Chronic Stress Animal Research Articles

Chronic stress induces Alzheimer's disease-like pathologies through DNA damage-Chk1-CIP2A signaling.

Stress is an important initiating factor in promoting Alzheimer's disease (AD) pathogenesis. However, the mechanism by which stress induces AD-like cognitive impairment remains to be clarified. Here, we demonstrate that DNA damage is increased in stress hormone Corticotropin-releasing factor (CRF)-treated cells and in brains of mice exposed to chronic restraint stress. Accumulation of DNA damage drives activation of cell cycle checkpoint protein kinase 1 (Chk1), upregulation of cancerous inhibitor of PP2A (CIP2A), tau hyperphosphorylation, and Aβ overproduction, eventually resulting in synaptic impairment and cognitive deficits. Pharmacological intervention targeting Chk1 by specific inhibitor and DNA damage by vitamin C, suppress DNA damage-Chk1-CIP2A signaling pathway in chronic stress animal model, which in turn attenuate AD-like pathologies, synaptic impairments and cognitive deficits. Our study uncovers a novel molecular mechanism of stress-induced AD-like pathologies and provides effective preventive and therapeutic strategies targeting this signaling pathway.

Brain matrix metalloproteinase-9 activity is altered in the corticosterone mouse model of depression

Major depressive disorder is a highly prevalent psychiatric condition. Metalloproteinase 9 (MMP-9), a gelatinase involved in synaptic plasticity, learning and memory processes, is elevated in both chronic stress animal models and human peripheral blood samples of depressed patients. In this study we have evaluated the MMP-9 activity and protein expression in brain areas relevant to depression using the chronic corticosterone mouse model of depression. These mice show a depressive- and anxious-like behaviour. The MMP-9 activity and protein levels are significantly elevated in both the hippocampus and the cortex, and nectin-3 levels are lower in these brain areas in this model. In particular, these mice display an increased gelatinase activity in the CA1 and CA3 subfields of the hippocampus and in the internal layer of the prefrontal cortex. Moreover, the immobility time in the tail suspension test presents a positive correlation with the cortical MMP-9 activity, and a negative correlation with nectin-3 levels.In conclusion, the chronic corticosterone model of depression leads to an increase in the protein expression and activity of MMP-9 and a reduction of its substrate nectin-3 in relevant areas implicated in this disease. The MMP-9 activity correlates with behavioural despair in this model of depression. All these findings support the role of MMP-9 in the pathophysiology of depression, and as a putative target to develop novel antidepressant drugs.

Citrate Synthase and OGDH Protein Targets are Linked with Adrenomegaly Under Atherosclerosis Combined with Chronic Stress

Background: Pathological changes of the adrenal gland and the possible underlying molecular mechanisms are currently unclear in the case of atherosclerosis (AS) combined with chronic stress (CS). Methods and Findings: New Zealand white rabbits were used to construct a CS and AS animal model. Proteomics and bioinformatics were employed to identify hub proteins in the adrenal gland related to CS and AS. Hematoxylin–eosin staining was used to visualize pathological changes in adrenal gland. Hub proteins were detected using immunohistochemistry, immunofluorescence assays, and Western blotting. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression of hub protein mRNAs. In addition, a neural network model was constructed. The quantitative relationships among the results of the three techniques were inferred by cubic spline interpolation. Two most significant hub proteins (citrate synthase and OGDH) that were related to CS and AS were identified in the adrenal gland using numerous bioinformatic methods. The hub proteins were mainly enriched in mitochondrial proton transport ATP synthase complex, ATPase activation, and the AMPK signaling pathway. Compared with the control group, the adrenal glands were larger and more disordered, irregular, and necrotic in the AS+CS group. The expression of citrate synthase and OGDH were higher in the AS+CS group than the control group, both at the protein and mRNA levels (P<0.05). There were strong correlations among the cross-sectional areas of adrenal glands, citrate synthase, and OGDH (P<0.05) via Spearman’s rho analysis, receiver operating characteristic curves, a neural network model, and cubic spline interpolation. Conclusions: AS combined with CS plays an important role in the hypothalamic–pituitary–adrenal (HPA) axis, promotes adrenomegaly, increases the release of GC, and may enhance ATP synthesis and energy metabolism in the body through citrate synthase and OGDH gene targets, providing a potential research direction for future, related explorations into this mechanism. Funding: The present study was funded by the National Key R&D Program of China (Grant no. 2020YFC2003000), Chinese Academy of Medical Sciences, CAMS Innovation Fund for Medical Sciences (Grant no. 2018-I2M-1-002.), National Natural Science Foundation of China (Grant no. 31271097, and 51672030). Declaration of Interest: The authors declare that they have no competing interests Ethical Approval: All experiments were approved by Animal Care and Use Committee of the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College. All institutional and national guidelines for the care and use of laboratory animals were followed.

