Cold-stressed Mice Research Articles

Evaluation of the antioxidant peptide SS31 for treatment of burn-induced insulin resistance

After severe burn injury and other major traumas, glucose tolerance tests demonstrate delayed glucose disposal. This 'diabetes of injury' could be explained by insulin deficiency, and several studies have shown that soon after trauma (ebb phase) insulin concentrations are reduced in the face of hyperglycemia. After resuscitation of trauma patients (flow phase), β-cell responsiveness normalizes and plasma insulin levels are appropriate or even higher than expected, however, glucose intolerance and hyperglycemia persist. In the acute care setting, several approaches have been used for treating insulin resistance, including insulin infusion, propranolol and glucagon-like-peptide-1 (GLP-1). Recently, it was demonstrated that a tetrapeptide with antioxidant properties D-Arg-Dmt-Lys-Phe-NH2 (SS31), but not its inactive analogue Phe-D-Arg-Phe-Lys-NH2 (SS20) attenuates insulin resistance in mice maintained on a high fat diet. In this report the effects of SS31 and SS20 on burn-induced insulin resistance was studied in mice. Oral glucose tolerance tests (OGTT) were performed in 4 groups of 6 mice with thermal injury with or without pre-treatment with SS31 or SS20 and sham controls. In addition, biodistribution of 18FDG was measured in burned mice with and without SS31 treatment and shams (subsets of these animals were also studied by µPET). For comparison purposes, groups of 6 cold-stressed mice with and without SS31 treatment were also studied. The results of these studies demonstrate that SS31 but not SS20 ameliorated burn-induced insulin resistance. In addition, SS31 treatment resulted in marked reduction in the increased 18FDG uptake by brown adipose tissue (BAT) in burned but not cold-stressed animals; suggesting that the stressors act by different mechanisms. Overall, these studies confirmed that SS31 can be used to reverse burn-induced insulin resistance and provide a firm pre-clinical basis for future clinical trials of SS31 for the treatment of insulin resistance in patients with burn injury.

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue

Aims/hypothesisIncreasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues.MethodsMethylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding.ResultsWe show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter–reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment.Conclusions/interpretationThe results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-010-1701-4) contains supplementary material, which is available to authorised users.

Specific Alternation of Rhythm in Temperature (SART) Stress-Induced Irritable Bowel Syndrome-Like Changes in Mice and Effects of Drugs

Stress is closely associated with the manifestation and progress of irritable bowel syndrome (IBS). For the purpose of establishing experimentally the relationship between IBS and stress, the transportation capacity of the small intestine in specific alternation of rhythm in temperature (SART)-stressed animals was studied using charcoal transportation method. The charcoal suspension was administered orally into the stomach of fasting mice. Mice were sacrificed after a certain time and %charcoal transit (%CT) of the small intestine was measured. The %CTs in SART-stressed mice were greater than those in unstressed or continuously cold-stressed mice. This increase in %CT remained for 1 week after discontinuation of SART stress loading. Cholinergic blockers decreased %CTs in SART-stressed mice. Increases in %CT by a cholinesterase inhibitor were less in SART-stressed mice than in unstressed mice. Increases of %CT in SART-stressed mice were suppressed by Neurotropine. These results suggested that the parasympathetic hypertonicity, not just cold, played a role in the increases in the transportation capacity in SART-stressed mice and that these animals can be a useful tool for elucidation of the mechanism of IBS.

Processed Aconite Root Prevents Cold-Stress-Induced Hypothermia and Immuno-Suppression in Mice

Processed aconite root (PA) is a crude drug used in traditional Chinese or Japanese medicine to generate heat in interior body and dispel cold. We evaluated the effects of PA on hypothermia and reduction in the activity of natural killer (NK) cells in mice exposed to chronic cold stress. Male mice were reared at 4 degrees C, and powdered PA was administered for 10 d as a food additive. Core body temperature of mice significantly decreased by approximately 1 degrees C after rearing in a cold environment, and PA administration significantly restored the reduction in core body temperature in a dose-dependent manner. After 10 d, splenic NK-cell activity of cold-stressed mice was significantly reduced, and the reduction was dose-dependently recovered by PA administration. An aconitine-type alkaloid fraction prepared from PA was ineffective when administered to cold-stressed mice, and the thermogenic effect on hypothermic mice was present in the fraction containing low-molecular-weight compounds without alkaloids. In cold-stressed mice, the weight of brown adipose tissue (BAT) and uncoupling protein (UCP)-1 level in BAT increased, whereas the weight of white adipose tissue decreased. The increase in UCP-1 level in BAT of cold-stressed mice was further augmented by PA treatment. These results indicate that PA exhibited a thermogenic effect on hypothermia induced by cold stress in mice by additional upregulation of UCP-1 level in BAT, which was already enhanced by hypothermia, and that the active ingredients present in PA are non-alkaloidal low-molecular-weight compounds.

