Restraint-stressed Mice Research Articles

Antidepressive synergism between crocin and D-AP5 in acute restraint-stressed mice.

Emerging evidence suggests that crocin rescues stress-induced depressive symptoms in mice via stimulation of hippocampal neurogenesis. Glutamate modulators mainly involving N-methyl- d -aspartate (NMDA) receptors (NMDARs) have highlighted a role in neural development, synaptic plasticity, and depression. The research presented here was designed to appraise the interaction between NMDAR agents and crocin on depressive-related behaviors in the NMRI male mice exposed to acute restraint stress (ARS) for a period of 4 h. The mice were submitted to the splash test, forced swimming test, and tail suspension test to evaluate depressive-like behavior. The ARS decreased the grooming duration in the splash test and increased immobility time in the forced swimming test and tail suspension test, suggesting a depressive-like phenotype. NMDA (0.25 and 0.5 μg/mouse, intracerebroventricular) did not alter depression-related profiles in both non-acute restraint stress (NARS) and ARS mice, while the same doses of NMDAR antagonist D-AP5 potentiated the antidepressive-like activities in the ARS mice compared with the NARS mice. Moreover, a low dose of NMDA did not change depression-related parameters in the crocin-treated NARS or ARS mice, while D-AP5 enhanced the crocin response in the NARS and ARS mice. Isobologram analysis noted a synergism between crocin and D-AP5 on antidepressive-like behavior in the NARS and ARS mice. Collectively, the combination of crocin and D-AP5 was shown to mitigate depression symptoms and can be potentially used for the treatment of depression disorders.

Alterations in Blaood Profiles and Spleen Tissue Following Purple Sweet Pota-to Anthocyanin Extract Treatment in Restraint-Stressed Mice

Stress is associated with broad changes in blood and immune cell dysfunction, which are correlated with the spleen as a secondary lymphoid organ. Anthocyanin, a plant bioactive compound from purple sweet potatoes, has previously been reported to have a beneficial effect on stress response behavior. However, the limited studies evaluated anthocyanin's effect on blood profiles and spleen tissue under stress. Therefore, this study aimed to explore the effect of total anthocyanin extract (ANC) from purple sweet potatoes (PSP) on the blood profiles and spleen tissue of restraint stressed mice. A total of 20 male adult BALB/c mice were divided into control (CTRL), stress (STR), stress + ANC 10 mg/kgBW (STR+ANC10), stress + ANC 20 mg/kgBW (STR+ANC20), and stress + ANC 40 mg/kgBW (STR+ANC10). Restraint stress was applied two hours/day for 14 days. An automatic blood analyzer was used for blood profiling. Immunoblotting of caspase-3 and hematoxylin-eosin-stained histological slides were observed for spleen tissue analysis. Combination of STR and ANC treatment reduced the erythrocyte count, hemoglobin level, and leucocyte count. The ANC-treated groups had a higher percentage of lymphocytes and a lower percentage of granulocytes. The expression of caspase-3 declined among ANC-treated groups. Spleen extramedullary erythropoiesis, vacuolization, and white pulp involution were demonstrated in stress and ANC-treated animals. In summary, restraint stress modified the blood cell profiles and spleen tissue in the mice model. Further research is necessary to evaluate the mechanism of ANC during stress on blood cells and splenic tissue repair enhancement

Tibetan mineral-herbal medicine Zuotai alleviates the depressive-like behaviors in chronic restraint-stressed mice while regulating stress hormone, inflammation and monoamine

