Core Body Temperature In Mice Research Articles

Adipose Tissue-Resident Sphingomonas Paucimobilis Suppresses Adaptive Thermogenesis by Reducing 15-HETE Production and Inhibiting AMPK Pathway.

Obesity represents a low-grade chronic inflammation status, which is associated with compromised adaptive thermogenesis. However, the mechanisms underlying the defective activation of thermogenesis in chronic inflammation remain unclear. Here, a chronic inflammatory model is first estabolished by injecting mice with low-dose lipopolysaccharide (LPS) before cold exposure, and then it is verified that LPS treatment can decrease the core body temperature of mice and alter the microbial distribution in epididymal white adipose tissue (eWAT). An adipose tissue-resident bacterium Sphingomonas paucimobilis is identified as a potential inhibitor on the activation of brown fat and browning of inguinal WAT, resulting in defective adaptive thermogenesis. Mechanically, LPS and S. paucimobilis inhibit the production and release of 15-HETE by suppressing its main metabolic enzyme 12 lipoxygenase (12-LOX) and 15- Hydroxyeicosatetraenoic acid (15-HETE) rescues the impaired thermogenesis. Interestingly, 15-HETE directly binds to AMP-activated protein kinase α (AMPKα) and elevates the phosphorylation of AMPK, leading to the activation of uncoupling protein 1 (UCP1) and mitochondrial oxidative phosphorylation (OXPHOS) complexes. Further analysis with human obesity subjects reveals that individuals with high body mass index displayed lower 15-HETE levels. Taken together, this work improves the understanding of how chronic inflammation impairs adaptive thermogenesis and provides novel targets for alleviating obesity.

Characterising core body temperature response of free-moving C57BL/6 mice to 1.95 GHz whole-body radiofrequency-electromagnetic fields.

The present study investigated the core body temperature (CBT) response of free-moving adult male and female C57BL/6 mice, during and following a 2-h exposure to 1.95 GHz RF-EMF within custom-built reverberation chambers, using temperature capsules implanted within the intraperitoneal cavity and data continuously logged and transmitted via radiotelemetry postexposure. Comparing RF-EMF exposures (WBA-SAR of 1.25, 2.5, 3.75, and 5 W/kg) to the sham-exposed condition, we identified a peak in CBT within the first 16 min of RF-EMF exposure (+0.15, +0.31, +0.24, +0.37°C at 1.25, 2.5, 3.75, and 5 W/kg respectively; statistically significant at WBA-SAR ≥ 2.5 W/kg only), which largely dissipated for the remainder of the exposure period. Immediately before the end of exposure, only the CBT of the 5 W/kg condition was statistically differentiable from sham. Based on our findings, it is apparent that mice are able to effectively compensate for the increased thermal load at RF-EMF strengths up to 5 W/kg. In addition, the elevated CBT at the end of the exposure period in the 5 W/kg condition was statistically significantly reduced compared to the sham condition immediately after RF-EMF exposure ceased. This would indicate that measures of CBT following the end of an RF-EMF exposure period may not reflect the actual change in the CBT of mice caused by RF-EMF exposure in mice.

Feeding behavior modifies the circadian variation in RR and QT intervals by distinct mechanisms in mice.

Rhythmic feeding behavior is critical for regulating phase and amplitude in the ≈24-h variation of heart rate (RR intervals), ventricular repolarization (QT intervals), and core body temperature in mice. We hypothesized changes in cardiac electrophysiology associated with feeding behavior were secondary to changes in core body temperature. Telemetry was used to record electrocardiograms and core body temperature in mice during ad libitum-fed conditions and after inverting normal feeding behavior by restricting food access to the light cycle. Light cycle-restricted feeding modified the phase and amplitude of 24-h rhythms in RR and QT intervals, and core body temperature to realign with the new feeding time. Changes in core body temperature alone could not account for changes in phase and amplitude in the ≈24-h variation of the RR intervals. Heart rate variability analysis and inhibiting β-adrenergic and muscarinic receptors suggested that changes in the phase and amplitude of 24-h rhythms in RR intervals were secondary to changes in autonomic signaling. In contrast, changes in QT intervals closely mirrored changes in core body temperature. Studies at thermoneutrality confirmed that the daily variation in QT interval, but not RR interval, primarily reflected daily changes in core body temperature (even in ad libitum-fed conditions). Correcting the QT interval for differences in core body temperature helped unmask QT interval prolongation after starting light cycle-restricted feeding and in a mouse model of long QT syndrome. We conclude feeding behavior alters autonomic signaling and core body temperature to regulate phase and amplitude in RR and QT intervals, respectively.NEW & NOTEWORTHY We used time-restricted feeding and thermoneutrality to demonstrate that different mechanisms regulate the 24-h rhythms in heart rate and ventricular repolarization. The daily rhythm in heart rate reflects changes in autonomic input, whereas daily rhythms in ventricular repolarization reflect changes in core body temperature. This novel finding has major implications for understanding 24-h rhythms in mouse cardiac electrophysiology, arrhythmia susceptibility in transgenic mouse models, and interpretability of cardiac electrophysiological data acquired in thermoneutrality.

