Background-Hibernation is an adaptation to extreme environments known to provide organ protection... more Background-Hibernation is an adaptation to extreme environments known to provide organ protection against ischemia-reperfusion (I/R) injury. An unbiased systems approach was utilized to investigate hibernation-induced changes characteristic of the hibernator cardioprotective phenotype, by comparing the myocardial proteome of winter hibernating arctic ground squirrels (HIB AGS), summer active (SA) AGS, and rats subjected to I/R, and further correlating with targeted metabolic changes. Methods-In a well-defined rodent model of I/R by deep hypothermic circulatory arrest followed by 3h or 24h of reperfusion or sham, myocardial protein abundance in AGS (HIB, SA) and rats (n=4-5/group) was quantified by label-free proteomics (n=4-5/group), and correlated with metabolic changes. Results-Compared to rats, HIB AGS displayed markedly reduced plasma levels of Troponin I, myocardial apoptosis, and left ventricular contractile dysfunction. Of the 1,320 rat and 1,478 AGS proteins identified, 545 were differentially expressed between HIB AGS and rat hearts (47% upregulated, 53% downregulated). Gene ontology analysis revealed downregulation in HIB AGS hearts of most proteins involved in mitochondrial energy transduction, including electron transport chain complexes, acetyl CoA biosynthesis, Krebs cycle, glycolysis and ketogenesis. Conversely, fatty acid oxidation enzymes and Sirtuin-3 were upregulated in HIB AGS, with preserved †
... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gju... more ... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gjumrakch Aliev, Takafumi Wataya, Shun Shimohama, Kelly Drew ... 7. Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N. Oxidative damage in Alzheimers ...
... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gju... more ... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gjumrakch Aliev, Takafumi Wataya, Shun Shimohama, Kelly Drew ... 7. Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N. Oxidative damage in Alzheimer s ...
Alzheimer disease (AD) is defined pathologically and diagnostically defined by amyloid- senile p... more Alzheimer disease (AD) is defined pathologically and diagnostically defined by amyloid- senile plaques and neurofibrillary tangles (NFT) composed of tau. From the time of their original description nearly a century ago, a major focus has been to understand the role that these lesions play in the pathogenesis of the disease. The majority favors the notion that these lesions cause the disease and therefore attempts at therapeutic intervention are focused on preventing lesions formation. However, this rationale may be misguided since new evidence from our laboratories and others suggest that the lesions not only occur as a by-product of the fundamental disease process but also that they may be protective.
Hibernating animals are very tolerant of trauma to the central nervous system such that dramatic ... more 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.
Using quantitative microdialysis in hibernating Arctic ground squirrels (AGS), striatal glutamate... more Using quantitative microdialysis in hibernating Arctic ground squirrels (AGS), striatal glutamate concentrations ([glu](dia)) progressively increased to approximately 200 microM after 3 days of microdialysis in euthermic but not hibernating ground squirrels. Initially, the progressive increase in [glu](dia) was thought to be related to greater tissue response in euthermic animals. Alternatively, given the vastly different body temperatures between the two groups (37 vs. 3 degrees C), glutamate might have originated from microbes, replicating at a faster rate in the warmer animals. To test these hypotheses, microdialysis was repeated using sterile technique and tissue response surrounding the probe tract was assessed in hematoxylin and eosin stained sections. Using sterile microdialysis technique, traumatic tissue response was greater in euthermic compared to hibernating tissue. However, sterile microdialysis abolished the progressive increase in glutamate. To confirm the microbial origin of glutamate we monitored [glu](dia) collected in vitro from probes immersed in glutamine-rich liquid medium incubated at 37 degrees C. In vitro, [glu](dia) increased as much as in vivo. Two bacteria isolated from in vitro dialysate and liquid medium were both identified as Ralstonia pickettii. Growth of these isolates as well as glutamate release was enhanced when glutamine rather than NH(4)NO(3) was added to the medium suggesting the bacteria utilize glutamine preferentially over ammonium as a nitrogen source.
