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Title page
Contents
ABBREVIATIONS 5
ABSTRACT 7
INTRODUCTION 9
MATERIALS AND METHODS 13
Animals 13
MSC isolation and cultivation from canine bone marrow 13
Flow-cytometric determination of the cell-surface antigen profile 16
Differentiation protocols 16
SCI model 18
Transplantation of MSCs after SCI 21
Behavioral analysis based on the Olby score 22
MRI 22
CSF analysis 23
Postmorterm examination, histopathological and immunohistoche-mical analysis 23
Neurotrophic factor expression in the SCI lesion 25
Statistical analysis 26
RESULTS 30
Characterization of canine MSCs 30
Osteogenic differentiation of canine MSCs 33
Neuronal differentiation of canine MSCs 35
Behavioral analysis based on the Olby score 40
MRI analysis 42
CSF analysis 45
Postmorterm examination, histopathological and immunohistochemical 47
Neurotrophic factor expression in the SCI lesion 62
DISCUSSION 65
CONCLUSION 72
국문요약 73
REFERENCES 75
ACKNOWLEDGEMENT 83
Table 1. The Olby scoring system for pelvic limb function 27
Table 2. Primer sequences used in the RT-PCR of neurotrophic factors 29
Table 3. The size of injured spinal cord area at 1 week and 5 weeks after SCI in three groups based on MRI findings 44
Table 4. Percentage of vacuolar and cystic cavity formation area in transverse section of the epicenter of injured spinal cord at 2 and 5 weeks after SCI 55
Fig. 1. Bone marrow aspiration from the greater tubercle of the humerus 15
Fig. 2. Photographs of the balloon compression procedure of SCI used in this study 20
Fig. 3. Photomicrographs of canine bone marrow-derived MSCs 31
Fig. 4. Flow-cytometric determination of the cell-surface antigen profiles of canine MSCs 32
Fig. 5. ALP staining before and after osteogenic induction 34
Fig. 6. Photomicrographs following neuronal differentiation 37
Fig. 7. Immunocytochemical photomicrographs of canine MSCs following neuronal differentiation and staining with NF-M, NSE, NeuN, and GFAP 38
Fig. 8. Double staining immunofluorescent photomicrographs of canine MSCs following neuronal differentiation induced by IBMX/dbcAMP 39
Fig. 9. Graphical illustration of the assessment of pelvic limb function 41
Fig. 10. T2-weighted sagittal MR images of the spinal cord of dogs from control, autologous and allogenic group 44
Fig. 11. CSF cytology after autologous and allogenic MSC transplantation 46
Fig. 12. Gross findings at 2 weeks after SCI 50
Fig. 13. Gross findings at 5 weeks after SCI 51
Fig. 14. Low-power and high-power H & E image of transverse spinal cord section at injured lesion (2 weeks after SCI) 53
Fig. 15. Low-power and high-power H & E image of transverse spinal cord section at injured lesion (5 weeks after SCI) 55
Fig. 16. Graphical illustrations of intensities of the prelabeled MSCs on the epicenter of injured spinal cord in autologous and allogenic groups 56
Fig. 17. Immunohistochemical analysis of samples from clinically normal dog and spinal injured dog by using laser scanning confocal microscopy 57
Fig. 18. Immunofluorescence colocalization studies of autologous group at 1 week after MSC transplantation 58
Fig. 19. Immunofluorescence colocalization studies of autologous group at 4 weeks after MSC transplantation 59
Fig. 20. Immunofluorescence colocalization studies of allogenic group at 1 week after MSC transplantation 60
Fig. 21. Immunofluorescence colocalization studies of allogenic group at 4 weeks after MSC transplantation 61
Fig. 22. Results of RT-PCR (A) and densitometric analysis (B) of neurotrophic factor expression at 2 weeks after SCI (at 1 week after MSC transplantation) 63
Fig. 23. Results of RT-PCR (A) and densitometric analysis (B) of neurotrophic factor expression at 5 weeks after SCI (at 4 weeks after MSC transplantation) 64
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