N6-cyclohexyladenosine is better than meperidine and buspirone at suppressing metabolism during TTM32 but does not improve outcome after cardiac arrest.

N6-clyclohexyladenosine (CHA) is an adenosine A1 receptor agonist that inhibits thermogenesis. Cardiovascular side effects however, limit use of CHA as a therapeutic. We and others have shown that this can be reversed by administering 8-p-(sulfophenyl)theophylline (8-SPT), a nonspecific antagonist that does not cross the BBB. Other evidence shows that CNS actions of CHA may contribute to bradycardia through enhanced vagal tone and other mechanisms. Here we test the hypothesis that 8-SPT pretreatment alone is sufficient to prevent hypotension caused by CHA. To test this hypothesis, we pretreated rats with 8-SPT alone, and in combination with other antagonists to test the hypothesis that direct action of CHA on the heart is the primary mechanism by which CHA induces bradycardia and hypotension. Results show that pretreatment with 8-SPT alone is not sufficient to prevent CHA-induced hypotension. Pretreatment with 8-SPT or atropine alone did not prevent the fall in mean arterial pressure (MAP) and heart rate (HR), however, pretreatment with 8-SPT (25 mg/kg) and atropine (1 mg/kg) 15 min before CHA (1 mg/kg) preserves MAP and HR baseline values after CHA administration. We next asked if blood pressure was managed during the transition into a hypometabolic state, would prolong CHA-mediated inhibition of metabolism after cardiac arrest improve outcome better than anti-shivering medications meperidine and buspirone. We found that CHA-mediated hypotension can be mitigated by pretreatment with atropine and 8-SPT. This combination administered after cardiac arrest facilitated temperature management and metabolic suppression better than meperidine and buspirone, however, did not improve survival.

Pre-hibernation diet alters skeletal muscle relaxation kinetics, but not force development in torpid arctic ground squirrels.

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as - 3 °C), during which they are mostly physically inactive. Once Tb reaches ~ 15 °C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~ 35 °C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω-6 and the ω-6:ω-3 PUFA ratio correlate with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. We hypothesized that diet (increased ω-6:ω-3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. (1) Standard rodent chow with an ω-6:ω-3 ratio of 5:1, or (2) a balanced diet with an ω-6:ω-3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4 °C, 15 °C, 25 °C, and 37 °C to determine functional resilience. Our data show that torpid animals fed standard rodent chow have faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω-6:ω-3 PUFA ratio improves skeletal muscle performance during decreased temperatures in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.

Remodelling of Skeletal Muscle Myosin Metabolic States in Hibernating Mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Cardiac Rhythms and Variation in Hibernating Arctic Ground Squirrels.

AbstractThe dramatic decrease in heart rate (HR) during entrance into hibernation is not a mere response to the lowering of core body temperature (Tb) but a highly regulated fall, as the decrease in HR precedes the drop in Tb. This regulated fall in HR is thought to be mediated by increased cardiac parasympathetic activity. Conversely, the sympathetic nervous system is thought to drive the increase of HR during arousal. Despite this general understanding, we lack temporal information on cardiac parasympathetic regulation throughout a complete hibernation bout. The goal of this study was to fill this gap in knowledge by using Arctic ground squirrels implanted with electrocardiogram/temperature telemetry transmitters. Short-term HR variability (root mean square of successive differences [RMSSD]), an indirect measure of cardiac parasympathetic regulation, was calculated in 11 Arctic ground squirrels. RMSSD, normalized as RMSSD/RR interval (RRI), increased fourfold during early entrance (from 0.2±0.1 to 0.8±0.2, P<0.05). RMSSD/RRI peaked after HR dropped by over 90% and Tb fell by 70%. Late entrance was delineated by a decline in RMSSD/RRI while Tb continued to decrease. During arousal, HR started to increase 2 h before Tb, with a concurrent decrease in RMSSD/RRI to a new minimum. As Tb increased to a maximum during interbout arousal, HR declined, and RMSSD/RRI increased. These data suggest that activation of the parasympathetic nervous system initiates and regulates the HR decrease during entrance into hibernation and that withdrawal of parasympathetic activation initiates arousal. We conclude that cardiac parasympathetic regulation persists throughout all phases of a hibernation bout-a feature of the autonomic nervous system's regulation of hibernation that was not appreciated previously.

