Sources of axonal calcium loading during in vitro ischemia of rat dorsal roots.

A detailed understanding of injury mechanisms in peripheral nerve fibers will help guide successful design of therapies for peripheral neuropathies. This study was therefore undertaken to examine the ionic mechanisms of Ca 2+ overload in peripheral myelinated fibers subjected to chemical inhibition of energy metabolism. Myelinated axons from rat dorsal roots were co-loaded with Ca 2+ -sensitive (Oregon Green BAPTA-1) and Ca 2+ -insensitive (Alexa Fluor 594) dextran-conjugated fluorophores and imaged using confocal laser scanning microscopy. Axoplasmic regions were clearly outlined by the Ca 2+ -insensitive dye, from which axonal Ca 2+ -dependent fluorescence changes (F Ca.ax ) were measured. Block of Na + -K + ATPase (ouabain), opening of Na + channels (veratridine), and inhibiting energy metabolism (iodoacetate + NaN 3 ) caused a rapid rise in F Ca.ax to 96% above control after 30 min. Chemical ischemia (iodoacetate + NaN 3 ) caused a more gradual increase in F Ca.ax (54%), which was almost completely dependent on bath Ca 2+ , indicating that most of the Ca 2+ accumulation occurred via influx across the axolemma. Na + channel block (tetro-dotoxin) reduced ischemic F Ca.ax rise (14%); however, inhibition of L-type Ca 2+ channels (nimodipine) had no effect (60%). In contrast, Na + -Ca 2+ exchange inhibition (KB-R7943) significantly reduced ischemic F Ca.ax rise (18%). Together our results indicate that the bulk of Ca 2+ overload in injured peripheral myelinated axons occurs via reverse Na + -Ca 2+ exchange, driven by axonal Na + accumulation through voltage-gated tetrodotoxin-sensitive Na + channels. This mechanism may represent a viable therapeutic target for peripheral neuropathies.