GCN2 is required to maintain core body temperature in mice during acute cold.

Nonshivering thermogenesis in rodents requires macronutrients to fuel the generation of heat during hypothermic conditions. In this study, we examined the role of the nutrient sensing kinase, general control nonderepressible 2 (GCN2) in directing adaptive thermogenesis during acute cold exposure in mice. We hypothesized that GCN2 is required for adaptation to acute cold stress via activation of the integrated stress response (ISR) resulting in liver production of FGF21 and increased amino acid transport to support nonshivering thermogenesis. In alignment with our hypothesis, female and male mice lacking GCN2 failed to adequately increase energy expenditure and veered into torpor. Mice administered a small molecule inhibitor of GCN2 were also profoundly intolerant to acute cold stress. Gcn2 deletion also impeded liver-derived FGF21 but in males only. Within the brown adipose tissue (BAT), acute cold exposure increased ISR activation and its transcriptional execution in males and females. RNA sequencing in BAT identified transcripts that encode actomyosin mechanics and transmembrane transport as requiring GCN2 during cold exposure. These transcripts included class II myosin heavy chain and amino acid transporters, critical for maximal thermogenesis during cold stress. Importantly, Gcn2 deletion corresponded with higher circulating amino acids and lower intracellular amino acids in the BAT during cold stress. In conclusion, we identify a sex-independent role for GCN2 activation to support adaptive thermogenesis via uptake of amino acids into brown adipose.NEW & NOTEWORTHY This paper details the discovery that GCN2 activation is required in both male and female mice to maintain core body temperature during acute cold exposure. The results point to a novel role for GCN2 in supporting adaptive thermogenesis via amino acid transport and actomyosin mechanics in brown adipose tissue.

Mechanisms and physiological function of daily haemoglobin oxidation rhythms in red blood cells.

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro, or taken from humans in vivo, and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

Social isolation increases metabolic rate in the transition of light- to dark- phase and advances Rev-erb-α expression in brown adipose tissue to regulate daily rhythm of core body temperature in mice

Mammals use social thermoregulation to maintain the core body temperature (Tc) at a lower energy cost. Brown adipose tissue (BAT) plays a crucial role in thermoregulation. This study tested the hypothesis that social isolation induces alterations in thermogenesis and metabolism to maintain the rhythm of Tc throughout the day. Adult male mice (C57BLJ/6) were maintained in groups of 4–5 (group-housed) or isolated (single-housed) for 28 days. Telemetric probes recorded spontaneous locomotor activity (SLA) and Tc to analyze SLA and Tc daily rhythms. Body weight was measured weekly. VO2 consumption was analyzed at zeitgeber time (ZT) 1–3 (beginning of light phase) or ZT10–14 (beginning of dark phase). The expression of thermogenic-related and clock genes in the BAT occurred at ZT2, ZT8, ZT14, and ZT20. The β3 adrenergic agonist CL-316,243 was i.p. injected in the group- or single-housed mice. Social isolation increased the amplitude of Tc rhythm but decreased the amplitude of SLA oscillation. Nevertheless, in single-housed mice, the circadian peak of Tc and SLA was unaltered, with reduced body weight gain, increased VO2 consumption, and BAT Ucp1 expression at ZT14. Isolation advanced the BAT Rev-erb-ɑ peak of expression and phased-shift the peak of expression in BAT Bmal1, while it abolished the daily BAT Per2 oscillation. Isolation also abolished the BAT Ucp1 and hormone-sensitive lipase (Hsl) expression induced by stimulation of the β3ADR, but it increased Rev-erb-ɑ and Pgc1α. Thus, alterations in Ucp1, Reverb-α, Bmal1, and Per2 daily expression may favor an increased metabolic rate at the beginning of the dark, which, in turn, contributes to maintaining the daily Tc rhythm at the expense of reduced body weight gain in isolated mice. In addition, at least at the transcription level, the response of BAT from isolated mice to adrenergic agonist seems to be via a non-canonical mechanism involving Rev-erb-α and Pgc1α.

