Preliminary Research On The Mechanism Of P38 MAPK Pathway In Depression Like Behavior Of Rats After MCAO Induced By CRS

BackgroundPSD is one of the common complications after stroke,which is mainly manifested by emotional disturbances such as loss of interest,sleep disturbance,slow thinking,low mood and even hopeless thoughts and behaviors.Chronic restraint stress（CRS）behavioral restriction mimics the behavioral restriction of hemiplegia in clinical stroke patients,gradually causing depression and anxiety-like manifestations such as depression and mood disorders,and down-regulating synaptic plasticity in the cortex and hippocampus.After cerebral ischemia injury in rats,the activation and regulation of p38 MAPK signaling pathway participates in the hypoxia ischemic response and inflammatory response of brain nervous system,and affects the post injury repair of brain function and morphology,which may be one of the molecular mechanisms of PSD occurrence and development.Objectives1.The rat model of PSD was established by chronic restraint stress combined with MCAO operation,and the depression like behavior,learning and memory of rats were observed.2.To investigate the role of p38 MAPK signaling pathway in depressive-like behavior in post-stroke rats exposed to CRS.Methods1.Construction of PSD animal model rats: 6-week-old male SD rats with a body weight of 200-220 g were selected,and then randomly stratified into healthy control（Ctrl）group,stroke（MCAO）group,post-stroke depression（PSD,MCAO+CRS）group and chronic restraint stress（CRS）group;SD rats in both MCAO and PSD groups were treated with left middle cerebral artery occlusion model（MCAO）method to construct middle cerebral artery.The rat model of ischemia-reperfusion was used.Longa score was used to evaluate the neurological damage of rats after MCAO.If the score was ≥ 1 and < 4,SD rats with an evaluation score of 1-3 were selected into the group;the Ctrl group was selected.No suture was inserted,and no surgical treatment was performed;the rats in the PSD group were given CRS intervention after 1 week of MCAO recovery;the intensity and duration of chronic restraint stress were the same in the CRS group and the PSD group.Behaviors such as body weight measurement,sucrose preference test（SPT）,Forced Swimming Test（FST）,open-field test（OFT）,and Y-maze（Y-maze）were selected.The changes of general physical state,vital signs,loss of hedonic behavior,hopeless state,anxiety and depression-like behavior,exploratory exercise ability,learning and memory ability,etc.After the rats in the PSD group were successfully constructed,follow-up experimental behaviors such as gene detection,protein expression and morphology were carried out.2.Western Blot method was used to detect the proteins of MKK3,MKK6,p38 MAPK,phospho-p38 MAPK,Bax,Bcl-2 and synaptic proteins SYP and PSD95 in the left hippocampus,left cortex,and left prefrontal region of SD rats in each group.Changes in the level of protein expression,etc.3.Nissl staining and TUNEL staining were used to compare the morphological and structural changes,damage degree and apoptosis of neurons in the cortex and hippocampus（CA1,CA3,DG）of rats in each group.Results1.Behavioral measurement results: After 1 week of normal adaptation,there was no statistical difference in the baseline measurement indicators of behavior among SD rats in each group（P > 0.05）.The rats in the MCAO group and the PSD group developed functional impairments such as behavioral limitation within 1 week after the MCAO operation,and then gradually recovered one week later.The body weight of the rats in the PSD group decreased significantly（P < 0.01 vs Ctrl）,the sucrose water preference index of the rats in the PSD group was significantly decreased（P < 0.01 vs Ctrl）;the immobility time of the rats in the PSD group was significantly prolonged in the forced swimming test（P < 0.01 vs Ctrl）,the total range of activities in the forced swimming test was significantly decreased（P < 0.05 vs Ctrl）,and the total distance and range of activities of horizontal movement in the open field test results of the rats in the PSD group were significantly decreased（P < 0.01 vs Ctrl）,the PSD group The Y-maze time and total activity of the rat Y-maze decreased,but there was no statistical difference（P > 0.05 vs Ctrl）.2.Western blot detection results: The expressions of MKK6 and MKK3 proteins in the left parietal cortex of the PSD group were significantly decreased（P < 0.05 vs Ctrl）,the expression of p-p38 protein was increased（P < 0.05 vs Ctrl）,and the expression of p38 protein was increased,but not statistically significant（P > 0.05）;the protein expressions of MKK6（P < 0.05 vs Ctrl）and MKK3 in the left hippocampus of rats in the PSD group were decreased,and the protein expression of p-p38 was increased（P < 0.05 vs Ctrl）.The expression was not statistically significant（P > 0.05）.The expression of p38 in the left prefrontal lobe of rats in the PSD group was increased（P < 0.05 vs Ctrl）,while the expression of p-p38 protein was not statistically significant（P > 0.05 vs Ctrl）,The expression of apoptosis-related protein Bax/Bcl-2 in the left parietal cortex,left hippocampus,and left forehead of rats in PSD group increased（P < 0.05 vs Ctrl）,the left parietal cortex,left hippocampal synapse-related protein synaptophysin（SYP）and PSD95 expression was decreased（P < 0.05 vs Ctrl）,and synaptophysin（SYP）and PSD95 were decreased in the left prefrontal lobe,but not statistically significant（P > 0.05）.3.Nissl staining results: In the Ctrl group,the neurons in the hippocampal cortex were full in shape,with clear edges,dense and neat arrangement,and full and lightly stained nuclei;In the MCAO group,the PSD group and the CRS group,the morphological and structural abnormalities of neurons in the hippocampus and cortex were abnormal,and some marginal cells were atrophied,deformed,loose,and the number of cells decreased.The damage in the PSD group was more severe than that in the MCAO group and the CRS group.4.The results of TUNEL staining showed that apoptotic cells could be seen in the hippocampal DG area and cortical area of the rats in the PSD group.CRS combined with MCAO could induce neuronal damage and apoptosis in the rat brain tissue.Conclusions1.CRS could induce depressive and anxiety-like behavior in rats after MCAO.2.CRS combined with MCAO induced depression like behavior may be activated via p38 MAPK pathway.