Resealing of transected myelinated mammalian axons in vivo: Evidence for involvement of calpain

The mechanisms underlying resealing of transected myelinated rat dorsal roo t axons were investigated in vivo using an assay based on exclusion of a hy drophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (< 5 mu m outer diameter) resealed faster than larger axons. Resealing was Ca2 + dependent, requiring micromolar levels of extracellular [Ca2+] to proceed , and further accelerated in 1 mM Ca2+. Two hours after transection, 84% of axons had resealed in saline containing 2 mM Ca2+, 28% had resealed in sal ine containing no added Ca2+ and only 3% had resealed in the Ca2+ buffer BA PTA (3 mM). The enhancing effect of Ca2+ could be overcome by both non-spec ific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca2+-activated proteases. Resealing in 2 mM Ca2 was not inhibited by an inhibitor of phospholipase A(2). Resealing in low [Ca2+] was not enhanced by agents which disrupt microtubules, but was enhan ced by dimethylsulfoxide (0.5-5%). These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca2+] contributes importantly to membrane reseali ng in transected myelinated mammalian axons in vivo. (C) 1999 IBRO. Publish ed by Elsevier Science Ltd.