Sustained effect of habitual feeding time on daily rhythm of core body temperature in mice.

Background and aimCircadian clocks in most peripheral tissues are entrained mainly by feeding. Therefore, this study aimed to investigate whether the daily rhythm of core body temperature (CBT), including the effect of diet-induced thermogenesis, varies according to habitual feeding time.MethodsWild-type and uncoupling protein 1 (UCP1) knockout mice were fed only during the first 4 h (Breakfast group) or the last 4 h of the dark period (Dinner group) for 17 days. On day 18, both groups were fed twice for 2 h, at the same starting times. Locomotor activity and CBT were measured continuously during the experiment.ResultsOn day 18, CBT increased at the beginning of each feeding period, regardless of the group and strain. However, the CBT increase induced by the first meal decreased sharply in the Breakfast group and mildly in the Dinner group; the opposite was observed after the second meal. In UCP1 knockout, but not wild-type, mice, the total amount of CBT was significantly lower in the Dinner group than in the Breakfast group. These effects were mostly independent of the locomotor activity and food intake.ConclusionThese results reveal that the effect of habitual feeding time on the daily rhythm of CBT is sustained at least until the following day. These effects may be mediated by both UCP1-dependent and -independent mechanisms.

Influence of Cold-TRP Receptors on Cold-Influenced Behaviour.

The transient receptor potential (TRP) channels, TRPA1 and TRPM8, are thermo-receptors that detect cold and cool temperatures and play pivotal roles in mediating the cold-induced vascular response. In this study, we investigated the role of TRPA1 and TRPM8 in the thermoregulatory behavioural responses to environmental cold exposure by measuring core body temperature and locomotor activity using a telemetry device that was surgically implanted in mice. The core body temperature of mice that were cooled at 4 °C over 3 h was increased and this was accompanied by an increase in UCP-1 and TRPM8 level as detected by Western blot. We then established an effective route, by which the TRP antagonists could be administered orally with palatable food. This avoids the physical restraint of mice, which is crucial as that could influence the behavioural results. Using selective pharmacological antagonists A967079 and AMTB for TRPA1 and TRPM8 receptors, respectively, we show that TRPM8, but not TRPA1, plays a direct role in thermoregulation response to whole body cold exposure in the mouse. Additionally, we provide evidence of increased TRPM8 levels after cold exposure which could be a protective response to increase core body temperature to counter cold.

Primary role for melatonin MT2 receptors in the regulation of anhedonia and circadian temperature rhythm

Circadian rhythms disturbance is widely observable in patients with major depression (MD) and is also associated with depression vulnerability. Of them, disturbed melatonin secretion rhythm is particularly relevant to MD and is strongly phase-locked to core body temperature (CBT) rhythm.Here we aim to study the specific role of each melatonin receptor (MT1 and MT2) subtype in melatonin regulation of circadian CBT and its possible relationship with depressive-like behaviors.MT1-/- , MT2-/- and WT (C57BL/6) mice were used. Anhedonia, using the sucrose intake test, circadian CBT, environmental place preference (EPP) conditioning and vulnerability to chronic social defeat stress (CSDS) procedure were studied. Moreover, the antidepressant effects of reboxetine (15 mg/kg/day, i.p.) for three weeks or ketamine (15 mg/kg i.p. every four days, 4 doses in total) were studied. Further, exposure to ultra-mild stress induced by individual housing for several weeks was also studied in these mice.MT2-/- mice showed anhedonia and lower CBT compared to WT and MT1-/-. In addition, while reward exposure raised nocturnal CBT in WT this increase did not take place in MT2-/- mice. Further, MT2-/- mice showed an enhanced vulnerability to stress-induced anhedonia and social avoidance as well as an impaired acquisition of novelty seeking behavior. Both reboxetine and ketamine reverted anhedonia and induced a clear anti-helpless behavior in the tail suspension test (TST). Reboxetine raised CBT in mice and reverted ultra-mild stress-induced anhedonia.Our findings show a primary role for MT2 receptors in the regulation of circadian CBT as well as anhedonia and suggest that these receptors could be involved in depressive disorders associated to disturbed melatonin function.

