University of Alaska Fairbanks

Postdoctoral Research Fellow

Targeting Central A1 Adenosine Receptors Induces Therapeutic Hypothermia for Stroke

Changes in Bone Density and Lean Tissue Mass during Disuse and Torpor in the Arctic Ground Squirrel

Instructor for Chem 105, first semester chemistry course for STEM majors.

Intern with the Office of Intellectual Property and Commercialization at UAF

University of Alaska Fairbanks

Graduate Research Assistant

Biochemistry and Molecular Biology Program

Protective Mechanisms of Arctic Ground Squirrels against Global Ischemia and Multi Organ Failure caused by Ischemia/ Reperfusion (Cardiac Arrest and Hemorrhagic Shock Models)

1H NMR Metabolomic Analysis of Arctic Ground Squirrel Plasma and Liver Before and After Isobaric Hemorrhagic Shock

SomaLogic

Scientist

Bioinformatics

University of Colorado Anschutz Medical Campus

Postdoctoral Research Fellow

-LC-MS metabolomic analysis of hibernation state transitions.
-RNASeq to identify hibernation state specific mRNA editing in various brain regions and of hibernators.
-EDGE-tag transcriptomics to determine changes in mRNA profile over the physiological extremes of a hibernator's year.

Verizon

Data Scientist

Data Scientist for Cyber Security and Fraud.

Amgen Inc.

Sr. Associate

Analytical Sciences- Bio Analytical group for a late stage recombinant protein commercialization program. Responsible for:
- analytical method development, qualifications, transfers
- degradation characterization
- analytical support for drug substance and drug product process development
- supporting investigations in quality control and manufacturing

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Optimization of thermolytic response to A1 adenosine receptor agonists in rats

J. Pharmacology and Exp. Therapeutics

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Optimization of thermolytic response to A1 adenosine receptor agonists in rats

J. Pharmacology and Exp. Therapeutics

Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel.

PLoS One

Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. 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 systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season. METHODS: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment. RESULTS: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage. CONCLUSIONS: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Optimization of thermolytic response to A1 adenosine receptor agonists in rats

J. Pharmacology and Exp. Therapeutics

Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel.

PLoS One

Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. 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 systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season. METHODS: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment. RESULTS: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage. CONCLUSIONS: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Effects of combined insulin-like growth factor 1 and macrophage colony-stimulating factor on the skeletal properties of mice.

In Vivo

Background/Aim: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. Materials and Methods: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). Results: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (−13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. Conclusion: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Optimization of thermolytic response to A1 adenosine receptor agonists in rats

J. Pharmacology and Exp. Therapeutics

Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel.

PLoS One

Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. 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 systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season. METHODS: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment. RESULTS: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage. CONCLUSIONS: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Effects of combined insulin-like growth factor 1 and macrophage colony-stimulating factor on the skeletal properties of mice.

In Vivo

Background/Aim: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. Materials and Methods: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). Results: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (−13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. Conclusion: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.

1H-NMR metabolomic biomarkers of poor outcome after hemorrhagic shock are absent in hibernators.

PLoS One

Hemorrhagic shock (HS) following trauma is a leading cause of death among persons under the age of 40. During HS the body undergoes systemic warm ischemia followed by reperfusion during medical intervention. Ischemia/reperfusion (I/R) results in a disruption of cellular metabolic processes that ultimately lead to tissue and organ dysfunction or failure. Resistance to I/R injury is a characteristic of hibernating mammals. The present study sought to identify circulating metabolites in the rat as biomarkers for metabolic alterations associated with poor outcome after HS. Arctic ground squirrels (AGS), a hibernating species that resists I/R injury independent of decreased body temperature (warm I/R), was used as a negative control. Male Sprague-Dawley rats and AGS were subject to HS by withdrawing blood to a mean arterial pressure (MAP) of 35 mmHg and maintaining the low MAP for 20 min before reperfusing with Ringers. The animals' temperature was maintained at 37 ± 0.5 °C for the duration of the experiment. Plasma samples were taken immediately before hemorrhage and three hours after reperfusion. Hydrophilic and lipid metabolites from plasma were then analyzed via 1H-NMR from unprocessed plasma and lipid extracts, respectively. Rats, susceptible to I/R injury, had a qualitative shift in their hydrophilic metabolic fingerprint including differential activation of glucose and anaerobic metabolism and had alterations in several metabolites during I/R indicative of metabolic adjustments and organ damage. In contrast, I/R injury resistant AGS, regardless of season or body temperature, maintained a stable metabolic homeostasis revealed by a qualitative 1H-NMR metabolic profile with few changes in quantified metabolites during HS-induced global I/R. An increase in circulating metabolites indicative of anaerobic metabolism and activation of glycolytic pathways is associated with poor prognosis after HS in rats. These same biomarkers are absent in AGS after HS with warm I/R.

