Restraint-induced Stress Research Articles

Biochemical state of brain-liver axis of rats under restraint-induced stress and 2-oxoglutarate impact

Environmental factors play a significant role in affecting the overall health of organisms, with stress being a notable contributor. The process of urbanization and globalization in modern society introduces additional stressors, exacerbating population health issues. Consequently, there is a need for thorough examination, analysis, and exploration of strategies to mitigate the adverse effects of stress. 2-Oxoglutarate, an essential intracellular metabolite and mediator with metabolite trophic properties, emerges as a promising candidate for intervention. In this study, we aimed to evaluate the combined impact of restraint-induced stress and 2-oxoglutarate on the oxidative-reducing balance, antioxidant system effectiveness, and the functional status of the liver and brain in rats. Restraint-induced stress was found to elevate oxidative stress levels, as evidenced by increased concentrations of malonic dialdehyde and oxidative-modified proteins, particularly in the brain. Additionally, signs of lactic acidosis were observed in the liver, indicating physiological changes in response to stress. Furthermore, restraint-induced stress significantly altered bioenergy components, with decreased superoxide dismutase activity and increased cytochrome C concentration, potentially indicating mitochondrial dysfunction and increased membrane permeability. The incorporation of a 2% solution of 2-oxoglutarate into the diet demonstrated a reduction in malonic dialdehyde and carbonylated protein formation, leading to more effective restoration of oxidative-reducing balance in the brain compared to the liver. Additionally, normalization of the lactate/pyruvate concentration ratio and decreased lactate dehydrogenase activity, alongside elevated alanine aminotransferase levels, suggested a decrease in oxidative stress in the liver. Moreover, exogenous 2-oxoglutarate exhibited a positive effect on superoxide dismutase activity and cytochrome C concentration, indicating a reduction in oxidative tension in the liver and progressive mitochondrial function recovery. Based on these findings, exogenous 2-oxoglutarate emerges as a promising metabolitotrope and adaptogen for managing oxidative stress and improving mitochondrial function.

The defects of the hippocampal ripples and theta rhythm in depression, and the effects of physical exercise on their amelioration

Adverse environmental stress causes depressive symptoms with the impairments of memory formation, cognition, and motivation, however, their underlying neural bases have not been well understood, especially based on the observation of living animals. In the present study, therefore, the mice model of restraint-induced stress was examined electrophysiologically to investigate the alterations of hippocampal sharp wave ripples (SWRs) and theta rhythms. In addition, the therapeutic effects of physical exercise on the amelioration of those hippocampal impairments were examined in combination with a series of behavioral tests. The data demonstrated that chronic restraint stress caused the reductions of occurrence and amplitude of hippocampal SWRs and the decreases of occurrence, duration, and power of theta rhythms, while physical exercise significantly reverted them to the levels of healthy control. Furthermore, hippocampal adult neurogenesis and microglial activation, previously reported to be involved in the etiology of depression, were histologically examined in the mice. The results showed that the impairment of neurogenesis and alleviation of microglial activation were induced in the depressed mice. On the other hand, physical exercise considerably ameliorated those pathological conditions in the affected brain. Consistently, the data of behavioral tests in mice suggested that physical exercise ameliorated the symptomatic defects of motivation, memory formation, and cognition in the depressed mice. The impairments of hippocampal SWRs and theta rhythms in the affected hippocampus are linked with the symptomatic impairments of cognition and motivation, and the defect of memory formation, respectively, in depression. Taken together, this study demonstrated the implications of impairment of the hippocampal SWRs and theta rhythms in the etiology of depression and their usefulness as diagnostic markers of depression.

