Ca2+/calmodulin‐dependent protein kinase II and Dimethyl Sulfoxide affect the sealing frequencies of transected hippocampal neurons

Traumatic injury often results in axonal severance, initiating obligatory Wallerian degeneration of distal segments, whereas proximal segments often survive. Calcium ion (Ca2+) influx at severed proximal axonal ends activates pathways that can induce apoptosis. However, this same Ca2+‐influx also activates multiple parallel pathways that seal the plasmalemma by inducing accumulation and fusion of vesicles at the lesion site that reduce Ca2+‐influx and enhance survival. We examined whether various inhibitors of Ca2+/calmodulin‐dependent protein kinases (CaMKs), and/or dimethyl sulfoxide (DMSO), a common solvent for biologically active substances, affected the ability of a hippocampal‐derived neuronal cell line (B104 cells) to seal membrane damage following axotomy. Axolemmal sealing frequencies were assessed at different transection distances from the axon hillock and at various times after Ca2+‐influx (PC times) by observing whether transected cells took‐up fluorescent dyes. Inhibition of CaMKII by tatCN21 and KN‐93, but not inhibition of CaMKI and CaMKIV by STO‐609, affected axonal sealing frequencies. That is, CaMKII is a component of previously reported parallel pathways that induce membrane sealing, whereas CaMKI and CaMKIV are not involved. The effects of these CaMKII inhibitors on plasmalemmal sealing depended on their mechanism of inhibition, transection distance, and PC time. DMSO at low concentrations (90 µM–28 mM or 0.00064%–0.2% v/v) significantly increased membrane‐sealing frequencies at most PC times and transection distances, possibly by permeabilizing the plasmalemma to Ca2+. Inhibition of CaMKII, DMSO, PC time, and the transection distance significantly affect plasmalemmal sealing that is critical to somal survival in traumatic lesions. This paper examines substances that increase or decrease the probability that a B104 cell will seal (left) or will not seal (right) at various times after adding calcium, and at various transection distances (large arrow) from the axon hillock (delineated by small arrowheads).