Ca(2+) involvement in activation of extracellular-signal-regulated-kinase 1/2 and m-calpain after axotomy of the sciatic nerve

Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells (SCs), are poorly understood. Here, we investigated axotomy-induced activation of extracellular-signal-regulated kinase-1/2 (ERK1/2; important for proliferation) and m-calpain in vitro, and the relation to Ca(2+) deletion and Schwann cell proliferation and death after rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis(β-aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA). In some experiments, 5-bromo-2’-deoxyuridine (BrdU) was added during the last 24 hours to detect proliferating cells and propidium iodide (PI) was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2. The experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A significant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca(2+), activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca(2+) deprivation also significantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca(2+) seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca(2+) levels are also required for m-calpain activation and up-regulation in axons. Thus, regulation of Ca(2+) levels is likely to be a useful method to promote SC proliferation.