Elsevier

Journal of Thermal Biology

Volume 84, August 2019, Pages 228-235
Journal of Thermal Biology

Evaporative cooling and vasodilation mediate thermoregulation in naked mole-rats during normoxia but not hypoxia

https://doi.org/10.1016/j.jtherbio.2019.07.011Get rights and content

Highlights

  • Naked mole-rats reduce body temperature and metabolic rate in hypoxia but the underlying mechanism(s) are unknown.

  • We hypothesized that heat shunting and/or vasodilation would augment heat loss through evaporative cooling in hypoxia.

  • Body temperature and metabolic rate changes in hypoxia were unaffected by a vasoconstrictive agent or high humidity.

  • We conclude that evaporative cooling and vasodilation do not contribute to thermoregulatory changes during hypoxia.

Abstract

Naked mole-rats are among the most hypoxia-tolerant mammals but have a poor thermoregulatory capacity due to their lack of insulating fur and fat, and small body size. In acute hypoxia, naked mole-rat body temperature (Tb) decreases to ambient temperature (Ta) but the mechanisms that underlie this thermoregulatory response are unknown. We hypothesized 1) that naked mole-rat blood vessels vasodilate during hypoxia to shunt heat toward the body surface and/or 2) that they augment heat loss through evaporative cooling. Using open-flow respirometry (indirect calorimetry) we explored metabolic and thermoregulatory strategies of naked mole-rats exposed to hypoxia (7% O2 for 1 h) at two relative humidities (RH; 50 or 100% water saturation), and in two Ta's (25 and 30 °C), alone, and following treatment with the vasoconstrictor angiotensin II (ANGII). We found that Tb and metabolic rate decreased in hypoxia across all treatment groups but that neither RH nor ANGII effected either variable in hypoxia. Conversely, both Tb and metabolic rate were reduced in 100% RH or by ANGII treatment in normoxia at 25 °C, and therefore the absolute change in both variables with the onset of hypoxia was reduced when vasodilation or evaporative cooling were prevented. We conclude that naked mole-rats employ evaporative cooling and vasodilation to thermoregulate in normoxia and in 25 °C but that neither mechanism is involved in thermoregulatory changes during acute hypoxia. These findings suggest that NMRs may employ passive strategies such as reducing thermogenesis to reduce Tb in hypoxia, which would support metabolic rate suppression.

Introduction

Animals that inhabit hypoxic environments have evolved complicated suites of physiological adaptations that enable them to thrive in such low oxygen niches (Bickler and Buck, 2007; Buck and Pamenter, 2018; Dzal et al., 2015; Hochachka et al., 1996). The key to tolerating prolonged hypoxia is to match metabolic demand to reduced energy supply (i.e., reduced oxygen supply; Buck and Pamenter, 2006), and hypoxia-tolerant animals typically exhibit robust decreases in metabolic rate when oxygen supplies are limited (Dzal et al., 2015; Guppy and Withers, 1999; Hochachka, 1986). Conversely, hypoxia-intolerant animals are generally unable to sufficiently reduce their metabolic rate during hypoxia to accommodate reduced oxygen supply.

