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

The mechanisms underlying resealing of transected myelinated rat dorsal root axons were investigated in vivo using an assay based on exclusion of a hydrophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (<5μm outer diameter) resealed faster than larger axons. Resealing was Ca²⁺ dependent, requiring micromolar levels of extracellular [Ca²⁺] to proceed, and further accelerated in 1mM Ca²⁺. Two hours after transection, 84% of axons had resealed in saline containing 2mM Ca²⁺, 28% had resealed in saline containing no added Ca²⁺ and only 3% had resealed in the Ca²⁺ buffer BAPTA (3mM). The enhancing effect of Ca²⁺ could be overcome by both non-specific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca²⁺-activated proteases. Resealing in 2mM Ca²⁺ was not inhibited by an inhibitor of phospholipase A₂. Resealing in low [Ca²⁺] was not enhanced by agents which disrupt microtubules, but was enhanced by dimethylsulfoxide (0.5-5%).These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca²⁺] contributes importantly to membrane resealing in transected myelinated mammalian axons in vivo. Copyright (C) 1999 IBRO.