Abstract
A mild ischemic load applied after a lethal ischemic insult reduces the subsequent ischemia–reperfusion injury, and is called ischemic postconditioning (PostC). We studied the effect of ischemic PostC on synaptic glutamate release using a whole-cell patch-clamp technique. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) from CA1 pyramidal cells in mouse hippocampal slices. The ischemic load was perfusion of artificial cerebrospinal fluid (ACSF) equilibrated with mixed gas (95% N2 and 5% CO2). The ischemic load was applied for 7.5 min, followed by ischemic PostC 30 s later, consisting of three cycles of 15 s of reperfusion and 15 s of re-ischemia. We found that a surging increase in sEPSCs frequency occurred during the immediate-early reperfusion period after the ischemic insult. We found a significant positive correlation between cumulative sEPSCs and the number of dead CA1 neurons (r = 0.70; p = 0.02). Ischemic PostC significantly suppressed this surge of sEPSCs. The mitochondrial KATP (mito-KATP) channel opener, diazoxide, also suppressed the surge of sEPSCs when applied for 15 min immediately after the ischemic load. The mito-KATP channel blocker, 5-hydroxydecanoate (5-HD), significantly attenuated the suppressive effect of both ischemic PostC and diazoxide application on the surge of sEPSCs. These results suggest that the opening of mito-KATP channels is involved in the suppressive effect of ischemic PostC on synaptic glutamate release and protection against neuronal death. We hypothesize that activation of mito-KATP channels prevents mitochondrial malfunction and breaks mutual facilitatory coupling between glutamate release and Ca2+ entry at presynaptic sites.
Highlights
Excessive accumulation of spontaneous excitatory post-synaptic currents (sEPSCs) tended to increase the number of death neurons We examined whether the accumulation of synaptic glutamate release would cause neuronal death at the immediate-early stage of the post-ischemic reperfusion period
Because neuronal death is caused by ischemia-reperfusion injury and by the slice preparation procedure in this experiment, we used two membrane-impermeable dyes for nuclear stain with different fluorescent wave lengths to detect exclusively the cells that have died during the period from the electrophysiological recording to 3 h after ischemic insult of 7.5 min (Fig 2A)
This positive correlation indicates that excessive accumulation of sEPSCs tended to cause neuronal death, even during the immediate-early stage of the post-ischemic reperfusion period
Summary
In the heart and brain, a brief sublethal ischemic load prior to lethal ischemia induces tolerance to the subsequent ischemic insult, a phenomenon known as ischemic preconditioning [1]. Ischemic preconditioning provides powerful protection against ischemia-reperfusion injury in both the heart and nervous system [2,3,4,5,6]. Preconditioning is an effective treatment for protection against cerebral and myocardial damage, its clinical use is limited, because the onset of an ischemic stroke or cardiac infarction is extremely difficult to predict. PostC consists of several repeated cycles of brief re-occlusion and reperfusion, which is started early in the reperfusion period after a lethal ischemic load [8]. Zhao and colleagues first reported that PostC reduces cerebral ischemia–reperfusion injury [10]
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