Thermoregulation is an energetically-expensive process, particularly in small mammals, and many species employ thermoregulatory strategies to reduce body temperature (Tb) and facilitate reduced metabolic demand in acute and prolonged hypoxia. These thermoregulatory strategies can be roughly divided into three categories: 1) behavioural (e.g., reductions in huddling behaviour, seeking cooler environments, passive heat loss through heat transfer via direct skin contact with moist soil, etc. (Okrouhlik et al., 2015)), 2) circulatory (e.g., vasodilation or the evolution of morphological features within the circulatory system that facilitate heat loss, such as arteriovenous anastomoses that provide increased blood flow to the skin), and 3) decreasing thermogenesis (e.g., turning off non-shivering and shivering thermogenesis, downregulating mitochondrial function) (Bicego et al., 2007; Ramirez et al., 2007; Staples, 2016; Steiner and Branco, 2002). In addition, many animals employ radiative heat loss and/or evaporative cooling through the evaporation of water molecules from the skin or surface membranes (e.g., sweating, panting) to prevent overheating. Similar processes may also facilitate decreases in Tb in hypoxia. For example, some reptiles spread urine on their skin to facilitate rapid heat loss in hypoxia (Tattersall and Gerlach, 2005). It is important to note that, although the cessation of active thermogenesis is the only process of the three categories described above that would confer direct energy savings, reductions of Tb through behavioural or circulatory means would nonetheless confer significant energy savings by systemically reducing the rate of cellular, molecular, and enzymatic activities through temperature-coefficient (Q10) related energy savings.

Naked mole-rats (Heterocephalus glaber) are among the most hypoxia-tolerant mammals identified and tolerate minutes of complete anoxia, hours at 3% O2, and days to weeks at 8% O2 (Chung et al., 2016; Pamenter et al., 2015, 2018; Park et al., 2017). The rate of oxygen consumption (V˙O2; an indirect measure of metabolic rate) of adult naked mole-rats decreases by up to 85% in severe hypoxia (3% O2). Metabolism decreases by ~70% in 7% O2, which is the level of hypoxia employed in the present study (Pamenter et al., 2015, 2018, 2019). Although this degree of V˙O2 suppression is not remarkable among hypoxia-tolerant species (Guppy and Withers, 1999), it is important to note that other mammals that are capable of similar or more extreme metabolic rate suppression in severe hypoxia typically enter into a coma- or torpor-like state until oxygen levels are restored (Guppy and Withers, 1999; Hayden and Lindberg, 1970). Conversely, naked mole-rats remain awake and active in hypoxia, albeit to a reduced degree (Houlahan et al., 2018; Ilacqua et al., 2017; Kirby et al., 2018). Therefore, understanding physiological mechanisms that support reduced metabolic demand during hypoxic periods, despite the avoidance of torpor, is of interest in elucidating the underlying adaptations that support hypoxia-tolerance in this remarkable species.

Naked mole-rats are poor thermoregulators due to their lack of insulating fur and fat (Daly and Buffenstein, 1998), and their small body size (Sumbera, 2019). In normoxia, and as a result of this poor ability to retain heat, naked mole-rats exhibit a mesothermic thermoregulatory phenotype in isolation such that at temperatures well below their thermoneutral zone, they are unable to effectively maintain thermal homeostasis. However, their metabolic rate increases substantially in the cold, which indicates that they do attempt to thermoregulate, even at substantial metabolic cost (Kirby et al., 2018; Mcnab, 1966; Withers and Jarvis, 1980). Naked mole-rats are able to ameliorate this cost to some degree in normoxia by moving to warmer environments (Kirby et al., 2018), by huddling to help conserve heat in their crowded natural burrow systems (Yahav and Buffenstein, 1991), or if they are provided with insulation (Withers and Jarvis, 1980). Both huddling and the provision of insulation decrease the amount of surface area exposed per animal and lower individual metabolic demand (V˙O2) (Withers and Jarvis, 1980; Yahav and Buffenstein, 1991). These observations suggest that other physiological adaptations that reduce heat loss may also confer metabolic savings in hypoxia.

Recently, we have begun to explore thermoregulatory responses to acute hypoxia in naked mole-rats. During hypoxia, naked mole-rat Tb decreases to near ambient temperatures (Ta) (Ilacqua et al., 2017; Kirby et al., 2018; Pamenter et al., 2019), suggesting the realization of thermoregulatory-related energy savings. Our investigations to date have focused upon behavioural strategies and we have found that naked mole-rats do not employ behavioural thermoregulation per se. Specifically, naked mole-rats decrease overall behavioural activity in hypoxia but when given the option of choosing between different environmental temperatures when oxygen is limited, they prefer warm temperatures and avoid colder environments (Ilacqua et al., 2017; Kirby et al., 2018). Similarly, naked mole-rat huddling behaviour is unchanged in acute hypoxia (Houlahan et al., 2018). Taken together, these data suggest that naked mole-rats do not employ anapyrexic strategies in response to low environmental oxygen.