4.P4. Selection for high basal metabolic rate compromises immune response in cold-stressed laboratory mice

4.P4. Selection for high basal metabolic rate compromises immune response in cold-stressed laboratory mice

ELOVL3 Is an Important Component for Early Onset of Lipid Recruitment in Brown Adipose Tissue

During the recruitment process of brown adipose tissue, the mRNA level of the fatty acyl chain elongase Elovl3 is elevated more than 200-fold in cold-stressed mice. We have obtained Elovl3-ablated mice and report here that, although cold-acclimated Elovl3-ablated mice experienced an increased heat loss due to impaired skin barrier, they were unable to hyperrecruit their brown adipose tissue. Instead, they used muscle shivering in order to maintain body temperature. Lack of Elovl3 resulted in a transient decrease in the capacity to elongate saturated fatty acyl-CoAs into very long chain fatty acids, concomitantly with the occurrence of reduced levels of arachidic acid (C20:0) and behenic acid (C22:0) in brown adipose tissue during the initial cold stress. This effect on very long chain fatty acid synthesis could be illustrated as a decrease in the condensation activity of the elongation enzyme. In addition, warm-acclimated Elovl3-ablated mice showed diminished ability to accumulate fat and reduced metabolic capacity within the brown fat cells. This points to ELOVL3 as an important regulator of endogenous synthesis of saturated very long chain fatty acids and triglyceride formation in brown adipose tissue during the early phase of the tissue recruitment.

Stress- and aging-associated modulation of macrophage functions

Effects of environmental (cold) stress and aging on cells in monocyte/macrophage lineage were investigated. We demonstrated that immune suppressive states seen in acute cold-stressed mice (8-10 weeks of age) is attributable to FcγRII(bright) suppressor macrophages. Serum corticosterone levels were markedly increased in acute cold-stressed mice. In addition, expression of glucocorticoids (GC) receptor mRNA was observed in FcγRII(bright) cells from these mice. The increase of FcγRII(bright) cells in peritoneal exudate cells caused by acute cold stress was inhibited by adrenalectomy or administration of a saturating amount of the GC antagonist RU 38486 (mifepristone). On the contrary, administration of the GC agonist, dexamethasone, markedly increased the proportion of FcγRII(bright) cells in peritoneal exudate cells of control mice. These results suggest that the generation of FcγRII(bright) suppressor cells of monocyte/macrophage lineage by acute cold stress was mediated by action of GC through the GC receptor. We likewise found that the proportion of FcγRII(bright) suppressor macrophages is increased in aged mice (22-24 months of age). Meanwhile, activated macrophages which function as antigen presenting cells were decreased in aged rats. Both the basal corticosterone concentrations in serum and the expression of mRNA for GC receptor in peritoneal macrophages increased significantly in aged animals, suggesting that these populational and functional changes of macrophages in aged animals were mediated, in part, by the increased basal levels of GC. This is probably being responsible for immunosenescence.

Stress- and Aging-Associated Modulation of Macrophage Functions.

Effects of environmental (cold) stress and aging on cells in monocyte/macrophage lineage were investigated. We demonstrated that immune suppressive states seen in acute cold-stressed mice (8–10 weeks of age) is attributable to FcγRIIbright suppressor macrophages. Serum corticosterone levels were markedly increased in acute cold-stressed mice. In addition, expression of glucocorticoids (GC) receptor mRNA was observed in FcγRIIbright cells from these mice. The increase of FcγRIIbright cells in peritoneal exudate cells caused by acute cold stress was inhibited by adrenalectomy or administration of a saturating amount of the GC antagonist RU 38486 (mifepristone). On the contrary, administration of the GC agonist, dexamethasone, markedly increased the proportion of FcγRIIbright cells in peritoneal exudate cells of control mice. These results suggest that the generation of FcγRIIbright suppressor cells of monocyte/macrophage lineage by acute cold stress was mediated by action of GC through the GC receptor. We likewise found that the proportion of FcγRIIbright suppressor macrophages is increased in aged mice (22–24 months of age). Meanwhile, activated macrophages which function as antigen presenting cells were decreased in aged rats. Both the basal corticosterone concentrations in serum and the expression of mRNA for GC receptor in peritoneal macrophages increased significantly in aged animals, suggesting that these populational and functional changes of macrophages in aged animals were mediated, in part, by the increased basal levels of GC. This is probably being responsible for immunosenescence.

