Microglia In The Anterior Cingulate Cortex Are Involved In Regulating Pain And Depression Behavior In A Depression And Pain Comorbidities Mouse Model

Objective: Chronic restraint stress combined with pain stress to establish a depression and pain comorbidities mouse model,and to investigate whether the activation of microglia in the anterior cingulate cortex and brain-derived neurotrophic factor are involved in regulating pain-depression behavior in mice.Methods:(1)Mouse were randomly divided into 3 group:SNI+CRS(n=4),SNI(n=4),Control(n=4),Then the mouse of SNI+CRS group were performed spared nerve injury surgery and applied chronic restraint stress over a period of time,the mouse of SNI group were performed spared nerve injury surgery,while Control group were left untreated.Paw withdrawal mechanical threshold and paw withdrawal thermal latency were measured at 1,3,5,7,14 and 18 days after spared nerve injury surgery;Sucrose preference test and tail suspension test were perform at17,18 days after spared nerve injury surgery.By perform those experiments we can evaluate whether it’s successful at constructing a depression and pain comorbidities model.(2)Microglia marker ionized calcium binding adapter molecule 1 was stained by immunofluorescence in brain tissue sections of mice in the SNI+CRS group,the activation of microglia in the anterior cingulate cortex was measured by integrated optical density of ionized calcium binding adapter molecule 1,and the difference was compared with that of the Control group.(3)Mouse were randomly divided into 3 group: PLX3397(n=5),Saline(n=5),Control(n=5),Then PLX3397 group and Saline group were performed spared nerve injury surgery and chronic restraint stress to construct depression and pain comorbidities mouse model,left Control group untreated.Day 19 after spared nerve injury surgery,brain stereotactic injection was used to deliver corresponding drugs to anterior cingulate cortex,PLX3397 group was injected microglia inhibitor PLX3397,Saline group was injected normal saline,left Control group untreated.Paw withdrawal mechanical threshold was measured at 1,3,5,7,14,18 and 21 days after spared nerve injury surgery;Sucrose preference test was perform at 17,22 days after spared nerve injury surgery;Tail suspension test was perform at18,21 days after spared nerve injury surgery.By perform those experiments we can evaluate whether the pain and depression behavior of mice changed after microglia inhibitor injection.(4)Microglia marker ionized calcium binding adapter molecule 1 was stained by immunofluorescence in brain tissue sections of mice in the PLX3397 group to observe the changes of microglia after microglia inhibitor injection.(5)Brain sections of mice from SNI+CRS group,Control group,PLX3397 group and Saline group were stained for brain-derived neurotrophic factor by immunofluorescence staining,then evaluate the expression and distribution of brain-derived neurotrophic factor in anterior cingulate cortex.(6)Brain sections of mice from SNI+CRS group and Control group were stained for NLRP3 by immunofluorescence staining,then evaluate the expression of NLRP3 in anterior cingulate cortex.Results:(1)Chronic restraint stress combined with spared nerve injury pain stress construct depression and pain comorbidities mouse model successfully,the mice showed mechanical hyperalgesia,extended tail immobility time,decreased sucrose preference ratio.(2)Microglia are activated in the anterior cingulate cortex of depression and pain comorbidities mouse model,and the integrated optical density of the microglia activation marker ionized calcium binding adapter molecule 1was increased.(3)Microglia inhibitor was successfully injected into the anterior cingulate cortex,and microglia death was observed at the injection site.(4)Microglia inhibitor injected into the anterior cingulate cortex relieved mechanical hyperalgesia and depression-like behavior in a depression and pain comorbidities mouse model.(5)The content of brain-derived neurotrophic factor in anterior cingulate cortex increased in depression and pain comorbidities mouse model.After microglia inhibitor was injected,brain-derived neurotrophic factor levels in the region where microglia were inhibited is lower,while on the edge of that region showed higher brain-derived neurotrophic factor levels.(6)There was no significant difference in NLRP3 content between SNI+CRS group and Control group in the anterior cingulate cortex.Conclusions:Abnormally activated microglia in region Ⅱ of the anterior cingulate cortex are involved in promoting mechanical hyperalgesia and depression-like behavior In a depression and pain comorbidities mouse model.Brain-derived neurotrophic factor may play an important role in microglia regulation of the anterior cingulate cortex.NLRP3 may not be involved in microglia regulation of the anterior cingulate cortex.