How murine models of human disease and immunity are influenced by housing temperature and mild thermal stress

ABSTRACT

At the direction of The Guide and Use of Laboratory Animals, rodents in laboratory facilities are housed at ambient temperatures between 20°C and 26°C, which fall below their thermoneutral zone (TNZ). TNZ is identified as a range of ambient temperatures that allow an organism to regulate body temperature without employing additional thermoregulatory processes (e.g. metabolic heat production driven by norepinephrine), thus leading to mild, chronic cold stress. For mice, this chronic cold stress leads to increased serum levels of the catecholamine norepinephrine, which has direct effects on various immune cells and several aspects of immunity and inflammation. Here, we review several studies that have revealed that ambient temperature significantly impacts outcomes in various murine models of human diseases, particularly those in which the immune system plays a major role in its pathogenesis. The impact of ambient temperature on experimental outcomes raises questions regarding the clinical relevance of some murine models of human disease, since studies examining rodents housed within thermoneutral ambient temperatures revealed that rodent disease pathology more closely resembled that of humans. Unlike laboratory rodents, humans can modify their surroundings accordingly – by adjusting their clothing, the thermostat, or their physical activity – to live within the appropriate TNZ, offering a possible explanation for why many studies using murine models of human disease conducted at thermoneutrality better represent patient outcomes. Thus, it is strongly recommended that ambient housing temperature in such studies be consistently and accurately reported and recognized as an important experimental variable.

Introduction

The Guide for the Care and Use of Laboratory Animals, produced by the National Research Council, sets guidelines for the ethical and humane housing of laboratory rodents [1]. These guidelines are adopted internationally and therefore standardize nearly every aspect of animal housing in research facilities, including the temperature at which they are housed. The Guide describes proper housing temperatures for research animals to be “necessary for animal well-being,” and identifies the thermoneutral zone (TNZ) – the range of ambient temperatures that do not require metabolic heat production or evaporative processes to regulate body temperature [2,3] – for mice under laboratory conditions as between 26 and 34°C [1]. However, it is recommended in The Guide that mice instead be housed at vivarium temperatures below their TNZ “to avoid heat stress,” and that mice should be provided additional resources, such as nesting material, to prevent cold stress. As such, the standard temperature (ST) at which research mice are housed is between 20 and 26°C [1]. Numerous studies have revealed that housing at ST subjects laboratory mice to a mild, but chronic cold stress and suggest that this stress may worsen when additional stressors, such as tumor implantation, are added, thereby significantly impacting outcomes in murine models of human disease [4–6].

While The Guide does acknowledge that the standard housing temperature for mice is lower than their preferred thermoneutral temperature and recommends that mice be provided with nest-building materials, other studies have demonstrated that nesting materials alone may not prevent the loss of heat [1,7]. As a result, mice housed at ST continuously rely on heat generated by a process known as non-shivering thermogenesis, elicited by the release of norepinephrine (NE) by the sympathetic nervous system in brown adipose tissue (BAT) [6,8]. However, NE release is not confined to BAT, and systemic release of NE has the potential to alter numerous cell types through activation of ubiquitously expressed β-adrenergic receptors, and therefore can alter many normal physiological processes. Unlike laboratory mice, humans can modify their environment to operate at their appropriate thermoneutral zone [9]. So, what does this mean for murine disease models and the translatability of pre-clinical findings obtained from mice that are housed at a single, subthermoneutral temperature? In this review, we highlight several studies which indicate that there are significant differences in the severity and phenotype of the pathology being modeled due to ambient temperature. Based on this continuously growing body of work, it is clear that, while no one housing temperature is consistently optimal for mice, the use of a single housing temperature is a major variable that can affect outcome and data interpretation from murine experiments that are conducted to model human disease.

