of tissue injury, secondary hyperalgesia after incision injury is present only to mechanical, not thermal, stimuli ( Pogatzki et al 2000 ). The incision-induced primary and secondary hyperalgesia results from characteristic peripheral, spinal, and supraspinal mechanisms ( Zahn and Brennan 1999 , Pogatzki et al 2002 ). The conversion of mechanically insensitive “silent nociceptors” to mechanically responsive fibers may play an important role in the maintenance of primary mechanical hyperalgesia ( Pogatzki et al 2002 ). Release of ATP from injured cells is considered to play an important role in the induction of mechanical allodynia after a skin incision ( Tsuda et al 2001 ).
The incision-induced spontaneous activity in primary afferent fibers plays a critical role in maintaining wide–dynamic range neurons in the dorsal horn in a sensitized state. In contrast to the central mechanisms of hyperalgesia following other forms of cutaneous injury where N -methyl- D -aspartate (NMDA) receptors play a critical role, the hyperalgesia that results from an incision is characterized by distinct pharmacological mechanisms that are not dependent on NMDA receptors.

Role of the Sympathetic Nervous System in Inflammation
Nociceptors normally do not respond to sympathetic stimulation. In addition, sympathectomy plus depletion of catecholamine stores with reserpine has no effect on acute inflammation. In contrast, sympathectomy reduces the severity of injury in chronic adjuvant-induced arthritis (see Raja 1995 , Jänig et al 1996 for reviews). Inflammation may lead to catechol sensitization of cutaneous nociceptors. Sympathetic stimulation and close arterial injection of norepinephrine (NE) also excite 35–40% of C-polymodal nociceptors in chronically inflamed rats ( Sato et al 1993 ). This adrenergic activation of nociceptors was blocked by α 2 - but not by α 1 -adrenergic antagonists. Sympathetic efferent fibers are also thought to play a role in neurogenic inflammation.
In human skin sensitized by the topical application of capsaicin, hyperalgesia persists longer at sites where exogenous NE was administered, and this α-adrenoceptor–mediated effect was independent of the vasoconstrictor response ( Drummond 1995 , 1996 ). Additionally, local administration of an α-adrenergic antagonist reduced the spontaneous pain and hyperalgesia resulting from the intradermal injection of capsaicin ( Kinnman et al 1997 ). However, physiological modulation of sympathetic vasoconstrictor activity by whole-body warming or cooling does not alter the intensity or spatial distribution of capsaicin-evoked spontaneous pain and mechanical hyperalgesia ( Baron et al 1999 ). Anatomical studies indicate that SP and NMDA receptor mRNA is up-regulated in preganglionic sympathetic neurons after paw inflammation in rats ( Ohtori et al 2002 ). These changes are postulated to possibly be evidence of a role of the sympathetic nervous system in inflammatory hyperalgesia.

Secondary Hyperalgesia
An understanding of secondary hyperalgesia is important not only with regard to understanding the neural mechanisms of acute pain but also with regard to understanding many aspects of chronic pain. In this section we consider the nature of secondary hyperalgesia and its possible peripheral and central mechanisms.

Secondary Hyperalgesia to Mechanical but Not Heat Stimuli
Primary hyperalgesia is characterized by the presence of enhanced pain in response to heat and mechanical stimuli, whereas secondary hyperalgesia is characterized by enhanced pain in response to only mechanical stimuli (e.g., Ali et al 1996 ). In one study in which the sensory changes that occur in the zones of primary and secondary hyperalgesia were compared ( Raja et al 1984 ), burn injuries were induced in two locations on the glabrous skin of the hand in human subjects ( Fig. 1-14 ). Within minutes of the injury, lightly touching the skin at the site of the two burns, as well as in a large area surrounding the burns, caused pain. The decrease in the pain threshold to von Frey hairs in the primary (injured) zone was similar to that in the area of secondary hyperalgesia ( Fig. 1-14 B). Marked hyperalgesia to heat was observed in the area of primary hyperalgesia (site A, the injury site, Fig. 1-14 C). In the uninjured region between the two burns, however, the painfulness of the heat stimuli actually decreased ( Fig. 1-14 D). Notably, the area between the burns was hypo-algesic to heat while being hyperalgesic to mechanical stimuli.


Figure 1-14 Hyperalgesia to mechanical and heat stimuli develops at the site of injury (zone of primary hyperalgesia), whereas hyperalgesia to mechanical but not heat stimuli develops in the uninjured area surrounding an injury (zone of secondary hyperalgesia). A, Two burns (53°C, 30 seconds) were applied to the glabrous skin of the hand (sites A and D). Mechanical thresholds for pain and ratings of pain in response to heat stimuli were recorded before and after the burns at one of the injury sites (site A), in the uninjured skin between the two burns (site B), and at an adjacent site (site C). The areas of flare and mechanical hyperalgesia following the burns in one subject are also shown. In all subjects, the area of mechanical hyperalgesia was larger than the area of flare. Mechanical hyperalgesia was present even after the flare disappeared. B, Mean mechanical thresholds for pain before and after burns. The mechanical threshold for pain was significantly decreased following the burn. The mechanical hyperalgesia was of similar magnitude at each of the three test spots (A, B, C). C–E, Mean normalized ratings of the painfulness of heat stimuli (same as described in Fig. 1-5 ) before and after burns. C, At burn site A, all the characteristics of heat hyperalgesia (i.e., decrease in pain threshold, increased pain in response to suprathreshold stimuli, and spontaneous pain) were observed after the burns. D, In the uninjured area between the two burns (site B), pain ratings decreased after the burns. Thus, heat hypalgesia was observed. E, At site C, pain ratings before and after the burns were not significantly different. (Note that a different scale is used in C .) (Reproduced with permission from Raja SN, Campbell JN, Meyer RA 1984 Evidence for different mechanisms of primary and secondary hyperalgesia following heat injury to the glabrous skin. Brain 107:1179–1188.)

Spreading Sensitization of Nociceptors Does Not Occur
Activation of nociceptors leads to a flare response (discussed in more detail below). This response is neurogenic in the sense that it depends on intact innervation of the skin by nociceptors. The flare response extends well outside the area of initial injury. One explanation for the flare response is that it involves spreading activation of nociceptors. Activation of one nociceptor leads to the release of chemicals that activate neighboring nociceptors, which leads to further release of chemicals and activation of additional nociceptors. Lewis (1942) believed that a similar mechanism, which he termed spreading sensitization , accounted for secondary hyperalgesia. Activation and sensitization of one nociceptor lead to spread of this sensitization to another nociceptor, possibly because of the effects of a sensitizing substance released from the nociceptor initially activated. Another theoretical possibility is that coupling between nociceptors increases after injury.
Several lines of evidence indicate that spreading sensitization does not occur:

•  A heat injury to half the receptive field of nociceptors does not alter the sensitivity of the other half to heat stimuli ( Thalhammer and LaMotte 1983 ).
•  A mechanical injury adjacent to the receptive field of nociceptors fails to alter the responses of CMHs in the monkey ( Campbell et al 1988a ) and rat ( Reeh et al 1986 ).
•  Antidromic stimulation of nociceptive fibers in the monkey ( Meyer et al 1988 ) and rat ( Reeh et al 1986 ) does not cause sensitization.
•  Application of mustard oil to one part of the receptive field of C-fiber nociceptors in humans does not lead to sensitization of other parts of the receptive field ( Schmelz et al 1996 ).
Other differences exist between flare and secondary hyperalgesia ( LaMotte et al 1991 ):

•  The zone of secondary hyperalgesia is generally larger than the zone of flare.
•  Flare can be induced without causing secondary hyperalgesia (for example, with histamine), and secondary hyperalgesia can be induced without a flare response.
•  Secondary hyperalgesia does not spread beyond the body’s midline, whereas the flare response does.

Central Mechanisms of Secondary Hyperalgesia
If peripheral sensitization does not account for secondary hyperalgesia, the mechanisms noted in Figure 1-10C– F should be examined in the CNS. Indeed, it has been relatively easy to demonstrate enhanced responsiveness of CNS neurons to mechanical stimuli after cutaneous injury (e.g., Simone et al 1991b ). Substantial evidence favors the following important tenet: the peripheral signal for pain does not reside exclusively with nociceptors. Under pathological circumstances, other receptor types, which are normally associated with the sensation of touch, acquire the capacity to evoke pain. This principle applies not only to secondary hyperalgesia but also to neuropathic pain states in general. This condition arises in part through augmentation of the responsiveness of central pain-signaling neurons to input from low-threshold mechanoreceptors, a phenomenon often termed central sensitization .
Many of the insights acquired about secondary hyperalgesia have been gained from studies with capsaicin. Investigators have been drawn to the use of capsaicin as the “injury” stimulus for several reasons:

•  Capsaicin selectively activates nociceptors ( Szolcsányi 1990 ).
•  Capsaicin causes intense pain and a large zone of secondary hyperalgesia when applied topically or intradermally to the skin ( Simone et al 1989 ).
•  Injection of capsaicin into the skin does not produce any apparent tissue injury.
•  The characteristics of hyperalgesia resemble those for heat or cut injuries. Immediately around the injection site, heat and mechanical hyperalgesia is present. Outside this area of primary hyperalgesia is a large zone of secondary hyperalgesia characterized by mechanical hyperalgesia but not heat hyperalgesia ( Ali et al 1996 ).
LaMotte and colleagues performed a number of pivotal experiments to determine the relative importance of peripheral and central sensitization in secondary hyperalgesia ( LaMotte et al 1991 ). To test whether peripheral nerve fibers are sensitized, capsaicin was administered under conditions of a proximal nerve block, and the magnitude of hyperalgesia was determined after the effects of the anesthetic dissipated. When the relevant nerve is blocked proximal to the capsaicin injection site, the CNS is spared the nociceptive input generated at the time of injection. The effects of capsaicin on the peripheral nervous system are not affected (e.g., a flare develops) since the nerve block is proximal to the area of capsaicin application. Figure 1-15 shows the results of this experiment in one subject. No hyperalgesia was present after the block had worn off. Thus, when the CNS is spared the input of nociceptors at the time of the acute insult, hyperalgesia does not develop ( LaMotte et al 1991 , Pedersen et al 1996 ).


Figure 1-15 A proximal nerve block prevents the development of secondary hyperalgesia. A, After blockade of the lateral antebrachial nerve with 1% Xylocaine, capsaicin (100 μg in 10 μL) was injected into the anesthetic skin. A flare (dashed line) developed within 5 minutes. No hyperalgesia was present 180 minutes after the capsaicin injection when the local anesthetic block had dissipated. B, On the control arm, normal flare and hyperalgesia in response to stroking (dotted line) and punctate (solid line) stimuli developed within 5 minutes. Hyperalgesia to punctate stimuli was still present 180 minutes after the capsaicin injection. (Adapted from LaMotte RH, Shain CN, Simone DA, et al 1991 Neurogenic hyperalgesia: psychophysical studies of underlying mechanisms. Journal of Neurophysiology 66:190–211.)
Additional evidence that central sensitization, not peripheral sensitization, plays a major role in secondary hyperalgesia includes the following:

•  Electrical stimulation of the skin can be used to produce a large zone of secondary hyperalgesia ( Koppert et al 2001 ). Electrical stimulation directly activates the axon and therefore bypasses a peripheral receptor mechanism.
•  When an anesthetic strip is produced in the skin, electrical stimulation on one side of the anesthetic strip produces a flare only on that side of the strip, thus indicating that the strip has blocked the axon reflexive flare; secondary hyperalgesia develops symmetrically around the stimulation site and extends well beyond the anesthetic strip ( Klede et al 2003 ).
•  Secondary hyperalgesia following injection of capsaicin within the territory of a given nerve spreads into the territory of an adjacent nerve ( Sang et al 1996 ).

Different Mechanisms for Stroking and Punctate Hyperalgesia
Two distinct forms of mechanical hyperalgesia are observed in the zone of secondary hyperalgesia: punctate hyperalgesia and stroking hyperalgesia. Hyperalgesia to blunt pressure is not observed in the secondary zone ( Koltzenburg et al 1992 ). We will first consider stroking hyperalgesia (also called allodynia). Stroking hyperalgesia appears to be mediated by activity in low-threshold mechanoreceptors. When a pressure cuff was used to selectively block myelinated fibers, the pain in response to stroking disappeared at a time when touch sensation was lost but heat and cold sensations were still present ( LaMotte et al 1991 , Koltzenburg et al 1992 ). This is also true in patients with stroking hyperalgesia from neuropathic pain ( Campbell et al 1988b ). In another series of experiments, Torebjörk and colleagues (1992) performed intraneural microstimulation in awake human subjects. As shown in Figure 1-16 , stimulation of primary afferent fibers normally concerned with tactile sensibility evoked pain when (but not before) secondary hyperalgesia was produced.


Figure 1-16 Microneurographic evidence that large-diameter myelinated fibers are involved in the pain observed in the zone of secondary hyperalgesia. A, Intraneural electrical stimulation of the superficial peroneal nerve at a fixed intensity and frequency evoked a purely tactile (non-painful) sensation projected to a small area of skin on the dorsum of the foot (dark blue area). B, After intradermal injection of capsaicin (100 μg in 10 μL) adjacent to the projected zone (at the site indicated by the open circle), a zone of secondary hyperalgesia (indicated by light blue area) developed that overlapped the sensory projection field. Now, intraneural stimulation at the same intensity and frequency as in A was perceived as a tactile sensation accompanied by pain. C, When the zone of secondary hyperalgesia no longer overlapped the sensory projection field, the intraneural stimulation was again perceived as purely tactile, without any pain component. (Adapted from Torebjörk HE, Lundberg LER, LaMotte RH 1992 Central changes in processing of mechanoreceptive input in capsaicin-induced secondary hyperalgesia in humans. Journal of Physiology [London] 448:765–780.)
Punctate hyperalgesia is manifested by heightened pain associated with the application of small, stiff, or sharp probes to the skin (e.g., von Frey monofilaments). Several lines of evidence indicate that punctate hyperalgesia has a different neural mechanism than stroking hyperalgesia does and is mediated by central sensitization to activity in nociceptors:

•  The area of punctate hyperalgesia is consistently larger than that of stroking hyperalgesia.
•  Stroking hyperalgesia after capsaicin injection lasts 1–2 hours, whereas punctate hyperalgesia lasts more than 12 hours ( LaMotte et al 1991 ).
•  Punctate hyperalgesia, not stroking hyperalgesia, developed after intradermal capsaicin injection into the arm of a patient with a severe large-fiber neuropathy ( Treede and Cole 1993 ). This evidence suggests that punctate hyperalgesia is mediated by small-diameter (presumably nociceptive) fibers.
•  The pain produced by touching the skin with different wool fabrics was greatly increased in the region of secondary hyperalgesia ( Cervero et al 1994 ). The pain was proportional to the prickliness of the fabrics. Since nociceptors and not low-threshold mechanoreceptors exhibit a differential response to different wool fabrics ( Garnsworthy et al 1988 ), activity in nociceptors probably contributes to this form of secondary hyperalgesia to wool fabrics.
•  When the area of primary hyperalgesia is anesthetized or cooled, stroking hyperalgesia is eliminated but punctate hyperalgesia persists ( LaMotte et al 1991 ). Therefore, stroking hyperalgesia has an ongoing dependence on input from the sensitized area, whereas punctate hyperalgesia is more enduring and less dependent on ongoing discharge from the sensitized area.
The pain in response to a controlled punctate stimulus does not vary significantly across the zone of secondary hyperalgesia but decreases precipitously at the border ( Huang et al 2000 ). This suggests that the sensitization responsible for secondary hyperalgesia is an all-or-nothing phenomenon. In addition, subjects were able to grade the magnitude of pain from stimuli of different intensity. Interestingly, although the threshold for pain in response to punctate stimuli decreases in the zone of secondary hyperalgesia ( Magerl et al 1998 ), the threshold for touch detection increases ( Magerl and Treede 2004 ).

Model for Stroking Hyperalgesia
From the above we know that secondary hyperalgesia to stroking stimuli appears to be due to sensitization of central pain-signaling neurons to the input from low-threshold mechanoreceptors ( Fig. 1-17 ). In normal skin, activity in low-threshold mechanoreceptors signals touch sensation ( Fig. 1-17 A). As a result of the barrage of activity in nociceptors, sensitization occurs in the CNS such that input from low-threshold mechanoreceptors gains access to the pain system ( Fig. 1-17 B). Now, light touching of the skin is painful. Plasticity in the response of second-order neurons in the dorsal horn appears to be a major factor that accounts for this central sensitization (see Chapter 6 for detailed discussion). However, another possibility that involves plasticity in primary afferents is that mechanoreceptors gain access to nociceptive neurons by means of a presynaptic link ( Cervero et al 2003 ; see discussion below on primary afferent depolarization).


Figure 1-17 Central sensitization accounts for secondary hyperalgesia. A, Nociceptors signal acute pain. Noxious stimuli selectively activate nociceptors that project to central pain-signaling neurons (CPSNs) in the spinal cord. The CPSNs project to higher centers, where pain is perceived. Low-threshold mechanoreceptors convey the sensation of touch. B, Injury or inflammation leads to the sensitization of primary afferent nociceptors. The enhanced responsiveness or sensitization of primary afferents accounts for primary hyperalgesia. Spontaneous activity also develops in the nociceptors and drives the development of sensitization of the CPSNs. This central sensitization involves enhanced connectivity between low-threshold mechanoreceptors and CPSNs. Now, signals from low-threshold mechanoreceptors gain access to the pain pathway, which leads to the development of secondary hyperalgesia to mechanical stimuli.

Model for Punctate Hyperalgesia
Punctate hyperalgesia appears to be mediated by central sensitization to nociceptor input. However, most nociceptors respond to heat stimuli. Why is there not hyperalgesia to heat stimuli in the secondary hyperalgesic zone? One possibility is that this central sensitization involves a mechano-specific channel. In this model, punctate hyperalgesia is mediated by mechano-specific nociceptive afferents that project via sensitized mechano-specific interneurons to central pain-signaling neurons. Support for this hypothesis comes from experiments in which the skin was pretreated with topical capsaicin to eliminate epidermal nerve fibers that are sensitive to heat ( Nolano et al 1999 ). Such treatment led to a lack of pain in response to heat stimuli; however, secondary hyperalgesia to punctate stimuli developed after the injection of capsaicin into nearby untreated skin ( Fig. 1-18 ; Fuchs et al 2000 ). Additional experiments with selective nerve fiber blocks revealed that the punctate hyperalgesia disappeared when Aδ fibers were blocked ( Magerl et al 2001 ). Thus, punctate hyperalgesia appears to be signaled by Aδ-fiber afferents that are insensitive to capsaicin and heat.


Figure 1-18 Secondary hyperalgesia to punctate mechanical stimuli occurs in skin that has been pretreated with topical capsaicin, which desensitizes unmyelinated, epidermal nerve fibers. A, Capsaicin was applied to a 2 × 2-cm area on the volar aspect of the forearm to produce desensitization of the skin to heat stimuli. A nearby vehicle-treated area served as control. Two days later, capsaicin was injected intradermally between the two treatment areas and produced a large, symmetrical zone of secondary hyperalgesia to punctate mechanical stimuli. B, Pain ratings in response to a sharp probe increased dramatically 60 minutes after the capsaicin injection. The average pain ratings at the capsaicin pretreatment area (left panel) were not significantly different from those at the vehicle treatment area (right panel). (Adapted from Fuchs PN, Campbell JN, Meyer RA 2000 Secondary hyperalgesia persists in capsaicin desensitized skin. Pain 84:141–149.)
One well-studied form of central sensitization, termed wind-up , is characterized by a slowly increasing response of central neurons to repeated C-fiber stimulation at rates greater than 0.3 Hz (e.g., Mendell and Wall 1965 ). The perceptual correlate of wind-up is temporal summation ( Price et al 1977 ). The finding that temporal summation does not change in the zone of secondary hyperalgesia argues against wind-up as a mechanism for secondary hyperalgesia ( Magerl et al 1998 ).

Effect of Aging on Nociceptive Properties
Aging induces changes in the properties of unmyelinated nociceptive afferents ( Namer et al 2009 ). Thus, the percentage of mechanosensitive afferents decreased whereas the percentage of mechano-insensitive afferents increased, and some fibers showed signs of sensitization, desensitization, and spontaneous activity, features previously observed in patients with neuropathic pain. In addition, changes in the conductive properties of nociceptive afferents were also observed. The mechanisms underlying these changes are unknown, but they may, for example, be due to a diminished supply of neurotrophic factors. It has been hypothesized that age-related changes could render nociceptive afferents more susceptible to neuropathy-inducing insults ( Namer et al 2009 ).

Efferent and Trophic Functions of Nociceptors
Nociceptors, apart from signaling pain, serve regulatory and trophic functions. An efferent role for nociceptors was suggested by several investigators almost a century ago (see Lynn 1996 for historical review). Two efferent cutaneous phenomena have been considered to be dependent on the integrity of afferent nociceptive fibers and are part of the so-called neurogenic inflammation: vasodilatation, which becomes visible as a flare surrounding a site of injury, and plasma extravasation, which is manifested as a wheal at the site of injury. Several peptides have been identified in the peripheral terminals of sensory neurons, including SP and other tachykinins such as neurokinins A and K, CGRP, SST, and vasoactive intestinal polypeptide. The presence and release of SP and CGRP from capsaicin-sensitive sensory nerve endings in experimental animals, their ability to induce many of the signs of acute inflammation, including vasodilatation and plasma extravasation, and inhibition of neurogenic vasodilatation by selective neuropeptide antagonists suggest that they may be the principal mediators of neurogenic inflammation and axon reflexive flare. SP-induced vasodilatation and plasma extravasation may result from a direct effect on the vasculature or be due to release of histamine by degranulation of mast cells by SP. Differences in neurogenic inflammation and peptide release in rat and human skin have, however, been observed. Electrical stimulation results in release of CGRP and SP in rat and human skin. However, unlike rat skin, the endogenous release of peptides after strong chemical or electrical stimulation is not associated with neurogenic protein extravasation or release of mast cell mediators in human skin (see Schmelz and Petersen 2001 for review).
SP and CGRP are also reported to play a role in immunological processes (e.g., migration of leukocytes to sites of tissue injury), and they stimulate epidermal cells (e.g., keratinocytes and Langerhans cells) necessary for the maintenance and repair of skin integrity (for reviews see Maggi and Meli 1988 , Holzer 1998 ). Other efferent actions of nociceptors that are mimicked by vasoactive neuropeptides are contraction of smooth muscles, stimulation of mucous secretion from airways, and leukocyte adhesion. Some efferent functions of nociceptors and the chemical mediators involved are shown in Figure 1-19 .


Figure 1-19 Efferent actions of nociceptors. A noxious stimulus leads to action potentials in nociceptive fibers that propagate not only to the central nervous system but also antidromically into peripheral branches. These antidromic action potentials lead to the release of neuropeptides such as substance P, calcitonin gene–related peptide (CGRP), and neurokinin A (NKA). These substances can stimulate epidermal cells (1) and immune cells (2) or lead to vasodilatation (3), plasma extravasation (4), and smooth muscle contraction (5). (Artwork by Ian Suk, Johns Hopkins University.)
Flare is thought to be due to a peripheral axon reflex. Activation of one branch of a nociceptor by a noxious stimulus results in the antidromic invasion of action potentials into adjacent branches of the nociceptor, which in turn causes the release of vasoactive substances from terminals of the nociceptor. Capsaicin-sensitive A- and C-fiber nociceptors are thought to be involved in the flare reaction. However, the extent of the flare far exceeds the size of the receptive fields of conventional nociceptors. A possible explanation for this discrepancy is that the flare is mediated, at least in part, by a subpopulation of chemosensitive nociceptive fibers with large receptive fields. Some C-fibers with large, complex receptive fields have been reported ( Meyer et al 1991 , Schmelz et al 1997 ). Transcutaneous electrical stimulation studies in the skin of human volunteers suggest that the axon reflex flare, measured by laser Doppler imaging, was mediated via mechano-insensitive C-fiber nociceptors ( Schmelz et al 2000a ).
Several lines of evidence indicate that the neural substrates for vasodilatation and the perception of pain are different. (1) The magnitude of the vasodilatation induced by a noxious stimulus does not always increase with the intensity of pain ( Koltzenburg and Handwerker 1994 ). (2) Low activity (<1 Hz) in C fibers can generate significant vasodilatation ( Lynn and Shakhanbeh 1988 ), but in humans does not cause any conscious sensation. (3) Histamine can produce a large flare with little or no pain. Possible explanations are that different discharge patterns are needed for pain versus flare in a given fiber population or certain classes of afferents are better designed for flare than for pain and vice versa.
Similarly, pain sensations and plasma extravasation have independent mechanisms. The ability of inflammatory mediators such as bradykinin, histamine, and serotonin to induce plasma extravasation and excite nociceptors, as assessed by pain ratings and flare response, has been examined in humans. In healthy human skin, no clear relationship between nociceptor activation and plasma extravasation could be established. For example, bradykinin induced protein extravasation without pain or flare, and serotonin evoked pain and flare at concentrations that did not induce plasma extravasation. These observations suggest that the plasma extravasation induced by these mediators is mostly non-neurogenic in mechanism ( Lischetzki et al 2001 ).
Antidromic activity involved in the effector responses can also originate from the spinal cord. A series of studies in a model of acute arthritis indicated that primary afferent input to the spinal cord activates multisynaptic central neuronal pathways, which in turn influence the development of neurogenic inflammation (for review see Sluka et al 1995 ). Activation of primary afferent fibers may result in depolarization of the central terminals of other afferent fibers (primary afferent depolarization [PAD]). If PAD is large enough (e.g., under peripheral inflammatory conditions), the depolarization can be sufficient to initiate action potentials at the central terminals that are conducted antidromically in the primary afferent fibers (dorsal root reflexes [DRRs]). It is postulated that the antidromic impulses (DRRs) triggered by PAD result in the release of neuropeptides in the joint from peripheral terminals of the afferents and contribute to the inflammatory process. DRRs have been recorded in C, Aδ, and Aβ fiber types in rat models of acute arthritis. The joint inflammation and the DRRs were attenuated by prior dorsal rhizotomy.
Cervero and Laird proposed that the DRRs may also explain secondary hyperalgesia ( Cervero et al 2003 ). According to this hypothesis, the action potentials initiated in primary afferent fibers as a result of enhanced PAD propagate peripherally to produce flare and centrally to evoke pain sensation. As evidence to support this hypothesis, they reported that light stroking of the skin leads to an increase in blood flow in the zone of secondary hyperalgesia, but not in normal skin.
Nociceptive innervation of the skin has been suggested to also play a critical role in wound healing. Sensory denervation by capsaicin injection impairs cutaneous wound healing in rats ( Smith and Liu 2002 ). Skin denervation decreases keratinocyte proliferation and leads to decreased skin thickness ( Hsieh and Lin 1999 ). The role of cutaneous nociceptors in wound healing may be due to neuromodulatory actions of the sensory peptides SP and CGRP, which when injected at skin wound sites, promote wound healing in aged rats ( Khalil and Helme 1996 ).

Nociceptors and Neuropathic Pain
A well-known axiom in the field of pain is that injury to the nociceptive pathways, whether it be in the peripheral nervous system or CNS, carries with it the liability that pain may result. This is paradoxical in the sense that lesions should, one would think, lead to deficits in function. The ongoing pain in patients is frequently associated with enhanced pain in response to natural stimuli, a phenomenon termed hyperalgesia . Hyperalgesia may be prominent in neuropathic conditions such as post-herpetic neuralgia, certain cases of diabetic or human immunodeficiency virus–associated neuropathy, and certain cases of traumatic nerve injury. In this section we consider the role of altered function of nociceptors in neuropathic pain.
In considering inflammatory pain it was noted earlier in this chapter that primary hyperalgesia is explained by sensitization of nociceptors whereas secondary hyperalgesia is due to central sensitization. In the case of secondary hyperalgesia, the input of low-threshold mechanoreceptors, normally concerned only with touch sensibility, leads to pain because the synaptic links with central pain-signaling cells in the dorsal horn are strengthened. A similar mechanism of central sensitization appears to also explain the allodynia seen with neuropathic pain states. This was demonstrated in human subjects by selectively blocking the neural activity in large fibers (touch fibers) with an ischemic block. When touch sensation was eliminated and the functions of other nerve fibers were still preserved, the allodynia disappeared ( Campbell et al 1988b ).
The relative role of central and peripheral mechanisms in neuropathic pain is not well understood and probably varies not only with the disease but also with factors such as genetic differences. In many cases, however, the abnormal input of neural activity from nociceptive afferents plays a dynamic and ongoing role in maintenance of the pain state.
Understanding of neuropathic pain involves two key concepts: (1) inappropriate activity in nociceptive fibers (injured and uninjured) and (2) central changes in sensory processing that arise from these abnormalities. To consider how these mechanisms generate heightened pain we discuss in some depth the simplest of neuropathic pain models: the sequelae of severing a nerve.

Ectopic Sensitivity Develops in Injured Fibers
When a nerve is severed, the nociceptors are also severed. The injured (transected) nociceptors could in principle function abnormally at the site of nerve transection (the neuroma). Indeed, abnormal spontaneous activity has been observed in A and C fibers originating from a neuroma (see Chapter 64 ). Given that a substantial proportion of C-fiber afferents are nociceptors, it is likely that this spontaneous activity is in fact occurring in nociceptive afferents. In patients with a painful neuroma and hyperalgesia, locally anesthetizing the neuroma may eliminate the pain and hyperalgesia ( Gracely et al 1992 ). Thus, ongoing activity arising from nociceptive fibers in the neuroma contributes to the ongoing pain and hyperalgesia after nerve injury and may contribute to phantom limb pain (see Chapter 67 ).
Ectopic mechanical sensitivity also develops in experimental neuromas. Tapping at the site of a neuroma leads to a neural response. This may account for the observation that tapping on a neuroma is quite often found to be painful (Tinel’s sign). Superficial neuromas, which are more prone to accidental mechanical stimulation, or neuromas that are in locations associated with high mechanical stress are more likely to be painful. One strategy to alleviate neuroma pain is to resect the neuroma and move the nerve to a deep location. Since neuromas form when a nerve is cut, removing a neuroma in essence is a neuroma relocation operation. Neuroma relocation effectively relieves pain enduringly in at least some cases ( Burchiel et al 1993 ).

The Role of the Intact Nociceptor
Several lines of evidence suggest that uninjured, intact nociceptors that share the nerve of the injured fibers play a role in neuropathic pain. Much of the evidence comes from animal models of neuropathic pain in which the L5 spinal nerve is cut and ligated ( Kim and Chung 1992 ). This injury leads to behavioral signs of hyperalgesia to mechanical and heat stimuli applied to the ipsilateral foot. Using this model, we have learned the following: (1) Dorsal rhizotomy of the lesioned L5 root does not reverse the hyperalgesia regardless of whether this is done pre-emptively (before the L5 spinal nerve is severed) or after the lesion. Thus, interruption of input from the injured L5 spinal nerve fails to reverse the hyperalgesia in the foot, which indicates that ectopic activity from the injured nerve is not essential for the development of neuropathic pain ( Li et al 2000 ). (2) Electrophysiological recordings from the uninjured L4 spinal nerve (the root that most overlaps the innervation territory of the L5 root) reveal abnormal spontaneous activity in C-fiber nociceptors. The spontaneous activity appears to emanate at least in part from the skin ( Wu et al 2001 ). (3) Molecules related to pain (e.g., CGRP, brain-derived neurotrophic factor [BDNF], VR1) are up-regulated in L4 DRGs ( Fukuoka et al 2000 ). (4) Expression of the TTX-resistant sodium channel Na v 1.8 increases in the sciatic nerve ( Gold et al 2003 ).
Additional evidence for a contribution of non-axotomized nociceptors comes from clinical studies demonstrating that distal therapies are effective in neuropathic pain states. Capsaicin causes degeneration of the cutaneous terminals of nociceptors that express TRPV1 ( Nolano et al 1999 ). Capsaicin applied to the skin can alleviate the pain associated with nerve injuries (e.g., Robbins et al 1998 ). This clinical effect can be understood only by invoking a role of cutaneous nociceptors that survive the injury. Moreover, since the toxicity of capsaicin appears to be restricted to the skin, it is the cutaneous terminal of the nociceptor that must be generating pain.

Wallerian Degeneration and Neuropathic Pain
When the L5 spinal nerve is cut, the axons distal to the cut undergo wallerian degeneration. In the peripheral nerve, axons from intact nerve roots are in close proximity to degenerating axons and thus are exposed to diffusible factors released into the endoneurial space or at the nerve terminals. These factors could be derived from Schwann cells or macrophages and could affect nociceptive terminals directly or indirectly by an alteration in the cell bodies of nociceptors. As illustrated in Figure 1-20 , wallerian degeneration may play a role in neuropathic pain by producing sensitization of primary afferent nociceptors and/or by leading to the development of central sensitization.


Figure 1-20 Wallerian degeneration in distal nerves may account for the development of hyperalgesia in patients with neuropathic pain. When a nerve is cut, the nerve fibers distal to the cut undergo wallerian degeneration. Adjacent uninjured fibers are exposed to a dramatically altered endoneurial environment. The inflammatory milieu may include chemokines, cytokines, and growth factors. This may lead to the development of peripheral or central sensitization. A, Peripheral sensitization. Wallerian degeneration may lead to the sensitization of primary afferent nociceptors. Now, mechanical stimulation of the nociceptors results in an enhanced response, which accounts for the mechanical hyperalgesia. B, Central sensitization. Wallerian degeneration may lead to the development of spontaneous activity in nociceptors. This spontaneous activity may produce a state of central sensitization similar to that described for secondary hyperalgesia (see Fig. 1-17 B). Now, mechanical stimulation of low-threshold mechanoreceptors activates the sensitized central pain-signaling neurons, which accounts for the mechanical hyperalgesia. CNS, central nervous system; DRG, dorsal root ganglion; PNS, peripheral nervous system.

Nociceptors and the Sympathetic Nervous System
Activity in nociceptors induces an increase in sympathetic discharge. This increased discharge is associated with the rise in blood pressure in acute pain states. Usually, the converse is not true: sympathetic activity does not affect the discharge of nociceptive neurons. In certain patients with pain, however, nociceptors acquire sensitivity to NE released by sympathetic efferents. Pain dependent on activity in the sympathetic nervous system is referred to as sympathetically maintained pain (SMP).
SMP may or may not be an important pain mechanism overall in patients, but in at least some individuals SMP may be the driving pathophysiological basis for the pain (for review see Drummond 2010 ). SMP in particular is noted in many cases of complex regional pain syndrome (reflex sympathetic dystrophy, causalgia). This condition is usually triggered by trauma to an extremity, with varying combinations of edema, allodynia and hyperalgesia, vasomotor and sudomotor abnormalities, and motor disturbances developing in the extremity. For SMP, procedures that interrupt the function of the sympathetic nervous system can alleviate the pain and hypersensitivity.

Sympathetically Maintained Pain Is a Receptor Disorder
Clinical studies support the concept that catechol sensitivity may develop in nociceptors after partial nerve injury. For example, intraoperative stimulation of the sympathetic chain induces pain in patients with causalgia ( Walker and Nulson 1948 , White and Sweet 1969 ). Also, physiological activation of sympathetic vasoconstrictor neurons leads to enhanced spontaneous pain and hyperalgesia in patients with SMP ( Baron et al 2002 ). Pain is increased in the majority of patients with complex regional pain syndrome when sympathetic nervous system activity is evoked by a loud startling noise or by cooling their forehead ( Drummond et al 2001 ). Injection of NE around stump neuromas or in the skin of patients with post-herpetic neuralgia induces an increase in spontaneous pain ( Chabal et al 1992 , Choi and Rowbotham 1997 , Lin et al 2006 ). In SMP, anesthetic blockade of the sympathetic nervous system relieves the pain and hyperalgesia; intradermal injection of NE into the previously hyperalgesic area induces pain ( Ali et al 2000 ). NE injected into normal subjects evokes little or no pain. This suggests that SMP does not arise from too much NE, but rather from the presence of adrenergic receptors in the skin that are coupled to nociceptors. Therefore, in SMP, the NE that is normally released from sympathetic terminals acquires the capacity to evoke pain.

Nerve Injury Induces Catechol Sensitization in Nociceptors
In addition to spontaneous activity, adrenergic sensitivity develops in nociceptors after nerve injury. In a primate model of an L6 spinal nerve lesion ( Ali et al 1999 ), intact nociceptors innervating the foot exhibited spontaneous activity and a response to the α 1 -adrenergic agonist phenylephrine applied to the receptive field. In monkeys in which no spinal nerve lesion was applied, little or no catechol sensitivity and spontaneous activity were present. Using a somewhat different injury model, studies in rabbits have also demonstrated catechol sensitization of intact nociceptors after injury to companion nerve fibers ( Sato and Perl 1991 ). C fibers ending in a neuroma also display adrenergic sensitivity (e.g., Häbler et al 1987 ). Thus adrenergic mechanisms may play a role in activating injured nociceptors as well.

α 1 -Adrenergic Agonists Activate Nociceptors Leading to Central Sensitization
Clinical studies support the postulate that SMP arises from expression of α-adrenergic receptors on the terminals of nociceptors. Systemic phentolamine, an α-adrenergic antagonist, relieves pain when given to patients with SMP ( Raja et al 1991 ). Topical application of clonidine, an α 2 -adrenergic agonist, to the painful skin of patients with SMP relieved hyperalgesia in the painful area ( Davis et al 1991 ). Activation of α 2 -adrenoceptors located on sympathetic terminals by clonidine blocks the release of NE. When phenylephrine, a selective α 1 -adrenergic agonist, was applied to the clonidine-treated area, pain was rekindled in patients with SMP ( Davis et al 1991 ). Thus, clinical data, as well as primate physiological data, suggest that in SMP, release of NE from the sympathetic terminals activates nociceptors that express α 1 -adrenoceptors. The spontaneous activity and excitation of nociceptors by NE lead to central sensitization. Whether a change in phenotype or some other molecular change explains this nociceptor chemical sensitization is unanswered. Of some interest is the finding that the density of α 1 -adrenoceptors in the epidermis of hyperalgesic skin of patients with complex regional pain syndrome is increased as measured with quantitative autoradiographic techniques ( Drummond et al 1996 ). For SMP, procedures that reduce or eliminate excitation of α 1 -adrenergic receptors lessen nociceptor activity and therefore lessen the hyperalgesia. Other potential central mechanisms that modulate nociception and emotional responses by adrenergic facilitation of nociceptive transmission in the dorsal horn or thalamus and/or by depletion of bulbospinal opioids have also been postulated ( Drummond 2010 ).

Acknowledgment
We appreciate the technical assistance of T. V. Hartke and Ian Suk for contributing original artwork. This work was supported by National Institute of Health grants P01 NS 47399, NS-14447, and NS-26363.
The references for this chapter can be found at www.expertconsult.com .

