Effects Of Chronic Stress On The Expression Of CRHR1 In Hippocampus Of Stroke Rats

Background Post-stroke depression(PSD)is the most common neuropsychiatric disorder after stroke.It affects the recovery of daily living ability and neurological deficits of patients,and the disability and mortality rates also gradually increase.The body is exposed to external stress to activate the hypothalamic-pituitary-adrenal(HPA)axis and the corticotropin-releasing hormone(CRH)in the hypothalamic paraventricular nucleus in a short period of time with positive feedback corrosotropin-releasing hormone receptor1(CRHR1)expression is up-regulated in a way that CRHR1 transmits signals by coupling with the alpha subunit(Gs-α)of the stimulating G protein,leading to adenylate Cyclase stimulation produces a second messenger,cyclic adenosine monophosphat(cAMP).The accumulation of cAMP in turn stimulates the activation of cyclic-AMP dependent protein kinase A(PKA),phosphorylates the cAMP response element binding protein(CREB)protein,and makes cAMP located on the nuclear CRH promoter response element(CRE)phosphorylation induces changes in the cognitive function of the pCREB-BDNF-TrkB pathway.Objective This study intends to explore the effects of chronic stress on the behavioral level and expression of CRHR1,pCREB,BNDF,and TrkB in stroke rats,and to explore the mechanism of CRHR1 in the process of chronic stress after stroke.Methods 1.Preparation of PSD model rats: 180-200 g male SPF-grade SD rats were randomly divided into a sham operation group,a stroke group,a depression group,and a PSD group;the stroke group and the PSD group were treated with left middle cerebral artery embolization middle cerebral artery occlusion model(MCAO)method was used to prepare a model of middle cerebral artery ischemia-reperfusion,and rats with Longa score to evaluate neurological impairment ≥1 and < 4 were included in the group;in the sham operation group,only the left neck was isolated.Arteries and internal carotid arteries were not used for the suture method.The depression group was modeled with simple chronic unpredictable mild stimulation.The PSD group was given chronic unpredictable mild stress(CUMS)1 week after MCAO.Weight changes,sucrose preference test,elevated cross-maze,open-field test(OFT),and Y-maze were used to evaluate the lack of pleasure,depression,anxiety-like behavior,motor exploration ability,and learning in rats cognitive behaviors such as memory.By comparing the behavior with the model rats in the depression group,the PSD group model rats successfully prepared by chronic stress for post-stroke stimulation were screened for protein,gene and morphology experiments.2.Real-time fluorescent quantitative PCR(q-PCR)was used to detect the expression levels of CRHR1,CREB,BDNF and TrkB mRNA in hippocampus of each model rats.3.Western blot method was used to detect the expression levels of CRHR1,pCREB and CREB,BDNF,TrkB in the hippocampus of each group of model rats.4.Nissl staining was used to observe the morphological structure and damage of neurons in hippocampal CA1 area of model rats.Results 1.Behavioral results: There was no statistical difference in behavioral tests between model rats in each group on day 7(baseline)(P > 0.05).In the stroke group and the PSD group,the corresponding cognitive dysfunction occurred 1 week after the MCAO operation,and gradually recovered in the later stage.On day 42(1 week after MCAO +CUMS stimulation for 4 weeks),the weight change in the PSD group was significantly lower than that in the sham operation group and the stroke group(P < 0.01),which was different from the depression group(P < 0.05);The sucrose water preference index waslower than that of the sham operation group(P < 0.05),and the sucrose water preference index of the PSD group was significantly lower than that of the sham operation group,stroke group,and depression group(P < 0.05).The behavior evaluation scores of PSD group were significantly lower than those of sham operation group and stroke group(P <0.05).The time ratio of the elevated cross labyrinth into the open arm in the PSD group was significantly lower than that in the sham operation group,stroke group,and depression(P < 0.05).The horizontal motion distance in the open field test of the PSD group was significantly lower than that of the sham operation group and the stroke group(P < 0.05).The percentage of correct corrective alternations of Y labyrinth in the PSD group decreased significantly in the sham operation group,stroke group,and depression group,and the percentage of correct corrective alternations of Y maze in the different arm in the depression group decreased significantly(P < 0.05).2.The results of q-PCR showed that the expression of CRHR1 mRNA in hippocampus of PSD group was significantly higher than that of sham operation group and stroke group(P < 0.01).The CREB gene expression level in the PSD group was significantly lower than that in the sham operation group(P < 0.01).Compared with the sham operation group,the CREB gene expression level in the stroke group was significantly lower(P < 0.05).The CREB gene expression level in the PSD group was lower than that in the stroke group,but no Statistical significance(P < 0.05).The relative expression level of BDNF mRNA in PSD group was significantly lower than that in sham operation group and stroke group(P < 0.01),and the expression level of BDNF mRNA in stroke group was significantly lower than that in sham operation group(P < 0.05).The TrkB mRNA expression level in the PSD group was significantly lower than that in the sham operation group(P < 0.01),and the TrkB mRNA expression level in the stroke group was lower than that in the sham operation group(P < 0.05).3.Western Blot test results showed that CRHR1 expression level in PSD group was significantly higher than that in sham operation group and stroke group;pCREB proteinexpression level in PSD group was significantly lower than that in sham operation group and stroke group(P < 0.05).The expression level of CREB protein in hippocampus of PSD group was lower than that of sham operation group(P < 0.05).The expression of BDNF protein in PSD group was significantly lower than that in sham operation group and stroke group(P < 0.01).Compared with the sham operation group,the expression of TrkB protein in the stroke group and the PSD group was reduced(P < 0.05).The expression of the TrkB protein in the PSD group was lower than that in the stroke group,but it was not statistically significant(P > 0.05).4.The results of Nissl staining showed that the vertebral body layer of the stroke group and the PSD group had different degrees of looseness,and the morphological structure of the neurons was obviously abnormal compared with the sham operation group.The number of cells in the stroke group was reduced to a lesser extent,and the cell gap Slightly increased;the nucleus of the PSD group was significantly reduced,the cytoplasm was atrophy,there was a hollow,the axon dendrite structure was incomplete,and even the cell structure disappeared,the arrangement of Nissl bodies was severe,the number was seriously reduced,and the neuron damage was serious.Conclusion Chronic stress stimulation after stroke in rats may activate the expression of CRHR1 in the hippocampus of stroke rats and activate its downstream pathway pCREB-BDNF-TrkB,leading to neuronal damage and cognitive function in post-stroke depression rats decline.