Chronic stress

Sympathetic nervous system physiology

Stress is defined as an event or stimulus that disrupts an organism’s homeostasis, or as the response to an unexpected or unpleasant event [10]. Acute stress is stress that lasts for minutes or hours, while chronic stress lasts for several hours per day, for weeks or longer [11]. Though the source of the stress may vary – psychological, physical, social, or homeostatic – the subsequent physiological response is the same: increased release of the catecholamines epinephrine and NE by the sympathetic nervous system (SNS) [10]. In response to stress, the adrenal medulla releases these catecholamines into the bloodstream, perpetuating the “fight or flight” response [12]. Stress also stimulates the release of NE from sympathetic nerve endings that directly innervate various tissues throughout the body, including both primary (bone marrow and thymus) and secondary immune organs (lymph nodes and spleen) [12–14]. Within innervated tissues, NE binds to alpha- or beta-adrenergic receptors (α- or β-ARs) present on most cell types, including immune cells. Interestingly, β₂-ARs have been established as the primary adrenergic receptor subtype expressed on immune cells and are therefore identified as responsible for mediating the effects of NE on the cellular immune response [15–17]. Activation of β-AR signaling in immune cells initiates a signaling cascade that modulates transcription factor activity and gene expression, in turn affecting major cellular processes including cytokine production, proliferation, differentiation, effector function, and survival, and thus, the SNS plays an important role in modulating the immune response [13,18].

Impact of chronic stress on the immune system and inflammation

While acute stress has been shown to elicit an immune response that is protective against pathogens or wounding [19], chronic stress is often associated with chronic low-grade inflammation, leading to exacerbated symptoms of autoimmune disorders [11,20]. Conversely, chronic activation of the SNS as a result of chronic stress has also been shown to dampen the effects of cytotoxic immune cells responsible for pathogen or tumor elimination and control, while simultaneously enhancing the function of immunosuppressive cells such as tumor associated macrophages or T regulatory cells [11,17,21]. In line with this, many murine models of human disease with a known immune component, including cardiovascular disease, type II diabetes, Parkinson’s disease, metabolic disorders, viral infection, and cancer, have revealed that chronic stress influences the susceptibility to disease onset and/or progression [21–24]. Likewise, chronic stress in humans has long been associated with worse disease outcome. Individuals who report generally high levels of stress or who experienced stressful life events are more likely to experience a heart attack, heart disease, allergic inflammatory responses, develop type II diabetes, or report overall poorer health [25,26]. Those who regularly experience stressful situations have also been reported to be at increased risk for various diseases or a dampened immune response. For example, air traffic controllers at large airports are more likely to develop peptic ulcers, while caregivers of Alzheimer’s patients or medical students who reported high levels of stress had poorer immune responses to vaccinations of influenza or hepatitis B, respectively [27]. Interestingly, individuals who exhibit increased resilience – the ability to overcome stress and recover from stressful events – may even heal and recover faster from diseases such as colon cancer and have better outcomes in cardiovascular disease [28].