In the present study we sought to examine the second category of potential thermoregulatory responses in hypoxia (i.e., circulatory strategies). Specifically, we comprehensively evaluated potential roles for peripheral vasodilation and evaporative cooling in thermoregulatory and metabolic responses to acute hypoxia. Arterioles and venules are connected via arteriovenous anastomoses in naked mole-rat dorsal skin, and thus capillary networks are brought close to the surface of the skin and are believed to mediate cooling of the blood in normoxia (Daly and Buffenstein, 1998). When the skin is instead chilled, the capillaries constrict, reducing the flow of blood to the surface of the skin and thereby conserving heat. These observations suggest that naked mole-rats could shunt blood to their skin while in hypoxia to dump heat and facilitate whole body metabolic cooling and thus reduce metabolic demand through the Arrhennius effect (Schulte, 2015). Conversely, naked mole-rats lack subcutaneous sweat glands and are therefore unable to utilize the common evaporative cooling strategy of sweating (Daly and Buffenstein, 1998). However, naked mole-rats may utilize moisture found in their environment or even bodily fluids to disperse heat through evaporative means in hypoxia (Tattersall and Gerlach, 2005).

We hypothesized that naked mole-rats utilize circulatory strategies to rapidly decrease Tb in acute hypoxia and predicted that abrogation of these abilities by injection of a vasoconstrictor (angiotensin II, ANGII) and/or exposure to an H2O-saturated environment (100% relative humidity, RH), respectively, would impair their ability to reduce Tb, and in turn metabolic rate, in acute hypoxia. To test our hypothesis, we exposed naked mole-rats to 1 h of hypoxia (7% O2) at two ambient temperatures (Ta's; 25 and 30 °C), in either 50 or 100% RH, and also following injection of ANGII, and measured metabolic rate (O2 consumption and CO2 production, V˙O2 and V˙CO2, respectively) and Tb.

Animals. Naked mole-rats were group-housed in interconnected multi-cage systems at 30°C and 21% O2 in 50% humidity with a 12L:12D light cycle. Animals were fed fresh tubers, vegetables, fruit, and Pronutro cereal supplement ad libitum. Animals were not fasted prior to experimental trials. All experimental procedures were approved by the University of Ottawa Animal Care Committee in accordance with the Animals for Research Act and by the Canadian Council on Animal Care. All experiments were performed during daylight working hours in the middle of the animals’ 12L:12D light cycle. Naked mole-rats that are housed within colony systems do not exhibit circadian rhythmicity of general locomotor activity (Riccio and Goldman, 2000b), and exhibit inconsistent rhythmicity of Tb and metabolic rate (Riccio and Goldman, 2000a); however, significant changes in these latter parameters were only reported in animals during the nocturnal phase of their circadian cycle with no significant changes observed during the daylight period of this cycle. Therefore, since we only ran experimental trials during the daylight period, we do not expect our results to be influenced by circadian rhythms. We examined physiological responses to environmental hypoxia in non-breeding naked mole-rats that were 1–2 years old. Non-breeding (subordinate) naked mole-rats do not undergo sexual development or express sexual hormones and thus we did not take sex into consideration when evaluating our results (Holmes et al., 2009).

