Our previous studies have shown that fibroblasts genetically modified to express Brain Derived Neurotrophic Factor (BDNF) promote long distance regeneration of rubrospinal axons and partial recovery of motor function when transplanted into a subtotal hemisection spinal cord injury in adult rats. The proposed experiments will test the idea that additional populations of supraspinal neurons important for locomotion will regenerate in response to transplants of fibroblasts engineered to express BDNF and neurotrophin-3 (NT3).
Our aim i s to demonstrate that these transplants will be effective not only when provided at the time of injury but also after a delay and can therefore be applied to models of chronic spinal cord injury. The first set of experiments will test the hypothesis that fibroblasts genetically modified to express BDNF or NT3 or a combination of these fibroblasts will elicit regeneration of corticospinal, vestibulospinal, and rubrospinal axons when transplanted acutely into a cervical hemisection. Anterograde and retrograde tracing techniques will provide a quantitative and qualitative characterization of the regeneration, including the numbers of neurons that regenerate, their path, length and targets. Immunocytochemical methods will identify host dorsal roots that grow into the transplants and serotonergic and noradrenergic axons originating in the brainstem that grow into the transplants and caudal host spinal cord. The second set of experiments will test the idea that engineered fibroblasts that promote the greatest amount of regeneration in rats with acute hemisections, presumably a combination of BDNF- and NT3-expressing fibroblasts, will also elicit regeneration of supraspinal axons when transplanted into a hemisection after delays as long as 12 weeks (chronic injury). We will maximize the survival of axotomized neurons by introducing the antiapoptotic Bcl-2 gene by injection of plasmid both at the time of the initial injury and at the time of transplantation. Retrograde and anterograde tracing studies and immunocytochemical methods will permit analysis of the regeneration. The third set of experiments will examine the effects of these transplants in acute and chronic spinal cord transections. Transection is the most challenging but also the least ambiguous experimental spinal cord injury, and regeneration in this model will represent compelling confirmation of the effectiveness of these transplants. A battery of tests will evaluate motor and sensory function in each series of experiments, and re-lesion rostral to the initial injury will indicate whether regenerated axons contribute to recovery. Physiological assessment of the recovered functions will be studied as a component of two Projects. The success of these strategies will be an important step toward developing an effective treatment in humans.
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