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Chapter 2
Molecular Biology of Sensory Transduction

Michael S. Gold


SUMMARY
The perception of pain arising from a noxious stimulus starts with conversion of the energy of the stimulus into an electrical signal in the primary afferent neurons innervating the site of the stimulus. This process of energy conversion is called transduction. The three general modalities of noxious stimuli that impinge on the body are chemical, thermal, and mechanical, although each of these groups can be broken down into the specific nature of the stimulus, including the type of chemical, the temperature, or unique properties of the mechanical stimulus such as torque, sheer, or stretch. Specific proteins or groups of proteins called transducers underlie the process of transduction. There has been tremendous progress over the past decade in the identification and characterization of transducers responsive to all three modalities of stimuli. Our understanding of chemotransduction has progressed the farthest with the detailed maps that are now available for some chemotransducers of chemical binding sites and the conformational changes in protein structure with ligand binding. Putative transducers responsive to temperatures ranging from noxious cold to noxious hot have been identified, as have transducers responsive to a variety of mechanical stimuli, but it is now clear that still more of both types of transducers have yet to be identified. Although transduction of noxious stimuli was once thought to be an intrinsic property of nociceptive afferents, mounting evidence indicates that transduction also occurs in a variety of cells surrounding the afferent terminals; we are only just beginning to tease apart the impact of the interplay between these direct and indirect transduction processes. The critical interaction between transducers and the ion channels that control the excitability of afferent terminals has long been appreciated. However, the molecular identity of many of these channels has now been determined. Finally, despite evidence that there are still transducers to be identified, the contribution of many transducers to injury-induced changes in sensitivity has now been characterized. Advances on all three of these fronts have suggested novel approaches for the treatment of pain that are being actively pursued.


Introduction
Pain, a sensory and emotional experience , is in the brain. As discussed elsewhere in this textbook, there are clearly cases, such as stroke, where pain can originate from within the central nervous system. However, the vast majority of the pain that we experience, including chronic pain associated with peripheral nerve injury and inflammatory disorders, arises from activity in primary afferent neurons. Moreover, the vast majority of this activity is due to the impact of thermal, chemical, and/or mechanical stimuli. Afferent activity may arise spontaneously under pathological conditions as a result of changes in the relative balance of ionic currents in the membrane ( Liu et al 2000 , 2002 ; Amir et al 2002 ), although even “spontaneous” activity may ultimately depend on membrane depolarization driven by a mechanical, thermal, or chemical stimulus impinging on the afferent, even though the source of the stimulus may not be readily apparent ( Gold 2000a ). The focus of this chapter is on the mechanisms that enable thermal, mechanical, and chemical stimuli to initiate neural activity.
By definition, sensory transduction is conversion of the energy of a stimulus into an electrical signal. For the special senses (vision, audition, olfaction, taste), transduction occurs in specialized organs via cellular events specific to the stimuli associated with these senses. Sensory information arising from the body, referred to as somatosensation, may also involve specialized sense organs. For example, Golgi tendon organs and Meissner’s corpuscles are involved in the transduction of tension on tendons and low-threshold mechanical stimuli on glabrous skin, respectively. The primary afferents or sensory neurons innervating these structures tend to have rapidly conducting myelinated axons and anatomically distinct, specialized endings that often incorporate non-neuronal cells ( Caterina et al 2005 ). In contrast, afferents referred to as nociceptors respond to noxious or potentially tissue-damaging stimuli that are normally perceived as painful. Axons of these neurons tend to have slowly conducting unmyelinated (C fibers) or thinly myelinated (Aδ fibers) axons with peripheral terminals that are not associated with specific structures or cell types ( Caterina et al 2005 ). Thus, nociceptors are said to have free nerve endings. Although recent data, discussed below, have forced investigators to rethink the contribution of other cell types to sensory transduction, an important implication of the free nerve ending is that the molecular machinery necessary for transduction of noxious stimuli must be intrinsic to the nociceptive afferents. The subsequent demonstration that subpopulations of isolated sensory neurons are responsive to thermal (both hot and cold) ( Cesare and McNaughton 1996 ; Reichling and Levine 1997 ; Reid and Flonta 2001a , 2001b ; Viana et al 2002 ; Thut et al 2003 ), mechanical ( McCarter et al 1999 , Drew et al 2004 ), and a variety of algogenic chemical stimuli ( Gold and Gebhart 2010 ) is consistent with the idea that transduction is an intrinsic property of nociceptive afferents.
The available evidence indicates that the resting membrane potential of nociceptive afferents is negative to −40 mV, with values at the cell body ranging between −50 and −75 mV ( Baccaglini and Hogan 1983 , Gold et al 1996 ). Evidence from study of the putative nociceptive afferent somata in vitro suggests that the action potential threshold is relatively high at greater than −35 mV ( Gold et al 1996 ; Petruska et al 2000 , 2002 ; Flake et al 2005 ; Harriott et al 2006 ; Harriott and Gold 2009b ). Thus, because action potential generation is necessary for propagation of sensory information to the central nervous system, transduction of nociceptive stimuli must ultimately result in membrane depolarization. The membrane depolarization resulting from a transduction event is called a generator potential.
A generator potential can be initiated in three primary ways. The first and most direct way involves the opening of an ion channel with an ion permeability ratio such that the equilibrium potential for the net charge movement through the channel is depolarized to the action potential threshold. In this case, sufficient activation of this ion channel will drive the membrane potential above threshold and thereby result in an action potential that can be propagated toward the central nervous system. Activation of the transient receptor potential vanilloid type 1 (TRPV1) channel is an example of such a transduction mechanism. As discussed below, the TRPV1 channel is activated or opened by thermal (heat) stimuli ( Caterina et al 1997 ), as well as by a variety of chemical stimuli ( Caterina et al 2005 ). It is a non-selective cation channel that is permeable to Ca 2+ , Na + , and K + such that the equilibrium potential, or the potential at which there is no net flux of charge, is approximately 0 mV for this channel. Because this equilibrium potential is above the action potential threshold, activation of enough TRPV1 channels can ultimately result in action potential generation ( Fig. 2-1 ).


Figure 2-1 At least three mechanisms underlie the initiation of a generator potential. A, One involves activation of an ion channel such as transient receptor potential vanilloid 1 (TRPV1) with a permeability ratio such that the equilibrium potential for ions flowing through the channel (E channel ) is above the action potential threshold (AP Thresh ). Sufficient activation of such a channel will drive the membrane above the AP threshold. This generator potential is passively propagated to an action potential initiation site with a high density of voltage-gated Na + channels. Decay of the generator potential is determined by the passive electrophysiological properties of the nociceptor terminal, which is dynamically influenced by the density, distribution, and activity of leak K + channels such as the two-pore K + channels TREK-1, TASK, and others. If the generator potential is above the AP threshold at the spike initiation site, an AP will be generated that is propagated toward the central nervous system. B, Another mechanism involves closing of a K + channel such as a two-pore K + channel. In this scenario there must be at least one other channel open, such as a persistent Na + channel with an E channel above the AP threshold. Closing the K + channels results in an increase in the relative permeability of the membrane for the depolarizing current, which may be sufficient to drive the membrane potential above the AP threshold. As in A, this generator potential must also be passively propagated toward the spike initiation zone. C, A third mechanism involves activation of a channel that has an equilibrium potential threshold. This appears to be the case for GABA A receptors in some nociceptive afferents. If this channel is close to a low-threshold voltage-activated channel, such as a low-voltage-activated Ca 2+ channel, the depolarization driven by activation of the transducer may be sufficient to activate the voltage-gated channel and push the membrane potential above the AP threshold. As in A, this generator potential must also be passively propagated toward a spike initiation site. GABA, γ-aminobutyric acid. leak K + channels; voltage-gated K + channels; voltage-gated Na 2+ channels.
A second mechanism underlying the generator potential involves the closing of a channel responsible for a hyperpolarizing current. K + channels are the only channels capable of contributing such a current in nociceptive afferents. This is because of the distribution of ions inside and outside nociceptive afferents. That is, interstitial fluid has a relatively high concentration of Na + , Ca 2+ , and Cl − and a low concentration of K + . In contrast, in nociceptive afferents, the intracellular concentration of K + is high, that of Cl − is relatively high ( Rocha-Gonzalez et al 2008 ), and that of Na + and Ca 2+ is low. Closing of a K + current is clearly an indirect mechanism of sensory transduction since it will result in a generator potential only if a resting depolarizing current is simultaneously active with the hyperpolarizing K + current. Relatively high K + conductance in the face of relatively low Na + conductance will still enable the neuron to maintain a resting membrane potential in the expected range. If the decrease in K + channels is sufficient in such a neuron, the result will be a generator potential capable of driving the membrane potential above the action potential threshold (see Fig. 2-1 ).
The third mechanism underlying a generator potential is also indirect, but in contrast to the second mechanism, it is dependent on a relatively close association between an ion channel capable of driving membrane depolarization and a low-threshold voltage-gated ion channel capable of pushing the membrane potential above the action potential threshold. That is not to say that the localization of ion channels is not critical for the ultimate success (i.e., generation of action potentials) of transduction via all three mechanisms, but this is particularly true for Cl − because of the unique regulation of Cl − in sensory neurons. There is evidence in some neurons that the concentration of intracellular Cl − may be high enough that the Cl − equilibrium potential is above the action potential threshold. Consequently, activation of a Cl − channel in these neurons, such as bradykinin-induced activation of the Ca 2+ -dependent Cl − channel TMEM16, may result in a generator potential sufficient for generation of an action potential ( Liu et al 2010 ). Though depolarized relative to the resting membrane potential in almost all sensory neurons, the Cl − equilibrium potential is still below the action potential threshold in many sensory neurons. However, if there are low-threshold voltage-activated channels such as the T-type Ca 2+ channel Ca v 3.2 in close association with Cl − channels, activation of a Cl − channel may still be sufficient for action potential generation even if the Cl − equilibrium potential is below the action potential threshold (see Fig. 2-1 ).
Many chemical stimuli act on the G protein–coupled receptors (GPCRs) expressed on nociceptors. In these cases, as discussed below, subsequent intracellular signaling cascades are needed to modify ion channel activity and drive initiation of the generator potential.

Chemo-, Thermo-, and Mechanotransducers
Transducers are often categorized according to the stimuli to which they are responsive. This is a useful way to think about transducers, particularly in the context of a particular type of pain or altered sensitivity such as cold allodynia or heat hyperalgesia, because it is reasonable to assume that these “types” of pain are due to afferent activity evoked with a specific stimulus. However, many, if not most of the putative thermo- and mechanotransducers respond to more than one stimulus modality and are therefore said to be polymodal. Given evidence that a variety of transducers are present and functional in non-neural tissue, it is also reasonable to categorize transducers according to whether they are intrinsic or extrinsic to the primary afferent. Furthermore, given evidence that at least one putative transducer (TRPV1) may be present and functional on subcellular organelles ( Castro et al 2009 ), it is at least worth considering transducer localization despite evidence that the vast majority of transducers are membrane bound. Nociceptive afferents and consequently transducers are present throughout the body. Although a pinprick and noxious stretch are both mechanical stimuli, visceral afferents such as those innervating the colon are far more sensitive to stretch (i.e., colon distention) than other forms of mechanical stimuli ( Ness and Gebhart 1990 ), whereas pinprick is a highly effective stimulus for activating nociceptive afferents innervating the skin ( Caterina et al 2005 ). Consequently, it is important to consider the nature of the stimulus and the tissue being affected. Finally, even though a number of chemotransducers are activated by noxious chemicals in the environment, the majority, if not all, are responsive to endogenous chemicals. Therefore, it is also important to consider the source of the stimulus.

Chemotransducers
Of the three primary modalities of somatosensory stimuli, the process of chemotransduction is the most well understood. Specificity for one chemical over another is achieved through binding sites in the transducer that are unique, or at least relatively so, for a particular chemical. In the most direct form of chemotransduction, the transducer has a binding site, or receptor, for the chemical stimulus and is also an ion channel. Binding of the chemical to the receptor drives a conformational change in the transducer protein that opens the ion channel (e.g., see Mayer 2011 ). Thus, these transducers are also referred to as ionotropic receptors ( Fig. 2-2 ). This is the most rapid form of chemical transduction, with signaling possible on the microsecond time scale. There is also an indirect form of chemotransduction whereby the conformational change in the transducer driven by chemical binding results in the activation of an intracellular signaling cascade. These transducers are referred to as metabotropic receptors (see Fig. 2-2 ). This form of chemical transduction is slower and occurs on a time scale of milliseconds to minutes. Guanine nucleotide–binding proteins, or GPCR receptors, are by far the most common type of metabotropic receptor, with the type of G protein being responsible for both initiation of the cellular signaling cascade and the type of cascade initiated ( Bunnett and Cottrell 2010 ). Additional metabotropic receptors found in sensory neurons include receptors bearing intrinsic protein tyrosine kinase domains (i.e., Trk receptors), receptors that associate with cytosolic tyrosine kinases (i.e., non–tyrosine kinase receptors such as cytokine receptors, integrins), and protein serine/threonine kinases (i.e., transforming growth factor-α [TGF-α] receptors) (see Gold 2005 for review). This second form of signaling is very widespread and responsible for changes in the regulation of a variety of cellular processes, including ion channel properties ( Fitzgerald et al 1999 ), cellular properties such as the regulation of intracellular Ca 2+ ( Werth et al 1996 ) and neurite extension ( Yasuda et al 1990 , Jones et al 2003 ), and gene expression ( Huang and Reichardt 2003 ). Second-messenger signaling is complex with multiple points of convergence and interaction ( Gold and Gebhart 2010 ). However, to keep this chapter tractable, I will consider only metabotropic receptor–mediated transduction events that are coupled to an ion channel that may initiate a generator potential.


Figure 2-2 Ionotropic receptors are directly coupled to ion channels. The purinergic P2X 3 receptor is composed of three subunits. Binding of adenosine triphosphate (ATP) drives a conformational change in the subunit assembly that leads to opening of an Na + channel. The result is rapid membrane depolarization. Metabotropic receptors initiate a second-messenger cascade. The purinergic P2Y 2 receptor is a G protein–coupled receptor able to drive an action potential. The ion channel coupled to P2Y 2 receptors has yet to be identified, but as a G q -coupled G protein–coupled receptor, it could drive the activation of phospholipase C, which has been shown to activate transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel, secondary to cleavage of phosphatidylinositol 4,5-bisphosphate (PIP 2 ).
At least three additional factors have an impact on the efficacy of chemoreceptor signaling. First, the spatial distribution of transducers relative to other ion channels in the membrane, particularly those responsible for action potential initiation, is a critical determinant of whether a generator potential will result in an action potential. To add to the complexity of metabotropic receptor signaling, there is evidence that second-messenger coupling is dynamic and changes in response to a number of different conditions, including hormonal status ( Dina et al 2001 ) and history of prior stimulation ( Parada et al 2005 ). Furthermore, it is dependent not only on the appropriate localization of transducers and targeted ion channel but also on the appropriate cellular signaling machinery. Consequently, metabotropic receptor activation may not always result in a generator potential. Second, allosteric modulation of chemotransducers, where a second chemical binding site is located at a site different from that of the chemical that activates the receptor, is common and can result in profound changes in chemotransducer activity. An extreme example is the N -methyl- D -aspartate (NMDA) type of ionotropic glutamate receptor: for glutamate to activate the receptor, it must also be bound to glycine ( Ren and Dubner 1999 , Dubner 2004 , Salter 2005 ). Third, a number of chemotransducers may be activated by several distinct chemicals. In the case of TRPV1, a transducer activated by protons and capsaicin (the pungent component of chili peppers), the binding sites or receptors for these compounds are on distinct parts of the protein ( Gavva et al 2005 ).

Ionotropic Receptor Families

Acid-Sensing Ion Channels
Ionotropic receptors are generally classified according to their structure and genetic homology. The acid-sensing ion channels (ASICs) are, as their name implies, activated by extracellular protons, although this is true for only three of the four genes encoding ASIC channel subunits (ASIC1, 3, and 4) since ASIC2 does not appear to be activated by protons ( Lingueglia 2007 ). All four subunits are detected in sensory neurons ( Alvarez de la Rosa et al 2002 , Hughes et al 2007 , Bohlen et al 2010 ), including the two splice variants of ASIC1 (1a and 1b) and ASIC2 (2a and 2b). The ASICs are trimeric proteins with homology to the epithelial Na + channel (ENaC)/degenerin family that can form homomeric or heteromeric channels ( Qadri et al 2012 ). ASIC3 was originally thought to be specific to dorsal root and trigeminal ganglion neurons ( Chen et al 1998 ), where it is enriched in nociceptive afferents, but it has subsequently been shown to be more widely expressed ( Sanchez-Freire et al 2011 , Sole-Magdalena et al 2011 ). It is the most sensitive to protons—activated by a decrease of less than 0.2 pH units—and this sensitivity is dramatically enhanced with lactate ( Immke and McCleskey 2001 ). Intense muscle use produces lactic acidas, a metabolic produce that is thought to contribute to exercise and ischemic muscle pain. Thererfore, ASIC3 is one of several transducers present in muscle afferents that may also be referred to as metaboreceptors ( Molliver et al 2005 ). ASIC3 is enriched in specific subpopulations of afferents, including those innervating the heart ( Benson et al 1999 ) and dura ( Yan et al 2011 ), where it has been suggested to contribute to the pain associated with coronary ischemia and migraine, respectively. Evidence from null mutant mice suggests that ASIC3 contributes to hyperalgesia of an inflamed muscle (primary hyperalgesia) whereas ASIC1 contributes to the hyperalgesia observed at sites distant from the inflamed muscle (secondary hyperalgesia) ( Walder et al 2010 ), although the details underlying the basis for this distinct pattern have yet to be fully clarified. Recent data indicating that venom from the western coral snake can directly activate ASIC1 ( Bohlen et al 2010 ) further support a role for this subunit in the response to pain-producing stimuli and suggest that these channels are responsive to a wider variety of stimuli than originally thought. The observation that the pH sensitivity of ASIC2a was dramatically increased by coral snake venom also led to the suggestion that this subunit may function as a coincidence detector for as yet to be identified compounds. Along these same lines, recent evidence suggests that ASIC3 and P2X 5 may act as coincidence detectors since binding of adenosine 5′-triphosphate (ATP) to P2X 5 dramatically increases the sensitivity of ASIC3 to protons ( Birdsong et al 2010 ).

Cys-Loop Family of Ligand-Gated Ion Channels

ACh/5-HT 3 /GABA A : One of the larger families of ligand-gated ion channels is the Cys-loop family, so named because of the characteristic loop in the extracellular N-terminal domain of the α subunit formed by a disulfide bond between two cysteine (Cys) residues ( Tsetlin et al 2011 ). Members of three of the four major subfamilies of Cys-loop receptors are present in sensory neurons. These include nicotinic acetylcholine receptors (nAChRs), serotonin type 3 (5-HT 3 ) receptors, and type A γ-aminobutyric acid (GABA) receptors (GABA A ). All Cys-loop receptors contain five subunits, which may be homomeric or heteromeric, depending on the receptor subtype and subunit composition.
Both homomeric (α 7 ; Shelukhina et al 2009 ) and heteromeric (α 3–6 , β 2–4 ; Xiao et al 2002 , Rau et al 2005 , Spies et al 2006 ) nAChRs composed of two α and three β subunits are present in sensory neurons, with evidence that both forms contribute to nociceptive processing ( Carstens et al 1998 , Schmelz et al 2003 ). Although there are several potential non-neural sources of acetylcholine (ACh) in the periphery, it is important to note that ACh levels are increased in inflamed tissue ( Wessler et al 2003 , Gahring et al 2010 ). Furthermore, peripheral administration of ACh- or nAChR-selective agonists results in burning pain (although recent evidence suggests that at least some of the pain associated with nicotine may be due to activation of TRPA1 ( Talavera et al 2009 ). Interestingly, recent evidence also suggests that the relative contribution of nAChR subtypes to peripheral pain may depend on the target of innervation. That is, α 7 nAChRs appear to suppress activity in colonic dorsal root ganglion (DRG) neurons ( Abdrakhmanova et al 2010 ), whereas the burning pain associated with the application of nAChR agonists to the skin is probably mediated by heteromeric receptors ( Carstens et al 1998 , Schmelz et al 2003 ).
The 5-HT 3 receptor is the only ionotropic serotonin receptor. It may exist as a homomeric receptor composed of five 5-HT 3A subunits or as a heteromeric receptor composed of a 5-HT 3A subunit with one of the other four 5-HT 3 subunits (5-HT 3B–3E ) ( Thompson and Lummis 2007 ). It was originally thought to have a relatively limited role in nociception because of its expression pattern in afferents with a medium to large cell body diameter ( Tecott et al 1993 ). Subsequent analysis, however, suggested that this receptor is critical for both serotonin-induced pain and, more importantly, full expression of the second phase of pain behavior in the formalin test ( Zeitz et al 2002 ), a behavior thought to reflect spontaneous inflammatory pain. Given that the gastrointestinal (GI) tract is the largest source of serotonin in the body, it should not be surprising that this receptor plays an even more important role in visceral pain. In fact, several 5-HT 3 receptor antagonists have been approved for use in treating the pain associated with irritable bowel and other visceral pain syndromes ( Fayyaz and Lackner 2008 ).
Unlike the nAChRs and 5-HT 3 receptors, which have ion channels permeable to Na + , K + , and to varying degrees, Ca 2+ , GABA A receptors have an ion pore selective for anions (primarily Cl − and to a less degree HCO 3 − ) ( Michels and Moss 2007 , Picton and Fisher 2007 ). Like nAChRs and 5-HT 3 receptors, GABA A receptors may exist as homomeric or heteromeric proteins. The homomeric proteins are composed of one of the three ρ subunits (originally called GABA C receptors) and have been most studied extensively in the retina ( Qian 1995–2005 ). Recent evidence suggests that ρ subunit–containing receptors are present in sensory neurons. However, heteromeric GABA A receptors are far more common throughout the nervous system, including sensory neurons. Although 19 subunits of the GABA A receptor have been identified, the number of possible receptors is constrained by a subunit stoichiometry that consists of two α subunits, two β subunits, and one of the remaining seven non-ρ subunits. GABA A receptor subunit composition determines the pharmacological and biophysical properties of the receptor, as well as its cellular distribution ( Michels and Moss 2007 ).
Given that GABA A receptors underlie the vast majority of fast inhibitory synaptic transmission in the adult central nervous system, it may be surprising to find these receptors among a list of chemotransducers. However, because, as noted above, the concentration of intracellular Cl − is maintained at levels considerably higher in nociceptive afferents than in neurons in the central nervous system ( Rocha-Gonzalez et al 2008 ), GABA A receptor activation results in membrane depolarization in nociceptive afferents ( Price et al 2009 ), which may still be inhibitory in the absence of tissue injury. However, following tissue injury, GABA A receptor activation may result in action potential generation in nociceptive afferents ( Willis 1999 ). This can occur at the central terminals of nociceptive afferents, a process referred to as the dorsal root reflex. More importantly in the context of the present discussion, GABA A receptor activation in the periphery can excite nociceptive afferents ( Carlton et al 1999 , Carr et al 2010 ). It should be noted that although there do not appear to be sources of peripheral GABA necessary to achieve the concentrations needed for activation of low-affinity synaptic receptors, recent evidence suggests that high-affinity receptors, which may be activated by GABA concentrations close to the resting levels observed in extracellular fluid, are present in sensory neurons ( Lee et al 2012 ). Thus, even though there is evidence that a shift in GABA A signaling in the spinal cord contributes to both the initiation and maintenance of inflammatory hypersensitivity, high-affinity GABA A receptors in the periphery may also contribute to ongoing afferent drive in the presence of inflammation.

Glutamate Receptors
Ionotropic glutamate receptors play a critical role in fast excitatory synaptic transmission in the central nervous system and are therefore generally studied in the context of synaptic transmission. These receptors have historically been classified according to their response to three selective agonists: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, and NMDA ( Mayer 2011 ). Subsequent analysis has indicated that these receptors are composed of heteromeric combinations of four subunits, with AMPA receptors being composed of GluR1–4, kainate receptors being composed of GluR5–7 (GluK1–3) and/or KA1–2 (GluK4–5), and NMDA receptors being composed of a combination of NR1, NR2A–D, and/or NR3A–B. All three types of receptors are present and functional in primary afferent neurons ( Miller et al 2011 ). Interestingly, even though these receptors are present on the central terminals of primary afferents, where they appear to contribute to synaptic transmission in the spinal cord, they are also present in the periphery, where they can drive action potential generation ( Cairns et al 1998 , 2001b ; Lam et al 2009a , 2009b ) and pain ( Cairns et al 2001a ). There are a number of potential sources of peripheral glutamate, including the afferent itself, where peripherally released glutamate could serve as a form of feedback excitation to amplify injury-induced activation of nociceptive afferents.

HCN Channels
Hyperpolarization-activated, cyclic nucleotide–gated (HCN) ion channels are not typical chemotransducers since they are not directly activated by extracellular ligand binding ( Wickenden et al 2009 ). Furthermore, because these non-selective cation channels are activated by membrane hyperpolarization and close with membrane depolarization, their biophysical properties argue against a role in the initiation of a generator potential capable of driving the membrane above the action potential threshold. Under resting conditions, these channels are activated only during the increase in membrane potential associated with the after-hyperpolarization that follows an action potential, where they provide a depolarizing drive for subsequent spike initiation ( Ingram and Williams 1994 ). There are four HCN family members (HCN1–4), which consistent with their homology to Kv family members, form homomeric tetramers ( Wickenden et al 2009 ). mRNA for all four HCN channels is detectable in sensory neurons, with HCN1 being differentially distributed in large- and small-diameter neurons and HCN3 enriched in small-diameter neurons ( Chaplan et al 2003 , Kouranova et al 2008 ).
These channels have been included in the list of chemotransducers for several reasons. The voltage dependence of channel activation is regulated by intracellular cyclic adenosine monophosphate (cAMP) such that an increase in cAMP drives a depolarizing shift in the voltage dependence of channel activation. This shift can be sufficiently large that HCN channels contribute to the depolarization of resting membrane potential and, more relevantly, to a depolarizing drive that facilitates action potential generation ( Emery et al 2011 ). The channel is also a putative target for a number of metabotropic receptors coupled to second-messenger pathways that result in an increase in cAMP. There is an increase in HCN current density in large-diameter neurons following traumatic peripheral nerve injury, where the increase appears to be responsible for ectopic activity ( Chaplan et al 2003 ). Recent evidence from null mutant mice suggests that HCN2 in Na v 1.8-expressing neurons plays a particularly important role in the generation of inflammatory thermal hyperalgesia, as well as peripheral nerve injury–induced thermal and mechanical hypersensitivity ( Emery et al 2011 ).

Purinergic Receptors—P2X
Ionotropic purinergic, or P2X, receptors are activated by ATP. The functional receptor is a trimer. Seven subunits have been identified, P2X 1–7 ; all but P2X 6 can form functional homomers, and each appears to be able to form a heteromeric protein with at least one other subunit ( Burnstock 2006 , Jarvis and Khakh 2009 ). At least six, if not all seven, subunits are present in sensory neurons, with all but P2X 4 being differentially distributed among subpopulations of DRG neurons ( Kobayashi et al 2005 ). P2X 3 , which forms a heteromer with P2X 2 , has been the most extensively studied member of this family with respect to nociceptor activation ( Jarvis 2003 ). This subunit was originally shown to be enriched in a subpopulation of neurons with a small cell body diameter that did not express the neuropeptides substance P or calcitonin gene–related peptide (CGRP) ( Bradbury et al 1998 ), the so-called non-peptidergic afferents. Although the distribution of P2X 3 receptors among specific subpopulations of sensory neurons was subsequently shown to depend on the target of innervation (e.g., see Ambalavanar et al 2005 ), pharmacological and molecular biological analysis confirmed that this subunit plays a dominant role in mediating the nociceptive response to the application of ATP, as well as the response to a variety of noxious stimuli applied to various tissues ( Jarvis 2003 ). Interestingly, this subunit appears to mediate the activation of nociceptive afferents observed following damage to neighboring cells ( Cook and McCleskey 2002 ), thus making this transducer a critical player in the initial pain observed in response to tissue injury. As noted above, more recent work has highlighted a potential role for P2X 5 in the sensitization of ASIC currents in sensory neurons and consequently the pain associated with muscle ischemia ( Birdsong et al 2010 ).

Transient Receptor Potential Channels
The TRP channels involved in chemotransduction come from a large family of ion channels that encompass eight subfamilies ranging from TRPA (for ankyrin) to TRPV (for vanilloid), with TRPC (for canonical), TRPM (for melastatin), TRPML (for mucolipins), TRPP (for polycystins), and TRPN (for NO-mechanopotential C) in between ( Nilius and Owsianik 2011 ). Unifying features of this family include a channel protein formed from four subunits, each with a structure analogous to that for voltage-gated K + ion channel subunits with a six-transmembrane segment and a pore loop between transmembrane segments 5 and 6. Specific to TRP channels are ankyrin domains on the intracellular N-terminus and proline-rich domains on the intracellular C-terminus. All TRP family members form Ca 2+ -permeable cation channels. Many of the family subunits have a voltage sensor in segment 4 and consequently exhibit voltage-sensitive properties. As discussed below, several of the channels are activated by different stimulus modalities, with the biophysical properties of the channel activity appearing to depend on how the channel is activated. For example, capsaicin-induced activation of TRPV1 desensitizes in the presence of extracellular Ca 2+ , whereas heat-evoked activation of TRPV1 does not ( Caterina et al 1997 ). The channels also appear to be used differentially across phyla. For example, TRPA1 functions as a cold receptor among other modalities of transduction in mammals ( Story et al 2003 , Karashima et al 2009 ) but underlies infrared detection in snakes ( Gracheva et al 2010 ). The relative contribution of various TRP channels to nociceptive processing is an active area of investigation.

TRPA1: TRPA1 was originally identified through a combined bioinformatic and expression strategy designed to identify additional TRP family members ( Story et al 2003 ). It is the only member of its subfamily defined by the exceptional number (14) of N-terminal ankyrin repeats. Although TRPA1 was first thought to function primarily as a cold transducer (see below), subsequent analysis has indicated that TRPA1 is responsive to a variety of noxious compounds. This list first included allyl isothiocyanates (i.e., mustard oil) and cannabinoids ( Jordt et al 2004 ) and was rapidly expanded to include the pungent ingredients of garlic ( Bautista et al 2005 ), cinnamon, wintergreen oil, clove oil, and ginger ( Bandell et al 2004 ), as well as a variety of environmental irritants such as acrolein, CO 2 , and formalin ( McNamara et al 2007 ). TRPA1 is also activated by a number of endogenous mediators, including products of oxidative stress ( Andersson et al 2008 ) and cyclooxygenase-dependent fatty acid metabolites ( Materazzi et al 2008 , Taylor-Clark et al 2008 ). TRPA1 appears to be expressed in a subset of TRPV1-expressing neurons and, like TRPV1, appears to be a target of endogenous algogenic compounds such as bradykinin ( Bandell et al 2004 ). There is also evidence that TRPA1 may interact with TRPV1 at the level of a complex ( Akopian 2011 ). As a result, activation of one channel influences the response resulting from activation of the other. Interestingly, despite its role in mediating the acute painful response to a variety of chemicals, a gain-of-function mutation in TRPA1 is not associated with ongoing pain. Rather, individuals with the TRPA1 mutation experience episodic upper body pain triggered by fasting or physical stress ( Kremeyer et al 2010 ).

TRPM2: TRPM2 is widely expressed throughout the body but is particularly enriched in immune cells such as macrophages ( Harteneck 2005 , Jiang et al 2011 ). It was originally suspected that TRPM2 would have enzymatic properties in addition to its putative function as a Ca 2+ channel based on the presence of a Nudix box on its C-terminus, a motif common to enzymes, particularly those that degrade nucleoside diphosphates ( Perraud et al 2001 ). It is likely that this motif serves as a binding site for adenosine diphosphate (ADP)-ribose, which was subsequently shown to activate the channel. It was soon realized, however, that TRPM2 plays a significant role in mediating the cellular response to stress since it is activated by reactive oxygen species such as hydrogen peroxide ( Takahashi et al 2011 ). The channel has recently been shown to be present in sensory neurons, where it enables sensitivity to hydrogen peroxide ( Naziroglu et al 2011 ). Recent data indicate that the channel plays a prominent role in facilitating the inflammatory response to infection ( Yamamoto et al 2008 ) and is likely to contribute to inflammatory hypersensitivity. However, though present in sensory neurons ( Naziroglu et al 2011 , Özgül and Naziroglu 2012 ), the pro-nociceptive role of this channel is likely to be indirect via facilitation of the release of chemokines such as CXCL2 from immune cells and microglia.

TRPM3: TRPM3 is another member of the melastatin subfamily of TRP receptors implicated in nociception. This TRP channel is expressed in a variety of different tissues, including a relatively broad population of sensory neurons ( Vriens et al 2011 ). The most potent known agonist for TRMP3 is pregnenolone sulfate. Although this compound acts at a number of receptors and ion channels, it has been shown to induce nociceptive behavior when administered to mice, behavior that is eliminated in TRPM3 null mutant mice ( Vriens et al 2011 ).

TRPM8: The discovery of TRPM8 was reported almost simultaneously by two different groups that used complementary strategies of bioinformatics ( Peier et al 2002a ) and expression cloning ( McKemy et al 2002 ) to identify novel TRP channels. This channel was shown to be responsive to both cooling and menthol. The channel is present in a small subpopulation of non-peptidergic afferents that does not overlap with TRPV1/TRPA1-expressing neurons ( McKemy et al 2002 , Peier et al 2002a ). Because the sensation of menthol is not usually described as painful, this transduction mechanism would generally be grouped with those associated with the transduction of non-noxious stimuli. It is included here for the sake of completeness and, as noted below, because of evidence that the channel contributes to the perception of cold pain ( Knowlton et al 2011 ). Interestingly, recent genome-wide association studies have linked TRPM8 to migraine without aura (ref PMID:21666692 and 22683712), although it remains to be determined how a polymorphism in this channel contributes to the increased risk for the presence of migraine.

TRPV1: The field of sensory transduction broke open in 1997 with the discovery of TRPV1, a TRP channel activated by capsaicin, the pungent compound in chili peppers ( Caterina et al 1997 ). This receptor had long been sought because of an extensive body of both preclinical and clinical data supporting a link between the actions of capsaicin and pain ( Holzer 1991 ). Interestingly, although the only sensation associated with capsaicin in the short term is pain ( Schmelz et al 2000 ), in the long term, treated tissue can become desensitized to subsequent noxious stimuli ( Nolano et al 1999 ). Probably because of species differences in the distribution of TRPV1 among subpopulations of nociceptive afferents, the modality specificity of this desensitization appears to depend on the species being studied; it appears to be specific to noxious heat in the mouse ( Cavanaugh et al 2009 ), but it encompasses heat, mechanical, and possibly chemical stimuli in the rat, dog, and primate ( Nolano et al 1999 ). Various preparations of TRPV1 agonist and administration routes are still being explored for the treatment of chronic pain ( Wong and Gavva 2009 , Anand and Bley 2011 ).
A number of interesting features of TRPV1 have been revealed since its original discovery. As discussed below, it is not just a receptor for capsaicin but is activated by noxious heat and plays a critical role in the manifestation of thermal hyperalgesia ( Caterina et al 2000 ). In addition to capsaicin, TRPV1 is responsive to a number of different pungent compounds found in plants, including resiniferatoxin, piperine, and camphor ( Tominaga et al 1998 ), as well as endogenous mediators such as protons (though with considerably lower potency than ASIC3) and lipids (e.g., oxidized linoleic acid metabolites; Patwardhan et al 2009 , 2010 ). There is evidence that phosphatidylinositol 4,5-bisphosphate (PIP 2 ) can both activate and inhibit TRPV1 ( Vriens et al 2009 ). In a model in which PIP 2 is inhibitory, it was proposed that algogenic compounds such as bradykinin are able to activate TRPV1 subsequent to the activation of phospholipase C, which frees TRPV1 from inhibition following PIP 2 hydrolysis ( Prescott and Julius 2003 ). The channel is a target for a number of different protein kinases, thereby resulting in dynamic regulation of channel properties and membrane distribution ( Gold and Gebhart 2010 ). Channel translation and expression are also dynamically regulated and enable the channel to contribute to both the initiation and maintenance of persistent pain. Prolonged activation of the receptor results in a process referred to as pore dilation, where the size of the channel increases to the point that it becomes permeable to large macromolecules ( Chung et al 2008 ). A comparable process has recently been described in TRPA1 ( Chen et al 2009 ). Although the physiological consequences of this process have yet to be fully elucidated, it may be possible to exploit this property for therapeutic purposes and deliver molecules such as membrane-impermeable local anesthetics to provide selective blockade of nociceptive afferents ( Binshtok et al 2007 ).

TRPV4: TRPV4 was first thought to function as an osmosensor responsive to cell swelling (see below) ( Strotmann et al 2000 ). Subsequent analysis has indicated that the channel is also a thermosensor (see below) responsive to warming ( Guler et al 2002 ). Data from knockout or knockdown experiments suggest that the channel contributes to inflammatory pain and sensitivity ( Alessandri-Haber et al 2005 , 2006 ), particularly of visceral structures such as the bladder ( Everaerts et al 2010 ), although this appears to be due to its function as a mechanotransducer (see below). Nevertheless, like other TRP channel family members, TRPV4 is responsive to a plant derivative, as well as to artificial ligands such as 4-α-PDD ( Vriens et al 2007 ), thus raising the possibility that there are endogenous ligands that have yet to be identified.

Two-Pore Potassium Channels
Two-pore potassium (K2P) channels are a large family of resting, or background, K + channels that when active, inhibit cell excitability ( Honore 2007 ). The channels are widely distributed throughout the body. Family members present in sensory neurons include TREK-1–2, TRAAK, TASK1–3, and TRESK, although recent data suggest that additional family members are detectable in mRNA extracted from whole ganglia ( Marsh et al 2012 ). The name for TREK-1 comes from TWIK-related K + channel, where TWIK stands for a tandem of P domains in a weak inward rectifier K + channel ( Patel and Honore 2001 ). TREK-1 was cloned in 1996 based on homology to TWIK and was first shown to be activated by arachidonic acid. TREK-2 and TRAAK (TWIK-related arachidonic acid–activated K + channel) are also activated by arachidonic acid. TWIK-related acid-sensitive K + channels (TASK) are present in putative nociceptive afferents ( Rau et al 2006 ) and are inhibited by protons and serotonin ( Hopwood and Trapp 2005 ). The S-type K2P TRESK activity appears to be regulated primarily via second-messenger signaling cascades, with activity being increased by calcineurin and inhibited by kinase activity, including protein kinase A, and by 14–3–3 adaptor protein docking ( Czirjak and Enyedi 2010 ). That inhibition of K2P activity is sufficient to drive afferent activation is suggested by the observation that K2P channel blockers such as sanchool can drive activity in isolated DRG neurons ( Bautista et al 2008 ). Moreover, DRG neurons from TRESK null mutant mice are hyperexcitable ( Dobler et al 2007 ) and hyper-responsive to noxious stimuli. TRESK is also down-regulated in primary afferents following peripheral nerve injury, and this down-regulation is associated with an increase in afferent excitability ( Tulleuda et al 2011 ). Most recently, a dominant negative mutation in TRESK was linked to a form of familial migraine with aura ( Lafreniere et al 2010 ). Finally, there is evidence that TREK-1 and -2 are down-regulated in colonic afferents in a mouse model of colitis ( La and Gebhart 2011 , La et al 2011 ).

