Mechanisms of ischaemic damage to central white matter axons: a quantitative histological analysis using rat optic...

The mechanism of ischaemic injury to white matter axons was studied by transiently depriving rat optic nerves in vitro of oxygen and glucose. Light and electron microscopic analysis showed that increasing periods of oxygen/glucose deprivation (up to 1 h) caused, after a 90-min recovery period, the appearance of increasing numbers of swollen axons whose ultrastructure indicated that they were irreversibly damaged. This conclusion was supported by experiments showing that the damage persisted after a longer recovery period (3 h). To quantify the axonal pathology, an automated morphometric method, based on measurement of the density of swollen axons, was developed. Omission of Ca2+ from the incubation solution during 1 h of oxygen/glucose deprivation (and for 15 min either side) completely prevented the axonopathy (assessed following 90 min recovery). Omission of Na+ was also effective, though less so (70% protection). The classical Na+ channel blocker, tetrodotoxin (1 microM), provided 92% protection. In view of this evidence implicating Na+ channels in the pathogenesis of the axonal damage, the effects of three different Na+ channel inhibitors, with known neuroprotective properties towards gray matter in in vivo models of cerebral ischaemia, were tested. The compounds used were lamotrigine and the structurally-related molecules, BW619C89 and BW1003C87. All three compounds protected the axons to varying degrees, the maximal efficacies (observed at 30 to 100 microM) being in the order: BW619C89 (>95% protection) > BW1003C87 (70%) > lamotrigine (50%). At a concentration affording near complete protection (100 microM), BW619C89 had no significant effect on the optic nerve compound action potential. Experiments in which BW619C89 was added at different times indicated that its effects were exerted during two distinct phases, one (accounting for about 50% protection) was during the early stage of oxygen/glucose deprivation itself and the other (also about 50%) during the first 15 min of recovery in normal incubation solution. The results are consistent with a pathophysiological mechanism in which Na+ entry through tetrodotoxin-sensitive Na+ channels contributes to Na+ loading of the axoplasm which then results in a lethal Ca2+ overload through reversed Na(+)-Ca2+ exchange. The identification of BW619C89 as a compound able to prevent oxygen/glucose deprivation-induced injury to white matter axons without affecting normal nerve function opens the way to testing the importance of this pathway in white matter injury in vivo.