Microdialysis is a widely used in vivo sampling technique commonly used to monitor extracellular ... more Microdialysis is a widely used in vivo sampling technique commonly used to monitor extracellular levels of a variety of molecules including neurotransmitters and metabolites. To facilitate interpretation of microdialysis results, this study critically examines changes in synaptic morphology induced by microdialysis. Tissue surrounding microdialysis probes was examined using light and electron microscopy at three distances from the probe tract. Microdialysis probes were implanted into rat striatum, and after 40 h of post-operative recovery were perfused with a modified Ringer's solution. Light microscope analysis revealed tissue disruption up to 1.4 mm from the probe site. Axonal damage indicative of non-excitotoxic insult was also seen as far away from the probe as was examined. The presence of dark-degenerating neurons was also noted and estimates of neuronal densities revealed loss up to 400 mm from the probe tract. This study, the first qualitative ultrastructural investigation of neuropil surrounding the probe site, indicated swollen processes up to 1.4 mm from the probe tract. Swollen mitochondria and bloated endoplasmic reticulum suggest intracellular chemical disruption. Tissue damage resulting in synaptic and neuronal disruption may affect neurotransmitter efflux or extracellular concentrations of metabolites.
Brains of hibernating mammals are protected against a variety of insults that are detrimental to ... more Brains of hibernating mammals are protected against a variety of insults that are detrimental to humans and other nonhibernating species. Such protection is associated with a number of physiological adaptations including hypothermia, increased antioxidant defense, metabolic arrest, leukocytopenia, immunosuppression, and hypocoagulation. It is intriguing that similar manipulations provide considerable protection as experimental treatments for central nervous system injury. This review focuses on neuroprotective mechanisms employed during hibernation that may offer novel approaches in the treatment of stroke, traumatic brain injury, and neurodegenerative diseases in humans.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2002
Ground squirrels tolerate up to 90% reductions in cerebral blood flow during hibernation as well ... more Ground squirrels tolerate up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousal from torpor without apparent neurological damage. Thus, hibernation is studied as a model of tolerance to cerebral ischemia and other types of brain injury. Metabolic suppression likely plays a primary adaptive role that allows hibernating species to tolerate dramatic fluctuations in blood flow. Several other aspects of hibernation physiology are also consistent with tolerance to ischemia and reperfusion suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work is to review evidence for enhanced antioxidant defense systems during hibernation, with a focus on ascorbate, and discuss potential roles of these antioxidants during hibernation. In concert with dramatic decreases in blood flow, nutrient and oxygen delivery, plasma concentrations of the antioxidant ascorbate w(Asc)px increase 3-5-fold during hibernation. In contrast, during re-warming, wAscxp declines at a relatively rapid rate that peaks at the time of maximal O consumption. This peak in O consumption also 2 2 coincides with a brief rise in plasma urate concentration consistent with a surge in reactive oxygen species production. Overall, data suggest that elevated concentration of plasma ascorbate is poised for distribution to metabolically active tissues during the surge in oxidative metabolism that accompanies re-warming during hibernation. This pool of ascorbate, as well as increased expression of other antioxidant defense systems, may protect vulnerable tissues from oxidative stress during hibernation and re-warming from hibernation. Better understanding of the role of ascorbate in hibernation may guide use of ascorbate and other antioxidants in treatment of stroke, head trauma and neurodegenerative disease.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2002
In this review, we consider comparative aspects of the biology and pathology of oxygen radicals i... more In this review, we consider comparative aspects of the biology and pathology of oxygen radicals in neurodegenerative disease and how these findings have influenced our concept of oxidative stress. The common definition of oxidative stress is a breach of antioxidant defenses by oxygen radicals leading to damage to critical molecules and disrupted physiology. Inherent in this definition is that oxidative stress is an unstable situation, for if there is net damage, viability of the system decreases with time, leading to disequilibria and death. While this circumstance defines acute conditions, such as stroke and head trauma which result in dysfunction and death, it does not fit physiological situations or chronic diseases closely aligned to normal physiology. Therefore, we propose that oxidative modifications in Alzheimer disease may actually serve as a homeostatic response to stress resulting in a shift of neuronal priority from normal function to basic survival. This phenomenon is comparable to normal physiological conditions of metabolic decrease, such as those seen in hibernation and estivation. Thus, Alzheimer disease could be seen as part of normal aging that includes additional pathology due to inadequate homeostatic response.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2002
