Repetitive Restraint Stress Research Articles

Maternal stress during pregnancy alters circulating small extracellular vesicles and enhances their targeting to the placenta and fetus

BackgroundMaternal psychological distress during pregnancy can negatively impact fetal development, resulting in long-lasting consequences for the offspring. These effects show a sex bias. The mechanisms whereby prenatal stress induces functional and/or structural changes in the placental-fetal unit remain poorly understood. Maternal circulating small extracellular vesicles (sEVs) are good candidates to act as “stress signals” in mother-to-fetus communication. Using a repetitive restraint-based rat model of prenatal stress, we examined circulating maternal sEVs under stress conditions and tested whether they could target placental-fetal tissues.ResultsOur mild chronic maternal stress during pregnancy paradigm induced anhedonic-like behavior in pregnant dams and led to intrauterine growth restriction (IUGR), particularly in male fetuses and placentas. The concentration and cargo of maternal circulating sEVs changed under stress conditions. Specifically, there was a significant reduction in neuron-enriched proteins and a significant increase in astrocyte-enriched proteins in blood-borne sEVs from stressed dams. To study the effect of repetitive restraint stress on the biodistribution of maternal circulating sEVs in the fetoplacental unit, sEVs from pregnant dams exposed to stress or control protocol were labeled with DiR fluorescent die and injected into pregnant females previously exposed to control or stress protocol. Remarkably, maternal circulating sEVs target placental/fetal tissues and, under stress conditions, fetal tissues are more receptive to sEVs.ConclusionOur results suggest that maternal circulating sEVs can act as novel mediators/modulators of mother-to-fetus stress communication. Further studies are needed to identify placental/fetal cellular targets of maternal sEVs and characterize their contribution to stress-induced sex-specific placental and fetal changes.

Peripheral CCL2-CCR2 signalling contributes to chronic headache-related sensitization.

Migraine, especially chronic migraine, is highly debilitating and still lacks effective treatment. The persistent headache arises from activation and sensitization of primary afferent neurons in the trigeminovascular pathway, but the underlying mechanisms remain incompletely understood. Animal studies indicate that signalling through chemokine C-C motif ligand 2 (CCL2) and C-C motif chemokine receptor 2 (CCR2) mediates the development of chronic pain after tissue or nerve injury. Some migraine patients had elevated CCL2 levels in CSF or cranial periosteum. However, whether the CCL2-CCR2 signalling pathway contributes to chronic migraine is not clear. Here, we modelled chronic headache with repeated administration of nitroglycerin (NTG, a reliable migraine trigger in migraineurs) and found that both Ccl2 and Ccr2 mRNA were upregulated in dura and trigeminal ganglion (TG) tissues that are implicated in migraine pathophysiology. In Ccl2 and Ccr2 global knockout mice, repeated NTG administration did not evoke acute or persistent facial skin hypersensitivity as in wild-type mice. Intraperitoneal injection of CCL2 neutralizing antibodies inhibited chronic headache-related behaviours induced by repeated NTG administration and repetitive restraint stress, suggesting that the peripheral CCL2-CCR2 signalling mediates headache chronification. We found that CCL2 was mainly expressed in TG neurons and cells associated with dura blood vessels, whereas CCR2 was expressed in subsets of macrophages and T cells in TG and dura but not in TG neurons under both control and disease states. Deletion of Ccr2 gene in primary afferent neurons did not alter NTG-induced sensitization, but eliminating CCR2 expression in either T cells or myeloid cells abolished NTG-induced behaviours, indicating that both CCL2-CCR2 signalling in T cells and macrophages are required to establish chronic headache-related sensitization. At cellular level, repeated NTG administration increased the number of TG neurons that responded to calcitonin-gene-related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP) as well as the production of CGRP in wild-type but not Ccr2 global knockout mice. Lastly, co-administration of CCL2 and CGRP neutralizing antibodies was more effective in reversing NTG-induced behaviours than individual antibodies. Taken together, these results suggest that migraine triggers activate CCL2-CCR2 signalling in macrophages and T cells. This consequently enhances both CGRP and PACAP signalling in TG neurons, ultimately leading to persistent neuronal sensitization underlying chronic headache. Our work not only identifies the peripheral CCL2 and CCR2 as potential targets for chronic migraine therapy, but also provides proof-of-concept that inhibition of both peripheral CGRP and CCL2-CCR2 signalling is more effective than targeting either pathway alone.