SAT270 Parity Increases Corticosterone Response To Restraint-induced Stress After Mild Blast Traumatic Brain Injury In Mice

Abstract Disclosure: S. Anisowicz: None. M. Bodemeier Loayza Careaga: None. T. Wu: None. Background: Traumatic brain injury (TBI) is a common problem worldwide, with an estimated incidence of 69 million injuries per year. In the US military, one of the most common forms of TBI during operational deployments after Sep 11, 2001 was the blast TBI, thought to be responsible for over 80% of TBI sustained in theater. One consequence of TBI is dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis. HPA axis dysregulation is linked to increased risk for psychiatric disorders. Furthermore, prior research has shown that TBI may dysregulate the HPA axis in a sex-dependent manner. Throughout a female’s life span, there are several sentinel events of hormonal consequence, including pubarche, menopause, and parity, which are known to be times of changes in risks for psychiatric disorders. Objectives: The role of parity on TBI response has not yet been studied, though parity has been shown to decrease fear-related behaviors and decreased corticosterone load in multiparous mice when compared to nulliparous (NP) controls. Our objective is to determine if parity alters corticosterone (CORT) response to restraint, a mild psychological stressor, in mice exposed to mild blast TBI (mbTBI). Methods: One hundred thirteen NP or primiparous (PP) C57Bl6/J mice were randomly assigned to receive sham or mbTBI. Among those that received mbTBI (19.2 ± 0.12 psi), 32 were NP and 24 were PP. Among sham mice, 32 were NP and 26 were PP. All mice received isoflurane anesthesia prior to sham or mbTBI treatment. At 7-8 days after mbTBI, half the mice within each treatment group were subjected to a mild 20-min restraint before being anesthetized by CO2 followed by collection of trunk blood. Serum was harvested and analyzed for CORT concentrations by ELISA (Enzo Life Sciences). Results: All mice that received mbTBI had a longer (p<0.05) righting time compared to those that did not. There was no main effect of parity in the righting time (p>0.1). PP mice who experienced mbTBI demonstrated a greater (p<0.05) CORT response to restraint (157.3 ± 21 pg/mL) when compared to NP mice (95.9 ± 7.2 pg/mL). CORT response to restraint did not differ (p>0.1) between NP and PP mice who did not experience mbTBI (96.1 ± 7.6 vs 109.2 ± 16 pg/mL). Conclusions: Our data indicates that mbTBI alters HPA axis reactivity in PP mice compared to NP mice, increasing the CORT response to restraint induced stress. Parity alone did not alter CORT response to restraint stress or baseline CORT levels. The results of our finding highlight the importance of parity as a factor in an individual’s lifespan. Presentation: Saturday, June 17, 2023

Stress aggravates imiquimod-induced psoriasiform inflammation by promoting M1 macrophage polarization

Psychological stress has long been considered to cause the aggravation and recurrence of psoriasis, but the underlying mechanism remains largely unknown. Here, we used a mouse model of restraint-induced stress and imiquimod (IMQ)-induced psoriasiform inflammation to investigate the crosstalk between stress and the skin immune system and their functions in the pathogenesis of psoriasis. We found that stress aggravated skin inflammation and elevated serum corticosterone (CORT) levels in mice. Stress also increased the number of macrophages and glucocorticoid receptor (GR) expression in IMQ-treated mouse skin. GR agonist CORT upregulated the phosphorylation of STAT1 to promote M1 macrophage polarization in vitro. Additionally, GR deletion in macrophages and pharmacologic inhibition of GR improved skin inflammation and reduced M1 macrophage polarization under stress. Taken together, these results indicate that stress aggravates psoriasiform inflammation by promoting CORT/GR signaling-induced M1 macrophage polarization, suggesting that blocking the GR signaling has great potential as an adjuvant treatment for psoriasis patients with chronic stress.

Experimental investigations on early crack development in fully restrained reinforced concrete members with active zero-displacement control

The development of restraint-induced stress in reinforced concrete members at an early age can be tested using setups applying either passive or active displacement control. To provide results relevant for the practical design of minimum reinforcement to limit the crack width, an actively controlled test setup for large specimens was developed to perform a systematic experimental campaign focusing on the early crack development under well-defined mechanical and thermal boundary conditions. The full degree of restraint achieved by actively preventing any displacement of the member allowed for the theoretical interpretation of early restraint-induced stress evolution, deformation behaviour in the cracked and uncracked regions, and reinforcement stress for a variety of parameters, including hydration rates, member height, bar diameters and subsequently reinforcement ratio. The cracking process, starting with primary cracks accompanied by secondary cracks within the effective reinforcement area, is monitored in combination with the development of the tensile strength and Young’s modulus of the concrete. Furthermore, the time of cracking and the forces released upon crack occurrence, considering the effect of self-equilibrating stresses, are determined, analysed, and compared with the results of crack-force based design rules to determine the minimum amount of reinforcement that ensures the crack width limitation.

