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J Neurotrauma. 2012 Nov 1;29(16):2555-63. doi: 10.1089/neu.2012.2473. Epub 2012 Aug 29.

Calpastatin overexpression protects axonal transport in an in vivo model of traumatic axonal injury.

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Department of Emergency Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.


Traumatic brain injury (TBI) causes substantial morbidity and mortality worldwide. A key component of both mild and severe TBI is diffuse axonal injury. Except in cases of extreme mechanical strain, when axons are torn at the moment of trauma, axonal stretch injury is characterized by early cytoskeletal proteolysis, transport disruption, and secondary axotomy. Calpains, a family of Ca(2+)-dependent proteases, have been implicated in this pathologic cascade, but direct in vivo evidence is lacking. To test the hypothesis that calpains play a causal role in axonal stretch injury in vivo, we used our rat optic nerve stretch model following adeno-associated viral (AAV) vector-mediated overexpression of the endogenous calpain inhibitor calpastatin in optic nerve axons. AAV vectors were designed for optimal expression of human calpastatin (hCAST) in retinal ganglion cells (RGCs). Calpain inhibition by the expressed protein was then confirmed in primary cortical cultures. Finally, we performed bilateral intravitreal injections of AAV vectors expressing hCAST or the reporter protein ZsGreen 3 weeks prior to unilateral optic nerve stretch. Immediately after stretch injury, Fluoro-Gold was injected into the superior colliculi for assessment of retrograde axonal transport. Rats were euthanized 4 days after stretch injury. Both hCAST and ZsGreen were detected in axons throughout the optic nerve to the chiasm. Calpastatin overexpression partially preserved axonal transport after stretch injury (58.3±15.6% reduction in Fluoro-Gold labeling relative to uninjured contralateral controls in ZsGreen-expressing RGCs, versus 33.8±23.9% in hCAST-expressing RGCs; p=0.038). These results provide direct evidence that axonal calpains play a causal role in transport disruption after in vivo stretch injury.

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