Research On The Role And Mechanism Of P2Y12 Receptor In Spinal Microglia For Neuropathic Pain

Part Ⅰ Spinal P2Y12 receptor expression changes and its effects on pain behavior in neuropathic pain ratsObjective To detect the changes in the expression of P2Y12 receptor protein in spinal cord of neuropathic pain rats in spinal nerve ligation model(SNL)and the corresponding changes in pain behavior.Methods(1)Establish SNL neuropathic pain model by ligating spinal nerve roots of L5 in rats;(2)Observe the expression of P2Y12 protein in the spinal cord of SNL model rats by western blot.(3)Observe the changes of P2Y12 protein expression in the spinal cord of SNL model rats by confocal immunofluorescence histochemistry.(4)Observe the co-localization of P2Y12 protein with neurons,microglia,astrocytes and oligodendrocytes in the spinal dorsal horn of SNL rats by confocal immunofluorescence histochemical double-labeling method.(5)Construct an intrathecal canal model in rats to facilitate intrathecal drug administration;(6)Observe the changes in mechanical pain threshold of rats using von Frey fibrosis,compare the behavioral changes in mechanical pain threshold of the sham operation group,the SNL group,the SNL+ intrathecal injection of P2Y12 inhibitor MRS2395 group,and the SNL+ oral administration of P2Y12 inhibitor clopidogrel group.(7)Observe the changes of thermal pain threshold in rats by Hargreaves’ thermal radiation stimulation method,compare the thermal pain threshold changes of the sham operation group,the SNL group,the SNL+ intrathecal injection of P2Y12 inhibitor MRS2395 group,and the SNL+ oral administration of P2Y12 inhibitor clopidogrel group.(8)Western blot analysis was used to observe the spinal expression of iba-1 protein,an activation marker of microglia cells,after intrathecally injected P2Y12 inhibitor MRS2395 in the spinal cord of SNL rats.(9)Confocal immunofluorescence histochemistry was used to observe the expression of P2Y12 protein in spinal cord after intrathecal injection of P2Y12 inhibitor MRS2395 in SNL rats,and the expression of iba-1 protein,an active marker of microglial cells was observed.Results(1)A stable SNL neuropathic pain model was successfully established after spinal nerve ligation(L5)operation.The rats showed hyperalgesia symptoms.(2)Western blot results showed that the expression of P2Y12 protein in the ipsilateral side of spinal cord in the SNL model rats was significantly increased on the 3rd,7th and 14 th days after the operation compared with the sham operation group.(3)The quantitative statistics of confocal immunofluorescence histochemical fluorescence intensity showed that the expression of P2Y12 protein in the spinal cord of the ipsilateral side of the SNL rats was significantly increased on the third day and the seventh day after the operation compared with the sham operation group.(4)The results of confocal immunofluorescence histochemical double-labeling showed that P2Y12 protein in the dorsal horn of the spinal cord in the SNL rats was obviously co-labeled with microglia cells,and had not co-labeled with neurons,astrocytes and oligodendrocytes.(5)The rat model of intrathecal canal was successfully established,and a small amount of lidocaine could induce reversible bilateral posterior limb paralysis in rats.(6)The mechanical pain threshold of rats observed by Von Frey filaments showed that the mechanical pain threshold of the SNL group was significantly reduced than the sham operation group.Compared with the SNL surgery group,the SNL+ intrathecal injection of P2Y12 inhibitor MRS2395 group and the SNL+ oral administration of P2Y12 inhibitor clopidogrel group can partially alleviate the mechanical pain threshold reduction caused by SNL surgery.(7)Hargreaves’ thermal radiation stimulation was used to observe the thermal pain threshold of rats.The results showed that the thermal pain threshold of the SNL operation group was significantly lower than that of the sham operation group.Compared with the SNL surgery group,the SNL+ intrathecal injection of P2Y12 inhibitor MRS2395 group and the SNL+ oral administration of P2Y12 inhibitor clopidogrel group can partially alleviate the reduction of thermal pain threshold caused by surgery.