The Role Of KCC2 In Spinal Dorsal Horn In Chronic Postoperative Pain Induced By SMIR In Rats

BackgroundChronic post-surgery pain(CPSP)is a persistent postoperative pain.CPSP delays the recovery from surgery,prolongs the hospital stay,reduces the patient’s quality of life,and is a serious problem in clinical pain management.At present,the pathogenesis and development mechanism of CPSP is still unclear.The spinal dorsal horn(SDH)is an important starting site for pain transmission.In the dorsal horn of the spinal cord,it produces central sensitization,inhibitory interneuron loss and microglia activation,which mediate the occurrence and development of pain.Potassium chloride ion cotransporter 2(KCC2)is responsible for maintaining low chloride ion concentration in central nervous system(Central Nervous System,CNS)neurons..The post-synaptic inhibition of GABAA and glycine receptors is necessary,so KCC2 is the core of pain gating theory.At present,the loss of activity of this transporter has become a key mechanism for several neurological and psychiatric diseases,including epilepsy,motor cramps,stress,anxiety,schizophrenia,hyperalgesia caused by morphine and related chronic pain.However,there are still few studies on its mechanism in chronic postoperative pain.This study intends to evaluate the role of KCC2 in CPSP and its molecular mechanism,indicating that the clinical treatment of CPSP is expected to adopt methods based on the mechanism of KCC2.ObjectiveIn this study,an animal model of chronic postoperative pain in rats caused by the Skin/Muscle Incision and Retraction(SMIR)was selected.First,the postoperative L4-5 spinal cord(SP)was observed.The expression changes of KCC2,and through in situ microinjection of the spinal dorsal horn(SDH)adeno-associated virus(AAV)overexpression of KCC2 to explore the role of KCC2 in the occurrence and development of chronic postoperative pain,and to explore KCC2 mediates the molecular mechanism of CPSP.Methods1 The expression of KCC2 in the spinal cord of rats after skin/muscle incision and stretchingTwenty-seven male SD rats were divided into 3 groups(n=9)operation group(SMIR group),sham operation group(Sham group)and blank control group(Naive group)by random number table method.The mechanical pain withdrawal threshold(PWT)of rats was measured 3 days before the skin/muscle incision and traction operation to observe the mechanical hyperalgesia of rats.Paw withdrawal latency(PWL)was measured to observe thermal hyperalgesia in rats.The measurement lasted 32 days after SMIR.In addition,model and use Western Blot technology to detect the expression level of KCC2 in SP(n=5),q-PCR to detect the change of mRNA(n=5)and immunofluorescence technology to detect the expression of KCC2in L4-5 SDH Change(n=3).2 The role of KCC2 in the dorsal horn of the spinal cord in the occurrence and development of chronic postoperative pain in rats induced by SMIR surgeryThirty-six male SD rats were divided into 4 groups(n=9)Naive group,SMIR group,SMIR+overexpression virus group(AAV-KCC2 group)and SMIR+virus empty vector group(AAV-Contrl group)by random number table method.The rats in each group received L4-5 SDH in situ microinjection of AAV-KCC2-HA,AAV-HA or normal saline 4 weeks before modeling.PWT values were measured at different time points after modeling to observe the development of CPSP in rats.In addition,L4-5 SP was modeled and taken on the 12th day,and Western Blot was used to detect the expression of KCC2 protein in each group of SP(n=5),and the expression of KCC2 in L4-5 SDH was detected by immunofluorescence staining technology(n=3).This proves the role of KCC2 in the spinal dorsal horn in the occurrence and development of CPSP.3 Molecular mechanism of KCC2 in the spinal cord mediating chronic postoperative pain in ratsAfter SMIR modeling(n=5),Western Blot was used to detect the expression changes of Calpain Ⅰ and Calpain Ⅱ in SP of CPSP model rats.36 male SD rats were randomly divided into 4 groups(n=9):Naive+normal saline(Naive)group,SMIR+normal saline(SMIR)group,SMIR+calpain inhibitor MDL28170(SMIR+M)group and SMIR+solvent DMSO(SMIR+D)group.Rats in each group received intrathecal catheterization 5 days before modeling,and