Adolescent intermittent ethanol exposure enhances adult stress effects in male rats

Binge patterns of alcohol use, prevalent among adolescents, are associated with a higher probability of developing alcohol use disorders (AUD) and other psychiatric disorders, like anxiety and depression. Additionally, adverse life events strongly predict AUD and other psychiatric disorders. As such, the combined fields of stress and AUD have been well established, and animal models indicate that both binge-like alcohol exposure and stress exposure elevate anxiety-like behaviors. However, few have investigated the interaction of adolescent intermittent ethanol (AIE) and adult stressors. We hypothesized that AIE would increase vulnerability to restraint-induced stress (RS), manifested as increased anxiety-like behavior. After AIE exposure, in adulthood, animals were tested on forced swim (FST) and saccharin preference (SP) and then exposed to either RS (90 min/5 days) or home-cage control. Twenty-four hours after the last RS session, animals began testing on the elevated plus maze (EPM), and were re-tested on FST and SP. A separate group of animals were sacrificed in adulthood after AIE and RS, and brains were harvested for immunoblot analysis of dorsal and ventral hippocampus. Consistent with previous reports, AIE had no significant effect on closed arm time in the EPM (anxiety-like behavior). However, in male rats the interaction of AIE and adult RS increased time spent in the closed arms. No effect was observed among female animals. AIE and RS-specific alterations were found in glial and synaptic markers (GLT-1, FMRP and PSD-95) in male animals. These findings indicate AIE has sex-specific effects on both SP and the interaction of AIE and adult RS, which induces a propensity toward anxiety-like behavior in males. Also, AIE produces persistent hippocampal deficits that may interact with adult RS to cause increased anxiety-like behaviors. Understanding the mechanisms behind this AIE-induced increase in stress vulnerability may provide insight into treatment and prevention strategies for alcohol use disorders.

Prenatal Restraint Stress-Induced Maternal Exposure and Clomifene Citrate Administration Modulates Reproductive Programming in Female Wistar Rats.

&lt;b&gt;Background and Objective:&lt;/b&gt; Infertility and maternal stress studies have become major public health problems in most societies and this has attracted urgent global attention. This study examined the modulatory role of restraint-induced stress and clomiphene citrate (CC) administration on the reproductive programming of Wistar rats. &lt;b&gt;Materials and Methods:&lt;/b&gt; A total of 119 healthy Wistar rats weighing between 150-200 g were used and assigned into four major groups. Stress was induced for 1, 2, 4 and 6 hrs daily by exposure of the rats to a restraint plastic chamber for 1 and 2 weeks, respectively. At the end of each experimental protocol, the animals were euthanized by cervical dislocation and serum samples were collected for hormonal assay. The average gestational length, litter size and pup weight were examined. Data were expressed as Mean±SEM and mean differences were analyzed using One-way (ANOVA) and LSD &lt;i&gt;post hoc&lt;/i&gt; Test with SPSS 23 at p<0.05 level of significance. &lt;b&gt;Results:&lt;/b&gt; These findings showed that restraint stress caused a significant elevation in corticosterone level, while estrogen, progesterone, follicle-stimulating hormone and luteinizing hormone were significantly reduced when compared with control. Gestation length and litter size were also significantly reduced by stress while pup weights were not significantly affected. The CC increased litter size in unstressed rats when compared to litters of stressed rats that were significantly reduced, although, CC was able to increase the litter size of stressed rats towards normal. &lt;b&gt;Conclusion:&lt;/b&gt; Evidence indicated that stress alters reproductive potential in female rats and also, reduces the effectiveness of CC in inducing ovulation.

Glucocorticoid-Regulated Kinase CAMKIγ in the Central Amygdala Controls Anxiety-like Behavior in Mice.