Jiang Gui Fang activated interscapular brown adipose tissue and induced epididymal white adipose tissue browning through the PPARγ/SIRT1-PGC1α pathway.

Gui Zhi Tang, a well-known Chinese herbal formula recorded in the Eastern Han Dynasty, has been widely used to treat exogenous cold for thousands of years. Recent studies have shown that Gui Zhi Tang has the effect of regulating the body temperature. Because of its effect on heat production, protecting vital organs of the body and avoiding damage from the cold environment, Jiang Gui Fang (JG) was obtained from the Department of Traditional Chinese Medicine at the General Hospital of Northern Theatre Command where it has been used clinically for many years and has exhibited favourable efficacy. Based on research on Gui Zhi Tang, the principles of traditional Chinese medicine and survey of a large number of studies, this empirical formula was developed. The composition of JG included Dried ginger, Cassia twig, and Liquorice in Gui Zhi Tang, which play a major role in the treatment of exogenous cold, and combined these components with other Chinese medicines, such as Pueraria, Spatholobus, Acanthopanacis cortex, Evodiae fructus, and Codonopsis pilosula. To promote the core body temperature and prevent invasion of the major organs from the cold environment, we studied the effect of JG on the core body temperature of mice and then explored its regulation of interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) and the possible mechanism. Finally, we determined the phytochemical composition of JG that plays a role in heat production. In vivo study, we performed a 4-week treatment of JG in acute cold environment at -20 °C and chronic cold exposure at 4 °C. The core temperature, adipose tissue weight, serum parameters, and morphological observation of adipocytes, liver and kidney were measured. Then we investigated the expression levels of adipogenic factors, thermogenic factors and lipoprotein. In vitro, we determined the lipid droplet content, ATP content, and the maximum oxygen consumption of mitochondria. JG treatment promoted core temperature, inhibited eWAT weight, protected liver, and reduced glucose and lipids in Kunming (KM) mice. JG also increased the expression of BAT-associated thermogenic factors, including uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α). The levels of the lipogenic factor peroxisome proliferate-activator receptor gamma (PPARγ) and the lipolytic protein hormone-sensitive triglyceride lipase (HSL) in eWAT were elevated. The results of H&E and immunohistochemistry showed that JG significantly reduced the size of iBAT and eWAT and increased the content of UCP1. In vitro, JG reduced the content of lipid droplets and ATP in brown fat cells. The maximum oxygen consumption capacity of mitochondria and the expression levels of UCP1, PGC1α and silent mating type information regulation 2 homologue 1 (SIRT1) were enhanced after JG treatment. In vivo and in vitro studies, the results demonstrated that JG obviously increased the core temperature of mice by activating iBAT and inducing eWAT browning, which proved the mechanism is closely related to the PPARγ/SIRT1- PGC1α pathway. In this paper, we will provide a reference for further study of iBAT activation and eWAT browning.

The Anti-obesity Effects of Direct Delivery of Resveratrol-encapsulated Liposomes to Inguinal White Adipose Tissue in Male APOE*3Leiden.CETP Transgenic Mice (P21-006-19)

