Arctic ground squirrel hippocampus tolerates oxygen glucose deprivation independent of hibernation season even when not hibernating and after ATP depletion, acidosis, and glutamate efflux

Abstract

Cerebral ischemia/reperfusion (I/R) triggers a cascade of uncontrolled cellular processes that perturb cell homeostasis. The arctic ground squirrel (AGS), a seasonal hibernator resists brain damage following cerebral I/R caused by cardiac arrest and resuscitation. However, it remains unclear if tolerance to I/R injury in AGS depends on the hibernation season. Moreover, it is also not clear if events such as depletion of ATP, acidosis, and glutamate efflux that are associated with anoxic depolarization are attenuated in AGS. Here, we employ a novel microperfusion technique to test the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is independent of the hibernation season and persists even after glutamate efflux. Acute hippocampal slices were harvested from summer euthermic AGS, hibernating AGS, and interbout euthermic AGS. Slices were subjected to oxygen glucose deprivation (OGD), an in vitro model of I/R injury to determine cell death marked by lactate dehydrogenase (LDH) release. ATP was assayed using ENLITEN ATP assay. Glutamate and aspartate efflux was measured using capillary electrophoresis. For acidosis, slices were subjected to pH 6.4 or ischemic shift solution (ISS). Acute hippocampal slices from rats were used as a positive control, susceptible to I/R injury. Our results indicate that when tissue temperature is maintained at 36°C, hibernation season has no influence on OGD-induced cell death in AGS hippocampal slices. Our data also show that tolerance to OGD in AGS hippocampal slices occurs despite loss of ATP and glutamate release, and persists during conditions that mimic acidosis and ionic shifts, characteristic of cerebral I/R. Read the Editorial Comment for this article on page 10.

Keywords: in vitro; hibernation; hippocampal brain slices; ischemia; lactate dehydrogenase release; oxygen glucose deprivation.

Figure 1. AGS brain tolerates OGD regardless of hibernation season

(A) seAGS are better protected from OGD than rats. Slices from rats and seAGS were subjected to 30 min OGD and time course of LDH release was monitored (OGD: rat, n=30 slices; seAGS, n= 30 slices; aCSF: rat, n=25 slices; seAGS, n= 28 slices, *p<0.05 rat aCSF vs rat OGD, ⁺p<0.05 rat vs AGS OGD, ^#p<0.05 seAGS aCSF vs seAGS OGD). (B) AGS slices exposed to 0.1% Triton-X as a positive control (n= 4 slices from 2 AGS). *p<0.05 vs. seAGS aCSF (C) AGS slices obtained based on hibernation season were subjected to 30 min OGD and time course and total LDH release was monitored. *p<0.05 seAGS vs. 20h ibeAGS, ⁺p<0.05 4h ibeAGS vs. 20h ibeAGS, ^#p<0.05 hAGS vs. 20h ibeAGS. (D) AGS slices obtained based on hibernation season were subjected to aCSF treatment. Grey bar indicates 30 min treatment period. Data shown are means ± SEM.

Figure 2. AGS brain is tolerant to ionic shift solution (ISS) or low pH injury

(A) Shows time course of LDH release in acute hippocampal slices from rat and seAGS exposed to ISS, pH 6.3 (n= 3) or aCSF, pH 7.3 (n=3). *p< 0.05 rat aCSF vs. rat ISS (pH 7.4), ⁺p< 0.05 rat ISS vs seAGS ISS (pH 6.3). (B) Shows time course of LDH release exposed to low pH 6.3 (n= 7) or aCSF, pH 7.3 (n=7). *p< 0.05 rat aCSF pH 7.4 vs. rat aCSF pH 6.3, ⁺p< 0.05 rat aCSF pH 6.3 vs seAGS aCSF pH 6.3. Grey bar indicates treatment period. Means ± SEM.

Figure 3. ATP declines in rat and seAGS following OGD

Levels of whole tissue ATP were determined over time following bath application of treatment in slices from (A) rat (aCSF n= 4, OGD n= 4) and (B) seAGS (aCSF n= 3, OGD n= 3) subjected to either 30 min of aCSF or OGD followed by 3 h reperfusion. Grey bar indicates insult period. Data shown are means ± SEM, *p< 0.05 versus aCSF.

Figure 4. OGD induces efflux of glutamate and aspartate in both seAGS and rat

Time-dependent excitatory neurotransmitter (glutamate and aspartate) efflux in rat and seAGS hippocampal slices induced by 30 min OGD insult. (A) and (B) illustrates glutamate efflux, (C) and (D) illustrate aspartate efflux in rat and seAGS hippocampal slices during OGD insult (n=17 slices from 6 rats, n= 14 slices from 5 seAGS). Grey bar indicates insult period. *p<0.05 for OGD versus aCSF group. Data shown are means ± SEM.