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

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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.
Publié le : lundi 1 janvier 2007
Lecture(s) : 40
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Source : DOCSERV.UNI-DUESSELDORF.DE/SERVLETS/DERIVATESERVLET/DERIVATE-4108/DRUCKVERSION_DRARBEIT_2007.PDF
Nombre de pages : 144
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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 ………………………………………………………………………… 111
4.6.1 Glial Scar ………………………………………………………………………. 111
4.6.2 The Collagen IV containing Fibrous Scar ………………………………….. 111

4.7 Axonal Degeneration and Regrowth ………………………………………………… 116
4.7.1 Axonal Degeneration …………………………………………………………. 116
4.7.2 Axonal Regrowth ……………………………………………………………… 117
4.7.3 Traced Fibre Tracts ………………………………………………….............. 121


5.0 References ……………………………………………................. 123

Danksagung…………………………………………………………………………………….. 141

Curriculum vitae………………………………………………………………………………… 142

Summary and Perspectives…………………………………………………………………… 143

Zusammenfassung …………………………………………………………………………….. 144
8ABBREVIATIONS


8-Br-cAMP 8-Brome-cyclic Adenoside MonoPhosphate
AB Antibody
ABC Avidine-Biotine-Complex
AH Anterior Horn
ASIA American Spinal Cord Injury Association
AST Anti-Scarring Treatment
tube filled with Matrigel/Schwann Cell mélange plus AST
Avi-Alexa Avidin-Alexa 488
BBB Blood-Brain Barrier
BDA Biotinylated Dextrane-Amine
BDNF Brain derived Nerve Growth Factor
BM Basal Membrane
BPY-DCA 2,2`- Bipyridine-5,5`-Dicarboxylic Acid (Iron Chelator)
BWSTT Body-weight supported Treadmill Training
C3 C. botulinum
2+Ca Calcium
CC Central Canal
CNS Central Nervous System
Coll IV Collagen type IV
CSF Cerebrospinal Fluid
CSPG Chondroitinsulphate Proteoglycane
CST Corticospinal Tract
DAB Diaminobenzidine
DC Dorsal Column
DMEM Dulbecco's Modified Eagle's Medium
DPX a Xylole-containing mounting fluid
DRG Dorsal Root Ganglia
ECM Extracellular Matrix
eGFP Green Fluorescent Protein
Elvax Ethylene Vinyle Acetate Copolymer
EtOH Ethanol
FCS Fetal Calf Serum
FES Functional Electrical Stimulation
FF Free-Floating (staining method)
Fig. Figure
G Gauge
9G&R Goat anti Rabbit
GAG Glycosaminoglycane
GCW Guidance Channel Wall
GDNF Glial Cell-Line Derived Neurotrophic Factor
GFAP Glial Fibrillary Acidic Protein
gt Goat
H&M Horse anti Mouse
HCl Hydrochloric Acid
HE Hematoxyline-Eosine staining
HEMA Hydroxyethyl - Methylacrylat
hs Horse
+K Potassium
MAG Myelin associated Gylcoprotein
ME-008 Guidance Channel material
ME-021
Med Medium
MetOH Methanol
MG Matrigel®
MG/Med Tube filled with Matrigel/Medium mélange
MG/SC d with Matrigel/Schwann Cell mélange
n Number
+Na Sodium
Na HPO Di-sodium Hydrogenphosphate Anhydrous 2 4
Na HPO x 2 H0 Sodium di-Hydrogenphosphate 2 4 2
NaCl Chloride
NaH PO x H0 di-Hydrogenphosphate Monohydrate 2 4 2
NaOH Sodium Hydroxide solution
NGF Nerve Growth Factor
NGF, BDNF, NT-3, GDNF Trophic Factors
NgR Nogo Receptor
NTF Neurotrophic Factor
OEC Olfactory Ensheathing Cell
OMgp Oligodendrocyte Myelin Glycoprotein
ORFs Open Reading Frames
PAM Pan-axonal Marker
PAN/PVC Poly(acrylonitrile) poly(vinyl chloride)
PB Phosphate Buffer
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
10

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