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Optimization of thermolytic response to A1 adenosine receptor agonists in rats

J. Pharmacology and Exp. Therapeutics

Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel.

PLoS One

Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. 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 systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season. METHODS: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment. RESULTS: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage. CONCLUSIONS: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Effects of combined insulin-like growth factor 1 and macrophage colony-stimulating factor on the skeletal properties of mice.

In Vivo

Background/Aim: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. Materials and Methods: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). Results: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (−13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. Conclusion: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.

1H-NMR metabolomic biomarkers of poor outcome after hemorrhagic shock are absent in hibernators.

PLoS One

Hemorrhagic shock (HS) following trauma is a leading cause of death among persons under the age of 40. During HS the body undergoes systemic warm ischemia followed by reperfusion during medical intervention. Ischemia/reperfusion (I/R) results in a disruption of cellular metabolic processes that ultimately lead to tissue and organ dysfunction or failure. Resistance to I/R injury is a characteristic of hibernating mammals. The present study sought to identify circulating metabolites in the rat as biomarkers for metabolic alterations associated with poor outcome after HS. Arctic ground squirrels (AGS), a hibernating species that resists I/R injury independent of decreased body temperature (warm I/R), was used as a negative control. Male Sprague-Dawley rats and AGS were subject to HS by withdrawing blood to a mean arterial pressure (MAP) of 35 mmHg and maintaining the low MAP for 20 min before reperfusing with Ringers. The animals' temperature was maintained at 37 ± 0.5 °C for the duration of the experiment. Plasma samples were taken immediately before hemorrhage and three hours after reperfusion. Hydrophilic and lipid metabolites from plasma were then analyzed via 1H-NMR from unprocessed plasma and lipid extracts, respectively. Rats, susceptible to I/R injury, had a qualitative shift in their hydrophilic metabolic fingerprint including differential activation of glucose and anaerobic metabolism and had alterations in several metabolites during I/R indicative of metabolic adjustments and organ damage. In contrast, I/R injury resistant AGS, regardless of season or body temperature, maintained a stable metabolic homeostasis revealed by a qualitative 1H-NMR metabolic profile with few changes in quantified metabolites during HS-induced global I/R. An increase in circulating metabolites indicative of anaerobic metabolism and activation of glycolytic pathways is associated with poor prognosis after HS in rats. These same biomarkers are absent in AGS after HS with warm I/R.

Comparative Tissue Transcriptomics Highlights Dynamic Differences among Tissues but Conserved Metabolic Transcript Prioritization in Preparation for Arousal from Torpor

Journal of Comparative Physiology B

During the hibernation season, 13-lined ground squirrels spend days to weeks in torpor with body temperatures near freezing, then spontaneously rewarm. The molecular drivers of the drastic physiological changes that orchestrate and permit torpor are not well understood. Although transcription effectively ceases at the low body temperatures of torpor, previous work has demonstrated that some transcripts are protected from bulk degradation in brown adipose tissue (BAT), consistent with the importance of their protein products for metabolic heat generation during arousal from torpor. We examined the transcriptome of skeletal muscle, heart and liver to determine the patterns of differentially expressed genes in these tissues, and whether, like BAT, a subset of these were relatively increased during torpor. EDGE-tags were quantified from five distinct physiological states representing the seasonal and torpor-arousal cycles of 13-lined ground squirrels. Supervised clustering on relative transcript abundances with Random Forest separated the two states bracketing prolonged torpor, entrance into and aroused from torpor, in all three tissues. Independent analyses identified 3347, 6784, and 2433 differentially-expressed transcripts among all sampling points in heart, skeletal muscle, and liver, respectively. There were few differentially expressed genes in common across all three tissues; these were enriched in mitochondrial and apoptotic pathway components. Divisive clustering of these data revealed unique cohorts of transcripts that increased across the torpor bout in each tissue with patterns reflecting various combinations of cycling within and between seasons as well as between torpor and arousal. Transcripts that increased across the torpor bout were likewise tissue specific. These data shed new light on the biochemical pathways that alter in concert with hibernation phenotype and provide a rich resource for further hypothesis-based studies.