Chronic stress increases the tyrosine phosphorylation in female reproductive organs: An experimental study.

Background Changes in tyrosine-phosphorylated (TyrPho) protein expressions have demonstrated stress in males. In females, chronic stress (CS) is a major cause of infertility, especially anovulation. However, the tyrosine phosphorylation in the female reproductive system under stress conditions has never been reported.Objective To investigate the alteration of TyrPho protein expression in ovary, oviduct, and uterus of CS rats.Materials and MethodsIn this experimental study, 16 female Sprague-Dawley rats (5 wk: 220-250 gr) were divided into control and CS groups (n = 8/group). Every day, the CS animals were immobilized within a restraint cage and individually forced to swim in cold water for 60 consecutive days. Following the stress induction, the ovary, oviduct, and uterus of all rats were observed for their morphologies. The total protein profiles of all tissues were revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) before detecting TyrPho proteins using western blot. Intensity analysis was used to compare the expression of proteins between groups.ResultsThe results showed that the morphology and weights of ovary and oviduct in the CS group were not different from control. In contrast, the CS significantly increased the uterine weight as compared to control. Moreover, the expressions of TyrPho proteins in the ovary (72, 43, and 28 kDas), oviduct (170, 55, and 43 kDas), and uterus (55, 54, and 43 kDas) were increased in CS group as compared to those of control.ConclusionThe increased expressions of TyrPho proteins in ovary, oviduct, and uterus could be potential markers used to explain some machanisms of female infertility caused from chronic stress.

Citrate Synthase and OGDH as Potential Biomarkers of Atherosclerosis under Chronic Stress.

Background Pathological changes of the adrenal gland and the possible underlying molecular mechanisms are currently unclear in the case of atherosclerosis (AS) combined with chronic stress (CS). Methods New Zealand white rabbits were used to construct a CS and AS animal model. Proteomics and bioinformatics were employed to identify hub proteins in the adrenal gland related to CS and AS. Hub proteins were detected using immunohistochemistry, immunofluorescence assays, and Western blotting. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression of genes. In addition, a neural network model was constructed. The quantitative relationships were inferred by cubic spline interpolation. Enzymatic activity of mitochondrial citrate synthase and OGDH was detected by the enzymatic assay kit. Function of citrate synthase and OGDH with knockdown experiments in the adrenal cell lines was performed. Furthermore, target genes-TF-miRNA regulatory network was constructed. Coimmunoprecipitation (IP) assay and molecular docking study were used to detect the interaction between citrate synthase and OGDH. Results Two most significant hub proteins (citrate synthase and OGDH) that were related to CS and AS were identified in the adrenal gland using numerous bioinformatic methods. The hub proteins were mainly enriched in mitochondrial proton transport ATP synthase complex, ATPase activation, and the AMPK signaling pathway. Compared with the control group, the adrenal glands were larger and more disordered, irregular, and necrotic in the AS+CS group. The expression of citrate synthase and OGDH was higher in the AS+CS group than in the control group, both at the protein and mRNA levels (P < 0.05). There were strong correlations among the cross-sectional areas of adrenal glands, citrate synthase, and OGDH (P < 0.05) via Spearman's rho analysis, receiver operating characteristic curves, a neural network model, and cubic spline interpolation. Enzymatic activity of citrate synthase and OGDH increased under the situation of atherosclerosis and chronic stress. Through the CCK8 assay, the adrenal cell viability was downregulated significantly after the knockdown experiment of citrate synthase and OGDH. Target genes-TF-miRNA regulatory network presented the close interrelations among the predicted microRNA, citrate synthase and OGDH. After Coimmunoprecipitation (IP) assay, the result manifested that the citrate synthase and OGDH were coexpressed in the adrenal gland. The molecular docking study showed that the docking score of optimal complex conformation between citrate synthase and OGDH was -6.15 kcal/mol. Conclusion AS combined with CS plays a significant role on the hypothalamic–pituitary–adrenal (HPA) axis, promotes adrenomegaly, increases the release of glucocorticoid (GC), and might enhance ATP synthesis and energy metabolism in the body through citrate synthase and OGDH gene targets, providing a potential research direction for future related explorations into this mechanism.