Metabotropic Receptors
Seemingly endless lists of metabotropic receptors have been identified in sensory neurons, including a long list of GPCRs and neurotrophin receptors and a growing list of receptors for cytokines and chemokines. As discussed above, there is evidence that the activity of a number of chemotransducers and ion channels may be regulated directly by activation of metabotropic receptors. For example, TRPV1 can be activated by metabotropic receptors coupled to phospholipase C ( Chuang et al 2001 ). More commonly, however, metabotropic receptor activation results in “sensitization” of a transducer or ion channel such that the ion channel is more readily activated by natural stimuli such as a change in membrane potential (as for HCN channels) or temperature (as for TRPV1 channels). Thus, under the appropriate conditions, all metabotropic receptors are theoretically capable of acting as a chemotransducer. Not surprisingly, although there are a number of cases in which the ion channels underlying a metabotropic receptor–mediated generator potential have been identified, such as the Ca 2+ -dependent Cl − channel TMEM16A (or ONO1) underlying the actions of bradykinin in a subpopulation of DRG neurons ( Liu et al 2010 ), in many cases the ion channels responsible for the generator potential are unknown.
Despite the large number of metabotropic receptors expressed in sensory neurons, the number that normally activates nociceptive afferents is relatively small. These include the B 1 and B 2 receptors for bradykinin ( Mense 1982 ), the H 1 receptor for histamine ( Fu et al 1997 , Jafri et al 1997 , Schmelz et al 2003 ), P2Y 2 receptors for ATP ( Molliver et al 2002 , Stucky et al 2004 ), the endothelin A receptor for endothelin-1 ( Gokin et al 2001 , Namer et al 2008 ), the protease-activated receptor 2 (PAR-2) for extracellular proteases ( Patwardhan et al 2006 ), and IP receptors for prostacyclin ( Birrell et al 1991 ). Understanding signaling via metabotropic receptors in sensory neurons is complicated by two factors. First, there are metabotropic receptor homologues for many of the ionotropic receptors activated by endogenous ligands. For example, afferents express both ionotropic and metabotropic receptors for ACh ( Spies et al 2006 , Nandigama et al 2010 ), ATP ( Molliver et al 2002 ), glutamate ( Willcockson and Valtschanoff 2008 , Carlton et al 2009 ), and serotonin ( Pierce et al 1996 , Zeitz et al 2002 ). The result may be a unique pattern of activity as has been observed for ATP, with the initial burst of activity being mediated by the ionotropic receptor and a more slowly developing, longer-lasting burst being driven by the metabotropic receptor ( Molliver et al 2002 ) ( Fig. 2-3 ). Second, the metabotropic receptors may be coupled to inhibitory second-messenger pathways, which can result in yet another pattern of activity. A variety of GPCRs are discussed further in Chapter 3 of this volume.


Figure 2-3 Action potentials ( A ) and currents ( B ) evoked in a sensory neuron by adenosine triphosphate (ATP) and its analogues. A single application of ATP evoked two series of action potentials: a brief train occurred immediately after the application of ATP, and then a much longer train occurred after a delay and then persisted for many seconds after removal of ATP. Application of alpha-beta methylene ATP (αβme-ATP), which selectively stimulates sensory neuron ATP-gated ion channels (P2X receptors), evoked the initial train of action potentials but not the prolonged one. Application of uridine triphosphate (UTP), which activates G protein–coupled ATP receptors (P2Y), evoked the delayed, persistent train but not the rapid, brief one. ATP and αβme-ATP both evoked the large currents typical of P2X 3 channels, but UTP evoked only a very small inward current; KCNQ has been shown to underlie much of this current. (Reproduced from Molliver DC, Cook SP, Carlsten JA, et al 2002 ATP and UTP excite sensory neurons and induce CREB phosphorylation through the metabotropic receptor, P2Y2. European Journal of Neuroscience 16:1850–1860. Copyright 2002 with permission from Blackwell Publishing Ltd.)

Chemotransducer Ligands
A common feature of the chemotransducers in sensory neurons is their ability to respond to both exogenous and endogenous compounds. Given the array of noxious chemicals and pathogens in the environment, it makes intuitive sense that the body would adapt ways of detecting the presence of these potential sources of threat or injury. Some classes of receptors for structurally conserved components of bacteria, fungi, viruses, and other organisms, such as the toll-like receptors, are specialized to detect ligands derived from exogenous sources. Many of these receptors, including 7 of the 13 known toll-like receptors, are present in sensory neurons ( Ochoa-Cortes et al 2010 ), and although the second-messenger pathways activated by these receptors could result in a generator potential, these receptors appear to be responsible primarily for afferent sensitization rather than activation (e.g., see Diogenes et al 2011 ). On the other hand, a number of chemotransducers are clearly specialized to detect endogenous signaling molecules, an extreme example being the glutamate receptors specialized to subserve fast synaptic transmission. The TRP channels are prime examples of receptors with the capacity to respond to both exogenous and endogenous compounds. It should therefore not be surprising that identification of endogenous and exogenous ligands for chemotransducers in sensory neurons is an active area of investigation. Notable in this regard are the creative strategies used. In one compelling example, researchers reasoned that the intense and prolonged pain associated with a burn injury may be due to mediators released in response to the heating of tissue. This led to the discovery of oxidized linoleic acid metabolites, which turned out to be potent endogenous agonists for TRPV1 ( Patwardhan et al 2009 , 2010 ). A second compelling example is the use of microdialysis probes in patients with painful oral cancers ( Hardt et al 2011 ). In this case, the researchers again reasoned that oral cancers are particularly painful because of the compounds released within the cancer. Although this research team has only just begun to identify the endogenous source of this form of cancer pain, it appears to involve an increase in proteolytic activity that drives the activation of PARs ( Lam and Schmidt 2010 ). A third example involves a screen of toxins associated with intense pain in humans. This led to the discovery of a toxin in western coral snake venom that acts on ASIC channels as described above ( Bohlen et al 2010 ). This first example highlights the possibility that transduction of any stimulus may involve a multiple-step process whereby the initial stimulus, such as heat or a bacterial cell wall, results in the activation of another cell type, such as a resident immune cell in the case of the bacteria, that releases the chemical ultimately responsible for the generator potential in sensory neurons.

Thermotransduction


The Molecular Thermometer (TRPA1–TRPV2)
The cloning of TRPV1 was not just a watershed moment for the understanding of chemotransduction in nociceptive afferents; it also facilitated the understanding of thermotransduction as well: TRPV1 was not only the capsaicin receptor but was additionally a thermotransducer activated by noxious heat ( Caterina et al 1997 ). Within 6 years of the cloning of TRPV1 six different TRP channels had been identified that together had the biophysical properties necessary to build a molecular thermometer that spanned the full range of human thermosensory perception ( Fig. 2-4 ). TRPA1 was found to be a transducer for noxious cold that encodes drops in temperature from approximately 18 to 4°C ( Story et al 2003 ). TRPM8 had the properties of a cool receptor that was responsive to small decreases in temperature lower than 30°C ( McKemy et al 2002 ). TRPV3, though present in keratinocytes rather than sensory neurons, was responsive to innocuous increases in temperature above 37°C ( Peier et al 2002b , Xu et al 2002 ). TRPV4 was also activated by innocuous warming and was clearly expressed in sensory neurons ( Guler et al 2002 ). TRPV1 was responsive to noxious heat with a threshold for activation roughly comparable to that associated with a heat pain threshold ( Caterina et al 1997 ). Finally, TRPV2 was responsive to heat with a threshold for activation (>48°C) close to that associated with heat pain tolerance in psychophysical studies ( Caterina et al 1999 ). The observation that TRPM8, TRPV4, TRPV1, and TRPV2 are present in different populations of neurons was consistent with in vivo single-unit electrophysiological and psychophysical data ( Bessou and Perl 1969 , Iggo 1969 , Hensel and Iggo 1971 , Darian-Smith et al 1973 , Dubner et al 1975 , LaMotte and Campbell 1978 , Campbell and LaMotte 1983 , Treede et al 1995 ) and was used to provide compelling support for the argument that a population code (i.e., activity in different populations of afferents) is used for thermosensation and that there was a “labeled line,” at least from the periphery, for heat pain. Furthermore, the overlap between TRPV1 and TRPA1 ( Story et al 2003 ) was used to explain the psychophysical observation that noxious cold is perceived as burning pain. This idea has proved overly simplistic, however, as discussed below.


Figure 2-4 Thermosensitive channels respond to a wide range of temperatures. This diagram depicts mammalian channels that have been demonstrated or proposed to underlie the neural response to thermal stimuli, arranged according to their temperature response profiles when examined in heterologous expression systems. Listed below each channel are their reported thermal thresholds and the range of temperatures to which they respond. Recent data suggest that there may be additional channels that contribute to thermosensation. Most problematic is evidence in support of a role for TRPV2 in thermosensation since null mutant mice have no detectable thermal phenotype. Also problematic is TPRA1, which clearly functions as a thermoreceptor for noxious cold in heterologous systems and in isolated sensory neurons but appears to contribute to the response to noxious cold only in the presence of tissue injury. Additional channels have also been identified that appear to contribute to thermosensation. These include the two-pore K + channels, TREK-1, TREK-2, and TRAAK, which are activated at between approximately 28 and 42°C. This property enables these channels to contribute to the response to both warming (via inhibition of afferent activity) and cooling (via a decrease in inhibition). TRPM3 has a relatively low threshold for activation (≈30°C) but appears to contribute to the response to noxious heat. Finally, TRPC5 is activated with decreases in temperature from 37 to 25°C and may contribute to the response to cooling. (Modified from Jordt SE, McKenny DD, Julius D 2003 Lessons from peppers and peppermint: the molecular logic of thermosensation. Current Opinion in Neurobiology 13:487–492. Copyright 2003 Elsevier Ltd.)

TRPA1: Concerns over the molecular thermometer model were raised almost immediately, with some of the most contentious disagreements being focused on TRPA1. In heterologous expression systems, cold-evoked TRPA1 responses were transient, which differed from the sustained responses to noxious cold observed in isolated sensory neurons ( Thut et al 2003 ), single-unit electrophysiology ( Bessou and Perl 1969 , Iggo 1969 , Hensel and Iggo 1971 ), and psychophysical studies ( Kenshalo and Scott 1966 , Johnson et al 1973 ). Data from some of the initial studies of cold transduction in isolated sensory neurons implicated the closing of K + channels as a contributing ( Viana et al 2002 ) if not primary mechanism ( Reid and Flonta 2001a ). Others failed to detect a response to noxious cold with TRPA1, even in comparable heterologous expression systems ( Jordt et al 2004 ). Subsequent data with TRPA1 null mutant mice suggested that the contribution of TRPA1 to the response to acute noxious cold stimuli was minimal, if detectable at all ( Bautista et al 2006 , Kwan et al 2006 ). Data derived from TRPA1-selective antagonists confirmed the negative behavioral data obtained in TRPA1 null mutant mice, thus suggesting that the channel has no detectable influence on cold sensitivity in naïve animals ( Chen et al 2011 ). With some distance from this particular debate and time for more detailed analysis, it is clear that TRPA1 is gated by noxious cold ( Karashima et al 2009 ). It is also clear that the channel contributes to injury-induced cold hypersensitivity ( del Camino et al 2010 ). Finally, as yet unidentified mechanisms must contribute to the response to acute noxious cold stimuli.

TRPM8: Data from a number of different lines of investigation support the notion that TRPM8 contributes to the response to innocuous cooling. This includes single-cell polymerase chain reaction of isolated sensory neurons ( Nealen et al 2003 ), single-unit electrophysiology, and behavioral data from null mutant mice ( Bautista et al 2007 , Knowlton et al 2011 ). The only point of contention over TRPM8 is whether this channel also contributes to nociceptive behavior. In the absence of tissue injury, the answer to this question appears to be no because the channel does not code well into the noxious range, it is present in a subpopulation of neurons that have properties of non-nociceptive afferents ( Nealen et al 2003 , Bautista et al 2007 ), and there is little change in behavior in response to noxious cold stimuli in TRPM8 null mutant mice ( Bautista et al 2007 , Babes et al 2011 ). However, high-dose menthol applied topically in humans is used to generate hyperalgesia. Moreover, in the presence of tissue injury, cold hypersensitivity is attenuated in TRPM8 null mutant mice ( Xing et al 2007 ) and with a TRPM8 antagonist ( Knowlton et al 2011 ). Nonetheless, several additional lines of evidence argue against a role for TRPM8 in the cold sensitivity associated with nerve injury ( Katsura et al 2006 , Caspani et al 2009 ). Whether the putative role for TRPM8 in nociception is due to de novo expression of TRPM8 in nociceptive afferents ( Djouhri et al 2004 ; but see Caspani et al 2009 ) and/or changes in the central nervous system such that input via cool-responsive neurons is able to engage a nociceptive circuit remains to be determined.

TRPV3 and TRPV4: Unfortunately, because warm fibers are generally absent in rodents, progress in our understanding of the relative contribution of TRPV3 and TRPV4 to the response to warmth has been slow. Whether these channels contribute to the response to innocuous warming in other species remains to be determined. However, results from null mutant mice and the use of non-selective TRP channel blockers ( St Pierre et al 2009 ) indicate that these channels have little role in the afferent or behavioral response to innocuous warm or noxious heat. Because rodents appear to have the ability to discriminate temperatures in the innocuous warm range, these observations suggest that other channels must contribute to warmth transduction.

TRPV1: There has also been little dispute over whether TRPV1 is activated by noxious heat. The response to heating in some isolated sensory neurons is blocked by TRPV1 antagonists and absent in TRPV1 null mutant mice ( Caterina et al 2000 ). The single-unit response to noxious heat ramps may be attenuated but is not lost in null mutant mice ( Caterina et al 2000 ), and the behavioral response to noxious heat is also not lost with TRPV1 antagonists ( Wong and Gavva 2009 ) and in null mutant mice ( Caterina et al 2000 ). The most pronounced phenotype, at least with respect to thermosensation, in null mutant mice is the absence of inflammatory heat hyperalgesia ( Caterina et al 2000 ). Together, these observations have several important implications. The complete loss of a heat response in isolated sensory neurons from TRPV1 null mutant mice in the face of heat sensitivity that persists in vivo suggests that (1) there are other thermotransducers and (2) TRPV1 may not be present in primary afferents that contribute to thermosensation. The possibility that these other transducers act in concert with TRPV1 to generate a “normal” response to heat is suggested by the decrease in the slope of the single-unit stimulus–response function ( Caterina et al 2000 ). Subsequent data indicating the presence of a subpopulation of afferents deficient in TRPV1 immunoreactivity that were responsive to noxious heating and still present in TPRV1 null mutant mice confirmed that there were additional heat transducers ( Woodbury et al 2004 ). Finally, the prominent role of TRPV1 in inflammatory heat hyperalgesia supported the conclusion that activity in specific subpopulations of afferents mediates specific types of pain.

TRPV2: The thermal threshold for TRPV2, as well as its presence in a subpopulation of DRG neurons with a “medium-diameter” cell body, was an excellent fit with the available single-unit and psychophysical data ( Treede et al 1995 ). Previous data from the primate had revealed two types of nociceptive afferents with axons conducting in the Aδ range. Type II AMHs (A fibers responsive to mechanical and heat stimuli) a higher threshold for activation than did heat-responsive C fibers but a very short utilization time (i.e., were activated very rapidly). These fibers were thought to be primarily responsible for the behavioral responses to acute noxious stimuli. Data from rodents indicated that there was a rough correlation between cell body diameter and axon conduction velocity and suggested that neurons giving rise to axons conducting in the Aδ range should be medium diameter ( Lawson 2002 ). Thus, TRPV2 had the right biophysical properties and was in the right population of afferents. Closer inspection of these correlational studies, however, reveals that there is no correlation between cell body size and axon conduction velocity for neurons with a medium-diameter cell body ( Lawson 2002 ). More problematic is that TRPV2 null mutant mice appear to have no deficits in heat sensitivity, even with very intense stimuli ( Park et al 2010 ). Therefore, if there is another heat transducer in this population of afferents, it has yet to be identified.

Other Thermotransducers

K2P Channels: Consistent with the evidence described above that there must be other thermotransducers contributing to temperature sensation, at least four other channels have been identified that are gated by temperature. The first of these were the K2P channels TREK-1 and -2 and TRAAK ( Noel et al 2009 ). These channels are activated by increases in temperature over a range from approximately 28 to 42°C. Because they are active at rest, the converse is also true, with channel activity being decreased over the same range. TASK has comparable temperature sensitivity but far less dramatic changes in activity associated with warming ( Noel et al 2009 ). There is also evidence that a 4-aminopyridine–sensitive K + channel contributes to cold transduction ( Viana et al 2002 ), but this channel has yet to be identified. As noted above, all four thermosensitive K2P channels are present in sensory neurons. The involvement of a leak K + conductance in mediating the response to cooling is consistent with the underlying mechanism predicted in one of the first characterizations of cooling-evoked responses in isolated sensory neurons ( Reid et al 1999 ). Although the increase in K2P channel activity associated with warming should result in a decrease in afferent activity potentially, thereby explaining the therapeutic value of heat for the treatment of inflammatory pain, one would predict that these channels do not contribute the warming-induced increase in activity in warm fibers. Consistent with this prediction, double-null mutant mice deficient in TRAAK and TREK-1 have an increased response to heating and an inflammatory heat hyperalgesia that is fully intact ( Noel et al 2009 ). The cooling phenotype in these mice is a little harder to interpret because the animals show an increase in cold sensitivity following injury. This is in contrast to the prediction that the loss of a channel that normally closes as a means to enable a response to cooling should be associated with an increase in the response to cooling in the absence of injury.

TRPM3
Recent evidence indicates that TRPM3 is also a heat-sensitive channel ( Vriens et al 2011 ). It has a threshold for activation of about 30°C, which can be sensitized by the co-application of pregnenolone sulfate. Like TRPA1 and V1, the thermal sensitivity of TRPM3 appears to reflect a temperature-dependent shift in the voltage dependence of channel gating. Interestingly, in contrast to TRPM2, 4, and 5, which also have thermal sensitivity in a temperature range comparable to TRPM3, TRPM3 null mutant mice exhibit a deficit in noxious heat sensitivity ( Vriens et al 2011 ). Although the majority of TRPM3-expressing neurons also express TRPV1, the observation that TRPM3 is present in a subpopulation of TRPV1-negative neurons suggests that this transducer may contribute to the heat sensitivity observed in afferents from TRPV1 null mutant mice. Nevertheless, the observation that heat sensitivity is still detectable in neurons from TRPM3 null mutant mice in the presence of TRPV1 blockers suggests that at least one more heat transducer has yet to be identified.

TRPC5
TRPC5 is the most recent addition to the thermosensitive family of ion channels ( Zimmermann et al 2011 ). It is proposed to function as a cool receptor with channel activity that is steeply temperature sensitive between 37 and 25°C, where interestingly, only the homomeric channel is cold sensitive whereas the TRPC5/TRPC1 heteromeric channel is not. The channel is detectable in approximately 32% of DRG neurons in a proportion that mirrors the size distribution of the entire population of DRG neurons. The protein appears to be targeted to peripheral terminals, many of which terminate in the superficial layers of the skin. The channel does not contribute to cold-evoked currents in isolated sensory neurons from TRPC5 null mutant mice, possibly because of preferential targeting in the periphery ( Zimmermann et al 2011 ). There are several compensatory changes in the afferent properties of the TRPC5 null mutant, which was used to explain the paradoxical increase in cooling-evoked activity in mechanosensitive C fibers. Thus, absence of the expected cooling phenotype in these animals (i.e., the loss of a cooling response) may be due to compensatory changes. Alternatively, it was suggested that TRPC5 could play a role in responses to cooling that are not tied to behavior (i.e., regulation of peripheral blood flow). Additional data will be needed to further define the role of this putative cold transducer.

Na v 1.8
In contrast to other transduction modalities, transduction of noxious cold stimuli is entirely dependent on the biophysical properties of the voltage-gated Na + channels that underlie action potential initiation ( Zimmermann et al 2007 ). As already touched on above, the ultimate fate of a generator potential depends on a number of factors, not the least of which are the density, relative distribution, and biophysical properties of the voltage-gated Na + channels that ultimately transform the passive depolarization initiated by the transducer into an action potential. In nociceptive afferents, the voltage-gated Na + channel Na v 1.8 plays a particularly important role in this process. This channel has a number of unique features, including relatively slow kinetics of activation and inactivation but recovery from inactivation that is exceptionally fast ( Elliott and Elliott 1993 , Flake et al 2004 ), a high threshold for activation, and a voltage dependence of inactivation curve that is relatively depolarized ( Akopian et al 1996 ). These properties alone can account for many of the unique properties of nociceptive afferents, such as a high threshold for activation and the ability to continue to fire action potentials in the presence of sustained membrane depolarization ( Gold 2000b ). An additional feature of Na v 1.8 is that the channel is not inactivated by noxious cold temperatures ( Zimmermann et al 2007 ). This is in contrast to other voltage-gated Na + channels responsible for action potential initiation in low-threshold afferents. This difference accounts for why cold tissue feels both “numb” and “on fire” at the same time.

Mechanotransduction
Despite the fact that mechanosensation is the dominant modality of somatosensation and that mechanical hypersensitivity is far more common than thermal or even chemical hypersensitivity (e.g., see Backonja and Stacey 2004 ), mechanotransduction remains the most poorly understood of the stimulus modalities that activate the somatosensory system. This lack of understanding is not due to a dearth of putative mechanotransducers since a number of ion channels have been shown to be gated by mechanical stimuli. Rather, the problem appears to be due to the fact that data from parallel lines of evidence are not internally consistent. Not only are there differences between the results obtained with isolated neurons, isolated organ preparation, and behavioral assays, but there are also differences between behavioral assays engaging different body regions. Nevertheless, at least two conclusions can be drawn from this data set at present. First, the process of mechanotransduction appears to involve several different mechanotransducers, and second, the underlying mechanisms are likely to vary as a function of both the type of stimulus (stretch versus pressure) and the tissue stimulated.


ASIC Channels
Although ASICs have yet to demonstrate intrinsic mechanosensitivity when expressed in heterologous expression systems, several lines of evidence suggest that these channels may play a role in mechanotransduction. First, the channels are members of a larger family of ion channels, degenerins, identified in Caenorhabditis elegans in genetic screens for mechanosensory defects ( Goodman et al 2002 , O’Hagan et al 2005 ). The closest mammalian homologue, ENaCs, have been shown to possess intrinsic mechanosensitivity in lipid bilayers ( Ismailov et al 1997 ) and in heterologous expression systems ( Kizer et al 1997 ). Second, like degenerins, ASICs are also sensitive to amiloride and related compounds ( Kizer et al 1997 ). Third, the channels are present in peripheral terminals ( Price et al 2000 , 2001 ; Garcia-Anoveros et al 2001 ). Unfortunately, there is considerably more conflicting evidence or evidence against a role for ASICs in mechanotransduction than for it. Mechanically evoked currents in isolated sensory neurons from ASIC2/3 double-null mutant mice are fully intact ( Drew et al 2004 ). The mechanical stimulus–response properties of afferents from ASIC1 null mutant mice were comparable to those in wild-type mice when studied in a skin nerve preparation ( Page et al 2004 ). A reduction in the slope of the stimulus–response function with no change in threshold was observed in one study of rapidly adapting low-threshold mechanosensitive afferents from an ASIC2 null mutant ( Price et al 2000 ); however, comparable changes were not observed in a subsequent study ( Roza et al 2004 ). Even more confusing was the observation that in ASIC3 null mutant mice there was a decrease in the mechanosensitivity of Aδ mechanosensitive afferents but an increase in the mechanosensitivity of low-threshold, rapidly adapting afferents. An increase in inflammatory mechanical hypersensitivity was also observed in these mice ( Price et al 2001 ). In contrast to the role of ASICs in the skin, ASIC1, 2, and 3 appear to contribute to the mechanosensitivity of visceral afferents. ASIC1 appears to inhibit mechanosensitivity such that afferents of the GI tract are even more excitable in ASIC1 null mutant mice ( Page et al 2004 , 2005 ). The response properties of some subpopulations of GI afferents were increased whereas others were decreased in ASIC2 null mutant mice ( Page et al 2005 ). Most striking, however, was the suppression of mechanosensitivity observed in all but one subpopulation of GI afferents defined by the response properties to various mechanical stimuli ( Jones et al 2005 , Page et al 2005 ).

Piezo1 and 2
The most recent additions to the list of channels with intrinsic mechanosensitivity are Piezo1 and 2 ( Coste et al 2010 ). These channels were identified through a heroic expression cloning approach in a heterologous expression system in which “hits” were identified by the cell’s response to a “poke” with a small pipette ( Fig. 2-5 ). Both channels are widely expressed across phyla, as well as in a number of tissues in rodents. Piezo2 appears to play a particularly important role in the rapidly activating and rapidly inactivating “poke”-evoked currents in isolated sensory neurons. The contribution of these channels to mechanosensation in vivo has yet to be determined, but Piezo does appear to be a bona fide mechanotransducer. That is, when expressed it is sufficient to carry an inward current when the cell is mechanically stimulated. There are probably many ancillary proteins that modulate the mechanically induced currents, several discussed here, but this is not a sufficient basis to consider them mechanotransducers.


Figure 2-5 Piezo2 underlies rapidly activating and rapidly inactivating mechanically activated (MA) currents in Neuro2A (N2A) cells and sensory neurons. A, Representative traces of MA inward currents expressed in N2A cells. Cells were subjected to a series of mechanical steps consisting of 1-μm movements with a stimulation pipette (inset drawing, arrow) in the whole-cell patch configuration at a holding potential of −80 mV. B, Average current–voltage relationships of MA currents in N2A ( n = 11) cells. The inset shows representative MA currents evoked at holding potentials ranging from −80 to +40 mV (applied 0.7 second before the mechanical step). A comparable current is detectable in sensory neurons that is selectively reduced with small interfering RNA against Piezo2. Sustained MA currents are also detected in sensory neurons ( C ). However, the sustained current is completely blocked by the TRPA1-selective antagonist HC-030031. Results from these studies indicate that there are several mechanotransducers in sensory neurons. ( A and B, From Coste B, Mathur J, Schmidt M, et al 2010 Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330:55–60; C, from Vilceanu D, Stucky CL 2010 TRPA1 mediates mechanical currents in the plasma membrane of mouse sensory neurons. PLoS One 5:e12177.)

TRPA1

TRP Channels
One of the most controversial putative mechanotransducers is TRPA1. The first suggestion that the channel might function as a mechanotransducer came from observations that the channel is present in hair cells, where it is localized in the hair tips, and that protein knockdown results in the inhibition of receptor cell function ( Corey et al 2004 ). Subsequent data from TRPA1 null mutant mice ruled out a role for TRPA1 as the mechanotransducer in cochlea hair cells ( Kwan et al 2006 ). In isolated cells, there is evidence both for and against a role for TRPA1 in mechanically activated (MA) currents. In ND-C cells, a cell line derived from neonatal rat DRGs, MA currents are cationic, rapidly activating, and slowly inactivating. TRPA1 is not present in these cells normally, thus suggesting that other channels underlie the MA evoked currents ( Rugiero and Wood 2009 ). Furthermore, transfecting ND-2 cells with TRPA1 failed to alter the MA currents in these cells. In contrast, slowly inactivating MA currents are completely absent in neurons from TRPA1 null mutant mice and completely blocked with the TRPA1 antagonist HC-030031 ( Vilceanu and Stucky 2010 ) (see Fig. 2-5 ). Interestingly and at least partially consistent with results obtained in ND-C cells, heterologous expression of TRPA1 in HEK293 cells failed to alter the MA currents in these cells, which led the authors to suggest that TRPA1 alone was not sufficient to mediate MA evoked currents.
In isolated skin nerve preparations from TRPA1 null mutant mice, the entire mechanical stimulus–response function in C fibers and only the top of the stimulus–response function in Aδ fibers were suppressed ( Kwan et al 2009 ), with no change in threshold in either population. Surprisingly, the stimulus–response function for slowly adapting Aβ fibers is also suppressed in TRPA1 null mutant mice, whereas that for rapidly adapting Aβ fibers and D hairs is increased. Evidence of TRPA1 in keratinocytes was offered as one potential explanation for the widespread impact of the loss of TRPA1. However, in contrast to the original descriptions of TRPA1 in which it was indicated that channel expression was restricted to a subpopulation of small-diameter TRPV1-expressing sensory neurons ( Story et al 2003 ), evidence of TRPA1 expression was detected in all sizes of DRG neurons, as well as in the terminals of low-threshold afferents. These results suggested that changes in firing properties were due to the actions, or lack thereof, of TRPA1 in all populations of sensory neurons. The subsequent observation that a TRPA1 antagonist produced a suppression of activity in C fibers confirmed a role for TRPA1 in the response to mechanical stimulation in these neurons ( Kerstein et al 2009 ). However, failure of the TRPA1 antagonist to influence the mechanical response properties of Aδ fibers or the response of wide–dynamic range neurons to low-intensity mechanical stimuli ( McGaraughty et al 2010 ) raises the possibility that at least some of the results obtained with the null mutant mouse are due to compensatory changes rather than the loss of TRPA1 itself. Of note, the observation that TRPA1 expression in primary afferents may be more generalized than originally thought has yet to be repeated. Interestingly, TRPA1 appears to contribute to the response of all types of visceral afferents, including vagal afferents, to punctate mechanical stimuli but not to stretch ( Brierley et al 2009 ), again highlighting the importance of both the type and site of stimulation on the relative contribution of a putative transducer.
The behavioral data are consistent with a role for TRPA1 in the response to noxious mechanical stimuli. The response to punctate mechanical stimuli (i.e., the von Frey test) is attenuated in TRPA1 null mutant mice, as is the response to noxious paw pressure in rats following administration of the TRPA1 antagonist HC-030031 ( Wei et al 2009 ). However, one would have predicted that the response to colonic distention would be unaffected by the loss of TRPA1 given the selective deficit in the response to punctate mechanical stimuli in TRPA1 null mutants. Nevertheless, this was observed in only one of two studies in which TPRA1 null mutant mice were used ( Cattaruzza et al 2010 ). In a second study, the response to distention was attenuated in the null mutant. The response to distention was also attenuated following antisense knockdown of TRPA1 ( Kondo et al 2009 ). Unfortunately, interpretation of the behavioral data has been complicated by evidence that TRPA1 acts at the central terminal of nociceptive afferents, where it facilitates nociceptive signaling. Consequently, spinal block of TRPA1 is antinociceptive ( McGaraughty et al 2010 , Wei et al 2010 ). Although TRPA1 remains a target of active investigation, if conclusions can be drawn from the data available on TRPA1 and its potential role as a mechanotransducer, they are the following: First, the role of TRPA1 in mechanotransduction is limited to modulation of evoked activity. This implies that other channels or transducers play a dominant role in the process. Second, consistent with this modulatory role, TRPA1 plays a significant role in the injury-induced increase in mechanosensitivity, as has been demonstrated in models of both somatic and visceral inflammation, as well as in several models of peripheral nerve injury ( Petrus et al 2007 , Eid et al 2008 , Wei et al 2009 , da Costa et al 2010 , McGaraughty et al 2010 ).

TRPV4
TRPV4 was known to function as an osmolality-gated ion channel ( Strotmann et al 2000 ) before the realization that the channel also functions as a thermoreceptor. The channel is activated by hypertonic solutions, presumably because of the mechanical stress associated with cell shrinkage. The channel is widely distributed in a number of different cell types, including primary afferents, probably reflecting the fact that the ability to respond to changes in tonicity is essential to most cell types, particularly epithelial cells. However, in sensory neurons the channel is essential for the pain behavioral response associated with hypertonic solutions ( Alessandri-Haber et al 2005 ). Interestingly, although TRPV4 is responsive to swelling, chemical, and thermal stimuli, each modality activates the channel via distinct pathways ( Vriens et al 2004 ). Like other TRP channels, TRPV4 may play a more important role in mechanosensation in the presence of injury, as suggested by the observation that mechanical hypersensitivity is attenuated in TRPV4 null mutant mice ( Alessandri-Haber et al 2004 , Chen et al 2007 , Cenac et al 2008 , Zhang et al 2008 ).

K2P
There are at least three K2P channels present in sensory neurons that have been shown to have mechanosensitivity: TREK-1, TREK-2, and TRAAK ( Maingret et al 1999a , 1999b ; Bang et al 2000 ). TREK-1 was first described as a chemotransducer activated by arachidonic acid ( Fink et al 1998 ) and inhibited by cAMP ( Fink et al 1996 ). It was subsequently shown to be activated by osmotic swelling, stretch, and membrane crenators ( Maingret et al 1999b ). TREK-2 and TRAAK have the same mechanosensitive properties as TREK-1. Despite evidence that the channels are differentially regulated in the presence of tissue injury in a manner consistent with a role in the injury-induced hypersensitivity model ( Marsh et al 2012 ), the relative contribution of K2P channels to mechanosensitivity remains to be determined.

T-Type Ca 2+ Channel (Ca v 3.2)
Transient or T-type Ca 2+ channels are members of the voltage-activated Ca 2+ channel family that have a low threshold for activation ( Catterall et al 2005 ). They are therefore also referred to as low-threshold voltage-activated channels. The α subunit of these channels is a large molecule with four homologous domains, each of which has six transmembrane segments with a pore loop between segments 5 and 6 and a voltage sensor in segment 4. Thus, the α subunit has all the components necessary for a functional channel. Three α subunits for the low-threshold channel have been identified and designated Ca v 3.1–3.3. Of these, Ca v 3.2 is enriched in a subpopulation of sensory neurons that appear to innervate D hairs ( Shin et al 2003 ). More importantly, the response to mechanical stimulation of the receptive field of D-hair units is selectively attenuated with the T-type channel blocker mibefradil. There is evidence that T-type currents are also enriched in a subpopulation of nociceptive afferents and that sensitization of these channels results in a decrease in the mechanical threshold ( Todorovic and Jevtovic-Todorovic 2006 ). However, data from a Ca v 3.2 null mutant mouse suggest that the mechanical response properties of nociceptive afferents are minimally altered, thus indicating a minor role for this channel in these afferents. This is in contrast to the response of D-hair fibers in this knockout line, which is reduced by more than 50%, largely as a result of an increase in the mechanical threshold and utilization time ( Shin et al 2003 ). Although data in support of a role for T-type channels in the mechanical response properties of D hairs are compelling, the channel has received the most attention for its role in mediating the hypersensitivity observed in response to a number of pain-producing manipulations, including hydrogen sulfide injection, diabetic and post-traumatic neuropathy (including a compressed DRG model; Wen et al 2006 ), and a model of irritable bowel syndrome ( Marger et al 2011 ). Consistent with these observations is that a number of small-molecule inhibitors of T-type channels have antinociceptive efficacy in a variety of animal models of persistent pain ( Zamponi et al 2009 ).

Polymodality
It is clear from the preceding discussion that in primary afferents, transducer specificity is rare. Many transducers are activated by several stimulus modalities. Even though the functional implications of this polymodality are still being worked out, the chemosensitivity of many of the thermo- and mechanotransducers makes interpretation of results from intact preparations difficult, at least with respect to the contribution of a specific transducer to the response to a specific stimulus. For example, it will be difficult to distinguish the relative contribution of the mechanosensitive properties of the transducer from its chemosensitive properties if it is possible that chemicals that activate the transducer are released from other cells in response to mechanical stimuli. Evidence abounds that chemicals are released from thermally ( Patwardhan et al 2010 ) and mechanically ( Burnstock 2009 ) stimulated tissue, thus making this a serious technical hurdle.

Indirect Signaling Pathways
The focus up until now has been on transduction in sensory neurons. However, it is becoming increasingly clear that many putative transducers are not only present but also functional in other cell types. The bladder epithelium, or urothelial cells, are probably the best characterized in this regard. A wide variety of chemo-, thermo-, and mechanotransducers are reportedly present on these cells, including nAChRs, bradykinin receptors (B 1 and B 2 ), and TRP channels (TRPA1, TRPM8, and TRPV1–2, 4) (see Birder 2011 for review). The only transducer present in urothelial cells minimally represented in sensory neurons ( Hermanstyne et al 2008 ) is the ENaC, which is related to ASIC channels (see above). Urothelial cells are activated by thermal, mechanical, and chemical stimuli, and they release a variety of mediators that are able to activate and/or sensitize afferents. These mediators include ACh, ATP, reactive oxygen species (nitric oxide), peptides, neurotrophins, cyclooxygenase metabolites, and cytokines ( Birder 2011 ). Of these, ATP has received the most attention because it was the first mediator shown to be released in response to bladder stretch. This observation provided a mechanism for mechanical transduction via ATP binding P2X receptors on primary afferents that terminate in close contact to urothelial cells. Consistent with this model, there is an increase in release of ATP from the urothelium of both animals ( Birder et al 2003 ) and humans ( Sun and Chai 2002 ) with interstitial cystitis, a painful inflammation of the bladder. TRPV1 is another channel that has received a lot of attention both because it appears to contribute to normal bladder function (i.e., bladder afferents from TRPV1 null mutant mice have a lower response to bladder distention) ( Birder et al 2002 ) and because TRPV1 agonists can be used to produce afferent desensitization and provide some relief for patients with pain associated with bladder hypersensitivity disorders ( Cruz 1998 ).
The bladder urothelium is proving to be far from unique with respect to its potential role in sensory transduction inasmuch as similar roles have been implicated for epithelial cells lining the GI tract ( Wynn et al 2004 ) and airway ( Button et al 2007 ). Epithelial cell signaling appears to be even more complex in the skin, where keratinocytes have been shown to express not only a wide variety of transducers but also channels that could serve to facilitate signaling, such as voltage-gated Na + channels ( Zhao et al 2008 , Dussor et al 2009 , Hou et al 2011 ). Like the bladder, there is considerable heterogeneity among keratinocytes with regard to the expression of various transducers and ion channels. Consistent with the fact that skin consists of stratified epithelium, there is also heterogeneity in the distribution of channels between layers. Interestingly, this pattern appears to be disrupted in the presence of tissue injury and under pathological conditions ( Zhao et al 2008 ), thus raising the possibility that these changes contribute to the associated alterations in sensation. Much remains to be determined, however, with respect to the role of these cells in signal transduction, not the least of which is the answer to how modality specificity is achieved in the nervous system if all modalities of stimuli result in the release of common mediators such as ATP.

