| ObjectiveOpioids are the preferred analgesics in general anesthesia. However, it was claimed that remifentanil provided analgesic effect, at the same time it increased sensitivity to noxious stimuli of patients called opioid-induced hyperalgesia (OIH).Remifentanil is a ?-opioid receptor agonist and it has the properties of rapid onset,short half-time and metabolising not depending on liver and kidney. So it has been used widely in the maintenance of general anesthesia. It was confirmed that the incidence of remifentanil-induced hyperalgesia is relatively higher than other opioids.As a highly selective a2-adrenergic receptor (?2AR) agonist, dexmedetomidine possesses sedative, analgesic and anxiolytic properties. It is used as an adjuvant in general anesthesia. Some clinical studies claimed that dexmedetomidine had a certain preventive effect on OIH, while the concrete mechanism is still needed to discuss. In this study, we evaluated the changes of NMDA receptor subunits expression,trafficking, function as well as the expression and phosphorylation of PKCy and CaMK ? ? in remifentanil-induced hyperalgesic rats caused by dexmedetomidine. We had conducted a thorough research on the the influence of dexmedetomidine on remifentanil-induced hyperalgesia and possible mechanisms.MethodsPart 1: Ninety-six male Sprague-Dawley rats which were caudal vein cathetered,were randomly divided into 6 groups (n=16 each): blank control group (group C),remifentanil group (group R), incisional pain group (group ?), dexmedetomidine group (group D), remifentanil+incisional pain group (group R+I), dexmedetomidine+remifentanil+incisional pain group (group D+R+I). The incisional pain rat model was established by plantar incision. Remifentanil was infused at a rate of 1.2?g· kg-1·min-1 for 90min via the caudal vein. Dexmedetomidine was administered subcutaneously at a dose of 50ug/kg 30 min before plantar incision. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured at 24h(T0) before and 2h,6h,24h,48h(T1?4) after remifentanil infusion. The rats were sacrificed after the last behavioral test and L4-6 segments of the spinal cord were removed to determine the expression of NR1, NR2A and NR2B subunits in cell membrane(m) and total(t) protein by western blot analysis. The ratios of mNR1/tNR1?mNR2A/tNR2A and mNR2B/tNR2B were calculated. The expression of NR1, NR2A and NR2B subunits in spinal doral horn were detected by immunohi stochemi stry.Part 2: Forty-eight newborn male Sprague-Dawley rats were randomly divided into 6 groups (n=8 each): blank control group (group C), remifentanil group (group R), dexmedetomidine group (group D), dexmedetomidine+remifentanil group 1-3(group D1-3). The lumbar segments of spinal cord were immediately removed, sliced and incubated. Spinal cord slices from group C were incubated in artificial cerebrospinal fluid (ACSF) for 90 min; spinal cord slices from group R were incubated in ACSF with 4 nM remifentanil for 90 min; spinal cord slices from group D were incubated in ACSF with 4 nM dexmedetomidine for 90 min; spinal cord slices from group D1, D2, D3 were incubated in ACSF with 4 nM remifentanil and 2 nM, 4 nM, 6 nM dexmedetomidine for 90 min respectively. The whole cell patch clamp technique was used to investigate the amplitude and time interval of NMD A receptor-mediated miniature excitiatory postsynaptic currents (mEPSCs) in spinal dorsal horn neurons.Part 3: Forty male Sprague-Dawley rats which were caudal vein cathetered, were randomly divided into 5 groups (n=8 each): blank control group (group C),remifentanil + incisional pain group (group R+I), dexmedetomidine + remifentanil +incisional pain group (group D+R+I), dexmedetomidine+remifentanil+incisional pain+phorbol myristate acetate (PMA) +DMSO group (group D+R+I+P+DMSO),dexmedetomidine+remifentanil+incisional pain+DMSO group (group D+R+I+DMSO). The incisional pain rat model was established by plantar incision.Remifentanil was infused at a rate of 1.2?g·kg-1 · min-1 for 90min via the caudal vein. Dexmedetomidine was administered subcutaneously at a dose of 50 ug/kg 30min before plantar incision. PMA and DMSO were intrathecally injected at a dose of 10 ?l. The value of PWL and PWT were measured at 24h(T0) before and 2h,6h,24h,48h(T1?4) after remifentanil infusion. The rats were sacrificed after the last behavioral test and the L4-6 segments of spinal cord were removed to determine the expression and phosphorylation of spinal PKCy and CaMK ?? by western blot analysis.ResultsPart 1: Compared with group C, PWL was significantly shortened and PWT was significantly decreased except T0, the expression of mNR1, tNR1, mNR2B, tNR2B were up-regulated, and the ratios of mNR1/tNR1 and mNR2B/tNR2B were significantly increased in other groups except group D (P<0.01). Compared with group R or group I or group D, PWL was significantly shortened and PWT was significantly decreased except T0, the expression of mNRl, tNRl, mNR2B, tNR2B were up-regulated, and the ratios of mNR1/tNRl and mNR2B/tNR2B were significantly increased in group R+I (P<0.01). Compared with group R+A, PWL was significantly prolonged and PWT was significantly increased except T0, the expression of mNR1, tNR1, mNR2B, tNR2B were down-regulated, and the ratios of mNR1/tNR1 and mNR2B/tNR2B were significantly decreased in group D+R+I (P<0.01). However, there was no significant difference in the expression of mNR2A,tNR2A and the ratio of mNR2A/tNR2A in each group (P>0.05).Part 2: Compared with group C, the amplitude of mEPSCs were significantly increased, while the time interval were significantly decreased in group R, D1, D2,D3(P<0.01). Compared with group R, the amplitude of mEPSCs were significantly decreased, while the time interval were significantly increased in group D1, D2, D3(P<0.01). Compared with group D1, the amplitude of mEPSCs were significantly decreased, while the time interval were significantly increased in group D2, D3(P<0.01). Compared with group D2, the amplitude of mEPSCs were significantly decreased, while the time interval were significantly increased in group D3(P<0.01).Part 3: Compared with group C, PWL was significantly shortened and PWT was significantly decreased except To, and the expression of PKCy, CaMK ? ? and pCaMK ? ? were up-regulated in the other groups (P<0.01). Compared with group R+I, PWL was significantly prolonged and PWT was significantly increased except T0, the expression of PKCy, CaMK ? ? and pCaMK ? ? were down-regulated in group D+R+I and group D+R+I+DMSO (P< 0.01). Compared with group D+R+I,PWL was significantly shortened and PWT was significantly decreased except T0, the expression of PKCy, CaMK ? a and pCaMK ? a were up-regulated in group D+R+I+P+DMSO (P < 0.01). Compared with group D+R+I+P+DMSO, PWL was significantly prolonged and PWT was significantly increased except T0, the expression of PKCy, CaMK ? ? and pCaMK ? ? were down-regulated in group D+R+I+DMSO (P<0.01).ConclusionDexmedetomidine administration could decrease the expression and trafficking of NR1 and NR2B subunits from cytoplasm to membrane as well as the expression and phosphorylation of PKCy and CaMK ? a in spinal cord in remifentanil-induced hyperalgesic rats; dexmedetomidine could dose-dependently reduce NMDAR electrophysiological function enhanced by remifentanil in spinal dorsal horn neurons,which might be related to the mechanism of preventive effect of dexmedetomidine on remifentanil-induced hyperalgesia. |
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