Introduction:Zuotaiis an ancient mineral-herbal mixture containing β-HgS in Tibetan medicine. It is used to treat nervous system diseases, similar to Chinese medicine cinnabar and Indian Ayurveda medicineRasasindura. However, one of the key problems faced byZuotaiis that its indications are ambiguous. Our previous study found thatZuotaiexhibited the activity of ameliorating depressive-like behaviors in a chronic mild stress model. However, due to the inherent limitations of animal models in simulating human disease, clear results often require more than one model for confirmation.Methods:Therefore, another depression model, chronic restraint stressed (CRS) mice, was used to validate the antidepression effect ofZuotai. Prophylactic treatment was conducted for 21 consecutive days while mice were subjected to chronic restraint stress.Results:It was observed thatZuotaiand β-HgS alleviated anhedonia, behavioral despair, stereotype behavior, and reduced exploratory and spontaneous movement in CRS mice.Zuotaiand β-HgS also reversed the increases of stress hormone corticosterone (Cort) in serum and pro-inflammatory cytokines in serum and brain, and increased the serotonin in cortex in CRS mice, with positive dose-effect relationship. The number of Ki67-positive cells in the dentate gyrus and the level of brain-derived neurotrophic factor (BDNF) in the hippocampus were slightly elevated in CRS mice treated withZuotai; however, there was no statistically significant difference. AlthoughZuotaiincreased the total Hg concentration in main organs, the levels remained below those needed to result in observed adverse effect, at least for kidney and liver; andZuotaishowed no observed adverse effect on the brain histopathology, the cell proliferation in dentate gyrus, as well as the hippocampal and cortical organ coefficients.Conclusion:Zuotaiexhibited the alleviation of depressive-like behaviors in CRS mice, accompanying with ameliorating stress hormone, peripherical and cerebral inflammation, and monoamine neurotransmitter.

Sex differences in androgen receptor, estrogen receptor alpha, and c-Fos co-expression with corticotropin releasing factor expressing neurons in restrained adult mice

Gonadal hormone actions through androgen receptor (AR) and estrogen receptor alpha (ERα) regulate sex differences in hypothalamic-pituitary-adrenal (HPA) axis responsivity and stress-related behaviors. Here we tested whether corticotropin releasing factor (CRF) expressing neurons, which are widely known to regulate neuroendocrine and behavioral stress responses, co-express AR and ERα as a potential mechanism for gonadal hormone regulation of these responses. Using Crh-IRES-Cre::Ai9 reporter mice we report high co-localization of AR in CRF neurons within the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST), medial amygdala (MeA), and ventromedial hypothalamus (VMH), moderate levels within the central amygdala (CeA) and low levels in the paraventricular hypothalamus (PVN). Sex differences in CRF/AR co-expression were found in the principal nucleus of the BST (BSTmpl), CeA, MeA, and VMH (males>females). CRF co-localization with ERα was generally lower relative to AR co-localization. However, high co-expression was found within the MPOA, AVPV, and VMH, with moderate co-expression in the arcuate nucleus (ARC), BST, and MeA and low levels in the PVN and CeA. Sex differences in CRF/ERα co-localization were found in the BSTmpl and PVN (males>females). Finally, we assessed neural activation of CRF neurons in restraint-stressed mice and found greater CRF/c-Fos co-expression in females in the BSTmpl and periaqueductal gray, while co-expression was higher in males within the ARC and dorsal CA1. Given the known role of CRF in regulating behavioral stress responses and the HPA axis, AR/ERα co-expression and sex-specific activation of CRF cell groups indicate potential mechanisms for modulating sex differences in these functions.

Up-regulation of BDNF/TrkB signaling by δ opioid receptor agonist SNC80 modulates depressive-like behaviors in chronic restraint-stressed mice

Depressive disorder is a psychiatric disease characterized by its main symptoms of low mood and anhedonia. Due to its complex etiology, current clinical treatments for depressive disorder are limited. In this study, we assessed the role of the δ opioid receptor (δOR) system in the development of chronic-restraint-stressed (CRS)-induced depressive behaviors. We employed a 21-day CRS model and detected the c-fos activation and protein levels' changes in enkephalin (ENK)/δOR. It was found that the hippocampus and amygdala were involved in CRS-induced depression. The expression of pro-enkephalin (PENK), the precursors of the endogenous ligand for δOR, was significantly decreased in the hippocampus and amygdala following CRS. We then treated the mice with SNC80, a specific δOR agonist, to examine its anti-depressant effects in the tail suspension test (TST), forced swimming test (FST), and sucrose preference test (SPT). SNC80 administration significantly reversed depressive-like behaviors, and this antidepressant effect could be blocked by a TrkB inhibitor: ANA-12. Although ANA-12 treatment had no significant effect on the expression of ENK/δOR, it blocked the promoting effects of brain-derived neurotrophic factor (BDNF)/tyrosine kinase B(TrkB) signaling by SNC80 in the hippocampus and amygdala. Therefore, the present study demonstrates that SNC80 exerts anti-depressant effects by up-regulating the BDNF/TrkB signaling pathway in the hippocampus and amygdala in CRS-induced depression and provides evidence that δOR's agonists may be potential anti-depressant therapeutic agents.