ObjectivesBrowning of white adipose tissue (WAT) is a promising strategy for preventing and treating obesity. Trans-resveratrol (R) has the potential to increase the energy expenditure via inducing WAT browning, but its application in obesity is limited by a low level of aqueous solubility, a high level of hepatic metabolism and toxicity. We have successfully synthesized biocompatible and biodegradable R encapsulated liposomes (Rlipo). The objective of this project is to deliver Rlipo directly to the inguinal WAT (iWAT) in mice with the goal of maximizing anti-obesity efficacy while minimizing side effects and toxicity. MethodsRlipo were prepared using R and soy phosphatidylcholine (soy-PC) dissolved in ethanol. After mixing and drying them under nitrogen, deionized water was added followed by sonication. Unencapsulated R was removed by ultrafiltration. The void liposomes (Vlipo) were prepared using only soy-PC.The particle size and polydispersity index (PI) of Rlipo and Vlipo were measured using a Brookhaven BI-MAS particle size analyzer. R’s encapsulation efficiency and solubility were measured using a liquid chromatography-mass spectrometry.Male APOE*3Leiden.CETP mice having human-like lipoprotein metabolism were fed with a high-fat diet (45% of calorie from fat) for 10 weeks. After treating the high-fat diet for 4 weeks, mice received iWAT injection of Rlipo, Vlipo, free R and saline (control) once a week. R concentration was 17.5 mg/kg body weight/week. Body weight and food intake were measured weekly. Body composition of mice was measured using an EchoMRITM every other week. Core body temperatures of mice were determined using a Type T thermometer. Glucose tolerance test (GTT) was performed at the 4th week of treatment. ResultsRlipo increased the aqueous solubility of R by more than 100 times. The size of Rlipo and Vlipo was 170 ± 6 nm and 140 ± 7 nm, respectively. Their PI values were less than 0.2. The encapsulation efficiency of Rlipo was 99.8%. The post-treatment core body temperatures for control, free R, Vlipo and Rlipo groups were 36.2°C, 36.2°C, 36.6°C, and 37.0°C, respectively. Food intake was similar among 4 groups. Compared to other groups, the Rlipo-treated mice had the lowest body weight, body fat mass, and body fat percentage. The Rlipo group had the lowest area under the curve in the GTT among 4 groups. ConclusionsNanoencapsulation increased R’s solubility and enhanced its anti-obesity efficacy. Funding SourcesNIH/NCCIH (Grant R15AT008733).

Abstract 2440: Potentiation of the action of chemotherapeutic drugs by the hypoxia-activated prodrug SN30000 in multicellular tumor spheroids

Abstract Introduction: Chemotherapy is often limited by resistant subpopulations of tumor cells. Tumor hypoxia contributes to resistance of tumor cells to clinical drugs via multiple mechanisms. Hypoxia-activated prodrugs selectively target hypoxic cells, among which SN30000 is a second generation of tirapazamine analogue with superior diffusion coefficient and hypoxia selectivity. Here we hypothesize that SN30000/chemotherapy drug combinations will provide improved activity. Methods: HCT116 and SiHa spheroids were used to screen schedules of SN30000 in combination with 4 clinical drugs (cisplatin, doxorubicin, gemcitabine, paclitaxel) under 5% O2 and 20% O2, with endpoints of spheroid growth delay and clonogenic cell survival. The schedules were tested on HCT116 and SiHa monolayers under 5% O2. HCT116 monolayers and spheroids exposed to SN30000 or the clinical drugs were dissociated for clonogenic survival assay. Tumor growth delay using the same schedules of the most promising combination (SN30000 + gemcitabine) were then tested in HCT116 xenografts in NIH-III mice. Body weight loss and core body temperature of mice in response to the treatments were monitored. Results: Exposing to 25 µM SN30000 and 25 µM cisplatin/5 µM doxorubicin/10 µM gemcitabine simultaneously for 2 hr, and SN30000 3 hr before gemcitabine under 5% O2 significantly delayed HCT116 and SiHa spheroid growth, but their combined activity was compromised under 20% O2. Dosing SN30000 and cisplatin simultaneously, and SN30000 3 hr before gemcitabine significantly delayed HCT116 and SiHa monolayer growth. Clonogenic survival assay demonstrated that HCT116 monolayers were more sensitive than spheroids to doxorubicin, but not the other 3 clinical compounds, consistent with the known limited diffusion of doxorubicin into spheroids.Dosing SN30000 3 hr before gemcitabine or at the same time, but not SN30000 3 hr after gemcitabine significantly delayed tumor growth, which was consistent with the spheroid data. Body weight measurements showed that the combinations of SN30000 and gemcitabine added little additional toxicity while SN30000/doxorubicin was more toxic when administered simultaneously. SN30000 caused hypothermia in mice with recovery after 3 hr. Discussion: Screening schedules of SN30000 in combination with 4 clinical drugs in HCT116/SiHa spheroids and monolayers demonstrated schedule and oxygen dependence of the activity of SN30000 and gemcitabine combinations, which was further demonstrated in HCT116 xenografts. The mechanism of the promising interaction between gemcitabine and SN30000 is being further investigated by flow cytometry and immunohistochemical staining to explore the hypoxic fraction and cell proliferation following treatments, and by evaluating the plasma pharmacokinetics of SN30000 and gemcitabine to investigate the implications of SN30000-induced hypothermia. Citation Format: Xinjian Mao, Sarah McManaway, William Robert Wilson, Kevin Owen Hicks. Potentiation of the action of chemotherapeutic drugs by the hypoxia-activated prodrug SN30000 in multicellular tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2440.