The expression of the Calcium/Calmodulin-Dependent Protein Kinase I gamma (encoded by the Camk1g gene) depends on the activation of glucocorticoid receptors (GR) and is strongly regulated by stress. Since Camk1g is primarily expressed in neuronal cells of the limbic system in the brain, we hypothesized that it could be involved in signaling mechanisms that underlie the adaptive or maladaptive responses to stress. Here, we find that restraint-induced stress and the GR agonist dexamethasone robustly increase the expression of Camk1g in neurons of the amygdalar nuclei in the mouse brain. To assess the functional role of Camk1g expression, we performed a virally induced knock-down of the transcript. Mice with bilateral amygdala-specific Camk1g knock-down showed increased anxiety-like behaviors in the light-dark box, and an increase in freezing behavior after fear-conditioning, but normal spatial working memory during exploration of a Y-maze. Thus, we confirm that Camk1g is a neuron-specific GR-regulated transcript, and show that it is specifically involved in behaviors related to anxiety, as well as responses conditioned by aversive stimuli.

Effects of Concurrent Exposure to Chronic Restraint-Induced Stress and Total-Body Iron Ion Radiation on Induction of Kidney Injury in Mice.

Concurrent exposure to ionizing radiation (IR) and psychological stress (PS) may affect the development of adverse health consequences in scenarios such as space missions, radiotherapy and nuclear accidents. IR can induce DNA damage and cell apoptosis in the kidneys, thus potentially leading to renal fibrosis, which is the ultimate outcome of various chronic progressive nephropathies and the morphological manifestation of a continuous coordinated response after renal injury. However, little is known regarding the effects of concurrent IR exposure and PS on renal damage, particularly renal fibrosis. In this study, using a chronic restraint-induced PS (CRIPS) model, we exposed Trp53-heterozygous mice to total body irradiation with 0.1 or 2 Gy 56Fe ions on the eighth day of 28 consecutive days of a restraint regimen. At the end of the restraint period, the kidneys were collected. The histopathological changes and the degree of kidney fibrosis were assessed with H&E and Masson staining, respectively. Fibronectin (FN) and alpha smooth muscle actin (α-SMA), biomarkers of fibrosis, were detected by immunohistochemistry. Analysis of 8-hydroxy-2 deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, was performed with immunofluorescence, and terminal deoxynucleotidyl transferase-mediated nick end labeling assays were used to detect apoptotic cells. Histopathological observations did not indicate significant structural damage induced by IR or CRIPS + IR. Western blotting revealed that the expression of α-SMA was much higher in the CRIPS + IR groups than the CRIPS groups. However, no differences in the average optical density per area were observed for FN, α-SMA and 8-OHdG between the IR and CRIPS + IR groups. No difference in the induction of apoptosis was observed between the IR and CRIPS + IR groups. These results suggested that exposure to IR (0.1 and 2 Gy 56Fe ions), 28 consecutive days of CRIPS or both did not cause renal fibrosis. Thus, CRIPS did not alter the IR-induced effects on renal damage in Trp53-heterozygous mice in our experimental setup.

Enhanced Effects of Chronic Restraint-Induced Psychological Stress on Total Body Fe-Irradiation-Induced Hematopoietic Toxicity in Trp53-Heterozygous Mice.

Humans are exposed to both psychological stress (PS) and radiation in some scenarios such as manned deep-space missions. It is of great concern to verify possible enhanced deleterious effects from such concurrent exposure. Pioneer studies showed that chronic restraint-induced PS (CRIPS) could attenuate Trp53 functions and increase gamma-ray-induced carcinogenesis in Trp53-heterozygous mice while CRIPS did not significantly modify the effects on X-ray-induced hematopoietic toxicity in Trp53 wild-type mice. As high-linear energy transfer (LET) radiation is the most important component of space radiation in causing biological effects, we further investigated the effects of CRIPS on high-LET iron-particle radiation (Fe)-induced hematopoietic toxicity in Trp53-heterozygous mice. The results showed that CRIPS alone could hardly induce significant alteration in hematological parameters (peripheral hemogram and micronucleated erythrocytes in bone marrow) while concurrent exposure caused elevated genotoxicity measured as micronucleus incidence in erythrocytes. Particularly, exposure to either CRISP or Fe-particle radiation at a low dose (0.1 Gy) did not induce a marked increase in the micronucleus incidence; however, concurrent exposure caused a significantly higher increase in the micronucleus incidence. These findings indicated that CRIPS could enhance the deleterious effects of high-LET radiation, particularly at a low dose, on the hematopoietic toxicity in Trp53-heterozygous mice.

