Effects of neurotrophin-3 (NT-3) administration on gene expression and dorsal root ganglion neuron loss and repair following axotomy in adult rats
Following sciatic nerve transection in adult rats, a proportion of injured dorsal root ganglion (DRG) neurons die, through apoptosis, over the following 6 months. Axotomy also has effects on expression of neurotrophins (NGF, BDNF, and NT-3) and their receptors (trkA, trkB, trkC, and p75NTR) in DRG as shown by in situ hybridisation or Northern blotting. Previous groups showed that administration of neurotrophin-3 (NT-3) to the proximal stump or intrathecally appears to prevent neuronal loss and functional impairment after axotomy. This thesis tests the hypothesis that (1) Axotomy may cause certain cells to differentiate into DRG neurons and NT-3 may stimulate this process. (2) Systemic NT-3 may produce morphological and biochemical changes in DRG that may assist regeneration. During the course of the study, the 4th and 5th lumbar DRGs were examined up to 8 weeks after right sciatic nerve transection and ligation. Stereology was used to estimate neuronal numbers, and morphological and immunohistochemical techniques were used to examine the incidence of neuronal apoptosis. Antibodies for p-III tubulin, trkA, trkC and CGRP were applied to characterise nestin-immunoreactive cells. Real-time quantitative PCR was used to investigate the effects of axotomy and systemic NT-3 on the mRNA expression of neurotrophins, their receptors and nestin in injured DRGs at various time points. In addition, the effects of axotomy and NT-3 treatment on neuronal genes were investigated by microarray. The results obtained suggest that: (1) Axotomy led to 16% neuronal loss in L4 and L5 DRGs 4 weeks after injury administration of NT-3 systemically for 4 weeks prevented neuronal loss, but did not reduce neuronal apoptosis. The appearance of nestin- and p-III tubulin- immunoreactive cells in these DRGs suggests an axotomy-initiated replacement mechanism, which was enhanced by systemic NT-3 treatment. (2) The changes of neurotrophin and neurotrophin receptor mRNA suggest a higher overall responsiveness of DRG neurons to neurotrophins after axotomy and NT- 3 treament. (3) Microarray data showed the up-regulation of a few genes relevant to neuronal lineage commitment of progenitor cells following axotomy and that some signalling pathways were activated after axotomy and NT-3 administration. NT- 3 may stimulate axonal regeneration after axotomy.