Lessons Learned from Injury-Induced Changes
Our understanding of the molecular mechanisms of transduction is complicated by the apparent paradox between naïve and injured tissue with regard to the relative contribution of putative transducers to sensation since the contribution often appears to be greater in the presence of tissue injury. This may be due to the fact that in some tissues (e.g., skin), the relative contribution of C-fiber activity is minimal under normal conditions and becomes apparent only in the presence of injury-induced hypersensitivity ( Khasar and Levine 1996 ). This differential contribution of fiber types may also explain why putative transducers appear to contribute more significantly to the response of naïve visceral tissue, given the relative dearth of myelinated fibers that innervate visceral structures. Of course, the implication of such a suggestion is that we have yet to identify transducers underlying the response of the more rapidly conducting Aβ and Aδ fibers thought to dominate the response to acute noxious stimulation of naïve cutaneous tissue. The paradox may also reflect the fact that many of the known transducers are dramatically up-regulated in the presence of tissue injury. Injury-induced changes in TRPV1 are an excellent example of transducer up-regulation. The channel is sensitized via an array of second-messenger pathways, including those involving protein kinase A (PKA), protein kinase C (PKC), phosphatidylinositol-3′-kinase (PI3K), calcium-calmodulin–dependent kinase II protein (CaMKII), and p38/mitogen-activated protein kinase (MAPK), which results in an increase in channel activity, a decrease in desensitization, and/or an increase in receptor density as a result of translocation to the membrane ( Gold and Gebhart 2010 ). On a slower time scale, there is evidence that TRPV1 protein is increased via post-transcriptional mechanisms ( Ji et al 2002 ) and that the distribution of the channel is increased in DRG neurons, thus suggesting alterations in transcriptional machinery as well ( Breese et al 2005 ). Following nerve injury there is evidence that the channel is even expressed in A fibers ( Rashid et al 2003 ). These observations highlight the importance of both the time course of changes after injury and injury-induced changes in the relative contribution of afferent subpopulations to pain after injury.
Changes in the properties of ion channels regulating the passive and active electrophysiological properties of the afferent are also likely to contribute to the increase in the relative impact of putative transducers on the generation of afferent activity in the presence of tissue injury. That is, the fate of the generator potential depends on both the passive and active electrophysiological properties of the afferent terminal, with passive properties that include resting membrane potential and input resistance influencing the magnitude of the generator potential and the distance over which it is passively spread. The resting membrane potential will also influence the availability of many of the ion channels underlying active electrophysiological properties. Channels that contribute to passive properties in primary afferents include the P2K channels, which are dynamically regulated by a variety of mediators (see above), as well as over the long term by changes in expression ( Marsh et al 2012 ). Voltage- and Ca 2+ -modulated ion channels underlie active electrophysiological properties, and these will determine the action potential threshold, the amplitude and duration of the action potential, the amplitude and duration of the afterpotential, and more stimulus–response properties such as the interspike interval and burst duration ( Harriott and Gold 2009a ).
Because the generator potential provides the underlying drive for activation of these other ion channels, the interaction between the amplitude and duration of the generator potential with the biophysical properties of the channels underlying the active electrophysiological properties can have a profound influence on the output of the neuron. For example, very slow depolarization in a neuron in which initiation of an action potential is dependent on a voltage-gated Na + channel subject to steady-state inactivation may drive the inactivation of Na + channels before initiation of the action potential. Similarly, a large and rapid depolarization associated with activation of TRPV1 may drive the membrane potential to 0 mV, the reversal potential for TRPV1, and enable the generation of few if any action potentials before a depolarization-induced inactivation of voltage-gated Na + channels. Another critical point of interaction between the transducer and ion channels underlying the active electrophysiological properties is at the level of the permeant ions. That is, TRPV1 is highly permeable to Ca 2+ , whereas ASIC3 is far more selective for Na + . The presence of a high density of Ca 2+ -dependent ion channels that contribute to determination of the action potential threshold and/or burst duration should respond very differently to stimuli engaging TRPV1 than to those engaging ASIC3. Conversely, Ca 2+ influx via voltage-gated Ca 2+ channels is not only important for peripheral transmitter release and therefore the efferent function of afferents but can also facilitate the desensitization of channels such as TRPV1 ( Vyklicky et al 2008 ).
The literature is now full of descriptions of injury-induced changes in an array of ion channels that underlie active electrophysiological properties. This includes changes in a variety of voltage-gated K + channels, Na + channels, Ca 2+ channels, and Ca 2+ -dependent K + and Cl − channels in a manner consistent with an increase in afferent excitability ( Harriott and Gold 2009a ). Changes in all channel types are associated with both the acute actions of inflammatory mediators and longer-term changes in channel distribution and gene expression. Importantly, as noted above, the nature and timing of the changes depend on a number of factors, including the type of injury, the site of injury, the previous history of the injured tissue, age, and sex.
Finally, data from nerve injury models have highlighted the importance of transducer distribution on the emergence of ectopic activity. There is evidence that transducers may be inserted into the axon membrane following nerve injury and thereby result in the emergence of mechanical, thermal, and presumably chemical sensitivity at sites along the axon ( Michaelis et al 2000 , Grossmann et al 2009 , Janig et al 2009 ). This process appears to occur much more readily in muscle afferents. The process is also likely to occur within ganglia and contribute to the emergence of ectopic activity arising from within the ganglia following traumatic nerve injury ( Devor 1999 ). The emergence of sources of activity at locations remote from the site of injury or even the painful tissue can add to the difficulty in treating neuropathic pain with peripherally targeted interventions.

Conclusion
The past 15 years have yielded an explosion of information regarding the molecular mechanisms of sensory transduction. An array of putative transducers have been identified, as have details regarding mechanisms underlying their activation. As is true of many aspect of science, the more we learn about something, the more complicated it becomes. This has been particularly true of our understanding of thermal transduction, which seemed so clear 5 years ago but is considerably less so now. This complexity has clearly proved to be a barrier to the development of novel therapeutic approaches for the treatment of pain. With multiple channels working in parallel and/or differentially contributing to the response in one fiber type or after a particular type of injury, it should not be surprising that an effective blocker of a particular channel has not emerged as the next silver bullet for the treatment of pain. Nevertheless, although it is clear that there is still much to learn about sensory transduction, novel approaches are on the horizon that should provide relief for many in need.
The references for this chapter can be found at www.expertconsult.com .

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Chapter 3
Inflammatory Mediators and Modulators of Pain

John M. Dawes, David A. Andersson, David L.H. Bennett, Stuart Bevan, and Stephen B. McMahon


SUMMARY
Disease and injury frequently result in pain and hyperalgesia. These abnormal sensory events arise in part from the action of inflammatory mediators on the peripheral terminals of nociceptive neurons. In this chapter we begin by reviewing the different ways in which such mediators bring about the activation or sensitization of nociceptive terminals. We then consider the biological effects and potential importance of different inflammatory mediators. The list of mediators has steadily been increasing and includes not only traditionally recognized molecules such as arachidonic acid metabolites and bradykinin but also other small molecules such as adenosine triphosphate and nitric oxide. Additionally, evidence has accumulated for an important role of a series of inflammatory cytokines and chemokines, such as tumor necrosis factor-α and interleukin-1β, and growth factors, particularly nerve growth factor, which are all capable of changing the response properties of pain-signaling neurons. They achieve this in a variety of ways, including activation or sensitization of nociceptive terminals, as well as regulation of gene expression by nociceptors. Immune cells are an important source of inflammatory mediators, cytokines, and some growth factors. Recently, it has become clear that they modulate pain processing not just by release of mediators into peripherally damaged or diseased tissue but also by release of the same mediators into the central nervous system.


Introduction
A long-standing interest for pain scientists has been the identification of chemical mediators released into injured or diseased tissues that are responsible for the abnormal pain states associated with these disorders. For some time, attention was focused on a small number of molecules such as prostaglandins and bradykinin. These factors were known to be produced as a result of tissue damage or inflammation and were thought to be responsible for activation and sensitization of peripheral pain-signaling sensory neurons; that is, they were seen as the principal peripheral pain mediators. During the past decade or so, evidence has emerged for many novel pain mediators. The old ones have not disappeared, although their roles have been redefined in some cases. Prostaglandin E 2 (PGE 2 ), for instance, is now recognized as playing a prominent role in central nervous system (CNS) as well as peripheral tissues. The newly identified mediators include a variety of factors produced and released from non-neuronal cells, often immune and glial cells. There is now a rapidly expanding evidence base that these are important mediators of persistent pain states and can act at a number of loci.
This chapter focuses on the cellular characteristics of nociceptive afferent neurons, their ion channels, and their signal transduction pathways and discusses the ways in which inflammatory mediators impinge on these basic properties. In particular, we first review the cellular mechanisms of activation and sensitization of nociceptors. Then we discuss the roles and actions of particular immune cells and specific pain mediators, starting with a group of small molecules often rapidly released into damaged tissue. We conclude with a review of the actions of another group of peripheral pain mediators and modulators: the pro-inflammatory cytokines, some chemokines, and some neurotrophic factors, which in addition to their traditionally recognized roles, are all capable of changing the response properties of pain-signaling neurons. The topic of neuro-immune interactions within the CNS is considered in Chapter 4 .

Overview Of Inflammatory Mediator Actions
A large number of endogenously generated factors produce pain when injected into peripheral tissue. Many of these substances can also sensitize nociceptors. That is, they reduce the threshold for activation of nociceptors by one or more stimulus modalities and/or increase the responsiveness of nociceptors to suprathreshold stimulation. This process of sensitization is recognized as being of critical importance in many chronic pain states; it is precisely this aberrant excitability of nociceptors that causes a large part of the sensory abnormality. Some features of the sensitization process are described in Chapter 1 . Here we first review the cellular mechanisms by which sensitization occurs.

Receptors and Effectors
Sensory nerves express a variety of receptors for inflammatory mediators. Different classes of nociceptors express distinct patterns of receptors. The receptors fall into three main classes: G protein–coupled receptors (GPCRs), ligand-gated ion channels, and the cytokine receptors or receptor tyrosine kinases ( Fig. 3-1 ).


Figure 3-1 Peripheral sensitization of nociceptive neurons. A, Some of the different stimuli (and the receptors that they act on) that can lead to activation and sensitization of the peripheral terminals of nociceptive neurons. B and C show the main effector mechanisms and second-messenger cascades underlying sensitization, respectively. ASIC, acid-sensing ion channel; DAG, diacylglycerol; ERK, extracellular signal–regulated kinase; IP 3 , inositol triphosphate; MEK, mitogen-activated protein/ERK kinase; NGF, nerve growth factor; PGE 2 , prostaglandin E 2 ; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; TRPV1, transient receptor potential vanilloid 1. Many changes are effected by phosphorylation of receptors or channels (P).

G Protein–Coupled Receptors
Many mediators produced during inflammation, such as bradykinin, serotonin, prostaglandins, and chemokines, act via GPCRs. These receptors elicit a specific biochemical response that depends on the type of G protein that is activated. Activation of G s stimulates adenylate cyclase to raise the level of cyclic adenosine monophosphate (cAMP) and activate protein kinase A (PKA) in the neuron, whereas G i inhibits the activity of adenylate cyclase to lower cAMP levels. Although many cAMP effects are mediated by PKA, other mechanisms may be operative. For example, cAMP can activate Epac (exchange protein directly activated by cAMP), a guanine nucleotide exchange factor, which leads to activation of the ε isoform of protein kinase C (PKC-ε). Stimulation of G q/11 activates phospholipases, notably phospholipase C (PLC), which generates inositol triphosphate (IP 3 ) and diacylglycerol (DAG) from the membrane lipid precursor phosphatidylinositol 4,5-bisphosphate (PIP 2 ). G q activation can also stimulate PLA 2 , which cleaves membrane phospholipids at the sn-2 position to produce the prostaglandin precursor arachidonic acid. G-protein control of cellular function can also involve direct action of βγ subunits on ion channels and enzymes, such as PLC (see Smrcka 2008 , Zylbergold et al 2010 ).

Ion Channels
Some inflammatory mediators act by directly gating the ion channels expressed by sensory neurons. Notable examples in this class are adenosine triphosphate (ATP; acting via P2X channels), protons (acting via acid-sensing ion channels [ASICs] and transient receptor potential vanilloid 1 [TRPV1]), and the lipid activators of TRPV1. All these ion channels are cation selective and are permeable to either sodium ions or both monovalent and divalent cations. In all cases the ion flow evoked by channel opening depolarizes the sensory neurons and leads to neuronal firing.

Receptor Tyrosine Kinases
The third general type of receptor includes cytokine receptors activated by mediators such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α) and the receptor tyrosine kinases for neurotrophic factors, such as the receptors for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line–derived neurotrophic factor (GDNF), and artemin. Both classes of receptors have monomers derived from a single transmembrane segment with a large extracellular ligand-binding domain. The cytosolic domain of receptor tyrosine kinases contains an intrinsic protein tyrosine kinase catalytic site, whereas the cytosolic domain of cytokine receptors is generally associated with a separate protein kinase that is recruited to the complex either directly or via adapter proteins. The functional receptors are either dimers or trimers, which either exist normally or are formed by cross-linking of adjacent monomers by the ligand. In either case, ligand binding activates kinase pathways that affect gene transcription and can also elicit acute effects on neuronal function.

Nitric Oxide and Cyclic Guanosine Monophosphate
In addition to receptor-mediated signaling, cells also signal via nitric oxide (NO). NO is an important intercellular mediator and is produced by many cells that have close physical association with neurons both in the periphery and within the spinal cord. NO is formed from L -arginine following activation of the enzyme nitric oxide synthase (NOS) by calcium and other co-factors, including calmodulin. Three forms of NOS have been identified: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), each with a distinct physiological role. nNOS and eNOS are both Ca 2+ /calmodulin dependent and are present in both the spinal cord and brain, whereas iNOS is functionally Ca 2+ independent and normally expressed in macrophages, inflammatory cells, and glia (for review see Benarroch 2011 ). NO diffuses to its site of action, where it stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP). In turn, cGMP modifies intracellular processes, including activation of protein kinases, ion channels, and phosphodiesterases. NO can also act in other ways, for example, by activating cyclooxygenase (COX) enzymes and by S -nitrosylation of proteins ( Tegeder et al 2011 ).

Intracellular Signaling Pathways
Sensory nerves are activated and sensitized by inflammatory mediators in several ways (see Fig. 3-1 B). Some mediators directly activate cation channels and thus depolarize neurons toward the voltage for initiation of an action potential. Other receptors activate intracellular pathways and influence neuronal sensitivity and excitability indirectly. These mechanisms include GPCR-mediated production of the second-messenger molecules NO, COX, and lipoxygenase products of arachidonic acid. Phosphorylation or dephosphorylation of membrane proteins often regulates the transduction and transmission of sensory signals ( Fig. 3-1 C), and this can occur via PKA-, PKC-, mitogen-activated protein kinase (MAPK)-, or phosphatidylinositol-3′-kinase/Akt-mediated phosphorylation or by dephosphorylation via protein phosphatases such as calcineurin. In addition to phosphorylation, some of the mediators that act on nociceptors can stimulate biochemical processes such as methylation and lipid modification of proteins, and these pathways may be important in nociceptive neurons.
In general, the effect of sensitization is to increase the probability that a given stimulus (ligand or voltage) will activate the target receptor or ion channel or increase the probability that the neuron will be excited. Protein phosphorylation is a well-known mechanism for controlling the activity of ion channels. For example, activity of the heat-sensitive ion channel TRPV1 is modified by both PKC- and PKA-mediated phosphorylation ( Bhave et al 2003 , Mohaptra and Nau 2005 ), and the level of membrane expression is regulated by src-mediated phosphorylation ( Zhang et al 2005b ). Control of transduction channel activity can also be regulated by hydrolysis of PIP 2 and removal of the tonic inhibition caused by PIP 2 binding to the ion channel (see, e.g., Dai et al 2007 ). Ion channels that control the excitability and firing frequency of sensory neurons are also substrates for regulation by PIP 2 ( Suh and Hille 2008 ) and phosphorylation ( Gold 1999 , Baker 2005 , Beyak and Vanner 2005 , Stamboulian et al 2010 , Emery et al 2011 ).
Neuronal sensitization can occur through changes in the level of protein expression, either by transcriptional control altering the production of proteins or by changing the trafficking such that an altered amount of the protein is functionally expressed. Transcriptional control is an important long-term mechanism underlying the effects of neurotrophin receptor activation. In some cases, sensitization has been associated with the de novo expression of molecules important for nociception in neurons that do not normally express the protein ( Hudson et al 2001 , Vellani et al 2004 ).

Specific Pain Mediators

Bradykinin
There is a considerable body of evidence that kinins contribute to the pathophysiological processes accompanying both acute and chronic inflammation. Bradykinin and the related peptide kallidin (Lys 0 -bradykinin) are formed from kininogen precursor proteins following the activation of plasma or tissue kallikrein enzymes during inflammation, tissue damage, or anoxia. The activity of these kinins is terminated by several degradative enzymes. Kininase I liberates the biologically active metabolites des-Arg 9 -bradykinin and des-Arg 10 -kallidin, whereas kininase II and endopeptidases form inactive metabolites ( Calixto et al 2000 , Marceau and Regoli 2004 ). The biologically active kinins activate two distinct types of G protein–linked receptors. Bradykinin and kallidin act preferentially at the B 2 receptor, whereas des-Arg 9 -bradykinin and des-Arg 10 -kallidin act with much higher affinity at the B 1 receptor than at the B 2 receptor.
B 2 receptors are expressed constitutively on a wide range of cell types, including nociceptive sensory nerves, and administration of bradykinin evokes pain and sensitizes polymodal nociceptors (see Mizumura et al 2009 ). Bradykinin acts directly on sensory nerves and can also act indirectly by evoking the release of other inflammatory mediators from non-neuronal cells. There is good pharmacological evidence that the acute and some of the long-term effects of bradykinin are mediated via the B 2 receptor. For example, peptide and non-peptide B 2 receptor antagonists have analgesic and anti-hyperalgesic actions in animal models of inflammatory pain ( Dray and Perkins 1993 ; Perkins and Kelly 1993 , 1994 ; Asano et al 1997 ; Burgess et al 2000 ; Cuhna et al 2007 ; Valenti et al 2010 ), as well as in some neuropathic pain models ( Werner et al 2007 , Luiz et al 2010 ). Interestingly, thermal hypersensitivity is still evoked by complete Freund’s adjuvant (CFA)-induced inflammation in mice lacking the B 2 receptor ( Boyce et al 1996 , Rupniak et al 1997 , Ferreira et al 2001 ), but carrageenan-evoked thermal hypersensitivity is reduced ( Boyce et al 1996 , Rupniak et al 1997 ).
In contrast to B 2 receptors, B 1 receptors are not normally expressed at significant levels in normal tissue, except in some vascular beds, but their expression is induced by tissue injury and infection. This up-regulation of B 1 receptors requires de novo protein synthesis ( Regoli et al 1978 , Bouthillier et al 1987 , DeBlois et al 1991), and there is evidence that the induction is stimulated by the release of cytokines such as IL-1β and TNF-α from immunocompetent cells in the damaged tissue ( Calixto et al 2004 , Cuhna et al 2007 ). Some effects of B 1 agonists are mediated via non-neuronal cells, where activation of the B 1 receptor evokes the release of PGE 2 and PGI 2 , NO, and various cytokines ( Leeb-Lundberg et al 2005 , Kuhr et al 2010 ). There is also immunocytochemical and autoradiographic evidence that the B 1 receptor is expressed in a subset of sensory neurons ( Wotherspoon and Winter 2000 , Ma 2001 , Petcu et al 2008 ) and that the level of expression is increased during inflammation ( Fox et al 2003 ). The mechanisms regulating expression of the B 1 receptor in sensory neurons are not well understood but are likely to involve cytokines, as found in other cell types, and neurotrophins. Functional expression of sensory neuron B 1 receptors is up-regulated by exposure to the neurotrophins GDNF and neurturin. Under such conditions, B 1 receptor activation evokes sustained enhancement of the heat-gated current mediated by TRPV1 ( Vellani et al 2004 ).
There is good pharmacological evidence that B 1 receptors have an important role in the hyperalgesia associated with persistent inflammation. Although B 1 agonists do not normally affect nociceptive thresholds in animals, they evoke hyperalgesia following inflammation ( Davis and Perkins 1994 , Perkins and Kelly 1994 , Fox et al 2003 ). Furthermore, peptide B 1 antagonists such as des-Arg 10 -HOE140 and des-Arg 8 Leu 9 -bradykinin ( Perkins and Kelly 1993 , Perkins et al 1993 , Campos and Calixto 1995 , Rupniak et al 1997 , Fox et al 2003 ), as well as non-peptide B 1 antagonists ( Fox et al 2005 , Hawkinson et al 2007 ), inhibit thermal or mechanical hyperalgesia in models of joint, paw, or tail inflammation. These data are consistent with the finding that mice lacking the B 1 receptor show reduced thermal ( Ferreira et al 2001 ) and mechanical ( Fox et al 2005 ) hyperalgesia after CFA treatment.
The relative importance of the changes in subtypes of bradykinin receptors is variable and depends on the inflammatory condition, with evidence of a shift toward a dominant role of B 1 receptors in chronic conditions in which B 1 receptor expression is up-regulated (see, e.g., Cuhna et al 2007 ). Although many studies have focused on the peripheral role of kinin receptors, there is also evidence from studies involving selective antagonists and knockout mice that B 1 and B 2 receptors expressed in the spinal cord influence spinal processing of nociceptive signals in inflammatory conditions ( Pesquero et al 2000 ; Ferriera et al 2001 , 2002 ).

Bradykinin Receptor Signaling
B 1 and B 2 receptors couple through G q α to stimulate PLC, which results in phosphoinositide hydrolysis, DAG production, and mobilization of intracellular Ca 2+ from intracellular stores. They can also act through G i α to inhibit adenylate cyclase and stimulate the MAPK pathways ( Leeb-Lundberg et al 2005 , Cheng and Ji 2008 ). A significant body of evidence supports the idea that bradykinin activates sensory neurons via a DAG–PKC pathway. Bradykinin causes the translocation of a specific PKC isoform, PKC-ε, from the cytoplasm to the plasma membrane of dorsal root ganglion (DRG) neurons ( Cesare et al 1999 ), and the excitatory effects of bradykinin are inhibited by the PKC inhibitor staurosporine ( Burgess et al 1989 ), which also attenuates the responses of skin afferents ( Dray et al 1992 ). Furthermore, the bradykinin responses of many, but not all, neurons are reduced or abolished when PKC activity is down-regulated by prolonged exposure to phorbol esters ( Rang and Ritchie 1988 , Burgess et al 1989 ).
PKC activators depolarize sensory neurons by opening a cation-permeable ion channel ( Burgess et al 1989 , McGehee and Oxford 1991 ), and several pieces of information indicate that bradykinin exerts its effects, in part, by sensitizing or opening the heat-sensitive TRPV1 ion channel. Bradykinin activates ion channels in DRG neurons with properties similar to those of TRPV1 channels ( Premkumar and Ahern 2000 ); this agonistic effect requires the presence of PKC-ε and is blocked by PKC inhibitors ( Cesare et al 1999 , Premkumar and Ahern 2000 ). Bradykinin also increases the capsaicin sensitivity of TRPV1 and reduces the temperature threshold for activation from approximately 42°C toward or below normal body temperature via a PKC mechanism ( Vellani et al 2001 , Sugiura et al 2002 ).
Activation of TRPV1 cannot explain all the excitatory effects of bradykinin inasmuch as activation of vagal and visceral afferents by bradykinin is retained in TRPV1 knockout mice ( Kollarik and Undem 2004 , Rong et al 2004 ) and bradykinin can stimulate DRG neurons from TRPV1 −/− mice ( Katanosaka et al 2008 ). Bradykinin can also act via PLC to activate TRPA1 ( Bandell et al 2004 ), and bradykinin-evoked responses were significantly attenuated in sensory neurons from both TRPV1 and TRPA1 knockout mice ( Bautista et al 2006 ). One possibility is that TRPV1 and TRPA1 act in concert. In this scenario ( Bautista et al 2006 ), activation of PLC evokes TRPV1 gating and calcium influx. Because TRPA1 is often co-expressed with TRPV1 and because TRPA1 can be activated by increases in the intracellular calcium concentration ( Doerner et al 2007 , Zuborg et al 2007), a small calcium influx through TRPV1 may activate TRPA1.
Failure to inhibit bradykinin responses in all sensory neurons with staurosporine or prolonged exposure to phorbol esters ( Burgess et al 1989 , Rang and Ritchie 1988 ) suggests that excitation can be mediated by a PKC-independent mechanism. Other evidence points to different phospholipase-linked mechanisms resulting in activation of TRPV1. One proposal is that binding of PIP 2 to TRPV1 inhibits channel activity ( Prescott and Julius 2003 ) and its hydrolysis by B 2 receptor–mediated activation of PLC potentiates channel opening by removing this tonic inhibition ( Chuang et al 2001 ). However, the inhibitory influence of PIP 2 on TRPV1 has been challenged, and there is evidence that PIP 2 binding potentiates rather than inhibits TRPV1 ( Klein et al 2008 , Yao and Qin 2009 , Sowa et al 2010 ). Phosphoinositide binding may have both inhibitory and potentiating effects on TRPV1, depending on the level of stimulation ( Lukacs et al 2007 ).
B 2 receptor activation also stimulates the 12-lipoxygenase pathway and leads to the production of endogenous TRPV1 agonists (e.g., 12-hydroperoxyarachidonate [HPETE] and leukotriene B 4 [LTB 4 ]. Bradykinin-evoked activation of TRPV1-like currents, neuronal firing, and behavioral responses are blocked by lipoxygenase inhibitors, consistent with a contribution of this pathway ( Shin et al 2002 , Carr et al 2003 , Calixto et al 2004 , Wu and Pan 2007 ). Other data point to a role of COX products since the COX inhibitor flurbiprofen inhibits the heat sensitization induced by bradykinin in a skin–nerve preparation ( Petho et al 2001 ).
Two other ionic mechanisms have recently been proposed for bradykinin-evoked activation of DRG neurons. Depolarization resulting from inhibition of M-type potassium currents and activation of a calcium-activated chloride current, encoded by TMEM16A, have been proposed as important PLC-linked mechanisms for the excitatory actions of bradykinin ( Liu et al 2010 ).

Arachidonic Acid Metabolites
The enzymatic breakdown of arachidonic acid yields a variety of bioactive lipid molecules that have diverse physiological roles, including important actions in inflammation and pain. These molecules are not stored but are synthesized de novo from membrane lipids. The first step is release of arachidonic acid from phospholipids by the action of PLA 2 enzymes. Arachidonic acid is then metabolized to prostaglandins via the COX enzymes; to leukotrienes, 5-HPETE, and 5-hydroxyeicosatetraenoic acid (HETE) via 5-lipoxygenase; to 12-HPETE and 12-HETE via 12-lipoxygenase; to lipoxins via 15-lipoxygenase; and to epoxyeicosatetraenoic acids via the action of cytochrome P450.

Prostaglandins
Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX enzymes, are the most widely used and effective drugs for the clinical treatment of inflammatory pain and hyperalgesia. NSAIDs have no obvious effect on normal pain thresholds but attenuate the abnormal pain responses in inflammatory conditions. Two COX enzymes, COX-1 and COX-2, are responsible for the first steps in prostaglandin synthesis. These enzymes have two catalytic enzymatic activities: a COX activity responsible for the production of PGG 2 from arachidonic acid and a peroxidase activity that reduces PGG 2 to form PGH 2 , the first steps in prostanoid biosynthesis.
In general, COX-1 is considered to have a “housekeeping” role in almost all tissues mediating physiological responses. In contrast, COX-2 is not constitutively expressed (except in the kidney, vas deferens, and importantly, the brain) but is induced in inflammatory conditions. In the periphery, COX-2 expression is induced in cells involved in inflammation (macrophages, monocytes, and synoviocytes) and is primarily responsible for synthesis of the prostaglandins involved in acute and chronic inflammatory states. COX-2 expression is induced in peripheral tissues in animal models of arthritis, and up-regulated expression is seen in human rheumatoid arthritic joints, although relatively little expression has been noted in human osteoarthritic joints. Both COX-1 and COX-2 are expressed constitutively in DRG neurons and in the spinal cord. Normally, COX-1 is expressed in small and medium-sized DRG neurons and in neurons and astrocytes in the spinal cord. Enzyme expression in both neuronal and non-neuronal cells in the spinal cord is up-regulated after peripheral inflammation and nerve injury (see Samad et al 2002 , Svensson and Yaksh 2002 ), and intraspinal release of PGE 2 is enhanced during peripheral inflammation ( Yang et al 1996 , Ebersberger et al 1999 ).
The important roles of spinal cord COX enzymes are not discussed in detail here but are covered in Chapter 28 . The available information indicates that COX inhibition at both peripheral and central sites can contribute to the anti-hyperalgesic effects, with the predominant clinical effect being mediated centrally. Certainly, prostaglandins produced in the periphery after tissue injury can sensitize peripheral nerves and induce hyperalgesia in animal models of inflammation, thus suggesting that a component of hyperalgesia could be due to a peripheral action. However, the finding that intrathecal administration of COX-2–selective inhibitors suppresses experimentally induced inflammatory hyperalgesia also argues for a central site of action ( Samad et al 2001 ). The observations that COX-2 inhibitors have clinical efficacy similar to that of non-selective NSAIDs and that COX-2 inhibitors exert a rapid effect after surgery also argue that they act in these conditions at central sites where COX-2 is constitutively expressed.
PGH 2 is metabolized by different prostaglandin synthetases to a range of prostaglandins. Prostaglandins such as PGE 2 , PGD 2 , and PGI 2 are produced during inflammation and act with some specificity on different prostanoid receptors, termed EP, DP, and IP, respectively. Each of the prostanoid receptors has distinct coupling to G proteins, and the pattern of coupling determines the biochemical consequence of receptor activation. Four major types of EP receptors (EP 1–4 ) have been described, and splice variants of the EP 3 subclass have also been identified, which probably explains the multiplicity of transduction pathways that have been associated with this receptor. In situ hybridization studies have shown the presence of mRNA for IP, EP 1 , EP 3 , and EP 4 receptors in DRG neurons. About half the neurons express EP 3 receptor mRNA; 40%, IP mRNA; 30%, EP 1 mRNA; and 20%, EP 4 mRNA, with some degree of co-expression ( Sugimoto et al 1994 , Oida et al 1995 ). Of these, EP 1 , EP 4 , IP, and some splice variants of EP 3 receptors (EP 3B and EP 3C ) couple positively via G s to stimulate adenylate cyclase and raise cAMP levels.
A major peripheral effect of PGE 2 and PGI 2 is to sensitize afferent neurons to noxious chemical, thermal, and mechanical stimuli (see, for example, Mizumura et al 1987 , Schaible and Schmidt 1988 , Birrell et al 1991 ). In contrast, PGD 2 shows little or no such activity ( Rueff and Dray 1992 ). The importance of these receptor subtypes in the periphery is confirmed by the findings that EP 3 −/− and IP −/− mice show reduced hyperalgesia after lipopolysaccharide (LPS) administration ( Ueno et al 2001 ). In contrast, intrathecal administration of PGE 2 induced normal mechanical allodynia in wild-type and EP 3 −/− mice but not in EP 1 −/− mice, thus illustrating that the EP 1 receptor plays a role in prostaglandin-induced spinal sensitization ( Minami et al 2001 ).

Lipoxygenase Products
The potential role of lipoxygenase products in inflammatory pain is less clear, and although the levels are increased in inflammatory conditions, evidence of a direct role in nociception is lacking. The major effect of these lipids is to recruit immune cells and alter microvascular permeability. Intradermal injection of LTB 4 or 8R,15S-diHETE decreases mechanical and thermal thresholds in rats ( Levine et al 1984 , 1985 , 1986a ; Martin et al 1987 ; Martin 1990 ) and humans ( Bisgaard and Kristensen 1985 ), and LTB 4 sensitizes dental afferents ( Madison et al 1992 ). The sensitizing actions of LTB 4 require the presence of polymorphonuclear (PMN) leukocytes and are thus likely to be indirect ( Levine et al 1984 , 1985 ). 8R,15S-diHETE reduces the thermal and mechanical thresholds of C fibers ( Taiwo et al 1989 , White et al 1990 ) and excites some C-fiber neuromas ( Devor et al 1992 ). A role of LTB 4 in experimental antigen (ovalbumin)-induced mechanical hyperalgesia has been shown by using the LTB 4 antagonist CP10596 ( Cunha et al 2003 ). More recently, the cysteinyl-leukotriene receptor CysLT2 was found to be expressed in about 40% of rat DRG neurons, preferentially in small-diameter neurons. Intraplantar administration of the CysLT2 agonist LTC 4 strongly enhanced the nocifensive response evoked by the P2X 3 agonist αβ-me-ATP but was without effect on thermal sensitivity, thus suggesting a lack of effect on TRPV1 channels ( Okubo et al 2010 ).
One probable action for some lipoxygenase products is to activate TRPV1 channels inasmuch as 12S-HPETE, 15S-HPETE, 5S-HETE, and LTB 4 all open TRPV1 channels in DRG neurons ( Hwang et al 2000 ). The behavioral effects of 8R,15S-diHETE noted earlier are unlikely to be due to such an action since this lipid shows very weak agonist effects on TRPV1.

Other Fatty Acid Metabolites
In addition to the leukotrienes, lipoxygenases can also convert eicosapentaenoic and docosahexaenoic acids into active signaling molecules. Formation of some of these metabolites requires the sequential action of COX-2 or cytochrome P450 followed by lipoxygenase-mediated oxidation ( Bannenberg and Serhan 2010 ). The resulting molecules have been named resolvins because of the roles that they are thought to play in the resolution phase of inflammation, and they have attracted interest for their analgesic potential ( Ji et al 2011 ). The resolvins RvE1 and RvD1 potently reduce thermal and mechanical hypersensitivity in inflammatory pain models. Resolvins produce these effects by stimulating G i/o -coupled GPCRs located both on DRG neurons and in the spinal cord, thereby effectively inhibiting the activity of the sensory neuron ion channels TRPA1 and TRPV1, as well as C-fiber evoked long-term potentiation in the spinal cord ( Xu et al 2010 , Park et al 2011 ).
Linoleic acid is converted into several hydroxyl and carbonyl derivatives (9-HODE, 13-HODE, 9-oxoODE, and 13-oxoODE) by both lipoxygenase pathways and non-enzymatic lipid peroxidation reactions. In experimental situations, formation of these mediators is increased by depolarization of the spinal cord with a high-K + solution ( Patwardhan et al 2009 ). Extended exposure to heat also significantly increases the tissue concentration of these oxidized linoleic acid metabolites in mouse skin biopsy samples. Application of 9-HODE to cultured trigeminal neurons stimulates TRPV1, and administration in vivo evokes nocifensive behavior and thermal hypersensitivity, which is absent in Trpv1 − / − mice, thereby demonstrating that TRPV1 mediates the nociceptive effect of 9-HODE (Patwardhan et al 2010). Thus, oxidized linoleic acid metabolites, such as the endocannabinoid anandamide and several lipoxygenase products formed from arachidonic acid, can act as direct TRPV1 agonists ( Zygmunt et al 1999 , Hwang et al 2000 ).
During conditions characterized by oxidative stress, such as inflammation or reperfusion after ischemia, a range of lipid peroxidation products are formed in reactions between free radicals and membrane lipids. Many of the lipids formed are well-known reactive, electrophilic molecules that bind covalently to proteins such as hydroxynenonal, cyclopentenone prostaglandins, isoprostanes, and related species. The covalent modification of redox-sensitive transcription factors initiates specific signaling cascades that may act to modify or protect against oxidative conditions, but the electrophilic lipids also stimulate nociceptive sensory neurons directly by activating TRPA1 ( Trevisani et al 2007 , Andersson et al 2008 ).

Protease-Activated Receptors
Four types of G protein–coupled protease-activated receptors (PARs) have been identified (PAR1–4). These receptors are activated by a unique mechanism whereby extracellular, soluble, or surface-associated proteases cleave at specific residues in the extracellular N-terminal domain of the G protein to expose a novel N-terminal sequence that acts as a tethered ligand and activates the receptor by binding to other regions of the protein. These agonist effects can be mimicked by short synthetic peptides based on the sequence of the tethered ligands of the different PARs. PAR1, PAR3, and PAR4 are activated by thrombin produced during the blood-clotting cascade, whereas PAR2 activation is triggered by tryptase, which is known to be released from mast cells in inflammatory conditions, as well as by the blood-clotting factors VIIa and Xa and the cysteine protease cathepsin S ( Soh et al 2010 , Cattaruzza et al 2011 ). In this way PARs are activated as a result of tissue damage and inflammation. Because activation involves an irreversible enzymatic cleavage, restoration of PAR sensitivity requires internalization of the receptors and insertion of new receptor into the plasma membrane.

Protease-Activated Receptor Signaling
Activation of PARs can trigger a variety of intracellular signaling pathways. PAR1 and PAR2 couple to either G q/11 α, G 12/13 α, or G i α; PAR3 signals through G q/11 α activation; and PAR 4 through either G q/11 α or G 12/13 α ( Russo et al 2009 , Soh et al 2010 ). In this way, activation of PAR1 and PAR2 may stimulate PLC-β to activate the DAG–PKC and IP 3 –Ca 2+ pathways (G q11 α), Rho and Rho-kinase (G 12/13 α), and the MAPK cascade and inhibit adenylate cyclase (G i α).

Protease-Activated Receptor Expression
PARs were initially detected in platelets, endothelial cells, and fibroblasts, but they are also expressed in the nervous system. All four PARs are expressed on peripheral sensory neurons. Expression of PAR2 is almost exclusively restricted to small-diameter unmyelinated neurons in rat and mouse DRG neurons, a majority of which are also positive for calcitonin gene–related peptide (CGRP) expression ( Zhu et al 2005 , Vellani et al 2010 ).
Studies using PAR subtype–selective peptide agonists and knockout mice suggest that the hyperalgesic effect of PAR activation is mediated primarily through PAR2, although in vitro, PAR1 and PAR4 receptor activation can sensitize TRPV1-mediated heat responses ( Vellani et al 2010 ). Intraplantar injection of PAR2 synthetic agonists, as well as tryptase, evokes prolonged thermal and mechanical hyperalgesia and c-fos expression in laminae I and II in the spinal cord ( Kawabata et al 2001 , 2002 ; Vergnolle et al 2001 ). This hyperalgesia occurs with low concentrations of agonists that do not cause overt inflammation, and it is not seen in mice lacking the neurokinin 1 (NK1; substance P) receptor or in the presence of centrally acting NK1 receptor antagonists. Mast cells are known to be closely associated with sensory nerves in normal as well as inflammatory conditions ( Stead et al 1997 ), and the hyperalgesia evoked by the mast cell–degranulating agent 48/80 is significantly reduced in PAR2 −/− mice ( Vergnolle et al 2001 ). These findings suggest a direct action of PAR2 activation on sensory nerve function. Such a direct action has been demonstrated in isolated DRG neurons, where activation of PAR2 sensitizes TRPV1 and TRPA1 to agonist stimulation. The sensitizing effect of PAR2 activation on TRPV1 appears to be mediated by PKC since it is inhibited by PKC inhibitors and a PKC-ε translocation inhibitor ( Amadesi et al 2004 , Dai et al 2004 ). In contrast, PAR2-mediated sensitization of TRPA1 is independent of PKC and instead depends on activation of PLC and subsequent reduction of PIP 2 levels ( Dai et al 2007 ). In vivo, administration of a selective PAR2 agonist enhances the nocifensive responses evoked by the TRPA1 agonists AITC and cinnamaldehyde in the rat ( Dai et al 2007 ). An important role for TRPV1 in vivo is also shown by the finding that the thermal hyperalgesia, mechanical allodynia, and spinal cord c-fos expression evoked by the intraplantar injection of a PAR2 agonist peptide are significantly attenuated in TRPV1 −/− mice ( Amadesi et al 2004 , Dai et al 2004 ).
Activation of PAR1 may have complex effects on nociception. Sub-inflammatory doses of PAR1 agonists have been reported to increase nociceptive thresholds and significantly reduce the inflammatory hyperalgesia induced by carrageenan ( Asfaha et al 2002 ). However, higher doses of PAR1 agonists are pro-nociceptive, and it is possible that stimulation of PAR1 on different neuronal populations (small TRPV1-containing nociceptors and larger non-nociceptive neurons) can explain these apparently contradictory observations ( Vellani et al 2010 ). The pro-nociceptive effect of PAR1 stimulation appears to depend on PKC-ε and sensitization of TRPV1.

