Biological and Pharmaceutical Bulletin
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Repeated Cold Stress Enhances the Acute Restraint Stress-Induced Hyperthermia in Mice
Tomoyoshi MiyamotoYoshinori FunakamiErika KawashitaAi NomuraNanako SugimotoHaruka SaekiMaho TsubotaSeiji IchidaAtsufumi Kawabata
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2017 Volume 40 Issue 1 Pages 11-16

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Abstract

The rodents exposed to repeated cold stress according to a specific schedule, known as specific alternation of rhythm in temperature (SART), exhibit autonomic imbalance, and is now used as an experimental model of fibromyalgia. To explore the susceptibility of SART-stressed animals to novel acute stress, we tested whether exposure of mice to SART stress for 1 week alters the extent of acute restraint stress-induced hyperthermia. Mice were subjected to 7-d SART stress sessions; i.e., the mice were alternately exposed to 24 and 4°C at 1-h intervals during the daytime (09:00–16:00) and kept at 4°C overnight (16:00–09:00). SART-stressed and unstressed mice were exposed to acute restraint stress for 20–60 min, during which rectal temperature was monitored. Serum corticosterone levels were measured before and after 60-min exposure to restraint stress. SART stress itself did not alter the body temperature or serum corticosterone levels in mice. Acute restraint stress increased the body temperature and serum corticosterone levels, both responses being greater in SART-stressed mice than unstressed mice. The enhanced hyperthermic responses to acute restraint stress in SART-stressed mice were significantly attenuated by SR59230A, a β3 adrenoceptor antagonist, but unaffected by diazepam, an anxiolytic, mifepristone, a glucocorticoid receptor antagonist, or indomethacin, a cyclooxygenase inhibitor. These results suggest that SART stress enhances the susceptibility of mice to acute restraint stress, characterized by increased hyperthermia and corticosterone secretion, and that the increased hyperthermic responses to acute stress might involve accelerated activation of sympathetic β3 adrenoceptors, known to regulate non-shivering thermogenesis in the brown adipose tissue.

Fever is one of the most common complaints in daily life. Apart from infectious fever resulting from activation of the immune system, there exists stress-induced hyperthermia that is often referred to as ‘emotional fever.’1,2) A variety of stresses including psychological stress raise body temperature in mammals, such as rats,3,4) mice57) and humans.812) The acute stress-induced rise in body temperature appears to involve the excitation of the sympathetic nervous system which regulates non-shivering thermogenesis in brown adipose tissue.1315) It is hypothesized that stress-induced hyperthermia and infection-induced fever are two distinct processes mediated largely by different neurobiological mechanisms,4) although they result in similar clinical signs, i.e., higher body temperature accompanied by shivering and cutaneous vasoconstriction.1) On the other hand, the influence of chronic or long-term stress on thermoregulation still remains largely unknown. This should be investigated, since chronic stress, rather than acute stress, has great impact on the development of various diseases including neurodegenerative, mental, cardiovascular and gastrointestinal diseases.16,17)

Kita et al.18) have optimized a repeated cold stress procedure, known as specific alternation of rhythm in temperature (SART), by which various pathological symptoms resulting from autonomic imbalance can be induced in rodents. SART-stressed animals show depressive and anxiety symptoms,19,20) hyperalgesia,21) hypotension,22) abnormal hemostasis,23) etc. Recent studies have shown that the mouse exposed to repeated cold stress according to the essentially same protocol with minor modifications is useful as an experimental model of fibromyalgia.24,25) It is to be noted that SART-stressed animals reveal remarkable increase in plasma noradrenaline levels.26) Given evidence that chronic stress can alter the susceptibility of the mammalian body including the brain to acute stress,27,28) we hypothesized that SART-stressed animals might show distinct responses to different types of acute stress. We thus examined whether SART stress for a week alters the acute restraint stress-induced hyperthermia in mice.

MATERIALS AND METHODS

Experimental Animals

Male ddY mice (4–5 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan) or Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan). The animals were used in accordance with ethical procedures following the guidelines for the care and use of laboratory animals issued by the Japanese government and Japanese Pharmacological Society. All procedures employed in the present study were approved by the Committee for the Care and Use of Laboratory Animals at Kindai University. The animals were housed in a room maintained at 23–25°C under a 12-h light/dark cycle (light phage, 08:00–20:00) and had free access to food (MF, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water.

Procedure for SART Stress Loading

Mice were exposed to SART stress, as reported previously.18,23) The mice were moved at 1-h intervals between two cages; one was prepared in a room maintained at 24°C and the other was in a room at 4°C room during the period from 09:00 to 16:00, and kept in the cage in the 4°C room during the period from 16:00 to 09:00 overnight. These stress loading procedures were repeated for 7 d. On the final day, the animals were kept in the 24°C room for 1 h and then in the 4°C room for 1 h during 09:00–11:00, and used for experiments in the 24°C room at least 30 min after the final movement.

