This study addresses the effects of calcium activated neutral protease inhibition by the tripeptide, leupeptin, on muscle and peripheral nerve after 1) primary and 2) delayed primary, epineural neurorrhaphy (nerve repair) in a primate Capuchin monkey model (Cebus apella). Our combined histological, ultrastructural, biochemical, toxicological and functional studies are designed to confirm and expand on leupeptin's in vivo inhibitory effects on muscle and neural calcium activated neutral protease. This enzyme has been convincingly implicated in mediating both Wallerian degeneration and secondary denervation muscle atrophy. Inhibition of this enzyme after peripheral nerve repair has potential for enhancement of neuromuscular recovery. We propose to investigate leupeptin's inhibition of the degenerative events after primary and delayed primary epineural nerve repair by a) standard techniques in light and electron microscopy; b) by immunohistochemical techniques for loci of calcium activated neutral protease; and c) by immunohistochemical techniques for fibronectin associated to basal laminae in myofibers and Schwann cells. Also, d) biochemical studies are designed to answer whether leupeptin's inhibition of the protease in muscle will enhance the appearance of a specific form of end-plate acetylcholinesterase (16S) which is a marker for efficient reinnervation of muscle. Further, e) Motor and sensory nerve conduction velocity evaluations are planned as functional assessment of reinnervation after primary and delayed primary nerve repair and leupeptin treatment. Finally, f) serial toxicological and absorption studies after leupeptin administration are planned. The goal of our studies is to further test leupeptin as an adjunctive therapy to peripheral primary and delayed primary nerve repair in facilitating an earlier and more efficient return to function.
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