Serotonin
Serotonin is one of many mediators released from platelets (rats and humans) and mast cells (rats) in injured and inflamed tissues. In humans, intradermal dialysis of 5-hydroxytryptamine (5-HT) evokes burning pain (Lischetski et al 2001), and intramuscular injection of 5-HT elicits pain and sensitization to pressure stimuli ( Ernberg et al 2000 , Ernberg et al 2006 ). In situ hybridization studies have shown that DRG neurons normally express mRNA for 5-HT 1B , 5-HT 1D , 5-HT 2A , 5-HT 2B , 5-HT 3B , and 5-HT 4 receptors ( Nicholson et al 2003 ), with other evidence for the expression of 5-HT 7 receptors ( Amaya-Castellanos et al 2011 ). Expression of some of these receptor subtypes (5-HT 2A , 5-HT 3 , 5-HT 4 , and 5-HT 7 ) is increased with inflammation ( Wu et al 2001 , Liu et al 2005 ).
Some of the excitatory actions of serotonin have been ascribed to activation of the 5-HT 3 receptor/ion channel. 5-HT 3 receptor agonists enhance the excitability of unmyelinated C-fibers ( Moalem et al 2005 , Lang et al 2006 ), and relatively selective 5-HT 3 antagonists reduce the pain evoked by peripheral administration of serotonin or carrageenan in rats (Eschalier et al 1985, Richardson et al 1985, Sufka et al 1992 ).
Serotonin can also activate and sensitize nociceptors by actions on G protein–coupled 5-HT receptors. 5-HT 2A receptors are expressed mainly in small-diameter (Aq- and C-fiber) peptidergic and non-peptidergic sensory neurons, and there is significant overlap with TRPV1 expression ( Okamoto et al 2002 , van Steenwinckel et al 2009 ). 5-HT 2A receptors play a significant role in inflammatory thermal hypersensitivity. Intraplantar administration of 5-HT 2A agonists into rats produces thermal hyperalgesia ( Abbott et al 1996 , Tokunaga et al 1998 ), and activation of peripheral 5-HT 2A receptors induces Fos expression in dorsal horn neurons, indicative of sensory neuron excitation (Doi-Saika et al 1997). Conversely, peripheral administration of 5-HT 2A receptor antagonists reduces the thermal hyperalgesia induced by either CFA or carrageenan ( Okamoto et al 2002 , Wei et al 2005 , Huang et al 2009 ). In addition to the strong evidence for a role of 5-HT 2A receptors, there is also pharmacological evidence that 5-HT 2B receptors play a role in inflammatory mechanical hypersensitivity but not in thermal hyperalgesia ( Lin et al 2011 ). The cellular mechanisms responsible for these effects are unclear. 5-HT 2 receptors are usually linked to the PLC pathway, and the sensitization mechanism or mechanisms may be attributable to PKC-mediated modulation of ion channels.
Relatively few data are available on the roles of peripheral 5-HT 4 and 5-HT 7 receptors in inflammatory conditions, although some pharmacological evidence indicates that these receptor subtypes have roles in the longer-term (days) mechanical allodynia following intraplantar administration of formalin ( Godinez-Chapiro et al 2011 ). These receptors are positively coupled to adenylate cyclase, and receptor activation stimulates cAMP production. An increase in cAMP can result in a PKA-mediated modification of ion channel function, notably, increased activity of tetrodotoxin (TTX)-resistant sodium channels ( Cardenas et al 2001 , Scroggs 2011 ).

Nitric Oxide
Although the actions of NO on nociceptive processes are primarily spinal and evident after intrathecal administration of drugs, there is controversial evidence of a peripheral action of NO. The cellular source of NO is unclear, and both neuronal and non-neuronal sources are likely. NO is produced in the periphery during inflammation (see Toriyabe et al 2004 ). nNOS appears to be responsible for synthesis in the early phase of inflammation and nNOS and iNOS at later phases ( Omote et al 2001 ). Experimentally, intradermal and intravascular injection of NO evokes a concentration-dependent pain in human volunteers ( Holthusen and Arndt 1994 , 1995), whereas topical administration of NO donors is antinociceptive. The site of action appears to be important. Studies in rats have shown that intradermal administration of the NO precursor L -arginine or an NO donor (3-[4-morphinolinyl]-sydnonimine hydrochloride [SIN-1]) evokes mechanical hypersensitivity. In contrast, subcutaneous injection of these agents had little effect on baseline mechanical thresholds but reversed PGE 2 -induced hypersensitivity ( Vivancos et al 2003 ) via an NO/cGMP pathway ( Sachs et al 2004 ). A pro-nociceptive action of NO in inflammatory conditions is supported by the finding that local administration of the NOS inhibitor N ω -nitro- L -arginine methyl ester ( L -NAME) reduces both mechanical and thermal hyperalgesia, as well as the inflammation induced by carrageenan ( Lawand et al 1997 , Nakamura et al 1996 ). Similarly, co-injection of another NOS inhibitor, N G -methyl- L -arginine ( L -NMA), inhibited PGE 2 -induced mechanical hyperalgesia, whereas intradermal injection of the NOS substrate L -arginine or the NO donor SIN-1 evoked mechanical hyperalgesia ( Aley et al 1998 ). In peripheral nerves the NO-sensitive (soluble) guanylate cyclase is expressed by non-neuronal cells and not by sensory neurons (Schmidtko et al 2007), so the sensory neuron effects of activating the NO/cGMP pathway are likely to be indirect. NO can also nitrosylate ion channels, and this may be a more important mechanism for any direct pro- or antinociceptive NO effects. NO can stimulate DRG neurons by activation of both TRPA1 and TRPV1, and studies of genetically modified mice show that the thermal hyperalgesia elicited by injection of an NO donor is largely dependent on TRPV1 expression. In addition, both TRPA1 and TRPV1 appear to play roles in the acute nociceptive behavioral response to NO donor injection after pre-activation of the PLC/PKA pathways ( Miyamoto et al 2009 ). Conversely NO activates ATP-sensitive K + channels ( Kawano et al 2009 ) and inhibits voltage-gated sodium channels ( Renganathan et al 2002 ) in DRG neurons; both actions will inhibit neuronal firing and could contribute to antinociception. Many of the peripheral effects of NO or NOS inhibition are likely to involve other cells and mediators, including alterations in cytokine levels ( Chen et al 2010b ).

ATP and Adenosine

ATP, P2X, and P2Y Receptors
There has been considerable debate about the role of ATP in activation of peripheral nerves, especially in inflammatory conditions. ATP is released from damaged cells, and ATP levels are elevated in damaged and inflamed tissues ( Gordon 1986 , Cook and McCleskey 2002 ). It has also been proposed that ATP has a role in the genesis of pain associated with malignancy inasmuch as ATP levels at tumor sites are higher than those in normal tissues ( Pellegatti et al 2008 ). In humans, application of ATP to the skin evokes the sensation of pain ( Bleehen and Keele 1977 , Coutts et al 1981 ), which is enhanced after ultraviolet irradiation ( Hamilton et al 2000 ), and intracutaneous administration of ATP excites human C fibers ( Hilliges et al 2002 ). Similar pain behavior has been noted in animals, with nocifensive behavior being evoked by intraplantar administration of ATP ( Bland-Ward and Humphrey 1997 , Hamilton et al 1999 , Jarvis et al 2001 ), and this is augmented by treatment with PGE 1 and the inflammatory agent carrageenan ( Sawynok and Reid 1997 , Hamilton et al 1999 ). These behavioral responses are probably mediated by Aδ and C fibers because these fibers are excited by ATP both in vivo ( Dowd et al 1998 ) and in isolated nerve preparations ( Hamilton et al 2001 ) and the pain response evoked by ATP in human skin is markedly reduced after the topical application of capsaicin to functionally desensitize the TRPV1-expressing fibers ( Hamilton et al 2000 ).
The receptors responsible for this excitation are likely to contain the P2X 3 receptor subtype (i.e., P2X 3 homomeric or P2X 2/3 heteromeric receptors) because sensory fibers are excited by the P2X 3 agonist α,β-me-ATP (see Irnich et al 2002 ). Furthermore, P2X 3 receptor expression is restricted to small-diameter sensory afferents ( Vulchanova et al 1997 , Bradbury et al 1998 ), and their expression is up-regulated in experimental inflammatory conditions ( Xu and Huang 2002 , Shinoda et al 2005 ). One mechanism for this up-regulation is an increased supply of the growth factors NGF and GDNF in sensory nerves during inflammation since administration of both these growth factors (by intrathecal administration) increased P2X 3 receptor immunoreactivity in rat DRG neurons ( Ramer et al 2001 ). Similarly, P2X 3 receptor expression is elevated following injection of NGF into skeletal muscle ( Liu et al 2011 ). Inflammatory mediators may also increase ATP sensitivity via PKA- and PKC-mediated phosphorylation of P2X 3 -containing receptors ( Paukert et al 2001 , Fabbretti et al 2006 ).
A role of P2X 3 receptors in inflammatory pain is supported by the finding that intrathecal delivery of antisense oligonucleotides or small interfering RNA (siRNA) directed against P2X 3 mRNA, which reduces P2X 3 protein expression by about 50%, partially reverses inflammatory thermal and mechanical hyperalgesia ( Barclay et al 2002 , Honore et al 2002 , Dorn et al 2004 ). In addition, reversal of inflammatory thermal and mechanical hyperalgesia, as well as thermal and mechanical hyperalgesia after nerve injury, is seen after the administration of selective antagonists (A317491 and AF-353) ( Jarvis et al 2002 , Oliveira et al 2009 , Ford 2012 ). AF-353 is also effective in models of bone cancer pain, where it reversed mechanical hypersensitivity and improved weight bearing on the affected limb ( Kaan et al 2010 ). The marked effects of antisense oligonucleotide treatment and pharmacological antagonism contrast with the relatively mild phenotypic changes seen in P2X 3 -null mice ( Cockayne et al 2000 , Souslova et al 2000 ), which display a modest reduction in the behavioral response to intraplantar administration of formalin. The paradoxical finding that P2X 3 -null mice show increased thermal hyperalgesia after injection of CFA suggests that some adaptive processes occur when the P2X 3 receptor is ablated.

P2Y Receptors
ATP can also stimulate sensory neurons by activating G protein–coupled P2Y receptors. Of the known P2Y receptors, mRNA for the G q/11 α-linked receptors P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 is expressed in sensory ganglia. P2Y 1 and P2Y 2 receptors, which are expressed by sensory neurons ( Molliver et al 2002 , Kobayashi et al 2006 ), have received the most attention. Expression of P2Y 2 is increased during inflammation induced by CFA, whereas P2Y 1 , P2Y 4 , and P2Y 6 are reduced ( Malin et al 2008 ). Both P2Y 1 and P2Y 2 receptors are G q11 linked and signal via IP 3 –DAG pathways, which is consistent with the finding that activation of either receptor subtype evokes a rise in intracellular calcium levels and an increase in excitability in DRG neurons that is blocked by PLC and PKC inhibition ( Usachev et al 2002 , Malin and Molliver 2010 , Yousuf et al 2011 ). On the other hand, stimulation of the G i/o -coupled receptors P2Y 12–14 reduced the excitation of DRG neurons in a pertussis toxin–sensitive fashion. In vivo, peripheral administration of P2Y 13 and P2Y 14 agonists reduced the inflammatory hyperalgesia induced by CFA ( Malin and Molliver 2010 ). P2Y receptor activation in DRG neurons also activates the transcription factor cAMP response element–binding protein (CREB), which is likely to lead to longer-term changes in the cell phenotype ( Molliver et al 2002 ). P2Y receptor activation by the P2Y 2 /P2Y 4 agonist uridine triphosphate (UTP) evokes sustained action potential firing in capsaicin-sensitive C fibers and some Aδ fibers ( Stucky et al 2004 ). This effect is probably mediated through P2Y 2 receptors since these receptors appear to be expressed at very low levels by sensory neurons ( Sanada et al 2002 ).
The mechanisms underlying P2Y receptor–mediated excitation involve sensitization of TRPV1 and modulation of ion channels that regulate the firing frequency of action potentials. P2Y 2 receptor activation potentiates the capsaicin-evoked TRPV1 currents and [Ca 2+ ] i responses in isolated sensory neurons, and this potentiation is lost in P2Y 2 -null mice ( Moriyama et al 2003 , Malin et al 2008 ). P2Y 1 receptor activation also lowers the heat activation threshold for TRPV1 in rat DRG neurons ( Tominaga et al 2001 ) and increases sensitivity to the TRPV1 agonist capsaicin ( Yousuf et al 2011 ).
P2Y 1/2 receptor activation can also increase the excitability of DRG neurons by inhibiting K v 7 potassium channels ( Yousuf et al 2011 ), which is also a mechanism described for bradykinin sensitization. Furthermore, P2Y 2 activation sensitizes mechanotransduction channels ( Lechner and Lewin 2009 ) and purinergic P2X 2 and P2X 3 channels ( Chen et al 2010a ) and may underlie the ATP-induced potentiation of TTX-resistant sodium channel (Na v 1.8) currents ( Baker 2005 ).

Adenosine
It is almost certain that some of the effects of ATP in vivo are mediated by adenosine diphosphate (ADP) ( Bleehen and Keele 1977 , Coutts et al 1981 ), AMP, and adenosine ( Bleehen and Keele 1977 ) formed by rapid sequential ectonucleotidase cleavage of ATP. All these agents produce pain when applied to human skin. However, the underlying mechanisms probably differ because the nocifensive response to ADP seen in animal studies differs from that evoked by ATP ( Bland-Ward and Humphrey 1997 ).
During inflammation, adenosine is released from a variety of cell types (endothelial cells, mast cells, neutrophils, and fibroblasts), in addition to release from neurons. The effects of adenosine are complex, with evidence of both pro-nociceptive and analgesic effects (see Sawynok and Liu 2003 ) mediated through various receptor subtypes (A 1 , A 2A , A 2B , and A 3 ) at peripheral and spinal sites. Although some of the effects are probably directly on nerves, others are more likely to be mediated via activation of adenosine receptors on other cell types, such as mast cells. Nevertheless, there is clear evidence that adenosine can activate sensory nerves since intravenous administration of adenosine produces pain in human volunteers ( Sylven 1989 ) and application of adenosine sensitizes cat myelinated and unmyelinated vagal afferents ( Cherniak et al 1987 ). Isolated segments of human nerve are also depolarized by ATP; the pharmacological properties are consistent with an effect mediated by adenosine acting on G s -coupled A 2B receptors ( Irnich et al 2002 ). In other experiments, A 1 agonists have been reported to activate C fibers in the rat ( Esquisatto et al 2001 , Sawynok et al 2000 ), and stimulation of A 1 receptors induces an inward current and action potential firing in guinea pig jugular and spinal esophageal TRPV1-positive nociceptors ( Ru et al 2011 ). In contrast to the predominating pro-nociceptive peripheral effects produced by adenosine, intrathecal administration of adenosine has well-recognized analgesic effects mediated by A 1 receptor activation ( Sawynok and Liu 2003 ). Accordingly, intrathecal administration of the ectonucleotidase prostatic acid phosphatase (PAP) has been shown to produce long-lasting antinociception and anti-hyperalgesia mediated by hydrolysis of extracellular AMP to adenosine, which in turn stimulates adenosine A 1 receptors ( Zylka et al 2008 ).

Low pH
The pH of the extracellular environment is known to fall in a number of pathophysiological conditions, such as hypoxia and anoxia, as well as with inflammation and tumors. Acidic conditions can have direct effects on sensory nerves. Low-pH solutions evoke prolonged activation of sensory nerves and produce a sharp stinging pain in humans ( Lindahl 1962 , Steen and Reeh 1993 , Jones et al 2004 ). Several mechanisms are thought to underlie the neuronal excitation observed. One key effect of acid solutions is activation and sensitization of the thermosensitive ion channel TRPV1 ( Tominaga et al 1998 , McLatchie and Bevan 2001 , Leffler et al 2006 ). A second mechanism is direct activation of ASICs (see Deval et al 2010 ), notably ASIC3, which is expressed in the sensory innervation of the heart and activated by modest reductions in extracellular pH (to about pH 7). ASIC3 has been proposed to be the sensor in cardiac nociceptors that triggers cardiac pain in response to myocardial acidity ( Sutherland et al 2001 ) and may play a role in sensing acidic conditions in other tissues such as skin ( Deval et al 2008 ) and skeletal muscle ( Sluka et al 2003 ). Finally, low pH can augment or stimulate neuronal firing by inhibiting K + channel activity ( Baumann et al 2004 ).

Immune Cells and Pain
It is now well established that the immune system, as well as the factors that it produces, can alter sensory processing and play a pivotal role in the development and maintenance of persistent pain ( Marchand et al 2005 , Ren and Dubner 2010 ). For example, not only are cytokines and chemokines an important means of communication between immune cells, but such factors can also act as pain mediators and have a direct sensitizing action on nociceptors. The importance of the immune system is not restricted to inflammatory pain states but extends to neuropathic conditions since nerve injury evokes a profound immune response. Many of the pain mediators discussed below are closely linked to this system through either their release by or their action on different immune cells. We discuss the role of particular immune cells in different pain states below and summarize these actions in Table 3-1 .

Table 3-1
Contribution of Peripheral Immune Cells to Animal Models of Persistent Pain
CELL TYPE *
INFLAMMATORY PAIN †
NEUROPATHIC PAIN ‡ Macrophage ↑ Infiltration (joint, muscle) ↓ Mechanical, spontaneous ↑↑ Infiltration (nerve) ↓/↔ Mechanical, ↓/↔thermal, ↓ spontaneous Dendritic cell/Langerhans cell − ↑ Infiltration/activation (skin, nerve) Mast cell ↑ Degranulation (skin) ↓ Mechanical, thermal, spontaneous, visceral ↑ Degranulation (skin, nerve) ↓ Mechanical, thermal Neutrophils ↑↑ Infiltration (skin, joint) ↓ Mechanical, thermal ↑ Infiltration (nerve) ↓ Thermal T cells ↑ Infiltration (joint) ↓ Mechanical ↑ Infiltration (nerve) ↓ Mechanical, thermal Natural killer cells − ↑ Infiltration (nerve) B cells − ↑ Infiltration (nerve) ↔ Mechanical
This table highlights the involvement of immune cells in both inflammatory and neuropathic pain by using data from animal models. Following the injection of an inflammogen or damage to a peripheral nerve (either traumatic or drug induced), various immune cells infiltrate the relevant peripheral tissue and/or alter their response state. In addition, via genetic, chemical, or pharmacological approaches, certain immune cell populations can be depleted, their infiltration suppressed, or their activation prevented, thereby leading to the attenuation of persistent pain. The data in this table is a summary of the studies discussed in this section.
* Although microglia are important in the development and/or maintenance of persistent pain, they are central nervous system immune cells and therefore have not been mentioned in this table. Work regarding these cells is discussed in Chapter 4 .
† Inflammatory models include complete Freund’s adjuvant, carrageenan, zymosan, nerve growth factor, lipopolysaccharide, formalin, collagen- or antigen-induced arthritis, and acetic acid.
‡ Neuropathic pain models include partial sciatic nerve ligation, chronic constriction injury, spinal nerve ligation, spared nerve injury, vincristine, paclitaxel, and streptozocin.

Mast Cells
Mast cells are found in areas of the body that interact with the external environment, such as the skin and mucosal layers, and these cells are normally situated in close proximity to blood vessels and nerves. Mast cell granules contain numerous chemicals, including histamine, and they can also synthesize and release many cytokines and chemokines ( Metcalfe et al 1997 ).
Mast cells can be degranulated by the compound 48/80, which when applied to human skin causes thermal hyperalgesia, thus indicating that chemicals in the granules of mast cells are pro-algesic ( Drummond 2004 ). Chronic treatment with this compound prevents re-granulation of these cells, and in this state some common models of inflammatory pain, including those precipitated by injection of acetic acid or zymosan and the second phase of the formalin test, show reduced pain-like behavior ( Ribeiro et al 2000 , Parada et al 2001 ). Treatment with 48/80 to deplete mast cell granules also reduces both the thermal and mechanical hyperalgesia produced by CFA ( Woolf et al 1996 ). One of the mechanisms by which NGF induces thermal hyperalgesia (see below) is thought to be mediated via its action on mast cells ( Lewin et al 1994 ). Thus, NGF was not able to sensitize nociceptors to thermal stimuli in mice deficient in these cells ( Rueff and Mendell 1996 ), and these mice do not fully develop pain-like symptoms in a model of cystitis, which also seems to be strongly dependent on the release of mast cell mediators ( Rudick et al 2008 ).
Mast cells are also present in the sciatic nerve. Following partial sciatic nerve ligation (PSNL, a model of neuropathic pain), very few intact mast cells remain at the site of injury or directly distal to it, thus suggesting that the majority have released the contents of their granules. Stabilization of these cells increases the presence of intact mast cells and reduces the development of both mechanical and thermal hyperalgesia ( Theodosiou et al 1999 , Zuo et al 2003 ). Although the chemicals released by mast cells may act directly to sensitize nociceptors, such agents may also act to recruit and activate other immune cells within the injured nerve. Histamine is one of the mediators released by mast cells. However, the analgesic effect of antihistamine treatment is modest, and in some neuropathic pain models such agents have limited effects on mechanical pain–related hypersensitivity. Stabilization of mast cells with cromoglycate can reduce neuropathic hypersensitivity. Some of this action is likely to be indirect since such treatment also reduced both neutrophil and macrophage infiltration into the injured nerve ( Zuo et al 2003 ). Mast cells can produce NGF ( Leon et al 1994 ), and this might also contribute to the pro-algesic action of these cells.

Neutrophils
Neutrophils are PMN granulocytes and make up around 60% of the circulating white blood cells, which puts them in an ideal position to react, in large numbers, to pathogens or tissue injury. Rodent models of inflammatory pain are commonly induced by the local injection of an antigen, such as zymosan, LPS, or carrageenan, and subsequent activation of the innate and adaptive immune system ( Cunha et al 2008a , 2008b ; Guerrero et al 2008 ; Ting et al 2008 ). Accumulation of neutrophils occurs in all these models and can be reduced by blocking receptors that mediate the rolling, attachment, and transmigration of these cells from blood into tissue. Complement component 5a (C5a), a complement activation product, is a potent chemotactic factor for neutrophils ( Shin et al 1968 ). Following injection of zymosan into the paw, pharmacological inhibition of the C5a receptor attenuated both mechanical hypersensitivity and neutrophil influx ( Ting et al 2008 ). The chemokine receptors CXCR1 and CXCR2 are both important in neutrophil migration and activation in numerous inflammatory states ( Bizzarri et al 2006 ). Dual inhibition of these receptors was able to significantly reduce the accumulation of neutrophils and abnormal sensory behavior induced by zymosan, carrageenan, and LPS ( Cunha et al 2008a ). More recently, specific antagonism of the CXCR2 receptor via the small molecule SB225002 reduced both pain-related hypersensitivity and neutrophil accumulation in the carrageenan model ( Manjavachi et al 2010 ). Other factors with strong chemotactic effects on neutrophils include the lipoxygenase product LTB 4 ( Ford-Hutchinson et al 1980 ). Both pharmacological and genetic inhibition of the action of LTB 4 reduced the hypersensitivity produced by joint inflammation ( Guerrero et al 2008 ). In agreement with these data, chemical depletion of neutrophils decreased their accumulation in skin after both zymosan and carrageenan treatment and prevented full development of the abnormal sensory behavior in these models ( Ting et al 2008 ). Although recruitment of these cells is important, blockade of the C5a receptor in the LPS and carrageenan models did not affect neutrophil recruitment but did attenuate pain-like behavior, thus suggesting that certain molecules such as C5a may, in some instances of inflammation, be more important for activation than for direct recruitment of these cells ( Ting et al 2008 ). In naïve animals, intradermal injection of neutrophil chemotactic factors such as LTB 4 , N -formylmethionyl-leucyl-phenylalanine (fMLP), C5a, and chemokine C-X-C motif ligand 1 (CXCL1) induces pain-related hypersensitivity ( Levine et al 1985 , 1986a ; Cunha et al 2008a ). Interestingly the prominent pro-algesic properties of NGF are also reported to depend on neutrophil recruitment ( Bennett et al 1998b ). Recently, IL-17 has been shown to be a pro-nociceptive cytokine, particularly in the setting of antigen-induced arthritis, where neutralization of its effect reduced pain-related hypersensitivity and neutrophil recruitment in a TNF-α–dependent manner ( Pinto et al 2010 ). In addition, intraplantar injection of this cytokine produces both thermal and mechanical hypersensitivity associated with the accumulation of neutrophils in the dermis ( Kim and Moalem-Taylor 2011b , McNamee et al 2011 ). However, it must be stated that neutrophil attraction alone may not be sufficient to cause pain-like behavior because the activation status of these cells is also likely to be important. The chemotactic factor glycogen results in neutrophil recruitment but does not cause any significant pain-like hypersensitivity ( Levine et al 1985 ). Nevertheless, systemic depletion of neutrophils significantly reduced the pain-like behavior elicited by LTB 4 , C5a, fMLP, and NGF administration, thus suggesting that activated neutrophils are crucial in the pro-algesic properties of these and other factors ( Levine et al 1985 , 1986a ; Bennett et al 1998b ; Ting et al 2008 ). In vitro experiments have shown that in a co-cultured system, dissociated DRG neurons increase their excitability following neutrophil activation, which suggests that neutrophils do release factors that can act directly on nociceptive neurons ( Shaw et al 2008 ). Clinically, it seems that neutrophils play an important role in inflammatory diseases; in particular, they are present in the joint fluid and synovial membrane of patients with rheumatoid arthritis (RA) ( Wright et al 2010 ). Interestingly, therapies used to treat RA, such as antibodies against TNF-α, reduce pain scores in these patients and decrease the influx of neutrophils into the joint ( den Broeder et al 2003 ).
Neutrophils are normally completely absent from the naïve sciatic nerve. However, in animals in which the nerve has been injured to induce neuropathic pain–like behavior, substantial neutrophil infiltration takes place ( Perkins and Tracey 2000 , Zuo et al 2003 , Kim and Moalem-Taylor 2011a ). In addition, cytokine recruitment of neutrophils into the non-injured nerve can recapitulate this pain-like behavior ( Kim and Moalem-Taylor 2011b ). Some of the strongest evidence for a role of these cells in the development of neuropathic pain–like behavior comes from depletion studies. Systemic depletion of neutrophils before injury reduced the development of thermal hypersensitivity ( Perkins and Tracey 2000 ). However, an attempt to deplete neutrophils 8 days after injury had no effect on pain behavior ( Perkins and Tracey 2000 ), a finding suggestive of an important role in the initiation rather than the maintenance of neuropathic pain. Neutrophils can release numerous chemokines ( Scapini et al 2000 ), and it is likely that their algogenic effects, like those of mast cells, may partly be due to the subsequent recruitment and activation of other immune cells such as macrophages.

Macrophages
Macrophages are leukocytes and represent a heterogeneous group of cells resident in the majority of tissues. They are continually being replenished from a circulating peripheral blood mononuclear cell population, which itself originates from bone marrow. These cells have homeostatic actions in their tissue of residence, such as clearing cell debris, as well as repairing and remodeling tissue following damage and inflammation. Macrophages derive from monocytes, which also generate a range of other specialized cells contributing to innate immunity, including microglia in the CNS, alveolar macrophages in the lung, Langerhans cells in the skin, osteoclasts in bone, Kupffer cells in the liver, and histocytes in connective tissue, as well as resident cells in the spleen, gastrointestinal tract, and the peritoneum ( Gordon and Taylor 2005 ). Following tissue damage or infection, the macrophage population is augmented by blood-derived monocytes. The resident as well as the infiltrating macrophages react to endogenous danger signals released by necrotic cells or exogenous signals such as factors produced by microorganisms and appropriately release cytokines to orchestrate the innate and adaptive immune response. A strong body of evidence suggests a role of macrophages in the development of both inflammatory and neuropathic pain.
Intraperitoneal injection of acetic acid or zymosan is used as a model of visceral pain and induces overt pain-like behavior in rodents in the form of a writhing response. This behavior can be exacerbated by increasing the macrophage population ( Ribeiro et al 2000 ). Inhibiting the production of inflammatory mediators by macrophages through treatment with either anti-inflammatory cytokines or pentoxifylline (which reduces activation of these cells via a poorly defined mechanism) has been shown to reduce inflammatory pain ( Vale et al 2003 , 2004 ). Mice deficient in the purinergic receptor P2X 4 demonstrate reduced mechanical hyperalgesia following either CFA or carrageenan application. This effect is attributed to a reduction in the release of PGE 2 from tissue-resident macrophages, which would normally occur in a P2X 4 -dependent manner, and in agreement, injection of ATP-stimulated macrophages from wild-type mice into P2X 4 -deficient mice was able to induce mechanical hyperalgesia ( Ulmann et al 2010 ).
Macrophages have an important role in the development and maintenance of neuropathic pain. Traumatic injury to a peripheral nerve results in degeneration of axons separated from their cell bodies and breakdown of the associated myelin sheath in a process termed wallerian degeneration. Macrophages have an important role in phagocytosing and clearing myelin debris; because such debris is inhibitory to axon regeneration, clearance is vital for effective nerve repair. Chronic constriction injury (CCI) of the sciatic nerve in mice results in an increase in macrophage infiltration over a 28-day period that is strongly associated with neuropathic pain–like behavior ( Myers et al 1996 ). Naturally occurring mutant mice that exhibit slow wallerian degeneration display delayed macrophage recruitment and reduced cytokine production in injured nerves ( Sommer and Schafers 1998 ). Consistent with this attenuated inflammatory response, such mice also show delayed onset/reduced mechanical and thermal pain–related hypersensitivity ( Myers et al 1996 , Ramer et al 1997 ). Systemic depletion of macrophages also reduced both thermal and mechanical hyperalgesia in the PSNL model of neuropathic pain ( Liu et al 2000 , Barclay et al 2007 ).
Another means of inhibiting the pro-algesic actions of macrophages is to reduce their recruitment from the circulation to the injured nerve. An important molecule for macrophage chemotaxis is CCL3, blockade of which reduces macrophage infiltration, as well as thermal and mechanical pain–related hypersensitivity ( Kiguchi et al 2010 ). The toll-like receptors (TLRs) are pattern recognition receptors that respond to structural motifs on pathogens and the products of tissue injury. They have an important role in macrophage recruitment and activation. Mice lacking TLR2 demonstrate absent macrophage recruitment and reduced neuropathic pain–like behavior ( Shi et al 2011 ).
Another option for modulating the functional properties of these cells is to alter their functional status and thereby reduce the production of pro-inflammatory cytokines ( Kiguchi et al 2010 ). This can be achieved by treatment with anti-inflammatory cytokines such as IL-10 ( Wagner et al 1998 ). Trying to change the phenotype of macrophages from a pro- to an anti-inflammatory state may be a better therapeutic option than trying to globally inhibit their recruitment to or function within the injured nerve because they are essential for effective nerve repair ( Barrette et al 2008 ).

Dendritic Cells
Dendritic cells (DCs) are closely related to macrophages; they are primarily antigen-presenting cells but also have phagocytic capabilities and can release cytokines and chemokines. Some of the pro-nociceptive effects of IL-17 may be mediated by these cells ( Ruts et al 2010 , Kim and Moalem-Taylor 2011b ). In the epidermis these cells are referred to as Langerhans cells (LCs). Following traumatic nerve injury, epidermal nerve fiber density is decreased. However, spared fibers that intermingle with degenerating axons share innervation territories, and these spared axons have an important role in the generation of neuropathic pain. The endings of these spared axons show increased association with LCs after nerve injury ( Lin et al 2001 , Lindenlaub and Sommer 2002 ). In chemotherapy-induced neuropathic pain–like states these LCs express OX-6, a marker of activation associated with pro-inflammatory cytokine production ( Siau et al 2006 ). This same phenomenon has been seen in skin biopsy samples taken from patients with complex regional pain syndrome ( Calder et al 1998 ), and in diabetic patients with small-fiber neuropathy, the number of LCs is increased in the skin in comparison to control samples ( Casanova-Molla et al 2012 ). DCs also infiltrate the injured sciatic nerve distal to and at the site of injury. This infiltration is delayed and occurs 1 week after the initial injury when a robust thermal and mechanical pain–related hypersensitivity takes place ( Kim and Moalem-Taylor 2011a ). Although these cells respond to a number of different types of tissue injury, there is as yet no direct evidence that they contribute to persistent pain states.

T Cells
T cells are lymphocytes activated by the presentation of antigens. They mediate cellular immunity either by directing the immune response via the release of cytokines to activate innate immune cells or through the destruction of infected cells. More than most, T cells represent a heterologous group of immune cells loosely divided into CD4 + (helper) and CD8 + (cytotoxic) with type 1 and type 2 subsets. Th1 cells release pro-inflammatory cytokines that activate neutrophils, macrophages, and natural killer (NK) cells, whereas Th2 cells release anti-inflammatory cytokines that activate humoral immunity and strongly deactivate macrophages ( O’Garra and Arai 2000 ).
T cells have a pivotal role in autoimmune diseases. RA represents one such disease that is commonly associated with persistent pain, T-cell infiltration, and cytokine production ( Toh and Miossec 2007 ). Abatacept is a fusion protein that prevents the activation of T cells and decreases pain in patients with RA. In models of neuropathic pain induced by either CCI or PSNL, the number of T cells is significantly increased in the injured nerve in comparison to controls ( Cui et al 2000 , Kim and Moalem-Taylor 2011a ). This increase is associated with both thermal and mechanical pain–related hypersensitivity ( Cui et al., 2000 ). Neuropathic pain–related behavior is reduced in T cell–deficient mice or in mice that lack the ability to produce mature T cells ( Moalem et al 2004 , Kleinschnitz et al 2006 , Cao and Deleo 2008 , Costigan et al 2009 ). Passive transfer of Th1 cells into mature T cell–deficient mice is able to restore full neuropathic pain–like behavior, and this pain behavior can be attenuated by Th2 cells in immune-competent mice ( Moalem et al 2004 ). Guillain-Barré syndrome is an autoimmune disorder affecting the peripheral nervous system. The syndrome is a form of peripheral neuropathy and is commonly associated with abnormal pain ( Ruts et al 2010 ). Rats with experimental autoimmune neuritis model this syndrome, and these animals display robust neuropathic pain–like behavior and significant expression of T cells in the affected nerves ( Moalem-Taylor et al 2007 , Ruts et al 2010 ).

Other Immune Cells
Many other cells have immune functions and orchestrate both innate and adaptive immunity. NK cells are lymphocytes and constitute up to 20% of the mononuclear cells found in the blood and spleen. They target and kill infected or “stressed” cells, thereby playing a major role in tumor rejection, and can release many pro-inflammatory cytokines. NK cell activity is not altered in the CCI model of neuropathic pain ( Tsai and Won 2001 ), and although their numbers do increase in some nerve injuries, this is not associated with the development of hyperalgesia ( Cui et al 2000 ). B lymphocytes mediate the humoral arm of the adaptive immune response and produce specific antibodies against presented antigens. A recent study looking at immune cell infiltration into the sciatic nerve found a significant increase in the number of B cells 3–14 days after injury, particularly at the site of damage ( Kim and Moalem-Taylo, 2011a ). However, there does not seem to be any direct evidence linking B cells to the development or maintenance of persistent pain. In fact, full neuropathic pain–like behavior develops in B cell–deficient mice following nerve injury ( Costigan et al 2009 ). Eosinophils and basophils are, like neutrophils, PMN cells that play a role in both parasitic infection and the body’s response to allergens. There is little evidence linking these cells to pain modulation. Both these cell types can release a variety of pro-algesic factors, and further study is required to elucidate any possible role in pain pathophysiology.

Production of Immune Mediators by Non-immune Cells
Many immune cells in their quiescent state do not appear to interact with nociceptive systems. However, following tissue injury some cells undergo a profound phenotypic change that results in the release of cytokines and chemokines. These factors can recruit more immune cells and may also act as pain mediators. Non-immune cells can play an important role in initiating this process. An example is keratinocytes found within the epidermis, an important innervation target in which the naked endings of nociceptors terminate. Following injury or disease, keratinocytes can release an array of cytokines, chemokines, and growth factors ( Pastore et al 2006 , Li et al 2011 ), which can have sensitizing actions on nociceptors, as well as endogenous opioids, which can have an analgesic action ( Khodorova et al 2003 ). A further example is Schwann cells, which in normal nerves are intimately associated with axons: myelinating Schwann cells wrap around peripheral axons to form the myelin sheaths that facilitate axonal conduction. There are also non-myelinating Schwann cells, which in nerve fibers ensheathe small-diameter unmyelinated axons, usually in groups called Remak bundles ( Griffin and Thompson 2008 ). In the process of wallerian degeneration these cells de-differentiate and proliferate. They produce a variety of pro-algesic factors such as NGF ( Bandtlow et al 1987 , Heumann et al 1987 , Matsuoka et al 1991 ); cytokines such as TNF-α, IL-1β, and IL-6 ( Bolin et al 1995 , Shamash et al 2002 , Wagner and Myers 1996 ); and chemokines such as CCL2 ( Fu et al 2010 ). Such factors may act directly by sensitizing the remaining intact axons within injured nerves ( Sorkin et al 1997 ) and may also have a role in the recruitment of immune cells ( Tofaris et al 2002 , Perrin et al 2005 ), thereby contributing to the development of neuropathic pain.
Clearly, then, there is a large body of evidence showing that immune cells are important contributors to the development of multiple types of persistent pain. Immunosuppressive strategies, however, are in general not useful in treating pain because, of course, many aspects of inflammation are of use in promoting tissue repair. A more productive strategy is likely to be the identification of mediators produced by immune cells that lead to activation and sensitization of nociceptors. Below we consider the evidence for such specific mediators.

