Restraint-stressed Mice Research Articles

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concomitantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling.

Sex differences in the effects of housing conditions in the behavior of restraint-stressed mice on the elevated plus-maze test

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Enhanced Resistance of Restraint-Stressed Mice to Sepsis

Sepsis remains a major health concern across the world. The effects of stress on host resistance to sepsis are still not very clear. To explore the effects of chronic stress on sepsis(') we examined the impact of restraint stress on the resistance of mice to sepsis. Interestingly, it was found that restraint stress enhanced the antisepsis resistance of mice and the concentrations of the proinflammatory cytokines IL-1, IL-6, IL-12, and TNF-alpha in the blood of stressed mice were dramatically reduced post Escherichia coli infection or LPS treatment as compared with that of controls (p < 0.05). In addition, the mRNA expressions of glucocorticoid-induced leucine zipper (GILZ) were up-regulated in the spleen and peritoneal macrophages of mice receiving restraint stress or dexamethasone treatment. These results demonstrate that restraint stress enhances the resistance of mice to sepsis, supporting corticotherapy for sepsis and proposing restraint-stressed mouse as an animal model to elucidate mechanisms of stress-associated, antisepsis resistance.

Microglial cells from psychologically stressed mice as an accelerator of cerebral cryptococcosis

Severe stress decreases the resistance of hosts exposed to microbial infections. As compared with two groups of control mice (normal mice, food-and-water-deprived mice [FWD mice]), restraint-stressed mice (RST mice) were shown to be greatly susceptible to intracerebral growth of Cryptococcus neoformans. The susceptibility of FWD mice to cerebral cryptococcosis increased to the level shown in RST mice, when these groups of mice were inoculated with microglial cells from the brains of RST mice. However, the susceptibility of FWD mice to cerebral cryptococcosis was not influenced by the adoptive transfer of microglial cells from normal mice or FWD mice. Microglial cells from RST mice produced CC-chemokine ligand-2 (CCL-2/monocyte chemoattractant protein 1), but not microglial cells from FWD mice. The resistance of RST mice to cerebral cryptococcosis was improved to the extent shown in FWD mice, when they were treated with anti-CCL-2 antibody. However, the susceptibility of normal mice and FWD mice to cerebral cryptococcosis increased to that shown in RST mice, when they were treated with rCCL-2. Microglial cells from RST mice were discriminated from the same cell preparations derived from FWD mice by their abilities to produce CCL-2, to phagocytize C. neoformans cells and to express Toll-like receptor 2. These results indicate that the resistance of RST mice to cerebral cryptococcosis is diminished by CCL-2 produced by microglial cells that are influenced by restraint stress.

Morphine delays wound healing in restraint stressed mice

Morphine delays wound healing in restraint stressed mice

Acute restraint stress enhances calcium mobilization and proliferative response in splenic lymphocytes from mice

Calcium (Ca2+ ) plays an essential role in lymphocyte activation and maturation. Acute and chronic stress has been shown to modulate the lymphocyte immune response; but the relationship between cytosolic free Ca2+ concentration ([Ca2+ ]i) and the immune response in lymphocytes following exposure to stress has not been examined. In the present study, we investigated the effects of acute restraint stress on [Ca2+ ]i and the proliferation of splenic lymphocytes from mice. We observed that 2 h of restraint significantly increased plasma corticosterone levels in mice. On examining [Ca2+ ]i and the proliferation ex vivo of splenic lymphocytes isolated from restraint-stressed mice using fura-2 and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, respectively, we found that acute restraint stress caused a significant increase in resting [Ca2+ ]i and significantly enhanced the ability of concanavalin A (Con A; a T-cell-selective mitogen) to increase [Ca2+ ]i but not that of lipopolysaccharide (LPS; a B-cell-selective mitogen). In addition, acute restraint stress significantly enhanced Con A-stimulated but not LPS-stimulated lymphocyte proliferation. Overall, there was a positive correlation between [Ca2+ ]i and T-cell proliferation following acute restraint stress. The enhancements of [Ca2+ ]i and T-cell proliferation were completely suppressed by verapamil (a Ca2+ channel blocker). These results suggest that acute restraint stress enhances Con A-stimulated T-cell proliferation by increasing [Ca2+ ]i via stimulation of Ca2+ entry.

Obesity enhances the induction of plasminogen activator inhibitor-1 by restraint stress: a possible mechanism of stress-induced renal fibrin deposition in obese mice.

