Sardella, Thomas Christian Peter
Promoting bridging axonal regeneration after spinal cord injury.
PhD thesis, University of Glasgow.
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Olfactory ensheathing cells (OECs) are amongst the best candidates for cell transplantation into lesions of the central nervous system. These specialised glial cells are found in the olfactory system and are associated with the physiological restoration of neuronal circuitry in the first cranial nerve of the adult. There is conflicting evidence on the extent to which OECs promote long distance axonal regeneration when transplanted into spinal cord lesions. In the first chapter of Results (chapter 3) we aimed to determine the potential of OEC transplantation in promoting axonal regeneration beyond the injury. This was investigated by producing a transection of the dorsal columns at the lumbar 3-lumbar 4 (L3-L4) border using a wire knife device that cut ascending primary sensory afferents and immediately after, transplanting olfactory bulb derived OECs into the lesion. Regeneration was assessed in these fibres by injecting the neurotracer biotin dextran amine (BDA) into the left L4 and L5 spinal roots and sacrificing the animals 6 weeks after the lesion. We detected axonal labelling in the lumbar and thoracic cord rostral to the dorsal column lesion; but the labelled axons included spared fibres. For this reason it was necessary to develop a quantification method that included only convincing regeneration and excluded sparing or potential sprouting. With care it was possible to detect convincing axonal regeneration of dorsal root ganglia (DRG) neurons across the lesion in animals transplanted with OECs. Although there have been several claims of axonal regeneration following OEC transplantation in spinal cord pathways information is still lacking on the spatial relationship between regenerating axons and transplanted OECs. To understand this, OECs were transduced before transplantation with a lentiviral vector to express green fluorescent protein (GFP). With this technique it was possible to detect the OECs in the transplanted spinal cord and determine that tract traced axons preferentially regenerated though continuous tracts of GFP labelled OECs in continuity with the transplanted lesion, while they did not normally regenerate through the host spinal cord in areas devoid of OECs.
Regeneration beyond the lesion in OEC transplanted animals was very limited in terms of both distance and numbers of axons. The conditioning lesion of the sciatic nerve is a procedure which appears to prime DRG neurons for growth. We next asked whether the regeneration observed after an OEC transplant alone could be maximised by a combined treatment paradigm that included OEC transplantation and conditioning lesions. Conditioning lesions were performed by transecting the left sciatic nerve at mid thigh level and the spinal cord was lesioned at the L3-L4 level. Long distance bridging axonal regeneration was strongly potentiated (58 fold). As seen in OEC transplanted animals, axons in the distal spinal cord were located preferentially in areas where OECs were also present. Regeneration was also studied at earlier and later time points. The number of regenerating axons was significantly greater if survival time was of 2 weeks from the lesion/transplantation compared to the 6 week survival, while it did not vary significantly between 6 weeks and 17 weeks survival.
Direct comparison of the growth promoting effects exerted by OECs and Schwann cells have not so far been performed on transection type lesions, but only on other lesion models where sparing cannot be ruled out. To understand if these two cell types share similar properties with reference to promoting long distance axonal regeneration, in the second chapter of Results (chapter 4) we consider sciatic nerve derived Schwann cell transplantation alone or in combination with conditioning lesions following the same lesion paradigm in which OECs were transplanted. Interestingly, no significant difference was found between the growth promoting properties of the two glial cells. Furthermore, with Schwann cells as with OECs, regenerating axons in the distal spinal cord were associated mostly with the transplanted cells. To understand whether the long distance axonal regeneration observed was occurring irrespective of the cell type injected, we transplanted sciatic nerve derived fibroblasts in combination with conditioning lesions. With this lesion paradigm significantly less regeneration was detected indicating that fibroblasts are less effective than OECs and Schwann cells in terms of their regeneration promoting properties. On the other hand we determined that OECs, Schwann cells and fibroblasts all preserved the lesion integrity by preventing the lesion from dilating. Importantly, rats are now being used as a model of study for syringomyelia, a pathology that leads to progressive central canal cavitation. We have found that dilation of the central canal normally develops also in the wire knife dorsal column lesion model; however transplantation of OECs or Schwann cells significantly reduced the central canal dilation compared to non-transplanted animals.
Any clinical use of cell transplantation will likely involve a delay between injury and treatment, and since a glial scar will develop and the regenerative response of the axotomised fibres may subside, it is not clear to what extent regeneration will occur. In the third chapter of results (chapter 5) we have therefore investigated whether delayed OEC transplantation might promote a significant level of regeneration. Surprisingly, the distribution of OECs following delayed transplantation was similar to that after an acute transplant and OECs formed tracts that were in continuity with the lesion transplant. The presence of these OEC tracts allowed regenerating axons to bridge the lesion and grow for relatively long distances on the other side. Delayed conditioning lesions were combined with delayed OEC transplantation and boosted axonal growth 9 times compared to delayed OEC transplanted animals.
||OEC, olfactory ensheathing cells, Schwann cells, fibroblasts, spinal cord injury, wire knife, conditioning lesions, transplant, delayed transplant
||Q Science > Q Science (General)
||College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
||Riddell, Dr. John S. and Barnett, Prof. Susan C.
|Date of Award:
Mr Thomas Sardella
||Copyright of this thesis is held by the author.
||16 Mar 2010
||10 Dec 2012 13:39
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