Downregulation Of KCC2 In Anterior Cingulate Cortex Participates In SMIR-mediated Chronic Post-surgical Pain In Rats

BackgroundChronic post-surgical pain(CPSP)has become an increasingly important health problem in the world.The high incidence of chronic post-surgical pain seriously affects the prognosis of patients,prolongs the hospital stay,increases medical expenditures,and reduces the quality of life of patients after surgery.Therefore,the problem of how to better prevent and treat chronic postoperative pain needs to be solved urgently.Central sensitization caused by peripheral injury is the main mechanism of chronic pain.Studies have shown that anterior cingulate cortex(ACC),as the most important cerebral cortex for pain activation,receives pain signals from the spinal dorsal horn and mediates the occurrence and development of pain.The decrease in ACC inhibitory transmission function(disinhibition)plays an important role in chronic pain.Disinhibition leads to increased excitability of excitatory projection neurons and increased pain transmission signal conduction.The increase in excitability of central projection nerve cells mainly relies on potassium chloride co-transporter 2(KCC2).KCC2 is the main regulator of chloride ion balance in the central nervous system.KCC2 can transport Cl-out of the cell and maintain a low concentration of Cl-in the cell,thereby maintaining the synaptic inhibition mediated by GABA receptor and glycine receptor.When KCC2 expression is down-regulated,it will cause Cl accumulation in nerve cells,Cl-is transported out of the cell through GABAA receptors,nerve cells change from hyperpolarization to depolarization,cell excitability increases,pain signal transmission increases,leading to chronic pain.Studies have found that Calpain in the cerebral cortex can regulate the expression of KCC2,enhance the excitability of central neurons and increase the transmission of pain signals to mediate the occurrence and maintenance of epilepsy and neuropathic pain in rats.However,whether the downregulation of KCC2 in the anterior cingulate cortex(ACC)is involved in the occurrence and development of chronic postoperative pain has not yet been clearly reported.ObjectiveIn this study,a rat model of chronic post-surgical pain induced by skin/muscle incision and retraction(SMIR)was used to explore the role and its molecular mechanism of anterior cingulate cortex(ACC)KCC2 in chronic post-surgical pain.Experimental methods1 Expression changes and cell distribution types of KCC2 of ACC in rats after skin/muscle incision and traction(SMIR)Male SD rats were divided into 3 groups(n=8)by random number table:experimental group(SMIR group),sham operation group(Sham group)and control group(Naive group).Mechanical paw withdrawal threshold(Paw withdrawal threshold,PWT)and Paw withdrawal latency(PWL)were measured 3 d before the SMIR and 1-32 d after SMIR of three groups of rats.The changes in the transcription level and protein expression level of KCC2 in the anterior cingulate cortex(ACC)were detected with qRT-PCR(n=4)and Western Blot(n=6)technology.The expression changes of KCC2 and cell distribution types in ACC was detected by Immunofluorescence technology.2 The role of KCC2 of ACC in chronic post-surgical pain induced by SMIR in ratsMale SD rats were divided into 4 groups(n=8)by random number table method:Na(?)ve group,SMIR group,SMIR-AAV-CON group and SMIR-AAV-KCC2 group.SMIR-AAV-CON group and SMIR-AAV-KCC2 group received AAC in situ microinjection of adeno-associated virus and adeno-associated virus control(AAV2/9-Null overexpression control、AAV2/9-CMV-r-Slc12a5-HA-Null)4 weeks before SMIR.And the adeno-associated virus transfection and KCC2 expression changes in L-ACC were detected by immunofluorescence technology.Using qRT-PCR(n=4)and Western Blot(n=6)technology to detect the changes in the transcription level and protein expression level of KCC2 in the left anterior cingulate cortex(L-ACC).The PWT value was measured 3 days before SMIR and 1-25 days after SMIR.Prove the role of KCC2 in ACC in chronic post-surgical pain.3 Calpain-2 regulate KCC2 of ACC participates in SMIR-induced chronic post-surgical pain in ratsWestern Blot(n=6)technology was used to detect the changes in the expression of Calpain-2 protein levels in the left anterior cingulate cortex(L-ACC)of the SMIR model rats.Male SD rats were randomly divided into 4 groups(n=8):Naive+Vehicle group,SMIR group,SMIR+Vehicle group and SMIR+MDL28170 group.Rats in Na(?)ve+Vehicle group,SMIR+Vehicle group and SMIR+MDL28170 group underwent intrathecal catheterization 5 days before SMIR,and intrathecal injection of DMSO and calpain inhibitors MDL28170 