The acute phase response and soman-induced status epilepticus: temporal, regional and cellular changes in rat brain cytokine concentrations

Johnson, Kan

Description

Neuroinflammation occurs following brain injury, including soman (GD) induced status epilepticus (SE), and may contribute to loss of neural tissue and declined behavioral function. However, little is known about this important pathological process following GD exposure. Limited transcriptional information on a small number of brain-expressed inflammatory mediators has been shown following GD-induced SE and even less information on protein upregulation has been elucidated. The purpose of this study is to further characterize the regional and temporal progression of the neuroinflammatory process following acute GD-induced SE. Methods The protein levels of 10 cytokines was quantified using bead multiplex immunoassays in damaged brain regions (i.e., piriform cortex, hippocampus and thalamus) up to 72 hours following seizure onset. Those factors showing significant changes were then localized to neural cells using fluorescent IHC. Results A significant concentration increase was observed in all injured brain regions for four acute phase response (APR) induction cytokines: interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor (TNF)-α. Increases in these APR cytokines corresponded both temporally and regionally to areas of known seizure damage and neuronal death. Neurotoxic cytokines IL-1α and IL-1β were primarily expressed by activated microglia whereas the potentially neuroprotective cytokine IL-6 was expressed by neurons and hypertrophic astrocytes. Conclusions Increases in neurotoxic cytokines likely play an active role in the progression of GD-induced SE neuropathology though the exact role that these and other cytokines play in this process require further study.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2010
• Nombre de lectures: 97
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Johnson and Kan Journal of Neuroinflammation 2010, 7:40 JOURNAL OF
http://www.jneuroinflammation.com/content/7/1/40
NEUROINFLAMMATION
RESEARCH Open Access
ResearchThe acute phase response and soman-induced
status epilepticus: temporal, regional and cellular
changes in rat brain cytokine concentrations
Erik A Johnson* and Robert K Kan
Abstract
Background: Neuroinflammation occurs following brain injury, including soman (GD) induced status epilepticus (SE),
and may contribute to loss of neural tissue and declined behavioral function. However, little is known about this
important pathological process following GD exposure. Limited transcriptional information on a small number of
brain-expressed inflammatory mediators has been shown following GD-induced SE and even less information on
protein upregulation has been elucidated. The purpose of this study is to further characterize the regional and
temporal progression of the neuroinflammatory process following acute GD-induced SE.
Methods: The protein levels of 10 cytokines was quantified using bead multiplex immunoassays in damaged brain
regions (i.e., piriform cortex, hippocampus and thalamus) up to 72 hours following seizure onset. Those factors
showing significant changes were then localized to neural cells using fluorescent IHC.
Results: A significant concentration increase was observed in all injured brain regions for four acute phase response
(APR) induction cytokines: interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor (TNF)-α. Increases in these APR
cytokines corresponded both temporally and regionally to areas of known seizure damage and neuronal death.
Neurotoxic cytokines IL-1α and IL-1β were primarily expressed by activated microglia whereas the potentially
neuroprotective cytokine IL-6 was expressed by neurons and hypertrophic astrocytes.
Conclusions: Increases in neurotoxic cytokines likely play an active role in the progression of GD-induced SE
neuropathology though the exact role that these and other cytokines play in this process require further study.
Background ronal cell loss in the piriform cortex, hippocampus,
Chemical warfare nerve agents (CWNA) were developed amygdala and thalamus [5,6]. Excitotoxic neural damage
during World War II but remain a significant threat following GD exposure activates a neuroinflammatory
through deployment by hostile nations or by terrorist response [7-10], which may contribute to the neuropa-
organizations [1]. CWNA, such as soman (pinacolyl thology.
methylphosphonofluoridate, GD), rapidly and irrevers- The extent to which neuroinflammation contributes to
ibly bind to acetylcholinesterase, causing excess acetyl- cell loss following central nervous system (CNS) injury
choline accumulation in the central and peripheral largely depends on many factors, such as local environ-
nervous systems. GD exposure can cause intense tonic- ment, concentration of the inflammatory mediators, the
clonic convulsions, respiratory paralysis and possibly responding immune cell phenotype and the timing of
death [2]. Following exposure, the ensuing cholinergic their interaction with damaged neural cells [11,12]. In
crisis leads to the development of status epilepticus (SE) severe and progressive CNS injuries, increased neuroin-
that can continue unabated for many hours [3]. SE flammatory activity appears detrimental since anti-
induces neuroinflammatory gliosis [4] and profound neu- inflammatory treatments are successful in reducing brain
pathology in animal models of CNS injury [13,14]. Fol-
