Axonal regeneration in biohybrid nerve guidance channels following implantation into rat spinal cord [Elektronische Ressource] / vorgelegt von Caterina Schulte-Eversum

Caty Schucany

Sujets

• Gesundheit

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2007
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 11 Mo

Extrait

Aus der Neurologischen Klinik des Universitätsklinikums
der Heinrich-Heine-Universität



Direktor: Prof. Dr. Hans-Peter Hartung





Axonal Regeneration
in Biohybrid Nerve Guidance Channels following
Implantation into Rat Spinal Cord






Dissertation



zur Erlangung des Grades eines Doktors der
Medizin

Der Medizinischen Fakultät der Heinrich-Heine Universität
Düsseldorf

vorgelegt von



Caterina Schulte-Eversum

2007
























Als Inauguraldissertation gedruckt mit Genehmigung der Medizinischen Fakultät der
Heinrich-Heine Universität Düsseldorf


gez.: Univ.-Prof. Dr. med. Dr. rer. nat. Bernd Nürnberg

Referent: Prof. Dr. Hans-Werner Müller

Korreferent: Prof. Dr. Guido Reifenberger









„Damit das Mögliche entsteht,
muß immer wieder das
Unmögliche versucht werden.“

Hermann Hesse











für meine Großmutter
CONTENTS


Abbreviations ………………………………………………………………………………………. 9

1.0 Introduction ……………………………………………………… …12
1.1 History on Nerve Injury ………………………………………………………..………... 12

1.2 Axonal Regeneration in the PNS versus CNS ……………………………………. …. 12

1.3 Pathophysiology of Spinal Cord Injury ……………………………………………..…. 13

1.4 Human Spinal Cord Injury …………………………………………………………... …. 14
1.4.1 Epidemiology ………………………………………………………………… …. 14
1.4.2 Classification …………………………… …... 15

1.5 Therapies of Spinal Cord Injury ……………………………………………………….. 16
1.5.1 Current Standard Therapies ……………………………………………….. …. 16
1.5.2 Therapies in Clinical Trial ………………………………………………….. …. 17

1.6 Different Approaches in Spinal Cord Injury Research ………………………………. 18

1.7 Regeneration Inhibiting Barrier – the Lesion Site ……………………………………. 20
1.7.1 Fibrous Scar …………………………………………………………………….. 21
1.7.2 Glial Scar ……………………………… 22
1.7.3 Anti-Scarring Treatment ……………………………………………………….. 22

1.8 Entubulation ……………………………………………………………………………… 24
1.8.1 History of Entubulation ………………………………………………………….
1.8.2 Qualities of a Guidance-Channel …………………………………………….. 27

1.9 Schwann Cells ……………………………………………………………………………28

1.10 Rodent Models in Spinal Cord Injury…….. …………………………………………… 29

1.11 Aim of this Thesis ……………………………………………………………………….. 31


42.0 Material and Methods ……………..………………………………32

2.1 Cell Culture ………………………………………………………………………………. 32
2.1.1 …………………………………………………………… 32
2.1.2 Retroviral Overexpression of eGFP in vitro ……………………….. 32

2.2 Buffers and Antibodies …………………………………………………………………. 33
2.2.1 Buffers and Other Solutions …………………………………………………… 33
2.2.2 Antibodies ……………………………………………………………………….. 35
2.2.2.1 Primary Antibodies ………………………………………………… 35
2.2.2.2 Secondary …………………………………………….. 36
2.2.2.3 Reagents and Tracers ……………………………………………. 36

2.3 Guidance Channels …………………………………………………………………….. 36
2.3.1 Tube Material ……………………………………………………………………. 36
2.3.2 Improvement of the Guidance Channel Filling Procedure …………………. 37

2.4 Experimental Settings ………………………………………………………………….. 39

2.5 Surgery …………………………………………………………………………............... 40
2.5.1 Optimisation of the Surgery ……………………………………………………. 40
2.5.2 Protocol of the Optimised Operating Technique ……………………………. 41
2.5.3 Post-Operative Care ……………………………………………………………. 43
2.5.4 Animal Sacrifice ………………………………… 43

2.6 Tracing …………………………………………………………………………………… 43
2.6.1 Stereotaxic Methods ……………………………………………………...........
2.6.2 Injection of BDA into Layer V of the Sensomotoric Cortex ………………… 44
2.6.3 Anterograde Tracing of the Dorsal Column at Th11 with BDA …………….. 44

2.7 Tissue Processing ………………………………………………………………………. 45
2.7.1 Paraffin Sections ……………………………………………………………….. 45
2.7.2 Freezing-Microtome Sections …………………………………………………. 46

2.8 Staining Protocols ………………………………………………………………………. 46
2.8.1 Histological Stainings …………………………………………………………... 46
2.8.2 Immunohistochemical Stainings ……………………………………………… 48
2.8.2.1 Paraffin Sections ………………………………………………….. 48
2.8.2.2 Fluorescent Staining of Freezing-Microtome Sections ………… 50