Effect of chronic stress on expression and secretion of seminal vesicle proteins in adult rats.

Chronic stress (CS) is known to affect men's health especially fertility by reducing semen quality. Although the effects of CS on testicular function and sperm parameters are documented, changes of substances and secreting proteins in the seminal vesicle (SV) have never been reported. This study aimed to demonstrate the alterations of contents and expressions of proteins in seminal vesicle fluid (SVF) under CS. Fourteen adult rats were divided into control and CS groups (n=7/each). Control rats were not exposed to stressor, while the CS animals were immobilised by restraint cage (4hr/day) and followed by forced swimming (15min/day) for consecutive 60days. Biochemical substances and malondialdehyde (MDA) level in SVF were examined. Expressions of heat-shock protein 70 (Hsp70), caspases (Casp) 3 and 9, and tyrosine-phosphorylated (TyrPho) proteins were investigated in seminal vesicle tissue (SVT) and SVF. It was found that CS caused reductions of seminal epithelial height and secreted substance levels. Significantly, MDA levels in SVF and expressions of Hsp70, Casp and TyrPho proteins were increased in of CS animals. It was concluded that CS affected seminal secretion. Low quality of CS seminal plasma may associate with increase of MDA and expressions of secreted proteins.

Sympathetic nervous system contributes to enhanced corticosterone levels following chronic stress

Exposure to chronic stress often elevates basal circulating glucocorticoids during the circadian nadir and leads to exaggerated glucocorticoid production following exposure to subsequent stressors. While glucocorticoid production is primarily mediated by the hypothalamic–pituitary–adrenal (HPA) axis, there is evidence that the sympathetic nervous system can affect diurnal glucocorticoid production by direct actions at the adrenal gland. Experiments here were designed to examine the role of the HPA and sympathetic nervous system in enhancing corticosterone production following chronic stress. Rats were exposed to a four-day stress paradigm or control conditions then exposed to acute restraint stress on the fifth day to examine corticosterone and ACTH responses. Repeated stressor exposure resulted in a small increase in corticosterone, but not ACTH, during the circadian nadir, and also resulted in exaggerated corticosterone production 5, 10, and 20min following restraint stress. While circulating ACTH levels increased after 5min of restraint, levels were not greater in chronic stress animals compared to controls until following 20min. Administration of astressin (a CRH antagonist) prior to restraint stress significantly reduced ACTH responses but did not prevent the sensitized corticosterone response in chronic stress animals. In contrast, administration of chlorisondamine (a ganglionic blocker) returned basal corticosterone levels in chronic stress animals to normal levels and reduced early corticosterone production following restraint (up to 10min) but did not block the exaggerated corticosterone response in chronic stress animals at 20min. These data indicate that increased sympathetic nervous system tone contributes to elevated basal and rapid glucocorticoid production following chronic stress, but HPA responses likely mediate peak corticosterone responses to stressors of longer duration.

Neuroprotective effects of Asiaticoside.

In the central nervous system, Asiaticoside has been shown to attenuate in vitro neuronal damage caused by exposure to β-amyloid. In vivo studies demonstrated that Asiaticoside could attenuate neurobehavioral, neurochemical and histological changes in transient focal middle cerebral artery occlusion animals. In addition, Asiaticoside showed anxiolytic effects in acute and chronic stress animals. However, its potential neuroprotective properties in glutamate-induced excitotoxicity have not been fully studied. We investigated the neuroprotective effects of Asiaticoside in primary cultured mouse cortical neurons exposed to glutamate-induced excitotoxicity invoked by N-methyl-D-aspartate. Pretreatment with Asiaticoside decreased neuronal cell loss in a concentration-dependent manner and restored changes in expression of apoptotic-related proteins Bcl-2 and Bax. Asiaticoside pretreatment also attenuated the upregulation of NR2B expression, a subunit of N-methyl-D-aspartate receptors, but did not affect expression of NR2A subunits. Additionally, in cultured neurons, Asiaticoside significantly inhibited Ca2+ influx induced by N-methyl-D-aspartate. These experimental findings provide preliminary evidence that during excitotoxicity induced by N-methyl-D-aspartate exposure in cultured cortical neurons, the neuroprotective effects of Asiaticoside are mediated through inhibition of calcium influx. Aside from its anti-oxidant activity, down-regulation of NR2B-containing N-methyl-D-aspartate receptors may be one of the underlying mechanisms in Asiaticoside neuroprotection.