Immune Cell and Neurotrophic Factors as Pain Mediators and Modulators

Cytokines
One well-recognized consequence of inflammation is the production of various prostanoids, but the limited efficacy of NSAIDs that target COX enzymes—and therefore prostanoid production—strongly suggests a role for other inflammatory mediators. The inflammatory process, triggered by the recruitment of immunocompetent cells and the generation of free radicals, leads to the release of several other algogenic mediators. NGF is one such mediator, and the biology of this factor is discussed at some length below since anti-NGF therapies are now being tested in the clinic. TNF-α and IL-1β are two inflammatory cytokines that also contribute to inflammatory pain. Administration of small doses of TNF-α or IL-1β to adult animals and humans can produce pain and hyperalgesia that start within minutes in some cases and typically persist for several hours (see Watkins and Maier 2003 , McMahon and Cafferty 2004 , Sommer and Kress 2004 ). Both these factors are capable of activating and sensitizing peripheral nociceptive neurons and thereby contribute to ongoing pain and hyperalgesia. There is evidence demonstrating that neutralization or block of IL-1β and TNF-α is also effective in preventing abnormal pain behavior in some inflammatory pain models (see Sommer and Kress 2004 ). Antibodies against TNF-α and IL-1β are now in clinical use for the treatment of inflammatory arthritis and are proving very successful both in treating disease symptoms, including pain, and in modifying the course of the disease ( Fleischmann et al 2004 , Iannone et al 2007 , Laas et al 2009 ). The analgesic effects of blocking TNF-α are also seen in patients with osteoarthritis (OA) of the hand, for which they have been shown to significantly reduce spontaneous as well as pressure-evoked pain ( Fioravanti et al 2009 ).
Sensory neurons are known to express receptor components capable of transducing extracellular TNF-α ( Pollock et al 2002 ) and IL-1β ( Gardiner et al 2002 ). Intraneural injection of either TNF-α or IL-1β can induce both thermal and mechanical hyperalgesia ( Zelenka et al 2005 ), and blocking either of these factors peripherally following nerve injury attenuates such pain behavior ( Lindenlaub et al 2000 , Schafers et al 2003 , Kiguchi et al 2010 ). For TNF-α these effects seem to be mediated via TNFR1 and not TNFR2 ( Sommer et al 1998 ). In addition, intraplantar injection of TNF-α ( Cunha et al 1992 , Perkins et al 1994 ) or IL-1β ( Safieh-Garabedian et al 1995 , Amaya et al 2006 ) can induce hypersensitivity to both thermal and mechanical stimuli. These effects can be mediated directly on nociceptors; both TNF-α and IL-1β have been shown to increase the excitability of nociceptive neurons by enhancing TTX-resistant sodium channel currents via the activation of intracellular cascades involving p38 MAPK ( Jin and Gereau 2006 , Binshtok et al 2008 ). TNF-α can also enhance the sensitivity of TRPV1 to contribute to thermal hypersensitivity ( Nicol et al 1997 , Jin and Gereau 2006 ). Intriguingly, trimers of TNF-α have been reported to insert into membranes and form functional voltage-dependent sodium channels ( Kagan et al 1992 ), which may allow generalized sensitization of sensory neurons in the absence of functional TNF-α receptors. In addition to these direct actions on sensory neurons, it is clear that a large proportion of the algogenic effect of TNF-α and IL-1β is mediated via NGF. Neutralizing antisera or other molecules blocking NGF prevent the pain produced by these inflammatory cytokines. Mast cells also express trkA ( Horigome et al 1993 ) and in response to NGF proliferate, degranulate, and release inflammatory mediators, including TNF-α ( Woolf et al 1996 ). Because mast cells also release NGF, there is the possibility of a vicious circle of events promoting pain.
Leukemia inhibitory factor (LIF) and IL-6 both belong to a family of neuropoietic cytokines defined by their binding to the common receptor gp130. Other members include IL-11, ciliary-derived neurotrophic factor, oncostatin M, and cardiotrophin-1. LIF signals via a receptor complex of the LIF receptor-β and gp130 and is retrogradely transported by a population of small-diameter DRG cells ( Thompson et al 1997 ). Levels of LIF are normally very low. However, following nerve injury, LIF expression increases at the site of injury ( Banner and Patterson 1994 ). Nerve injury also results in a large increase in expression of the neuropeptide galanin within sensory neurons. Evidence from both animals deficient in LIF ( Sun and Zigmond 1996 ) and the administration of exogenous LIF ( Thompson et al 1998 ) indicates that this cytokine is responsible for up-regulation of galanin. LIF may also be implicated in the sprouting of post-ganglionic sympathetic neurons that occurs around DRG cell bodies following nerve injury ( Thompson and Majithia 1998 ).
The actions of LIF are not restricted to nerve injury but also extend to inflammatory conditions ( Banner et al 1998 ). LIF levels increase during inflammation. LIF knockout mice have an enhanced inflammatory reaction. Conversely, administration of exogenous LIF can attenuate both the hyperalgesia and the increased NGF expression that normally occur during inflammation. Confusingly, the effects of exogenous LIF may be dose dependent in that another study has found that administration of this factor to naïve animals may itself produce hyperalgesia ( Thompson et al 1996 ). Endogenous LIF, however, appears to have an interesting role as a mediator that suppresses the inflammatory reaction possibly at an early stage by negatively regulating the expression of IL-1β and NGF.
IL-6 can exert its biological effect through the binding of either a membrane-bound IL-6 receptor or a soluble receptor subunit, both of which need to form a complex with gp130 for signal transduction. Sensory neurons, which constitutively express gp130 ( Gardiner et al 2002 ), lack the IL-6 membrane receptor, and it is therefore likely that direct actions of IL-6 on these cells involve it and the soluble form of the receptor binding to a cell. Intraplantar injection of IL-6 induces a dose-dependent mechanical hyperalgesia ( Cunha et al 1992 ). Mice deficient in IL-6 show both reduced thermal and mechanical hyperalgesia following inflammation or nerve injury ( Xu XJ et al 1997 , Ramer et al 1998 ). By measuring release of CGRP, it seems that IL-6, in combination with its soluble receptor, can sensitize nociceptors in the skin to thermal stimuli ( Obreja et al 2002 ), and electrophysiological experiments have also reported that this cytokine can sensitize joint afferents to mechanical stimulation ( Brenn et al 2007 ). The effect of IL-6 on pain behavior could be direct since IL-6 can elicit calcium transients in around 33% of DRG neurons in vitro ( von Banchet et al 2005 ). This evidence is suggestive of a role of IL-6 as a peripheral pain mediator, and this notion has been highlighted recently by a study using an antigen-induced arthritis model. Here, local neutralization of the IL-6/soluble IL-6 receptor complex with soluble gp130 in the knee joint significantly attenuated mechanical hyperalgesia to a greater extent than did repeated systemic delivery (Boetteger et al 2010). Like IL-1β and TNF-α, therapies that block IL-6 are used clinically and have been shown to reduce pain scores in RA patients ( Smolen et al 2008 , Burmester et al 2011 ).
These same immune-related factors and others can also act as pain mediators in the CNS, and such actions are discussed in Chapter 4 .

Chemokines
Chemokines are chemotactic cytokines and have a key role in immune cell recruitment. They are small molecules (8–10 kDa) and are structurally related with four conserved cysteine residues. They signal via GPCRs, and a level of complexity is added by the fact that multiple chemokines may signal via one receptor. Like a number of cytokines, there is good preclinical evidence to suggest that some chemokines, particularly CCL2 and CX3CL1, are able to modulate pain processing at the level of the spinal cord. However, there is also evidence suggesting that this family of chemotactic cytokines can act as peripheral pain mediators. For example, inflammation or tissue injury can up-regulate numerous chemokine ligands, and the application of such factors can induce pain-related behavior (as highlighted in Table 3-2 and in Fig. 3-2 A). These actions can be achieved either through direct actions on nociceptive neurons or through the recruitment of immune cells (as shown in Fig. 3-2 B) and the subsequent release of other algogenic factors.

Table 3-2
Chemokines as Peripheral Pain Mediators
GENE NAME
FC IN RAT
FC IN HUMAN CXCL5 51.3 (20.5–128.2) * 82.5 (45.4–150.0) * iNOS 34.3 (3.1–385.1) ‡ −1.1 (−2.2–1.8) IL-24 32.7 (8.3–128.8) * 63.7 (44.5–91.3) * CXCL2 24.6 (3.1–198.4) * 12.0 (8.0–18.0) * CCL4 15.4 (6.6–35.8) * 2.5 (1.4–4.5) ‡ IL-6 14.8 (4.1–53.9) * 54.7 (30.3–99.0) * CCL2 14.6 (5.3–40.6) * 5.1 (3.8–7.0) * CCL7 14.2 (6.2–32.6) * 13.8 (4.2–44.8) * CXCL7 14.0 (2.8–70.5) § 4.0 (1.8–8.6) CCL11 11.6 (5.9–22.9) * 4.2 (1.1–16.6) IL-10 10.7 (5.4–21.2) * 8.0 (4.1–158) * IL-3 9.0 (3.4–23.3) § ND G-CSF 7.1 (−1.2–62.2) 25.0 (10.7–58.5) * IL-19 6.2 (3.0–12.6) § ND CCL3 6.0 (2.9–18.9) § 16.6 (10.7–25.7) * CXCL4 6.0 (3.9–9.2) * 2.1 (−1.1–5.0) KGF 5.8 (3.2–10.5) * 4.3 (2.9–6.5) * CXCL1 5.4 (1.9–15.5) § 18.9 (12.6–28.4) * IL-1β 5.0 (1.3–18.7) ‡ 10.3 (5.9–17.8) * COX-2 4.6 (2.0–10.6) § 5.3 (3.0–9.5) *
Numerous chemokines can be up-regulated by tissue injury or inflammation, and one such example is ultraviolet B (UVB) irradiation. In both human and rat skin the transcriptional expression of various chemokines is increased at the peak of UVB-induced hyperalgesia when compared with control skin.
* P < 0.001.
‡ P < 0.05.
§ P < 0.01; mean FC (±1 SD range).
From Dawes JM, Calvo M, Perkins JR, et al 2011 CXCL5 mediates UVB irradiation–induced pain. Science Translational Medicine 3(90):90ra60, Table 1.


Figure 3-2 Chemokines as peripheral pain mediators. A, When injected into naïve rats, the chemokine, CXCL5, was able to recapitulate ultraviolet B–induced mechanical hypersensitivity. B, In addition, the increase in mechanical sensitivity was associated with the infiltration of numerous neutrophils and macrophages into the chemokine-treated skin. (From Dawes JM, Calvo M, Perkins JR, et al 2011 CXCL5 mediates UVB irradiation–induced pain. Science Translational Medicine 3(90):90ra60, Fig. 3A–C.)
A number of chemokines have been shown to modulate peripheral pain pathways. This was first shown with the intraplantar injection of exogenous human CXCL8, also known as IL-8, which induced dose-dependent mechanical hyperalgesia in rats ( Cunha et al 1991 ). Interestingly, there is no direct rodent ortholog of this chemokine, but CXCL1 seems to elicit similar effects when given to naïve rats, and antiserum against it attenuates carrageenan-induced mechanical hyperalgesia ( Lorenzetti et al 2002 , Qin et al 2005 ). In terms of the influence that chemokines have in persistent pain states, the majority of work suggests a prominent role for the CCL2/CCR2 axis. CCL2 is up-regulated in peripheral tissues in neuropathic ( Perrin et al 2005 , Fu et al 2010 ) and inflammatory pain states such as intraplantar CFA injection ( Jeon et al 2008 ). Indeed, up-regulation in a number of different pain models was recently emphasized in a meta-analysis of micro-array studies ( LaCroix-Fralish et al 2011 ). When injected into the paw, CCL2 is able to elicit both thermal and mechanical hyperalgesia ( Abbadie et al 2003 , Qin et al 2005 , Bogen et al 2009 ). In addition to these findings, ablation of CCR2, the predominant receptor for CCL2, prevents the development of mechanical hyperalgesia following nerve injury ( Abbadie et al 2003 ). CCL2 therefore acts as a peripheral pain mediator in the setting of nerve injury and/or inflammation. Another closely related chemokine, CCL3, can produce pain-related hypersensitivity when applied peripherally ( Zhang et al 2005a , Eijkelkamp et al 2010 ) and is able to recapitulate neuropathic pain–like behavior when given intraneurally ( Kiguchi et al 2010 ). One possible mechanism by which chemokines may influence the perception of nociceptive input is via direct interaction with sensory afferents. A number of chemokines can directly act on DRG neurons, as seen with calcium imaging ( Oh et al 2001 ). These same chemokines were also able to induce pain-related behavior when injected into the paw. Subsequent to this work it was shown that CCL3 was capable of sensitizing TRPV1 on DRG neurons and that sodium currents in cultured sensory neurons could be enhanced by incubation with CXCL1 ( Wang et al 2008 ). In addition, CCL3 might also be able to desensitize opioid receptors on sensory neurons, thereby preventing the analgesic effects of endogenous opioids released following tissue injury ( Zhang et al 2004 ). The idea that chemokines can act directly on sensory neurons requires that appropriate receptors be expressed by these cells. Following nerve injury the chemokine receptors CCR2, CCR5, and CXCR4 can be expressed by DRG neurons, and cells in this condition have been shown to increase their responsiveness to a number of chemokine ligands, including CCL2, CCL5, CXCL10, and CXCL12 ( White et al 2005 ; Sun et al 2006 ; Bhangoo et al 2007 , 2009 ; Jung et al 2008 ). One study using in vivo electrophysiological techniques showed that after a chronic compression injury of the spinal nerve, DRG neurons were depolarized by CCL2 and lowered their threshold for activation with this ligand ( Sun et al 2006 ). Although this effect was clear in DRG neurons from injured animals, some responses were also measured in neurons from uninjured ganglia ( Sun et al 2006 ). Via an immunohistochemical approach in naïve rats, expression of both CCR1 and CXCR2 has been found in a high proportion of sensory neurons from naïve animals ( Zhang et al 2005a , Wang et al 2008 ). In addition, Oh and colleagues (2001) detected mRNA for CXCR4, CX3CR1, CCR4, and CCR5 in DRGs, as well as staining for CXCR4 and CCR4 in vitro. Therefore, a range of chemokines released by either resident or infiltrating cells into damaged tissue could act directly on nociceptor nerve terminals to enhance pain perception.
Chemokines may also act indirectly to induce pain-related hypersensitivity. For example, the pro-algesic actions of both CXCL8 and CXCL1 have been attributed to their ability to induce the release of sympathetic amines from resident cells ( Cunha et al 1991 , 1992 , 2005 ; Ben-Baruch et al 1995 ; Lorenzetti et al 2002 ), which can act to directly sensitize nociceptors. The majority of these indirect effects, however, are most likely to involve the recruitment of immune cells, cells that are known to infiltrate areas of damaged tissue. CXCL1-induced hyperalgesia is associated with neutrophil recruitment into the treated peripheral tissue ( Cunha et al 2008a , 2008b ). This chemokine attracts neutrophils predominantly through activation of the CXCR2 receptor. Systemic treatment with a CXCR1/2 inhibitor or specific antagonism of CXCR2 is able to attenuate the mechanical hyperalgesia induced by peripheral injection of carrageenan, zymosan, LPS, and CFA and that caused by nerve injury ( Cunha et al 2008a , Manjavachi et al 2010 ). These analgesic effects are associated with reduced neutrophil infiltration. The ultraviolet B (UVB) model of inflammatory pain is associated with both neutrophil and macrophage infiltration and up-regulation of numerous chemokines at the peak of both thermal and mechanical hyperalgesia ( Dawes et al 2011 ). Neutralization of one of the most overexpressed chemokines, CXCL5, which also acts via the CXCR2 receptor, was able to reduce the UVB-induced mechanical hyperalgesia and infiltration of immune cells. In addition, the pro-algesic properties of this chemokine in naïve animals involved the recruitment of both neutrophils and macrophages ( Dawes et al 2011 ).
A number of chemokines are up-regulated in injured peripheral nerves. One of these, CCL2, is particularly pivotal in the recruitment of macrophages ( Toews et al 1998 )—cells that seem crucial for the full development of neuropathic pain (see the previous section on immune cells; Liu et al 2000 , Barclay et al 2007 ). With the use of bi-transgenic reporter mice in a focal demyelination model of neuropathic pain it has been suggested that a large proportion of the pro-nociceptive effects of CCL2 occur in peripheral nerves because of its action on CCR2-expressing leukocytes ( Jung et al 2009 ). In this same model, disruption of this interaction with a CCR2 antagonist significantly attenuates neuropathic pain–like behavior ( Bhangoo et al 2007 ). CCL3 is also up-regulated in injured nerves ( Perrin et al 2005 , Kiguchi et al 2010 ). Local neutralization of CCL3 was able to attenuate both thermal and mechanical hyperalgesia following nerve damage, and this was associated with a reduction in the level of macrophages (Kiuchi et al 2010).

Resolution of Inflammation
The inflammatory cascade is under complex regulatory control, and regulatory factors include anti-inflammatory cytokines (IL-4, IL-10, IL-13, transforming growth factor [TGF-β]), promoters of resolution (lipoxins, neuroprotectins, maresins, resolvins), endocannabinoids, and inhibitors of pro-inflammatory signaling pathways (inhibitor of the nuclear factor NF-κB, complement inhibitors, IL-1R antagonist, co-stimulatory molecules, and MAPK phosphatases). These systems may be exploited to terminate inflammation, and a number of these mediators have been shown to have analgesic actions. An example is the resolvins, which are endogenous lipid mediators derived from ω-3 fatty acids. These factors promote resolution of inflammation through inhibition of leukocyte recruitment and can directly modulate sensory transduction and synaptic plasticity within the dorsal horn ( Serhan et al 2002 , Xu et al 2010 , Park et al 2011 ). They have been shown to have potent analgesic actions in inflammatory pain states ( Xu et al 2010 , Park et al 2011 ).

Neurotrophic Factors
In 1996 a study was published on the genetic basis of the congenital insensitivity to pain observed in a single family (Indo et al 1996). A mutation was identified in the gene encoding a tyrosine kinase receptor known as trkA. This protein is the high-affinity receptor for a single trophic factor, NGF, and the mutation disrupts the normal signaling of NGF. This single example provides a startling example of the importance of trophic factors in general and NGF in particular for normal nociceptive functioning. Several clinical trials have indicated the analgesic efficacy of blocking NGF, and we now have a good understanding of the mechanisms by which NGF interacts with pain-signaling systems, which we review here.
Neurotrophic factors can be defined as factors that regulate the long-term survival, growth, or differentiated function of discrete populations of nerve cells. There are many neurotrophic factors, and multiple factors can affect a single population of neurons. However, trophic factors fall into a smaller number of families, with members being related by high levels of structural homology or by the common or related receptors that they use in exerting biological actions. The number of factors identified as affecting sensory processing is limited. Most data relate to just two families of factors: the neurotrophin family and the GDNF-related family. Here we primarily consider one member of the neurotrophin family—NGF. Other members of the neurotrophin family are BDNF, neurotrophin-3, and neurotrophin-4/5 ( Gotz et al 1994 , Lindsay 1996 ). In general, these members share around 50% of their amino acid sequence. At physiological concentrations, the neurotrophins exist as homodimers. The neurotrophins are initially expressed as pre-pro-precursors, which when processed, yield highly basic mature proteins of around 13 kDa (120 amino acids). These pre-pro-precursors themselves might be biologically active, and currently there is much discussion whether some of the pro-forms of neurotrophins are secreted and act on specific receptors ( Teng et al 2010 ). Binding studies have demonstrated the presence of both high- and low-affinity binding sites for NGF on responsive cell lines ( Bothwell 1995 ). Two different classes of neurotrophin receptor have now been characterized (for reviews see Barbacid 1995 , Chao and Hempstead 1995 ). The first to be cloned was the p75 or low-affinity NGF receptor LNGFR, which binds all the neurotrophins more or less equally with relatively low affinity ( Chao et al 1986 ). Additionally, there is a family of high-affinity receptors, trks, that are tyrosine kinase receptors ( Kaplan et al 1991 ). The p75 receptor is thought to play several roles and may serve as the preferred receptor for pro-NGF. It can also interact with the trk receptors and modulate the specificity and sensitivity of neurotrophin interaction. There are three known members of the trk family of receptors, trkA, trkB, and trkC, and all show different specificities for the neurotrophins. NGF is the preferred ligand for trkA, BDNF and neurotrophin-4/5 are the preferred ligands for trkB, and neurotrophin-3 is the preferred ligand for trkC ( Ip et al 1993 ).
A great deal of the information about signal transduction following trk activation comes from the study of events following activation of trk by NGF in PC12 cells. After NGF binding, receptor dimerization occurs, which is critical for receptor activation ( Clary et al 1994 ). The tyrosine kinase domain of the receptor is activated and a number of substrates are phosphorylated; autophosphorylation of the receptor also occurs. There is now a large body of evidence demonstrating that neurotrophin receptors are expressed in specific populations of DRG cells. With double-labeling techniques it has been possible to relate receptor expression to different functional classes of DRG cells. Multiple approaches have demonstrated that approximately 40% of DRG cells express the NGF receptor trkA ( Verge et al 1989 , 1992 ; McMahon et al 1994 ; Averill et al 1995 ; Kashiba et al 1995 ; Molliver et al 1995 ; Wetmore and Olson 1995 ), and cells that express trkA are principally of small cell diameter. TrkA is expressed principally in the peptidergic population of small-diameter DRG cells, whereas very few non-peptidergic (isolectin B4-binding) small-diameter DRG cells express trkA ( Averill et al 1995 , Molliver et al 1995 ). Some of the myelinated DRG cells (i.e., those that express neurofilament 200) do express trkA (around 20%). TrkA-immunoreactive terminals within the spinal cord are present within laminae I and II outer . TrkA is therefore expressed in small-diameter DRG cells that express CGRP and project to the superficial laminae of the spinal cord. These are all characteristic of nociceptive afferents. Thus, about half of the nociceptors in adult animals express both p75 and trkA and are therefore likely to be sensitive to NGF. The other half of the nociceptor population does not express any trk receptor, nor p75. Rather, they express receptors for members of the GDNF family of receptors. Interestingly, these two populations of C fibers have different central terminations, even though it is not yet clear whether they have distinct functional roles (although the receptors that they express do indicate that they can be activated by different putative pain mediators).
During development it appears that functionally distinct groups of sensory neurons depend on different neurotrophins for survival. Animals with a gene deletion of either NGF or trkA are born with DRGs lacking virtually all small-diameter primary sensory neurons, including the peptidergic neurons expressing CGRP and substance P (Crowley et al 1994). These animals are, as expected, profoundly hypo-algesic. In utero deprivation of NGF, achieved by antibody treatment, produces similar effects ( Johnson et al 1980 , Ruit et al 1992 ), and this phenotype is equivalent to that seen in patients with loss-of-function mutations in trkA, which also leads to loss of peripheral pain-signaling neurons.
The developmental dependence of nociceptors on NGF for survival is lost in the postnatal period, some time before the second week of life in the rat and presumably in the first few years of life in humans. However, NGF continues to exert profound effects on adult nociceptors. Adult DRG neurons can be cultured in the absence of added trophic factors ( Lindsay 1988 ). If NGF is then added to these cultures, extensive neurite outgrowth of trkA-positive cells is promoted. NGF in these cultures also regulates expression of the neuropeptides substance P and CGRP ( Lindsay and Harmar 1989 ). In addition, NGF regulates the chemical sensitivity of cultured sensory neurons. For example, the sensitivity of cultured sensory neurons to the potent algogen capsaicin is increased by NGF, as is their sensitivity to protons and to γ-aminobutyric acid (GABA) ( Winter et al 1988 , Bevan and Winter 1995 ). Expression of bradykinin binding sites in cultured sensory neurons has also been shown to be regulated by NGF ( Petersen et al 1998 ), apparently in a p75 receptor–dependent manner. This marked regulation of the chemosensitivity of cultured sensory neurons by NGF is interesting in relation to the association between NGF and inflammatory pain, and the in vivo effects of NGF are discussed here.
The effects of NGF extend from the peripheral to the central terminals of sensory neurons, and many are mediated via altered gene expression in neurons expressing trkA. These effects are summarized in Figure 3-3 .


Figure 3-3 Summary of the biological effects of exogenous nerve growth factor (NGF) on pain-signaling systems in normal animals ( A ) and in animals with nerve injury ( B ). In normal animals, NGF causes peripheral sensitization of some nociceptors, in part directly as a result of NGF binding to receptors on nociceptors and in part indirectly by the release of algogens from other cell types. NGF also regulates the expression of many genes in trkA-expressing neurons, ranging from transmitters and modulators to ion channels and receptors. In the spinal cord, NGF produces central sensitization via altered expression of putative neuromodulators, particularly brain-derived neurotrophic factor (BDNF). CGRP, calcitonin gene–related peptide; TRPV1, transient receptor potential vanilloid 1.
Administration of small doses of NGF to adult animals and humans can produce pain and hyperalgesia. In rodents, thermal hyperalgesia is present within 30 minutes of systemic NGF administration and both thermal and mechanical hyperalgesia after a couple of hours (Lewin et al 1993). Subcutaneous injections of NGF also produce both thermal and mechanical hyperalgesia at the injection site. In humans, intravenous injections of very low doses of NGF produce widespread aching pain in deep tissues and hyperalgesia at the injection site ( Petty et al 1994 ). Detailed quantitative sensory testing in human volunteers has demonstrated long-lasting mechanical and thermal hypersensitivity following the intradermal injection of NGF ( Rukweid et al 2010 ). The rapid onset of some of these effects and their localization to the injection site strongly suggest that they arise, at least in part, from a local effect on the peripheral terminals of nociceptors. This has been substantiated by the observation that acute administration of NGF can sensitize nociceptive afferents to thermal and chemical stimuli ( Rueff and Mendell 1996 ). Cutaneous nociceptors chronically exposed to elevated NGF levels (in an NGF-overexpressing mouse) show marked heat sensitization ( Stucky et al 1998 ). Recordings of primary afferents innervating porcine skin following NGF application have demonstrated reduced activity-dependent slowing in mechanically insensitive afferents and increased ongoing activity, thus emphasizing the potential effects of NGF on axonal excitability ( Obreja et al 2011 ).
NGF produces sensitization of nociceptors by several mechanisms. Some of these mechanisms are direct (that is, they follow activation of trkA on nociceptors), and some are indirect and mediated by NGF inducing the release of other algogens from a variety of peripheral cell types. The direct mechanisms involve both altered gene expression and post-translational regulation of receptors and ion channels. There are now multiple examples of post-translational changes induced by NGF that involve phosphorylation of receptors and ion channels, although other actions are possible, such as altered trafficking of receptors. The heat sensitization of nociceptors induced by NGF is prominent and rapid. Phosphorylation of particular residues on TRPV1 receptors appears to account for most of the effect. However, the intracellular cascades responsible are disputed, with published data supporting a critical role for PKA, PKC, MAPK ERK1/2, or a mechanism involving inhibition of PIP 2 (see Bonnington and McNaughton 2003 ). NGF has also been shown to enhance mechanically activated currents in cultured sensory neurons ( Di Castro et al 2006 ). The post-translational modification of some ion channels, particularly TTX-resistant sodium channels, by NGF may likewise contribute to the sensitization of nociceptors by this agent (see Zhang et al 2002 and references therein).
Because cellular elements other than nociceptors in peripheral tissues express trkA, some of the sensitization of nociceptors by NGF may arise indirectly, and some of these elements have already been discussed. Mast cells contain a number of inflammatory mediators known to excite primary afferents, including histamine and serotonin ( Leon et al 1994 ), and some types of mast cells express trkA receptors ( Horigome et al 1994 ). NGF can produce mast cell degranulation ( Mazurek et al 1986 , Horigome et al 1994 ) and increase the proliferation of mast cells resident in tissue. In peritoneal mast cell cultures, NGF induces the expression of a number of cytokines ( Bullock and Johnson 1996 ). Mast cell degranulators and serotonin antagonists have both been demonstrated to partially prevent the thermal but not the mechanical hyperalgesia ( Lewin et al 1994 , Woolf et al 1996 ) that occurs in response to NGF. These degranulators can significantly reduce hyperalgesia (both thermal and mechanical) and the up-regulation of NGF expression induced by CFA ( Woolf et al 1996 ).
In skin, NGF may also affect keratinocytes, some of which express p75 receptors. NGF increases the proliferation of keratinocytes in culture ( Paus et al 1994 , Fantini et al 1995 ), and this process may contribute to tissue remodeling after inflammation. In addition, NGF may also target eosinophils and convert circulating eosinophils into tissue-type eosinophils ( Hamada et al 1996 ), and it has been reported to increase B- and T-cell proliferation ( Otten et al 1989 ), thus suggesting a role for NGF as an immunoregulatory factor.
There may be an interaction between NGF and sympathetic neurons during inflammation. Sympathetic efferents also possess the trkA receptor ( Smeyne et al 1994 ). Surgical or chemical sympathectomy can reduce the short-latency thermal and mechanical hyperalgesia evoked by NGF ( Andreev et al 1995 , Woolf et al 1996 ). Production of eicosanoids by sympathetic efferents within the skin has been suggested to contribute to hyperalgesia in some inflammatory conditions ( Levine et al 1986b ). However, a role for eicosanoids in NGF-induced hyperalgesia is unlikely since it is unaffected by treatment with the NSAID indomethacin ( Amann et al 1996 ).

Effects of NGF on Gene Expression and the Phenotypic Properties of Sensory Neurons
Not all the algogenic and hyperalgesic effects of NGF can readily be explained by peripheral processes. Some aspects of NGF actions are centrally mediated via altered gene expression in nociceptors. There appear to be a group of peptides that are constitutively expressed in trkA cells and whose expression is controlled mainly by NGF, with an increase following NGF supplementation and a decrease following NGF depletion (resulting, for instance, from peripheral axotomy). CGRP and substance P belong to this group ( Goedert et al 1981 , Otten et al 1984 , Verge et al 1995 ). Based on NGF’s ability to reverse some axotomy-induced increases in peptide, it would appear that there is also a group of peptides whose production is partly inhibited by neurotrophins; vasoactive intestinal peptide, cholecystokinin, neuropeptide Y, and galanin belong to this group. NGF also represses expression of the transcription factor ATF3 (Averill et al 2004). In addition to an effect on substance P and CGRP, NGF has been shown to produce a dramatic up-regulation of BDNF mRNA and protein in trkA-expressing DRG cells ( Apfel et al 1996 , Michael et al 1997 ). This is interesting since there is good evidence to suggest that BDNF may serve as a central regulator of excitability ( Pezet et al 2002 , Coull et al 2005).
NGF also regulates the expression of some of the receptors expressed by nociceptors. Capsaicin sensitivity is increased in vivo by NGF. NGF-sensitive nociceptors (i.e., those expressing trkA) have the highest levels of TRPV1 ( Tominaga et al 1998 ). Because TRPV1 is sensitive to heat and also to protons, regulation by NGF is likely to have consequences for the responsiveness of nociceptors to noxious stimuli. NGF can also positively regulate the expression of other ligand-gated ion channels in nociceptors, including several ASICs (Mamet et al 2003) and the ATP receptor P2X 3 . In addition, NGF may alter the excitability of sensory neurons by changing the expression of several voltage-gated ion channels, for instance, the sodium channel Na v 1.8 ( Black and Waxman 1996 , Boucher et al 2000 ). Because some forms of nociceptor sensitization appear to be mediated through this channel ( Gold and Levine 1996 ), this provides another potential mechanism by which NGF might regulate pain signaling.

Actions on Spinal Processing of Nociceptive Information: Central Sensitization
NGF given systemically fails to penetrate into the spinal cord. There is also little trkA expression in the spinal cord, with the receptor largely being restricted to the terminals of primary afferent nociceptors ( Averill et al 1995 ). One might therefore imagine that exogenously administered NGF would have little effect on spinal nociceptive processes. However, several of the biological effects of NGF described earlier are capable of leading to secondary effects on the spinal cord. First, activation and sensitization of primary afferent nociceptors may lead to sufficient afferent activity to trigger central changes. Second, the altered chemistry of afferent neurons produced by NGF may lead to increased neurotransmitter or neuromodulator release from nociceptive afferent terminals ( Malcangio et al 1997 ). Release of some sensory neuropeptides can contribute to the induction of central sensitization. One might therefore expect that peripheral NGF treatment could lead to the induction of central sensitization.
It has been shown that several hours after systemic NGF treatment, C-fiber stimulation produces greater than normal amounts of central sensitization, seen as wind-up of ventral root reflexes ( Thompson et al 1995 ). A fibers also develop the novel ability to produce wind-up. Such changes in the central processing of nociceptive information may occur during inflammation secondary to expression of substance P within A fibers ( Neumann et al 1996 ). There is considerable evidence that the ability of NGF to up-regulate BDNF expression in some nociceptors may prove to be the most significant mechanism by which NGF regulates the sensitivity of spinal processing of noxious stimuli.

Functional Role of NGF
Because mice with NGF or trkA deletions rarely survive past the first postnatal week, most of what we know about endogenous NGF function in the adult has been determined by the use of blocking agents. A number of studies have used autoimmune models of NGF deprivation or infusions of NGF antisera to study the effects of NGF in normal adult animals. Local infusion of trkA–IgG (an NGF-neutralizing reagent; Shelton et al 1995) into the rat hindpaw leads to thermal hypo-algesia and a decrease in CGRP content in DRG neurons projecting to the infused area ( McMahon et al 1995 ). These changes take several days to develop. In addition, there is a decrease in the thermal and chemical sensitivity of nociceptors projecting to the area and a reduction in epidermal innervation density ( Bennett et al 1998a ). These results provide strong evidence that NGF continues to play an important role in regulating the function of small, peptidergic sensory neurons in the adult.
By far the most extensively studied area of endogenous NGF function in the adult is in models of relatively persistent inflammatory pain (lasting at least several days). NGF is found in many cell types in tissues subjected to an inflammatory insult, and much evidence now supports the hypothesis that up-regulation of NGF levels is a common component of the inflammatory response that relates to hyperalgesia. Elevated NGF levels have been found in a variety of inflammatory states in humans, including in the bladder of patients with cystitis ( Lowe et al 1997 ), and increased levels are present in the synovial fluid of patients with arthritis ( Aloe et al 1992 ) and in the cerebrospinal fluid of fibromyalgia patients (Sarchielli et al 2007). In animal studies, NGF is found in the exudate produced during blistering of the skin ( Weskamp and Otten 1987 ) and is elevated in skin after inflammation induced by CFA ( Donnerer et al 1992 , Woolf et al 1994 ), IL-1β ( Safieh-Garabedian et al 1995 ), ultraviolet light ( Gillardon et al 1995 ), or TNF-α ( Woolf et al 1997 ).
There is now widespread agreement that blocking NGF can impede many of the effects of inflammation on sensory nerve function. For instance, intraplantar injection of carrageenan produces an acute inflammatory reaction, which has previously been widely used to study the analgesic effects of NSAIDs. When the trkA–IgG molecule was co-administered with carrageenan, it could almost completely prevent development of the thermal hyperalgesia that normally occurs ( McMahon et al 1995 ; Fig. 3-4 A). The properties of nociceptive afferents innervating carrageenan-inflamed skin have also been studied. Following carrageenan inflammation, there was a marked increase in spontaneous activity in these afferents and increased thermal and chemical sensitivity ( Fig. 3-4 B). This probably represents the multiple peripheral actions of NGF described earlier. Administration of the trkA–IgG molecule could largely prevent these changes ( Bennett et al 1996 ), and similar results have now been found in a number of different inflammatory models (see Pezet and McMahon 2006 ).


Figure 3-4 The role of nerve growth factor (NGF) in inflammation as revealed by use of the NGF-sequestering protein trkA–IgG. A, The thermal hyperalgesia that develops in rats in the hours following intraplantar carrageenan. The ordinate plots the ratio of the withdrawal times to radiant noxious heat applied to the inflamed paw and the non-inflamed contralateral paw. Most of the expected thermal hyperalgesia fails to develop in animals inflamed and concurrently treated with trkA–IgG. B, Effects of carrageenan inflammation on the properties of primary afferent nociceptors. Recordings were made from an isolated skin–nerve preparation a few hours after the inflammatory stimulus was given in vivo, and afferents were tested for their responses to a ramp increase in skin temperature. In inflamed skin, some nociceptors develop spontaneous activity and show thermal sensitization. In animals inflamed with carrageenan and concurrently treated with trkA–IgG, the thermal sensitization of nociceptors is completely blocked. The inset on the right shows the average stimulus–response functions for nociceptors in these groups. (After McMahon SB, Bennett DL, Priestley JV, et al 1995 The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule. Nature Medicine 1:774–780; and Koltzenburg M, Bennett DL, Shelton DL, et al 1999 Neutralization of endogenous NGF prevents the sensitization of nociceptors supplying inflamed skin. European Journal of Neuroscience 11:1698–1704.)
The increased NGF levels observed after inflammatory stimuli result from increased synthesis and release of NGF from a variety of cell types, including keratinocytes, smooth muscle cells, and Schwann cell ( Heumann et al 1987 , Raychaudhuri et al 1998, Freund et al 2002). IL-1β and TNF-α have been shown to drive changes in NGF expression during inflammation in vivo, and the hyperalgesia produced by these cytokines can be prevented by NGF antagonism.

Role of NGF in Clinical Pain States
Findings from the sequestration of endogenous NGF and the administration of exogenous NGF suggest that this factor is important in modulating the sensitivity of the sensory nervous system to noxious stimuli. The evidence that NGF levels increase during inflammation, which is derived from studies using NGF antagonism, makes a strong case for NGF being a critical mediator of inflammatory pain. NGF clearly has a powerful neuroprotective effect on small-diameter sensory neurons, and NGF levels have been shown to change in a number of models of nerve injury. The idea that NGF does act as a mediator in persistent pain states has recently been tested in a series of clinical trials with encouraging results. The persistent pain associated with OA has a strong peripheral drive and represents an ideal platform to test whether NGF may act as a peripheral pain mediator in this context. In a 16-week study of patients with OA of the knee, tanezumab, a humanized monoclonal antibody directed against NGF, given at 8-week intervals dose-dependently reduced the pain associated with walking ( Lane et al 2010 ). This effect lasted for the whole study period and was maximal with the highest dose, which on average reduced pain scores by about 75% ( Fig. 3-5 ). The analgesic effects of blocking NGF are not limited to OA. Tanezumab also significantly reduced pain scores in patients with lower back pain and pain arising from inflammation of the bladder ( Evans et al 2011 , Katz et al 2011 ). In addition, in patients with lower back pain, tanezumab outperformed the NSAID naproxen ( Katz et al 2011 ). These findings represent clear evidence of the important role that a signal mediator can play in patients with persistent pain. However The development of tanezumab and other anti-NGF antibodies for widespread use was halted because of adverse events observed in a phase II trial involving OA. Here, worsening OA developed in 16 tanezumab-treated patients and joint replacement therapy was required. The mechanisms behind these osteonecrotic events are unclear but potentially suggest a modulatory role of NGF on joint homeostasis. Alternatively, some level of hyponociception caused by excessive NGF neutralization may have resulted in accelerated progression of OA because of the overuse of damaged joints. However, it seems that in more than half the cases, the bone necrosis occurred in previously unaffected joints. Many other trials in which NGF was neutralized have not observed such severe adverse events, and it has been reported that repeated doses of tanezumab induce a favorable side effect profile ( Schnitzer et al 2011 ). The block on NGF clinical trials was lifted in early 2012, and it is likely that analgesic efficacy will now be examined in multiple clinical trials.


Figure 3-5 Neutralizing nerve growth factor in patients with osteoarthritis significantly reduces pain scores. At baseline, visual analog scale scores for patients were obtained to assess the level of pain experienced while walking. Patients then received either placebo or varying doses of tanezumab (10, 25, 50, 100, 200 μg/kg) at the start of the study and again after 8 weeks. A decrease in the score represents a reduction in pain scores. (Data from Lane NE, Schnitzer TJ, Birbara CA, et al 2010 Tanezumab for the treatment of pain from osteoarthritis of the knee. New England Journal of Medicine 363:1521–1531.)