Suppression of milk-derived miR-148a caused by stress plays a role in the decrease in intestinal ZO-1 expression in infants

Milk-derived miR-148a-3p (miR-148a), which is abundant in breast milk, has been shown to be associated with the development of infants' intestines. Although it is well known that stress during lactation changes milk constituents in terms of lipid and protein, no studies have examined the influence of stress on miR-148a expression in breast milk. The objective of this study is to investigate the relationship between stress and miR-148a expression in milk, and to evaluate whether the changes in milk-derived miR-148a expression-caused by the mother's exposure to stress-influence intestinal ZO-1 expression in infants. The participants of this study were healthy Japanese women who were nursing. Psychological stress evaluation of the subjects was conducted using a short form of the Profile of Mood State Second Edition-Adult (POMS-2). Additionally, miR-148a expressions in restraint stressed nursing mice were investigated using quantitative real-time PCR. The levels of a tight junction protein zonula occludens-1 (ZO-1) and DNA methyltransferase 1 (DNMT1), which is a direct target of miR-148a, in ileum in neonatal mice breastfed by stressed nursing mice were investigated using Western blot. Furthermore, to investigate the influence of miR-148a on ZO-1 expression within the intestine, the levels of ZO-1 and DNMT1 in human intestinal epithelial Caco-2cells with lentivirus-mediated miR-148a overexpression were evaluated. A significantly negative correlation was observed between relative miR-148a expression in breast milk and the total mood disturbance T-score. Each T-score on negative mood subscales of anger-hostility, confusion-bewilderment, depression-dejection, fatigue-inertia, and tension-anxiety was significantly negatively correlated with relative miR-148a expression in breast milk: a positive mood subscale vigor-activity T-score was significantly positively correlated with relative miR-148a expression in breast milk. A positive mood friendliness T-score, estimated separately from other scores, was significantly positively correlated with relative miR-148a expression in breast milk. Additionally, the relative expression of miR-148a in the milk obtained from stressed mice was significantly lower than that of control mice. The relative level of ZO-1 in ileum of neonatal mice nursed by stressed mice was significantly lower than that of neonatal mice nursed by control mice. Additionally, the relative level of DNMT1 in ileum of neonatal mice nursed by stressed mice was significantly higher than that of neonatal mice nursed by control mice. Furthermore, the relative level of ZO-1 in miR-148a-overexpressed Caco-2cells was significantly higher than that in control cells. The relative level of DNMT1 in miR-148a-overexpressed Caco-2cells was significantly lower than that in control cells. Mothers' exposure to stress during lactation may cause miR-148a expression in breast milk. Additionally, stressed-induced suppression of miR-148a expression in breast milk may cause a decrease in intestinal ZO-1 level via the increase in DNMT1 in infants' intestines. These observations are beneficial information for breastfeeding mothers and their families and perinatal medical professionals. Our findings encourage monitoring maternal psychological stress during lactation to promote breastfeeding and adequate infant nutrition.

Probiotics (Bacillus clausii and Lactobacillus fermentum NMCC-14) Ameliorate Stress Behavior in Mice by Increasing Monoamine Levels and mRNA Expression of Dopamine Receptors (D1 and D2) and Synaptophysin.