O-Mannosylation of Proteins Enables Histoplasma Yeast Survival at Mammalian Body Temperatures.

ABSTRACTThe ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen Histoplasma capsulatum, elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection. Histoplasma yeast cells lacking the Pmt1 and Pmt2 protein mannosyltransferases, which catalyze O-linked mannosylation of proteins, are severely attenuated during infection of mammalian hosts. Cells lacking Pmt2 have altered surface characteristics that increase recognition of yeast cells by the macrophage mannose receptor and reduce recognition by the β-glucan receptor Dectin-1. Despite these changes, yeast cells lacking these factors still associate with and survive within phagocytes. Depletion of macrophages or neutrophils in vivo does not recover the virulence of the mutant yeast cells. We show that yeast cells lacking Pmt functions are more sensitive to thermal stress in vitro and consequently are unable to productively infect mice, even in the absence of fever. Treatment of mice with cyclophosphamide reduces the normal core body temperature of mice, and this decrease is sufficient to restore the infectivity of O-mannosylation-deficient yeast cells. These findings demonstrate that O-mannosylation of proteins increases the thermotolerance of Histoplasma yeast cells, which facilitates infection of mammalian hosts.

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Introduction: Heatstroke is a life-threatening illness caused by an elevation of the body temperature due to exposure to high temperature and/or humidity environments, and its incidence has been increasing in the summer in Japan. It is considered to be important to drink water to prevent the development of heatstroke. OS-1 is an oral rehydration solution which was designed to alleviate dehydration due to various conditions, such as cholera, and contains electrolyte ions such as sodium (50 mEq/l), potassium (20 mEq/l), chloride (50 mEq/l), magnesium (2 mEq/l), sulfate (2 mEq/l) and phosphate (2 mmol/l), as well as lactate (31 mEq/l) and glucose (18 g/l). Although drinking OS-1 is in the summer time, the effects of OS-1 supplementation on heatstroke have not been examined in detail. We produced a non-lethal experimental heatstroke model in mice, and then determined their core body temperature after supplying either pure water or OS-1. Methods: Experimental heatstroke was produced in a moisture chamber. The mean conditions of the chamber were an ambient temperature of 41 ± 0.5 ºC and relative humidity at 70 - 99% during the heat-exposure (HE). During the experiment, the ambient temperature, relative humidity and Wet-Bulb Globe Temperature (WGBT) were monitored every 1 minute. Male C57BL/6J mice had a thermometer implanted into the abdominal cavity three weeks before the experiments, and were maintained at a normal temperature (ambient temperature 24 ± 1ºC and relative humidity 20–40%) with free access to food and water. The animals were randomly divided into three groups: a no supplement group (control, n=6), 30 mg/kg water supplement group (Water, n=6) and 30 mg/kg OS-1 supplement group (OS-1, n=6) and were subsequently not allowed access to any drink for three hours. Then, the animals were subjected to HE for 60 min in the moisture chamber. The supplement was administered twice orally three hours after dehydration (before HE) and immediately after HE. The animals were then returned to a normal temperature environment and maintained for a further seven days with free access to food and water. It was previously reported that the core body temperature of mice rapidly elevated and reached the maximal temperature after the onset of HE, then it decreased immediately and severe hypothermia was induced after the HE. It took several hours to recover to the normal circadian rhythms. The level of hypothermia was correlated with the severity of HE in mice. In our experiments, the core body temperature was recorded every five minutes from one day prior to HE to five days post-HE in all the mice. The circadian rhythms of the core body temperatures before HE were recorded. The maximum core body temperature during the HE, minimum core body temperatures after the HE and the time to recover to normal circadian rhythm post-HE were compared among the groups. Results: Actual mean condition of the moisture chamber during HE was ambient temperature (39.0 ± 0.16 ºC), relative humidity (93.7 ± 0.11 %) and WGBT (41.6 ± 0.18 ºC). The control group was account for maximal temperature at 32 ± 2.5 min after onset of HE and measured at 41.1 ± 0.1 ºC. The maximal temperature in water and OS-1 groups was 40.9 ± 0.4 ºC and 39.7 ± 0.17 ºC, respectively. Maximal temperature in OS-1 group was significantly lower than that in control (p<0.05). Minimal body temperature which was account for 236 ± 33 min after onset of HE, was no difference between three groups (Control: 31.7 ± 1.45 ºC, water: 31.3kelou ºC, and OS-1: 33.3 ± 0.44 ºC). However, while the circadian rhythm of body temperature was lost during the HE, recovery of the rhythm in OS-1 group (495 ± 48 minutes) was significantly shorter than that in water group (961 ± 138 minutes, p<0.05) and was tend to shorter than control (881 ± 120 minutes, p = 0.056). Conclusions: These results suggest that OS-1 supplementation before and after HE decreased the dysregulation of body temperature, likely due to the efficient rehydration.