Other Neurotrophic Factors As Peripheral Pain Mediators
In addition to NGF, preclinical data also suggest that other neurotrophic factors can act as peripheral pain mediators. The neurotrophins NT3 and BDNF both induce thermal hypersensitivity in rats when injected into the hindpaw, and following nerve injury, neutralization of BDNF in the periphery can reduce the increase in thermal hypersensitivity ( Theodosiou et al 1999 ). In the adult system the non-peptidergic nociceptive fibers lack trkA but instead express c-Ret, the prominent receptor for GDNF ( Snider and McMahon 1998 ). When injected into the paw of naïve animals, GDNF is reported to lower thermal pain–related thresholds ( Malin et al 2006 ). However, this factor might not act as a pro-algesic mediator in persistent pain states since its application to nerve-injured rats is analgesic ( Boucher et al 2000 ). Artemin, a member of the GDNF family of ligands, is also able elicit thermal hypersensitivity when given intradermally. In inflammation induced by CFA injection, artemin is greatly up-regulated at a transcriptional level in the skin in the first 24 hours (Malin et al 2007). It has also been observed that in genetically modified mice that overexpress artemin in the skin, sensitivity to both thermal and cold stimuli is increased ( Elitt et al 2006 ).

Conclusion
The number of inflammatory pain mediators and modulators has grown steadily and now includes not only a variety of small molecules such as bradykinin, prostanoids, ATP, protons, and NO but also numerous cytokines, chemokines, and growth factors. The number of sources of such mediators has also increased and includes several or many immune cells, glial cells, and neurons. Finally, it has become clear that these mediators have diverse mechanisms and sites of action, including activation and sensitization of nociceptive terminals, regulation of primary nociceptive phenotype, and in the spinal cord, presynaptic control of nociceptor transmitter release and post-synaptic control of neuronal excitability. One of the most critical issues, of course, is to identify the relative importance of all these different mediators and mechanisms in particular pain states. This may seem a difficult job given the known interaction of many inflammatory mediators. However, the success of one series of new agents, TNF-α function–blocking molecules, as both disease-modifying and pain-relieving agents in several autoimmune disorders, including RA, and also the promise shown by anti-NGF antibodies give hope that this increased understanding of basic mechanisms will translate into effective new therapies for painful disorders.
The references for this chapter can be found at www.expertconsult.com .

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Suggested Readings

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Chapter 4
Microglia
Critical Mediators of Pain Hypersensitivity after Peripheral Nerve Injury

Simon Beggs and Michael W. Salter


SUMMARY
Neuron–glial interactions are increasingly recognized as being key for physiological and pathological processes in the central nervous system. Microglia in the spinal dorsal horn respond to injury to peripheral nerves by adopting a specific response state characterized by up-regulation of the purinergic receptor P2X 4 . In this P2X4R + state, microglia release brain-derived neurotrophic factor, which disinhibits neurons in the spinal nociceptive processing network. The transformation in processing caused by signaling of P2X4R + microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans.


Introduction

Historical Perspective
The world of pain research and therapy owes a great deal to the little-known German anesthetist and surgeon Carl Ludwig Schleich (1859–1922) for two specific contributions. First, Schleich was a pioneer of regional anesthesia and refined the technique considerably by introducing a new, safer method of infiltration anesthesia, as detailed in his book Schmerzlose Operationen ( Painless Operations ) ( Schleich 1899 ). However, within that book are contained his theories of brain function, which make for remarkable reading. In what would be an extremely prescient proposal, Schleich rejected the accepted neural network concept of the time being championed by Sigmund Exner (1894) and suggested an active role for glial cells. It was while listening to a piano recital that he was struck with inspiration and announced “glia as a damper pedal, an apparatus for switching registers … an inhibition regulator” ( Schleich 1921 ). Schleich postulated that glial cells control neuronal excitation in the brain, a theory now widely held and of intense research interest throughout neuroscience. Schleich lived in less enlightened times; both he and his theory were ignored, and he suffered the final ignominy of being described as though his own brain were “turning into glue” ( Schleich 1921 )—a reference to the Greek word for glue being the etymological root of glia.
Study of the nervous system has been a story of controversy since the first revelations of the inner structure of this “black box” were revealed, pioneered by discovery of the reazione nera or “black reaction,” the revolutionary tissue-staining technique of Camillo Golgi in 1873. Golgi’s silver staining revealed a new world of cellular structures, and it was immediately clear that other structures, distinct from neurons, were present in the tissue samples. These non-neuronal structures had no place in the prevalent theory of the day and were put to rest by one of the foremost physiologists of the time, Rudolf Virchow, who had previously dismissed these cells as Nervenkitt, or “nerve glue” ( Virchow 1862 ). The glue cells were deemed to not contribute to the “physiological explanation of mental phenomena” ( Exner 1894 ) and were subsequently ignored.
Though recognized as structural elements in their own right, this was the time of the advent of the “neuron doctrine.” This was established by the great Spanish histologist and founder of modern neuroanatomy Santiago Ramon y Cajal, who posited the nervous system as being made up of discrete individual cells. It was in opposition to Golgi himself who had developed his reticular theory proposing that every neuron in the entire nervous system is physically linked with its neighbors (ironically, a system that has more resonance with the structure of astrocytes than neurons). Cajal’s improvement of Golgi’s pioneering staining techniques showed clear differentiation of neurons from neuroglia (now known as astrocytes and the source of Schleich’s fascination), but it also revealed a further population of cells that he termed the “third element” ( Andres-Barquin 2002 ). It was to be the work of Cajal’s student Pio del Rio Hortega to unravel the mystery of this third element ( Penfield 1965 , Rezaie and Male 2002 ). By further refining the metallic impregnation techniques of Cajal, he was able to successfully stain this cell population and in 1919 identify and define it as two distinct populations that he named microglia and oligodendroglia ( Penfield 1965 ). This was an extremely controversial claim at the time, and debate raged between Rio Hortega and Cajal about the nature of the third element and stimulated a surge of research on the function of these enigmatic cells. However, this interest soon dwindled, and by the mid-20th century, microglia were once again the neglected cell population in the central nervous system (CNS) ( Rezaie and Male 2002 ). Interest was sparked anew toward the end of the century with the realization that microglia are the resident macrophage population and therefore the immune effectors of the CNS, and in more recent years the role of microglia in particular in CNS function and malfunction has been revisited ( Hanisch and Kettenmann 2007 , Ransohoff and Perry 2009 , Graeber 2010 , Kettenmann et al 2011 ). Given their immune role, microglia represent the first line of defense against damage to the CNS, and with the understanding that peripheral neuropathy is manifested as a pathological state of the CNS ( Costigan et al 2009b , Woolf 2010 ), there is intense interest in their role in pain pathophysiology.

Microglia in the Modern Era of Neuroscience
Microglia are now known to derive from a distinct macrophage population that comes from embryonic myeloid progenitors in the yolk sac ( Ginhoux et al 2010 ), and they invade and populate the CNS through the pial membranes ( Cuadros and Navascués 1998 , Ginhoux et al 2010 ). Microglia have been likened to the electricians of the CNS; they exist outside the neuronal circuit and are able to delve in and modulate the electrical activity within ( Graeber 2010 ). Unlike the reticular-like syncytial system of astrocytes throughout the CNS, microglia are not physically connected but reside within their own adjacent, non-overlapping microdomains within the brain and spinal cord ( Fig. 4-1 ). Such a spatially restricted system allows the microglial response to react in an anatomically precise fashion after pathology or damage. Under physiological conditions, quiescent microglia are not “resting” but are in a state of motility and surveillance, with cellular processes continuously scanning their microenvironment ( Exner 1894 , Wake et al 2009 ).


Figure 4-1 Microglia and their microdomains in the intact spinal cord. Two photon photomicrographs of dorsal horn microglia taken from the intact in situ spinal cord of CX3CR1GFP mice. Top left: Extended focus image showing the grid-like distribution of microglia in the parenchyma of the normal spinal cord dorsal horn. Top right: Four microglia surface-rendered with Volocity (Perkin Elmer) software to show the adjacent, non-overlapping microdomains of individual cells. The bottom panels are three-dimensional projections of the top panels.
How this grid-like network of microglia that extends throughout the CNS interacts and modulates the underlying cellular circuitry of the CNS is of considerable interest as a fundamental cellular mediator underlying the pathophysiology of neuropathic pain. The extensive toolbox that microglia possess in terms of cytokines, chemokines, neurotrophins, and neurotransmitters has made this population of glial cells a rich seam of research, and a wealth of knowledge now exists and also remains to be discovered.

Spinal Microglia as Intermediaries in the Pathobiology of Neuropathic Pain
Considerable progress has been made since microglia were heralded as “sensors of pathology” ( Kreutzberg 1996 ), but it is important to reject the inflexible notion of microglial “activation” as a causative factor underlying peripheral nerve injury (PNI)-induced pain behavior. The classic morphological and proliferative responses of microglia within the spinal cord following PNI ( Fig. 4-2 ) are a signifier of microglia reactivity but do not necessarily constitute a “pro-pain” phenotype. A common misconception is that resolving these microglial signatures following nerve damage will resolve the behavioral changes. That being said, microglia in the spinal dorsal horn do respond to and are critical mediators of the pathobiology of PNI; all existing neuropathic pain models now envisage some requisite degree of spinal microglial response ( Beggs and Salter 2006 , 2007 ) ( Fig. 4-3 ). However, such microglial changes are less evident in inflammatory and chemotoxic models of pain ( Honore et al 2000 , Clark et al 2007 , Lin et al 2007 ), and the role of microglia in the pain states that result from these insults remains to be elucidated ( Li et al 2010 ). As stated above, microglia exist throughout the neuraxis, including at the level of primary afferents, spinal nociceptive circuitry, and projections to the brain. For a definitive causal role of microglia in pain to be stated, tests of both sufficiency and necessity must be proven.


Figure 4-2 Proliferation of spinal microglia after peripheral nerve injury. Spinal microglia proliferate around the central terminals of peripherally axotomized primary afferents. A, Iba1-immunostained microglia (red) are densely packed around the central terminals of primary afferents and retrogradely labeled with fluorescent cholera toxin B subunit (CTB; green). B, Two photon microscopic images of microglia in the intact in situ spinal cord of CX3CR1GFP mice. Proliferative and morphological changes can be seen by 24 hours after peripheral nerve injury.


Figure 4-3 The P2X4R+ microglial phenotype mediates a core pain hypersensitivity cascade following peripheral nerve injury. Chronic neuropathic pain is generated in part by pathological amplification of input to the nociceptive network of the central nervous system. A growing literature has established that neuron–glial interactions within the spinal cord are responsible, at least in part, for the enhanced output of this network. Some of the key molecular components of these interactions are summarized here. A specific microglial phenotypic state characterized by up-regulated P2X4R expression (P2X4R+) is induced by peripheral nerve injury and has been shown to play a critical role in the pathological changes in nociceptive processing that underlie neuropathic pain. The lower panel shows the complex modulation of P2X4R expression by various elements of the parenchymal environment: extracellular matrix (fibronectin; Tsuda et al 2008a , 2009c ), infiltrating T cells ( Costigan et al 2009a , Tsuda al 2009b ), and cytokines and chemokines ( Zhang et al 2007 , Abbadie et al 2009 , Clark et al 2009 , Biber et al 2011 , Toyomitsu et al 2012 ).

What are the Upstream Regulators of the Spinal Microglia Response to Peripheral Nerve Injury?
The initial critical event for changes in the spinal cord microglial phenotype following PNI is elicited in the injured primary afferents themselves. It is known that the activity of nociceptors is sufficient to elicit a microglial response inasmuch as electrical stimulation of a peripheral nerve at C-fiber intensity will induce microglial proliferation in otherwise naïve animals ( Hathway et al 2009 ). This microgliosis is contiguous with the onset of mechanical hypersensitivity in these animals. Electrical stimulation at such intensity will stimulate all fibers, and Suter and colleagues further refined this effect to show that blockade of transient receptor potential vanilloid 1 (TRPV1)-expressing afferents (the majority of C fibers) did not diminish the microglial proliferation whereas complete blockade of all discharge activity in the nerve with bupivacaine did ( Wen et al 2007 , Suter et al 2009 ). It is possible that the residual non–TRPV1-expressing C fibers are responsible for the microglial response. However, a more parsimonious explanation would suggest a role for A-fiber activity ( Suter et al 2009 ). Corroborating evidence for afferent discharge activity driving microglial changes comes from the observation that the discrete boundaries of microglial proliferation map the anatomical boundaries of the central terminal fields of the injured nerve ( Beggs and Salter 2007 ). Spinal microglia express receptors for many neurotransmitters ( Pocock and Kettenmann 2007 ) and are therefore ideally placed to respond to sustained release of pro-inflammatory neurogenic factors following PNI. However, these features are not evidence of a causative role for spinal microglial proliferation in the development or maintenance of neuropathic pain, and it is perhaps more pertinent to describe the proliferative microgliosis as a signifier of peripheral nerve damage than in terms of being explicitly algogenic. Indeed, early studies of microglial proliferation following PNI suggested that microglia were involved in the transganglionic degenerative response ( Graeber et al 1988 , Eriksson et al 1993 , Svensson et al 1994 ).

Interpreting Findings with so-Called Glial Inhibitors
An important consideration in studying the potential role of microglia—or other types of glial cells—in pain is the use of compounds that are widely touted as “glial inhibitors.” The compounds most commonly used for their glia-inhibiting activity are minocycline and propentofylline. It is important to note that these are broad-spectrum agents with anti-inflammatory properties. Although these compounds are often described as being glial-specific inhibitors, it is perhaps too narrow a description of the activity of these drugs. It has been shown that administration of minocycline and propentofylline is more effective in preventing than in reversing nerve injury–induced chronic pain behavior ( Raghavendra et al 2003a , 2003b ; Ledeboer et al 2005 ). One interpretation could be that microglia have only a transient role in neuropathic pain. An alternative is that these compounds are active elsewhere, for example, attenuating the discharge activity of primary afferents following injury ( Gong et al 2010 ). In either case, the clear conclusion is to exercise caution in attributing erroneous cellular specificity to these compounds. These most commonly used “glial inhibitors” also function by probably non-specific anti-inflammatory activity.

The Critical Role of Microglial P2X 4 Receptors in Peripheral Nerve Injury–Induced Pain Hypersensitivity
Chronic neuropathic pain is characterized by altered afferent input to the spinal cord and amplification of that input within the nociceptive network in the spinal cord at the segmental level ( Woolf and Salter 2000 ). Sensory processing within the dorsal horn involves a complexly organized network of local and descending inhibitory and excitatory modulation ( Costigan et al 2009b ). Generation of pathological pain arises from a distorted output from the spinal cord to higher areas of the CNS involved in sensory and affective processing. The “distortion” is achieved through the suppression of inhibition and enhancement of excitatory transmission ( De Koninck 2007 ; see Chapter 6 ). Conventional neurocentric bias has concentrated on neuron–neuron signaling underlying these effects, but there is now intense interest in a neuroimmune contribution, and the cell population that represents the immune side is microglia ( Beggs and Salter 2010 ). How do neuron–glial interactions contribute to the enhancement of nociceptive output? There is now a considerable canon of literature detailing a plethora of potential molecular links between neurons and microglia and their involvement in the pathogenesis of neuropathic pain ( Andres-Barquin 2002 , Inoue and Tsuda 2009 , Beggs and Salter 2010 , Gosselin et al 2010 , Calvo and Bennett 2012 ). However, converging lines of evidence currently point to enhanced expression of the purinergic receptor P2X 4 (P2X4R) as playing a key role in neuropathic pain pathophysiology (see Fig. 4-3 ). Although many molecules have been implicated in mediating the microglial changes underlying the initiation and/or maintenance of chronic pain states, it is the P2X4R-expressing microglial phenotype, its intra- and intercellular signaling pathways, and consequent transformation of spinal output that have been systematically verified and describe a causal role of spinal microglia in the development of PNI-induced chronic pain ( Tsuda et al 2003 , 2008a ; Coull et al 2005 ; Ulmann et al 2008 ; Trang et al 2009 ; Beggs et al 2012 ).
The initial series of observations that identified P2X4Rs as a critical molecular element of the neuroglial signaling underlying neuropathic pain came from Tsuda and colleagues (2003) . The first indicator was a progressive increase in P2X4R protein in the ipsilateral spinal cord of rats with a PNI. Moreover, this increase correlated with the emergence of tactile allodynia ( Tsuda et al 2003 ). Of considerable surprise at the time, it was revealed immunohistochemically that increased expression of P2X4Rs was confined to microglia. An ongoing problem with parsing the actions of P2X subtypes has been the lack of functional pharmacological tools ( Jarvis and Khakh 2009 ). However, exploitation of the differences in pharmacological profiles of the P2X antagonists TNP-ATP and PPADs, the former reversing tactile allodynia in neuropathic rats and the latter having no effect, indicated that P2X4R was the active receptor mediating the pain behavior. Furthermore, because the behavioral allodynia could be transiently reversed by intrathecal administration of the antagonist, it could be surmised that ongoing P2X4R activation is required to maintain nerve injury–induced allodynia. The demonstration that P2X4R antisense oligonucleotide treatment had a similar action ( Tsuda et al 2003 ) provided further confirmation. The pharmacological and behavioral evidence was subsequently corroborated by a genetic approach: mice deficient in P2X4R have dramatically reduced pain behavior following PNI ( Ulmann et al 2008 , Tsuda et al 2009a ). This latter study also contained the surprising revelation that the proliferative response of microglia to PNI was undiminished in P2X4R −/− mice yet their behavioral responses were absent. In other words, although tonic P2X4R activation is required for maintenance of PNI-induced allodynia, proliferation and up-regulation of microglial markers in the spinal dorsal horn are independent. These experiments demonstrated the necessity of P2X4Rs as an active component in neuropathic pain but do not preclude the possibility of intermediary factors being required (i.e., sufficiency had not been demonstrated). This was definitively shown in experiments in which P2X4R-stimulated microglia were injected intrathecally into naïve rats and induced tactile allodynia similar to that seen in neuropathic rats ( Tsuda et al 2003 , Coull et al 2005 ). The pharmacological, genetic, and behavioral battery of experiments provided the requisite evidence to show sufficiency and necessity of P2X4Rs in mediating neuropathic pain behavior in rats and therefore logically a causative role. The question then turned to the effectors and effects of P2X4R up-regulation in the development and maintenance of neuropathic pain.

Modulators of P2X4R Expression and Function
For microglia resident within the protected confines of the spinal dorsal horn parenchyma to contribute to altered spinal output to the brain following peripheral nerve damage, there must be a signaling event or events between the injured primary afferent and the spinal environment. If a key central component of the neuroglial signaling pathway is P2X4R up-regulation in spinal microglia, what signals that up-regulation? There have been a number of advances recently that address this question, and several signaling molecules have been implicated, including members of the chemokine, cytokine, extracellular matrix molecule, and protease families. These include CCL21, a neuronally released chemokine ( de Jong et al 2005 , 2008 ; Biber et al 2011 ). Importantly, the authors showed this signaling event to be dependent on neuronal injury, and crucially, the details of transport of CCL21 from the neuronal to the microglial environment and signals via the chemokine receptor CXCR3 ( de Jong et al 2005 ) that precede P2X4R up-regulation have been elucidated. Of further interest to future translational studies, it has also been demonstrated that the same signaling event occurs with the human chemokine homologues ( Dijkstra et al 2004 ). The cytokine interferon-γ has been shown to transform quiescent spinal microglia into a P2X4R-expressing phenotype ( Tsuda et al 2009b ). P2X4R expression levels seem to be critically dependent on the extracellular matrix molecule fibronectin ( Nasu-Tada et al 2006 ; Tsuda et al 2008a , 2009c ). Further studies revealed the Lyn kinase signaling pathway as mediating this event, in turn modulating the transcriptional and post-transcriptional levels of microglial P2X4R expression ( Tsuda et al 2008b ). Mast cells have also been shown to modulate P2X4R expression through release of the protease tryptase, which activates proteinase-activated receptor 2 (PAR-2) in microglia ( Yuan et al 2010 ). Several other signaling pathways have been described that involve a primary afferent–microglial signaling component. These pathways include CCL2 (also known as monocyte chemoattractant protein 1 [MCP-1]), which is expressed on primary sensory neurons and is present in their central terminals and whose cognate receptor is present on microglia ( Zhang et al 2007 , Abbadie et al 2009 ), activation of which promotes membrane expression of P2X4Rs ( Toyomitsu et al 2012 ). Whether these examples represent converging pathways that are mechanistically intertwined or else exist alone as independent signaling pathways remains to be resolved.

Cell Biology of Microglial P2X4R Expression and Function
Although mechanistically relevant changes in receptor expression are often couched in the simplistic language of up- and/or down-regulation, the functionally meaningful activity of receptors such as P2X4R occurs on the cell surface, regulated by constitutive internalization and reinsertion of the receptors into the cell membrane ( Bobanovic et al 2002 , Toulmé et al 2006 , Fujii et al 2011 ). Much is known of P2X4R dynamics within the cell (which is beyond the scope of this review), but of considerable importance has been elucidation of the crystal structure of zebra fish P2X4R and subsequent details of the extracellular domain, putative adenosine triphosphate (ATP) binding site, transmembrane regions, and ion permeation pathway ( Kawate et al 2009 ). Of potentially greatest importance with respect to P2X4R signaling is discovery of the ability of the receptor to adopt two distinct structural conformations. Using fast-scanning atomic force microscopy, Shinozaki and colleagues (2009) showed that ATP stimulation in the presence of extracellular Ca 2+ causes the P2X4R to open a non-selective cation-permeable channel but that in the absence of extracellular Ca 2+ , the receptor undergoes pore dilatation and forms a macropore, which renders the receptor permeable to larger molecules. Importantly, microglial P2X4Rs have been shown to possess this ability to function in both conformations ( Bernier et al 2008 ). Clearly, the implications for enhanced signaling capability, especially given the phenotypic change to high P2X4R expression following PNI, are enormous. However, the question of whether this function is physiologically relevant in the etiology of neuropathic pain remains unanswered.

p38 Mitogen-Activated Protein Kinase Mediates Microglial Signaling
A critical question for microglial signaling mechanisms is what are the intracellular pathways that mediate the myriad molecular systems? Does convergence account for the parallel signaling events occurring through activation of different populations of receptors? Considerable evidence has identified the mitogen-activated protein kinases (MAPKs) as a candidate family of intracellular mediators ( Ji and Suter 2007 , Ji et al 2009 , Wen et al 2009 ). Of the MAPKs, p38 appears to be heavily implicated in microglia-mediated, post–nerve injury pain states ( Jin et al 2003 , Tsuda et al 2004 ) and has been identified as the intracellular mediator of P2X4R–brain-derived neurotrophic factor (BDNF) signaling in microglia ( Trang et al 2009 ).

P2X4R-Mediated Release of Brain-Derived Neurotrophic Factor
Parallel or converging pathways from a variety of cellular and molecular substrates regulate and influence microglial P2X4R expression. Considerable evidence points to p38 as a common intracellular mediator (see Fig. 4-3 ). The step from altered primary afferent input to spinal microglia modulation has been bridged. However, it is pertinent to reiterate that given the considerable interplay between neuronal and glial components at the spinal level, ultimately it is neuronal signaling, via spinal projection neurons to centers in the brain stem and brain, that completes the necessary “geography” of the pathway that ultimately leads from nerve injury in the periphery to “pain” in the brain. Again, it should be reiterated that a common misconception is to imbue altered microglial activity in the spinal dorsal horn with a causative role. This is too broad an interpretation and skews the logic of correlation and causation. A signaling component from microglia to second-order dorsal horn neuron is required to complete the circuit. In spinal microglia, influx of Ca 2+ through the P2X4R is a critical step that fulfils this requirement by linking stimulation of these receptors to the synthesis and release of BDNF ( Trang et al 2009 ). In vivo, P2X4R-deficient mice exhibit impaired microglial BDNF release and altered BDNF signaling in the spinal cord ( Ulmann et al 2008 ), and given the disruption of the P2X4R–BDNF pathway, mechanical allodynia does not develop in P2X4R null mice following PNI. Where the microglial transfer experiments earlier satisfied the conditions of sufficiency of microglial-derived BDNF to drive neuropathic pain behavior, studies by Ulmann and associates demonstrated that release of BDNF, driven by activation of P2X4R , is necessary for the development of neuropathic pain. Microglia as a source of BDNF within the spinal cord following PNI was countercurrent to contemporary thinking. Even though BDNF was considered a probable mediator of central cellular processes involved in pain states, primary afferents were the default source. Although a number of studies showed promising changes in expression of BDNF in spinal ganglia following PNI ( Obata et al 2003a , 2003b ; Pezet and McMahon 2006 ), it was subsequently demonstrated that primary afferent-evoked release of BDNF in the spinal cord was unaltered after nerve injury ( Lever et al 2003 ). Convincing support came from a conditional knockout mouse study in which selective deletion of BDNF from primary afferent neurons resulted in no effect on nerve injury–induced mechanical allodynia (but strong suppression of pain behavior in a number of inflammatory pain models) ( Zhao et al 2006 ). This presented something of a conundrum until a series of experiments provided the first evidence that in neuropathic pain states, it is indeed BDNF that affects nociceptive processing in the spinal cord but that the source of the BDNF was from microglia ( Coull et al 2005 ).
Previous studies had proposed that a central mechanism underlying pain behavior was disruption of anion homeostasis manifested as a depolarizing shift in the equilibrium potential of γ-aminobutyric acid (GABA)-mediated chloride currents ( Coull et al. 2003 ). This disruption, which affects the intrinsic circuitry of the spinal dorsal horn, results in a weakening of inhibitory tone within the nociceptive circuitry of the spinal cord. The underlying molecular mechanism was shown to be a rapid nerve injury–induced down-regulation of the neuronal chloride transporter KCC2 ( Coull et al 2003 , Prescott et al 2006 ). It was known that BDNF can down-regulate KCC2 levels under pathophysiological conditions in the hippocampus ( Rivera et al 2002 , 2004 ), therefore raising the possibility of a mechanistic link between P2X4R-dependent BDNF release and spinal KCC2 down-regulation. It was subsequently shown that intrathecal administration of P2X4R-stimulated microglia into naïve, uninjured rats causes a depolarizing shift in E anion in spinal lamina I neurons that was sufficient to reduce inhibition and, furthermore, that in approximately one-third of neurons, GABAergic responses became excitatory. The consequence of this altered inhibitory response, as will be described later, is to produce a phenotypic switch in spinal lamina I neurons such that they relay innocuous mechanical input, increase discharge when presented with a noxious stimulus, and display spontaneous activity ( Coull et al 2005 , Keller et al 2007 ). The signaling role of BDNF in this observation was shown by a series of findings. First, intrathecal BDNF mimicked both the mechanical allodynia and depolarized E anion caused by PNI or administration of P2X4R-stimulated microglia. Second, blocking BDNF-TrkB (tyrosine kinase receptor B) signaling either with a function-blocking antibody or by sequestration of free BDNF with TrkB–Fc fusion proteins prevented the mechanical allodynia evoked by P2X4R-stimulated microglia. Third and crucially, knockdown of BDNF expression in microglia with small interfering RNA (siRNA) abolished the effects of intrathecally administered ATP-stimulated microglia and resulted in an absence of both depolarized E anion and pain behavior. Taken together, these findings provide compelling evidence that BDNF from microglia is a critical signaling molecule mediating the central pathophysiological effects of PNI.

Transformation of Lamina I Output May Underlie Symptoms of Neuropathic Pain
The final stage in nociceptive processing within the dorsal horn involves the transmission of sensory information from the spinal segmental level rostrally to higher centers in the CNS. There are broadly two populations of dorsal horn neurons, located in lamina I and lamina V of the dorsal horn, that project to the brain stem and thalamus and provide a nociceptive output pathway from the spinal cord. Lamina I neurons differ from their deeper counterparts in that they receive limited direct input from low-threshold A fibers under normal conditions. This nociceptive bias and specificity (in control animals, less than 25% of lamina I projection neurons respond to low-threshold innocuous stimuli; Keller et al 2007 ) therefore ensures that in the normal situation, lamina I neurons preferentially encode noxious and thermal input only ( Bester et al 2000 ). However, tactile allodynia essentially requires that innocuous input elicit a nociceptive response. Because lamina I projection neurons are effectively nociceptive-specific output neurons, a mechanism is required that allows these neurons to become responsive to innocuous peripheral stimulation and elicit a noxious sensation supraspinally. Keller and co-workers (2007) showed that PNI causes a functional switch in lamina I projection neuron specificity such that the majority of the cells recorded, rather than being nociceptive specific, now responded to low-threshold tactile stimulation. The mechanism for this change has been the cause of some debate. It may represent an unmasking of polysynaptic connectivity within the dorsal horn such that low-threshold input can functionally activate lamina I projection neurons ( Baba et al 2003 , Kohno et al 2003 ) by either strengthening them, reducing their inhibition, inducing excitatory interneuron input, lowering the threshold of excitation of projection neurons via pre-existing subthreshold input, or switching the action of a subpopulation of inhibitory interneurons from inhibitory to excitatory ( Coull et al 2003 ). Compelling evidence for the latter comes from the observation that acute disruption of chloride homeostasis in naïve animals in vivo switches the phenotype of identified lamina I projection neurons from nociceptive specific to wide dynamic range, in essence unmasking innocuous afferent input to the nociceptive spinal circuitry ( Keller et al 2007 ). More pertinently, the same effect could be mimicked by acute spinal application of ATP-stimulated microglia ( Keller et al 2007 ). This finding is the principal evidence that microglia functionally affect the output characteristics of the spinal cord nociceptive network to higher centers in the CNS, a requirement for a causal role of microglia in the ontogeny of nerve injury–induced pain states ( Fig. 4-4 ).


Figure 4-4 Transformation of lamina I output neurons reveals the symptoms of neuropathic pain. A, Recording of single antidromically identified (1) lamina I projection neurons (2) in vivo. Neuronal responses to natural stimuli (brush, touch, pinch) (3) were made before and after adenosine triphosphate (ATP)-stimulated microglia were acutely administered into the spinal cord (4) ( Keller et al 2007 ). B, The top panel shows the transformation of a single lamina I projection neuron from nociceptive specific (NS; no responses to innocuous tactile stimuli) to wide dynamic range (WDR; emergence of responsivity to tactile stimuli) after acute exposure of the spinal cord of an anesthetized rat to ATP-stimulated microglia. These changes mimic those seen following peripheral nerve injury (PNI). The lower panel shows a reversal in the relative proportions of NS and WDR phenotypes of a population of identified lamina I projection neurons after either PNI or acute microglial exposure. C, Lamina I projections in naïve animals are generally quiescent and show little or no activity. However, following PNI or microglial treatment, these neurons start to fire spontaneously. The responsivity of previously NS neurons to innocuous stimuli and the development of spontaneous activity may represent physiological correlates to the allodynia and spontaneous pain experienced by patients with neuropathic pain.
Preclinical models of pain typically use evoked responses as behavioral readouts of mechanical and thermal sensitivity. However, clinically, neuropathic pain is characterized by ongoing spontaneous pain, as well as exaggerated evoked pain responses ( Baron et al 2010 ). Such a sensation requires ongoing, or episodic, activity at some point in the nociceptive pathway in the absence of an aberrant stimulus. Lamina I projection neurons are quiescent in the absence of nociceptive input ( Craig and Kniffki 1985 , Keller et al 2007 ) and display no spontaneous activity. However, following PNI, epileptiform-like burst activity can be seen in these neurons. This same activity can be reproduced in naïve animals by direct disruption of chloride homeostasis or by the acute spinal application of ATP-stimulated microglia ( Keller et al 2007 ). These observations of changes in the response properties, selectivity, and discharge activity of spinal cord lamina I neurons may provide a biologically plausible mechanism for the cardinal symptoms of neuropathic pain: hyperalgesia, allodynia, and spontaneous pain.

Conclusion
Microglia are able to respond in different ways to different stimuli and adopt an appropriate phenotype for a given stimulus. This phenotypic diversity includes proliferative, migrational, and phagocytic responses associated with a canon of expression of pro- and anti-inflammatory molecules. These responses are in addition to the immune role of microglia in antigen presentation and T-cell recruitment. The degree to which these phenotypes represent extant responses with discrete molecular signaling components is not clear, but a detailed picture of the molecular complexity involved is emerging ( Hanisch and Kettenmann 2007 ). The most important message is that microglial responses are complex and tuned to the nature of the stimulus. A common conceptual error in the role of glial responses in mechanistic studies of neuropathic pain is too broad an interpretation being drawn from these responses. Given the multimodal capability of microglia to respond to peripheral nerve damage, correlative and causative conclusions are often muddled. Advances in understanding the microglial molecular machinery have only highlighted the dearth of precise tools required to identify the specific processes and specific cell populations involved in the etiology of neuropathic pain. Furthermore, the possibility of over-interpretation through experimental design imprecision remains ever present. It is still the case that quantification and analysis of microglial changes in the literature are generally limited to a very small number of cellular proteins (e.g., iba1, ox42). Even though antibodies against these proteins generally have a high degree of sensitivity and specificity, thereby providing useful immunohistochemical tools for visualizing changes in morphology and cell density, it is unknown whether these proteins are surrogates for any specific functional change in microglial activity relevant to pain or any other function. On the contrary, any phenotypic diversity within a proliferating microglial population in pathological states would be masked by the homogenizing labeling with these markers. Sophisticated optical and genetic techniques allow imaging of microglia in vivo, thus providing insight into the morphological and motile reactivity of microglia in the living animal ( Exner 1894 , Davalos et al 2005 ). Yet it is ironic that although the great controversy between the discoverer of microglia Rio Hortega and his mentor Cajal was based on histological clarity, both literally and figuratively, it is the histological evaluation of microglial morphology that remains one of the foremost signifiers of pathological processes in the CNS.
The incredible complexity of microglia activity and responsivity may represent the adoption of stimulus-specific phenotypes. Any potential therapeutic intervention without defined specificity, such as a general glial inhibitor, is not necessarily desirable and, given their role in the immune system, potentially catastrophic. A case in point may be a recent report on propentofylline, purported to be a non-specific glial inhibitor, which was found to not affect pain intensity in patients with long-standing post-herpetic neuralgia ( Landry et al 2012 , Watkins et al 2012 ). Although the reason for this lack of efficacy is unclear, it may be that this drug did not suppress the requisite specific glial pathway. Whether glia play a role in clinical pain remains an open question. However, we anticipate that any advance in tackling chronic pain therapeutically by targeting microglia will need to involve the development of molecules or compounds capable of targeting a specific microglial phenotype with the ability to reverse, as well as prevent, the adoption of that specific phenotype. It is important to also consider the important roles that microglia have within the immune system of the non-pathological CNS and the potential deleterious effects of inhibiting their function. However, the remarkable plasticity and phenotypic diversity of microglia offer a compelling opportunity to identify the molecular mediators of stimulus-dependent microglial changes and to tailor therapies targeted at them.
It has been more than a century since Carl Gustav Schleich proposed a non-neuronal “switching mechanism which organized and regulated the ebb and flow of nervous excitation” ( Schleich 1921 , Anonymous 2006 ). The full impact of that statement is only now beginning to be fully realized.
The references for this chapter can be found at www.expertconsult.com .

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Suggested Readings

Abbadie C., Bhangoo S., De Koninck Y., et al. Chemokines and pain mechanisms. Brain Research Reviews . 2009;60:125–134.
Beggs S., Trang T., Salter M.W. The P2X4R + microglial state: an essential role in neuropathic pain. Nature Neuroscience . 2012. (In press)
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Clark A.K., Yip P.K., Malcangio M. The liberation of fractalkine in the dorsal horn requires microglial cathepsin S. Journal of Neuroscience . 2009;29:6945–6954.
Costigan M., Moss A., Latremoliere A., et al. T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain–like hypersensitivity. Journal of Neuroscience . 2009;29:14415–14422.
Coull J.A.M., Beggs S., Boudreau D., et al. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature . 2005;438:1017–1021.
Coull J.A.M., Boudreau D., Bachand K., et al. Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature . 2003;424:938–942.
Davalos D., Grutzendler J., Yang G., et al. ATP mediates rapid microglial response to local brain injury in vivo. Nature Neuroscience . 2005;8:752–758.
Hanisch U.-K., Kettenmann H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nature Neuroscience . 2007;10:1387–1394.
Hathway G.J., Vega-Avelaira D., Moss A., et al. Brief, low frequency stimulation of rat peripheral C-fibres evokes prolonged microglial-induced central sensitization in adults but not in neonates. Pain . 2009;144:110–118.
Jarvis M.F., Khakh B.S. ATP-gated P2X cation-channels. Neuropharmacology . 2009;56:208–215.
Ji R.-R., Gereau R.W., 4th., Malcangio M., et al. MAP kinase and pain. Brain Research Reviews . 2009;60:135–148.
Jin S.-X., Zhuang Z.-Y., Woolf C.J., et al. p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. Journal of Neuroscience . 2003;23:4017–4022.
Keller A.F., Beggs S., Salter M.W., et al. Transformation of the output of spinal lamina I neurons after nerve injury and microglia stimulation underlying neuropathic pain. Molecular Pain . 2007;3:27.
Kettenmann H., Hanisch U.-K., Noda M., et al. Physiology of microglia. Physiological Reviews . 2011;91:461–553.
Lin T., Li K., Zhang F.-Y., et al. Dissociation of spinal microglia morphological activation and peripheral inflammation in inflammatory pain models. Journal of Neuroimmunology . 2007;192:40–48.
Pocock J.M., Kettenmann H. Neurotransmitter receptors on microglia. Trends in Neurosciences . 2007;30:527–535.
Prescott S.A., Sejnowski T.J., De Koninck Y. Reduction of anion reversal potential subverts the inhibitory control of firing rate in spinal lamina I neurons: towards a biophysical basis for neuropathic pain. Molecular Pain . 2006;2:32.
Ransohoff R.M., Perry V.H. Microglial physiology: unique stimuli, specialized responses. Annual Review of Immunology . 2009;27:119–145.
Rivera C., Li H., Thomas-Crusells J., et al. BDNF-induced TrkB activation down-regulates the K + -Cl − cotransporter KCC2 and impairs neuronal Cl − extrusion. Journal of Cell Biology . 2002;159:747–752.
Suter M.R., Berta T., Gao Y.-J., et al. Large A-fiber activity is required for microglial proliferation and p38 MAPK activation in the spinal cord: different effects of resiniferatoxin and bupivacaine on spinal microglial changes after spared nerve injury. Molecular Pain . 2009;5:53.
Tsuda M., Kuboyama K., Inoue T., et al. Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays. Molecular Pain . 2009;5:28.
Tsuda M., Masuda T., Kitano J., et al. IFN-gamma receptor signaling mediates spinal microglia activation driving neuropathic pain. Proceedings of the National Academy of Sciences of the United States of America . 2009;106:8032–8037.
Ulmann L., Hatcher J.P., Hughes J.P., et al. Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain. Journal of Neuroscience . 2008;28:11263–11268.
Wen Y.-R., Suter M.R., Kawasaki Y., et al. Nerve conduction blockade in the sciatic nerve prevents but does not reverse the activation of p38 mitogen-activated protein kinase in spinal microglia in the rat spared nerve injury model. Anesthesiology . 2007;107:312–321.
Woolf C.J., Salter M.W. Neuronal plasticity: increasing the gain in pain. Science . 2000;288:1765–1769.
Zhang J., Shi X,., Echeverry S., et al. Expression of CCR2 in both resident and bone marrow–derived microglia plays a critical role in neuropathic pain. Journal of Neuroscience . 2007;27:12396–12406.
Chapter 5
Neuroanatomical Substrates of Spinal Nociception

Andrew J. Todd and H. Richard Koerber


SUMMARY
The spinal dorsal horn receives input from a wide variety of primary afferent axons, including nociceptors , which respond to tissue-damaging stimuli from the skin, muscles, joints, and viscera. The patterns of termination of primary afferents within the spinal cord are related to axonal diameter and receptive field modality. Most nociceptive primary afferents have slowly conducting fine myelinated or unmyelinated axons , and they terminate mainly in the superficial part of the dorsal horn. Primary afferents release a variety of chemical mediators, but all use glutamate as their principal neurotransmitter, and on entering the dorsal horn they form excitatory synapses with neurons located within it. These neurons include projection cells with axons that convey information to various parts of the brain and interneurons with axons that remain in the spinal cord and contribute to local neuronal circuits. Interneurons make up the great majority of the neuronal population in the dorsal horn and can be divided into two main functional classes: inhibitory interneurons, which use γ-aminobutyric acid (GABA) and/or glycine as a transmitter, and excitatory interneurons, which are glutamatergic. The organization of interneurons in the dorsal horn is very complex, and we still know little about the neuronal circuits in which they take part. Intrathecal administration of GABA A or glycine receptor antagonists can cause allodynia, in which brushing of the skin becomes an aversive stimulus. This suggests that one function of inhibitory interneurons is to suppress activity evoked by tactile afferents so that it is not perceived as painful.
The dorsal horn also receives input from descending axons that originate in various parts of the brain. An important group in terms of pain mechanisms consists of axons that release serotonin or norepinephrine. These axons are thought to play a role in controlling transmission of nociceptive information through the dorsal horn and contributing to stimulation-produced analgesia.