(8)Western blot analysis showed that,compared with the sham group,the expression of iba-1 protein,an active marker of microglia,was obviously increased in the SNL operation group.Compared with the simple operation group of SNL,the expression of iba-1 protein in the spinal cord was significantly reduced after intrathecal injection of P2Y12 inhibitor MRS2395 or oral administration of P2Y12 inhibitor clopidogrel in SNL model rats.(9)The quantitative results of confocal immunofluorescence histochemical fluorescence intensity showed that,compared with the sham rats,the expression of iba-1 protein,an active marker of microglia,was significantly increased in the SNL operation group,and the morphology of microglia changed from the resting state to the amoeba-like activation state.Compared with the SNL operation group,the expression of iba-1 protein in the spinal cord was significantly reduced after intrathecally injected P2Y12 inhibitor MRS2395 or oral administration of P2Y12 inhibitor clopidogrel in SNL model rats,and the morphology of microglia changed from amoeba-like activation state to branching resting state.Conclusions The mechanical pain threshold and thermal pain threshold of L5 spinal nerve ligation neuropathic pain model(SNL)rats can be partially reversed by P2Y12 inhibitors,the P2Y12 inhibitors can also suppress the expression of iba-1 protein in the spinal cord.Part Ⅱ Study on p38 MAPK and Rho A/ROCK2 signaling pathways’ relationship to P2Y12 receptor in the spinal cord of neuropathic pain ratsObjective To investigate the relationship between P2Y12 receptor and Rho A/ROCK2 or p38 MAPK signaling pathways in the spinal cord of neuropathic pain rats.Methods(1)Establish the SNL neuropathic pain model by ligating L5 spinal nerve;(2)Observe the co-localization of iba-1,an active marker of microglia cell,with several mitogen-activated protein kinase signaling pathway molecules(p38MAPK,p JNK,p ERK)in the spinal cord of SNL rats by confocal immunofluorescence histochemical doublelabeling.(3)Western blot analysis was used to observe the time-history expression of the activated mitogen-activated protein kinase signaling pathway in the spinal cord of SNL rats.(4)Western blot analysis was used to observe the effect of P2Y12 inhibitor MRS2395 on the activation of mitogen-activated protein kinase signaling pathway in the spinal cord of SNL rats.(5)Von Frey filaments was used to detect the effects of inhibitors of the activated mitogenactivated protein kinase signaling pathway in the rat spinal cord after SNL on the mechanical pain threshold.The mechanical pain threshold in the sham operation group,the SNL operation group,and the SNL+ intrathecal injection the inhibitor of the activated mitogenactivated protein kinase signaling pathway group were compared.(6)Hargreaves’ thermal radiation stimulation test was used to detect the effects of inhibitor of the activated mitogen-activated protein kinase signaling pathway in the spinal cord of SNL rats on the thermal pain threshold.The behavioral changes of thermal pain threshold in the sham operation group,the SNL operation group,and the SNL+ intrathecal injection inhibitor of the activated mitogen-activated protein kinase signaling pathway group were compared.(7)The time history of Rho A/ROCK2 signaling pathway protein in the spinal cord of neuropathic pain rats after spinal nerve ligation(SNL)was detected by western blot.(8)Western blot was used to check the effect of P2Y12 receptor inhibitor on the expression of Rho A/ROCK2 signaling pathway molecular protein in the spinal cord of SNL rats.(9)The effect of ROCK inhibitor on the expression of the activated mitogen-activated protein kinase signaling pathway molecules in the spinal cord of SNL rats was observed by western blot.(10)The influence of ROCK inhibitor on the mechanical pain threshold in SNL rats was detected by von Frey filaments.The changes of the mechanical pain threshold in the sham operation group,the SNL operation group,and the SNL+ intrathecal injection ROCK inhibitor group were compared.