intrathecal injection of MDL28170,DMSO or normal saline on the 12th day after modeling.PWT values were measured at 1h,2h,4h,8h and 12h after intrathecal injection.In addition,modeling and 8 hours after intrathecal injection of inhibitors were taken from L4-5 SP and subjected to Western Blot technology(n=5)to detect the expression of Calpain Ⅰ,Calpain Ⅱ and KCC2 in each group of SP,and immunofluorescence double staining technology was used to detect KCC2 and Co-localization of calpain.Result1 SMIR caused persistent mechanical hyperalgesia and down-regulation of KCC2 expression in the spinal cord in ratsPain behavior related tests showed that compared with the Naive group,the PWT value of the SMIR group began to decrease 1 day after the model was established,and reached a maximum on the 12th day,after which it began to recover,and returned to the basic value on the 32th day(P<0.05);The PWL value did not change significantly.The above results suggested that SMIR caused long-term mechanical hyperalgesia after surgery,but did not cause thermal hyperalgesia.Western Blot results showed that compared with 0 d,KCC2 was down-regulated in L4-5 SP on the operative side 1 d after SMIR,reached a peak at 12 d and continued to decrease until 25 d;in the q-PCR results,compared with 0 d,KCC2 was decreased on the operative side 1 d after SMIR The mRNA level of KCC2 in SP decreased and reached a peak on the 12th day.The results of immunofluorescence single staining showed that the number of KCC2 positive cells in the L4-5 SDH on the operation side decreased on the 12th day after SMIR compared with 0 d.The above results showed that SMIR caused continuous down-regulation of KCC2 in L4-5 SP on the operation side of rats.The results of immunofluorescence double staining showed that KCC2 can be co-labeled with Neun(mature neuron marker),suggesting that KCC2 is expressed in mature neurons in SDH.2 The role of KCC2 in SMIR-induced chronic postoperative pain.Behavioral data of mechanical pain showed that SDH in situ micro injection of AAV-KCC2-HA compared with SDH microinjection of normal saline 4 weeks before modeling showed that AAV-KCC2-HA can reduce the decrease in PWT induced by SMIR and promote PWT Value restoration.It shows that in the occurrence and development stage of CPSP,AAV-KCC2-HA can alleviate the persistent abnormal postoperative pain induced by SMIR.And Western Blot results showed that compared with the SMIR group,KCC2 was overexpressed in the L4-5 SP on the operation side of the AAV-KCC2 group.In conclusion,KCC2 overexpression therapy by in situ injection can alleviate SMIR-induced CPSP.3 Calpain Ⅰ participates in chronic postoperative pain by regulating the expression of KCC2Behavioral data of mechanical pain showed that,compared with the intrathecal solvent injection group,intrathecal injection of calpain inhibitor MDL28170 at 12 days after SMIR could reduce the decrease in SMIR-induced PWT value.Among them,intrathecal injection of the inhibitor 2 hours began to relieve mechanical Hyperalgesia,the effect reached its peak at 8 hours and returned to the level before intrathecal injection after 24 hours.Western Blot results showed that the protein expression level of calpain I in the ipsilateral L4-5 SP after SMIR was significantly increased while the protein expression level of calpain II remained unchanged.Compared with 0 d,the expression of calpain I in the ipsilateral L4-5 SP of rats was up-regulated on the 1st day after the model was established and maintained until 25 d,with the highest expression level at 12 d.Compared with SMIR group,intrathecal injection of calpain inhibitor MDL28170 can reverse the down-regulation of KCC2 expression.Immunofluorescence double staining showed that KCC2 and calpain I co-localized in SDH neurons.It shows that calpain Ⅰ can participate in chronic postoperative pain by regulating the expression of KCC2.ConclusionIn the chronic postoperative pain model established by SMIR,SMIR can cause the down-regulation of KCC2 expression and the up-regulation of the expression of calpain Ⅰ in L4-5 SP in rats.Calpain Ⅰ regulates the occurrence and development of chronic postoperative pain by mediating the down-regulation of KCC2.