Reactive oxygen species (ROS) generated by mitochondrial respiration and other processes are ofte... more Reactive oxygen species (ROS) generated by mitochondrial respiration and other processes are often viewed as hazardous substances. Indeed, oxidative stress, defined as an imbalance between oxidant production and antioxidant protection, has been linked to several neurological disorders, including cerebral ischemia-reperfusion and Parkinson's disease. Consequently, cells and organisms have evolved specialized antioxidant defenses to balance ROS production and prevent oxidative damage. Research in our laboratory has shown that neuronal levels of ascorbate, a low molecular weight antioxidant, are tenfold higher than those in much less metabolically active glial cells. Ascorbate levels are also selectively elevated in the CNS of anoxia-tolerant reptiles compared to mammals; moreover, plasma and CSF ascorbate concentrations increase markedly in cold-adapted turtles and in hibernating squirrels. Levels of the related antioxidant, glutathione, vary much less between neurons and glia or among species. An added dimension to the role of the antioxidant network comes from recent evidence that ROS can act as neuromodulators. One example is modulation of dopamine release by endogenous hydrogen peroxide, which we describe here for several mammalian species. Together, these data indicate adaptations that prevent oxidative stress and suggest a particularly important role for ascorbate. Moreover, they show that the antioxidant network must be balanced precisely to provide functional levels of ROS, as well as neuroprotection.
Ground squirrels withstand up to 90% reductions in cerebral blood flow during hibernation as well... more Ground squirrels withstand up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousals from torpor. Metabolic suppression likely plays a primary adaptive role which allows hibernating species to tolerate such phenomena. However, several other aspects of hibernation physiology are also consistent with tolerance to dramatic fluctuations in cerebral blood flow, suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work was to study the dynamics of the low molecular weight antioxidants, ascorbate and glutathione (GSH), during hibernation. Alterations in concentrations of ascorbate during hibernation and arousal in two species of hibernating ground squirrels suggest that it could play a protective role during hibernation or arousal. Samples were collected during the hibernation season from arctic ground squirrels (AGS; Spermophilus parryii) and 13-lined ground squirrels (TLS; S. tridecemlineatus) during prolonged torpor and in squirrels that did not hibernate or had not been hibernating for several weeks. We determined antioxidant levels in plasma, cerebrospinal fluid (CSF), and in frontal cortex, hippocampus and cerebellum using high-performance liquid chromatography (HPLC). Plasma ascorbate concentrations increased dramatically (3-4-fold) in both species during hibernation and rapidly returned to prehibernation levels upon arousal. By contrast, plasma GSH concentrations fell slightly or remained stable during hibernation. Ascorbate levels in the CSF doubled in hibernating AGS (not determined in TLS), while brain ascorbate content fell slightly (10-15%) in both species. Substantial increases in plasma and CSF ascorbate concentrations suggest that this antioxidant could play a protective role during hibernation and reperfusion upon arousal from hibernation.
Žw x. This study determined extracellular concentrations of g-aminobutyric acid GABA in striatum ... more Žw x. This study determined extracellular concentrations of g-aminobutyric acid GABA in striatum of non-hibernating and hibernating ecf ecf related to a decrease in GABA synthesis or reduced voltage dependent release. This paper demonstrates that measurement of extracellular concentrations of neurotransmitters in animals with vastly different body temperatures is possible using microdialysis techniques of extrapolation to zero flow or very slow flow rates that enable 100% recovery. Such quantitative techniques may prove valuable in the study of the neurochemistry of the cerebral mechanisms of hibernation and tolerance to cerebral ischemia exhibited by hibernating animals.
