ABSTRACT
Hibernating Ictidomys tridecemlineatus, 13-lined ground squirrels, are considered models of ischaemia–reperfusion tolerance, as both tissues and isolated mitochondria withstand anoxia followed by rapid re-oxygenation in vitro. This tolerance is likely adaptive, protecting against damage during the numerous arousals from torpor throughout the hibernation season. O2 availability is likely low during torpor, but suppressed metabolism lowers O2 demand, potentially mitigating hypoxic stress. During arousal to interbout euthermia (IBE), heart rate, blood pressure and ventilation increase rapidly, suggesting increased O2 availability, but tissue oxygenation has not been measured during arousal or IBE in 13-lined ground squirrels. Using pulse-oximetry collars, we characterized dramatic increases in O2 availability during arousal; carotid artery O2 saturation rose from as low as 35% early in arousal to 87% during IBE. These changes closely followed rising heart rate. Our results demonstrate that hibernating 13-lined ground squirrels survive profound O2 deprivation early in arousal and rapid O2 influx as arousal progresses.
INTRODUCTION
Hibernators evade winter's energetic demands through metabolic suppression. In ground squirrels, hibernation involves cycling between torpor and interbout euthermia (IBE). 13-Lined ground squirrels (Ictidomys tridecemlineatus) can experience up to 20 torpor bouts per season, lasting 6–15 days with 90% metabolic suppression and body temperatures (Tb) of 4–6°C (Muleme et al., 2006; Fig. 1A). Torpor is interrupted by arousals to IBE, where metabolism and Tb return to high values (similar to summer values) for 6–10 h before animals re-enter torpor (Fig. 1A). Low Tb alone typically induces a leftward shift in the haemoglobin–oxygen (Hb–O2) equilibrium curve, as P50, the partial pressure of O2 at which haemoglobin is 50% saturated, decreases with temperature. During torpor, P50 decreases in hibernating ground squirrels, corresponding with suppressed metabolism (Revsbech et al., 2013; Maginniss and Milsom, 1994; Revsbech and Fago, 2017). However, early in arousal, metabolic demand rises before Tb, increasing the risk of hypoxia, especially before shivering thermogenesis commences.
Physiological metrics that change throughout hibernation in 13-lined ground squirrels (Ictidomys tridecemlineatus). (A) Body temperature (Tb) in a 13-lined ground squirrel during hibernation. (B) Heart rate (fH; red dashed line) and Tb (solid black line) fluctuations during an arousal; adapted from MacCannell et al. (2018), with permission. (C) Comparison of cardiac output (from Popovic, 1964), breathing frequency (from Sprenger and Milsom, 2022) and whole-animal oxygen consumption (from Muleme et al., 2006) between torpor and interbout euthermia (IBE). Data are means±s.e.m.
Physiological metrics that change throughout hibernation in 13-lined ground squirrels (Ictidomys tridecemlineatus). (A) Body temperature (Tb) in a 13-lined ground squirrel during hibernation. (B) Heart rate (fH; red dashed line) and Tb (solid black line) fluctuations during an arousal; adapted from MacCannell et al. (2018), with permission. (C) Comparison of cardiac output (from Popovic, 1964), breathing frequency (from Sprenger and Milsom, 2022) and whole-animal oxygen consumption (from Muleme et al., 2006) between torpor and interbout euthermia (IBE). Data are means±s.e.m.
Hb concentration and haematocrit (Hct) are also important for O2 delivery; data on these values vary. For instance, one study reported declines in red blood cell count, Hb and Hct during torpor compared with summer (Spurrier and Dawe, 1973), but later research found no significant changes (Cooper et al., 2016a). Blood ion and metabolite concentration can help contextualize haematological changes; high blood Na+ may indicate dehydration, leading to overestimates of Hb and Hct as a result of decreasing plasma volume. Moreover, decreases in blood glucose may indicate anaerobic metabolism from reduced O2 availability (Abu et al., 2018).
During arousal, heart rate (fH), cardiac output and ventilation increase to accommodate increased metabolic rate (Fig. 1B,C). Blood pressure also increases during arousal, rising from 60/30 mmHg (systolic/diastolic) in torpor to 140/100 mmHg during IBE (Lyman, 1960), and blood flow to the heart of 13-lined ground squirrels increases 8-fold (Bullard and Funkhouser, 1962). During torpor in 13-lined ground squirrels, coagulation factor activity and platelet count decline (Cooper et al., 2016b), and fibrinolysis is 3-fold faster (Bonis et al., 2019), indicating minimal resistance to blood flow early in arousal.