Exposure to Acute Restraint Stress

A plastic cylinder designed to immobilize a mouse was used for exposure to restraint stress. The mouse was gently placed in the cylinder and kept for 20–60 min.

Measurement of Body Temperature

Rectal temperature was measured using a thermistor thermometer (Natsume Seisakusho Co., Ltd., Tokyo, Japan). A thermistor probe was inserted for a length of 2 cm into the rectum of a mouse, and held in the rectum for 20 s to determine stable rectal temperature. Before each experiment, baseline temperature was repeatedly measured in order to minimize the effect of handling stress on body temperature.

Determination of Corticosterone Levels in the Serum

Immediately after exposure to restraint stress, the mouse was anesthetized deeply with isoflurane, and blood was collected from the right atrium. The blood was allowed to stand for 30 min at room temperature, and centrifuged at 6000 rpm for 10 min. The supernatant was frozen and stored at −80°C. Serum corticosterone levels were determined using a Corticosterone EIA Kit (Cayman Chemical Company, MI, U.S.A.). According to the manufacturer’s instructions, the absorbance was detected at 412 nm using an automated plate reader, and the corticosterone concentration in each sample was calculated on the basis of the standard curve.

Measurement of Tissue Weight and Western Blot Analysis of Uncoupling Protein 1 (UCP1) in the Brown Adipose Tissue

In the mice anesthetized with intraperitoneal (i.p.) administration of urethane at 1.5 g/kg, the interscapular brown adipose tissue was removed rapidly and snap frozen in liquid nitrogen. The tissue samples were homogenized in a radio immunoprecipitation assay (RIPA) buffer (phosphate buffered saline (PBS), 1% Igepal CA-630, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS)) containing 0.1 mg/mL phenylmethylsulfonyl fluoride (PMSF), 0.15 U/mL aprotinin and 1 mM sodium orthocanadate. After the addition of 2-mercaptoethanol and bromophenol blue, the supernatant was denatured at 95–100°C for 5 min. The protein samples were separated by electrophoresis on 12.5% SDS-polyacrylamide gels (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and transferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA, U.S.A.). The anti-UCP1 goat polyclonal antibody (1 : 200) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit antibody (1 : 5000) (Santa Cruz Biotechnology, CA, U.S.A.) were used as primary antibodies, and horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) (1 : 5000) (Cell Signaling Technology, Beverly, MA, U.S.A.) and anti-goat IgG (1 : 1000) (Chemicon International, CA, U.S.A.) antibodies were used as secondary antibodies. Positive bands were visualized with an enhanced chemiluminescence detection regent (Nacalai Tesque, Kyoto, Japan) and detected by Image Quant 400 (GE Healthcare, Buckinghamshire, U.K.). Positive bands were quantified using ImageJ.29)

Drug Administration

Diazepam and indomethacin were purchased from Wako Pure Chemical Industries, Ltd., and SR59230A ((2S)-1-(2-ethylphenoxy)-3-{[(1S)-1,2,3,4-tetrahydronaphthalen-1-yl]amino}propan-2-ol) was from Sigma-Aldrich (St. Louis, MO, U.S.A.). Mifepristone was obtained from TCI Chemicals (Osaka, Japan). Diazepam and mifepristone were suspended in 0.5% carboxymethyl cellulose sodium. Indomethacin was dissolved in 0.5% sodium bicarbonate, and SR59230A was in saline. Diazepam at 4 mg/kg and mifepristone 50 mg/kg were administered per os (p.o.) 60 min before the onset of restraint stress. Indomethacin at 5 mg/kg and SR59230A at 5 mg/kg were administered subcutaneously (s.c.) and i.p., respectively, 30 min before exposure to restraint stress.

Statistical Analysis

Data are represented as the mean with standard error of the mean (S.E.M.). Statistical significance was analyzed by Student’s t-test for comparison between 2 groups and by ANOVA followed by Tukey’s test for multiple comparisons. Significance was set at a level of p<0.05.

RESULTS

Acute Restraint Stress-Induced Hyperthermia in SART-Stressed Mice

Exposure to restraint stress for 20–60 min induced significantly greater hyperthermic responses in SART-stressed mice than unstressed mice (Fig. 1).

Fig. 1. Effect of SART Stress on Acute Restraint Stress-Induced Hyperthermia in Mice

Data show the mean with S.E.M. for 11–16 mice. * p<0.05, ** p<0.01, vs. the non-SART group.