Efficacy of dual enkephalinase inhibition in a preclinical migraine model is mediated by activation of peripheral delta opioid receptors.

The aim of this study was to evaluate whether elevating levels of enkephalin by inhibiting their degradation can attenuate stress-induced migraine-like behaviors in mice. Previous studies in animals have suggested the delta opioid receptor (DOR) as a novel migraine target. The primary endogenous ligands for DOR are enkephalins and their levels can be increased by pharmacological inhibition of enkephalinases; however, it is not clear whether enkephalinase inhibition can be efficacious in preclinical migraine models through activation of DOR or whether other opioid receptors might be involved. Further, it is not clear whether opioid receptors in the central nervous system are necessary for these effects. This study used a model of repetitive restraint stress in mice that induces periorbital hypersensitivity and priming to the nitric oxide donor sodium nitroprusside (SNP; 0.1 mg/kg). Von Frey filaments were used to measure periorbital mechanical thresholds and grimace scores were evaluated by observing mouse facial features. Animals were treated with the dual enkephalinase inhibitor (DENKI) PL37. On day two post-stress, PL37 given to mice via either intravenous injection (10 mg/kg) or oral gavage (20 mg/kg) significantly attenuated stress-induced periorbital hypersensitivity and facial grimace responses. Additionally, both intravenous (10 mg/kg) and oral gavage (20 mg/kg) of PL37 prior to SNP (0.1 mg/kg) administration on day 14 post-stress significantly reduced SNP-induced facial hypersensitivity. Injection of the DOR antagonist naltrindole (0.1 mg/kg) but not the mu-opioid receptor antagonist CTAP (1 mg/kg) prior to PL37 treatment blocked the effects. Finally, pretreatment of mice with the peripherally restricted opioid receptor antagonist naloxone methiodide (5 mg/kg) blocked the effects of PL37. These data demonstrate that inhibiting enkephalinases, and thus protecting enkephalins from degradation, attenuates stress-induced migraine-like behavior via activation of peripheral DOR. Peripheral targeting of endogenous opioid signaling may be an effective therapeutic strategy for migraine.

Effects of NO modulators and antioxidants on endocrine and cellular markers in rats under repetitive restraint stress.

The aim of the study was to evaluate the effects of NO modulators and antioxidant treatments on endocrine (plasma corticosterone), cellular (heat shock protein 70 [HSP-70] and nuclear factor κB [NF-κB]), and oxidative stress markers in repetitively stressed rats. Repetitive (restraint) stress (RS 1hr/day × 21 days) enhanced the levels of cellular and endocrine stress markers in the rat blood and altered pro-oxidant-antioxidant balance differentially in the control and test groups. Exposure to repetitive RS enhanced malondialdehyde (MDA) levels, lowered reduced glutathione (GSH), and superoxide dismutase (SOD) levels as well as nitric oxide (NOx) levels. NO precursor L-arginine and NO synthase inhibitors were found to differentially modulate stress-induced mechanism in altering NF-κB, HSP-70, and corticosterone levels. The antioxidant L-ascorbic acid (L-AA) significantly suppressed RS(×21)-induced elevation of NF-κB and HSP-70 levels, depicting protective effects, as also evidenced by reversal of elevated corticosterone levels. The results suggest that NO modulators and antioxidants differentially influence repetitive stress-induced changes in endocrine and cellular markers, and the complex interaction between NO and cellular markers like HSP70 and NF-κB plays a crucial modulatory role in this phenomenon.

Exploratory study on neurochemical effects of low-intensity pulsed ultrasound in brains of mice.