Cardiovascular/thrombotic diseases are frequently induced by a variety of stressors. Obese patients are susceptible to thrombotic diseases associated with stress, but the underlying mechanism is still unknown. We have begun to investigate the expression of a primary inhibitor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1), in association with tissue thrombosis, using restraint-stressed obese mice. We analyzed the expression of PAI-1 after restraint (immobilization) stress in genetically obese mice in comparison with their lean counterparts. Dramatic increases in PAI-1 antigen in plasma and in tissue extracts were observed in the obese mice exposed to restraint stress. The induction of PAI-1 mRNA by stress in the tissues was also pronounced in the stressed obese mice as compared with the lean mice, especially in the hearts and adipose tissues. In situ hybridization analysis revealed that strong signals for PAI-1 mRNA were localized in the adipocytes, cardiovascular endothelial cells, and renal glomerular cells of the stressed obese mice. Histological examination revealed that renal glomerular fibrin deposition was detected only in the obese mice after 2 h of restraint stress. Obesity enhances the stress-mediated PAI-1 induction in the blood and tissues. This phenomenon may be associated with the increased risk of stress-induced renal fibrin deposition in obese subjects.

Impaired wound contraction and delayed myofibroblast differentiation in restraint-stressed mice

Previous research has shown that psychological stress delays wound closure by >25%. Gene expression of pro-inflammatory cytokines and the maturation of the epithelium were also impaired by stress (Mercado et al.). Wound contraction contributes to the speed of wound closure (Hunt and Hopf). In the current study, wound contraction was decreased by >45% ( p < .01) in restraint stressed mice. Fibroblast migration and differentiation into smooth muscle α-actin (SmαA) -expressing myofibroblasts were delayed in RST mice through day 7 post-wounding. In addition, there was a 25 ( p < .01), 48 ( p < .01), and 38% ( p < .05) decrease in SmαA mRNA levels at days 1, 3, and 5 post-wounding in RST mice, respectively. Cytokines that regulate fibroblast migration and differentiation include transforming growth factors-β1, -β2, and -β3 (TGF-βs). Although expression of TGF-β1 mRNA was downregulated by >25% ( p < .01) in RST mice on day 3 post-wounding, no significant differences were detected in active or total TGF-β1 protein levels. Stress did not alter the expression of TGF-β2 or -β3 through day 5 post-wounding. Thus, these data indicate that stress delays wound contraction and myofibroblast differentiation, which are likely independent of expression of TGF-β1, -β2, and -β3.

Inhibition of Decrease in Natural Killer Cell Activity in Repeatedly Restraint-Stressed Mice by a Biological Response Modifier Derived from Cultured Mycelia of the Basidiomycete Tricholoma matsutake

Objective: To develop a method to cope with stress-induced reduction in immunocompetence, we evaluated the immunomodulatory activities of a biological response modifier derived from the mycelia of the basidiomycete Tricholoma matsutake (CM6271) in mice under repeated restraint stress. Methods: C57BL/6 mice were inserted, one per tube, into 50-ml polypropylene tubes into which more than 30 ventilation holes had been drilled, and were restrained everyday for 20 days in this fashion for set periods of time. Natural killer (NK) cell activity and NK1.1-positive cell counts in the spleen, ACTH and corticosterone levels in the blood were determined. CM6271 was orally administered daily during the restraint stress period. Results: (1) When the mice were restrained in a confined space for 6 h per day for 20 days, the NK cell activity and the NK1.1-positive cell counts in the spleen significantly decreased after day 5 with an increase in the blood ACTH and corticosterone levels. (2) Oral administration of CM6271 during the restraint stress period significantly prevented the stress-induced decrease in NK cell activity. The effect was dependent on the timing, duration, and doses administered. (3) CM6271 did not significantly affect the splenic NK1.1-positive cell counts or the levels of blood ACTH and corticosterone in restraint-stressed mice. Conclusion: The above findings suggest that CM6271 inhibits the restraint stress-induced decrease of NK cell activity in a timing of administration and dose-dependent manner.

The effects of restraint stress on the neuropathogenesis of Theiler’s virus infection II: NK cell function and cytokine levels in acute disease

Psychological stress is thought to play an important role in multiple sclerosis. We have been investigating the role of restraint stress in Theiler’s virus infection in mice as a model for multiple sclerosis. We have previously determined that restraint stressed CBA mice had higher levels of mortality following infection with Theiler’s virus. We proposed that this was due to high levels of stress-induced corticosterone, which resulted in decreased numbers of circulating lymphocytes, decreased inflammatory cell infiltrates into the brain and consequently decreased viral clearance from the central nervous system (CNS). The effect of restraint stress on the innate immune response to Theiler’s virus is further investigated in the current study. Restraint stressed mice developed clinical signs of encephalitis, thymic atrophy, and adrenal hypertrophy. Decreased numbers of circulating lymphocytes and increased numbers of neutrophils were observed in the stressed mice. Stressed mice also had lower numbers of spleen cells which correlated with the decreased numbers of lymphocytes in circulation. Restraint stress caused elevations in serum tumor necrosis alpha (TNF-α). Virus-induced natural killer cell (NK) cytotoxic activity was significantly reduced in restrained mice at one day post infection which may account for the reduced viral clearance from the CNS. These data suggest that stress-induced immunosuppression of cytolytic NK cell activity may account in part for the reduced ability to clear virus from the CNS and increased mortality observed in this model.