Standard housing temperature

Murine thermoneutrality and adaptive thermogenesis

As endotherms, both mice and humans utilize heat produced by metabolic activity to maintain a stable internal body temperature when external temperatures fall below the TNZ. The TNZ of an organism is defined by the ambient temperature at which basal, or resting, metabolic rate is sufficient for maintaining core temperature [29]. This temperature range, referred to hereafter as thermoneutral temperature (TT), varies between organisms and with environmental conditions. For example, TT for clothed humans generally falls between 20°C and 22°C, while TT for mice under standard laboratory conditions is generally accepted to be between 29°C and 31°C [6]. However, the reported TT for laboratory mice can vary as greatly as 26–38°C depending on many internal and external conditions such as nesting material, number of mice per cage, strain, time of day, or even disease burden, as all of these environmental and biological factors influence thermal physiology [3,4,7,9,30–33]. Similar to mice, rat TT is most commonly accepted as 29–30°C, although many studies define TT from 26°C to 34°C depending on strain and environmental influences [34,35]. Thus, at standard housing temperatures (ST) of 20–22°C put forth by The Guide, laboratory rodents live their entire lives at subthermoneutral temperatures, likely to ensure comfort for researchers and veterinarian staff working in animal facilities [1,3]. When briefly exposed to ambient temperatures below TT, mice and other endotherms employ various mechanisms such as vasoconstriction, posture changes, and eventually, shivering thermogenesis to increase their core temperature [36]. However, mice are housed in subthermoneutral environments for their entire lives, and as a result must rely on an alternative mechanism of heat generation known as adaptive, or non-shivering thermogenesis (NST) – an energetically costly process, mediated by β-AR signaling, that generates heat from BAT [6,36]. Similar to chronic psychological stress, chronic cold stress induced by the subthermoneutral temperatures at ST activate the SNS, stimulating the release of NE from sympathetic nerves that innervate BAT [4,36,37]. Binding of NE to β-ARs on BAT cells results in the increased expression of uncoupling protein 1 (UCP-1), which in turn increases the production of heat, rather than ATP, from mitochondrial respiration, allowing mice to effectively maintain thermal homeostasis [4,36,38]. Not only does ST significantly increase NE production and β-AR signaling but it also requires that mice maintain energy expenditure that is approximated to be three times the basal metabolic rate. In humans, this energetic demand would equate to living nude at 5°C (41°F) or walking ~100 km (62 mi) every day [39]. Chronic elevation of β-AR signaling and energy expenditure, as a result of NST, effectively impairs immune cell metabolism, and reduces available energy for overall immune function (Figure 1) [4,39]. As such, the effects of prolonged activation of SNS in response to chronic ST-induced cold stress are not limited to NST in BAT [3–5,8,9,13,36,40,41]. Below, we discuss the findings of various studies that provide evidence that standard housing temperatures, as well as acute exposure to mild cold stress, significantly influence the outcomes, and likely the translatability, of several models of human disease.

Figure 1.

Mice housed at standard temperature utilize adaptive thermogenesis to increase heat production. Standard laboratory housing temperature requires mice to utilize adaptive thermogenesis to generate sufficient heat to maintain their core temperature. Norepinephrine (NE), produced by sympathetic nerves that innervate brown adipose tissue (BAT), binds to β-adrenergic receptors (β-ARs) on BAT cells. Signaling carried out by β-ARs results in increased UCP-1 protein expression, decreased ATP production, and increased mitochondrial heat production. Created with bioRender.

Murine models of human disease affected by cold stress

Fever, infection, & vaccine efficacy

Romanovsky et al. revealed that the ambient temperature at which lipopolysaccharide (LPS) injections, given to mimic systemic bacterial infection, are administered in rats will determine whether a bi- or triphasic febrile response can be detected. At a room temperature of 22°C, LPS injection resulted in an overall initial drop in core temperature, making the first febrile phase undetectable. At 30°C, LPS injection resulted in an increase in temperature during phase one, allowing for clear distinction between all three phases [42]. The same group also showed that temperature affects the phasic febrile response of male C57BL/6 mice, showing that mice injected with LPS at TT (31°C) exhibited a dose-dependent febrile response, which was lost at ST (26°C). Additionally, at ST, injection of higher doses of LPS was followed by late febrile phase hypothermia, suggesting that subthermoneutral conditions prevent the LPS-induced third febrile phase that would otherwise be seen at thermoneutral temperatures [43]. Another study revealed similar results, in which aged rats housed at 20°C and injected with a low dose of LPS mounted a significantly lower fever compared to rats housed at 30°C [44]. Therefore, studies examining the effects of bacterial infections on the febrile phase would benefit from considering housing temperature as a potential variable.