* Correspondence: [e-mail]
lowing injury, infiltrating leukocytes and activated mac-1 Research Division, Pharmacology Branch, US Army Medical Research Institute
rophages release many inflammatory proteins, includingof Chemical Defense (USAMRICD), Aberdeen Proving Ground, MD 21010, USA
Full list of author information is available at the end of the article
© 2010 Johnson and Kan; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original work is properly cited.Johnson and Kan Journal of Neuroinflammation 2010, 7:40 Page 2 of 9
http://www.jneuroinflammation.com/content/7/1/40
the acute phase response (APR) inducing cytokines IL-1, 3, 6, 12, 24, 48 or 72 hours after onset of convulsions. Fol-
IL-6 and TNF-α [15]. Though pluripotent, cytokines such lowing euthanasia, piriform cortex, hippocampus and
as IL-1 (α and β) and TNF-α are toxic to neural tissues in thalamus tissue was extracted and processed into lysate
vitro [16-18] and can exacerbate experimental CNS injury as previously described [25]. Briefly, the brain regions
in vivo [19-21]. were excised, rinsed with cold PBS and snap frozen in liq-
Evidence of neuroinflammation following GD-induced uid nitrogen. The tissues were weighed and homogenized
SE has been shown at the level of gene transcription [7- in ice-cold triple detergent lysis buffer containing a Com-
10], though data are limited on protein upregulation plete™ protease inhibitor cocktail (Roche Biochemicals,
[22,23]. Therefore, the purpose of this study was to inves- Indianapolis, IN) at a ratio of 1 ml buffer to 50 mg tissue.
tigate the extent and maturation of the neuroinflamma- Samples were allowed to stand at 4°C for at least 30 min-
tory response by examining cytokine protein increases utes before centrifugation at 8000 G for 5 minutes and
following GD exposure up to 72 hours after SE onset. removal of the lysate for assaying. Cytokine concentra-
Protein levels of ten cytokines were quantified using a tions were quantified using a rat cytokine multiplex bead
multiplex bead immunoassay in brain tissue lysates of SE- immunoassay kit containing IL-1α, IL-1β, IL-2, IL-4, IL-6,
injury susceptible regions (i.e., piriform cortex, thalamus IL-10, IL-12p70, IL-13, IL-17, and TNF-α (LINCO
and hippocampus). APR cytokines were markedly ele- Research, St. Charles, MO). The bead immunoassay pro-
vated in vulnerable brain regions and were localized to cedure used 25 μl of sample (94 ± 8 μg protein) per well
resident neural cells (i.e., neurons, astrocytes or micro- and was conducted according to the manufacturer's
glia). These data are the first to show concurrent cytokine instructions with each individual cytokine standard curve
protein upregulation and cellular origin of these factors and sample assayed in duplicate. The plate was read on a
following GD-induced SE. Luminex™ 100 instrument (Bio-Rad Laboratories, Hercu-
les, CA) and analyzed with either BioRad or STaRStation
software (Applied Cytometry, Sacramento, CA). TheMethods
Animals number of replicates for the experimental samples are as
Adult male Sprague-Dawley rats (Charles River Labora- follows: piriform cortex, n = 6 for each time point and
tories, Wilmington, MA; CRL: CD[SD]-BR, 250 - 350 g) naïve; hippocampus, n = 6 for each time point except for
were treated with HI-6 dichloride (Walter Reed Army naïve (n = 5), 6 hr (n = 5) and 24 hr (n = 7); thalamus, n =
Institute of Research, Silver Spring, MD; 125 mg/kg, i.p.) 5 for each time point and naïve except for 0.5 hr (n = 6), 6
30 minutes prior to GD administration and with atropine hr (n = 4), 12 hr (n = 3), 24 hr (n = 6) and 48 hr (n = 6).
methyl nitrate (AMN, Sigma-Aldrich, St. Louis, MO; 2.0 Time matched vehicle controls (n = 3 per time point)
mg/kg, i.m.) 1 minute after GD administration. Vehicle were analyzed individually and condensed into a single
control animals received HI-6, AMN and saline, while vehicle control comparison group when no significant
naïve animals received no injections. GD (GD-U-2323- difference was found between these samples over time by
CTF-N, purity 98.8 wt%) was diluted in saline at USAM- analyte or brain region.
RICD. GD (1.6 LD = 180 μg/kg) was administered sub-50
Immunohistochemistry (IHC)
cutaneously in the scruff of the neck and the rat was
Separate from the animals used in the multiplex bead
observed for convulsive activity. This dose of GD pro-
array immunoassay, experimental, vehicle control and
duces within minutes [24] a 100% generalized convulsive
naïve animals were deeply anesthetized, euthanized by
seizure rate that is maintained up to 24 hours [3]. The
decapitation at 12 hours after seizure onset and perfused
experimental protocol was approved by the Animal Care
with isotonic saline followed by 4% paraformaldehyde via
and Use Committee at the United States Army Medical
cardiac puncture. Brains were immediately frozen at -
Research Institute of Chemical Defense and all proce-
20°C, cut on a Leica CM3050 S cryostat (Thermo Shan-
dures were conducted in accordance with the principles
don, Inc.; Pittsburgh, PA) at 40 microns and stored in
stated in the Guide for the Care and Use of Laboratory
cryobuffer (30% each of glycerol, ethylene glycol and
Animals (National Research Council, 1996), and the Ani-
water, 10% 2 × phosphate buffer) until use. Free float fluo-
mal Welfare Act of 1966 (P.L. 89-544), as amended. The
rescent IHC labeling was conducted as previously
animal care program at this institute is fully accredited by
described [25]. Experimental and vehicle control s