Immunomodulatory Effects of Cold Stress on Mice Infected Intraperitoneally with a 50% Lethal Dose of Toxoplasma gondii

Cofactors such as stress have been suspected to play a role in the susceptibility to opportunistic infections. Toxoplasma gondii is one of the major opportunistic infectious agents in immunocompromised individuals, and infection can be modulated by external factors such as stress. Objective: The purpose of this study was to examine the in vivo and in vitro role of cold stress (CS) in the pathogenesis of T. gondii infection and its impact on regulatory cytokines in this model. Methods: Mice subjected to CS and control animals were infected intraperitoneally with an LD₅₀ of PD2 T. gondii tachyzoites, and the outcome of the infection was determined. In addition, peritoneal macrophages obtained from CS and non-stressed mice were infected in vitro with T. gondii. The number of infected macrophages, the number of intracellular parasites and the production of interferon (IFN)-γ, interleukin (IL)-12, and tumor necrosis factor (TNF)-α were determined. Results: CS applied before intraperitoneal inoculation increased susceptibility against T. gondii infection. Peritoneal cells from CS mice contained significantly higher numbers of intracellular parasites and infected macrophages compared to those from non-stressed animals. IFN-γ production was initially high in the CS group but decreased significantly after 36 h. Opposite results were found in the non-stressed group. Macrophages from CS mice persistently produced high levels of TNF-α and IL-12 and peaked after 36 h. Levels of these cytokines were lower or absent in the non-stressed group. Conclusion: These results suggest that CS increased the host susceptibility to intraperitoneal T. gondii infection by modulating the function of macrophages and the production of cytokines (IFN-γ) involved in the early control of infection.

Activation and Apoptosis of Murine Peritoneal Macrophages by Acute Cold Stress

Effects of acute cold stress (5°C for 24 h) on the functions of peritoneal macrophages and the mechanisms for controlling host homeostasis were investigated in mice. Phagocytic activity and expression of the cell surface adhesion molecule CD11b/CD18 were markedly increased in peritoneal exudate cells by acute cold stress. These alterations were attributable to an increased number and phenotypical changes of adherent cells from acute cold-stressed mice. On the other hand, a lipopolysaccharide-induced activity of src-family tyrosine kinase Fgr, an expression of interleukin-1β (IL-1 β) mRNA, and a bioactivity of IL-1 in the culture supernatants of adherent cells from acute cold-stressed mice were markedly lower than those from control mice. A time course study revealed that the number of adherent cells in peritoneal exudate cells was markedly increased in mice exposed to cold for 24 h but returned to normal numbers when mice were exposed to cold for 72 h. DNA fragmentation and Annexin-V+ cells were observed in peritoneal exudate cells from acute-cold stressed mice. Thus, cold stress activated macrophages but these macrophages were destined to be eliminated by apoptosis.

Anxiety-Like Behavior in Elevated Plus-Maze Tests in Repeatedly Cold-Stressed Mice

To clarify the relationship between SART (specific alternation of rhythm in temperature) stress (repeated cold stress) and anxiety, the effects of various types of stress on the behavior of mice were studied in elevated plus-maze tests and then the effects of anxiolytics were evaluated. The percentage of time spent in the open arms of the plus-maze apparatus decreased in mice subjected to SART stress without change in the total number of arm entries. No change was noted in mice subjected to other stresses, such as 1-h, 2-day and 5-day cold stress and 1-h, 15-h and 5 χ 15-h restraint stress. The reduction in the percentage of time spent in the open arms caused by SART stress was inhibited by single and repeated administrations of diazepam and alprazolam and by a single administration of buspirone, which have no influence on the percentage of time spent in the open arms in nonstressed mice, but not by flumazenil, WAY-100635 and chronic treatment with buspirone. The effects of diazepam and buspirone were antagonized by flumazenil and WAY-100635, respectively. The behavior of SART-stressed mice in the plus-maze would thus appear to arise from anxiety, to which benzodiazepine and serotonin receptors are related, but the diazepam binding inhibitor, an endogenous anxiogenic protein, is not. Thus SART-stressed animals may be useful for investigating the psychopharmacological and neuropharmacological basis of anxiety.