Stress is a physiological consequence of the body to adversity. The gut–brain axis and probiotics are gaining interest to provide better treatment for stress and other neurological disorders. Probiotic (Lactobacillus fermentum NMCC-14 and Bacillus clausii, 1010 colony-forming unit/day/animal, per oral) effects were investigated in acute (up to day 7) and subacute (days 8–14) restraint-stressed and normal mice through behavioral paradigms (elevated plus maze: EPM, light dark box/dark light box: LDB, and open field test: OFT). Time spent in the open arms of the EPM, time spent in the light compartment of the LDB, and movable time and time spent in the center of the OFT were significantly (p ≤ 0.05, n = 5) increased in probiotic-treated restraint-stressed mice. Enzyme-linked immunoassay determined blood cortisol and adrenocorticotropic hormone (ACTH) levels, which were reduced significantly (p < 0.05, n = 5) in probiotic-treated restraint-stressed mice. Hematoxylin and eosin-stained hippocampal slides also showed less or no neurodegeneration in the probiotic-treated animals. High-performance liquid chromatography and quantitative polymerase chain reaction were performed to determine the monoamine levels and mRNA expression of dopamine receptor subtypes (D1 and D2) and synaptophysin in the mice hippocampus (HC) and prefrontal cortex (PFC). The dopamine, serotonin, and norepinephrine levels were also significantly (p < 0.05, n = 5) increased in the HC and PFC of probiotic-treated animal brains. Fold expression of mRNA of D1 and D2 (except HC, LF-S, day 14) receptors and synaptophysin was also significantly (p < 0.05, n = 5) increased in the same brain parts of probiotic-treated restraint-stressed mice. Comparing mice in the Lactobacillus fermentum NMCC-14 and Bacillus clausii groups to mice in the normal group, only a significant (p < 0.05, n = 5) decrease was observed in the serum ACTH and cortisol levels on day 14 in Bacillus clausii-treated mice, where all other parameters also showed improvement. In comparison, Bacillus clausii showed greater stress suppressant activity than Lactobacillus fermentum NMCC-14. However, both probiotic bacteria can be a better and safer therapeutic alternative for ailments than currently available drugs.

Anxiolytic effects of inhibitors of endocannabinoid degrading enzyme on anxiety-like behavior in restraint-stressed mice.

In this study, we investigated the effects of endocannabinoid (eCB) degrading enzyme inhibitors on anxiety-like behavior and brain eCB levels in restraint-stressed mice.

The Effectiveness of Ecklonia Cava Kjellman Extract in Improving Menopausal Syndrome in Osteoporosis and Depression

Ecklonia cava (EC) is a natural material commonly used to decrease swelling, allergy, cancer, and sleep issues. Using EC has been reported to regulate hormones during ovarian failure in an aromatase inhibition rodent model. The aim of this study was to investigate EC’s benefits on ovariectomized female mice. Hormone replacement therapy is beneficial in menopause, but the risk of side effects increases. In the present study, alkaline phosphatase (ALP) activity and tryptophan hydroxylases (TPHs) expression were studied after the EC extracts were incorporated as elemental, phloroglucinol, eckol, dieckol, 6,6′-biekcol, and 8,8′-bieckol. In this in vivo study, the following seven groups of 10-week-old Balb/c female mice were evaluated over 8 weeks: normal mice (Sham), ovariectomized mice (OVX), ovariectomized and restraint stressed mice (OVX + R), ovariectomized and 17β-estradiol-treated mice (OVX + R + E2), ovariectomized and fluoxetine-treated mice (OVX + R + E2), and ovariectomized and EC-extract-treated mice (OVX + R + EC150 or OVX + R + EC300). The serum lipid profile, bone loss, and depressive symptoms were investigated in an ovariectomized and restraint-stressed mice model. In the in vitro models, ALP activity was dose-dependently upregulated by EC, including phloroglucinol, eckol, dieckol, 6,6′-biekcol, and 8,8′-bieckol, in RBL-2H3 cells. The transcripts of TPH1 and TPH2 were induced by EC and/or its elements (phloroglucinol, eckol, dieckol, 6,6′-biekcol, and 8,8′-bieckol) in RBL-2H3 cells. The re-uptake activity of serotonin (5-HT) was also decreased by EC and its ingredients such as phloroglucinol, eckol, dieckol, 6,6′-biekcol, and 8,8′-bieckol. In the models, the serum cholesterol and triglyceride levels were downregulated in OVX + R mice by EC treatment. The bone mineral density (BMD) was determined in EC-treated groups, and the bone metabolism markers, CTX and osteocalcin, were also reduced to normal levels. The depression experiments revealed that the immobility time was shortened in the forced-swimming test in OVX + R mice. Moreover, the serum serotonin level was promoted by EC treatment in OVX + R mice. These results showed that EC extract inhibits bone loss and depressive symptoms in a menopausal mouse model by modulating bone metabolism markers (CTX and osteocalcin) and serotonin level in OVX + R mice.

