The Effects Of High Frequency Repetitive Transcranial Magnetic Stimulation On The Rats With Neuropathic Pain And The Underlying Mechanisms

Part 1 The effects of different high frequency repetitive transcranial magnetic stimulations on neuropathic pain in ratsObjective To observe the effects of high frequency repetitive transcranial magnetic stimulation（rTMS）on the rats with neuropathic pain.And to explore which frequency is the best one according to comparing the results of different frequency rTMS on the rats with neuropathic pain.Methods Sixty Sprague-Dawley rats were included,and were divided into sham group,model group,TMS group and Control stimulation group,randomly.Then TMS group was subdivided into three groups:5Hz group,10Hz group,20Hz group.Rats in model group,control stimulation group and TMS group were received chronic constriction injury of sciatic nerve（CCI）.On the 3^rd day after operation,the rats in TMS group and Control stimulation group began to receive a 5-day rTMS treatment continuously,once a day.The pain was respectively evaluated at the day before the CCI operation,and the 3^rd,5^th,7^th day after the CCI operation.The evaluations included the paw withdrawal thermal latency（PWTL）and the paw withdrawal mechanical threshold（PWMT）.After finishing all the treatments and evaluations,rats were euthanized.（P<0.05 means the difference is significant.）Results 1.The results of 5 times rTMS on the paw withdrawal thermal latency（PWTL）:After CCI,model group and control stimulation group’s PWTL was decreased step by step,while TMS group was increased slowly since the 3^rd day after operation.Compared with sham group,all the PWTL values of model group,control stimulation group and TMS group were decreased after the operation,P<0.01.At the 3^rd day after CCI,model group,control stimulation group and TMS group had no notably difference,P>0.05.The values of both10Hz group and 20Hz group were higher than 5Hz group at the 5^th day after CCI,but it is not significant（P>0.05）.Also,it is obvious that 20Hz group’s PWTL value is higher than10Hz at the 5^th day after CCI,P<0.05.At the 7^th day after CCI,the PWTL values of both10Hz group and 20Hz group were significantly higher than 5Hz group（P<0.05）;and there is not obviously different between 10Hz group and 20Hz group（P>0.05）.There is no difference between model group and control stimulation group.2.The results of 5 times rTMS on the paw withdrawal mechanical threshold（PWMT）:After the operation,model group and control stimulation group’s PWMT were decreased gradually,while TMS group was increased slowly since the 3^rd day after CCI.Comparing with sham group,the PWMT values of model group,control stimulation group and TMS group were lower after operation,P<0.01.At the 3^rd day after CCI,there are no notably differences between model group,control stimulation group and TMS group,P>0.05.At the 5^th day after CCI,the values of 10Hz group is lower than 20Hz group but higher than 5Hz group,the differences among them are significant（P<0.05）.At the 7^th day after CCI,the PWMT values of both 10Hz group and 20Hz group were significantly higher than 5Hz group（P<0.05）;but there is not obviously different between 10Hz group and 20Hz group（P>0.05）.3.The results of 10 times rTMS on the paw withdrawal thermal latency（PWTL）:At the 12^th day after CCI,the PWTL values of model group and TMS group were significantly lower than sham group,P<0.01.Model group’s PWTL value was lower than TMS group,P<0.01.Though the PWTL value of 20Hz group was higher than 10Hz group,it is not significant,P>0.05.4.The results of 10 times rTMS on the paw mechanical threshold（PWMT）:At the 12^th day after CCI,the PWMT values of both model group and TMS group were significantly lower than sham group,P<0.01.Model group’s PWMT value was lower than TMS group,P<0.01.Though the PWMT value of 20Hz group was higher than 10Hz group,it is not significant,P>0.05.Conclusion Our results indict that high frequency rTMS can obviously relieve pain.There are significant differences among different frequency groups.For both PWTL and PWMT,the effects of 10Hz and 20Hz are equal,but better than 5Hz.Considering the incidence rate of side effects,we think the 10Hz is the best frequency to treat neuropathic pain with rTMS.Part 2 The underlying mechanisms of rTMS in treating neuropathic pain in ratsObjective To further observe the effects of rTMS in rats with neuropathic pain;and to explore the underlying mechanisms that how rTMS acts on neuropathic pain and relieve it,in the terms of microglia-neuron communications.Method Thirty Sprague-Dawley rats were included,and were divided into three groups at random: sham group,model group and 10 Hz group.All rats in model group,TMS group underwent chronic constriction injury of sciatic nerve.After two-day recovery phase,TMS group began to received a 10-day rTMS treatment,once a day.The pain intensity were evaluated at the day before CCI,3^rd,5^th,7^th,10 th,and 12^th day after CCI,respectively.The evaluated indexes include the paw withdrawal thermal latency（PWTL）and the paw withdrawal mechanical threshold（PWMT）.After completing all treatment and pain evaluation,rats were sacrificed.The expression of Iba1,P2X4,BDNF,KCC2 and GABAAR was detected by western blot and the quantitative polymerase chain reaction（q PCR）.The microglia activity and the local of P2X4 were observed by immunofluorescence double staining.（P<0.05 means it is significant.）Results 1.The results of pain intensity evaluation（1）PWTL: At the 12^th day after CCI,the PWTL values of model group and 10 Hz group were significantly lower than sham group,P<0.01.Model group’s PWTL value was lower than 10 Hz group,P<0.01.