Others have demonstrated that TT can have either protective or harmful impacts on the ability of rodents to fight infection, depending on the model and type of infection [4,6]. In two different models of sepsis, thermoneutrality appears to have opposing effects on mouse survival. TT housing (30°C) significantly improved survival of mice subjected to cecal ligation and puncture compared to mice housed at ST (22°C). TT also resulted in lower bacterial load and plasma levels of the inflammatory cytokine, IL-6 [45]. Conversely, exposure to TT failed to protect rats from shock-inducing doses of LPS or Escherichia coli, as indicated by significantly worsened abdominal organ dysfunction and decreased survival rates, compared to rats exposed to ST [46]. Important to consider is that these outcomes may be influenced by the mode of the infection, since cecal ligation and puncture mounts a less severe form of sepsis compared to systemic administration of shock-inducing doses of LPS or E. coli. Additional studies have revealed that increased ambient temperature enhances survival in other models of viral and bacterial infection. The mortality rate of mice infected with rabies was significantly reduced the longer they were housed in hyperthermic (35°C) conditions post-inoculation with the virus, compared to mice housed at ST (20°C) [47]. Another study showed that mice housed at TT (28°C) were able to mount a significantly stronger antigen-specific memory T cell response to Francisella tularensis following vaccination, leading to significantly longer survival of infected mice [48]. Together, these studies demonstrate both the importance of considering the impact of housing temperature on outcomes of mouse models of infection and reporting housing temperature in such studies.

Cancer

Recent studies indicate that tumors can attract nerves, which is essential for the growth and progression of these tumors, thus providing a direct conduit by which stress can influence the tumor microenvironment [49]. Our group has shown that housing mice at TT effects the growth of several different tumor models, both with and without the addition of therapeutic intervention. Specifically, housing mice at TT (30°C) reduced the growth rate of xenograft melanoma, breast, colon, and pancreatic tumors, as well as 3-methylcholanthrene-induced fibrosarcomas [31,37]. In mice housed at TT, CT26 colon adenocarcinoma tumors responded better to radiation therapy, growing significantly slower than in mice at ST (22°C) [50]. Thermoneutral housing also improved tumor response to multiple chemotherapies in mice innoculated with human or mouse pancreatic tumor cells, as well as patient lung tumor xenografts. As demonstrated in Eng et al., murine Pan02 pancreatic tumors responded better to cisplatin chemotherapy, while a human pancreatic tumor cell line and patient pancreatic tumor xenografts responded better to cisplatin, Apo2L/TRAIL, and nab-paclitaxel at TT, compared to ST. Additionally, patient lung tumor xenograft responses were improved at TT with the addition of the tyrosine kinase inhibitor, erlotinib [51]. Ami et al. also found that the addition of a common chemotherapeutic agent, paclitaxel (PTX), induced hypothermia in 4T1 tumor-bearing mice housed at ST for (23°C). When mice were housed at TT, the body temperature and blood flow of tumor-bearing mice was unchanged in response to PTX. Mice treated with PTX at ST also demonstrated significantly more lung metastases, as well as increased angiogenesis, while mice housed at TT did not [52]. Similarly, we have shown that when given the option of spending time at different temperatures (22, 28, 30, 34, or 38°C) within a thermal preference apparatus, non-tumor-bearing mice, not surprisingly, spend about half of the time in the TT chamber (30°C). However, when tumor-bearing mice are given the same options, we found that they spend more than half (~60%) of the time in the 38°C chamber, suggesting that tumor-bearing mice may actually have a TNZ several degrees higher than healthy mice [31].

In the last decade, immune checkpoint inhibitors, such as nivolumab (anti-programmed cell death protein-1, or αPD-1), have become a revolutionary advancement in the treatment of a wide variety of cancers, allowing some patients to achieve long-term remissions. However, these therapies appear to have limited efficacy in a majority of patients [53]. Our group has demonstrated that when mice are housed at ST, both B16-OVA and 4T1 tumors respond poorly or not at all, respectively, to αPD-1 therapy. However, at TT, αPD-1 significantly reduced tumor growth in both models, demonstrating the potential for reducing sympathetic nervous system stress in increasing the efficacy of these therapies [37]. Indeed, these findings, recapitulated in pre-clinical models using the pan beta-blocker propranolol, have led to a clinical trial, currently in phase II [NCT03384836], in which patients with advanced metastatic melanoma receive αPD-1 in addition to propranolol [54]. To build on these findings, our lab has also been interested in defining the changes in the immune landscape of the TME at TT. We have shown that, in mice housed at TT, both 4T1 and CT26 tumors had significantly more CD8⁺ T cells. CT26 tumor-bearing mice, specifically, were found to have significantly more antigen-specific CD8⁺ T cells present within both the tumor and in the tumor draining lymph nodes. In the same study, 4T1 tumors in mice housed at TT had significantly fewer immunosuppressive T regulatory cells [31].