Beyond the symptom: the biology of fatigue.

A workshop titled "Beyond the Symptom: The Biology of Fatigue" was held virtually September 27-28, 2021. It was jointly organized by the Sleep Research Society and the Neurobiology of Fatigue Working Group of the NIH Blueprint Neuroscience Research Program. For access to the presentations and video recordings, see: https://neuroscienceblueprint.nih.gov/about/event/beyond-symptom-biology-fatigue. The goals of this workshop were to bring together clinicians and scientists who use a variety of research approaches to understand fatigue in multiple conditions and to identify key gaps in our understanding of the biology of fatigue. This workshop summary distills key issues discussed in this workshop and provides a list of promising directions for future research on this topic. We do not attempt to provide a comprehensive review of the state of our understanding of fatigue, nor to provide a comprehensive reprise of the many excellent presentations. Rather, our goal is to highlight key advances and to focus on questions and future approaches to answering them.

Opportunities and barriers to translating the hibernation phenotype for neurocritical care.

Targeted temperature management (TTM) is standard of care for neonatal hypoxic ischemic encephalopathy (HIE). Prevention of fever, not excluding cooling core body temperature to 33°C, is standard of care for brain injury post cardiac arrest. Although TTM is beneficial, HIE and cardiac arrest still carry significant risk of death and severe disability. Mammalian hibernation is a gold standard of neuroprotective metabolic suppression, that if better understood might make TTM more accessible, improve efficacy of TTM and identify adjunctive therapies to protect and regenerate neurons after hypoxic ischemia brain injury. Hibernating species tolerate cerebral ischemia/reperfusion better than humans and better than other models of cerebral ischemia tolerance. Such tolerance limits risk of transitions into and out of hibernation torpor and suggests that a barrier to translate hibernation torpor may be human vulnerability to these transitions. At the same time, understanding how hibernating mammals protect their brains is an opportunity to identify adjunctive therapies for TTM. Here we summarize what is known about the hemodynamics of hibernation and how the hibernating brain resists injury to identify opportunities to translate these mechanisms for neurocritical care.

Translating PUFA omega 6:3 ratios from wild to captive hibernators (Urocitellus parryii) enhances sex-dependent mass-gain without increasing physiological stress indicators.

Omega 3 polyunsaturated fatty acids (PUFAs) are well-documented for their influence on health and weight loss. Recent studies indicate omega 3 PUFAs may exert a negative impact on cellular stress and physiology in some hibernators. We asked if physiological stress indicators, lipid peroxidation and mass gain in Arctic Ground Squirrels (AGS) were negatively influenced by naturally occurring dietary omega 3 PUFA levels compared to omega 3 PUFA levels found in common laboratory diets. We found plasma fatty acid profiles of free-ranging AGS to be high in omega 3 PUFAs with balanced omega 6:3 ratios, while standard laboratory diets and plasma of captive AGS are high in omega 6 and low in omega 3 PUFAs with higher omega 6:3 ratios. Subsequently, we designed a diet to mimick free-range AGS omega 6:3 ratios in captive AGS. Groups of wild-caught juvenile AGS were either fed: (1) Mazuri Rodent Chow (Standard Rodent chow, 4.95 omega 6:3 ratio), or (2) balanced omega 6:3 chow (Balanced Diet, 1.38 omega 6:3). AGS fed the Balanced Diet had plasma omega 6:3 ratios that mimicked plasma profiles of wild AGS. Balanced Diet increased female body mass before hibernation, but did not influence levels of cortisol in plasma or levels of the lipid peroxidation product 4-HNE in brown adipose tissue. Overall, as the mass gain is critical during pre-hibernation for obligate hibernators, the results show that mimicking a fatty acid profile of wild AGS facilitates sex-dependent mass accumulation without increasing stress indicators.