There is now a relatively large body of evidence suggesting a relationship between dysfunction of myelin and oligodendrocytes and the etiology of several neuropsychiatric disorders, including depression and schizophrenia, and also suggesting that ultrasound methods may alleviate some of the symptoms of depression. We have applied low-intensity pulsed ultrasound (LIPUS) to the brains of mice treated with the demyelinating drug cuprizone, a drug that has been used as the basis for a rodent model relevant to a number of psychiatric and neurologic disorders including depression, schizophrenia, and multiple sclerosis. Prior to conducting the studies in mice, preliminary studies were carried out on the effects of LIPUS in vitro in neuron-like SH-SY5Y cells and primary glial cells. In subsequent studies in mice, female C57BL/6 mice were restrained in plastic tubes for 20 min daily with the ultrasound transducer near the end of the tube directly above the mouse's head. LIPUS was used at an intensity of 25 mW/cm2 once daily for 22 days in control mice and in mice undergoing daily repetitive restraint stress (RRS). Behavioral or neurochemical studies were done on the mice or the brain tissue obtained from them. The studies in vitro indicated that LIPUS stimulation at an intensity of 15 mW/cm2 delivered for 5 min daily for 3 days in an enclosed sterile cell culture plate in an incubator increased the viability of SH-SY5Y and primary glial cells. In the studies in mice, LIPUS elevated levels of doublecortin, a marker for neurogenesis, in the cortex compared to levels in the RRS mice and caused a trend in elevation of brain levels of brain-derived neurotrophic factor in the hippocampus relative to control levels. LIPUS also increased sucrose preference (a measure of the attenuation of anhedonia, a common symptom of several psychiatric disorders) in the RRS model in mice. The ability of LIPUS administered daily to rescue damaged myelin and oligodendrocytes was studied in mice treated chronically with cuprizone for 35 days. LIPUS increased cortex and corpus callosum levels of myelin basic protein, a protein marker for mature oligodendrocytes, and neural/glial antigen 2, a protein marker for oligodendrocyte precursor cells, relative to levels in the cuprizone + sham animals. These results of this exploratory study suggest that future comprehensive time-related studies with LIPUS on brain chemistry and behavior related to neuropsychiatric disorders are warranted. Exploratory Study on Neurochemical Effects of Low Intensity Pulsed Ultrasound in Brains of Mice. Upper part of figure: LIPUS device and in-vitro cell experimental set-up. The center image is the LIPUS generating box; the image in the upper left shows the cell experiment set-up; the image in the upper right shows a zoomed-in sketch for the cell experiment; the image in the lower left shows the set-up of repetitive restraint stress (RRS) with a mouse; the image in the lower middle shows the set-up of LIPUS treatment of a mouse; the image in the lower right shows a zoomed-in sketch for the LIPUS treatment of a mouse.

Effects of Chronic Repetitive Restraint Stress on Acute and Long-Term Changes in Plasma Corticosterone Levels and Hypothalamic Neuronal Nitric Oxide Synthase Activity in Rats

Effects of Chronic Repetitive Restraint Stress on Acute and Long-Term Changes in Plasma Corticosterone Levels and Hypothalamic Neuronal Nitric Oxide Synthase Activity in Rats

Evaluation of oxidative stress and its modulation by L-Arginine and L-Ascorbic acid in repetitive restraint stress model in Wistar rats

BackgroundOxidative stress has been involved in pathophysiology of several cardiovascular, respiratory, neurological and metabolic diseases. Correction of oxidative stress with antioxidants may prove beneficial in several disease conditions. ObjectivesTo evaluate antioxidant effect of L-Arginine and L-Ascorbic acid in repetitive restraint stress induced oxidative stress in rats. MethodsIn the present animal study restrain stress for 1 h daily for 21 days was used to induce oxidative stress in Wistar rats. Antioxidant effect of L-Arginine and L-Ascorbic acid was evaluated using malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) levels and total antioxidant capacity levels. ResultThe results of our study demonstrated that repetitive restraint stress significantly enhance MDA levels and suppress SOD, GSH, catalase levels as well as total antioxidant capacity compared to vehicle group. L-Arginine (500 and 1000 mg/kg, I.P.) and L-Ascorbic acid (100 and 200 mg/kg, I.P) reversed changes of serum MDA, GSH, SOD, catalase levels and total antioxidant capacity level. ConclusionResults from our study demonstrated reversal of oxidative stress markers by L-Arginine and L- Ascorbic acid. They may be used therapeutically in disease associated with high oxidative stress.