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Optimization of thermolytic response to A1 adenosine receptor agonists in rats

J. Pharmacology and Exp. Therapeutics

Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel.

PLoS One

Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. 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 systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season. METHODS: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment. RESULTS: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage. CONCLUSIONS: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Effects of combined insulin-like growth factor 1 and macrophage colony-stimulating factor on the skeletal properties of mice.

In Vivo

Background/Aim: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. Materials and Methods: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). Results: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (−13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. Conclusion: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.

1H-NMR metabolomic biomarkers of poor outcome after hemorrhagic shock are absent in hibernators.

PLoS One

Hemorrhagic shock (HS) following trauma is a leading cause of death among persons under the age of 40. During HS the body undergoes systemic warm ischemia followed by reperfusion during medical intervention. Ischemia/reperfusion (I/R) results in a disruption of cellular metabolic processes that ultimately lead to tissue and organ dysfunction or failure. Resistance to I/R injury is a characteristic of hibernating mammals. The present study sought to identify circulating metabolites in the rat as biomarkers for metabolic alterations associated with poor outcome after HS. Arctic ground squirrels (AGS), a hibernating species that resists I/R injury independent of decreased body temperature (warm I/R), was used as a negative control. Male Sprague-Dawley rats and AGS were subject to HS by withdrawing blood to a mean arterial pressure (MAP) of 35 mmHg and maintaining the low MAP for 20 min before reperfusing with Ringers. The animals' temperature was maintained at 37 ± 0.5 °C for the duration of the experiment. Plasma samples were taken immediately before hemorrhage and three hours after reperfusion. Hydrophilic and lipid metabolites from plasma were then analyzed via 1H-NMR from unprocessed plasma and lipid extracts, respectively. Rats, susceptible to I/R injury, had a qualitative shift in their hydrophilic metabolic fingerprint including differential activation of glucose and anaerobic metabolism and had alterations in several metabolites during I/R indicative of metabolic adjustments and organ damage. In contrast, I/R injury resistant AGS, regardless of season or body temperature, maintained a stable metabolic homeostasis revealed by a qualitative 1H-NMR metabolic profile with few changes in quantified metabolites during HS-induced global I/R. An increase in circulating metabolites indicative of anaerobic metabolism and activation of glycolytic pathways is associated with poor prognosis after HS in rats. These same biomarkers are absent in AGS after HS with warm I/R.

Comparative Tissue Transcriptomics Highlights Dynamic Differences among Tissues but Conserved Metabolic Transcript Prioritization in Preparation for Arousal from Torpor

Journal of Comparative Physiology B

During the hibernation season, 13-lined ground squirrels spend days to weeks in torpor with body temperatures near freezing, then spontaneously rewarm. The molecular drivers of the drastic physiological changes that orchestrate and permit torpor are not well understood. Although transcription effectively ceases at the low body temperatures of torpor, previous work has demonstrated that some transcripts are protected from bulk degradation in brown adipose tissue (BAT), consistent with the importance of their protein products for metabolic heat generation during arousal from torpor. We examined the transcriptome of skeletal muscle, heart and liver to determine the patterns of differentially expressed genes in these tissues, and whether, like BAT, a subset of these were relatively increased during torpor. EDGE-tags were quantified from five distinct physiological states representing the seasonal and torpor-arousal cycles of 13-lined ground squirrels. Supervised clustering on relative transcript abundances with Random Forest separated the two states bracketing prolonged torpor, entrance into and aroused from torpor, in all three tissues. Independent analyses identified 3347, 6784, and 2433 differentially-expressed transcripts among all sampling points in heart, skeletal muscle, and liver, respectively. There were few differentially expressed genes in common across all three tissues; these were enriched in mitochondrial and apoptotic pathway components. Divisive clustering of these data revealed unique cohorts of transcripts that increased across the torpor bout in each tissue with patterns reflecting various combinations of cycling within and between seasons as well as between torpor and arousal. Transcripts that increased across the torpor bout were likewise tissue specific. These data shed new light on the biochemical pathways that alter in concert with hibernation phenotype and provide a rich resource for further hypothesis-based studies.