Synergistic Effects of Chronic Restraint-Induced Stress and Low-Dose 56Fe-particle Irradiation on Induction of Chromosomal Aberrations in Trp53-Heterozygous Mice.

Astronauts can develop psychological stress (PS) during space flights due to the enclosed environment, microgravity, altered light-dark cycles, and risks of equipment failure or fatal mishaps. At the same time, they are exposed to cosmic rays including high atomic number and energy (HZE) particles such as iron-56 (Fe) ions. Psychological stress or radiation exposure can cause detrimental effects in humans. An earlier published pioneering study showed that chronic restraint-induced psychological stress (CRIPS) could attenuate Trp53 functions and increase carcinogenesis induced by low-linear energy transfer (LET) γ rays in Trp53-heterozygous (Trp53+/-) mice. To elucidate possible modification effects from CRIPS on high-LET HZE particle-induced health consequences, Trp53+/- mice were received both CRIPS and accelerated Fe ion irradiation. Six-week-old Trp53+/- C57BL/6N male mice were restrained 6 h per day for 28 consecutive days. On day 8, they received total-body Fe-particle irradiation (Fe-TBI, 0.1 or 2 Gy). Metaphase chromosome spreads prepared from splenocytes at the end of the 28-day restraint regimen were painted with the fluorescence in situ hybridization (FISH) probes for chromosomes 1 (green), 2 (red) and 3 (yellow). Induction of psychological stress in our experimental model was confirmed by increase in urinary corticosterone level on day 7 of restraint regimen. Regardless of Fe-TBI, CRIPS reduced splenocyte number per spleen at the end of the 28-day restraint regimen. At 2 Gy, Fe-TBI alone induced many aberrant chromosomes and no modifying effect was detected from CRIPS on induction of aberrant chromosomes. Notably, neither Fe-TBI at 0.1 Gy nor CRIPS alone induced any increase in the frequency of aberrant chromosomes, while simultaneous exposure resulted in a significant increase in the frequency of chromosomal exchanges. These findings clearly showed that CRIPS could enhance the frequency of chromosomal exchanges induced by Fe-TBI at a low dose of 0.1 Gy.

Protective Effects of Borago officinalis (Borago) on Cold Restraint Stress-Induced Gastric Ulcers in Rats: A Pilot Study.

Stress is a typical body's natural defense to a generic physical or psychic change. A specific linking mechanism between ulcer onset and psycho-physical stress prolonged exposure has been reported. We decided to investigate the possible effects of Borago officinalis L. (Borago) in preventing physical (stress)-induced gastric ulcers in a rat model. Eighty male Sprague-Dawley rats were randomly divided into 16 groups, pretreated with a control solution, omeprazole (20 mg/kg), Borago methanolic extract (25, 50, 100, 250, and 500 mg/kg), Borago organic extract (50, 100, 250, and 500 mg/kg), Borago aqueous extract (5, 10, 20, 30, and 40 mg/kg), and D(-)-2-Amino-5-phosphonovaleric acid (AP5) (25 mg/kg) and kept in stressful conditions such as water immersion and restraint-induced stress ulcers. The animals were sacrificed and their stomach scored for the severity and the number of gastric ulcers. Methanolic extract (500 mg/kg) significantly reduced both ulcer parameters (***p < 0.001 and **p < 0.01, respectively). Aqueous and organic extract significantly decreased severity score at 5 and 10 mg/kg (**p < 0.01 and ***p < 0.001, respectively), and at 250 and 500 mg/kg (***p < 0.001), respectively, while gastric ulcers' resulted number significantly reduced only at 10 mg/kg (*p < 0.05) and at 500 mg/kg (**p < 0.01), respectively. On the other hand, aqueous extract significantly increased the mucosal gastric content of cAMP (*p < 0.05) and NR2A and NR2B subunits (*p < 0.05 and **p < 0.01, respectively) at 5 mg/kg. Organic extract showed also a significant cytotoxic effect at 500 and 1,000 mg/kg with a 3T3 cell viability reduction of 43.6% (**p < 0.01) and 92.1% (***p < 0.001), respectively. Borago aqueous extract at 10 mg/kg could be considered as a potential protective agent against stress-induced ulcers, and it is reasonable to possibly ascribe such protective activity to a modulation of the NR2A and NR2B subunit expression.