(11)The influence of ROCK inhibitor on the thermal pain threshold in SNL rats was detected by Hargreaves’ thermal radiation stimulation test.The behavioral changes of the thermal pain threshold in the sham operation group,the SNL operation group,and the SNL+ intrathecal injection ROCK inhibitor group were compared.Results(1)The results of confocal immunofluorescence histochemistry showed that the p38MAPK signaling pathway of mitogen-activated protein kinase in the spinal cord of SNL rats was significantly activated and double-labeling test showed the co-localization of iba-1,an active marker of microglia,with p38 MAPK.(2)Western blot results showed that the expression of phosphorylated p38 MAPK protein in the spinal cord of SNL model rats was significantly increased on the third day,the seventh day and the 14 th day after SNL operation compared with the sham operation group.(3)Western blot results showed that P2Y12 inhibitor MRS2395 significantly reduced the expression of the phosphorylated p38 MAPK protein in the spinal cord of SNL rats at 7 days after operation.(4)The mechanical pain threshold of rats was observed by Von Frey filaments.The results showed that SB203580,a p38 MAPK inhibitor,could significantly alleviate the tactile allodynia induced by SNL surgery in rats.Compared with the sham operation group,the mechanical pain threshold of the SNL group was significantly reduced.Compared with the SNL group,SNL+ intrathecal injection of p38 MAPK inhibitor SB203580 group can partially alleviate the mechanical pain threshold reduction caused by SNL surgery.(5)Hargreaves’ thermal radiation stimulation test was used to observe the thermal pain threshold of rats.The results showed that SB203580 could significantly increase the thermal pain threshold of rats induced by SNL surgery.Compared with the sham operation group,the thermal pain threshold of the SNL operation group was significantly reduced.Compared with the SNL group,the SNL+ intrathecal injection of p38 MAPK inhibitor SB203580 group can partially alleviate the reduction of thermal pain threshold caused by SNL surgery.(6)Western blot results showed that the expressions of GTP-Rho A and ROCK2 proteins in the spinal cord of SNL rats were significantly increased on the 3rd,7th and 14 th days after the operation compared with the sham operation group.(7)Western blot results showed that P2Y12 inhibitor MRS2395 significantly reduced the expression of ROCK2 protein in the spinal cord of rats at 7 days after SNL surgery.(8)Western blot results showed that ROCK inhibitor Y27632 significantly reduced the expression of phosphorylated p38 MAPK protein in the rat spinal cord at 7 days after SNL surgery.(9)The mechanical pain threshold results of rats observed by Von Frey filament showed that ROCK inhibitor Y27632 could significantly alleviate the tactile allodynia of rats induced by SNL surgery.(10)The thermal pain threshold of rats observed by Hargreaves’ thermal radiation stimulation test showed that ROCK inhibitor Y27632 could significantly improve the thermal pain threshold of rats induced by SNL surgery.Conclusions Both P38 MAPK and Rho A/ROCK2 signaling pathways were activated in the spinal cord of SNL rats.P2Y12 inhibitor MRS2395 can inhibit the activation of p38 MAPK and Rho A/ROCK2 signaling pathways in the spinal cord of SNL rats.ROCK inhibitor Y27632 can inhibit the activation of p38 MAPK,so ROCK is the upstream signalling pathway of p38 MAPK.P38MAPK inhibitor SB203580 or ROCK inhibitor Y27632 could alleviate the tactile allodynia and thermal hyperalgesia induced by SNL surgery.Part Ⅲ Research on the effect of P2Y12 gene knock out in pain behavior and excitability transmission changes in lamina Ⅱ neurons in the spinal cord of neuropathic pain miceObjective To observe the changes of pain behavior and spinal lamina Ⅱ neurons excitatory transmission in the partial sciatic nerve ligation(PSNL)model in P2Y12 gene knockout miceMethods(1)PCR was used to identify the mouse genotype.The wild-type homozygous and P2Y12 gene knockout homozygous were selected for experiments.Heterozygous were kept for breeding.(2)A stable PSNL neuropathic pain mouse model was established by ligating part of the sciatic nerve and the mice was divided into the wild-type sham operation group,wild-type PSNL operation group,P2Y12 gene knockout sham operation group and P2Y12 gene knockout PSNL operation group.