Hibernation is a unique and highly regulated physiological state characterized by profound, albei... more Hibernation is a unique and highly regulated physiological state characterized by profound, albeit periodically reversible, depression in body temperature, metabolism, and consciousness. Hippocampal synapses undergo pronounced remodeling in concert with torpor and arousal. During hibernation, the number of postsynaptic densities, apical dendritic branches, and spine densities decreases substantially in the hippocampus. Upon arousal these parameters increase beyond pre-hibernation levels and peak within 2-3 h. By 24 h after arousal, dendritic parameters remain elevated but have started to subside, consistent with pruning and differentiation. The present study examined the functional consequences of these natural changes in synaptic structure. Wild-caught Arctic ground squirrels (AGS) were trained in a hippocampal-dependent contextual fear conditioning task at 3 h, 24 h, or 4 weeks after arousal (warm-adapted euthermic control group). All groups acquired the fear conditioned response similarly on the training day. During a subsequent retention test session, AGS in the 24 h group exhibited enhanced expression of contextual fear compared to the other two groups. These data suggest that the morphological and biochemical changes occurring at 24 h after arousal from hibernation affect hippocampal-dependent learning and memory. The natural change in synaptic structure during hibernation may provide a unique opportunity to assess the neural substrates underlying cognitive enhancement.
Targeted temperature management (TTM) is standard of care for neonatal hypoxic ischemic encephalo... more Targeted temperature management (TTM) is standard of care for neonatal hypoxic ischemic encephalopathy (HIE). Prevention of fever, not excluding cooling core body temperature to • 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 e cacy 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.
During hemorrhagic shock (HS), the body undergoes global ischemia as blood pressure drops below t... more During hemorrhagic shock (HS), the body undergoes global ischemia as blood pressure drops below the threshold at which tissues can be adequately perfused. Resistance to ischemia/reperfusion (I/R) injury is a characteristic of hibernating mammals. The present study sought to determine if arctic ground squirrels (AGS) are protected from HS induced I/R and if any protection is dependent upon their hibernation season. Rats, euthermic AGS, and interbout arousal AGS were subject to HS by withdrawing blood to a mean arterial blood pressure (MAP) of 35 mmHg. Low MAP was maintained for 20 min and then the animals were reperfused with Ringers. The animals’ temperature was maintained at 36.5–37.5 °C. After reperfusion, animals were monitored for 3 hrs then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis. In addition, a group rats and AGS were monitored for three days after HS to access survival and physiological impairment. AGS were able t...
Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seaso... more Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seasonal changes is very evident. In late fall AGS decrease their metabolic demand followed by the entry into hibernation in winter. Previously in the lab it has been shown that N6‐cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, induces hibernation in AGS in a seasonally dependent manner. In winter AGS treated with CHA experience a decrease in metabolic demand and temperature as seen during natural hibernation. However, in summer CHA triggers a brief and transient metabolic suppression that does not lead to hibernation onset.As adenosine is known to promote sleep, we decided to investigate if the sleep‐arousal pathways were affecting the seasonal difference in response to CHA. The tuberomammillary nucleus (TMN) is a nucleus of the ascending arousal pathway characterized by fast firing rates to promote wakefulness. Previously we showed a decrease in neuronal activation in the TMN ...
D-serine and L-glutamate play crucial roles in excitotoxicity through N-methyl-D-aspartate recept... more D-serine and L-glutamate play crucial roles in excitotoxicity through N-methyl-D-aspartate receptor coactivation, but little is known about the temporal profile of efflux during cerebral ischemia. We utilized a newly designed brain slice microperfusion device coupled offline to capillary electrophoresis laser-induced fluorescence to monitor dynamic efflux of endogenous D-ser and Lglu in response to oxygen glucose deprivation (OGD) in single acute hippocampus slices. Efflux profiles with 2-min temporal resolution in response to 24-min OGD show that efflux of D-ser slightly precedes efflux of L-glu by one 2-min sampling interval. Thus both coagonists are available to activate NMDA receptors by the time when glu is released. The magnitude of D-ser efflux relative to baseline values is, however, less than that for L-glu. Peak efflux during OGD, expressed as pre-OGD baseline values, was as follows: D-ser 254% ± 24%, L-glu 1,675% ± 259%, L-asp 519% ± 128%, and L-thr 313% ± 33%. L-glutamine efflux was shown to decrease significantly in response to OGD. The microperfusion/CE-LIF approach shows several promising attributes for studying endogenous chemical efflux from single, acute brain slices.