Depressive State With Anxiety in Repeated Cold-Stressed Mice in Forced Swimming Tests

The effects of various types of stress and drugs were studied to assess mouse performance in forced swimming tests, following characterization of SART (specific alternation of rhythm in environmental temperature) stress. Immobility time in the test decreased in mice subjected to SART, acute cold and restraint stress. No change was noted due to chronic cold stress or repeated fasting. The shortened time did not recover even 24 hr after the end of SART and chronic restraint stress. The time in SART-stressed mice finally recovered at 5–7 days. Shortening of immobility time in SART-stressed mice was inhibited by diazepam and repeated imipramine but not by lithium carbonate. In chronic restraint-stressed mice, this time was inhibited by repeated lithium carbonate but not diazepam or imipramine. SART stress would thus appear related to anxiety and depression and may be useful for detecting new types of antidepressants.

Gastric ulcer formation in cold-stressed mice related to a central calcium-dependent-dopamine synthesizing system

Stress ulceration of the stomach in mice was investigated from the aspect of the calcium/calmodulin-dependent dopamine synthesizing system in the brain. Cold stress was induced in mice by restraining them at 4°C. Serum and brain calcium levels were increased by cold stress, and an increased brain calcium level was found to enhance dopamine synthesis and a successively increased brain dopamine level induced gastric ulcer formation. Development of gastric ulcers elicited by cold stress was significantly decreased by i.p. pretreatment with EDTA (1 μmol/mouse, 1 h before restraint) or α-methyltyrosine (a tyrosine hydroxylase inhibitor, 100 mg/kg, 24 h before restraint), and was further significantly increased by pretreatment with CaCl 2 (40 μmol/kg, 1 h before restraint). These findings suggest that the development of gastric ulcers in cold-stressed mice may be linked with the enhancement of calcium/calmodulin-dependent catecholamine synthesis in the brain.

Glucocorticoid-mediated generation of suppressor macrophages with high density Fc gamma RII during acute cold stress.

Acute cold stress induces adherent cells with suppressor function, resulting in immunosuppression. Glucocorticoids (GC) are known as an inhibitor of the immune system and are increased by stress through stimulation of the hypothalamic-pituitary-adrenal axis. To elucidate the mechanisms underlying acute cold stress-induced immunosuppression, functions and surface phenotypes of murine peritoneal cells of the monocyte/macrophage lineage from acute cold-stressed mice (5 C for 3 or 24 h) in addition to the role of GC in the immunomodulation were investigated. Flow cytometric analysis revealed that the proportion of MAC-1+ cells with a high density of Fc gamma RII (Fc gamma RIIbright cells) was markedly increased in the peritoneal exudate cells from acute cold-stressed mice. These Fc gamma RIIbright cells were also stained with F4/80, a monoclonal antibody directed specifically against the mouse mature macrophages. The prominent suppressor activity for Concanavalin A (Con A) responses of control spleen cells was found in Fc gamma RIIbright cells, whereas MAC-1+ cells, with a low density of Fc gamma RII (Fc gamma RIIdull cells), from the stressed mice did not suppress the Con A responses. Fc gamma RIIbright cells from control mice also suppressed the Con A responses; the inhibitory effect was considerably less than that of cells from acute cold-stressed mice. As was anticipated, serum corticosterone levels were markedly increased in acute cold-stressed mice. In addition, expression of GC receptor messenger RNA was observed in Fc gamma RIIbright cells from these mice. The increase in Fc gamma RIIbright cells in peritoneal exudate cells caused by acute cold stress was inhibited by adrenalectomy or administration of a saturating amount of the GC antagonist RU 38486 (mifepristone). On the contrary, administration of the GC agonist, dexamethasone, markedly increased the proportion of Fc gamma RIIbright cells in peritoneal exudate cells of control mice. These results suggest that the generation of Fc gamma RIIbright suppressor cells of monocyte/macrophage lineage by acute cold stress was mediated to a greater or lesser degree by the action of GC through the GC receptor.