Effects of postovulatory oviduct changes and female restraint stress on aging of mouse oocytes.

Postovulatory oocyte aging is one of the major causes for human early pregnancy loss and for a decline in the population of some mammalian species. Thus, the mechanisms for oocyte aging are worth exploring. While it is known that ovulated oocytes age within the oviduct and that female stresses impair embryo development by inducing apoptosis of oviductal cells, it is unknown whether the oviduct and/or female stress would affect postovulatory oocyte aging. By comparing aging characteristics, including activation susceptibility, maturation-promoting factor activity, developmental potential, cytoplasmic fragmentation, spindle/chromosome morphology, gene expression, and cumulus cell apoptosis, this study showed that oocytes aged faster in vivo in restraint-stressed mice than in unstressed mice than in vitro. Our further analysis demonstrated that oviductal cells underwent apoptosis with decreased production of growth factors with increasing time after ovulation, and female restraint facilitated apoptosis of oviductal cells. Furthermore, mating prevented apoptosis of oviductal cells and alleviated oocyte aging after ovulation. In conclusion, the results demonstrated that mouse oviducts underwent apoptosis and facilitated oocyte aging after ovulation; female restraint facilitated oocyte aging while enhancing apoptosis of oviductal cells; and copulation ameliorated oviductal apoptosis and oocyte aging.

Rapid action of Triiodothyronine on Mitochondrial H + , Ca 2+ and Mg 2+ -Dependent ion Transporters in Cortex, Hippocampus and Cerebellum of Restraint Mice

Thyroid hormones (TH) have a multitude of actions, mainly on development and differentiation during early life and play many vital roles in almost all tissues including neuronal tissues. TH rapidly alters the mitochondrial functions both by its genomic and direct actions on mitochondrial binding sites. The functional relationship between TH and mitochondrial ion transport during stress response has not yet been elucidated in mammals so far. Here, we report a rapid in vivo action of triiodothyronine (T 3 ) on mitochondrial ion transporter functions in the neuronal clusters of cortex, hippocampus and cerebellum of Swiss Albino mouse ( Mus musculus ) treated short-term with triiodothyronine (T 3 ; 200ng g-1) for 30 min either in non-stressed or in restraint-stressed (30 min each day for 7 days). The mH + -ATPase activity in the cortex decreased to significant levels after T 3 treatment in both non-stressed and restraint-stressed mice. On the contrary, the mH + -ATPase activity in the hippocampus and cerebellum increased to significant levels after T 3 treatment in both non-stressed and restraint-stressed mice. The mCa 2+ -ATPase activity in the cortex and cerebellum decreased to significant levels after T 3 treatment in both non-stressed and restraint-stressed mice. The mCa 2+ -ATPase activity in the hippocampus that increased to significant levels after T 3 treatment, showed a reversal after restraint-stress in T 3 -treated mice. The mitochondrial Mg 2+ -ATPase activity in the cortex decreased to significant levels after T 3 treatment in restraint-stressed mice. On the contrary, T 3 treatment in restraint stressed mice increased to significant levels the mitochondrial Mg 2+ -ATPase activity in the cerebellum. The mitochondrial Mg 2+ -ATPase activity in the hippocampus, which increased to significant levels after T 3 treatment in non-stressed mice, reversed its activity in T 3 -treated restraint-stressed mice. Spatial and differential action of T 3 on the mitochondrial ion transporters has been found in the present study that corroborates with a rapid modulatory action of T 3 on the transport of H + , Ca 2+ and Mg 2+ in the brain mitochondria of mice which appears to be sensitive to restraint stress.

Anti-stress effects of rosemary (Rosmarinus officinalis L.) leaf extract on intestinal goblet cells and immobility of forced-swimming test in BALB/c mice.

We investigated the anti-stress effect of rosemary (Rosmarinus officinalis L.) leaf extract (RLE) on restraint-stressed mice and found that RLE alleviated decreases in the number of intestinal goblet cells and amount of hepatic triglycerides. It also decreased the immobility time in the forced-swimming test and activation of microglia in the brain, suggesting that RLE has beneficial effects on stress-induced dysfunctions.