Comfortably Numb and Back: Plasma Metabolomics Reveals Biochemical Adaptations in the Hibernating 13-Lined Ground Squirrel.

J Proteome Res

Hibernation is an evolutionary adaptation that affords some mammals the ability to exploit the cold to achieve extreme metabolic depression (torpor) while avoiding ischemia/reperfusion or hemorrhagic shock injuries. Hibernators cycle periodically out of torpor, restoring high metabolic activity. If understood at the molecular level, the adaptations underlying torpor-arousal cycles may be leveraged for translational applications in critical fields such as intensive care medicine. Here, we monitored 266 metabolites to investigate the metabolic adaptations to hibernation in plasma from 13-lined ground squirrels (57 animals, 9 time points). Results indicate that the periodic arousals foster the removal of potentially toxic oxidative stress-related metabolites, which accumulate in plasma during torpor while replenishing reservoirs of circulating catabolic substrates (free fatty acids and amino acids). Specifically, we identified metabolic fluctuations of basic amino acids lysine and arginine, one-carbon metabolism intermediates, and sulfur-containing metabolites methionine, cysteine, and cystathionine. Conversely, reperfusion injury markers such as succinate/fumarate remained relatively stable across cycles. Considering the cycles of these metabolites with the hibernator's cycling metabolic activity together with their well-established role as substrates for the production of hydrogen sulfide (H2S), we hypothesize that these metabolic fluctuations function as a biological clock regulating torpor to arousal transitions and resistance to reperfusion during arousal.

Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons.

Physiological Reports

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. The present 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 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 (DXA) 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 (micro-computed 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.

Optimization of thermolytic response to A1 adenosine receptor agonists in rats

J. Pharmacology and Exp. Therapeutics

Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel.

PLoS One

Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. 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 systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season. METHODS: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment. RESULTS: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage. CONCLUSIONS: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Effects of combined insulin-like growth factor 1 and macrophage colony-stimulating factor on the skeletal properties of mice.

In Vivo

Background/Aim: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. Materials and Methods: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). Results: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (−13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. Conclusion: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.

1H-NMR metabolomic biomarkers of poor outcome after hemorrhagic shock are absent in hibernators.

PLoS One

Hemorrhagic shock (HS) following trauma is a leading cause of death among persons under the age of 40. During HS the body undergoes systemic warm ischemia followed by reperfusion during medical intervention. Ischemia/reperfusion (I/R) results in a disruption of cellular metabolic processes that ultimately lead to tissue and organ dysfunction or failure. Resistance to I/R injury is a characteristic of hibernating mammals. The present study sought to identify circulating metabolites in the rat as biomarkers for metabolic alterations associated with poor outcome after HS. Arctic ground squirrels (AGS), a hibernating species that resists I/R injury independent of decreased body temperature (warm I/R), was used as a negative control. Male Sprague-Dawley rats and AGS were subject to HS by withdrawing blood to a mean arterial pressure (MAP) of 35 mmHg and maintaining the low MAP for 20 min before reperfusing with Ringers. The animals' temperature was maintained at 37 ± 0.5 °C for the duration of the experiment. Plasma samples were taken immediately before hemorrhage and three hours after reperfusion. Hydrophilic and lipid metabolites from plasma were then analyzed via 1H-NMR from unprocessed plasma and lipid extracts, respectively. Rats, susceptible to I/R injury, had a qualitative shift in their hydrophilic metabolic fingerprint including differential activation of glucose and anaerobic metabolism and had alterations in several metabolites during I/R indicative of metabolic adjustments and organ damage. In contrast, I/R injury resistant AGS, regardless of season or body temperature, maintained a stable metabolic homeostasis revealed by a qualitative 1H-NMR metabolic profile with few changes in quantified metabolites during HS-induced global I/R. An increase in circulating metabolites indicative of anaerobic metabolism and activation of glycolytic pathways is associated with poor prognosis after HS in rats. These same biomarkers are absent in AGS after HS with warm I/R.