Background-Hibernation is an adaptation to extreme environments known to provide organ protection... more Background-Hibernation is an adaptation to extreme environments known to provide organ protection against ischemia-reperfusion (I/R) injury. An unbiased systems approach was utilized to investigate hibernation-induced changes characteristic of the hibernator cardioprotective phenotype, by comparing the myocardial proteome of winter hibernating arctic ground squirrels (HIB AGS), summer active (SA) AGS, and rats subjected to I/R, and further correlating with targeted metabolic changes. Methods-In a well-defined rodent model of I/R by deep hypothermic circulatory arrest followed by 3h or 24h of reperfusion or sham, myocardial protein abundance in AGS (HIB, SA) and rats (n=4-5/group) was quantified by label-free proteomics (n=4-5/group), and correlated with metabolic changes. Results-Compared to rats, HIB AGS displayed markedly reduced plasma levels of Troponin I, myocardial apoptosis, and left ventricular contractile dysfunction. Of the 1,320 rat and 1,478 AGS proteins identified, 545 were differentially expressed between HIB AGS and rat hearts (47% upregulated, 53% downregulated). Gene ontology analysis revealed downregulation in HIB AGS hearts of most proteins involved in mitochondrial energy transduction, including electron transport chain complexes, acetyl CoA biosynthesis, Krebs cycle, glycolysis and ketogenesis. Conversely, fatty acid oxidation enzymes and Sirtuin-3 were upregulated in HIB AGS, with preserved †
... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gju... more ... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gjumrakch Aliev, Takafumi Wataya, Shun Shimohama, Kelly Drew ... 7. Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N. Oxidative damage in Alzheimers ...
... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gju... more ... George Perry, Jesús Ávila, Gemma Casadesus, Akihiko Nunomura, Massimo Tabaton, Adam Cash, Gjumrakch Aliev, Takafumi Wataya, Shun Shimohama, Kelly Drew ... 7. Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N. Oxidative damage in Alzheimer s ...
Alzheimer disease (AD) is defined pathologically and diagnostically defined by amyloid- senile p... more Alzheimer disease (AD) is defined pathologically and diagnostically defined by amyloid- senile plaques and neurofibrillary tangles (NFT) composed of tau. From the time of their original description nearly a century ago, a major focus has been to understand the role that these lesions play in the pathogenesis of the disease. The majority favors the notion that these lesions cause the disease and therefore attempts at therapeutic intervention are focused on preventing lesions formation. However, this rationale may be misguided since new evidence from our laboratories and others suggest that the lesions not only occur as a by-product of the fundamental disease process but also that they may be protective.
Hibernating animals are very tolerant of trauma to the central nervous system such that dramatic ... more 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.
Using quantitative microdialysis in hibernating Arctic ground squirrels (AGS), striatal glutamate... more Using quantitative microdialysis in hibernating Arctic ground squirrels (AGS), striatal glutamate concentrations ([glu](dia)) progressively increased to approximately 200 microM after 3 days of microdialysis in euthermic but not hibernating ground squirrels. Initially, the progressive increase in [glu](dia) was thought to be related to greater tissue response in euthermic animals. Alternatively, given the vastly different body temperatures between the two groups (37 vs. 3 degrees C), glutamate might have originated from microbes, replicating at a faster rate in the warmer animals. To test these hypotheses, microdialysis was repeated using sterile technique and tissue response surrounding the probe tract was assessed in hematoxylin and eosin stained sections. Using sterile microdialysis technique, traumatic tissue response was greater in euthermic compared to hibernating tissue. However, sterile microdialysis abolished the progressive increase in glutamate. To confirm the microbial origin of glutamate we monitored [glu](dia) collected in vitro from probes immersed in glutamine-rich liquid medium incubated at 37 degrees C. In vitro, [glu](dia) increased as much as in vivo. Two bacteria isolated from in vitro dialysate and liquid medium were both identified as Ralstonia pickettii. Growth of these isolates as well as glutamate release was enhanced when glutamine rather than NH(4)NO(3) was added to the medium suggesting the bacteria utilize glutamine preferentially over ammonium as a nitrogen source.