Obesity and metabolic disorders

Many murine models of obesity and metabolic diseases examined at thermoneutrality have revealed a significant influence of housing temperature on disease severity and outcome. Mice housed in cooler, standard temperatures have more thermogenic BAT deposits and higher UCP-1 expression and as a result, significantly increased metabolic rates compared to mice housed at warmer, thermoneutral temperatures [35,36,55,56]. In line with this, it has been shown that mice housed below thermoneutrality are resistant to obesity, even when fed a high fat diet (HFD) [55,57,58]. Though it was previously hypothesized that this is due to the approximately twofold increase in basal metabolic rate, mediated by increased UCP-1 expression, UCP-1 ablation alone or in addition to a HFD does not induce obesity in mice housed at ST [55,59]. However, while Liu et al. also demonstrated that both WT and UCP-1-deficient mice housed at ST were resistant to HFD-induced obesity, increasing the housing temperature to 27°C led to weight gain in both strains and concluded that the resistance of WT mice to HFD-induced obesity was due to the nonshivering thermogenesis mediated by UCP-1 and induced by ST [57]. Similarly, Feldmann et al. found that UCP-1 ablated mice housed at TT were susceptible to obesity, regardless of diet. Their study showed a 50% increase in weight gain in control-fed UCP-1-deficient mice compared to WT mice, and an additional 50% increase in weight gain in mice fed a HFD. Altogether, these studies demonstrate that the twofold higher basal metabolic rate, mediated by UCP-1, of mice housed at ST protects them from HFD-induced obesity compared to mice at TT that do not engage in adaptive thermogenesis and therefore have a substantially lower basal metabolic rate [55,57,58]. These findings underscore a need to consider housing temperature when conducting clinically relevant studies of obesity. Indeed, it has been reported that the anti-obesity drug 2,4-dinitrophenol (DNP), which reduces weight by increasing total energy expenditure (TEE), yielded different results depending on the housing temperature [60]. While DNP-treated mice housed at ST did not demonstrate significant changes in total energy expenditure, adiposity, or body weight, DNP-treated mice housed at TT showed a ~17% increase in TEE and weighed 23% less compared to mice housed at TT that received the vehicle [60]. The authors concluded that this difference was due to the compensatory mechanisms absent in mice at thermoneutrality – that is, mice housed at ST responded to the increased energy generated by DNP-mediated uncoupling by reducing BAT-mediated uncoupling, resulting in no significant overall change in TEE and therefore no significant change in body weight. In contrast, mice housed at TT, that, like humans, rely very little on BAT-mediated uncoupling, exhibited an increase in TEE and subsequent weight loss in response to DNP treatment compared to vehicle-treated mice housed at TT.