Comparative Tissue Transcriptomics Highlights Dynamic Differences among Tissues but Conserved Metabolic Transcript Prioritization in Preparation for Arousal from Torpor

Journal of Comparative Physiology B

During the hibernation season, 13-lined ground squirrels spend days to weeks in torpor with body temperatures near freezing, then spontaneously rewarm. The molecular drivers of the drastic physiological changes that orchestrate and permit torpor are not well understood. Although transcription effectively ceases at the low body temperatures of torpor, previous work has demonstrated that some transcripts are protected from bulk degradation in brown adipose tissue (BAT), consistent with the importance of their protein products for metabolic heat generation during arousal from torpor. We examined the transcriptome of skeletal muscle, heart and liver to determine the patterns of differentially expressed genes in these tissues, and whether, like BAT, a subset of these were relatively increased during torpor. EDGE-tags were quantified from five distinct physiological states representing the seasonal and torpor-arousal cycles of 13-lined ground squirrels. Supervised clustering on relative transcript abundances with Random Forest separated the two states bracketing prolonged torpor, entrance into and aroused from torpor, in all three tissues. Independent analyses identified 3347, 6784, and 2433 differentially-expressed transcripts among all sampling points in heart, skeletal muscle, and liver, respectively. There were few differentially expressed genes in common across all three tissues; these were enriched in mitochondrial and apoptotic pathway components. Divisive clustering of these data revealed unique cohorts of transcripts that increased across the torpor bout in each tissue with patterns reflecting various combinations of cycling within and between seasons as well as between torpor and arousal. Transcripts that increased across the torpor bout were likewise tissue specific. These data shed new light on the biochemical pathways that alter in concert with hibernation phenotype and provide a rich resource for further hypothesis-based studies.

Comfortably Numb and Back: Plasma Metabolomics Reveals Biochemical Adaptations in the Hibernating 13-Lined Ground Squirrel.

J Proteome Res

Hibernation is an evolutionary adaptation that affords some mammals the ability to exploit the cold to achieve extreme metabolic depression (torpor) while avoiding ischemia/reperfusion or hemorrhagic shock injuries. Hibernators cycle periodically out of torpor, restoring high metabolic activity. If understood at the molecular level, the adaptations underlying torpor-arousal cycles may be leveraged for translational applications in critical fields such as intensive care medicine. Here, we monitored 266 metabolites to investigate the metabolic adaptations to hibernation in plasma from 13-lined ground squirrels (57 animals, 9 time points). Results indicate that the periodic arousals foster the removal of potentially toxic oxidative stress-related metabolites, which accumulate in plasma during torpor while replenishing reservoirs of circulating catabolic substrates (free fatty acids and amino acids). Specifically, we identified metabolic fluctuations of basic amino acids lysine and arginine, one-carbon metabolism intermediates, and sulfur-containing metabolites methionine, cysteine, and cystathionine. Conversely, reperfusion injury markers such as succinate/fumarate remained relatively stable across cycles. Considering the cycles of these metabolites with the hibernator's cycling metabolic activity together with their well-established role as substrates for the production of hydrogen sulfide (H2S), we hypothesize that these metabolic fluctuations function as a biological clock regulating torpor to arousal transitions and resistance to reperfusion during arousal.

Ischemia/reperfusion injury resistance in hibernators is more than an effect of reduced body temperature or winter season

Temperature

Hibernating mammals are resistant to injury following cardiac arrest. The basis of this protection has been proposed to be due to their ability to lower body temperature or metabolic rate in a seasonally-dependent manner. However, recent studies have shown that neither reduced body temperature nor hibernation season are components this protection.