The Mechanism Of Amygdala Astrocytes Apoptosis In Restraint Stress Rats

During the forensic practice,it is occurred frequently that the sustained binding or limit position cause the parties mental injury.Since there is no objective standard of judgements,the relationship between stress and mental injury is difficult to define in the forensic identification,which had caused a lot of adverse social influences.Therefore,it is very meaningful to uncover the molecular mechanism underlying the stress-releated mental impairments.Amygdala is involved in the emotional value of mood and cognition,and plays a critical role in fear and anxiety-related behaviors.Rats with damaged bilateral amygdala lack of fear resolution.Clinical imaging studies have found that patients with major depression and dementia,the amygdala volume change will happen.Glial cells consititute about 50% of brain cells,amygdala is no exception,astrocytes are known to play an important role in the normal functioning of the nervous system.Several studies have demonstrated that astrocyte apoptosis is a critical factor in the development of several central nervous system injury and degenerative diseases.Therefore,it is very meaningful to uncover the molecular mechanism of astrocytes apoptosis underlying the stress-releated mental impairments.Hypothalamic pituitary adrenal axis(HPA)activation followed by increased CRH,ACTH and GC in the serum is one of the neuroendocrine response induced by stress.Abundant glucocorticoid receptors(GR)and mineralocorticoid receptors(MR)are expressed in hypothalamus,hypophysis,prefrontal cortex and hippocampus,glucocorticoid(GC)could penetrate the blood-brain barrier and bind with GR and MR to provide a negative feedback to the overactivation of neuroendocrine response.However,the increasing GC in plasma has neurotoxticity.Abundant glucocorticoid receptors were present in cytoplasm of astrocytes of the amygdala,whether or not increased GC can penetrate the blood-brain barrier and bine with GR to induce astrocytes apoptosis is unknown.Recent studies suggest that endoplasmic reticulum stress(ERS/ER Stress)is a new signaling pathway of apoptosis.Overactivated ERS pathway will trigger apooptosis ultimately.It was proved that ER stress is linked to cell apoptosis in various neurodegenerative diseases,such as Alzheimer’s disease and PTSD.Whether or not ERS participates in the GC induced astrocytes apoptosis in amygdala has been undiscovered.Based on these findings,we proposed that increased GC might play an essential role in stress-induced astrocyte apoptosis through endoplasmic reticulum stress.In this study,we used the rat chronic restraint stress model and human astrocytes to investigate the effects of GC on astrocyte apoptosis in vivo and in vitro.Furthermore,we also examined the roles of endoplasmic reticulum stress in GC-induced astrocyte apoptosis.The present study results will provide theoretical basis for reveal the underlying mechanism of stress-releated mental impairments.Part one The injury effects of chroninc restraint stress on amygdala astrocytes in ratsObjective: Through rats chroninc restraint stress model,to explore the injury effects of chroninc restraint stress on amygdala astrocyte in rats.Methods: Establish a rat chroninc restraint stress(CRS)model.All rats were randomly divided into 2 groups: control group and CRS group;CRS group further divided into 4 groups: 1d,7d,14 d and 21 d.The body weights and adrenal weights were recorded daily.The behavior changes were evaluated using an elevated plus-maze(EPM).The plasma GC levels were tested by ELISA.The expressions of glucocorticoid receptor(GR)and glial fibrillary acid protein(GFAP)were determined by immunohistochemistry and Western blot.Ultrastructure changes of astrocyte were observed by transmission electron microscope(TEM).Annexin V and PI staining was used to detect the apoptosis rate.Results:1 Changes of body weights: Compared with control group,CRS group rats show a slow increase of body weight.2 Changes of adrenal: Compared with control group,the adrenal weights were increased in 7d,14 d and 21 d CRS rats,along with significantly widened adrenal cortical.3 Changes of glucocorticoid levels in serum by ELISA: A significantly increase of CRH level was observed in 7d,14 d and 21 d CRS group rats compared with control group.A significantly increase of ACTH level was observed in 21 d CRS group rats compared with control group.A significantly increase of GC level was observed in 14 d and 21 d CRS group rats compared with control group.4 Compared with control group,the CRS rats displayed a significantly decrease of standing time in open arms and of open arm entries from 7d to 21 d group,and especially in 21 d group.However,there was no significant difference in the total number of arm entries and total distance among the 5 groups,indicating that CRS has trifling impact on overall locomotion of rats.5 The expression of GR and GFAP by Western blot: Western blot indicated that the levels of GR were also gradually elevated in 7d,14 d and 21 d CRS group compared with control group,but the changes of GFAP displayed a reverse changes.6 The expression of GR and GFAP by Immunohistochemistry: Immunohistochemistry staining indicated that compared with control group,GR positive cells were gradually increased in the rat amygdala of 7d,14 d and 21 d CRS group rats.However,GFAP positive cells were gradually decreased in the rat amygdala of 7d,14 d and 21 d CRS group rats.7 Ultrastructure changes of glials in amygdala by TEM:Compared with control group,there is a variety of glial injuries were observed in CRS rats,including cell edema,widened nuclear-week gap,fusion of mitochondrial cristaes and membranes,fusion and degranulation of rough endoplasmic reticulum,condensation and margination of nuclear chromatin and nuclear pyknosis.8 The glial apoptosis in amygdala was determined by FCM with AnnexinV/PI staining: Compared to control group,the percentage of apoptosis cells was remarkably increased after CRS treatment.Conclusions: The present experiments successfully established chronic restraint stress model in rats.The rats displayed palpable anxiety-like behavioral changes,decreased numbers of GFAP positive astrocytes and apoptotic ultrastructural