Introduction
The dorsal horn of the spinal cord is the major receiving zone for primary afferent axons that transmit information from sensory receptors in the skin, viscera, joints, and muscles of the trunk and limbs to the central nervous system. Nociceptive primary afferent axons (i.e., those that respond to tissue-damaging stimuli) terminate almost exclusively in the dorsal horn, which is therefore the site of the first synapse in ascending pathways conveying the sensory information that underlies conscious perception of pain. In addition, it contains neuronal circuits involved in generating local reflexes.
In the Gate Control Theory of pain, Melzack and Wall (1965) proposed that inhibitory interneurons in the superficial part of the dorsal horn play a crucial role in controlling incoming sensory information before it is transmitted to the brain. This theory aroused a great deal of interest in organization of the dorsal horn. However, despite intensive study since then, our knowledge of the neuronal circuitry of the region remains limited. The dorsal horn contains four neuronal components: (1) central terminals of primary afferent axons, which arborize in different areas, depending on their diameter and the type of sensory stimulus that they respond to; (2) interneurons, with axons that remain in the spinal cord, either terminating locally or extending into other spinal segments; (3) projection neurons, with axons that pass rostrally in white matter to reach various parts of the brain; and (4) descending axons that pass caudally from several brain regions and play an important role in modulating the transmission of nociceptive information. In this chapter we review the anatomical organization of the mammalian dorsal horn, with particular emphasis on primary afferents and interneurons. Certain features of projection neurons are covered here, but they are described in more detail in Chapter 12 . Descending modulatory systems are dealt with in Chapter 8 , but here we discuss possible targets of the monoamine neurotransmitters released by axons projecting from the brain stem. Because many of the anatomical studies of the dorsal horn have been carried out on cats or rodents, our account is based on these species.

The Laminae of Rexed
Rexed (1952) divided the dorsal horn of the cat spinal cord into six parallel laminae based on differences in the size and packing density of neurons (cytoarchitectonics). This scheme has since been extended to other species, including human, monkey, and rat ( Fig. 5-1 ), and serves as a useful basis for describing its anatomical organization. Lamina II is often subdivided into two parts: inner (IIi) and outer (IIo). Laminae I and II, which are referred to as the superficial dorsal horn, constitute the main target for nociceptive primary afferents (see later). We concentrate our account on this region, partly because of its obvious importance in pain mechanisms and partly because more is known about its neuronal organization. However, the deeper laminae (III–VI) also have an important role in pain: some nociceptive primary afferents terminate in this region, and many neurons in these laminae (including some projection cells) are activated by noxious stimulation. In addition, low-threshold afferents that terminate in laminae IIi–V are at least partially responsible for the tactile allodynia (pain felt in response to touch) that occurs in certain pathological pain states ( Campbell et al 1988 ).


Figure 5-1 Rexed’s laminae applied to the rat spinal cord. A, Transverse section of the rat lumbar spinal cord (L4 segment) stained with antibody to NeuN, a neuronal nuclear protein. This results in immunostaining of all neurons in the spinal cord. B, The positions of Rexed’s laminae as applied to the rat lumbar spinal cord. Laminae I–VI constitute the dorsal horn. C, Higher-magnification view of laminae I–III stained with the NeuN antibody. Approximate positions of the laminar boundaries are shown with dashed lines. Laminae I and II contain numerous densely packed small neurons, whereas those in lamina III are generally slightly larger.
Lamina I, also known as the marginal layer, forms a thin sheet covering the dorsal aspect of the dorsal horn and contains both projection neurons and interneurons. Although this lamina contains the highest density of projection neurons in the dorsal horn, they are thought to make up only ≈5% of its neuronal population, with the remainder being interneurons ( Spike et al 2003 ). Most of the cells have dendrites that remain within the lamina. Lamina I neurons vary considerably in size and shape, with projection cells being larger than interneurons ( Al Ghamdi et al 2009 ). A few particularly large projection neurons, known as marginal cells of Waldeyer, can be recognized. Lamina II is also known as the substantia gelatinosa because the lack of myelinated fibers gives it a translucent appearance. Virtually all the neurons in this lamina are interneurons, and they are densely packed in its outer part. Lamina III also contains a high density of neurons. Most are interneurons and are generally somewhat larger than those of lamina II, but scattered large projection neurons are also present. Although Rexed’s scheme was based on cytoarchitectonic criteria, the border between laminae II and III can be identified more easily by the absence of myelinated axons in lamina IIi and their presence in lamina III. This can be seen with myelin stains or dark-field microscopy of unstained sections. It should be noted that the correlation between the substantia gelatinosa and Rexed’s lamina II, originally determined in cats, may differ in rodents ( Woodbury et al 2000 ) because lamina IIi receives abundant input from some large myelinated low-threshold mechanoreceptive afferent fibers in rodents but not in cats ( Brown 1982 , Woolf 1987 , Woodbury et al 2001 ). Laminae IV–VI are more heterogeneous, with neurons of various size, some of which are projection cells. The borders between these laminae are difficult to determine with certainty.

Primary Afferent Fibers
Primary sensory neurons provide constant feedback on the external environment, as well as the ongoing state of the body. The somata of those that innervate the limbs and trunk are located in sensory ganglia associated with spinal nerves (dorsal root ganglia). Their axons bifurcate within the ganglion and give rise to a peripheral branch that innervates various tissues and a central branch that travels through a dorsal root to enter the spinal cord, where it forms synapses with second-order neurons. The peripheral targets of these fibers provide a convenient means for classification. Fibers innervating skin are described as cutaneous sensory neurons. Likewise, those innervating abdominal or pelvic viscera are termed visceral afferents. Within these populations, fibers can respond to various sensory modalities, including mechanical, thermal, and chemical stimuli. Modality-specific groups are further divided according to the intensity of their adequate peripheral stimuli. Those that respond to gentle mechanical force or innocuous thermal stimuli are low-threshold mechanoreceptors or innocuous cooling or warming afferents. Fibers responding only to stimulus intensities considered tissue threatening or potentially tissue damaging are termed nociceptors.
As a group, primary sensory neurons exhibit a rich diversity in morphological and functional properties, including somatic membrane properties, laminar location of central projections, neurochemical content, and response properties of the central networks that they activate ( Koerber and Mendell 1992 , Djouhri et al 1998 ). The most common means of classifying primary sensory neurons is based on the conduction velocity of their peripheral axons, which is directly related to axon diameter and whether the axon is myelinated. From the distribution of these peripheral conduction velocities, primary sensory neurons are routinely divided into different groups: Aα/β, Aδ, and C.
The Aα/β group consists of large myelinated axons with the fastest peripheral conduction velocity, the Aδ group contains smaller fibers that are thinly myelinated and conduct at an intermediate velocity, and the C group consists of the smallest, unmyelinated, and most slowly conducting fibers. Within each group there is a wide range of functional types of primary afferents, as defined by sensory modality. Most sensory neurons with fibers conducting in the Aα/β range respond to innocuous mechanical stimuli, do not encode noxious stimulus intensities, and are classified as low-threshold mechanoreceptors. Some of these fibers, however, respond to relatively innocuous mechanical stimuli but also encode stimulus intensities in the noxious range and in some cases respond to noxious heating of the skin. This trend reverses with decreasing conduction velocity, with a majority of Aδ fibers and most C fibers being classified as nociceptors. The relative number of functional types in specific conduction velocity groups varies between species and the areas of the body that the fibers innervate. However, it is important to point out that both nociceptors and non-nociceptors exist in all three conduction velocity groups ( Fig. 5-2 ).


Figure 5-2 Schematic representation of the general properties of cutaneous afferent fibers. Inequality signs denote the relative numbers of afferent fibers within a conduction velocity that respond to innocuous (blue) or noxious (red) stimuli. The density of the terminals of each type of fiber is shown as varying stippling within each projection zone. Note that not all C fibers are nociceptors and not all Aβ fibers are low-threshold mechanoreceptors. HTMR, high-threshold mechanoreceptor.

Vesicular Glutamate Transporters
All primary afferents are thought to use glutamate as their principal neurotransmitter since the excitatory post-synaptic currents produced by these afferents can be blocked with antagonists of the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)-type glutamate receptor ( Yoshimura and Jessell 1990 ), glutamate is enriched in their central terminals ( Broman et al 1993 ), and these are associated with post-synaptic AMPA receptors ( Nagy et al 2004a ). Until recently it was difficult to identify glutamatergic axons, but this changed with the discovery of the vesicular glutamate transporters (VGLUTs). Two of these transporters, VGLUT1 and VGLUT2, are widely distributed in the spinal gray matter ( Varoqui et al 2002 , Todd et al 2003 ), whereas the third (VGLUT3) is present at much lower levels ( Seal et al 2009 ). Within the dorsal horn, VGLUT1 is largely restricted to the deeper part (laminae IIi–VI) and is apparently present in the central terminals of all low-threshold mechanoreceptive myelinated primary afferents, as well as in some descending glutamatergic axons ( Todd et al 2003 ). VGLUT2-immunoreactive axons are found throughout the gray matter, and most belong to local interneurons (see later). The central terminals of many myelinated nociceptors in lamina I also show strong VGLUT2 immunoreactivity. However, although most C fibers appear to express VGLUT2 ( Brumovsky et al 2007 ), it is present at very low or undetectable levels in their central terminals ( Todd et al 2003 ). For example, dorsal rhizotomy does not lead to detectable loss of VGLUT2 in the dorsal horn, but there is a substantial reduction in VGLUT1 ( Alvarez et al 2004 , Brumovsky et al 2007 ). Seal and colleagues (2009) recently identified a population of low-threshold mechanoreceptive unmyelinated primary afferents that express VGLUT3 and project mainly to the innermost part of lamina II and the dorsal part of lamina III.

Physiological Properties
Early studies of primary sensory neurons revealed heterogeneity in the shapes of somatic action potentials ( Yoshida et al 1978 , Gorke and Pierau 1980 ), and subsequent studies have shown strong correlation between these shapes and receptor function (e.g., Belmonte and Gallego 1983 , Koerber and Mendell 1988 ). Myelinated fibers that respond to innocuous mechanical stimulation of the skin have narrow somatic action potentials without breaks in the rising or falling phase. Unmyelinated and myelinated nociceptive fibers have broad somatic action potentials that most often have a distinct inflection on the falling phase. This correlation is very consistent for myelinated fibers. However, all unmyelinated fibers have broad inflected somatic action potentials regardless of their peripheral response properties.

Spinal Projections of Primary Sensory Neurons
The central projections of primary sensory neurons have been visualized with various labeling methods, including the Golgi technique, detection of degenerating axon terminals, bulk-labeling techniques in which tracer substances are administered to a nerve or peripheral tissue and transported by many primary afferents, and intracellular staining of individual identified fibers. In his landmark studies, Cajal (1909) used the Golgi technique and suggested that fine primary afferent fibers project to the superficial part of the dorsal horn. As newer bulk-labeling techniques were introduced, the organization of central projections of different fiber types was further refined (e.g., Light and Perl 1977 , Mesulam and Brushart 1979 ). However, the uncertainties inherent in bulk-labeling techniques left many questions unanswered. The use of intracellular labeling has clarified the specific central targets of different afferent fiber types (e.g., Light and Perl 1979 , Brown 1982 ). We will therefore focus on the findings of studies using these intracellular staining techniques.

Cutaneous Sensory Neurons

Myelinated Low - Threshold Mechanoreceptors
Low-threshold mechanoreceptive fibers enter the spinal cord and bifurcate into main ascending and descending branches that travel in the dorsal columns and migrate medially as they move away from their point of entry. Collateral fibers arise from these main branches, turn ventrally, and pass through the dorsal horn before terminating in dense arborizations that lie within a region extending from lamina IIi to lamina V. The morphological characteristics of these projections vary with the specific fiber type and the target tissue innervated, their relative position within the dorsal horn, and the distance from the point of entry into the spinal cord ( Brown 1982 , Woolf 1987 , Millecchia et al 1991 , Koerber et al 1995 ). In all cases the most superficial and dense central projections lie near the point of entry. Away from the entry zone they become more diffuse and occupy more ventral and medial positions ( Koerber et al 1995 ). Another consistent feature is that those innervating hair follicles terminate more superficially than do those innervating slowly adapting receptors. Among fibers innervating hair follicles, those conducting in the Aδ range (D-hair afferents) occupy the most superficial position of all low-threshold fibers and project extensively into lamina IIi ( Light et al 1979 , Woodbury and Koerber 2003 , Woodbury et al 2008 ).

Myelinated Nociceptive Afferent Fibers
Myelinated nociceptors, which are thought to signal fast pricking or sharp pain, were first identified in the pioneering studies of Burgess and Perl (1967) and have since been studied extensively by other groups (e.g., Campbell et al 1979 , Light and Perl 1979 , Reeh et al 1987 ). They span a very large range of conduction velocities, from the slowest in the Aδ spectrum to well into the Aβ range ( Campbell et al 1979 , Koerber and Mendell 1988 ). They respond to different stimulus modalities (e.g., mechanical and thermal) and have threshold stimulus intensities ranging from innocuous to noxious (e.g., Burgess and Perl 1967 , Fitzgerald and Lynn 1977 ). Recent studies also show that they exhibit different central morphologies and have a range of neurochemical phenotypes ( Light and Perl 1979 , Lawson et al 1997 , Lawson et al 2002 , Woodbury and Koerber 2003 , McIlwrath et al 2007 , Lawson et al 2008 ).
Most early studies examining individual myelinated nociceptors used extracellular recording techniques, either from peripheral nerves using microelectrodes ( Burgess and Perl 1967 ) or from nerve strands draped over metal electrodes ( Reeh et al 1987 ). With these techniques investigators are limited to using peripheral response properties, such as mechanical threshold, to identify myelinated nociceptors. Although it has long been known that some putative myelinated nociceptors could be activated by non-noxious moderate pressure ( Burgess and Perl 1967 ), threshold criteria were commonly used to avoid ambiguity, and thus neurons that code for both non-noxious and noxious mechanical stimuli have largely been overlooked.
The recent development of procedures that allow intracellular recordings from the cell soma combined with labeling of the central projections of the recorded fiber has increased the number of criteria that can be used to identify nociceptive sensory neurons ( Koerber and Woodbury 2002 , Woodbury and Koerber 2003 ). For example, myelinated nociceptors have broad inflected somatic action potentials that can easily be distinguished from those of low-threshold mechanoreceptors ( Koerber and Mendell 1988 , Djouhri et al 1998 ). Although relatively little information is available on the neurochemical properties of myelinated nociceptors, it is known that some contain neuropeptides, such as substance P and calcitonin gene–related peptide (CGRP), and express the high-affinity neurotrophin receptor TrkA, thus being responsive to nerve growth factor (NGF) ( Lawson et al 1997 , 2002 ). Others contain TrkC and are sensitive to neurotrophic factor 3 (NT3) ( McIlwrath et al 2007 ). In addition, these fibers often possess acid-sensing ion channel 3 (ASIC3) and the transient receptor potential vanilloid type 2 (TRPV2) receptor ( McIlwrath et al 2007 , Lawson et al 2008 ).
The central projections of myelinated nociceptors were first described by Light and Perl (1979) , who focused on fibers conducting in the Aδ range in cats. They found that on entry into the spinal cord, the main branches were located laterally in the dorsal column, often in or near Lissauer’s tract. Terminal arbors from these afferents were centered primarily on laminae I and IIo, with some passing ventrally to terminate in lamina V. However, some of the faster-conducting fibers had collateral branches that penetrated deeply into the dorsal horn and then recurved dorsally and projected into the ventral part of lamina IV. The findings of this landmark study led to the widespread belief that myelinated nociceptors project only to laminae I and V.
More recently, Woodbury and Koerber used an ex vivo preparation consisting of isolated spinal cord and attached innervated skin to examine the projections of these and other afferent types in neonatal and adult mice ( Woodbury et al 2003 , 2008 ). Two distinct morphological types of myelinated nociceptor were observed. The first closely resembled the thinly myelinated nociceptors described in the cat by Light and Perl (1979) . On entry into the spinal cord, their axons bifurcated to give rise to ascending and descending branches that extended over several segments. Some of these afferents gave rise to axons that ascended in the dorsal columns. However, most maintained a lateral position in the vicinity of Lissauer’s tract. The second type had main branches that ascended and descended in the dorsal columns and gave rise to numerous collaterals that penetrated ventrally through the depth of the dorsal horn before recurving dorsally, as seen with many low-threshold myelinated mechanoreceptive afferents. However, in marked contrast to the latter, the arbors of this group extended through the full depth of the dorsal horn, including laminae IIo and I ( Fig. 5-3 ). Once in lamina I, they typically turned to run along the rostrocaudal axis while continuing to arborize. In general, the arbors of these fibers were somewhat more diffuse than those of low-threshold mechanoreceptors with similar peripheral conduction velocities. Interestingly, those with central projections focused in laminae I and IIo had higher mechanical thresholds than did those with projections spanning the dorsal horn. Some fibers of each type also responded to noxious heating of the skin ( Woodbury and Koerber 2003 , Woodbury et al 2008 ).


Figure 5-3 Spinal projections of a myelinated cutaneous nociceptive fiber from a 3-week-old mouse showing novel morphology. A, Photomicrograph of a section of spinal cord dorsal horn showing the laminar distribution of a single recurving collateral. The dotted lines indicate the boundaries of laminae I–II. B, Camera lucida drawing of a different collateral from the same afferent fiber. Scale bar, 50 μm. (Modified from Woodbury CJ, Koerber HR 2003 Widespread projections from myelinated nociceptors throughout the substantia gelatinosa provide novel insights into neonatal hypersensitivity. Journal of Neuroscience 23:601–610. Copyright 2003 by the Society for Neuroscience.)

Unmyelinated Afferent Fibers
Cutaneous primary afferent neurons with unmyelinated peripheral axons are diverse in terms of response properties and neurochemical phenotypes. As a group, they exhibit a wide range in the stimulus intensity necessary for their activation. Some unmyelinated fibers respond to gentle brushing of the skin and/or innocuous cooling, whereas others require more intense mechanical or thermal stimulation in the noxious range ( Bessou and Perl 1969 ). Individual neurons can respond to different modalities of noxious stimuli, including mechanical, thermal, and chemical. Although some respond only to a single type of stimulus, others are equally responsive to two or more and are referred to as polymodal nociceptors ( Bessou and Perl 1969 ). Still other afferent fibers are normally insensitive to peripheral stimulation and become sensitized only after prolonged noxious stimulation or in response to injury (e.g., Meyer and Campbell 1988 ).
In terms of phenotypic diversity, unmyelinated cutaneous afferents can express a large number of neuroactive compounds and receptors. Frequently, C fibers are divided into two major groups based on the combination of neurochemical phenotype and sensitivity for different neurotrophins ( Snider and McMahon 1998 ). One group consists of axons that are sensitive to NGF, express TrkA, and usually contain neuropeptides such as CGRP, substance P, and galanin ( Averill et al 1995 , Molliver et al 1995 , Bennett et al 1996 , Zhang et al 1993 ). Neurons in the second group are responsive to members of the glial cell line–derived neurotrophic factors (GDNFs) and neurturin, express the receptor tyrosine kinase (RET), have binding sites for the lectin isolectin B4 (IB4, from Bandeiraea simplicifolia ), usually possess the purinergic receptors P2X 3 and P2Y 1 ( Molliver et al 1997 , Bennett et al 1998 , Bradbury et al 1998 , Vulchanova et al 1998 , Guo et al 1999 , Moriyama et al 2003 , Gerevich et al 2005 ), and lack CGRP and substance P ( Averill et al 1995 ). However, despite the obvious differences between these two populations, it is not clear whether they correspond to different functional types. For example, the capsaicin receptor (TRPV1), which responds to heat and protons ( Caterina et al 1997 ) and is believed to transduce noxious heat stimuli, is expressed in a variety of sensory neurons in the rat, including both TrkA/peptidergic and IB4-binding populations ( Guo et al 1999 , Michael and Priestley 1999 ). However, there is still debate over the location of the receptor in the peripheral and central projections of these fibers ( Guo et al 1999 ). The distribution of TRPV1 is different in mice because it does not appear to be expressed by the IB4-binding population ( Zwick et al 2002 ). Interestingly, TRPV1-knockout mice exhibit relatively modest changes in their behavioral response to noxious heat ( Caterina et al 2000 ). In addition, it has been shown that in these knockout mice, C fibers responding to both mechanical and heat stimuli have normal heat responses ( Woodbury et al 2004 ). Taken together, these results suggest that both populations of C fibers respond to heat stimuli and that TRPV1 is not the only receptor capable of transducing such stimuli. Recently, it has been shown that in naïve mice, cutaneous C fibers expressing TRPV1 are mechanically insensitive but respond robustly to heat stimuli (C heat, CH) whereas mice lacking TRPV1 also lack CH fibers, thus suggesting that TRPV1 is required for heat sensitivity in this fiber type ( Lawson et al 2008 ).
The central projections of these two different groups of unmyelinated primary afferents differ, with the IB4-positive (“non-peptidergic”) fibers projecting to the central part of lamina II (dorsal part of lamina IIi) and peptidergic fibers arborizing mainly in laminae I and IIo, but with scattered terminals in deeper laminae (IIi–V) ( Silverman and Kruger 1988 , Plenderlieth et al 1990 , Averill et al 1995 ; but also see Riberio-da-Silva et al 1986 , Woodbury et al 2000 ).
The central axons of individual unmyelinated fibers have been visualized with intracellular labeling techniques, most notably by Sugiura and colleagues ( Sugiura et al 1986 , 1989 ). They stained functionally identified C fibers from the guinea pig and reconstructed their central projections. Although relatively few fibers were examined, examples of low- and high-threshold mechanoreceptors, as well as polymodal nociceptors, were recovered. These fibers entered the spinal cord and ran rostrally and/or caudally along the surface of the dorsal funiculus near or in Lissauer’s tract while sending off collateral branches that penetrated ventrally into laminae I and II and ended in dense terminal fields. Although individual fibers had projections that were focused more or less in different parts of the superficial dorsal horn, most were found in both laminae I and II, with the primary focus usually being in lamina II ( Sugiura et al 1989 ). Recent studies in mice have largely confirmed these original reports by demonstrating that most nociceptive C fibers project extensively to lamina IIo and, to a more limited extent, to lamina I ( Woodbury et al 2004 , Albers et al 2006 ).
Advances in transgenic technology have recently been used to identify specific biomarkers for additional subsets of sensory fibers (e.g., Zylka et al 2005 , Seal et al 2009 ). The inclusion of fluorescent reporter constructs combined with intracellular recording and staining has allowed functional identification of some of these fiber types. For example, the Mas-related G protein–coupled receptor D (Mrgprd) is selectively expressed in unmyelinated cutaneous fibers that lack peptides and bind IB4 ( Zylka et al 2005 ). Intracellular recordings demonstrated that all these fibers were responsive to mechanical stimulation and that the vast majority were also sensitive to heat and occasionally cold stimuli ( Rau et al 2009 ). These predominantly polymodal fibers have central projections that precisely overlap the IB4-positive labeling within lamina II.

Afferent Fibers Innervating Muscle and Viscera
Afferent fibers innervating deep structures such as muscles, tendons, or viscera share many characteristics with those innervating skin. They have a large range of peripheral conduction velocities and respond to various stimulus modalities over a wide spectrum of intensities ( Hoheisel et al 1989 , Habler et al 1993 , Sengupta and Gebhart 1994 ). Myelinated fibers innervating muscles and tendons generally fall into two categories. One group innervates specific receptors (e.g., muscle spindles or Golgi tendon organs) and these afferents are generally referred to as proprioceptors. They have very low mechanical thresholds and their central projections are usually confined to the deeper parts of the dorsal horn (laminae IV–VI) and the ventral horn. Another group of myelinated mechanoreceptive fibers innervating muscle, tendon, or fascia is not associated with specialized endings. These fibers respond over a range of stimulus intensities and can be divided into low- and high-threshold groups ( Hoheisel et al 1989 ). High-threshold myelinated mechanoreceptors exhibit two different patterns of central termination. One type has projections confined exclusively to lamina I, whereas a second type projects to lamina I and also to laminae IV–V. Low-threshold mechanoreceptors have a different central morphology and generally cover a greater rostrocaudal extent in the dorsal horn. They project predominantly to lamina II, as well as to laminae IV and VI ( Hoheisel et al 1989 ) ( Fig. 5-4 ).


Figure 5-4 Schematic representation of the spinal projections of afferent fibers innervating muscle and viscera. Myelinated muscle afferents conveying information about innocuous muscle stimuli are myelinated and have widespread projections. Myelinated fibers that respond to noxious stimuli project to the deeper dorsal horn, whereas C fibers responding to painful stimuli project to the superficial (muscle) or superficial and deep (visceral) dorsal horn laminae. The relative density of fiber terminals among these fibers and with respect to cutaneous fiber projections (see Fig. 5-2 ) is depicted by the varying stippling.
Similarly, myelinated fibers innervating abdominal and pelvic viscera can be divided into low- and high-threshold mechanoreceptive groups, although many of the low-threshold group also encode into the noxious range (see Chapter 51 ). However, little is known about the morphology of their central projections. Results from bulk-labeling studies suggest that they are most likely to project to lamina I and/or laminae V–VI ( de Groat et al 1981 , Morgan et al 1981 ).
Unmyelinated fibers innervating muscle and viscera respond to a variety of noxious stimuli, including mechanical and chemical, and thus can be considered polymodal nociceptors ( Kumazawa 1996 ). These neurons contain many of the same neuroactive compounds and receptor types seen in their cutaneous counterparts ( Molander et al 1987 , O’Brien et al 1989 , Perry and Lawson 1998 ). However, one notable difference is that they rarely bind IB4, thus suggesting some fundamental differences between the populations ( Bennett et al 1996 , Perry and Lawson 1998 ). Early studies using bulk-labeling methods suggested that unmyelinated fibers innervating viscera ( de Groat et al 1981 , Morgan et al 1981 ) or muscle ( Craig and Mense 1983 , but see Brushart et al 1981 ) project to lamina I and deeper parts of the dorsal horn, but not to lamina II, as is the case with cutaneous C fibers. However, intracellular staining of individual unmyelinated fibers innervating these structures ( Sugiura et al 1989 , Ling et al 2003 ) has shown conclusively that these fibers do project to lamina II, as well as to the other parts of the dorsal horn.
Ling and colleagues (2003) examined the central projections of six unidentified unmyelinated afferents contained in the nerve to the lateral gastrocnemius muscle. These fibers were shown to enter the spinal cord and run rostrally and caudally in the superficial part of the dorsal funiculus. They gave rise to two different types of projections. One type had small focused projections in lamina I, the overlying white matter, and lamina IIi. They were less dense than those of cutaneous C fibers. The second type had projections throughout lamina II that were relatively sparse in comparison to cutaneous fibers. Both types also gave rise to sporadic projections into the dorsal part of lamina III. Additional studies are needed to determine whether these two morphological groups represent different functional types.
Individual unmyelinated fibers innervating abdominal viscera have also been examined ( Sugiura et al 1989 ). These fibers were identified by electrical stimulation of the celiac ganglion but were otherwise uncharacterized. On entering the spinal cord, they bifurcated and ran rostrally and caudally in the dorsal funiculus or Lissauer’s tract for several segments and gave rise to many collaterals that ramified in several different locations in the spinal cord, including laminae I–II, V, and X. The terminal ramifications formed relatively diffuse narrow sheets. Overall, comparison of unmyelinated fibers innervating different peripheral tissues shows that cutaneous fibers have the most focused and dense projections, visceral afferents have the most wide-ranging and diffuse projections, and those innervating muscle have projections that lie between these two extremes. These differences in central projection patterns could contribute to the difficulty in localizing muscle and visceral pain.

Summary of Spinal Projections
It is clear that both myelinated and unmyelinated afferent fibers that respond to noxious stimulation in the periphery project predominantly to the superficial dorsal horn. However, it is also clear that myelinated and unmyelinated fibers that signal the presence of innocuous mechanical and thermal stimuli also project to these same laminae. Therefore, the anatomical substrate for processing pain information cannot easily be distinguished from those involved in other functions such as homeostasis. Interestingly, although there is significant overlap in the projections of fibers signaling different stimulus intensities, there appears to be at least some degree of functional segregation at the post-synaptic level in the superficial laminae (e.g., Light and Willcockson 1999 , Andrew and Craig 2001 , Wilson et al 2002 ).

Receptors Associated with Primary Afferent Neurons
Primary afferent fibers possess a rich diversity of ligand-gated ionotropic, metabotropic, and tyrosine kinase receptors. Although a complete description of these receptors is beyond the scope of this chapter, several are present on the central terminals of primary afferent fibers, and since their activation appears to regulate the release of neurotransmitters, they merit consideration. These include both the AMPA and N -methyl- D -aspartate (NMDA) classes of ionotropic glutamate receptors ( Tachibana et al 1994 ) and metabotropic glutamate receptors ( Ohishi et al 1995 ). Both GABA A and GABA B receptors are expressed by sensory neurons; although GABA B receptors have been localized to presynaptic terminals ( Poorkhalkali et al 2000 ), little is yet known about the arrangement of GABA A receptor subunits on primary afferent terminals. The three main opioid receptors (μ, δ, and κ) are also found in primary sensory neurons, and μ- and δ-opioid receptors have been identified on fine-diameter primary afferent terminals ( Wang et al 2010 ). In addition, both nicotinic and muscarinic cholinergic receptors are present on afferent fibers ( Flores et al 1996 , Haberberger et al 1999 ). α 2 -Adrenergic receptors are also found in sensory neurons and are thought to be localized at the central terminals of peptidergic fibers ( Stone et al 1998 ).

Ultrastructure of Primary Afferent Terminals
Although most primary afferent boutons have relatively simple synaptic arrangements in the dorsal horn, some form complex structures involving several synapses and are known as synaptic glomeruli ( Fig. 5-5 ). Since the central axons of all synaptic glomeruli are of primary afferent origin ( Ribeiro-da-Silva 2003 ), this provides a convenient way of identifying primary afferent terminals with electron microscopy. Synaptic glomeruli consist of a central primary afferent bouton surrounded by several other profiles with which the central axon forms synapses. The peripheral profiles are either GABAergic axons, which are presynaptic to the primary afferent bouton at axo-axonic synapses, or dendrites. In most cases the dendrites are post-synaptic to the primary afferent bouton. However, some contain synaptic vesicles and form dendro-axonic synapses onto the central axon. These vesicle-containing dendrites, which are also GABAergic ( Todd 1996 ), may be involved in reciprocal synapses with the primary afferent bouton ( Fig. 5-5 A). Another type of arrangement that occurs in glomeruli is the synaptic triad, in which the central axon forms synapses with two peripheral profiles that are themselves linked by a synapse. It is clear from this description that synaptic glomeruli provide the basis for complex modulation of incoming sensory information, including GABAergic presynaptic inhibition of the primary afferent terminal by axo-axonic and dendro-axonic synapses.


Figure 5-5 Synaptic glomeruli in the superficial dorsal horn of the rat spinal cord. A, Type I glomerulus. The central axon (C) is indented and filled with vesicles of various sizes. It is surrounded by several profiles, including an axon (A) and dendrites, one of which is labeled (D). A vesicle-containing dendrite (V) is also present, and on an adjacent section this formed a reciprocal axodendritic/dendro-axonic synapse with the central axon (inset). Some of the synapses are indicated by arrows. B, The central axon (C) of this type II glomerulus contains several mitochondrial profiles and synaptic vesicles that are clustered at synapses. The central axon receives axo-axonic synapses from three other axons (A) and is presynaptic to two dendrites (D). Some of the synapses are shown with arrows. Scale bar for both parts, 1 μm. (Modified from Todd AJ 1996 GABA and glycine in synaptic glomeruli of the rat spinal dorsal horn. European Journal of Neuroscience 8:2492–2498, with permission from Blackwell Publishing Ltd.)
Ribeiro-da-Silva and Coimbra (1982) recognized two types of synaptic glomeruli in the rat spinal cord. Central axons of type I glomeruli typically have indented contours, are packed with synaptic vesicles of varying diameter, have few mitochondria, and receive a single axo-axonic synapse from a peripheral axon ( Ribeiro-da-Silva 2003 ) ( Fig. 5-5 A). They occupy a narrow band in the middle of lamina II, and their central axons usually belong to C fibers that lack neuropeptides. Type II glomeruli are located slightly more ventrally, on either side of the lamina II/III border. Their central axons are generally larger than those of type I glomeruli and are derived from Aδ D-hair afferents ( Rèthelyi et al 1982 ). Type II central axons contain clustered synaptic vesicles and several mitochondrial profiles, and they often receive several axo-axonic synapses ( Fig. 5-5 B), thus suggesting that they are under powerful presynaptic inhibitory control.
In the rat, most peptide-containing primary afferent terminals in the superficial dorsal horn form simple synaptic arrangements ( Ribeiro-da-Silva et al 1989 ), whereas in the monkey they may be involved in synaptic glomeruli ( Knyihar-Csillik et al 1982 ). The central terminals of peptidergic primary afferents also differ from those of non-peptidergic C fibers in that they receive very few axo-axonic synapses.
Central terminals of Aδ mechanical nociceptors have been identified in the cat and monkey ( Rèthelyi et al 1982 , Alvarez et al 1992 ). In lamina I, some of these nociceptors formed simple axodendritic synapses, but many had glomerular arrangements in which they were presynaptic to several dendrites and post-synaptic to GABAergic axons. Terminals of these afferents in lamina V had simpler synaptic arrangements.
The ultrastructure of several different types of Aβ low-threshold mechanoreceptive afferents has been studied in the cat ( Maxwell and Rèthelyi 1987 ). Most of them have a “non-glomerular” synaptic organization in which they are presynaptic to one or more dendrites and frequently receive axo-axonic GABAergic synapses.

Projection Neurons, Substance P, and the Neurokinin 1 Receptor
Neurons with axons that project to the brain are concentrated in lamina I and scattered through the deep dorsal horn (laminae III–VI) and the ventral horn. Those in lamina I, together with some of the projection cells in deeper laminae, have axons that cross the midline and ascend to several supraspinal targets, including the thalamus, midbrain periaqueductal gray (PAG), lateral parabrachial area (LPb) of the pons, and various parts of the medulla ( Craig 1995 , Villanueva and Bernard 1999 , Willis and Coggeshall 2004 ) (see Chapter 12 ). Quantitative studies on the rat spinal cord indicate that there are around 400 projection neurons on each side in lamina I in the L4 segment and that ≈95% of these neurons send axons to the LPb, around a third to the PAG, a quarter to the nucleus of the solitary tract, but <5% to the thalamus ( Al-Khater et al 2008 , Polgár et al 2010a , Todd 2010 ). However, spinothalamic lamina I neurons are far more numerous in the cervical region of the rat and in both lumbar and cervical enlargements in the cat and monkey ( Zhang et al 1996 , Zhang and Craig 1997 ). Many projection neurons send their axons to more than one supraspinal target.
In addition to their supraspinal targets, projection neurons also generate local axon collaterals and thus contribute to processing of information in the dorsal horn, as well as to segmental reflex pathways. For example, Szücs and colleagues (2010) have recently shown that the majority of lamina I projection neurons in the rat give rise to axon collaterals in the dorsal and/or ventral horn.
Many nociceptive primary afferents contain substance P ( Lawson et al 1997 ), and there is evidence that this peptide and the neurokinin 1 (NK1) receptor, on which it acts, have a significant role in spinal pain mechanisms. The NK1 receptor is present in the dorsal horn, with its highest concentration in lamina I (e.g., Nakaya et al 1994 ) ( Fig. 5-6 ). The receptor is expressed by some neurons in each lamina, including ≈80% of lamina I projection neurons ( Fig. 5-7 B). There is also a population of large NK1 receptor–immunoreactive projection neurons with cell bodies in laminae III–IV and dendrites that pass dorsally into lamina I ( Todd 2010 ) ( Fig. 5-7 E). These neurons are far less numerous than the lamina I projection neurons, with only ≈25 per side in the L4 segment. Immunocytochemical studies have shown that many NK1 receptor–immunoreactive neurons in laminae I, III, and IV internalize the receptor following acute noxious stimulation, presumably as a result of its activation by substance P released from nociceptive primary afferents ( Mantyh et al 1995 ).


Figure 5-6 Neurokinin 1 (NK1) receptor immunostaining in a section from the lumbar spinal cord of a rat. The receptor is found throughout the spinal cord but is present at the highest concentration in lamina I. (Modified from Nakaya Y, Kaneko T, Shigemoto R, et al 1994 Immunohistochemical localization of substance P receptor in the central nervous system of the adult rat. Journal of Comparative Neurology 347:249–274. Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley and Sons, Inc.)


Figure 5-7 Contacts formed by substance P (SP)-containing primary afferents and projection neurons that express the neurokinin 1 (NK1) receptor in the rat lumbar spinal cord. A-D show a lamina I neuron in a horizontal section. A, The cell was retrogradely labeled with the tracer Fluorogold (FG), which had been injected into the medullary reticular formation. B, It expresses the NK1 receptor (green), which outlines its cell body and dendrites. C and D show immunostaining with antibodies against SP (blue) and calcitonin gene–related peptide (CGRP, red). Axonal boutons that contain both peptides appear pink, and several of them are adjacent to the labeled cell. The presence of both these peptides identifies an axon as being an SP-containing (nociceptive) primary afferent. E, A sagittal section through the dorsal horn reveals two large NK1 receptor–immunoreactive neurons (asterisks) with cell bodies in lamina III and dendrites that extend up to lamina I. All cells of this type are projection neurons. Boxes indicate areas shown at higher magnification in F – I . In these images, SP (blue) and CGRP (red) are also shown, and it can be seen that dendrites of the two ce