Altered Kinetics of IL-1α, IL-1β, and KGF-1 Gene Expression in Early Wounds of Restrained Mice

Inflammatory processes that occur after injury contribute to wound closure. Previous studies showed that wounds of restraint-stressed (RST) mice had a reduced number of inflammatory cells and healed more slowly compared to controls. To investigate the molecular mechanisms between stress and wound healing, we studied cutaneous gene expression of IL-1α, IL-1β, and KGF-1. Female SKH-1 mice were restrained for 3 days before and for 5 days following placement of cutaneous wounds. Wounds were subjected to competitive RT–PCR. At day 1, RST mice had significantly lower IL-1β and KGF-1 mRNA than controls. At day 5, RST mice had significantly higher IL-1α and IL-1β mRNA than controls. Treatment of RST mice with the glucocorticoid receptor antagonist RU486 restored IL-1β mRNA expression at day 1. Our results suggest that stress induces alterations in the kinetics of cutaneous proinflammatory cytokine and growth factor gene expression which could impair the quality of healing tissues.

The Effects of Restraint Stress on the Neuropathogenesis of Theiler's Virus Infection: I. Acute Disease

Restraint stress was found to have a profound effect on the acute phase of Theiler's virus infection. Increased mortality rates were observed in restrained CBA mice infected with the BeAn strain of Theiler's virus. In addition, restrained mice developed higher CNS viral titers than infected/nonrestrained mice. Thymic atrophy was observed in both infected and uninfected restrained mice. Decreased microgliosis, perivascular cuffing, and astrocytosis were observed in restrained mice compared to nonrestrained infected mice at 7 days postinfection. Restraint-stressed mice also developed decreased numbers of lymphocytes and increased numbers of neutrophils in the blood. The mechanism proposed for these alterations involves stress-induced corticosterone, which causes immunosuppression, decreased trafficking of inflammatory cells in the CNS, and, consequently, increased viral replication.

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.

Parasite infection attenuates nonopioid mediated predator-induced analgesia in mice

Parasites have been shown to have a broad range of effects on host behavior, including alterations of host responses to predators. Response to the threat of predation consist of a number of defensive behaviors, including a reduction in pain sensitivity and the induction of analgesia. The present study examined the relationships between subclinical (i.e., nonpathological) infection with the naturally occurring, enteric, sporozoan (coccidian) parasite, Eimeria vermiformis, predator exposure, and nociceptive responses in male mice. Brief (30 s) exposure of nonparasitized mice to a predator (a cat) induced marked, relatively short-lived analgesia that was insensitive to naloxone and blocked by the serotonin-1A (5-HT 1A) agonist, 8-OH-DPAT. In contrast, mice acutely infected for 6 days with E. vermiformis, failed to show a predator-induced analgesia. The parasitized mice did display a naloxone-sensitive hypoalgesia or analgesia. However, restraint-stressed mice, which displayed a naloxone-sensitive hypoalgesia similar in amplitude to that of the infected mice, still exhibited a nonopioid mediated, predator-induced analgesia. These observations indicate that parasite infection attenuates 5-HT 1A-sensitive predator-induced analgesia and likely reduces the accompanying fear and anxiety related anticipatory defense reactions of the host to the predator.

Restorative effect of short term administration of thymulin on thymus-dependent antibody production in restraint-stressed mice

Thymulin is a well defined nonapeptide produced by thymic epithelial cells, and plays an important role in thymocyte differentiation. We investigated the restorative effect of thymulin on the stress-induced reduction in the production of antibodies against SRBC in mice. Antibody production in stressed mice was reduced to about 50% of that in normal mice. A 3-day period of administration of thymulin (1 ng/kg) restored antibody production to close to the normal level, whereas thymulin (1 ng−1 μg/kg) did not affect antibody production in normal mice. These results indicated that thymulin normalizes the altered thymus-dependent immune responses.