Hypothalamus-pituitary-adrenal axis imbalance and inflammation contribute to sex differences in separation- and restraint-induced depression

Whether social contact contributes to the underlying mechanisms of depression and the observed sex differences is unclear. In this study, we subjected young male and female mice to separation- and restraint-induced stress for 4 weeks and assessed behaviors, neurotransmitter levels, hormones, and inflammatory cytokines. Results showed that, compared with controls, male mice exposed to stress displayed significant decreases in body weight and sucrose preference after 1 week. In the fourth week, they exhibited a higher degree of anxiety (open field test) and depressive-like behavior (forced swim test). Moreover, the males showed significant decreases in monoamine neurotransmitters, including norepinephrine and dopamine in striatum, and an increase in pro-inflammatory cytokines, such as tumor necrosis factor α and interleukin 1β in serum. In contrast, females showed persistent loss of weight during stress and displayed significant decreases in sucrose preference after stress. Importantly, the females but not males showed activation of the hypothalamus-pituitary-adrenal (HPA) axis, with significantly higher levels adrenocorticotropic hormone. Additionally, mRNA level of c-fos and AVP showed there was significant interaction between stress and sex. Finally, we conclude that an imbalance of the HPA axis and inflammation might be important contributors to sex differences in separation/restraint-induced depressive behavior and that changes might be mediated by c-fos and AVP.

Characterization of brain dystrophins absence and impact in dystrophin-deficient Dmdmdx rat model.

Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disease caused by mutations in the DMD gene encoding dystrophin, expressed mainly in muscles but also in other tissues like retina and brain. Non-progressing cognitive dysfunction occurs in 20 to 50% of DMD patients. Furthermore, loss of expression of the Dp427 dystrophin isoform in the brain of mdx mice, the most used animal model of DMD, leads to behavioral deficits thought to be linked to insufficiencies in synaptogenesis and channel clustering at synapses. Mdx mice where the locomotor phenotype is mild also display a high and maladaptive response to stress. Recently, we generated Dmdmdx rats carrying an out-of frame mutation in exon 23 of the DMD gene and exhibiting a skeletal and cardiac muscle phenotype similar to DMD patients. In order to evaluate the impact of dystrophin loss on behavior, we explored locomotion parameters as well as anhedonia, anxiety and response to stress, in Dmdmdx rats aged from 1.5 to 7 months, in comparison to wild-type (WT) littermates. Pattern of dystrophin expression in the brain of WT and Dmdmdx rats was characterized by western-blot analyses and immunohistochemistry. We showed that dystrophin-deficient Dmdmdx rats displayed motor deficits in the beam test, without association with depressive or anxiety-like phenotype. However, Dmdmdx rats exhibited a strong response to restraint-induced stress, with a large increase in freezings frequency and duration, suggesting an alteration in a functional circuit including the amygdala. In brain, large dystrophin isoform Dp427 was not expressed in mutant animals. Dmdmdx rat is therefore a good animal model for preclinical evaluations of new treatments for DMD but care must be taken with their responses to mild stress.

Expression of VEGF and Nestin in Chronic Restraint-Induced Stress in the Livers of Male Albino Rats.

Background and Aim of work: Stress and its related comorbid diseases are responsible for many liver pathologies. Nestin, a protein detected in a wide range of progenitor cells, was implicated in hepatic remodeling. Moreover, vascular endothelial growth factor (VEGF) is involved in vessel repair. The aim of this study was to evaluate the effect of chronic restrain stress (CRS) on expression of hepatic VEGF and Nestin. Materials and methods: Twelve male rats were divided into two groups; negative control and CRS. Animals of CRS were restrained individually in ventilated glass tube restrainers daily for 6 hours/day for 3 weeks. The liver samples were stained by hematoxylin and eosin, Masson trichrome and immunohistochemical staining for Nestin and VEGF with morphometric analysis. Results: Exposure to chronic restraint stress for 3 weeks caused hepatic inflammation, deposition of collagen. Immunohistochemical staining showed a coinciding significantly higher positive reaction for VEGF and Nestin in the CRS group. Conclusion: Vascular endothelial growth factor and Nestin immunohistochemical staining were both increased in chronic restraint-induced hepatic stress coinciding with the induced increase in hepatic inflammation, deposition of collagen. Nestin & VEGF

Apoptosis Induction by Iron Radiation via Inhibition of Autophagy in Trp53+/- Mouse Testes: Is Chronic Restraint-Induced Stress a Modifying Factor?