Physical exercise-induced changes in the core body temperature of mice depend more on ambient temperature than on exercise protocol or intensity

The mechanisms underlying physical exercise-induced hyperthermia may be species specific. Therefore, the present study aimed to investigate the effects of exercise intensity and ambient temperature on the core body temperature (T core) of running mice, which provide an important experimental model for advancing the understanding of thermal physiology. We evaluated the influence of different protocols (constant- or incremental-speed exercises), treadmill speeds and ambient temperatures (T a) on the magnitude of exercise-induced hyperthermia. To measure T core, a telemetric sensor was implanted in the abdominal cavity of male adult Swiss mice under anesthesia. After recovering from the surgery, the animals were familiarized to running on a treadmill and then subjected to the different running protocols and speeds at two T a: 24 °C or 34 °C. All of the experimental trials resulted in marked increases in T core. As expected, the higher-temperature environment increased the magnitude of running-induced hyperthermia. For example, during incremental exercise at 34 °C, the maximal T core achieved was increased by 1.2 °C relative to the value reached at 24 °C. However, at the same T a, neither treadmill speed nor exercise protocol altered the magnitude of exercise-induced hyperthermia. We conclude that T core of running mice is influenced greatly by T a, but not by the exercise protocols or intensities examined in the present report. These findings suggest that the magnitude of hyperthermia in running mice may be regulated centrally, independently of exercise intensity.

Abstract 3517: Use of a novel synthetic biomaterial to induce mild whole body hyperthermia for the treatment of cancer in a preclinical model

