Hibernating Arctic ground squirrel (hAGS), Spermophilus parryii, survive profound decreases in cerebral perfusion during torpor and return to normal blood flow during intermittent rewarming periods without neurologic damage. Hibernating AGS tolerate traumatic brain injury in vivo, and acute hippocampal slices from hibernating animals tolerate oxygen and glucose deprivation. It remains unclear, however, if neuroprotection results from intrinsic tissue properties or from differences in response to acute trauma associated with slice preparation. The goal of this work was therefore to determine whether an intrinsic tissue tolerance persists in chronic culture of AGS hippocampal slices at 371C. A second goal was to address N-methyl-D-aspartate (NMDA) receptor involvement and channel arrest as potential mechanisms of intrinsic tissue tolerance. Baseline neuronal survival and tolerance to oxygen and nutrient deprivation (OND), an in vitro model of ischemia-reperfusion, were assessed in the CA1 region of hippocampal slices from juvenile, hAGS and interbout euthermic AGS (ibeAGS). Early in culture (insult onset at 3 h), slices from both hAGS and ibeAGS tolerate OND (4 h deprivation followed by 20 h recovery) and 500 lmol/L NMDA plus 20 mmol/L KCl. Later in culture (insult onset at 24 h), tolerance persists in slices from hAGS but not in slices from ibeAGS. Ouabain (Na + K + ATPase inhibitor) administered 24 h in culture enhances survival of slices from hAGS (assessed 24 h later). Thus, tolerance to OND in slices from hAGS is due to intrinsic tissue properties likely involving NMDA receptors and ion channel arrest.

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 371C. 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.

Hibernating animals are very tolerant of trauma to the central nervous system such that dramatic fluctuations in cerebral blood flow occur during hibernation and arousal without apparent damage. Indeed, it was demonstrated that Arctic ground squirrels (AGS) experience acute and severe systemic hypoxia along with the dramatic fluctuation in cerebral blood flow when the animals are aroused from hibernation. While initial hypotheses concerned protective mechanisms in the hibernating state, recent evidence of sustained elevation of HIF1alpha in euthermic AGS from our laboratory suggests that a preparatory program of protective gene expression is chronically expressed in euthermic AGS. In this study we evaluated potential neuroprotective adaptations by examining the alteration of intracellular MAPK pathways that may be modulated by hypoperfusion/reperfusion in AGS during hibernation and arousal. We found that ERK and JNK are activated in both euthermic and aroused AGS compared to the hibernating group which positively correlated with HIF1alpha levels. The activation of ERK and JNK associated with HIF1alpha may play an important role in mediating neuroprotective adaptations that is essential for successful hibernation. Interestingly, p38 is activated in euthermic AGS but not in aroused AGS, which shows strong correlation with iNOS induction. Therefore, the attenuation of p38 activation and iNOS induction in hibernating and aroused animals may contribute to the attenuation of inflammation that plays important neuroprotective roles during hibernation. Taken together, the differential modulation of the MAPK pathways may be critical for neuroprotection of AGS necessary for fluctuations in oxygen and nutrient delivery during hibernation.

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Heterothermic mammals such as ground squirrels tolerate ischemia and N-methyl-D-aspartate (NMDA) better than homeothermic mammals such as rats both in vivo and in vitro, and this tolerance is enhanced in the hibernating state. However, the cellular mechanisms underlying this tolerance remain unclear. NMDA receptors (NMDAR) play a key role in excitotoxicity. The purpose of the current study was therefore to test the hypothesis that NMDAR are down-regulated in hibernating Arctic ground squirrels (hAGS; Spermophilus parryii). To address this hypothesis, we used Western blot analysis to investigate NMDAR phosphorylation, an activator of NMDAR function, and internalization in naïve hippocampal tissue from hAGS, interbout euthermic AGS (ibeAGS), and rats. Furthermore, we used fura-2 calcium imaging to examine NMDAR function in cultured hippocampal slices from hAGS, ibeAGS, and rats. We report that phosphorylation of the NMDAR1 (NR1) subunit is decreased in hippocampal tissue from hAGS and that the NMDAR component of Glu-induced increase in [Ca 2+ ] i is decreased in hippocampal slices from hAGS. Moreover, the fraction of NR1 in the functional membrane pool in AGS is less than that in rats. Keywordshibernation; hippocampal slices; excitotoxicity; stroke; fura-2 Stroke is a primary cause of disability in the United States, and studies in traditional laboratory animals such as rats have produced a poor yield of pharmacotherapies (Sareen, 2002). Heterothermic mammals (e.g., ground squirrels) tolerate experimental hypoxia and ischemia significantly better than homeothermic mammals (e.g., rats) both in vivo and in vitro (Frerichs et al., 1998;Drew et al., 2004; Dave et al., 2006;Ross et al., 2006). Tolerance to central nervous system injury is enhanced in the hibernating state (Frerichs et al., 1998;Zhou et al., 2001;Ross et al., 2006). In addition, arousal from hibernation enhances learning in ground squirrels (Mihailovic et al., 1968;Weltzin et al., 2006). Nmethyl-D-aspartate receptors (NMDAR) play a key role in excitotoxicity and synaptic plasticity (Ascher and Nowak, 1986;Choi, 1995). Down-regulation of NMDAR has been shown to contribute to hypoxia and ischemia tolerance in developing brains of rats and piglets (Mishra et al., 2001;Fritz et al., 2002) Western painted turtles (Chrysemys picta;Bickler, 1998). The objective of this study was, therefore, to test the hypothesis that NMDAR function is down-regulated in hibernating AGS.Functional NMDAR are heteromeric and formed by NMDAR1 (NR1) subunits in various combinations with NMDAR2A-D (NR2A-D) subunits (Carroll and Zukin, 2002). NR1 is required to form functional NMDAR, whereas NR2 subunits play regulatory roles. Phosphorylation of NR1 and NR2 subunits enhances receptor function (Liu and Zhang, 2000). Internalization or altered insertion in the plasma membrane can also alter NMDAR function (Ehlers, 2000;Carroll and Zukin, 2002). To test the hypothesis that NMDAR function is down-regulated in hibernation, we first used Western blotting analysis to investigate N...

Ischemic-like insults decrease viability and increase cell death in cultures of human NPCs. Similar conditions have less affect on cell death and promote proliferation in AGS NPCs.

Hibernation is a unique phenotype displayed by a phylogenetically diverse group of organisms including several species of mammals and one species of primate. Here we review evidence for blood and tissue borne signaling molecules in hibernating animals, achievements in isolating and characterizing these molecules, and potential medicinal applications.