Thermoregulation in hibernating mammals: The role of the "thyroid hormones system".

Hibernation is a unique evolutionary adaptation to conserve energy. During the pre-hibernation (i.e. fall) season, a progressive decline in core body temperature and further decrease in metabolism underlie a seasonal modulation in thermoregulation. The onset of hibernation requires marked changes in thermoregulatory attributes including adjustment in body temperature and tissue specific increases in thermogenic capacity. The hibernation season is characterized by a regulated suppression in thermogenesis allowing the onset of torpor interrupted by periodic activation of thermogenesis to sustain interbout arousals. Thyroid hormones are known to regulate both body temperature and metabolism, and for this reason, the hypothalamic-pituitary-thyroid axis and thyroid hormones have been investigated as modulators of thermogenesis in the phenomenon of hibernation, but the mechanisms remain poorly understood. In this review, we present an overview of what is known about the thermogenic roles of thyroid hormones in hibernating species across seasons and within the hibernating season (torpor-interbout arousal cycle). Overall, the hypothalamic-pituitary-thyroid axis and thyroid hormones play a role in the pre-hibernation season to enhance thermogenic capacity. During hibernation, thermogenesis is attenuated at the level of sympathetic premotor neurons within the raphe pallidus and by deiodinase expression in the hypothalamus. Further, as recent work highlights the direct effect of thyroid hormones within the central nervous system in activating thermogenesis, we speculate how similar mechanisms may occur in hibernating species to modulate thermogenesis across seasons and to sustain interbout arousals. However, further experiments are needed to elucidate the role of thyroid hormones in hibernation, moving towards the understanding that thyroid hormones metabolism, transport and availability within tissues may be the most telling indicator of thyroid status.

Nitrogen recycling buffers against ammonia toxicity from skeletal muscle breakdown in hibernating arctic ground squirrels.

Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using 15N stable isotope methodology in arctic ground squirrels (Urocitellus parryii), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation.

Iodine Redistribution During Trauma, Sepsis, and Hibernation: An Evolutionarily Conserved Response to Severe Stress.

OBJECTIVE: We performed these studies to learn how iodine in the form of free iodide behaves during stress. DESIGN: Prospective observational trial using samples obtained from human trauma patients and retrospective observational study using remnant samples from human sepsis patients and arctic ground squirrels. Preclinical interventional study using hind-limb ischemia and reperfusion injury in mice. SETTING: Level I trauma center emergency room and ICU and animal research laboratories. SUBJECTS: Adult human sepsis and trauma patients, wild-caught adult arctic ground squirrels, and sexually mature laboratory mice. INTERVENTIONS: Ischemia and reperfusion injury was induced in mice by temporary application of tourniquet to one hind-limb. Iodide was administered IV just prior to reperfusion. MEASUREMENTS AND MAIN RESULTS: Free iodide was measured using ion chromatography. Relative to iodide in plasma from normal donors, iodide was increased 17-fold in plasma from trauma patients and 26-fold in plasma from sepsis patients. In arctic ground squirrels, iodide increases over three-fold during hibernation. And during ischemia/reperfusion injury in mice, iodide accumulates in ischemic tissue and reduces both local and systemic tissue damage. CONCLUSIONS: Iodide redistributes during stress and improves outcome after injury. Essential functions of iodide may have contributed to its evolutionary selection and be useful as a therapeutic intervention for human patients.

Seasonal decrease in thermogenesis and increase in vasoconstriction explain seasonal response to N6 -cyclohexyladenosine-induced hibernation in the Arctic ground squirrel (Urocitellus parryii).