Anxiolytic activity of Coriandrum sativum seeds aqueous extract on chronic restraint stressed mice and effect on brain neurotransmitters

Abstract Medicinal properties of Coriandrum sativum L. (coriander) are well documented. Its central nervous system related activities are mentioned in Iranian traditional medicine. However, studies evaluating its anxiolytic properties and the underlying mechanisms are lacking. We investigate anxiolytic activity of C. sativum L. seeds aqueous extract (CSE) induced by chronic restraint stress and its effect on the neurotransmitter system. The phytoconstituents were extracted using soxhlet apparatus and identified using LC-MS. The mice were orally administered with the standard drug diazepam/CSE daily and exposed to restraint stress for two weeks. Anxiolytic activity was assessed using elevated plus maze and light/dark transition test models on day 1 and 16. On the 16th day, brain regions were quantitatively assessed for neurotransmitters. CSE treatment improved exploratory activity in the animal models of anxiety, and restored monoamines and GABA levels to the respective baseline levels. Moreover, CSE reduced excitotoxic levels of glutamate in the hippocampus region.

Pharmacological Effects of Yokukansankachimpihange on Urinary Catecholamine in Restraint-stressed Mice

Herbal medicines, acupuncture and moxibustion are often used for unidentified complaints. It is well known that catecholamine secreted by the sympatho-adrenal medullary system primarily functions to increase cardiac output and raise glucose levels in the blood during acute stress. In the present study, the effects of yokukansankachimpihange (YKSKCH, a Kampo medicine) on urinary catecholamine in mice that were repeatedly stressed by restraining were examined. Restraint stress (240 min/d×3 d×3 cycles, daytime: 12:00-16:00) induced a marked increase in noradrenaline (NA) and adrenaline (A) levels in the urine. Oral administration of YKSKCH (750 mg/kg of body weight) significantly inhibited the increase in urinary NA and A levels in mice after repeated restraint stress. In addition, the NA/dopamine (physical stress) and A/dopamine (mental stress) ratios were lower in the 750 mg/kg YKSKCH-treated group than in the control group. The tail suspension test was also performed and locomotor activity was investigated. Oral administration of YKSKCH at 750 mg/kg significantly reduced the immobility time, which was longer in mice after repeated restraint stress. Furthermore, oral administration of YKSKCH at 750 mg/kg increased locomotor activity, which was lower in mice after repeated restraint stress. These results suggest that YKSKCH has positive effects on mental and physical stress after repeated restraint stress, without reducing locomotor activity.

Influence of MLC901 Alone and with Moderate Exercise on Pain Response Concurrent Due to Stress of Male Mice.

Background: Physical exercise is known to have a positive effect on pain responses induced by stress, while chronic stress causes a negative effect on cognitive abilities. Depending on the type, duration, and intensity of the stressor, it can induce analgesia or hyperalgesia. Furthermore, the beneficial effects of traditional Chinese medicine MLC901 on stress processes have been reported. Here, the effects of MLC901 and moderate physical activity on pain response in restraint-stressed mice was investigated. Materials and Methods: Male NMRI mice were used in this study and were restrained in plexiglass mesh restrainers for induction of chronic restraint stress. Treadmill exercise was carried out for moderated exercise, 5 days/week for 4 weeks. MLC901 was intraperitoneally administered in the experimental groups. The pain response of the adult NMRI mice was detected via the hot-plate test. Results: It was showed that intraperitoneal administration of MLC901 dose (0.4 but not 0.1 and 0.2 mg/kg; once/2 days; for 25 days) resulted in the decreased percentage of time in the hot plate, indicating hyperalgesia. Moreover, restraint stress for 3 but not 6 and 9 hours/day elicit hyperalgesia in mice. The data showed that subthreshold dose of MLC901 (0.1 mg/kg) reduced hyperalgesia in 3-day stressed mice. Moderate treadmill running (10 meters/min for 30 min/day, 5 days/ week) potentiated the effect of 6 and 9 days on pain (induced hyperalgesia) that was blocked by MLC901 (0.1 mg/kg). Conclusion: Our findings indicated that subthreshold dose of MLC901 alone or when it associated with moderate exercise decreased hyperalgesia induced by stress, indicating the protective effect of MLC901.