Discovery of blood transcriptomic markers for depression in animal models and pilot validation in subjects with early-onset major depression

Early-onset major depressive disorder (MDD) is a serious and prevalent psychiatric illness in adolescents and young adults. Current treatments are not optimally effective. Biological markers of early-onset MDD could increase diagnostic specificity, but no such biomarker exists. Our innovative approach to biomarker discovery for early-onset MDD combined results from genome-wide transcriptomic profiles in the blood of two animal models of depression, representing the genetic and the environmental, stress-related, etiology of MDD. We carried out unbiased analyses of this combined set of 26 candidate blood transcriptomic markers in a sample of 15–19-year-old subjects with MDD (N=14) and subjects with no disorder (ND, N=14). A panel of 11 blood markers differentiated participants with early-onset MDD from the ND group. Additionally, a separate but partially overlapping panel of 18 transcripts distinguished subjects with MDD with or without comorbid anxiety. Four transcripts, discovered from the chronic stress animal model, correlated with maltreatment scores in youths. These pilot data suggest that our approach can lead to clinically valid diagnostic panels of blood transcripts for early-onset MDD, which could reduce diagnostic heterogeneity in this population and has the potential to advance individualized treatment strategies.

Pre- and post-treatments with escitalopram protect against experimental ischemic neuronal damage via regulation of BDNF expression and oxidative stress

Selective serotonin re-uptake inhibitors (SSRI) have been widely used in treatment of major depression because of their efficacy, safety, and tolerability. Escitalopram, an SSRI, is known to decrease oxidative stress in chronic stress animal models. In the present study, we examined the neuroprotective effects of pre- and post-treatments with 20 mg/kg and 30 mg/kg escitalopram in the gerbil hippocampal CA1 region (CA1) after transient cerebral ischemia. Pre-treatment with escitalopram protected against ischemia-induced neuronal death in the CA1 after ischemia/reperfusion (I/R). Post-treatment with 30 mg/kg, not 20 mg/kg, escitalopram had a neuroprotective effect against ischemic damage. In addition, 20 mg/kg pre- and 30 mg/kg post-treatments with escitalopram increased brain-derived neurotrophic factor (BDNF) protein levels in the ischemic CA1 compared to vehicle-treated ischemia animals. In addition, 20 mg/kg pre- and 30 mg/kg post-treatments with escitalopram reduced microglia activation and decreased 4-hydroxy-2-nonenal and Cu,Zn-superoxide dismutase immunoreactivity and their levels in the ischemic CA1 compared to vehicle-treated ischemia animals after transient cerebral ischemia. In conclusion, these results indicated that pre- and post-treatments with escitalopram can protect against ischemia-induced neuronal death in the CA1 induced by transient cerebral ischemic damage by increase of BDNF as well as decrease of microglia activation and oxidative stress.

Differential regulation of hippocampal glucocorticoid receptors mRNA and fast feedback: relevance to post-traumatic stress disorder.