Cold stress-induced neuroinvasiveness of attenuated arboviruses is not solely mediated by corticosterone.

In previous studies we have shown that various stress paradigms can induce the penetration of noninvasive, attenuated viruses into the central nervous system (CNS). Since glucocorticoids levels are elevated during stress, we compared the effect of cold stress and corticosterone (CS) injection on neuroinvasiveness of a non-invasive encephalitic virus, WN-25 (West Nile). Exposure of inoculated mice to cold stress or CS resulted in high viremia and a marked increase in mortality when compared to control untreated mice. Exposure of WN-25 inoculated mice to cold treatment or CS injection led to high blood virus levels as compared to nontreated mice (3.2 and 3.1 vs > 1 log 10 PFU/ml). Cold stress or CS (5000 ng/mouse) treatment caused a mortality rate of 70% and 50% of the WN-25 inoculated mice respectively. No mortality was recorded in control inoculated groups (p < 0.05). Passive transfer serum from uninfected cold stressed mice to WN-25 inoculated nonstressed mice, resulted in similar mortality. The levels of CS in passive transferred serum from cold stressed animals was 500 ng/ml, only 2% (100 vs. 5000 ng) of the CS dose required to obtain a similar effect on viral penetration and mortality when CS was injected directly. Therefore, we concluded that CS was not the sole factor responsible for the cold stress effect on the viral infection outcome.

Acute cold stress induces suppressor macrophages in mice.

To elucidate mechanisms underlying acute cold stress-induced immunosuppression, functions of murine peritoneal cells of monocyte/ macrophage lineage from acute cold-stressed mice (exposed to 5 degrees C for 24 h) were investigated. Proliferative responses of spleen cells from control mice (reared at 25 degrees C) stimulated with concanavalin A (ConA) were significantly suppressed by adding peritoneal exudate cells from mice immediately after acute cold stress. The proportion of adherent cells was markedly increased in the peritoneal exudate cells from acute cold-stressed mice. These adherent cells from acute cold-stressed mice were shown to be the cells responsible for the suppressor activity for ConA responses of control spleen cells. Nonadherent cells did not suppress the ConA responses. The adherent cells in peritoneal exudate cells from control mice also suppressed the ConA responses, the inhibitory effect being considerably lower than that from acute cold-stressed mice. Addition of a nitric oxide synthase substrate analogue, NG-monomethyl-L-arginine, to the mixed cell cultures of normal spleen cells and adherent cells from acute cold-stressed mice inhibited nitric oxide release and completely abolished the suppressive effect of the adherent cells, suggesting that reactive nitrogen oxide released from the activated macrophages is apparently involved in the downregulation of proliferative responses of T cells. Thus the present findings suggest that acute cold stress induces macrophages with suppressor function and that this may contribute to the immune-suppressive state seen in spleen cells from acute cold-stressed mice.

Thermal stresses reduce natural killer cell cytotoxicity

The effects of different ambient temperatures (Ta) on the splenic natural killer (NK) cell activity, effector-target cell conjugation activity, and NK cell numbers were assessed in male inbred C3H/HeNCrj mice (7-10 wk old). The splenic NK cytotoxic activities were examined in a 4-h 51Cr release assay in mouse spleen cells that were obtained 1, 2, 4, 8, or 16 days after exposure to Ta of 22, 4, or 35 degrees C. The percentage of conjugating lymphocytes was calculated by counting the number of single lymphocytes bound to single target cells per 400 effector cells. The numbers of NK cells were expressed by the percentage of 5E6-positive cells. The 5E6 identifies only a subset of NK cells. It was found that the splenic NK cell activity, the effector-target cell conjugation activity, or the NK cell number began to fall 1 day after cold (Ta 4 degrees C) or heat (Ta 35 degrees C) stress. After a 16-day period of either cold or heat exposure, the fall in the splenic NK cell activity, the effector-target cell conjugation activity, or the number of 5E6-positive subsets of NK cells was still evident. Compared with those of the control group (Ta 22 degrees C), the cold-stressed mice had higher adrenal cortisol concentration and lower colonic temperature, whereas the heat-stressed animals had higher adrenal cortisol concentration and higher colonic temperature during a 16-day period of thermal exposure. However, neither cold nor heat stress affected both the body weight gain and the spleen weight in our mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Peak Sustained Metabolic Rate and Its Individual Variation in Cold-Stressed Mice