We used chronic restraint-induced stress (CRIS) and iron ionizing radiation (IR) to mimic human exposure to psychological stress (PS) and IR in a mouse model, and to investigate the relationship among endoplasmic reticulum stress (ERS), apoptosis and autophagy in testicular toxicity. Male Trp53+/- C57BL/6N mice were restrained for 6 h/day for 28 consecutive days, and total body irradiation with 0.1 or 2 Gy iron ion beam was performed on the day 8. Histopathological observation showed severely damaged spermatogenic cells, increased apoptotic cells, caspase-3 activation and cytochrome c release, indicating that IR and CRIS+IR induced testicular cell apoptosis. Upregulation of GRP78 (78-kDa glucose-regulated protein) suggested that IR and CRIS+IR induced ERS in the testes, and further analysis showed that apoptosis was enhanced by ERS through activation of the PERK/eIF2α/ATF4/CHOP pathway. Decreased expression of LC3II, Atg5 (autophagy related 5) and Beclin 1, and increased expression of p62, combined with ultrastructural changes seen under transmission electron microscopy, suggest that IR and CRIS+IR inhibit autophagosome formation. This process was related to inhibition of autophagy via activation of the PI3K/AKT/mTOR pathway under ERS. We showed that apoptosis was strengthened and autophagy was inhibited by ERS in mouse testes induced by IR and CRIPS+IR. These results showed that CRIS+IR had no difference in apoptosis induction and autophagy inhibition compared with IR alone. CIRS alone could induce apoptosis only in Leydig cells and its induction of pathological and molecular changes in testicular tissues was only a small extent as compared to those induced by IR. Of note, we showed that 28 consecutive days of CRIS did not exacerbate IR effects (no additive effect with IR). These findings also suggest that studies on the concurrent exposure to PS and IR should be done using different endpoints in both short and long-term CRIS models.

Agomelatine, venlafaxine, and running exercise effectively prevent anxiety- and depression-like behaviors and memory impairment in restraint stressed rats.

Several severe stressful situations, e.g., natural disaster, infectious disease out break, and mass casualty, are known to cause anxiety, depression and cognitive impairment, and preventive intervention for these stress complications is worth exploring. We have previously reported that the serotonin-norepinephrine-dopamine reuptake inhibitor, venlafaxine, as well as voluntary wheel running are effective in the treatment of anxiety- and depression-like behaviors in stressed rats. But whether they are able to prevent deleterious consequences of restraint stress in rats, such as anxiety/depression-like behaviors and memory impairment that occur afterward, was not known. Herein, male Wistar rats were pre-treated for 4 weeks with anti-anxiety/anti-depressive drugs, agomelatine and venlafaxine, or voluntary wheel running, followed by 4 weeks of restraint-induced stress. During the stress period, rats received neither drug nor exercise intervention. Our results showed that restraint stress induced mixed anxiety- and depression-like behaviors, and memory impairment as determined by elevated plus-maze, elevated T-maze, open field test (OFT), forced swimming test (FST), and Morris water maze (MWM). Both pharmacological pre-treatments and running successfully prevented the anxiety-like behavior, especially learned fear, in stressed rats. MWM test suggested that agomelatine, venlafaxine, and running could prevent stress-induced memory impairment, but only pharmacological treatments led to better novel object recognition behavior and positive outcome in FST. Moreover, western blot analysis demonstrated that venlafaxine and running exercise upregulated brain-derived neurotrophic factor (BDNF) expression in the hippocampus. In conclusion, agomelatine, venlafaxine as well as voluntary wheel running had beneficial effects, i.e., preventing the restraint stress-induced anxiety/depression-like behaviors and memory impairment.

Expression of VEGF and Nestin in Chronic Restraint-Induced Stress in the Livers of Male Albino Rats.