Murine models of obesity are also important for studying the benefits of exercise, as well as the impact of obesity on other diseases, such as cancer, nonalcoholic fatty liver disease (NAFLD), and type II diabetes. As such, it is important to consider the impact of mild chronic cold stress on obesity-related models as well. For example, Stemmer et al. found that C57BL/6 nude mice, which are commonly used for human xenograft tumor models, are not only resistant to HFD-induced obesity at ST but also at TT of 30°C, the temperature identified as within the TNZ of fur-bearing mice. C57BL/6 nude appeared to have an increased TT of 33°C, at which point they began to gain weight on a HFD, suggesting that housing temperature is an important variable to consider when studying the effects of obesity on cancer in human tumor xenograft models utilizing nude mice [32]. A murine model of NAFLD has also revealed significant differences in disease presentation. Giles et al. showed that housing mice at TT worsened HFD-induced NAFLD pathogenesis, including increased intestinal permeability, increased inflammatory pathways, and an altered microbiome, compared to mice housed at ST, and therefore more closely recapitulated human disease [61]. Pre-clinical studies aimed at better understanding the benefits of exercise also appear to better model human disease when conducted at TT [62,63]. Healthy, non-obese mice that engaged in voluntary wheel running at TT exhibited less browning of white adipose tissue, did not increase mitochondrial biogenesis, and blunted increases in glucose tolerance or metabolism compared to mice housed at ST, findings that more closely recapitulate what is seen in healthy human subjects [62,63].

Cardiovascular disease

Housing temperatures have also been shown to influence cardiovascular parameters and disease outcomes. Williams et al. found that housing mice at thermoneutral temperatures significantly reduced both mean arterial pressure (MAP) and resting heart rate (HR) [64]. Similar effects on resting heart rate were demonstrated by Swoap et al. who showed that at TT (30°C), murine resting HR is regulated by a dominant cardiac vagal tone, rather than cardiac sympathetic tone – a phenotype more closely modeling human physiology [65]. Interestingly, a recent study revealed that housing mice at TT reversed age-related reduction of HR and heart rate variability (HRV) [66]. Since humans experience age-related declines in HR and HRV, these findings suggest that our understanding of rodent age-related cardiovascular changes may be obscured by the fact that previous pre-clinical studies were conducted under subthermoneutral conditions [66]. In addition to changes in HR, HRV, and MAP, housing mice at TT has also been shown to potentiate and increase the severity of atherosclerosis. Thermoneutral housing and high-fat diets led to increased atherosclerotic plaques, inflammation in the vasculature, serum cholesterol, and macrophage-mediated inflammation of adipose tissue, further demonstrating a need for consideration of the impact of housing temperature on cardiovascular disease models [67,68].

Other inflammatory diseases

The impact of chronic mild cold stress also extends to murine models of human disease that are caused primarily by an exacerbated inflammatory response. One such disease is graft-versus-host disease (GvHD) – a common and dangerous complication of allogeneic hematopoietic stem cell transplantation (alloHCT) [69,70]. Our group has demonstrated that mice housed at ST are more resistant to developing GvHD, compared to mice housed at TT. Indeed, housing mice at TT lead to significantly more severe GvHD, suggesting that resistance to alloHCT-induced GvHD in mice is a result of the dampened immune response of mice at ST, and likely contributes to the difficulties in translating findings of murine GvHD studies to patients [69]. Additionally, we have shown that the increased severity of GvHD in mice housed at TT can be reduced by treatment with beta-agonists, without impairing graft-versus-leukemia, revealing a potential therapeutic intervention for patients undergoing alloHCT [69,70].

Various other studies conducted under thermoneutral conditions have revealed additional insight into the extent to which temperature impacts murine models of inflammatory diseases. Liao et al. demonstrated that TT housing reduced the number of inflammatory T helper 2 cells found in the lungs of mice with ovalbumin-induced asthma. They also showed that TT reduced the overall severity of asthmatic symptoms [71]. TT housing was also shown to reduce LPS-driven blood-brain barrier dysfunction in PI3Kγ-deficient mice, and therefore reduced the amount of neuroinflammation. The authors therefore concluded that murine models of neuroinflammation are likely biased if the housing temperature is not considered [72]. Furthermore, mice housed at TT exhibit increased intestinal disease tolerance compared to mice housed at ST, suggesting that temperature should also be considered when modeling inflammatory intestinal diseases (Figure 2) [73]. Though it has not been studied in mice housed at thermoneutrality, one study reported that cold-stressed mice (acclimated to 6°C) exhibited increased intestinal surface area and altered microbiome composition, an adaptive response to the increased energy demand of cold stress caused by acclimation to 6°C. Likewise, transfer of microbiota from the cold acclimated mice to mice housed at ST resulted in increased intestinal surface area and increased browning of white adipose tissue, suggesting that the microbiome may play an important role in the regulation of thermogenesis [74]. It is therefore worth considering the impact of ambient temperature on the gut microbiome and how this may impact immune function.