The Role of Redox Dysregulation in the Effects of Prenatal Stress on Embryonic Interneuron Migration.

Maternal stress during pregnancy is associated with increased risk of psychiatric disorders in offspring, but embryonic brain mechanisms disrupted by prenatal stress are not fully understood. Our lab has shown that prenatal stress delays inhibitory neural progenitor migration. Here, we investigated redox dysregulation as a mechanism for embryonic cortical interneuron migration delay, utilizing direct manipulation of pro- and antioxidants and a mouse model of maternal repetitive restraint stress starting on embryonic day 12. Time-lapse, live-imaging of migrating GAD67GFP+ interneurons showed that normal tangential migration of inhibitory progenitor cells was disrupted by the pro-oxidant, hydrogen peroxide. Interneuron migration was also delayed by in utero intracerebroventricular rotenone. Prenatal stress altered glutathione levels and induced changes in activity of antioxidant enzymes and expression of redox-related genes in the embryonic forebrain. Assessment of dihydroethidium (DHE) fluorescence after prenatal stress in ganglionic eminence (GE), the source of migrating interneurons, showed increased levels of DHE oxidation. Maternal antioxidants (N-acetylcysteine and astaxanthin) normalized DHE oxidation levels in GE and ameliorated the migration delay caused by prenatal stress. Through convergent redox manipula-tions, delayed interneuron migration after prenatal stress was found to critically involve redox dysregulation. Redox biology during prenatal periods may be a target for protecting brain development.

Differential mGluR5 expression in response to the same stress causes individually adapted hippocampal network activity

Individual differences in stress vulnerability and resilience have been observed even within a single cohort of inbred rats or mice. Stress phenotypes are typically quantified as changes in the behavior of experimental animals, which is the outcome of altered electrical activity of the brain network. Although mGluR5 is associated with individual vulnerability to stress and can act as a sensitive biomarker of stress adaptation, our understanding of mGluR5-dependent modifications to neural network activities in vivo remains limited. Here, we examined individual rats for changes in hippocampal mGluR5 expression induced by restraint stress and found that these changes cause accompanying changes in hippocampal electroencephalography (EEG) activity. We found six days of restraint stress caused variable changes in hippocampal mGluR5 expression, ranging from 20.9% to 210.7% of the control group. The low mGluR5 protein group (LE) showed increased methylation of the mGluR5 CpG island, reduced mGluR5 mRNA levels, and unaltered basal EEG theta spectral power between stress day 1 and 6. In contrast, the high mGluR5 protein group (HE) showed reduced methylation of CpG sites, increased mGluR5 mRNA expression, and reduced basal theta spectral power on stress day 6. We also found that injection of lentiviruses expressing mGluR5-specific shRNAs into the hippocampus rescued this reduction in baseline theta power in HE rats. These data suggest a causal relationship between individual differences in the changes in hippocampal mGluR5 expression induced by repetitive restraint stress and the accompanying changes in ensemble neural activity in the hippocampus.

Repetitive restraint stress changes spleen immune cell subsets through glucocorticoid receptor or β-adrenergic receptor in a stage dependent manner

Immune system is sensitive to stress. Spleen is the largest peripheral immune organ innervated with sympathetic nerves and controlled by adrenomedullary system in the body. However, the alterations and mechanism of spleen immune cell subsets caused by repetitive restraint stress (RRS) is poorly understood. In this study, we found that RRS reduced spleen index in mice, and induced an expansion of white pulp and involution of the red pulp. Meanwhile, the percentage of CD3+CD8+ T lymphocytes, CD11b+F4/80+ macrophages, CD11b+Ly-6G−Ly-6Chi monocytic myeloid derived suppressor cells (mMDSCs) and CD11b+Ly-6G+Ly-6Cint granulocytic myeloid derived suppressor cells (gMDSCs) in spleen were significantly changed by RRS. Mechanistically, we found that the expression of norepinephrine (NE) and β-adrenergic receptor (β-AR) in spleen were up-regulated after 21 days of RRS, but not 7 days. The expression of corticosterone (CORT) and glucocorticoid receptor (GR) in spleen were up-regulated after 7 days of RRS but were lower after 21 days of RRS, even though they were still higher than that in mice without stress. By treating the stressed mice with RU486 (antagonist of GR) or propranolol (antagonist of β-AR), we demonstrated that GR was responsible for the changes of spleen induced by 7 days of RRS and β-AR was for 21 days of RRS. Our data suggest that RRS changes spleen immune cell subsets through GR or β-AR in a stage dependent manner.