Involvement of β3 Adrenoceptors in the SART Stress-Induced Enhancement of Acute Restraint Stress-Induced Hyperthermia in Mice

Considering the evidence for involvement of β3 adrenoceptors in the brown adipose tissue (BAT) thermogenesis triggered by the sympathetic nerve excitation,30) we examined the effect of SR59230A, a selective β3 adrenoceptor antagonist, on the stress-induced hyperthermia in mice. Interestingly, SR59230A, preadministered i.p. at 5 mg/kg, significantly attenuated the SART stress-induced enhancement of restraint stress-induced hyperthermia in mice (Fig. 2a). The weight of BAT and the protein level of uncoupling protein 1 (UCP1), a prototypic brown adipocytes marker, in BAT tended to increase after 1-week exposure to SART stress (Figs. 2b, c).

Fig. 2. Effect of SR59230A, a β3 Adrenoceptor Antagonist, on the Enhancement of Acute Restraint Stress-Induced Hyperthermia by SART Stress, and Protein Levels of UCP1 in the Brown Adipose Tissue (BAT) of SART-Stressed Mice

(a) The mice received a single i.p. administration of SR59230A at 5 mg/kg or vehicle 60 min before the onset of restraint stress. The acute restraint stress-induced hyperthermia (ΔT) is presented as the difference of the body temperature before and after restraint stress for 0, 20, 40 or 60 min. (b) and (c) BAT weight, the one relative to the body weight (b) and protein levels of UCP1 in the BAT tissue, as assessed by Western blotting (c) in SART-stressed mice. Typical photographs for Western blotting and quantified data by densitometry are shown by densitometry (c). Data show the mean with S.E.M. for 10–14 or 4–5 (b, c) mice. ** p<0.01 vs. the vehicle-treated, non-SART group; p<0.05, ††p<0.01 vs. the vehicle-treated, SART group.

Effect of SART Stress on the Secretion of Corticosterone Caused by Acute Restraint Stress in Mice

Exposure to SART stress for a week itself did not change serum corticosterone levels in mice, whereas SART-stressed mice exhibited significantly greater increase in serum corticosterone levels following 60-min exposure to restraint stress than unstressed mice (Fig. 3).

Fig. 3. Effect of SART Stress on the Secretion of Corticosterone in Response to Acute Restraint Stress in Mice

Blood was collected before and after 60-min restraint stress in mice. Data show the mean with S.E.M. for 12–22 mice. ** p<0.01 vs. the non-SART group.

Lack of Involvement of Glucocorticoid Receptor Activation and Increased Anxiety on the SART Stress-Induced Increase in Hyperthermic Responses to Acute Restraint Stress

Although SART stress augmented the acute restraint stress-induced corticosterone secretion (see Fig. 3), the glucocorticoid receptor antagonist, mifepristone, when preadministered orally at 50 mg/kg, did not affect the elevated hypertherma in responses to acute restraint stress in SART-stressed mice (Fig. 4a). Despite the evidence that SART-stressed animals exhibit anxiety-related behavioral characteristics,31,32) oral preadministration of the anxiolytic, diazepam, at 4 mg/kg failed to inhibit the SART stress-evoked increase in hyperthermic response to acute restraint stress in mice (Fig. 4b).

Fig. 4. Influence of Mifepristone, a Glucocorticoid Receptor Antagonist, and Diazepam, Anxiolytic, on the Enhancement of Acute Restraint Stress-Induced Hyperthermia in SART-Stressed Mice

The mice received a single oral administration of mifepristone at 50 mg/kg (a) or diazepam at 4 mg/kg (b) 1 h before 20-min restraint stress. The acute restraint stress-induced hyperthermia (ΔT) is presented as the difference of the body temperature before and after 20-min restraint stress. Data show the mean with S.E.M. for 6–9 mice. * p<0.05, vs. the vehicle-treated, non-SART group.

The Increased Hyperthermia in Response to Acute Restraint Stress in SART-Stressed Mice Is Independent of Endogenous Prostanoid Generation in Mice

Although prostaglandin E2 plays roles in infectious fever development and also in stress responses,33) indomethacin, a cyclooxygenase (COX) inhibitor, when preadministered s.c. at 5 mg/kg, did not significantly affect the acute restraint stress-induced hyperthermia in SART-stressed mice (Fig. 5).

Fig. 5. Influence of Indomethacin, a Cyclooxygenase Inhibitor, on the Enhancement of Acute Restraint Stress-Induced Hyperthermia in SART-Stressed Mice

The mice received a single s.c. administration of indomethacin at 5 mg/kg or vehicle before the onset of restraint stress. The acute restraint stress-induced hyperthermia (ΔT) is presented as the difference of the body temperature before and after restraint stress for 0, 20, 40 or 60 min. Data show the mean with S.E.M. for 7–9 mice. * p<0.05, ** p<0.01 vs. the vehicle-treated, non-SART group.