2.9 Analysis and Documentation ………………………………………………………….. 50
53.0 Results ……………………………………………………………. ...51

3.1 Improvement of the Experimental Setting ……………………………………………. 51
3.1.1 Improvement of the Guidance Channel Filling Procedure …………………. 51
3.1.2 Optimisation of the Surgery ……………………………………………………. 51
3.1.3 Histological Phenomena observed in Nissl-Stainings …………………… 52
3.1.3.1 Guidance Channel Placement …………………………………… 52
3.1.3.2 Guidance Channel Stability ………………………………………. 53
3.1.3.3 Tissue Bridge ……….………………………………………………54
3.1.3.4 Guidance Channel Wall Presence ………………………………. 56
3.1.3.5 Bleeding Sites ……………………………………………………… 56
3.1.3.6 Cyst-like Structures ……………………………………………….. 57
3.1.3.7 Tearing apart of Tissue …………………………………………… 59

3.2 Material Properties of the Implant …………………………………………………….. 60
3.2.1 Tube Degradation ………………………………………………………………. 60
3.2.1.1 Quota of Luminal Sections ……………………………………….. 60
3.2.1.2 Tube Deformation …………………………………………………. 61
3.2.1.3 Cellular Infiltration of the Tube Wall ……………………………...

3.3 Characterisation of the Spinal Cord Lesion ………………………………………….. 63
3.3.1 Lesion Size ……………………………………………………………………… 63
3.3.1.1 Role of Inbleeding on the Lesion Size …………………………...
3.3.2 Cellular Reactions after Lesion ……………………………………………….. 65
3.3.2.1 Astrogliosis ……………………………………. 65
3.3.2.2 Inflammation …………………………………..67

3.4 Visualisation, Localisation and Survival of Implanted Schwann Cells …………….. 68
3.4.1 Visualisation of Implanted Schwann Cells …………………………………… 68
3.4.2 eGFP/DAPI Double-Staining ………………………………………………….. 69
3.4.3 Survival and Quantity of Implanted Schwann Cells ………………………… 70
3.4.4 Schwann Cell Localisation …………………………………………………….. 70

3.5 Cells Invading the Guidance Channel ………………………………………………… 72
3.5.1 Morphologically Schwann Cell-resembling Cells ……………………………. 72
3.5.2 Small Round Cells ……………………………………………………………… 73

3.6 Scar Formation ………………………………………………………………………….. 73
3.6.1 The Glial Scar …………………………………………………………………… 73
3.6.2 The Collagen IV containing Fibrous Scar ……………………………………. 74
3.6.2.1 Description of the Fibrous Scar in this Injury Model …………… 74
3.6.2.2 Effect of Schwann Cells on Scarring …………………………….. 76
6 3.6.2.3 Influence of the Anti-Scarring Treatment ……………………… 76
3.6.2.4 Development of the Scar with Time …………………………….. 78
3.6.3 Phenomena observed in Collagen IV-immunopositive Sections ………….. 79
3.6.3.1 Classification of the Fibrous Scar ………………………………... 79
3.6.3.2 Cyst or Ripped Tissue? …………………………………………… 81
3.6.3.3 Difficulties regarding the Application of Matrigel® …………….. 82

3.7 Axonal Degeneration and Regrowth ………………………………………………….. 83
3.7.1 Axonal Degeneration …………………………………………………………… 83
3.7.1.1 Description of Stainings with PAM in different Lesion Models .. 83
3.7.1.2 Axonal Retraction …………………………………………………. 84
3.7.1.3 Role of Inbleeding on Axon Stop ………………………………… 86
3.7.2 Axonal Regrowth into the Implant ……………………………………………. 88
3.7.2.1 Growth into Cell-free versus Schwann Cell-filled Tubes ……… 88
3.7.2.2 Untreated Animals versus Anti-Scarring Treated Animals ……. 91
3.7.2.3 Association of Schwann Cells with Regenerating Fibres ……… 93
3.7.2.4 Observations in Immunohistochemical Stainings with PAM ….. 94
3.7.3 Traced Fibre Tracts …………………………………………………………….. 95
3.7.3.1 Fibre Origin …………………………………………………………. 95
3.7.3.2 CST Tracing with BDA …………………………………………….95
3.7.3.3 Dorsal Column Tracing with BDA ………………………………... 97


4.0 Discussion …………………………………………………………..98

4.1 Improvement of the Experimental Setting ……………………………………………. 98
4.1.1 Improvement of the Guidance Channel Filling Procedure …………………. 98
4.1.2 Optimisation of the Surgery ……………………………………………………. 99
4.1.3 Histological Phenomena observed in Nissl-Stainings ………………………. 99

4.2 Material Properties of the Implant …………………………………………………… 102
4.2.1 Material Properties …………………………………………………………… 102
4.2.2 Tube Degradation …………………………………………………………… . 102

4.3 Characterisation of the Spinal Cord Lesion ………………………………………… 103
4.3.1 Lesion Size ……………………………………………………………............ 103
4.3.2 Cellular Reactions after Lesion ………………………………………. ……. 104
4.3.2.1 Astrogliosis ………………………………………………... ……. 104
4.3.2.2 Inflammation …………………………………………………….. 105

4.4 Visualisation, Localisation and Survival of Implanted Schwann Cells ………….. 106

74.5 Cells invading the Guidance Channel ………………………………………………. 109

4.6 Scar Formation ………………………