| Objective We established the rat model of complex regional pain syndrome type-I(CRPS-I)and studied the analgesic effects of different frequencies of electroacupuncture(EA)on the model.We further studied the effects of EA on TRPV1 expression on peripheral dorsal root ganglion(DRG)neurons and astrocyte and microglia activations in spinal cord dorsal horn(SCDH),with the purpose to unravel the mechanisms underlying EA's analgesic effect.Methods Part 1:The rat chronic post-ischemic pain(CPIP)model was established to mimic human CRPS-I.?Healthy male SD rats were randomly divided into Sham group and model group(CPIP group).CPIP was established by attaching a rubber ring on the right hind limb of the rat for 3 hours to induce ischemia-reperfusion.Before model establishment,the appearance of paw was recorded and the paw thickness and 50%paw withdrawal threshold(50%PWT)of rats in model group and control group were measured.The paw thickness was measured between 15 min and 72 hours after model establishment and 50%PWT was measured in 1,3,5,7,9,11 and 13 days after model establishment.?Healthy male SD rats were randomly divided into Sham+Veh group,CPIP+Veh group,Sham+Cap(Capsaicin,TRPV1 agonist)and CPIP+Cap group.Veh or Cap was injected into ipsilateral paw to observe nocifensive behavior(the accumulated time that the rats spent in licking,biting,and withdrawing their paws within 10 minutes right after the injection).?Healthy male SD rats were randomly divided into the control group(Sham group),model group(CPIP group),CPIP+2Hz EA group,CPIP+100Hz EA group,CPIP+2/100Hz EA group and CPIP+Sham EA group.The EA group and Sham EA group were treated on the 2nd day after model establishment.In EA group,bilateral acupoints "Zusanli(ST36)" and "Kunlun(BL60)" were used for EA treatment.The stimulation intensity of EA is 0.5-1.5mA(0.5mA first 0.5mA every 10min,a total of 30min)and EA was applied 30min each time.For Sham EA treatment,rats were inserted with needles subcutaneously into ST36 and BL60,but with no electrical discharge.50%PWT of bilateral hind paws were tested before and 1,3,5,7,9,11,13 days after model establishment.Part 2:?Male SD rats were randomly divided into Sham group,CPIP group,CPIP+100Hz EA group(Optimal EA frequency was selected for treatment)and CPIP+Sham EA group.DRGs of the ipsilateral side were harvested.The expression of TRPV1 in DRG of the ipsilateral side of the rats was detected by immunofluorescence.?Healthy male SD rats were randomly divided into Sham group,CPIP group,CPIP+2Hz EA group,CPIP+100Hz EA group and CPIP+Sham EA group.Spinal cord dorsal horn(SCDH)tissues of rats in each group were collected on the 13th day,and the expression levels of glial markers(GFAP-labeled astrocytes,OX42-labeled microglia)were detected by immunofluorescence.Results Part 1:? The CPIP model was established successfully:CPIP group rats showed obvious cyanosis in ipsilateral hind paw during model establishment and showed obvious hyperemia and swelling after the O-ring was cut.Compared with Sham group,the degree of ipsilateral hind paw swelling of CPIP rats increased significantly and returned to normal 48 hours later.Compared with Sham group,50%PWT of the rats in the CPIP group showed a significant decrease during 13 days observation period.?CPIP rats exhibited enhanced nocifensive responses to capsaicin.Vehicle injection induced a slight nocifensive response in the Sham group,whereas the CPIP group exhibited a much higher response than the Sham group.Moreover,capsaicin injection resulted in robust nocifensive response in the sham group.CPIP rats showed significantly higher responses to capsaicin injection compared to the Sham group.?The effect of EA on bilateral hind paws of CPIP rats showed frequency dependency.2Hz EA showed analgesic effect at the beginning of the treatment but the analgesic effect decreased gradually.In contrast,both 100 and 2/100 Hz showed significant and persistent analgesic effects.EA has similar analgesic effect on the mechanical hyperalgesia of the contralateral hind paw.Part 2:?100 Hz EA could reverse the overexpression of TRPV1 in DRGs of CPIP rats.Compared with Sham group,the proportion of TRPV1 positively expressed DRG neurons was significantly higher than that in Sham group,while the proportion of TRPV1 positively expressed DRG neurons in EA group was significantly lower than that in Sham EA group.?EA can interfere with the activation of glial cells in SCDH of CPIP rats.Compared with Sham group,the GFAP(astrocyte marker)immune activity and the number of GFAP positive cells in SCDH of CPIP group were significantly increased.Compared with Sham EA group,100Hz EA treatment significantly reduced the GFAP immunoactivity and the number of GFAP positive cells in the ipsilateral side of SCDH,while 2Hz showed no effect.Meanwhile,similar changes occurred in OX42,a marker of microglia.Conclusion ?The CPIP rats showed obvious swelling in the ipsilateral hind paw and obvious mechanical hyperalgesia.?CPIP rats showed enhanced nocifensive responses to capsaicin,a phenomenon mimicking human patients with CRPS-I.?2Hz EA showed analgesic effect at the beginning,but the analgesic effect decreased gradually.100Hz EA and 2/1 00Hz EA had the same and persistent analgesic effect.?In the peripheral level,the analgesic effect of 100Hz EA may be related to its ability to reverse the overexpression of TRPV1 in the ipsilateral DRGs of CPIP rats.In the level of spinal cord,different frequencies of EA may interfere with the activation of ipsilateral spinal glial cells of CPIP rats to different degrees:2Hz EA failed to alleviate the activation of glial cells in CPIP rats.However,100Hz EA significantly decreased the activation of spinal cord glial cells.This result is consistent with the results from animal behavioral tests. |
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