Microdialysis is a widely used in vivo sampling technique commonly used to monitor extracellular ... more Microdialysis is a widely used in vivo sampling technique commonly used to monitor extracellular levels of a variety of molecules including neurotransmitters and metabolites. To facilitate interpretation of microdialysis results, this study critically examines changes in synaptic morphology induced by microdialysis. Tissue surrounding microdialysis probes was examined using light and electron microscopy at three distances from the probe tract. Microdialysis probes were implanted into rat striatum, and after 40 h of post-operative recovery were perfused with a modified Ringer's solution. Light microscope analysis revealed tissue disruption up to 1.4 mm from the probe site. Axonal damage indicative of non-excitotoxic insult was also seen as far away from the probe as was examined. The presence of dark-degenerating neurons was also noted and estimates of neuronal densities revealed loss up to 400 mm from the probe tract. This study, the first qualitative ultrastructural investigation of neuropil surrounding the probe site, indicated swollen processes up to 1.4 mm from the probe tract. Swollen mitochondria and bloated endoplasmic reticulum suggest intracellular chemical disruption. Tissue damage resulting in synaptic and neuronal disruption may affect neurotransmitter efflux or extracellular concentrations of metabolites.
Brains of hibernating mammals are protected against a variety of insults that are detrimental to ... more Brains of hibernating mammals are protected against a variety of insults that are detrimental to humans and other nonhibernating species. Such protection is associated with a number of physiological adaptations including hypothermia, increased antioxidant defense, metabolic arrest, leukocytopenia, immunosuppression, and hypocoagulation. It is intriguing that similar manipulations provide considerable protection as experimental treatments for central nervous system injury. This review focuses on neuroprotective mechanisms employed during hibernation that may offer novel approaches in the treatment of stroke, traumatic brain injury, and neurodegenerative diseases in humans.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2002
Ground squirrels tolerate up to 90% reductions in cerebral blood flow during hibernation as well ... more Ground squirrels tolerate up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousal from torpor without apparent neurological damage. Thus, hibernation is studied as a model of tolerance to cerebral ischemia and other types of brain injury. Metabolic suppression likely plays a primary adaptive role that allows hibernating species to tolerate dramatic fluctuations in blood flow. Several other aspects of hibernation physiology are also consistent with tolerance to ischemia and reperfusion suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work is to review evidence for enhanced antioxidant defense systems during hibernation, with a focus on ascorbate, and discuss potential roles of these antioxidants during hibernation. In concert with dramatic decreases in blood flow, nutrient and oxygen delivery, plasma concentrations of the antioxidant ascorbate w(Asc)px increase 3-5-fold during hibernation. In contrast, during re-warming, wAscxp declines at a relatively rapid rate that peaks at the time of maximal O consumption. This peak in O consumption also 2 2 coincides with a brief rise in plasma urate concentration consistent with a surge in reactive oxygen species production. Overall, data suggest that elevated concentration of plasma ascorbate is poised for distribution to metabolically active tissues during the surge in oxidative metabolism that accompanies re-warming during hibernation. This pool of ascorbate, as well as increased expression of other antioxidant defense systems, may protect vulnerable tissues from oxidative stress during hibernation and re-warming from hibernation. Better understanding of the role of ascorbate in hibernation may guide use of ascorbate and other antioxidants in treatment of stroke, head trauma and neurodegenerative disease.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2002
In this review, we consider comparative aspects of the biology and pathology of oxygen radicals i... more In this review, we consider comparative aspects of the biology and pathology of oxygen radicals in neurodegenerative disease and how these findings have influenced our concept of oxidative stress. The common definition of oxidative stress is a breach of antioxidant defenses by oxygen radicals leading to damage to critical molecules and disrupted physiology. Inherent in this definition is that oxidative stress is an unstable situation, for if there is net damage, viability of the system decreases with time, leading to disequilibria and death. While this circumstance defines acute conditions, such as stroke and head trauma which result in dysfunction and death, it does not fit physiological situations or chronic diseases closely aligned to normal physiology. Therefore, we propose that oxidative modifications in Alzheimer disease may actually serve as a homeostatic response to stress resulting in a shift of neuronal priority from normal function to basic survival. This phenomenon is comparable to normal physiological conditions of metabolic decrease, such as those seen in hibernation and estivation. Thus, Alzheimer disease could be seen as part of normal aging that includes additional pathology due to inadequate homeostatic response.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2002
