Overexpression of TPA in the spinal cord promotes corticospinal axonal remodeling and motor behavioral recovery in mice after stroke

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Our previous studies demonstrated that axonal remodeling of the corticospinal tract (CST) in the spinal cord contributes to neurological recovery after stroke in rodents. In the present study, we designed a novel non-invasive peripheral approach, to over-express TPA in the denervated gray matter of the spinal cord mediated via recombinant adeno-associated virus (AAV) intramuscular injection. Here, TPA is not used as a fibrinolytic agent in the circulation system, but as a neurorestorative agent in the central nervous system. The study was performed in transgenic mice subjected to permanent middle cerebral artery occlusion (MCAo) or sham-MCAo, in which the CST axons are specifically and completely labeled with yellow fluorescent protein (YFP). One day after surgery, mice were randomly selected to receive AAV serotype 5 encoding RFP or TPA under neuron specific Synapsin promoter injected into the stroke-impaired forelimb wrist extensor and flexor muscles at a dose of 1x1010 viral particles in 5 μl total volume (n=10/group). The functional deficits and recovery were monitored with a series of foot-fault test and single pellet reaching performed prior to and on day 3 after stroke, and weekly thereafter. Mice were euthanized at day 28 after MCAo. The cervical enlargements were processed for vibratome traverse section to examine YFP labeling with a laser scanning confocal imaging system. Single layer confocal images were used to measure axonal densities in the central area of the gray matter on 30 consecutive sections. The ratio of axonal densities in the stroke-affected side and intact side on same sections was used as an index to evaluate axonal remodeling. Compared with AAV-RFP treated mice, intramuscular injection of AAV-tPA significantly enhanced behavioral recovery measured in both functional tests (p<0.01), as well as the index of CST axonal remodeling (p<0.001). Our results provided a fundamental basis for further development of therapeutic approaches aimed on promoting corticospinal innervation in the denervated spinal gray matter to enhance neurological recovery after stroke. If successful, this would greatly impact a large population of stroke patients with a significant unmet medical need of efficacious stroke treatment beyond the acute stage.




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