Experimental Design. Seventy (70) male and female subordinate adult naked mole-rats weighing 47.2 ± 6.9 g (mean ± s.d.) were divided into the following 9 experimental groups: (i) 30°C + 100% RH (n = 8), (ii) 30°C + 100% RH + ANGII (n = 8), (iii) 30°C + 0% RH (n = 8), (iv) 30°C + 0% RH + sham injection (n = 6), (v) 30°C + 0% RH + ANGII (n = 8), (vi) 25°C + 0% RH (n = 8), (vii) 25°C + 0% RH + ANGII (n = 8), (vii) 25°C + 100% RH (n = 8), and (ix) 25°C + 100% RH + ANGII (n = 8). For sham injection and ANGII treatment groups, animals received one intraperitoneal injection of either saline or ANGII (25  μg ml−1, total volume ~ 250  μL; Sigma Aldrich, USA). Intraperitoneal delivery of ANGII has been shown to increase vasomotor sympathetic drive for at least 2 h post-injection in other rodents (Zubcevic et al., 2017). Injections did not appear to impact the animals negatively in that they remained alert and active following injection and did not exhibit any signs of pain or discomfort.

At the start of the experiment (and following injection if appropriate), naked mole-rats were placed into a 500 ml cylindrical experimental chamber. All animals urinated and defecated shortly after being placed into the experimental chamber and the addition of moisture from this waste, combined with the low flow rate of gas through the chamber (see below), increased the RH from 0% (incurrent gas) to  ~ 50% (actual excurrent gas). Therefore, we considered our 0% RH data as being 50% saturated for the purpose of our data presentation and discussion (Fig. 1). Baseline recordings were obtained for 1 h in normoxia and then the incurrent gas composition was switched to 7% O2 for 1 h followed by 1 h in normoxia (recovery). Following experimentation, animals were returned to their colonies. Experiments were conducted in environmental rooms held at 25 or 30 °C and animals were acclimated for 2–3 h  at the appropriate temperature prior to commencing experimentation. These temperatures were selected since an Ta of 30 °C is the housing temperature of our colonies, and is near the thermoneutral zone of naked mole-rats (which spans from ~30.5 to 34 °C (Yahav and Buffenstein, 1991)); the 25 °C temperature was selected to increase the thermal scope within which the animals were able to respond through thermoregulatory adaptations to hypoxia. Naked mole-rats have a higher metabolic rate in colder temperatures relative to near their thermoneutral zone (Ilacqua et al., 2017; Kirby et al., 2018; Mcnab, 1966; Withers and Jarvis, 1980), and thus repeating our experiments in this temperature magnified the impact of our treatments on metabolic rate and Tb, and therefore our ability to detect any physiological changes in this condition.

Flow-through respirometry. The animal chamber was sealed and constantly ventilated with gas mixtures set to the desired fractional gas composition by calibrated rotameters (Praxair, Mississauga, ON, CA). The advantage of this open-flow system is that it prevents the depletion of O2 and accumulation of metabolic CO2 by flushing the animal chamber with fresh gas, and it allows for continuous and simultaneous monitoring of metabolic and ventilatory variables. Inflowing gas was provided at a flow rate of 85  ml min−1, as assessed by a calibrated mass flow meter (Q-G266 Flow Monitor, Qubit Systems). The gas flowing into the chamber first passed through either a bubbler or a drying column containing Drierite desiccant to achieve the conditions of ~100% or 0% RH, respectively. The bubbler or drying column was joined to the experimental chamber via the outflow tube. After passing through the chamber, the outflowing gas traveled to the inflow tube of a relative humidity sensor (RH-200 RH/Dewpoint Meter, Sable Systems Int., Las Vegas, NV, USA) and then through a drying column before entering a series of gas analyzers. The gas first passed through the flow analyzer, followed by the O2 analyzer (Q-S102, Qubit Systems) and finally, the CO2 analyzer (Q-S153, Qubit Systems). Gas analyzers were calibrated prior to each trial with 20.95% O2, 1.5% CO2, balanced with N2, and with 100% N2 gas mixes. The V˙O2 and V˙CO2 were calculated using equations 11.7 and 11.8 from (Lighton, 2008), and accounting for time lag of gas flow between the O2 and CO2 sensors. All metabolic variables are reported at standard temperature, pressure, dry (STPD).

