Cellular mechanisms by which lipoic acid confers protection during the early stages of cerebral ischemia: a possible role for calcium.

Lipoic acid (LA) is a naturally occurring compound and dietary supplement with powerful antioxidant properties. Although LA is neuroprotective in models of stroke, little is known about the cellular mechanisms by which it confers protection during the early stages of ischemia. Here, using a rat model of permanent middle cerebral artery occlusion (MCAO), we demonstrated that administration of LA 30 min prior to stroke, reduces infarct volume in a dose dependent manner. Whole-cell patch clamp techniques in rat brain slices were used to determine if LA causes any electrophysiological alterations in either healthy neurons or neurons exposed to oxygen and glucose deprivation (OGD). In healthy neurons, LA (0.005 mg/ml and 0.05 mg/ml) did not significantly change resting membrane potential, threshold or frequency of action potentials or synaptic transmission, as determined by amplitude of excitatory post synaptic currents (EPSCs). Similarly, in neurons exposed to OGD, LA did not alter the time course to loss of EPSCs. However, there was a significant delay the onset of anoxic depolarization as well as in the time course of the depolarization. Next, intracellular calcium (Ca(2+)) levels were monitored in isolated neurons using fura-2. Pretreatment with 0.005 mg/ml and 0.05 mg/ml LA for 30 min and 6 h did not significantly alter resting Ca(2+) levels or Ca(2+) response to glutamate (250 µM). However, pretreatment with 0.5 mg/ml LA for 6 h significantly increased resting Ca(2+) levels and significantly decreased the Ca(2+) response to glutamate. In summary, these findings suggest that LA does not affect neuronal physiology under normal conditions, but can protect cells from an ischemic event.

A novel method for inducing focal ischemia in vitro.

Current in vitro models of stroke involve applying oxygen-glucose deprived (OGD) media over an entire brain slice or plate of cultured neurons. Thus, these models fail to mimic the focal nature of stroke as observed clinically and with in vivo rodent models of stroke. Our aim was to develop a novel in vitro brain slice model of stroke that would mimic focal ischemia and thus allow for the investigation of events occurring in the penumbra. This was accomplished by focally applying OGD medium to a small portion of a brain slice while bathing the remainder of the slice with normal oxygenated media. This technique produced a focal infarct on the brain slice that increased as a function of time. Electrophysiological recordings made within the flow of the OGD solution ("core") revealed that neurons rapidly depolarized (anoxic depolarization; AD) in a manner similar to that observed in other stroke models. Edaravone, a known neuroprotectant, significantly delayed this onset of AD. Electrophysiological recordings made outside the flow of the OGD solution ("penumbra") revealed that neurons within this region progressively depolarized throughout the 75 min of OGD application. Edaravone attenuated this depolarization and doubled neuronal survival. Finally, synaptic transmission in the penumbra was abolished within 50 min of focal OGD application. These results suggest that this in vitro model mimics events that occur during focal ischemia in vivo and can be used to determine the efficacy of therapeutics that target neuronal survival in the core and/or penumbra.