During arousal, a 2-fold increase in both ventilation (Sprenger and Milsom, 2022) and cardiac output (Popovic, 1964) likely correspond with rapid increases in O2 delivery, which can increase oxidative damage in other animal models (Granger and Kvietys, 2015). Even 13-lined ground squirrels have higher oxidative damage to lipids and proteins in some tissues during IBE compared with torpor (Duffy and Staples, 2022). 13-Lined ground squirrel arousals are often considered similar to ischaemia–reperfusion (I–R) (Bonis et al., 2019; Otis et al., 2017; Kurtz and Carey, 2006). I–R is characterized by a restriction of tissue blood supply, followed by rapid restoration of blood flow and re-oxygenation, and is often associated with pathological conditions such as ischaemic stroke or myocardial infarction. In humans, an arterial oxygen saturation below 55% indicates ischaemia and severe O2 deprivation to tissue (Yu et al., 2020). While ischaemic hypoxia can cause significant injury itself, the damage is exacerbated during the reperfusion event, when sudden increases in O2 result in excess reactive oxygen species (ROS) production (Granger and Kvietys, 2015). Elevated ROS production rates can exceed antioxidant capacity, causing oxidative damage to cellular macromolecules, including proteins, lipids and DNA.
Resistance to I–R injury is a characteristic of hibernating mammals. For example, hepatic cells from the facultative hibernator Syrian hamster (Mesocricetus auratus) did not show DNA damage following I–R, whereas lab mice (C57BL/6) cells did (Pissas et al., 2025). At the whole-animal level, in the summer phenotype, arctic ground squirrels (Spermophilus parryii) survive haemorrhagic shock better than rats (Sprague–Dawley; non-hibernators) (Bogren et al., 2014). Within a species, the hibernation phenotype further enhances ischaemia–reperfusion tolerance. Livers from hibernating 13-lined ground squirrels tolerate cold ischaemia followed by warm reperfusion for longer and with less damage than livers from summer 13-lined ground squirrels or rats (Lindell et al., 2004). This resistance is hypothesized to persist because of acquired adaptations that allow small hibernators to endure dozens of arousals every hibernation season. However, the mechanisms of I–R resistance in hibernators remain unknown.
Despite the documented changes in cardiovascular function during arousal (Fig. 1C), whether tissues become hypoxic in 13-lined ground squirrels remains unclear. In torpid arctic ground squirrels, arterial O2 tension (PaO2) and arterial Hb O2 saturation (SaO2) are comparable to those in non-hibernating rats (Sprague–Dawley; Ma et al., 2005), indicating that reductions in O2 delivery are matched by reductions in tissue O2 demand in torpor. Conversely, lactate accumulates in red blood cells of arctic ground squirrels during torpor, suggesting anaerobic metabolism (Gehrke et al., 2019). Furthermore, in 13-lined ground squirrels, levels of hypoxia-inducible transcription factor-1 (HIF-1α) increase in skeletal muscle, brown adipose tissue and liver during torpor, indicating widespread hypoxia (Maistrovski et al., 2012).
While it has long been predicted that 13-lined ground squirrels experience a significant influx of O2 during arousal when fH (Fig. 1B), ventilation and blood pressure (Fig. 1C) increase rapidly, other variables, including Tb and metabolic rate, may confound this effect. We hypothesized that an increased O2 demand during arousal is matched by increased O2 supply and predicted that O2 availability would be low in early arousal but increase as arousal progresses. To test this hypothesis, we measured SaO2 during the transition from torpor to IBE using pulse oximetry of blood flow through the carotid arteries. Although changes in Hct and/or Hb could also affect O2 delivery during arousal, taking multiple blood samples from arousing animals was not feasible. We did, however, compare these values, as well as some blood ion and glucose concentrations, between 13-lined ground squirrels in deep torpor and in summer.
MATERIALS AND METHODS
13-Lined ground squirrels
All procedures were performed at Western University (London, ON, Canada, 251 m above sea level) following an approved animal use protocol (2020-034), conforming to the Canadian Council on Animal Care guidelines. The 13-lined ground squirrels, Ictidomys tridecemlineatus (Mitchill 1821), were live-trapped in Carman, MB, Canada (49°30′N, 96 98°01′W) or bred in captivity, following established husbandry protocols (Vaughan et al., 2006). Husbandry protocols followed the methodology previously outlined in Duffy and Staples (2022). Summer 13-lined ground squirrels were kept at 22±3°C, with a photoperiod adjusted weekly to match the conditions in Carman. Winter 13-lined ground squirrels were transferred to walk-in environmental chambers where the ambient temperature (Ta) was decreased by 1°C per day until it reached 4±2°C, and the photoperiod was set to 24 h of darkness to replicate fossorial (burrow) conditions.