Body temperature. Body temperature was measured using a handheld radio frequency identification (RFID) reader that scanned individual naked mole-rats instrumented with subcutaneous RFID microchips (Destron Fearing, Dallas, TX). The first measurement was taken immediately after placing the animal into the chamber and then subsequent measurements were taken every 10  min, as described previously (Ilacqua et al., 2017; Kirby et al., 2018). Measurements were taken when the body region containing the RFID microchip was not in contact with the chamber surface to avoid biased readings. The accuracy of these microchips for measuring Tb was confirmed in a separate set of experiments in which we took core Tb measurements using a thermocouple (Thermalert Model TH-8 temperature monitor, Physitemp, Clifton, NJ, USA) from animals held at 30 °C (n = 7). Temperatures measured by RFID vs. rectal probe were not significantly different (Tb(microchip)  = 3 2.34 °C, Tb(rectal)  =  32.42 °C).

Data collection and statistical analysis. Ambient temperature and incurrent and excurrent O2 and CO2 concentrations were recorded and analysed using Loggerpro software (Vernier, USA). We determined average Ta, Tb, V˙O2, V˙CO2, and RH values for the last 10–15  min of each O2 exposure (21% and 7% O2). Inflowing gas concentrations were measured before and after each O2 exposure. Gas flow was measured continuously throughout all experiments. Statistical analysis was performed to determine the effects of O2 level, Ta, RH, and ANGII injection. Statistical significance was determined using a two-way (treatment and O2 level) repeated measures analysis of variance (RM ANOVA) to analyze the final 10 min of each experimental stage (normoxia and hypoxia) (see statistical table in supplementary materials section). For comparisons between the magnitude of change between normoxia and hypoxia at Ta's of 25 and 30 °C, significance was evaluated using an ordinary one-way ANOVA. Dunnett's multiple comparison test was performed within groups while Tukey's multiple comparison test was performed between groups. All physiological and behavioural variables met the assumptions of normality, homogeneity of variances, linearity, and independence, and residuals from the statistical models were confirmed for normality. All results are presented as mean ± s.d., with statistical significance set as a < 0.05.

Section snippets

Results

Body temperature and metabolic rate are significantly reduced in acute hypoxia near the thermoneutral zone. Body temperature, V˙O2, and V˙CO2 were first measured at 30 °C, which is the temperature at which our animal colonies are housed, to assess the effects of acute hypoxia on thermoregulation and metabolic rate near the naked mole-rat thermoneutral zone. Changes in Tb, recorded every 10  min throughout the experimental period are presented in Fig. 2A (n = 6 for 50% RH + saline (sham

Discussion

Naked mole-rats exhibit a poor thermoregulatory capacity due to their lack of insulating fur and fat (Daly and Buffenstein, 1998), and due to their small body mass (Sumbera, 2019). We hypothesized that this functional deficit would in fact be beneficial in hypoxia and that evaporative cooling and vasodilation would facilitate heat loss and support metabolic rate suppression. We manipulated RH, Ta, and vascular tone to explore the roles of evaporative cooling and vasodilation on thermoregulatory

Funding

This work was supported by NSERC Discovery grants to MEP and GJT and a Canada Research Chair awarded to MEP.

Conflicts of interest

We have no competing interests.

Author contributions

MP and GT conceived of and designed the study. AV and AZ performed the physiology experiments. AV and MP analysed the data. MP conducted statistical analysis and MP, GT and AV wrote the manuscript. MP, GT, AV, and AZ edited the manuscript, gave final approval of the published version and agree to be accountable for all content therein. AK trained AV in the indirect calorimetry technique and provided logistical support to AV but did not make a direct contribution to this study.

Acknowledgements

We would like to thank the uOttawa animal care and veterinary services team for their assistance in animal handling and husbandry.

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