on the 12th day after SMIR.PWT values were measured at 1h,2h,4h,8h,12h and 24h after intrathecal injection,and Western Blot(n=6)technology was used to detect the changes in the expression of KCC2 and Calpain-2 protein levels in the left anterior cingulate cortex(L-ACC)8 h after intrathecal injection calpain inhibitors MDL28170.Immunofluorescence technology detects the co-localization of KCC2 and Calpain-2.Results1 SMIR causes mechanical hyperalgesia and down-regulates the expression of KCC2 and KCC2mRNA of L-ACC in ratsPain behavior data showed that compared with the Naive group,the PWT value of rats in the SMIR group began to decrease at 1 day after the SMIR,PWT was lowest at 12 days,and the basic PWT value was restored at 32 days after the SMIR;while the PWL value did not Significant changes occurred.The above results suggest that SMIR surgery induces mechanical hyperalgesia in rats.The results of qRT-PCR showed that,compared with the Na(?)ve group,the expression of KCC2 mRNA in the L-ACC of the rats was significantly down-regulated on the 12th day after the SMIR.Western Blot results showed that compared with the Na(?)ve group,the expression of KCC2 in the L-ACC was down-regulated on the 1st day after SMIR,and the expression level was the lowest on the 12th day.The above results suggest that SMIR can induce the downregulation the expression of KCC2 and KCC2mRNA in the L-ACC of rats.The results of immunofluorescence single staining showed that compared with Na(?)ve group,rats in SMIR group were significantly down-regulated KCC2 of L-ACC on the 12th day after SMIR.The results of immunofluorescence double staining showed that KCC2 can be co-labeled with NeuN(mature neuron marker)but not with GFAP(satellite glial cell marker).The results indicate that KCC2 in ACC is expressed in mature neurons cells.2 Overexpressing KCC2 by in situ injection of AAV2/9 in anterior cingulate can relieve chronic post-surgical pain induced by SMIRThe results of immunofluorescence double staining showed that KCC2 can be co-labeled with HA(viral label marker),and the results suggest that ACC in situ virus injection was successful.And the results of qRT-PCR showed that,compared with the SMIR-AAV-CON group,the expression of KCC2 mRNA in the L-ACC up-regulated in the SMIR-AAV-KCC2 group 12 days after SMIR.Western Blot showed that,compared with the SMIR-AAV-CON group,the expression of KCC2 in the L-ACC up-regulated in the SMIR-AAV-KCC2 group 12 days after the SMIR.Behavioral data of mechanical pain showed that L-ACC was injected with adeno-associated virus(AAV-KCC2)in situ 4 weeks before SMIR.Compared with the adeno-associated virus control group(SMIR-AAV-CON),SMIR-AAV-KCC2 could relieve mechanical hyperalgesia induced by SMIR.The results suggest that overexpressing KCC2 in the anterior cingulate cortex by in situ injection of adeno-associated virus can effectively alleviate the chronic postoperative pain induced by SMIR.3 Intrathecal injection of Calpain-2 inhibitor MDL28170 reversed the downregulation of KCC2 in the anterior cingulate cortex and relieved chronic-surgical pain induced by SMIRWestern Blot results showed that,compared with the Naive group,the expression of Calpain-2 in the L-ACC up-regulated on the 1st day after the SMIR,and the expression level was highest on the 12th day.Behavioral data of mechanical pain showed that compared with the SMIR+Vehicle group,rats in the SMIR+MDL28170 group were injected with calpain-2 inhibitor MDL28170 at 12 d after SMIR,and PWT began to increase after 2 h,and reached a peak at 8 h,then it began to fall and returned to the basic level before intrathecal injection 12 hours later.The results show that intrathecal injection of calpain-2 inhibitor MDL28170 can relieve SMIR-induced mechanical hyperalgesia.Western Blot results showed that 8 hours after intrathecal injection of calpain-2 inhibitor MDL28170 in rats,compared with SMIR+Vehicle group,the expression of Calpain-2 in the L-ACC of the SMIR+MDL28170 group down-regulated,and the expression of KCC2 up-regulated.Immunofluorescence single staining showed that the expression of Calpain-2 in L-ACC up-regulated after SMIR.Double immunofluorescence staining showed that KCC2 and Calpain-2 co-localized in ACC neurons.It shows that Calpain-2 may regulate the expression of KCC2 and participate in chronic post-surgical pain.ConclusionThe results of the study indicate that SMIR-induced chronic post-surgical pain model can cause the downregulation of KCC2 expression and the upregulation of Calpain-2 in ACC in rats,and Calapin-2 in ACC maybe participates in SMIR-mediated chronic postoperative pain by regulating the downregulation of KCC2 expression.