In hibernating animals, torpor bouts were tracked by monitoring the presence of un-shredded paper towels daily (Hutchinson et al., 2024). All squirrels used in this study were adults that had previously undergone at least one hibernation season. We used pulse oximetry to assay arousing 13-lined ground squirrels (three females and six males) in December 2022 and summer 13-lined ground squirrels (two females and two males) in June 2023. We collected blood samples from 17 torpid squirrels (nine females, eight males) in January 2024 and from a different group of 11 summer squirrels (six females, five males) in June 2024, which were also used for heart mass comparisons and separate studies on mitochondrial metabolism.
Pulse oximetry
During arousal from torpor to IBE and in the stable summer state, we measured fH and SaO2 using a MouseOx® Plus pulse oximeter collar (Starr Life Sciences, Oakmont, PA, USA; Harvard Apparatus Canada, Saint-Laurent, QC, Canada). Pulse oximetry measures the SaO2 of Hb molecules in red blood cells transcutaneously based on the principle that oxygenated and deoxygenated Hb absorb differing amounts of red and near-infrared (IR) light (Chan et al., 2013). The MouseOx® collar used in this study was secured around the ground squirrel's neck, obtaining a strong signal from the carotid arteries, which supply blood to the head. A limitation of this methodology is the reliance on stable fH for accurate measurements. The MouseOx® is optimized to work at fH between 90 and 900 beats min−1, but torpid 13-lined ground squirrels have fH as low as 2–4 beats min−1 (Fig. 2A; MacCannell et al., 2018). As a result, the collar could not identify fH or SaO2 during torpor but began taking measurements when fH reached approximately 100 beats min−1 early in arousal.
Increases in fH and oxygen saturation during arousal. (A,B) Increase in fH (A) and carotid artery haemoglobin (Hb) oxygen saturation (SaO2; B) with increasing Tb. (C) Change in SaO2 with increasing fH during arousal. Symbols represent mean and shading represents s.e.m., n=9 for each 1°C Tb increase. The red (A) and teal (B) lines represent mean summer fH and SaO2, respectively, n=5, with shading representing s.e.m. Solid black lines represent the relationships of fH and SaO2 with Tb, with dashed lines representing the 95% confidence interval, non-linear regression.
Increases in fH and oxygen saturation during arousal. (A,B) Increase in fH (A) and carotid artery haemoglobin (Hb) oxygen saturation (SaO2; B) with increasing Tb. (C) Change in SaO2 with increasing fH during arousal. Symbols represent mean and shading represents s.e.m., n=9 for each 1°C Tb increase. The red (A) and teal (B) lines represent mean summer fH and SaO2, respectively, n=5, with shading representing s.e.m. Solid black lines represent the relationships of fH and SaO2 with Tb, with dashed lines representing the 95% confidence interval, non-linear regression.
We removed torpid 13-lined ground squirrels from a walk-in environmental chamber (5°C) to an adjacent procedure room. Measurements of SaO2 were made at an ambient temperature of 22°C, similar to the housing temperature of summer animals. Hair was removed from the neck using electric clippers and Nair© hair remover for all animals. During arousal, Tb was measured continuously (once per second) throughout the experiment using a rectal probe, with data acquired using Expedata (Sable Systems, NV, USA). This procedure induced arousal in torpid animals. In summer 13-lined ground squirrels, Tb was measured at the beginning and end of the 40 min trials.
Haematological analysis
Torpid and a separate group of anaesthetized (5% isoflurane, 95% O2) summer squirrels were killed with cervical dislocation, and blood was drawn using a cardiac puncture to the right ventricle. We measured Hb, Hct, Na+, K+, Ca2+ and glucose in venous whole blood using a portable blood analyser (i-STAT-1; Abbott Laboratories, Abbott Park, IL, USA) equipped with CG8+ cartridges. Although the i-STAT system can measure blood gases, the very low blood pressure and flow during torpor made it impractical to collect blood in a gas-tight fashion, and the samples visibly changed colour within the syringe during sampling, indicating a change in oxygenation. Therefore, we excluded the blood gas data.
Data extraction and statistical analysis
Tb values were collected every second, and mean values for each 2.5 min interval were calculated. fH and SaO2 were collected every 0.2 s, with mean and s.e.m. values also calculated in 2.5 min intervals. The residuals of these data were normally distributed (Shapiro–Wilks test; Fig. S1A,B). To visualize the relationships among Tb, fH and SaO2, Tb was grouped in 1°C bins and analysed using non-linear regressions (second-order polynomial models) with least square fitting. Linear regression was also used to assess the effect of fH on SaO2.