Behavioral and neurobiological effects of prenatal stress exposure in male and female APPswe/PS1dE9 mice

Epidemiological evidence implies a role for chronic stress and stress-related disorders in the etiopathogenesis of sporadic Alzheimer's disease (AD). Although chronic stress exposure during various stages of life has been shown to exacerbate AD-related cognitive deficits and neuropathology in AD mouse models, the role of stress exposure during the prenatal period on AD development and progression remained to be investigated. The present study therefore explored the effects of prenatal maternal stress (PMS) in both male and female APPswe/PS1dE9 mouse offspring in terms of cognition, affect, and AD-related neuropathology. As prenatal perturbations are likely to mediate their effects via alterations in epigenetic regulation, changes in hippocampal DNA methyltransferase 3a, 5-methylcytosine and 5-hydroxymethylcytosine levels were assessed as underlying mechanisms. Repetitive restraint stress during the first week of gestation exerted a sex-dependent effect, with male PMS mice showing spatial memory deficits and a blunted hypothalamus-pituitary-adrenal axis response, while female PMS mice showed improved spatial memory performance, increased depressive-like behavior, as well as a decrease in hippocampal plaque load. In addition, sex differences were observed among APPswe/PS1dE9 mice, independent of PMS (i.e., female mice showed impaired spatial memory performance, higher hippocampal plaque load, altered amyloid precursor protein processing in the CA3 and lower DNA methyltransferase 3a immunoreactivity in the dentate gyrus when compared with male mice of the same age). In conclusion, PMS exposure impacts on the behavioral phenotype and neuropathology of APPswe/PS1dE9 mice. Moreover, given the remarkable sex differences observed, one should not overlook the impact of sex-specific responses to environmental exposures when investigating gene-environment interactions in AD.

Psychological stress accelerates the onset of tumour formation and alters the type and location of tumours in a DMBA mouse carcinogenesis model

AbstractPsychological stress is recognized as a factor that contributes to increased susceptibility to a number of diseases, including cancer. Psychological stress, via release of chemical mediators, can induce long‐term changes in the organism resulting in an altered responsiveness of the organism to external carcinogens. The present investigation sought to evaluate the impact of stress on polycyclic aromatic hydrocarbon (PAH)‐induced carcinogenesis. We utilized a repetitive restraint stress mouse model and the model PAH, 7,12‐dimethylbenz[a]anthracene (DMBA). Restraint stress was applied three times a week for 6 weeks and DMBA was administered intra‐gastrically once a week for 6 weeks and formation of skin, mammary and ovarian tumours was examined at 26 weeks by pathological analyses. The results indicate that stress accelerates the onset of tumour formation produced in response to DMBA and significantly influences the type and location of tumours. Taken together, these results demonstrate that stress enhances the carcinogenicity of DMBA. These results clearly indicate the need for further characterization of the impact of stress on carcinogen metabolism in oncology. Copyright © 2010 John Wiley & Sons, Ltd.

Phytonutrients: experimental effects on gastric lesions in rats exposed to acute repetitive restraint stress

