The Modulatory Effect Of Prefrontal Cortical Astrocytes On Diabetic Neuropathic Pain

Objective: Previous research showed that STZ-induced DNP rats were significantly activated in the prefrontal cortex astrocytes,the expression of proinflammatory cytokines TNF-? and IL-1? were significantly up-regulated.However,due to the inability to achieve astrocyte-specific modulation,the relationship with the pathogenesis of DNP remains unclear.Therefore,we used chemogenetics to specifically regulate astrocytes and neuronal activity in PFC of STZ-induced DNP rats and wild-type rats.To analyze the effects of astrocyte activity on proinflammatory cytokines and neuronal activity,and to elucidate the role of PFC astrocytes in DNP and its regulatory mechanisms.Methods: Stereotaxic surgery was performed to infuse adeno-associated virus expressed the inhibitory receptor hM4 Di carrying the GFAP promoter or the CaMKII promoter into bilateral prefrontal cortex of DNP rats induced by STZ.And adeno-associated virus expressed the excitatory receptor hM3 Dq carrying the GFAP promoter or the CaMKII promoter was infused into bilateral prefrontal cortex of wild-type rats.Experimental procedures started three weeks later.The exogenous ligand CNO was injected intraperitoneally to observe the effect of specific regulation of astrocyte or neuronal activity on pain.After the behavioral experiment,the brain tissue was frozen and sectioned,the viral infection site was confirmed under a fluorescence microscope.Fluorescence immunohistochemistry was used to detect the fluorescence intensity of GFAP and the positive immunoreactivity of Fos protein in rats prefrontal cortex.The levels of proinflammatory cytokines TNF-and IL-1? in rats prefrontal cortex were determined by ELISA.Results:(1)Chemogenetics specifically inhibited PFC astrocyte activity in DNP rats significantly improved STZ-induced mechanical allodynia;immunofluorescence histochemical assay showed that astrocytes activation,GFAP up-regulated and astrogliosis,astrocytes were recative state in DNP model rats in contrast to the control group,and the expression of Fos protein was significantly up-regulated;after chemogenetics treated the fluorescence of GFAP was decreased,astrocyte return to resting states and Fos protein expression decreased;ELISA results showed that the levels of proinflammatory cytokines TNF-? and IL-1? in DNP model group were notably enhanced in contrast to the control group and decreased after chemogenetics inhibited astrocyte activity;(2)Chemogenetics specifically inhibited PFC neurons activity in DNP rats significantly improved STZ-induced mechanical allodynia;immunofluorescence histochemical assay showed that compared with control group,the expression of Fos protein and GFAP in the PFC of DNP model group was significantly up-regulated,GFAP astrogliosis and astrocytes were recative state;chemogenetics inhibited the activity of PFC neurons could decrease Fos protein expression;the fluorescence intensity of GFAP decreased and the cells morphology tended to be resting states;(3)Chemogenetics activated wild-type rats PFC astrocytes induced mechanical allodynia;immunofluorescence histochemical assay showed that compared with control group,GFAP up-regulated and astrogliosis,astrocytes were recative state and Fos protein expression increased after chemogenetics activated astrocytes;ELISA results showed that the chemogenetics activated PFC astrocytes notably enhanced the levels of proinflammatory cytokines TNF-? and IL-1? in contrast to the control group;(4)Chemogenetics activated wild-type rats PFC neurons induced mechanical allodynia;immunofluorescence histochemical assay showed that compared with the control group,chemogenetics activated PFC neurons could increase Fos protein expression,GFAP up-regulated and astrogliosis,astrocytes were recative state.Conclusion: 1.Activation of astrocyte in prefrontal cortex induces diabetic neuropathic pain in diabetic rats;2.Activated astrocytes release TNF-? and IL-1?,causing activation of neurons,thereby regulating DNP.