Reactive oxygen species (ROS) generated by mitochondrial respiration and other processes are ofte... more Reactive oxygen species (ROS) generated by mitochondrial respiration and other processes are often viewed as hazardous substances. Indeed, oxidative stress, defined as an imbalance between oxidant production and antioxidant protection, has been linked to several neurological disorders, including cerebral ischemia-reperfusion and Parkinson's disease. Consequently, cells and organisms have evolved specialized antioxidant defenses to balance ROS production and prevent oxidative damage. Research in our laboratory has shown that neuronal levels of ascorbate, a low molecular weight antioxidant, are tenfold higher than those in much less metabolically active glial cells. Ascorbate levels are also selectively elevated in the CNS of anoxia-tolerant reptiles compared to mammals; moreover, plasma and CSF ascorbate concentrations increase markedly in cold-adapted turtles and in hibernating squirrels. Levels of the related antioxidant, glutathione, vary much less between neurons and glia or among species. An added dimension to the role of the antioxidant network comes from recent evidence that ROS can act as neuromodulators. One example is modulation of dopamine release by endogenous hydrogen peroxide, which we describe here for several mammalian species. Together, these data indicate adaptations that prevent oxidative stress and suggest a particularly important role for ascorbate. Moreover, they show that the antioxidant network must be balanced precisely to provide functional levels of ROS, as well as neuroprotection.
Ground squirrels withstand up to 90% reductions in cerebral blood flow during hibernation as well... more Ground squirrels withstand up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousals from torpor. Metabolic suppression likely plays a primary adaptive role which allows hibernating species to tolerate such phenomena. However, several other aspects of hibernation physiology are also consistent with tolerance to dramatic fluctuations in cerebral blood flow, suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work was to study the dynamics of the low molecular weight antioxidants, ascorbate and glutathione (GSH), during hibernation. Alterations in concentrations of ascorbate during hibernation and arousal in two species of hibernating ground squirrels suggest that it could play a protective role during hibernation or arousal. Samples were collected during the hibernation season from arctic ground squirrels (AGS; Spermophilus parryii) and 13-lined ground squirrels (TLS; S. tridecemlineatus) during prolonged torpor and in squirrels that did not hibernate or had not been hibernating for several weeks. We determined antioxidant levels in plasma, cerebrospinal fluid (CSF), and in frontal cortex, hippocampus and cerebellum using high-performance liquid chromatography (HPLC). Plasma ascorbate concentrations increased dramatically (3-4-fold) in both species during hibernation and rapidly returned to prehibernation levels upon arousal. By contrast, plasma GSH concentrations fell slightly or remained stable during hibernation. Ascorbate levels in the CSF doubled in hibernating AGS (not determined in TLS), while brain ascorbate content fell slightly (10-15%) in both species. Substantial increases in plasma and CSF ascorbate concentrations suggest that this antioxidant could play a protective role during hibernation and reperfusion upon arousal from hibernation.
Žw x. This study determined extracellular concentrations of g-aminobutyric acid GABA in striatum ... more Žw x. This study determined extracellular concentrations of g-aminobutyric acid GABA in striatum of non-hibernating and hibernating ecf ecf related to a decrease in GABA synthesis or reduced voltage dependent release. This paper demonstrates that measurement of extracellular concentrations of neurotransmitters in animals with vastly different body temperatures is possible using microdialysis techniques of extrapolation to zero flow or very slow flow rates that enable 100% recovery. Such quantitative techniques may prove valuable in the study of the neurochemistry of the cerebral mechanisms of hibernation and tolerance to cerebral ischemia exhibited by hibernating animals.