Is long-term sustained metabolic rate (SusMR) subject to a ceiling value? If so, what physiological or evolutionary factors impose that ceiling, and is the ceiling value independent of the type of energy stress? We determined food intake and daily digestible energy intake (DEI) as measures of SusMR, and resting metabolic rate (RMR), in mice that were energy stressed by being maintained at cold ambient temperatures. We compared these values with those from previous studies of mice that were energy stressed by peak lactation. We also measured intestinal brush-border nutrient uptakes, body lean and fat masses, and masses of the small intestine, heart, kidneys, and liver. Even with ad lib. quantities of a high-fat diet available, mice could not survive at temperatures below -15° C. Body mass declined at low temperatures because of depletion of body fat reserves. Food intake increased 2.5-fold, RMR 1.5-fold, and masses of the small intestine, heart, and kidneys by 30%-70% with a decrease in temperature from 23° C to -15° C. Intestinal hypertrophy served to restore the reserve capacity of intestinal nutrient transporters that would otherwise have been swamped by the increased food intake. The values reached by sustained metabolic scope (SusMR/RMR), food intake, and intestinal mass were still considerably below those of lactating mice, even though mice at temperatures below -15° C died in the presence of excess food. Thus, intestinal capacity was not the ultimate reason for an inability to survive at lower temperatures. Analysis of individual variation showed that those mice with unusually high food intake, DEI, or RMR tended to have unusually large hearts, kidneys, and intestines. Those organs are essentialfor high energy budgets (reflected in high DEIs), but they also incur large maintenance costs themselves (reflected in high RMRs).

Dehydroepiandrosterone protects mice inoculated with West Nile virus and exposed to cold stress.

The protective effect of pretreatment with dehydroepiandrosterone (DHEA) on stress-enhanced viral encephalitis was studied in mice exposed to cold following inoculation with West Nile virus (WNV). Exposure of WNV-inoculated mice to cold water (1 +/- 0.5 degrees C, 5 minutes/day for 8 days) resulted in a mortality rate of 83% as compared to 50% in nonstressed mice (p < 0.05). The effect of cold stress was more pronounced when mice were inoculated with WN-25, a noninvasive neurovirulent variant of WNV. Mice infected with WN-25 showed no mortality, whereas cold stressed mice inoculated with the same virus had a mortality rate of 67% (p < 0.05). The administration of DHEA (serial injections of 10-20 mg/kg with or without a loading dose of 1 gm/kg) resulted in a significant reduction in the mortality rate of stressed mice inoculated with either virus (p < 0.05). Virus levels in the blood and brain of the DHEA-treated mice, were significantly lower than in the control groups. DHEA also prevented the involution of lymphoid organs in stressed mice. The present study provides direct evidence of the protective effects of DHEA as an "anti-stress" agent. Its ability to prevent mortality associated with WNV or WN-25, and involution of lymphoid organs caused by stress-induced immunosuppression, supports the notion that its activity is based on the modulation of the host response.

Behavioral changes in cold-stressed mice related to a central calcium-dependent-catecholamine synthesizing system

An investigation was carried out regarding the mechanism of behavioral changes in mice elicited by cold stress. Cold stress was induced in adult male mice by restraining them from free action for 2 h at 4°C. As the control test, mice were restrained from free action for 2 h at room temperature. The locomotor counts in cold-stressed mice were found to be lower than in controls. The counts in cold-stressed mice were increased by IP pretreatment with EDTA or α-methyltyrosine (tyrosine hydroxylase inhibitor), and were further decreased by IP pretreatment with CaCl 2. On the other hand, serum calcium and brain calcium levels in cold-stressed mice were increased 15–30 min and 30 min, respectively, after restraint under cold temperatures, and returned to original levels 1 h after restraint. Also, the biochemical and immunohistochemical brain dopamine levels in cold-stressed mice were higher than in control mice. The increment of brain dopamine levels in the control mice was also observed by the administration of CaCl 2. Futhermore, the ability of cold stress to enhance the dopamine level in mice brains was attenuated by IP pretreatment with α-methyltyrosine. In light of previous reports that central calcium activates catecholamine-synthesizing enzymes via a calmodulin-dependent system, it is suggested that cold stress enhances the brain calcium level, and then increased calcium enhances dopamine synthesis in the brain through a central calcium-dependent catecholamine synthesizing system. Subsequently, increased dopamine induces behavioral changes.