Influence of MLC901 Alone and with Moderate Exercise on Pain Response Concurrent Due to Stress of Male Mice

Background: Physical exercise is known to have a positive effect on pain responses induced by stress, while chronic stress causes a negative effect on cognitive abilities. Depending on the type, duration, and intensity of the stressor, it can induce analgesia or hyperalgesia. Furthermore, the beneficial effects of traditional Chinese medicine MLC901 on stress processes have been reported. Here, the effects of MLC901 and moderate physical activity on pain response in restraint-stressed mice was investigated. Materials and Methods: Male NMRI mice were used in this study and were restrained in plexiglass mesh restrainers for induction of chronic restraint stress. Treadmill exercise was carried out for moderated exercise, 5 days/week for 4 weeks. MLC901 was intraperitoneally administered in the experimental groups. The pain response of the adult NMRI mice was detected via the hot-plate test. Results: It was showed that intraperitoneal administration of MLC901 dose (0.4 but not 0.1 and 0.2 mg/kg; once/2 days; for 25 days) resulted in the decreased percentage of time in the hot plate, indicating hyperalgesia. Moreover, restraint stress for 3 but not 6 and 9 hours/day elicit hyperalgesia in mice. The data showed that subthreshold dose of MLC901 (0.1 mg/kg) reduced hyperalgesia in 3-day stressed mice. Moderate treadmill running (10 meters/min for 30 min/day, 5 days/week) potentiated the effect of 6 and 9 days on pain (induced hyperalgesia) that was blocked by MLC901 (0.1 mg/kg). Conclusion: Our findings indicated that subthreshold dose of MLC901 alone or when it associated with moderate exercise decreased hyperalgesia induced by stress, indicating the protective effect of MLC901. [GMJ.2019;8:e1253]

Stress-Induced Suppression of Milk Protein Is Involved in a Noradrenergic Mechanism in the Mammary Gland.

Stress decreases milk components such as milk protein and milk yield. The objective of this study was to investigate whether noradrenaline (NA) in milk constituted a factor associated with stress-induced changes in milk proteins such as β-casein. Breast milk obtained from eight healthy, nursing women contained NA at concentrations ranging from 12.7 to 115.5 nM. The expression of tyrosine hydroxylase (TH), a rate-limiting enzyme of NA synthesis, was observed in primary normal human mammary epithelial cells (HMECs), and in MCF-12A and MCF-10A cell lines. The mean NA concentration in culture medium used by MCF-12A transfected with TH small interfering RNA (siRNA) was significantly lower than that of cells transfected with control siRNA. NA concentration in milk in restraint-stressed nursing mice was significantly higher than that in nonstressed nursing mice, owing to elevated TH expression in the mammary epithelium. The mean β-casein concentration in milk in restraint-stressed mice was significantly lower than that in nonstressed mice. NA treatment resulted in a concentration-dependent decrease in β-casein expression in HMECs. β2 adrenergic receptor (ADRB2) expression was observed in HMECs, MCF-12A, and MCF-10A, and immunohistochemical analysis of ADRB2 using mammary epithelium sections obtained from mice at day 10 of lactation showed that ADRB2 was expressed at the apical membrane of mammary epithelium. Treatment with salbutamol, an ADRB2 stimulant, decreased β-casein expression in a concentration-dependent manner in MCF-12A. Our results showed that endogenous NA derived from mammary epithelial cells likely comprises one of the factors involved in stress-induced changes in milk proteins such as β-casein.

Rubiscolin-6, a δ-Opioid Peptide from Spinach RuBisCO, Exerts Antidepressant-Like Effect in Restraint-Stressed Mice.

Rubiscolin-6 (Tyr-Pro-Leu-Asp-Leu-Phe) is produced by a pepsin digest of spinach d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and known to act as an agonist on δ-opioid receptor. Here, we showed that administration of rubiscolin-6 reduced immobility time in the tail suspension test in restraint-stressed mice without effect on locomotor activity. The antidepressant-like effect of rubiscolin-6 was blocked by a δ-opioid receptor antagonist, naltrindole. These results indicate that rubiscolin-6 exerts antidepressant-like effect through activation of δ-opioid receptor.

