Abstract TP281: Increased Susceptibility to Excitotoxic Injury in Rat Hippocampal Slices Exposed to Intermittent Hypoxia

Abstract

Introduction: The effect of sustained hypoxia (SH) on brain metabolism has been well studied. However less is known about intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), associated with increased risk for stroke, outcome severity and functional consequences. Hypothesis: Impaired glutamate homeostasis after IH may underlie increased brain vulnerability to stroke-induced excitotoxicity. Methods: P4 organotypic rat hippocampal slices cultured for 7 days, were exposed for 7 additional days to IH (alternating 2 min 5% O2 - 15 min 21% O2), SH (5% O2) or normoxia (RA; 21% O2), followed by 3 glutamate challenges (first and last 200 μM, 15 min, emulating a physiological stimulus; second, 10 mM, 10 min, emulating stroke-induced excitotoxicity). Viability was assessed by propidium iodide (PI) uptake at baseline then after each glutamate challenge to assess whether hypoxia impairs the response to physiological or excitotoxic glutamate release. Glial GFAP, neuronal MAP2, EAAT1 and EAAT2 glutamate transporters expression was assessed by immunohistochemistry. Spontaneous and evoked Ca2+ transient activity was assessed in Fluo-8LTM AM loaded slices, by optical recording of Ca2+ spikes proximal to a bipolar stimulating electrode, before and after each of 3 single 2 ms stimuli (0.6 mA). Ca2+ transients after high K+ were used to determine the total number of viable cells. Results: Viability, GFAP, MAP2, EAAT1 and EAAT2 expression and basal Ca2+ spikes activity significantly decreased in IH. The number of neurons with spikes evoked within 500 ms of stimuli was not significantly different, but RA evoked responses were more tightly clustered. Residual network activity, assessed by number of neurons with spikes 500 ms post stimulus, was significantly different RA>SH>IH. Overall number of spiking cells after high K+, representing total viable cells, confirmed the viability data obtained with PI staining. Conclusions: IH is more detrimental to cell survival and glutamate homeostasis than SH, suggesting that in addition to vascular changes, impaired glutamate homeostasis may increase OSA patients’ susceptibility to ischemic events.