Hibernation is a unique and highly regulated physiological state characterized by profound, albei... more Hibernation is a unique and highly regulated physiological state characterized by profound, albeit periodically reversible, depression in body temperature, metabolism, and consciousness. Hippocampal synapses undergo pronounced remodeling in concert with torpor and arousal. During hibernation, the number of postsynaptic densities, apical dendritic branches, and spine densities decreases substantially in the hippocampus. Upon arousal these parameters increase beyond pre-hibernation levels and peak within 2-3 h. By 24 h after arousal, dendritic parameters remain elevated but have started to subside, consistent with pruning and differentiation. The present study examined the functional consequences of these natural changes in synaptic structure. Wild-caught Arctic ground squirrels (AGS) were trained in a hippocampal-dependent contextual fear conditioning task at 3 h, 24 h, or 4 weeks after arousal (warm-adapted euthermic control group). All groups acquired the fear conditioned response similarly on the training day. During a subsequent retention test session, AGS in the 24 h group exhibited enhanced expression of contextual fear compared to the other two groups. These data suggest that the morphological and biochemical changes occurring at 24 h after arousal from hibernation affect hippocampal-dependent learning and memory. The natural change in synaptic structure during hibernation may provide a unique opportunity to assess the neural substrates underlying cognitive enhancement.
Targeted temperature management (TTM) is standard of care for neonatal hypoxic ischemic encephalo... more Targeted temperature management (TTM) is standard of care for neonatal hypoxic ischemic encephalopathy (HIE). Prevention of fever, not excluding cooling core body temperature to • 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 e cacy 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.
During hemorrhagic shock (HS), the body undergoes global ischemia as blood pressure drops below t... more During hemorrhagic shock (HS), the body undergoes global ischemia as blood pressure drops below the threshold at which tissues can be adequately perfused. Resistance to ischemia/reperfusion (I/R) injury is a characteristic of hibernating mammals. The present study sought to determine if arctic ground squirrels (AGS) are protected from HS induced I/R and if any protection is dependent upon their hibernation season. Rats, euthermic AGS, and interbout arousal AGS were subject to HS by withdrawing blood to a mean arterial blood pressure (MAP) of 35 mmHg. Low MAP was maintained for 20 min and then the animals were reperfused with Ringers. The animals’ temperature was maintained at 36.5–37.5 °C. After reperfusion, animals were monitored for 3 hrs then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis. In addition, a group rats and AGS were monitored for three days after HS to access survival and physiological impairment. AGS were able t...
Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seaso... more Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seasonal changes is very evident. In late fall AGS decrease their metabolic demand followed by the entry into hibernation in winter. Previously in the lab it has been shown that N6‐cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, induces hibernation in AGS in a seasonally dependent manner. In winter AGS treated with CHA experience a decrease in metabolic demand and temperature as seen during natural hibernation. However, in summer CHA triggers a brief and transient metabolic suppression that does not lead to hibernation onset.As adenosine is known to promote sleep, we decided to investigate if the sleep‐arousal pathways were affecting the seasonal difference in response to CHA. The tuberomammillary nucleus (TMN) is a nucleus of the ascending arousal pathway characterized by fast firing rates to promote wakefulness. Previously we showed a decrease in neuronal activation in the TMN ...
D-serine and L-glutamate play crucial roles in excitotoxicity through N-methyl-D-aspartate recept... more D-serine and L-glutamate play crucial roles in excitotoxicity through N-methyl-D-aspartate receptor coactivation, but little is known about the temporal profile of efflux during cerebral ischemia. We utilized a newly designed brain slice microperfusion device coupled offline to capillary electrophoresis laser-induced fluorescence to monitor dynamic efflux of endogenous D-ser and Lglu in response to oxygen glucose deprivation (OGD) in single acute hippocampus slices. Efflux profiles with 2-min temporal resolution in response to 24-min OGD show that efflux of D-ser slightly precedes efflux of L-glu by one 2-min sampling interval. Thus both coagonists are available to activate NMDA receptors by the time when glu is released. The magnitude of D-ser efflux relative to baseline values is, however, less than that for L-glu. Peak efflux during OGD, expressed as pre-OGD baseline values, was as follows: D-ser 254% ± 24%, L-glu 1,675% ± 259%, L-asp 519% ± 128%, and L-thr 313% ± 33%. L-glutamine efflux was shown to decrease significantly in response to OGD. The microperfusion/CE-LIF approach shows several promising attributes for studying endogenous chemical efflux from single, acute brain slices.
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Papers by Kelly Drew