PurposeThis study seeks to investigate the effect of palm‐based phytonutrient complex (PPC) on stress‐induced gastric lesions and accompanying changes in the gastric acidity and gastrin level.Design/methodology/approachA total of 40 rats were divided between a control group that was given a vitamin E deficient diet and a treatment group that was given a vitamin deficient diet with oral supplementation of PPC at 60 mg/kg body weight for 28 days. At the end of the treatment period half of the number of rats were subjected to restraint‐stress for two hours for four consecutive days. Following stress exposure, blood was taken for measurement of gastrin level, after which all the rats were disposed of. The gastric acid was collected for measurement of acid concentration, while the stomachs were opened along the greater curvature and examined for lesions.FindingsRats exposed to stress developed hemorrhagic gastric lesions. PPC supplemented rats had fewer gastric lesions compared with their respective control group. Stress without supplementation with PPC also caused a reduction in the gastric acid concentration and the serum gastrin levels. Compared with their corresponding controls, the pre‐ and post‐values of gastric acid and serum gastrin concentration in rats with PPC supplementation remained comparable.Originality/valueStress is an identified risk factor for the development of gastric lesions. The current study showed that PPC was able to reduce the development of gastric lesions induced by stress and blocks the stress‐induced changes in the gastric acid concentration and gastrin level. It is possible that part of the protective effect of PPC in stress is through maintenance of the normal gastrin level, which results in the maintenance of gastrin trophic action in the gastric mucosa.

Short- and long-term effects of glycyrrhizic acid in repetitive stress.

1. This study was carried out to determine the effect of short-term and long-term ingestion of glycyrrhizic acid on the response to 2 h of restraint stress by measuring locomotor activity and plasma corticosterone levels. 2. Male Sprague-Dawley rats were randomly assigned into four groups, each group having eight rats. Group 1 (control) was given ordinary tap water, while groups 2 (short term), 3 and 4 (both long term) were given tap water containing 1 mg/mL glycyrrhizic acid to drink for 10 days, 4 weeks and 9 weeks, respectively. All the rats were subjected to 2 h of restraint stress and the locomotor activity assessed using an activity test in an open field arena followed by blood sampling to determine the plasma corticosterone level. These procedures were repeated daily for 14 days. 3. The basal locomotor activity scores for rats given glycyrrhizic acid for 10 days or 4 weeks were similar to those of controls; however, that of the rats treated long term with glycyrrhizic acid was significantly lower (21.0 +/- 3.0 squares crossed; P < 0.0005). Following the first period of restraint stress there was a highly significant decrease in locomotor activity, which remained significantly lower until the seventh and subsequent periods, indicating an adaptation to the repeated stress had occurred. Although the decrease in locomotor activity was partially blocked and adaptation to repetitive stress was enhanced in the rats given glycyrrhizic acid for 10 days, this was not seen in rats treated with glycyrrhizic acid for 4 or 9 weeks. The corticosterone levels in control rats were significantly elevated for 4-5 days following the exposure to repetitive stress but decreased gradually from day 7 onwards. However, both short- and long-term glycyrrhizic acid-treated rats had higher plasma corticosterone levels than the controls (P < 0.05). 4. In conclusion, repetitive restraint stress caused decreased locomotor activity associated with increased plasma corticosterone levels, both of which, in normal rats, decreased with adaptation to stress. The stress response was partially blocked and adaptation enhanced in rats given glycyrrhizic acid for 10 days, but not in rats given glycyrrhizic acid for 4 and 9 weeks. Glycyrrhizic acid ingestion caused high plasma corticosterone.

Suppression of experimental autoimmune encephalomyelitis by restraint stress: Sex differences

We have recently reported that female Lewis rats exhibit significantly higher basal circadian levels of corticosterone (Cort) than male Lewis rats. The studies reported here were designed to explore whether male and female Lewis rats demonstrate a differential suppression of experimental autoimmune encephalomyelitis (EAE) following exposure to an identical regimen of repetitive restraint stress. Rats were restrained for 1 or 9 h/day beginning 5 days before myelin basic protein (MBP) challenge and extending through the recovery period (18 days post challenge). Both clinical signs and histopathological changes of EAE were more significantly suppressed in 9-h-stressed females relative to male Lewis rats. Investigation of the mechanism underlying the stress-induced suppression of EAE revealed that restraint stress did not alter the clinical course of EAE in rats challenged with MBP 68–88 encephalitogenic peptide, suggesting that restraint stress may affect processing and/or presentation of the MBP molecule. Stressed rats exhibited decreased interleukin-2 and interferon γ production, and the frequency of MBP-reactive lymphocytes was reduced in comparison to non-stressed rats. Finally, repetitive restraint stress had no effect on blood-spinal cord permeability during EAE. The results presented here underscore the importance of such experimental variables as sex, strain, time of day, and the kinetics of immune response development.