Hibernation is a seasonal phenomenon characterized by a drop in metabolic rate and body temperature. Adenosine A1 receptor agonists promote hibernation in different mammalian species, and the understanding of the mechanism inducing hibernation will inform clinical strategies to manipulate metabolic demand that are fundamental to conditions such as obesity, metabolic syndrome, and therapeutic hypothermia. Adenosine A1 receptor agonist-induced hibernation in Arctic ground squirrels is regulated by an endogenous circannual (seasonal) rhythm. This study aims to identify the neuronal mechanism underlying the seasonal difference in response to the adenosine A1 receptor agonist. Arctic ground squirrels were implanted with body temperature transmitters and housed at constant ambient temperature (2°C) and light cycle (4L:20D). We administered CHA (N6 -cyclohexyladenosine), an adenosine A1 receptor agonist in euthermic-summer phenotype and euthermic-winter phenotype and used cFos and phenotypic immunoreactivity to identify cell groups affected by season and treatment. We observed lower core and subcutaneous temperature in winter animals and CHA produced a hibernation-like response in winter, but not in summer. cFos-ir was greater in the median preoptic nucleus and the raphe pallidus in summer after CHA. CHA administration also resulted in enhanced cFos-ir in the nucleus tractus solitarius and decreased cFos-ir in the tuberomammillary nucleus in both seasons. In winter, cFos-ir was greater in the supraoptic nucleus and lower in the raphe pallidus than in summer. The seasonal decrease in the thermogenic response to CHA and the seasonal increase in vasoconstriction, assessed by subcutaneous temperature, reflect the endogenous seasonal modulation of the thermoregulatory systems necessary for CHA-induced hibernation. Cover Image for this issue: doi: 10.1111/jnc.14528.

Mechanisms of innate preconditioning towards ischemia/anoxia tolerance: Lessons from mammalian hibernators.

Hibernating mammals exhibit an innate physiological ability to withstand dramatic fluctuations in blood flow that occurs during hibernation and arousal or experimental models of ischemia reperfusion without significant damage. These innate adaptations are of significance particularly to organs that are highly susceptible to energy deprivation, such as the brain and the heart. Among vertebrates, the arctic ground squirrel (AGS) is a species that tolerates ischemic/anoxic insult. During the process of entering hibernation, a state of prolonged torpor, the AGS undergoes a profound decrease in respiratory rate, heart rate, blood flow, cerebral perfusion, and body temperature (Tb). The reduced level of blood flow during torpor resembles an ischemic state, albeit without energy deficit. During the process of arousal or emergence from torpor, however, when Tb, respiratory rate, heart rate, and blood flow rapidly returns to pre-torpid levels, the rapid return of cerebral blood flow mimics aspects of reperfusion such as is seen after stroke or cardiac arrest. This sublethal ischemic/reperfusion insult experienced by AGS during the process of arousal may precondition AGS to tolerate otherwise lethal ischemic/reperfusion injury induced in the laboratory. In this review, we will summarize some of the mechanisms implemented by mammalian hibernators to combat ischemia/anoxia tolerance.

Precise Control of Target Temperature Using N6-Cyclohexyladenosine and Real-Time Control of Surface Temperature.