Haematological data were normally distributed (Shapiro–Wilks test; Fig. S1C–H). Unpaired, two-tailed t-tests compared torpor and summer heart masses and blood metrics (Hct, Hb, sodium, potassium, calcium and glucose). All data analysis and plots were generated using Graph Pad Prism (version 9.5.1).
RESULTS AND DISCUSSION
Arousals involve transient hypoxia–reoxygenation
Beyond the low SaO2 values early in arousal, SaO2 declined further to 34% as Tb warmed to 10°C (Fig. 2B; Fig. S2B). This decline suggested that the early stages of arousal represented a phase where demand for O2, likely due to increased thermogenesis by brown adipose, outpaced O2 delivery. This decline in SaO2 also coincided with a fairly constant fH at the beginning of arousal (Fig. 2A; Fig. S2A). This delay in fH increase likely results from depressed myocardial contraction and relaxation kinetics at low temperatures, even though 13-lined ground squirrel hearts exhibit robust function at low temperatures that render non-hibernator hearts highly arrhythmic (Caprette and Senturia, 1984; Fedorov et al., 2005). Interestingly, the early decline in SaO2 during arousal found here aligns closely with another recent study in 13-lined ground squirrels, which found hyperventilation at the same Tb during arousal, suggesting decreased blood CO2 (Sprenger and Milsom, 2022). Additionally, the low Tb values experienced early during arousal would have challenged O2 offloading at the tissues, amplifying tissue hypoxia.
Throughout 80 min of arousal, SaO2 increased by 260%, which suggested 13-lined ground squirrels experienced hypoxia–reoxygenation. In IBE, when breathing room air, 13-lined ground squirrel SaO2 (87%) was similar to that of arctic ground squirrels (83%; Ma et al., 2005). In the wild, summer 13-lined ground squirrel burrow O2 levels are estimated to be 18.9% (Studier and Procter, 1971), compared with 20.9% in well-mixed air. To our knowledge, O2 levels in winter burrows have not been measured. However, snow cover could inhibit gaseous mixing in winter, so ambient O2 levels may be even lower during the hibernation season, further exacerbating hypoxia challenges. In other hypoxia-tolerant species, such low SaO2 levels have only been reported in animals exposed to severely hypoxic environments. For example, in high-altitude deer mice, ∼65% SaO2 was reached only when breathing 8% O2 (Ivy and Scott, 2021), and in naked mole rats, ∼40% SaO2 was reached only when breathing 3% O2 (Pamenter et al., 2019).
Haematological values differ slightly between torpor and summer
Heart mass was similar between torpor and summer 13-lined ground squirrels (T15=0.09, P=0.93; Fig. 3A). Body mass-corrected heart mass was 29.8% greater in torpor than in summer (T15=5.70, P<0.0001; Fig. 3B) due to the corresponding 29.8% lower body mass in torpor than in summer (T15=7.72, P<0.0001; Fig. 3C). Whole-blood Hct in torpor (49.76±1.01%) was 8.8% higher (T26=2.60, P=0.015; Fig. 3D) than summer values (45.82±1.07%), and Hb concentration showed similar trends (Fig. 3E).
Comparisons between torpid and summer haematological values. (A) Heart mass, (B) heart mass relative to body mass, (C) body mass, (D) haematocrit (Hct, as percentage packed cell volume, PCV) and concentrations of (E) haemoglobin (Hb), (F) sodium, (G) potassium, (H) calcium and (I) glucose in venous whole blood. Means±s.e.m. Asterisks indicate a significant difference, *P<0.05, ****P<0.0001, two-tailed t-tests.
Comparisons between torpid and summer haematological values. (A) Heart mass, (B) heart mass relative to body mass, (C) body mass, (D) haematocrit (Hct, as percentage packed cell volume, PCV) and concentrations of (E) haemoglobin (Hb), (F) sodium, (G) potassium, (H) calcium and (I) glucose in venous whole blood. Means±s.e.m. Asterisks indicate a significant difference, *P<0.05, ****P<0.0001, two-tailed t-tests.
Many experiments have previously characterized Hb concentration in hibernators with mixed results (reviewed in Milsom and Jackson, 2011). Our Hct results align closely with a recent study in 13-lined ground squirrels (Cooper et al., 2016a). The modest increase in torpor compared with summer is unlikely to affect blood flow significantly on its own, but Spurrier and Dawe (1973) noted that Hct tended to rise during arousal. This Hct increase, accompanied by low blood temperature early during arousal, could increase blood viscosity, further slowing rates of O2 delivery to tissues.