changes in amygdala glial cells.Part two The effects of glucocorticoid(GC)on amygdala astrocyte apoptosis and on the expression of marker protein of endoplasmic reticulum stress in chronic restraint stress ratsObjective: To observe the damage effects of GC on astrocyte in CRS rats amygdala,and to explore the related mechanism of endoplasmic reticulum stress.Methods: All rats were randomly divided into 4 groups: control group,CRS group,RU486 group and solvent control group(propylene glycol group).The behavior changes were assayed by an elevated plus-maze(EPM).The expressions of GFAP and β-subunit of S100(S100β)were determined by immunohistochemistry and Western blot,the expressions of GRP78、CHOP and Caspase 12 were determined by western blot.Annexin V and PI staining was used to detect the apoptosis rate.Results:1 The CRS rats displayed a significantly decrease of standing time in open arms and of open arm entries.Compared with the CRS,both standing time in open arms and of open arm entries were increased in RU486 group.There was no significant difference between the CRS and propylene glycol group animals.However,there was no significant difference in the total number of arm entries and total distance among the 4 groups.2 The expression of GFAP and S100β by Immunohistochemistry and Western blot: Immunohistochemistry staining indicated that GFAP and S100β positive cells were significantly decreased in the rat amygdala of restraint stress model.Therats treated with RU486 displayed a significantly increased percentage of GFAP and S100β positive cells compared to the CRS.However,there is no significant difference between the CRS and propylene glycol group.These results were further validated by Western blot analysis.The statistical results of Western blot showed that GFAP and S100β proteins level in the amygdala were significantly decreased in CRS and propylene glycol group compared to normal group rats,samples from RU486 treated rats displayed an obviously increased proteins levels of GFAP compared to the CRS and propylene glycol animalsl.However,there is no significant difference between the CRS and propylene glycol group.3 Western blot analyses demonstrated that CRS significantly increased the expression of GRP78,CHOP and caspase12 proteins levels in the amygdala.The GR antagonist RU486 reversed the CRS-induced increases in GRP78,CHOP and caspase12 levels.However,there is no significant difference between the CRS and propylene glycol group.4 The glial apoptosis in amygdala was determined by FCM with AnnexinV/PI staining: Compared to control group,the percentage of apoptosis cells was remarkably increased after CRS treatment,and the increased apoptosis was reduced by RU486 administration,there is no significant difference between the CRS and propylene glycol group.Summary: GC can induce astrocytes apoptosis in amygdala in chronic restraint stress challenged rat,which is related to ERS.Part three The ERS mechnisms of dexamethasone on human astrocytes apoptosis.Objective: To observe the apoptosis of human astrocytes(HA)by dexamethasone(DEX),and discuss the related mechanism of endoplasmic reticulum stress.Methods:1 Immumofluorescence method was used to examine the expression of GR in human astrocytes.2 The effects of different concentrations of DEX(10-9 M~10-5 M)on astrocytes survival rate were determined by MTT.3 The expression of GFAP,S100β,GRP78,CHOP,Caspase 12,p-PERK,p-eIF2αand ATF4 were detected by Western blot.Annexin V and PI staining was used to measure the apoptotic rate of astrocyte.4 Role of CHOP in DEX-induced cell apoptosis.The siRNA CHOP transfection was performed before the DEX exposure in human astrocytes.Western blot was performed to verify the efficiency of siRNA CHOP transfection.Annexin V and PI staining was used to measure the apoptotic rate of astrocyte.Results:1 The expression of GR in HA: GR was localized dominantly in the cytoplasm in HA.2 Cell viability was determined by MTT.This assay results indicated that compared with con group,DEX(10-9M~10-5M)treatment gradually decreased cell viability.3 Apoptosis was determined by flow cytometry.Compared with Control group,the apoptotic rate of HA was significantly increased after DEX treatment for 6h,but the apoptotic rate of HA was decreased after co-treated with RU486.However,there is no significant difference between the Control group and RU486 group.The percentages of apoptosis cells remarkably increased in DEX group,and the increased apoptosis was reduced by CHOP siRNA,there is no significant difference between the DEX and control siRNA group.4 The GRP78,CHOP and caspase12 expresssion was determined by Western blot.Western blot analysis demonstrated that DEX significantly increased the expression of GRP78,CHOP and caspase12.The GR antagonist RU486 reversed the DEX-induced increases in GRP78,CHOP and caspase12 levels.However,there is no significant difference between the DEX and Control group.5 The p-PERK,p-eIF2 αand ATF4 expresssion was determined by Western blot.Western blot analysis demonstrated that DEX significantly increased the expression of p-PERK,p-eIF2αand ATF4.The GR antagonist RU486 reversed the DEX-induced increases in p-PERK,p-eIF2αand ATF4 levels.However,there is no significant difference between the RU486 and Con group.6 Western blot analysis demonstrated that CHOP siRNA significantly decreased the expression of CHOP.Compared with Control group,the percentages of apoptosis cells remarkably increased in DEX group,and the increased apoptosis was reduced by CHOP siRNA,there is no significant difference between the DEX and control siRNA group.Summary:DEX can induce apoptosis of astrocytes independtely,and the increased apoptosis can be reduced by CHOP siRNA,which is related to the activation of endoplasmic reticulum stress and p-PERK/p-eIF2α/ATF4 /CHOP pathway through GR.Taken together,our studies provide important views in deeply understanding the effects of GC on astrocytes injury in rats CRS model and human astrocytes culture in vitro.The results proved that GC and ERS play an important role in restraint stress-induced astrocytes injury.It is very meaningful to evaluate the effects of GC,it will offer theoretical foundation for the forensic identification and treatments of stress-related mental impairments.