DISCUSSION

The present findings demonstrate that one-week exposure to SART stress enhances the susceptibility to acute restraint stress in mice, characterized by the hyperthermic responses and augmented corticosterone secretion. Our data also suggest that the β3 adrenoceptor-mediated sympathetic nerve/non-shivering thermogenesis in the brown adipose tissue may play a key role in the facilitation of restraint stress-induced hyperthermia by SART stress.

The neuronal pathways from the dorsomedial hypothalamus (DMH) to the rostral medullary raphe region (rMR) regulate shivering thermogenesis in skeletal muscles, non-shivering thermogenesis in brown adipose tissue and cutaneous vasomotion. The GABAergic inhibitory neurons in the hypothalamic medial preoptic area (MPO) project to the DMH-rMR thermoregulatory neurons. Prostaglandin E2 (PGE2) inhibits the GABAergic neurons in MPO through EP3 receptors, and consequently enhances the activity of the DMH-rMR thermoregulatory neurons, contributing to infectious fever.3436) The excitation of the DMH-rMR pathways appears to mediate the psychological stress-induced thermogenesis in brown adipose tissue and cardiovascular responses.37) EP3 receptors contribute to infectious fever but not stress-induced hyperthermia, because EP3 receptor-deficient mice show psychological stress-induced hyperthermia, but do not exhibit lipopolysaccharide (LPS)-induced fever.5) The prostanoid-independence of stress-induced hyperthermia is consistent to our finding that the inhibition of COX by indomethacin did not affect the SART stress-induced enhancement of acute stress-induced hyperthermia (see Fig. 5). There is evidence that stress-induced hyperthermia is associated with the level of anxiety1) and that SART-stressed animals have elevated anxiety levels.31,32) Nonetheless, the anxiolytic, diazepam, failed to reduce the enhanced hyperthermic responses to acute restraint stress in SART-stressed mice, suggesting that the increased anxiety levels are not involved in the increased hyperthermic responses.

It has been reported that social defeat stress-induced hyperthermia is suppressed by β3 adrenoceptor blockade,14) and that the repetitive intermittent immobilization stress enhances the extent of noradrenaline-induced increase in the temperature in brown adipose tissue.38) Our data that the facilitation of acute restraint stress-induced hyperthermia by SART stress was attenuated by β3 adrenoceptor blockade suggest that the sympathetic nerve-mediated, β3 adrenoceptor-dependent, non-shivering thermogenesis in the brown adipose may be functionally upregulated by SART stress for one week. This is consistent to the slight increase in the weight of BAT and the protein levels of UCP1 in the BAT (see Figs. 2b, c), and the previous report that SART-stressed animals show increased plasma noradrenaline levels.26) In the present study, we used relatively young mice to be exposed to SART stress, according to the previous studies.19,20,31,32) It would be interesting to study the changes in SART stress-induced physiological responses depending on the growth and/or aging.

It is well known that glucocorticoids inhibit the induction of COX-2 and subsequent PGE2 production39) that contributes to infectious fever,3436,40) whereas there is no evidence that glucocorticoids are associated with stress-induced hyperthermia. Considering the lack of inhibitory effect of the glucocorticoid receptor antagonist, mifepristone (see Fig. 4a), the enhancement of acute restraint stress-induced hyperthermia in SART-stressed mice is independent of the increased secretion of corticosterone in response to the acute restraint stress in SART-stressed mice (see Fig. 3).

That exposure to SART stress for one week itself did not increase serum corticosterone levels but enhanced the increase in serum corticosterone levels following acute restraint stress in mice (see Fig. 3) is in agreement with the previous evidence that repeated or chronic exposure to stress easily leads to habituation in hypothalamic–pituitary–adrenal (HPA) axis41,42) but may enhance HPA responses to a novel stressor.43,44) The HPA sensitization after chronic stress including SART stress may be associated with a reduced efficacy of negative glucocorticoid feedback45) and involve neurochemical alterations including extracellular signal-regulated kinase (ERK) activation in the hippocampus.46)

In conclusion, our data suggest that SART stress enhances the acute restraint stress-induced, β3-adrenocepor-dependent thermogenesis in mice, which is independent of endogenous prostanoids, glucocortigoids or increased anxiety levels.

Acknowledgment

This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan-Supported Program for the Strategic Research Foundation at Private Universities (2014–2018).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2017 The Pharmaceutical Society of Japan
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