Abstract Hyperthermia is currently being investigated as an alternative form or cancer therapy. Numerous basic and pre-clinical published reports have shown the antitumor effects of hyperthermia in various cancer models. High temperatures, low duration (heat shock) will have a direct killing effect on exposed cells. The limitation to this approach is the restriction of tumor location and associated side effects. Low temperature, high duration whole body hyperthermia (WBH) is generally considered to be the least toxic but requires long exposures that are generally uncomfortable or inconvenient to the patients. In the present study, we show the efficacy of a synthetic far-infrared emitting biomaterial to induce and evaluate its therapeutic potential when used alone or in combination with chemotherapy. We used BioRubber (BR, Yamamoto Bio-Technology Inc., Anaheim, CA), a state of the art biomaterial that emits far infrared radiation in the 4-25 μm spectral ranges. To determine the efficacy to induce mild whole body hyperthermia, athymic nude mice were house in BR lined cages. Core body temperatures of mice housed in BR-lined cages were significantly higher than mice housed in unlined cages and were maintained up to 2 weeks during continuous exposure (37.70oC vs. 36.75oC, p=0.038, respectively.). To determine the antitumor effects of WBH, KU7/GFP-bearing nude mice were housed in cages lined with BR for 28 days. Non-significant tumor growth inhibition was observed in tumors of mice housed in BR. Histological and immunohistochemical analysis revealed increased granulocyte activity in tumors of BR-exposed mice. Additionally, increased levels of C-GSF, GM-CSF, TNFα and IL-12p70 corresponded to exposure to WBH. To determine the long term safety and efficacy of mild WBH and chemotherapy in deep tumors, we carried out a survival study using immunodeficient mice orthotopically inoculated with KU7/GFP cells and treated doxorubicin (Dox) alone or in combination with BR. Although no treatment efficacy was observed in mice treated with BR with regards to tumor size (tumor size according to the expression of GFP was 350.5, 309.7, 357.4, 257.3 for control, WBH, Dox and WBH/Dox groups, respectively), survival was improved for mice exposed to BR. Survival rates were 50% (5/10), 70% (7/10), 10% (1/10) and 50% (5/10), for control, WBH, Dox and WBH/Dox groups, respectively. It is unclear if mild WBH has a direct antitumor effect; however, it appears that BR-induced mild WBH may be capable of inducing immuno-stimulatory that may improve survival in a preclinical model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3517. doi:1538-7445.AM2012-3517

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas. In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2. Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5. Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain In Vitro and In Vivo Cannabinoid 1 Receptor Affinity and Activity

BackgroundK2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ9-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).Methods/Principal FindingsJWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [3H]CP-55,940, and then for CB1R intrinsic efficacy using an [35S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting Ki values that were lower than or equivalent to Δ9-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ9-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ9-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.Conclusions/SignificanceUnlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ9-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.

Abstract 5492: The effects of whole body hyperthermia on tumor growth by a novel far infrared emitter

Abstract Whole body hyperthermia (WBH) has been used alone or as an adjunct to radio- and/or chemotherapy for the treatment of various malignant diseases. The therapeutic effect of WBH in these conditions has been hypothesized to involve direct or indirect activation of the immune system and heat shock proteins (HSPs). In this study, we investigate the effects of hyperthermia in vitro and in vivo using BioRubber (BR, Yamamoto Bio-Technology Inc., Anaheim, CA), a revolutionary far infrared emitter on tumor growth. BR is a pliable state-of-the-art biomaterial that emits FIR in the 4-25μm spectral ranges. For the in vitro experiments, cell growth assays and cell cycle analysis was performed in a total of twelve different cancer cell lines (HeLa, bladder, prostate and renal) sandwiched between BR. BR had no inhibitory effects on any of the tumor cells grown in vitro. To determine the effects of BR therapy on tumor and normal tissue in vivo, KU7/GFP-bearing nude mice were housed in cages lined with BR for 28 days. Tumor size and core body temperature were recorded weekly. The core body temperatures of mice exposed to BR increased significantly by 1oC (P&amp;lt;0.05) after 2 weeks of exposure to BR. Although not significant, tumors of mice exposed to BR showed a clear tendency in tumor size reduction. Tumors were collected and anlyzed for histopathology, TUNEL and PCNA immunohistochemical analysis. Proliferation and apoptotic rates in tumors from mice exposed to BR were similar to control mice. Further histopathological analysis showed that BR-exposed tumors had remarkable granulocyte infiltration accompanied by increased necrosis. This suggest that BR exposure was indeed able to induce and sustain mild hyperthermia in vivo with reduced tumor growth. However, no direct inhibitory on the tumor cells resulted form BR-induced hyperthermia but rather appeared to have been an indirect effect of immuno-stimulation achieved from sustained WBH. To further investigate the immunological component of hyperthermia-induced immune response, a syngeneic tumor model was carried out using mouse RENCA cells in immunocompetent and immunodeficient mice. Similar tumor growth inhibitory effects were seen in both immunocompetent and immunodeficient mice thus suggesting an innate immune response as a result of sustained WBH. Together, these data suggest that BR may be a promising biomaterial in for use hyperthermia therapy: however, more research has to be conducted to elucidate the specific mechanisms involved in immuno-stimulation and whether the effects from BR-induced hyperthermia can be maximized with combination therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5492.