Use of biomatrices to improve axon regeneration after chronic spinal cord injury in the rat [Elektronische Ressource] / vorgelegt von Veronica Estrada

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Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2010
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

Use of Biomatrices to
Improve Axon Regeneration after
Chronic Spinal Cord Injury in the Rat
Inaugural-Dissertation

zur Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf



vorgelegt von
Veronica Estrada
aus Neuss

Düsseldorf 2010



































Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf

Referent: Prof. Dr. H.W. Müller

Koreferent: Prof. Dr. D. Willbold

Tag der mündlichen Prüfung: 16.06.2010






“[SCIENCE IS] AN
IMAGINATIVE ADVENTURE
OF THE MIND SEEKING
TRUTH IN A WORLD OF
MYSTERY.”
Sir Cyril Herman Hinshelwood (1897-1967) TABLE OF CONTENS
ABBREVIATIONS ............................................................................................................................................ 6
1 INTRODUCTION .................................................................................................................................. 11
1.1 PATHOPHYSIOLOGY OF SPINAL CORD INJURY ..................................................................................................... 11
1.1.1 Spinal Cord Anatomy ..................................................................................................................... 11
1.1.2 The Injured Spinal Cord ................................................................................................................. 12
1.1.2.1 Outcome of Spinal Cord Injury ............................................................................................................ 12
1.1.2.2 Chronic Spinal Cord Injury ................................................................................................................... 13
1.1.2.3 Axonal Regeneration after Nerve Injury .............................................................................................. 14
1.2 THE LESION SCAR ! A BARRIER TO AXON REGENERATION ..................................................................................... 15
1.2.1 The Glial Scar Component ............................................................................................................. 17
1.2.2 The Fibrous Scar Component ........................................................................................................ 17
1.3 CURRENT APPROACHES IN THE TREATMENT OF SPINAL CORD INJURY ..................................................................... 20
1.4 BRIDGING THE INJURED SPINAL CORD .............................................................................................................. 21
1.4.1 Cellular Bridges .............................................................................................................................. 21
1.4.2 Acellular Scaffolds ......................................................................................................................... 22
1.4.2.1 Alginate Hydrogel ................................................................................................................................ 22
1.4.2.2 Collagen ............................................................................................................................................... 24
1.4.2.3 Matrigel ............................................................................................................................................... 24
1.4.2.4 Polyethylene Glycol ............................................................................................................................. 25
1.4.2.5 NeuroGel® ........................................................................................................................................... 26
1.4.2.6 Matrix Materials Applied in the Present Study ................................................................................... 26
1.5 THE ADULT RAT AS A MODEL SYSTEM FOR SPINAL CORD INJURY ........................................................................... 26
1.6 AIM OF THIS THESIS ...................................................................................................................................... 28
2 MATERIALS AND METHODS ................................................................................................................ 29
2.1 EXPERIMENTAL ANIMALS ............................................................................................................................... 29
2.2 BUFFERS AND ANTIBODIES ............................................................................................................................. 29
2.2.1 Buffers and Solutions .................................................................................................................... 29
2.2.2 Antibodies ..................................................................................................................................... 30
2.2.2.1 Primary Antibodies .............................................................................................................................. 30
2.2.2.2 Secondary Antibodies, Reagents and Tracer Substances .................................................................... 33
2.3 EXPERIMENTAL SETUP .................................................................................................................................. 34
2.3.1 Experimental Animal Groups ........................................................................................................ 34
2.3.2 Surgical Procedures ....................................................................................................................... 35
2.3.2.1 Dorsal Transection Lesion.................................................................................................................... 35
2.3.2.2 Scar Removal and Cavity Filling ........................................................................................................... 37
2.3.2.3 Matrix Preparation and Implantation .................................................................................................. 39
2.3.2.4 Anterograde Labeling of Axons in the Thoracic Spinal Cord ................................................................ 40
2.3.2.5 Anterograde Labeling of Corticospinal Tract Neurons ........................................................................ 41
2.3.3 Post!Operative Care ...................................................................................................................... 43
2.3.4 Animal Sacrifice ............................................................................................................................. 43
2.4 TISSUE PROCESSING ..................................................................................................................................... 43
2.4.1 Paraffin Sections ............................................................................................................................ 43
2.4.2 Cryo Sections ................................................................................................................................. 44
2.4.3 Freezing!Microtome Sections ....................................................................................................... 44
2.5 STAINING PROTOCOLS .................................................................................................................................. 45
2.5.1 Histological Stainings ..................................................................................................................... 45
2.5.1.1 Nissl Staining ....................................................................................................................................... 45
2.5.1.2 Masson Trichrome Staining ................................................................................................................. 45
3
TABLE OF CONTENS
2.5.2 Immunohistochemical Staining ..................................................................................................... 47
2.5.2.1 DAB!Staining of Paraffin Sections ........................................................................................................ 47
2.5.2.2 DAB!Staining of Cryo Sections ............................................................................................................. 48
2.5.2.3 Immunofluorescent Staining of Paraffin Sections ............................................................................... 49
2.5.2.4 Immunofluorescent Staining of Cryo Sections .................................................................................... 50
2.5.2.5 Immunofluorescent Staining of Freezing!Microtome Sections ........................................................... 51
2.6 ANALYSIS AND DOCUMENTATION .................................................................................................................... 51
2.6.1 Documentation of Immunohistologica