Background and Aim of work: Stress and its related comorbid diseases are responsible for many liver pathologies. Nestin, a protein detected in a wide range of progenitor cells, was implicated in hepatic remodeling. Moreover, vascular endothelial growth factor (VEGF) is involved in vessel repair. The aim of this study was to evaluate the effect of chronic restrain stress (CRS) on expression of hepatic VEGF and Nestin. Materials and methods: Twelve male rats were divided into two groups; negative control and CRS. Animals of CRS were restrained individually in ventilated glass tube restrainers daily for 6 hours/day for 3 weeks. The liver samples were stained by hematoxylin and eosin, Masson trichrome and immunohistochemical staining for Nestin and VEGF with morphometric analysis. Results: Exposure to chronic restraint stress for 3 weeks caused hepatic inflammation, deposition of collagen. Immunohistochemical staining showed a coinciding significantly higher positive reaction for VEGF and Nestin in the CRS group. Conclusion: Vascular endothelial growth factor and Nestin immunohistochemical staining were both increased in chronic restraint-induced hepatic stress coinciding with the induced increase in hepatic inflammation, deposition of collagen. Nestin & VEGF

Effects of chronic restraint-induced stress on radiation-induced chromosomal aberrations in mouse splenocytes

Both ionizing radiation (IR) and psychological stress (PS) cause detrimental effects on humans. A recent study showed that chronic restraint-induced PS (CRIPS) diminished the functions of Trp53 and enhanced radiocarcinogenesis in Trp53-heterozygous (Trp53+/−) mice. These findings had a marked impact on the academic field as well as the general public, particularly among residents living in areas radioactively contaminated by nuclear accidents. In an attempt to elucidate the modifying effects of CRIPS on radiation-induced health consequences in Trp53 wild-type (Trp53+/+) animals, investigations involving multidisciplinary analyses were performed. We herein demonstrated that CRIPS induced changes in the frequency of IR-induced chromosomal aberrations (CAs) in splenocytes. Five-week-old male Trp53+/+ C57BL/6J mice were restrained for 6h per day for 28 consecutive days, and total body irradiation (TBI) at a dose of 4Gy was performed on the 8th day. Metaphase chromosome spreads prepared from splenocytes at the end of the 28-day restraint regimen were painted with fluorescence in situ hybridization (FISH) probes for chromosomes 1, 2, and 3. The results obtained showed that CRIPS alone did not induce CAs, while TBI caused significant increases in CAs, mostly translocations. Translocations appeared at a lower frequency in mice exposed to TBI plus CRIPS than in those exposed to TBI alone. No significant differences were observed in the frequencies of the other types of CAs (insertions, dicentrics, and fragments) visualized with FISH between these experimental groups (TBI+CRIPS vs. TBI). These results suggest that CRIPS does not appear to synergize with the clastogenicity of IR.

Prenatal Stress+Morphine and Postnatal Re-exposure to Stress Alter Pentylenetetrazol-Induced Epileptic Manifestations in Rats

We studied effects of restraint-induced stress and morphine co-administration within the prenatal period and of re-exposure to stress at the end of infancy on the body mass and pentylenetetrazol-induced epileptic manifestations in rats. Pregnant rats were divided into six groups (control, restraint stressed, saline, morphine, stress+saline, and stress+morphine). In the stressed groups, pregnant rats were subjected to restraint stressing twice per day for three consecutive days (starting on pregnancy day 15). Rats in saline and morphine groups received saline and morphine subcutaneously on the same days. In the morphine/saline+stressed groups, rats were exposed to stress and received morphine/saline simultaneously. Control rats were used intact. The pups were weighed at postnatal days (PD) 1, 15, and 22. On P22, half of the pups were re-exposed to stress; then, pentylenetetrazol (PTZ)-induced seizures were recorded. The offspring body mass was significantly smaller in stressed, morphine, and stressed+morphine groups compared to the control. The time to onset of the first tonic-clonic seizure was shorter, while the duration and number of tonic-clonic attacks were significantly greater in the stressed+morphine group compared to other groups. Re-exposure to stress decreased the number of clonic seizures. The number of leg-opening and tail rigidity episodes was smaller in female offspring compared to male ones. Co-administration of restraint stress and morphine within the prenatal period reduces the offspring body mass and increases the seizure vulnerability more severely compared to the respective individual effects In addition, prenatal stress exerts stronger effects on the neural development and epileptic behaviors of the offspring than postnatal stress.