Targeted temperature management is standard of care for cardiac arrest and is in clinical trials for stroke. N6-cyclohexyladenosine (CHA), an A1 adenosine receptor (A1AR) agonist, inhibits thermogenesis and induces onset of hibernation in hibernating species. Despite promising thermolytic efficacy of CHA, prior work has failed to achieve and maintain a prescribed target core body temperature (Tb) between 32°C and 34°C for 24 hours. We instrumented Sprague-Dawley rats (n = 19) with indwelling arterial and venous cannulae and a transmitter for monitoring Tb and ECG, then administered CHA via continuous IV infusion or intraperitoneal (IP) injection. In the first experiment (n = 11), we modulated ambient temperature and increased the dose of CHA in an attempt to manage Tb. In the second experiment (n = 8), we administered CHA (0.25 mg/[kg·h]) via continuous IV infusion and modulated cage surface temperature to control Tb. We rewarmed animals by increasing surface temperature at 1°C h-1 and discontinued CHA after Tb reached 36.5°C. Tb, brain temperature (Tbrain), heart rate, blood gas, and electrolytes were also monitored. Results show that titrating dose to adjust for individual variation in response to CHA led to tolerance and failed to manage a prescribed Tb. Starting with a dose (0.25 mg/[kg·h]) and modulating surface temperature to prevent overcooling proved to be an effective means to achieve and maintain Tb between 32°C and 34°C for 24 hours. Increasing surface temperature to 37°C during CHA administration brought Tb back to normothermic levels. All animals treated in this way rewarmed without incident. During the initiation of cooling, we observed bradycardia within 30 minutes of the start of IV infusion, transient hyperglycemia, and a mild hypercapnia; the latter normalized via metabolic compensation. In conclusion, we describe an intravenous delivery protocol for CHA at 0.25 mg/(kg·h) that, when coupled with conductive cooling, achieves and maintains a prescribed and consistent target Tb between 32°C and 34°C for 24 hours.

The Raphe Pallidus and the Hypothalamic-Pituitary-Thyroid Axis Gate Seasonal Changes in Thermoregulation in the Hibernating Arctic Ground Squirrel (Urocitellus parryii).

Thermoregulation is necessary to maintain energy homeostasis. The novel discovery of brown adipose tissue (BAT) in humans has increased research interests in better understanding BAT thermogenesis to restore energy balance in metabolic disorders. The hibernating Arctic ground squirrel (AGS) offers a novel approach to investigate BAT thermogenesis. AGS seasonally increase their BAT mass to increase the ability to generate heat during interbout arousals. The mechanisms promoting the seasonal changes in BAT thermogenesis are not well understood. BAT thermogenesis is regulated by the raphe pallidus (rPA) and by thyroid hormones produced by the hypothalamic-pituitary-thyroid (HPT) axis. Here, we investigate if the HPT axis and the rPA undergo seasonal changes to modulate BAT thermogenesis in hibernation. We used histological analysis and tandem mass spectrometry to assess activation of the HPT axis and immunohistochemistry to measure neuronal activation. We found an increase in HPT axis activation in fall and in response to pharmacologically induced torpor when adenosine A1 receptor agonist was administered in winter. By contrast, the rPA neuronal activation was lower in winter in response to pharmacologically induced torpor. Activation of the rPA was also lower in winter compared to the other seasons. Our results suggest that thermogenic capacity develops during fall as the HPT axis is activated to reach maximum capacity in winter seen by increased free thyroid hormones in response to cooling. However, thermogenesis is inhibited during torpor as sympathetic premotor neuronal activation is lower in winter, until arousal when inhibition of thermogenesis is relieved. These findings describe seasonal modulation of thermoregulation that conserves energy through attenuated sympathetic drive, but retains heat generating capacity through activation of the HPT axis.

Arctic ground squirrel resist peroxynitrite-mediated cell death in response to oxygen glucose deprivation.

Cerebral ischemia-reperfusion (I/R) injury initiates a cascade of events, generating nitric oxide (NO) and superoxide(O2•-) to form peroxynitrite (ONOO-), a potent oxidant. Arctic ground squirrels (AGS; Urocitellus parryii) show high tolerance to I/R injury. However, the underlying mechanism remains elusive. We hypothesize that tolerance to I/R modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrative stress. Hippocampal slices (400µm) from rat and AGS were subjected to oxygen glucose deprivation (OGD) using a novel microperfusion technique. Slices were exposed to NO, O2.- donors with and without OGD; pretreatment with inhibitors of NO, O2.- and ONOO- followed by OGD. Perfusates collected every 15min were analyzed for LDH release, a marker of cell death. 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) were measured to assess oxidative and nitrative stress. Results show that NO/O2.- alone is not sufficient to cause ischemic-like cell death, but with OGD enhances cell death more in rat than in AGS. A NOS inhibitor, SOD mimetic and ONOO- inhibitor attenuates OGD injury in rat but has no effect in AGS. Rats also show a higher level of 3NT and 4HNE with OGD than AGS suggesting the greater level of injury in rat is via formation of ONOO-.

