Acidotoxicity via ASIC1a Mediates Cell Death during Oxygen Glucose Deprivation and Abolishes Excitotoxicity.

Ischemic reperfusion (I/R) injury is associated with a complex and multifactorial cascade of events involving excitotoxicity, acidotoxicity, and ionic imbalance. While it is known that acidosis occurs concomitantly with glutamate-mediated excitotoxicity during brain ischemia, it remains elusive how acidosis-mediated acidotoxicity interacts with glutamate-mediated excitotoxicity. Here, we investigated the effect of acidosis on glutamate-mediated excitotoxicity in acute hippocampal slices. We tested the hypothesis that mild acidosis protects against I/R injury via modulation of NMDAR, but produces injury via activation of acid sensing ion channels (ASIC1a). Using a novel microperfusion approach, we monitored time course of injury in acutely prepared, adult hippocampal slices. We varied the duration of insult to delay the return to preinsult conditions to determine if injury was caused by the primary insult or by the modeled reperfusion phase. We also manipulated pH in presence and absence of oxygen glucose deprivation (OGD). The role of ASIC1a and NMDAR was deciphered by treating the slices with and without an ASIC or NMDAR antagonist. Our results show that injury due to OGD or low pH occurs during the insult rather than the modeled reperfusion phase. Injury mediated by low pH or low pH OGD requires ASIC1a and is independent of NMDAR activation. These findings point to ASIC1a as a mediator of ischemic cell death caused by stroke and cardiac arrest.

Habituation of Arctic ground squirrels (Urocitellus parryii) to handling and movement during torpor to prevent artificial arousal.

Hibernation is a unique physiological adaptation characterized by periods of torpor that consist of repeated, reversible, and dramatic reductions of body temperature, metabolism, and blood flow. External and internal triggers can induce arousal from torpor in the hibernator. Studies of hibernating animals often require that animals be handled or moved prior to sampling or euthanasia but this movement can induce changes in the hibernation status of the animal. In fact, it has been demonstrated that movement of animals while they are hibernating is sufficient to induce an artificial arousal, which can detrimentally alter experimental findings obtained from animals assumed to be torpid. Therefore, we assessed a method to induce habituation of torpid hibernators to handling and movement to reduce inadvertent arousals. A platform rocker was used to mimic motion experienced during transfer of an animal and changes in respiratory rate (RR) were used to assess responsiveness of torpid Arctic ground squirrels (AGS, Urocitellus parryii). We found that movement alone did not induce a change in RR, however, exposure to handling induced an increase in RR in almost all AGS. This change in RR was markedly reduced with increased exposures, and all AGS exhibited a change in RR ≤ 1 by the end of the study. AGS habituated faster mid-season compared to early in the season, which mirrors other assessments of seasonal variation of torpor depth. However, AGS regained responsiveness when they were not exposed to daily handling. While AGS continued to undergo natural arousals during the study, occurrence of a full arousal was neither necessary for becoming habituated nor detrimental to the time required for habituation. These data suggest that even when torpid, AGS are able to undergo mechanosensory habituation, one of the simplest forms of learning, and provides a reliable way to reduce the sensitivity of torpid animals to handling.

Inhibition of NMDA-type glutamate receptors induces arousal from torpor in hibernating arctic ground squirrels (Urocitellus parryii).

Hibernation is an adaptation to overcome periods of resource limitation often associated with extreme climatic conditions. The hibernation season consists of prolonged bouts of torpor that are interrupted by brief interbout arousals. Physiological mechanisms regulating spontaneous arousals are poorly understood, but may be related to a need for gluconeogenesis or elimination of metabolic wastes. Glutamate is derived from glutamine through the glutamate-glutamine cycle and from glucose via the pyruvate carboxylase pathway when nitrogen balance favors formation of glutamine. This study tests the hypothesis that activation of NMDA-type glutamate receptors (NMDAR) maintains torpor in arctic ground squirrel (arctic ground squirrel (AGS); Urocitellus parryii). Administration of NMDAR antagonists MK-801 (5 mg/kg, i.p.) that crosses the blood-brain barrier and AP5 (5 mg/kg, i.p.) that does not cross the blood-brain barrier induced arousal in AGS. Central administration of MK-801 (0.2, 2, 20 or 200 µg; icv) to hibernating AGS failed to induce arousal. Results suggest that activation of NMDAR at a peripheral or circumventricular site is necessary to maintain prolonged torpor and that a decrease in glutamate at these sites may contribute to spontaneous arousal in AGS.

Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen-glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss.

Oxygen-glucose deprivation (OGD) initiates a cascade of intracellular responses that culminates in cell death in sensitive species. Neurons from Arctic ground squirrels (AGS), a hibernating species, tolerate OGD in vitro and global ischemia in vivo independent of temperature or torpor. Regulation of energy stores and activation of mitogen-activated protein kinase (MAPK) signaling pathways can regulate neuronal survival. We used acute hippocampal slices to investigate the role of ATP stores and extracellular signal-regulated kinase (ERK)1/2 and Jun NH(2)-terminal kinase (JNK) MAPKs in promoting survival. Acute hippocampal slices from AGS tolerated 30 mins of OGD and showed a small but significant increase in cell death with 2 h OGD at 37 degrees C. This tolerance is independent of hibernation state or season. Neurons from AGS survive OGD despite rapid ATP depletion by 3 mins in interbout euthermic AGS and 10 mins in hibernating AGS. Oxygen-glucose deprivation does not induce JNK activation in AGS and baseline ERK1/2 and JNK activation is maintained even after drastic depletion of ATP. Surprisingly, inhibition of ERK1/2 or JNK during OGD had no effect on survival, whereas inhibition of JNK increased cell death during normoxia. Thus, protective mechanisms promoting tolerance to OGD by AGS are downstream from ATP loss and are independent of hibernation state or season. Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1307-1319; doi:10.1038/jcbfm.2008.20; published online 9 April 2008.

Central nervous system regulation of mammalian hibernation: implications for metabolic suppression and ischemia tolerance.

Torpor during hibernation defines the nadir of mammalian metabolism where whole animal rates of metabolism are decreased to as low as 2% of basal metabolic rate. This capacity to decrease profoundly the metabolic demand of organs and tissues has the potential to translate into novel therapies for the treatment of ischemia associated with stroke, cardiac arrest or trauma where delivery of oxygen and nutrients fails to meet demand. If metabolic demand could be arrested in a regulated way, cell and tissue injury could be attenuated. Metabolic suppression achieved during hibernation is regulated, in part, by the central nervous system through indirect and possibly direct means. In this study, we review recent evidence for mechanisms of central nervous system control of torpor in hibernating rodents including evidence of a permissive, hibernation protein complex, a role for A1 adenosine receptors, mu opiate receptors, glutamate and thyrotropin-releasing hormone. Central sites for regulation of torpor include the hippocampus, hypothalamus and nuclei of the autonomic nervous system. In addition, we discuss evidence that hibernation phenotypes can be translated to non-hibernating species by H(2)S and 3-iodothyronamine with the caveat that the hypothermia, bradycardia, and metabolic suppression induced by these compounds may or may not be identical to mechanisms employed in true hibernation.