Alleviation of depressed immunity caused by restraint-stress, by the immunomodulator, lobenzarit disodium (disodium 4-chloro-2,2′-iminodibenzoate)

The immunomodulating effect of lobenzarit disodium (CCA, disodium 4-chloro-2,2′-iminodibenzoate), was examined in restraint-stressed mice which exhibited immunodeficient conditions mainly based on dysfunction of helper T cells. Primary direct plaque-forming cell (PFC) responses to T cell-dependent antigens such as sheep red blood cell (SRBC) and dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) were severely diminished by restraint-stress, whereas responses to T cell-independent antigens such as DNP-ficoll (DNP-Fic) and trinitrophenyl-lipopolysaccharide (TNP-LPS) were little reduced and sometimes were even increased. CCA, orally administered to mice at a dose of 50 mg/kg body weight, significantly restored anti-SRBC PFC response. Restraint-sterss also markedly decreased the proliferative response of splenic lymphocytes to concanavalin A (Con A), while it slightly augmented the response to lipopolysaccharide (LPS). CCA increased the response to Con A and suppressed the response to LPS. The numbers of T and B lymphocytes in the spleen were, however, reduced to the same degree by the stress and there was no striking difference in the susceptibility of these two subpopulations of splenic lymphocytes as analysed with fluorescein-activated cell sorter. CCA increased both T and B lymphocytes, but Lyt-1 + (helper/inducer T) cells recovered even more. It is noteworthy that in all these experiments CCA restored the weight of the thymus which is considered to be one of the organs most sensitive to stress. These results indicate that CCA promotes proliferation and differentiation of T cells, especially helper T cells, and restores immune functions depressed by restraint-stress.

Stress and Immune Responses I. Suppression of T Cell Function in Restraint-Stressed Mice

Effects of restraint stress on humoral immune responses were investigated in mice. Mice were restrained for 12 hours per day at nighttime and released at daytime for 2 consecutive days, either before or after sheep red blood cell (SRBC) immunization. The antibody response to SRBC was markedly suppressed in mice that were restrained before antigen injection. In contrast, the response was not significantly affected when the stress was loaded after immunization. Oral administration of 10 mg/kg diazepam prevented the stress-induced suppression of anti-SRBC antibody response. On the other hand, antibody responses to T cell-independent antigens such as trinitrophenylated (TNP)-FicolI and TNP-lipopolysac-charide were not suppressed. These results suggest that the restraint stress causes dysfunction of T cell populations in mice.

Stress and Immune Responses III. Effect of Restraint Stress on Delayed Type Hypersensitivity (DTH) Response, Natural Killer (NK) Activity and Phagocytosis in Mice

Several experiments were conducted to evaluate the influences of restraint stress on cell-mediated immune events in mice. Delayed type hypersensitivity response to sheep red blood cells was inhibited by the stress, regardless of the timing of restraint stress loading. The activity of phagocytosis of macrophages in vitro and in vivo were measured by using the zymosan-particle uptake method and the carbon clearance test, respectively. Both activities wero decreased in restraint-stressed mice. The suppressed carbon clearance rate in stressed mice, however, was recovered by the transfusion of serum from normal mice. Natural killer activity in spleen cells was decreased to 30-50% of the control in stressed mice However, no suppressor cells which could inhibit NK activity existed in the spleen from stressed mice. These results show that the restraint stress suppresses various kinds of cell-mediated immune events, which might play an important role in antitumor immunity.

Stress and Immune Responses IV. Adrenal Involvement in the Alteration of Antibody Responses in Restraint-Stressed Mice

We investigated the correlation between restraint stress-induced alteration of antibody responses and adrenal hormones. Adrenalectomy (Adx) blocked both the suppression of antibody response against T cell-dependent (TO) antigen and the enhancement of that against T cell-independent (TI) antigen in stressed mice. Adx also inhibited the atrophy of both thymus and spleen caused by restraint. Pre-treaiment of metyrapone, an inhibitor of adrenocortical steroid biosynthesis, had an effect that was similar to, but far weaker than that of Adx on stressed mice. The pre-administration of phenoxybenzamme, an α-adrenergic blocking agent, to mice prevented both the inhibition of antibody response to TD antigen and the decrease in spleen cell number of restrained mice. A similar effect was observed in mice pre-treated with 6-hydroxydopamine, an adrenergic neuron degenerating agent. However, no effects of these two agents were observed on the enhancement of antibody response to TI antigen The suppressive effect of the antibody response to TD antigen was augmented by the pre-administration of propranolpl, a β-adrenergic blocking agent. These results suggest that the suppression of the function of T cells in restrained mice are attributed to the released adrenocortical and adrenal medullary hormones and activated sympathetic nervous system and that the enhancement of B cell function is due to the adrenocortical hormones.

Effect of neurotropin (NSP) on the in vivo and in vitro antibody responses in mice.

Intraperitoneal administration of neurotropin (NSP) did not modify the antibody response to sheep red blood cells (SRBC) in normal BALB/c mice. However, the suppressed antibody responses to SRBC in restraint-stressed mice were favorably restored to normal level by NSP at a dose of 10 or 25 mg/kg either before or even after the stress loading. The suppressed antibody responses in hydrocortisone-treated mice were restored by NSP when it was administered after cortisone. Moreover, NSP augmented the in vitro antibody response to a T cell-dependent antigen, SRBC, but not to T cell-independent antigens such as dinitrophenylated-Ficoll and trinitrophenylated-lipopolysaccharide.