Hippocampal glucocorticoid receptors (GR and MR) play an important role in glucocorticoid negative feedback. Abnormalities in negative feedback are found in depression and in post-traumatic stress disorder (PTSD), suggesting that GR and MR might be involved in the pathophysiology of these disorders. Enhanced negative feedback, the PTSD-specific neuroendocrine abnormality, can be induced in animals using a single prolonged stress (SPS) paradigm (a number of different stressors in one prolonged session, 'no stress' interval and a testing session one week later). In the current study, we examined hippocampal GR and MR mRNA distribution in the same animals that exhibited altered negative feedback following the SPS. Seven groups of adult Sprague-Dawley male rats (seven animals each) were used in two studies, comparing unstressed controls to acutely stressed animals (SPS: 24 h group), SPS animals (seven and 14 days), and SPS + chronic stress animals. GR and MR mRNA distribution across hippocampal subfields was studied using in-situ hybridization with 35S-labelled cRNA probes. Acute stress produced down-regulation of GR and MR mRNA across all hippocampal subfields. Seven days later (SPS-7 group), there was a differential recovery, with GR mRNA reaching higher than the prestress levels, and MR mRNA remaining down-regulated. The same differential regulation was present in the 14-day group. Chronically stressed animals that exhibited normal fast feedback also had normalization in their GR and MR mRNA levels. The MR/GR ratio was decreased only in animals that had enhanced fast feedback. These findings suggest that the increase in GR, in hippocampus is involved in the fast feedback hypersensitivity observed in the SPS animals, and might also underlie enhanced dexamethasone sensitivity found in PTSD. Since differential activation of GR and MR can modulate memory, behavioural responsivity, anxiety and fear, change in MR/GR ratio might also explain other PTSD-related phenomena.

Changes in circulating and tissue angiotensin II during acute and chronic stress.

Changes of angiotensin II and cAMP in plasma, brain tissue, adrenal gland and cardiovascular tissue during the acute and chronic stress were studied in rats. The acute stress group was subjected to compulsive cold water swimming for 20 min, while the chronic stress group was exposed to an ambient temperature of 4-8 degrees C for 5 days. The results indicated that plasma angiotensin II levels were significantly increased in both stress groups, reaching up to 900% and 134% of the control in the acute and chronic groups, respectively. Angiotensin II contents in the anterior hypothalamus, medulla oblongata, myocardium, vasculature and adrenals were also elevated in both groups. With the exception of the adrenals, the contents of tissue angiotensin II in the chronic stress animals were significantly higher than those of the acute stress animals. In contrast, cAMP levels in plasma and tissue (hypothalamus and adrenals) and corticosterone levels in plasma in the acute stress group were all higher than those in the chronic stress animals, although the levels of the latter group were also increased compared with the control group. These results suggest that circulating and tissue angiotensin II may play an important role in the acute and chronic stress responses and that angiotensin II should be classified as a stress hormone.

MIF-1 is active in a chronic stress animal model of depression

MIF-1 was tested in an animal model of depression that used unpredictable chronic stress. In this paradigm, rats received either no stressors or a daily protocol of a variety of stressors for 20 days, during which time daily, intraperitoneal injections of various compounds were given. The tricyclic antidepressant imipramine (5 mg/kg) and low doses (0.1 and 1.0 mg/kg) of MIF-1 significantly increased activity and decreased defecation in an open field on day 21. No dose of naloxone (0.01–10.0 mg/kg) acted as an antidepressant. A high dose (10.0 mg/kg) of MIF-1 significantly increased the effects of chronic stress and produced hyperalgesia. Chronically-stressed rats were significantly more analgesic than controls. The results indicate that MIF-1 can act as an antidepressant in this model.

Pituitary Regulation in Endogenous Depression

This chapter describes the pituitary regulation of adrenocorticotropin hormone (ACTH) and b-endorphin in endogenous depression. The advantages and difficulties are very well exemplified in the study of the hypothalamo-pituitary-adrenal (HPA) axis in endogenous depression. The design and interpretation of the endocrine studies in depression depends upon the understanding of pituitary regulatory strategies in normal and disease states. The chapter reviews the general feedback regulatory strategies at the pituitary level. The chapter presents the basic science studies on pituitary regulatory strategies in chronic stress animal models and pituitary regulation in diseases of the HPA axis with special emphasis on Cushing's disease, one of the best-characterized pituitary regulatory diseases. With this, it is easy to explore the question of pituitary regulation in endogenous depression. This chapter also focuses on the factors controlling the release of adrenocorticotropic hormone (ACTH) and related peptides. The delineation of the ACTH precursor, the isolation and sequencing of CRF, and the development of sensitive radioimmunoassays for ACTH and related peptides to replace less sensitive bioassay methods, have lent great impetus to the exploration of the HPA axis. These new tools enable a new level of sophistication in the studies of pituitary regulation in normal and disease states.