Optimization of Thermolytic Response to A1 Adenosine Receptor Agonists in Rats.

Cardiac arrest is a leading cause of death in the United States, and, currently, therapeutic hypothermia, now called targeted temperature management (TTM), is the only recent treatment modality proven to increase survival rates and reduce morbidity for this condition. Shivering and subsequent metabolic stress, however, limit application and benefit of TTM. Stimulating central nervous system A1 adenosine receptors (A1AR) inhibits shivering and nonshivering thermogenesis in rats and induces a hibernation-like response in hibernating species. In this study, we investigated the pharmacodynamics of two A1AR agonists in development as antishivering agents. To optimize body temperature (Tb) control, we evaluated the influence of every-other-day feeding, dose, drug, and ambient temperature (Ta) on the Tb-lowering effects of N6-cyclohexyladenosine (CHA) and the partial A1AR agonist capadenoson in rats. The highest dose of CHA (1.0 mg/kg, i.p.) caused all ad libitum-fed animals tested to reach our target Tb of 32°C, but responses varied and some rats overcooled to a Tb as low as 21°C at 17.0°C Ta Dietary restriction normalized the response to CHA. The partial agonist capadenoson (1.0 or 2.0 mg/kg, i.p.) produced a more consistent response, but the highest dose decreased Tb by only 1.6°C. To prevent overcooling after CHA, we studied continuous i.v. administration in combination with dynamic surface temperature control. Results show that after CHA administration control of surface temperature maintains desired target Tb better than dose or ambient temperature.

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

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.

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.

Neural Signaling Metabolites May Modulate Energy Use in Hibernation.

Despite an epidemic in obesity and metabolic syndrome limited means exist to effect adiposity or metabolic rate other than life style changes. Here we review evidence that neural signaling metabolites may modulate thermoregulatory pathways and offer novel means to fine tune energy use. We extend prior reviews on mechanisms that regulate thermogenesis and energy use in hibernation by focusing primarily on the neural signaling metabolites adenosine, AMP and glutamate.

The effects of hibernation and forced disuse (neurectomy) on bone properties in arctic ground squirrels.

Bone loss is a well-known medical consequence of disuse such as in long-term space flight. Immobilization in many animals mimics the effects of space flight on bone mineral density. Decreases in metabolism are also thought to contribute to a loss of skeletal mass. Hibernating mammals provide a natural model of disuse and metabolic suppression. Hibernating ground squirrels have been shown to maintain bone strength despite long periods of disuse and decreased metabolism during torpor. This study examined if the lack of bone loss during torpor was a result of the decrease in metabolic rate during torpor or an evolutionary change in these animals affording protection against disuse. We delineated changes in bone density during natural disuse (torpor) and forced disuse (sciatic neurectomy) in the hind limbs of the arctic ground squirrel (AGS) over an entire year. We hypothesized that the animals would be resistant to bone loss due to immobilization and disuse during the winter hibernation season when metabolism is depressed but not the summer active season. This hypothesis was not supported. The animals maintained bone density (dual-energy X-ray absorptiometry) and most bone structural and mechanical properties in both seasons. This was observed in both natural and forced disuse, regardless of the known metabolic rate increase during the summer. However, trabecular bone volume fraction (microcomputed tomography) in the distal femur was lower in neurectomized AGS at the study endpoint. These results demonstrate a need to better understand the relationship between skeletal load (use) and bone density that may lead to therapeutics or strategies to maintain bone density in disuse conditions.