(3)1g von Frey filament was used as a specific mechanical stimulation to observe the frequency changes of leg lift in mice to reflect the mechanical pain threshold.The mechanical behavioral changes in the wild-type sham operation group,wild-type PSNL operation group,P2Y12 gene knockout sham operation group and P2Y12 gene knockout PSNL operation group were compared.(4)2.5 s’ radiant heat stimulation was used as a particular stimulus duration(adjust the basis of radiant heat intensity,make the leg lift latency period limited to 10 to 12 s in wild type mice).Thermal pain behavior changes were observed between wild type control mice,wild type PSNL mice,P2Y12 gene knock out control mice and P2Y12 gene knockout PSNL mice.(5)Using whole cell patch clamp technique to record the miniature excitatory postsynaptic currents(m EPSC)of lamina Ⅱ neurons in the spinal cord,to compare the m EPSC frequency and amplitude difference between the wild type control group,wild type PSNL group,P2Y12 gene knock out control group and P2Y12 gene knockout PSNL group.(6)Using whole cell patch clamp technique to record the resting membrane potentials of lamina Ⅱ neurons in the spinal cord,to compare the depolarization difference between the wild type control group,wild type PSNL group,P2Y12 gene knock out control group and P2Y12 gene knockout PSNL group.(7)Using whole cell patch clamp technique to record the amplitude of the single action potential evoked by an injection current of lamina Ⅱ neurons in the spinal cord,to compare the amplitude difference between the wild type control group,wild type PSNL group,P2Y12 gene knock out control group and P2Y12 gene knockout PSNL group.Results(1)A stable PSNL neuropathic pain mouse model was established by ligating part of the sciatic nerve.In the PSNL surgery group,the frequency of foot licking increased,and the foots reluctant to touch the ground.(2)1 g von Frey filament as a specific mechanical stimulation was tested in mice.The frequency changes of leg lifts reflect the mechanical pain threshold,compared with wild type control group,the mechanical pain threshold of wild type PSNL group and P2Y12 gene knockout PSNL group was reduced significantly,however,compared to wild type PSNL group,the mechanical pain threshold of P2Y12 gene knockout mice showed lighter allodynia.(3)2.5 s radiant heat stimulation as a particular heat stimulus duration(adjust the basal radiant heat intensity,which makes wild type controls’ basal time to 10 to 12 s)to observe the thermal pain threshold changes in mice,compared with wild type control group,the thermal pain threshold of wild type PSNL group and P2Y12 gene knockout PSNL group reduced significantly,however,compared to wild type PSNL group,the mechanical pain threshold of P2Y12 gene knockout mice showed lighter allodynia.(4)Compared to wild type control mice,the frequency and amplitude of the miniature excitatory postsynaptic currents(m EPSC)in the wild type PSNL mice has increased significantly in lamina Ⅱ neurons in the spinal cord.The frequency and amplitude of m EPSC in P2Y12 gene knockout PSNL group were significantly higher than those in P2Y12 gene knockout sham group.However,the increased frequency and amplitude of m EPSC in the P2Y12 gene knockout PSNL surgery group were smaller than those in the wild-type PSNL surgery group.(5)The resting membrane potential in wild type PSNL group was depolarized.In the P2Y12 gene knockout PSNL group,the resting membrane potential was also depolarized.However,there was no statistical difference in the resting membrane potential between the P2Y12 gene knockout PSNL group and the wild-type PSNL group,indicating that there was no difference in their degree of depolarization.(6)The action potential amplitude evoked by an injection current(2 ms、1000 p A)in wild type control group,wild type PSNL group,P2Y12 gene knock out control group,P2Y12 gene knockout PSNL group have no statistical difference.Conclusions Compared with wild-type PSNL mice,the pain behavior decreased in P2Y12 gene knockout mice.Postoperative P2Y12 gene knockout mice showed enhancement in excitatory synaptic transmission but smaller than wild type mice.Postoperative,resting membrane potential depolarization have taken place in wild type and P2Y12 gene knockout mice.No difference checked in single action potential induced by injection current in four different groups.