Development of strategies to overcome limitations to functional recovery after peripheral nerve injuries

Nerve regeneration after peripheral nerve injuries is relatively better than after injuries to the central nervous system. The difference in the regenerative capacity is attributed to the provision of a growth-supportive environment by the Schwann cells of the of the peripheral nervous system. However, functional recovery after peripheral nerve injuries is often very poor despite the regenerative capacity. Factors limiting functional recovery are unknown, and tremendous advancements in the microsurgical repair of injured nerves have not made significant improvement in functional outcome after nerve injuries. Hence, using rat models of nerve injury and repair, the objectives of this thesis were, (1) to study some of the cell-molecular mechanisms of poor functional recovery after peripheral nerve injuries, and (2) to develop experimental strategies to promote functional recovery. We studied the progressive changes in the capacity of injured motoneurons to regenerate axons under conditions that mimic the pathophysiology of nerve injuries in humans (i.e. after immediate and delayed nerve repairs), and how these changes relate to the functional state of the Schwann cells of the distal nerve stumps. The effects of transforming growth factor-beta (TGF-beta) on the capacity of chronically denervated Schwann cells to support motor axonal regeneration and that of the immunophilin, FK506, to promote motor axonal regeneration after delayed nerve repair, were explored as possible strategies to promote functional recovery after nerve injuries. Adult male Sprague-Dawley rats were used in all experiments under aseptic conditions. The common peroneal (CP) and tibial (TIB) branches of rat sciatic nerve were used in a cross-suture paradigm of nerve injury and repair. Briefly, the CP and TIB nerves were cut and either immediate or delayed TIB-CP cross-suture was performed to allow regeneration of TIB motoneurons into freshly or chronically denervated CP nerve stumps. Direct neuroanatomical estimation of the numbers of TIB motoneurons that regenerated axons was carried out using fluorescent neurotracers (Fluorogold or Fluororuby) to backlabel TIB neuronal cell bodies. Also, numbers of regenerated axons were counted and their myelination by the SCs was examined histomorphologically. Reverse-transcriptase polymerase chain reaction was used to determine changes in gene expression of SCs.; The major findings of this thesis include (i) delayed nerve repair (>4weeks) leads to progressive deterioration of the capacity of the Schwann cells of the distal nerve stumps to support motor axonal regeneration; (ii) this declining capacity of Schwann cells to support motor axonal regeneration is due, at least in part, to the progressive downregulation of the expression of glial-derived neurotrophic factor; (iii) 48 hour in vitro incubation of chronically denervated Schwann cells with TGF-beta dramatically improved their capacity to support motor axonal regeneration in vivo; and (iv) FK506 increased the rate of axonal regeneration and the number of motoneurons that regenerated axons, even after delayed nerve repair. In conclusion, the results of this thesis demonstrate that poor functional recovery after nerve injuries is primarily due to the detrimental effect of delayed reinnervation of the Schwann cells of the distal stumps of injured nerves, since they lose their capacity to support motor axonal regeneration. However, these detrimental effects of delayed nerve repair are reversible by cytokines such as TGF-beta, and preventable by FK506 which accelerates motor axonal regeneration and thereby, promotes timely reinnervation of Schwann cells.