（2）PWMT: At the 12^th day after CCI,the PWMT values of both model group and 10 Hz group were significantly lower than sham group,P<0.01.Model group’s PWMT value was lower than 10 Hz group,P<0.01.2.The influences of rTMS on microglia: The arrangement of the Iba1 expression from high to low is sham group,10 Hz group and model group at the 12^th day after CCI.The difference between model group and 10 Hz group is significant,P<0.05.There were no distinct difference between sham group and 10 Hz group,P>0.05.3.The expression of P2X4/BDNF at protein level（1）P2X4: At the 12^th day after CCI,the expression in model group was higher than both sham group and 10 Hz group（P<0.05）;the value of 10 Hz group was higher than sham group,but it is not obvious（P>0.05）.（2）BDNF: At the 12^th day after CCI,the expression of BDNF in model group was higher than both sham group and 10 Hz group（P<0.05）;the value of 10 Hz group was higher than sham group,but it is not obvious（P>0.05）.There were no distinct differences of both P2X4 and BDNF expression among sham group,10 Hz group at the 12^th day after CCI.4.The expression of P2X4/BDNF/KCC2/GABAAR at m RNA level（1）P2X4: At the 12^th day after CCI,the m RNA expression in model group was higher than both sham group and 10 Hz group（P<0.05）;the value of 10 Hz group was higher than sham group,but the differences were not significant,P>0.05.（2）BDNF: At the 12^th day after CCI,the expression in 10 Hz group was higher than sham group,lower than model group.The differences between sham group and 10 Hz group,nor model group and 10 Hz group had not significance,（P>0.05）.And the differences between sham group and model group was significant,P<0.05.（3）KCC2: At the 12^th day after CCI,the expression of KCC2 in 10 Hz group was higher than model group（P<0.05）;lower than sham group,but it is not obvious（P>0.05）.（4）GABAAR: At the 12^th day after CCI,the expression of GABAAR in 10 Hz group was lower than sham group,higher than model group.There is distinct differences between sham group and 10 Hz group（P<0.05）;but no evident difference in model group and 10 Hz group（P>0.05）.5.The Iba1/P2X4 /DAPI immunofluorescence double staining There are little Iba1/P2X4 positive cells of the left spinal cord dorsal horn in the sham group.The Iba1/P2X4 positive cells of the left spinal cord dorsal horn increased in model group.After rTMS treatment,the Iba1/P2X4 positive cells of the left spinal cord dorsal horn decreased in 10 Hz group.6.Correlation analysis between P2X4 receptor/Iba1 expression level and pain behavior scores There was strong negative correlation between expression of P2X4 receptor in the affected side of spinal and pain behavior scores,P<0.05.There were no significant correlation between Iba1 expression and pain behavior scores,or between the difference of Iba1/P2X4 receptor expression and the change of pain behavior scores,P>0.05,but there were negative correlation trend among them.Conclusion Our results suggest that the underlying mechanism of rTMS on neuropathic pain is related to microglia activity and microglia-neuron communication.rTMS can improve neuropathic pain by suppressing microglia activity,and adjusting P2X4/BDNF/KCC2 signaling pathway.Part 3 A validation study on the mechanism of effects of rTMS in treating neuropathic pain in ratsObjective To velidate the mechanism of rTMS on neuropathic pain in rats,whether it acts on pain through P2X4/BDNF/KCC2 signaling pathway.Methods Twenty Sprague-Dawley rats were included.Rats were divided into four groups: model group,model+ATP group,rTMS+ATP group,rTMS + NS group,randomly.All rats received chronic constriction injury of sciatic nerve（CCI）and intrathecal catheterization,while model group didn’t maintain intrathecal catheters in rats’ body.First,rats received intrathecal catheterization.After two-day recovery phase,the rats without spinal cord injury were selected to receive CCI.After two days’ rest,rTMS+ATP group and rTMS+NS group began to receive a 5-day 10 Hz rTMS treatment continuously,once a day.The pain evaluations were conducted at the day before operation,3^rd day（2 times,one before injection,and the other time after it）,5^th day and 7^th day after CCI,including PWTL and PWMT.After all treatment and evaluation finished,rats were euthanized.The expression of Iba1,P2X4 was detected by western blot.Results 1.The result of pain evaluation:（1）PWTL: At the 7^th day after CCI,the PWTL values of rTMS+ATP higher than model group（P<0.01）,lower than rTMS+NS group（P<0.01）;there is no significant difference between model group and model+ATP group（P>0.05）.（2）PWMT: At the 7^th day after CCI,the PWMT values of rTMS+ATP higher than model group（P<0.01）,lower than rTMS+NS group（P<0.01）;there is no significant difference between model group and model+ATP group（P>0.05）.2.The expression of Iba1/P2X4/KCC2 in protein levels:（1）Iba1: The Iba1 expression of model+ATP group is the highest comparing with other groups（P<0.05）;there are no significant differences among model group,rTMS+NS group and rTMS+ATP group,but the relative value of rTMS+ATP group is higher than rTMS+NS group,but lower than model group.（2）P2X4: The P2X4 expression of model+ATP group is the highest comparing with other groups（P<0.05）.There are no significant differences among model group,rTMS+NS group and rTMS+ATP group,but the relative value of rTMS+ATP group is higher than rTMS+NS group,but lower than model group.Conclusions When rTMS improving pain,rTMS can suppress microglia activity,and decrease P2X4 expression.The effects of rTMS on neuropathic pain may be related to microglia activity and P2X4/BDNF/KCC2 signaling pathway.