Figure 2.

Summary of the impact of housing temperature on murine models of human disease. Red arrows indicate worsened disease outcome as a result of housing temperature, while green arrows indicate improved disease outcomes. Created with bioRender.

Experimental considerations

As discussed above, in addition to the ambient temperature at which research rodents are housed, several other factors can reduce body temperature or impact TNZ and should therefore be considered as potential sources of experimental variabilities. One such factor is the housing density or mice per cage. Skop et al. clearly demonstrated that single-housed (SH) mice have a significantly higher expression of Ucp1 within BAT and significantly higher heat conductance (as determined by TEE/(core body temperature–ambient temperature)) compared to group-housed mice [75]. They also noted that differences in thermoregulation in SH conditions were greatly influenced by sex. While SH male mice experienced a significant reduction in body temperature and no change in TEE, female mice were able to maintain their body temperature by significantly increasing TEE, suggesting that male and female mice employ different thermoregulatory mechanisms under SH conditions [75]. Another housing condition known to influence body temperature is the available nesting material. Gaskill et al. showed that BALB/c mice, but not C57BL/6 mice, housed at ST and provided nesting material, had significantly lower expression of Ucp1 and increased radiated temperature compared to mice that were not provided nesting material [7]. As nesting material did not affect body temperature of either strain, this suggests that different strains, housed at identical temperatures, may utilize different behavioral and/or physiological thermoregulatory mechanisms to maintain body temperature. Female mice also appeared to be more sensitive to thermal stress than males, as the same amount of nesting material provided to male mice was insufficient to lessen signs of cold stress in female mice [7]. In line with this, Johnson et al. showed that adequate nesting material increased weight gain and respiration but not heat production, of three different strains of mice housed at ST, suggesting an overall improved energy balance [33]. Although it is not an amendable housing condition, fur may be an important consideration for an effective thermoneutral housing temperature. As described above, Stemmer et al. demonstrated that, like fur-bearing mice, C57BL/6 nude mice are protected from HFD-induced obesity [32]. However, unlike fur-bearing mice, nude mice remained resistant to HFD-induced obesity at 30°C, and only showed significant weight gain when housed at 33°C, suggesting that nude mice have a TNZ that is higher than fur-bearing mice [32]. These studies collectively demonstrate the importance of recognizing housing conditions – including number of mice per cage, ambient temperature, and nesting material – not only as sources of considerable stress on laboratory rodents but also as potential experimental variables in murine models of human disease (Figure 3).

Figure 3.

Effect of housing conditions on laboratory rodent core body temperature and stress. Increased ambient temperatures (T_a) of 26–38°C, three or more mice per cage, and sufficient nesting material decrease laboratory rodent stress. Conversely, standard housing conditions, including decreased T_a of 20–26°C, fewer mice per cage, and insufficient nesting material, increase laboratory rodent stress. Created with bioRender.

Outside of housing conditions, surgical preparation and procedures also appear to play a role in rodent thermoregulation. For example, surgical implantation of a telemetry device in C57BL/6, BALB/c, and CD-1 mice significantly reduced BAT weight and increased overall body weight by about 15%, perhaps due to the increased locomotive burden, and therefore increased energetic demand, placed on these animals with an extra 3.4 g of weight from the device [7]. Another group found that the type of aseptic scrub and rinse used to prepare mice for surgical procedures had a significant effect on core and surface temperatures [76]. Use of povidone-iodine with a saline rinse significantly reduced core body temperature for the duration of anesthesia. Conversely, isopropyl alcohol significantly reduced core body temperature upon application but resulted in rapid recovery of core temperatures, comparable to that of control mice that did not receive any surgical scrubs [76]. It has also been shown that anesthetizing mice with isoflurane rapidly induces adverse effects, including hypothermia, increased time until first movement, and depressed heart and respiratory rates, all of which could be stabilized to baseline levels with the use of a circulating-warm-water blanket maintained at 37.5°C [77].