Whole blood collected during torpor had 6.7% lower Na+ concentrations than in summer (T27=5.94, P<0.0001; Fig. 3F). K+ concentration did not differ between groups (T24=0.39, P=0.27; Fig. 3G), but Ca2+ was 17.8% higher in torpor than in summer (T27=2.45, P=0.021; Fig. 3H). Finally, glucose concentrations from torpid blood were 54.3% lower than in summer blood (T27=11.26, P<0.0001; Fig. 3I).
It seems that the modest increase in Hct and Hb during torpor cannot be attributed to dehydration, as plasma Na+ levels were actually lower in this state, consistent with recent reports which indicate that 13-lined ground squirrel blood osmolarity declines during torpor despite the absence of water intake (Feng et al., 2019). We found that Ca2+ levels were higher in torpor than in summer, even without a dietary source. This finding supports the hypothesis that hibernators have developed mechanisms to maintain calcium homeostasis (Arfat et al., 2020). Blood glucose levels were significantly lower during torpor compared with those in summer (Fig. 3I), and these values align closely with previous measurements in 13-lined ground squirrel blood (Burlington and Klain, 1967; Nizielski et al., 1986). Low glucose levels may suggest a reliance on anaerobic metabolism. However, the evidence of lactate accumulation during torpor varies by species and blood sample type; for example, lactate accumulates in the red blood cells of arctic ground squirrels during torpor (Gehrke et al., 2019), while concentrations remained low in whole blood of golden-mantled ground squirrels (Callospermophilus lateralis; Maginniss and Milsom, 1994) and in serum of 13-lined ground squirrels (Feng et al., 2019). Instead, the blood glucose levels likely reflect that, during hibernation, 13-lined ground squirrels do not typically feed; the concentrations we report for torpid 13-lined ground squirrels resemble those found in fasted rats (Arnall et al., 1988). However, the values we report for summer 13-lined ground squirrels are similar to those of diabetic rats (Yang et al., 2020), which is expected because of the high body fat content in summer 13-lined ground squirrels (MacCannell et al., 2017).
Possible mechanisms of I–R tolerance in hibernation
The resistance of hibernators to I–R has been attributed to many physiological adaptations that likely evolved as protection from O2 fluctuations during their torpor–IBE cycles, illustrated in Fig. 2B. Ground squirrel cardiac I–R tolerance is well defined in the literature (Bonis et al., 2019; Han et al., 2022; Xie et al., 2021; Salzman et al., 2017; reviewed in Drew et al., 2013).
Low glucose levels during torpor may indicate glycogenesis to supply energy during early arousal. One study found that by the third day of torpor, glycogen levels in 13-lined ground squirrel hearts were significantly higher than in summer, correlating with improved maintenance of cardiac function following ex vivo I–R in torpor (Heinis et al., 2015). Higher cardiac glycogen may help heart function during the early, hypoxic stages of arousal. Cardiac mitochondrial respiration is suppressed by 30% during torpor (Brown and Staples, 2014), which may help to conserve O2. ROS production during O2 fluctuations is also minimized in hibernator heart mitochondria by increased uncoupling proteins (Ballinger et al., 2017) to dissipate proton motive force and avoid reoxygenation-associated oxidative damage. There is also a hypothesis that repeated arousals themselves resemble ischaemic preconditioning, a condition in which previous bouts of tissue hypoxia prime tissues to resist later I–R insult (reviewed in Bhowmick and Drew, 2019). It is possible that repeated arousal throughout the hibernation season mimics preconditioning and mitigates oxidative stress by upregulating heat shock proteins and the HIF-1α pathway (reviewed in Jankovic et al., 2024).
Acknowledgements
The authors thank Dr Graham Scott for sharing his MouseOx® equipment and software. We also thank Sharla Thompson, Lauren Rego and Colleen Barghout for their help with animal care, and Marina Zhang for her help with haematological processing. Thanks to two anonymous referees for their constructive comments that helped to improve the manuscript.
Footnotes
Author contributions
Conceptualization: B.M.D.; Data curation: B.M.D.; Formal analysis: B.M.D.; Funding acquisition: J.F.S.; Investigation: C.M.I., B.M.D.; Methodology: C.M.I.; Supervision: J.F.S., C.M.I.; Writing – original draft: B.M.D.; Writing – review & editing: J.F.S., C.M.I.
Funding
This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant held by J.F.S. (RGPIN-2020-06421). Open Access funding provided by Western University. Deposited in PMC for immediate release.
Data availability
All relevant data and resources can be found within the article and its supplementary information.
References
Competing interests
The authors declare no competing or financial interests.