Antistress Effects of San-Huang-Xie-Xin Decoction on Restraint-Stressed Mice Revealed by 1H NMR-Based Metabolomics and Biochemistry Analysis.

San-Huang-Xie-Xin decoction (SHXXD), composed of Rhei Radix et Rhizoma, Coptidis Rhizoma, and Scutellariae Radix, is a representative antipyretic and detoxifying prescription in traditional Chinese medicine. In this study, we investigated the antistress effects and underlying mechanisms of San-Huang-Xie-Xin decoction (SHXXD) on restraint-stressed mice by 1H NMR-based metabolomics combined with biochemistry assay. A total of 48 male mice (5 weeks old, 18-22 g) were divided randomly into 6 groups (n = 8), including the normal group, restraint-stressed group, vitamin C group (positive drug, 17 mg/kg), and 3-dosage groups of SHXXD (200, 400, and 800 mg/kg). The stress model was induced by restraining mice in a polypropylene centrifuge tube for 6 h every day. The rotarod test was performed, and several biochemical indicators were measured. Moreover, other 24 animals were divided into 3 groups (n = 8) including the normal group, restraint-stressed group, and SHXXD group (800 mg/kg) for 1H NMR-based metabolomics analysis. Our results showed that SHXXD significantly increased the rotarod time, thymus index, spleen index, and the levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and interleukin- (IL-) 2, but decreased the levels of malondialdehyde (MDA), IL-1β, tumor necrosis factor- (TNF-) α, corticosterone (CORT), and adrenocorticotropic hormone (ACTH) in restraint-stressed mice. Moreover, the contents of eight endogenous metabolites that were changed by restraint stress were significantly reversed by SHXXD. The results of both metabolomics and biochemical analysis indicated that SHXXD (800 mg/kg, p.o.) could improve the biochemical changes and metabolic disorders in restraint-stressed mice by antioxidation and anti-inflammation, enhancing the body's immune function and restoring several disturbed metabolic pathways (i.e., lipid metabolism, glycolysis and gluconeogenesis, inflammatory injury, and energy metabolism). Taken together, these results indicated that SHXXD has a potential antistress effect in restraint-stressed mice and could be considered as a candidate drug for stress-related disorders.

SUN-476 Corticosterone-Mediated Suppression of the Reproductive Neuroendocrine Axis in Male Mice

It is well identified that psychosocial stress disturbs reproductive hormone secretion at multiple levels of the reproductive axis. The neural and hormonal mechanisms underlying this reproductive suppression and the role of reproductive brain neuropeptides in this process is not fully understood. We previously showed that in gonadectomized (GDX) male mice, a one-time acute restraint stress strongly suppressed in vivo luteinizing hormone (LH) pulsatility within 12-18 min of stress initiation. Furthermore, similarly stressed GDX mice showed a rise in circulating corticosterone (CORT) levels by 15 min. These correlative data suggest that CORT may be an important signaling factor involved in the stress-induced suppression of LH pulses. Thus, the objective of the current work is to identify the role of CORT in mediating stress inhibition of the reproductive neuroendocrine axis. First, we performed a dose-response and time course study to determine an effective exogenous CORT treatment paradigm to mimic endogenous CORT levels during psychosocial stress. Next, to assess CORT effects on LH pulses, GDX male mice were pulse bled every 6 min for 90 min, then injected with either an oil vehicle or 1 mg/kg CORT, and pulse bled every 6 min for another 90 min. CORT-injected mice showed a reduction in LH pulses, though not as robust or for as long as in restraint stressed mice. These data suggest that while CORT is sufficient to decrease LH pulsatile secretion, additional factors may also be involved to achieve maximal stress-induced suppression. To determine if upstream neuroendocrine signaling of stimulatory (kisspeptin) and inhibitory (RFRP-3) neurons to GnRH neurons are involved in this CORT-mediated suppression, in situ hybridization was conducted in brains of vehicle- and CORT-injected mice to determine changes in Kiss1 and Rfrp mRNA content and neuronal activation. These data are currently being analyzed. Our findings suggest that while acute CORT is capable of moderately suppressing reproductive hormone secretion, it is not the only mechanism involved during psychosocial stress exposure. Future studies will need to investigate additional factors that are important.