Concluding thoughts: Clinical & translation considerations

Here, we have focused on how just one variable, housing temperature, affects outcomes in several different murine models of human disease. A recent meta-analysis by Cait et al. detailed just how important the housing conditions of research animals are, identifying cool housing temperatures as one of several conditions contributing to the distressed and compromised health of research rodents [78]. Additional housing conditions they identified included number of mice per cage, provision of nesting or engaging/novel materials, cage size, and light exposure. Data from over 200 published studies not only identified ethical concerns raised by increased signs of anxiety and depression in rodents housed using conventional conditions, but also revealed simple changes to current housing practices that could improve the translatability of mouse models used in biomedical research. Cait et al. reported that conventional housing conditions likely prevent rodent models of disease from accurately representing disease in healthy humans, pointing out that 86–91% of drugs that successfully treat diseases in rodents ultimately fail in the clinic [78]. While subthermoneutral housing temperature may not be the sole condition responsible for the low clinical relevance of murine models of disease, it is, at least, sufficient to cause considerable variation in these models. As such, it is important for housing conditions, such as temperature and nesting material, to be consistently reported, both to improve reproducibility and to provide future investigators with a more complete understanding of the studies’ introduced variables that may be a significant source of stress (Figure 3). Particularly in studies that focus on understanding or reporting effects of thermoneutral housing and thermal stress, details on experimental setups, ambient temperature(s), and their relationship to the TNZ of the animal used in that setup should be reported (examples of this method can be found in Wanner et al. and Almeida et al.) [79,80]. That is, investigators should report the measured or previously reported TNZ of a rodent under specific experimental conditions (e.g. colonic measurement of body temperature of tumor-bearing mice in their home cage versus respirometry measurement of LPS-treated rats within a metabolic chamber) and whether the ambient temperature(s) is sub-, supra-, or neutral in reference to the TNZ for the experimental conditions being applied. As described above, the environment a rodent is in can shift its TNZ. Therefore, comparisons or analyses of experimental outcomes of studies that examine the effects of thermoneutrality or thermal stress on murine models of human disease should acknowledge and take into consideration the impact of the experimental setup.

Even more imperative, perhaps, is that we start to rethink how our research animals are housed. As indicated above, and in many other studies, adjusting housing temperatures to the appropriate TNZ of the research animal being utilized more often than not more closely resembles human development, pathology, or response to therapies [6,9,39]. Providing research animals with the necessary environmental conditions, such as sufficient nesting material, may therefore increase the generalizability and clinical relevance of our pre-clinical animal models of disease. This is not to say, however, that data generated from chronically cold stressed rodents should be entirely written off – for example, utilizing our current standards for housing research rodents could potentially prove useful for understanding the impact of chronic stress on disease and how we might address this in chronically stressed patients. Subthermoneutral housing may also serve to replace standard methods of inducing stress in our animal models, such as chronic restraint stress or forced swim, introducing a more humane method of studying the effects of chronic stress. Undoubtedly, effectively making these transitions will heavily rely on more consistent and accurate reporting of housing variables by investigators, ambient temperature included.

Funding Statement

This work was supported by the National Cancer Institute [P30CA016056]; the National Institutes of Helath [R01AI155499], [R01CA205246], [R01CA236390]; and the T32 Multidisciplinary Approaches to Tumor Immunology Research Training Grant [T32CA085183].

Disclosure statement

No potential conflict of interest was reported by the author(s).