ObjectiveTo study the distribution and expression of NMDA receptor subunit 2A in the spinal cord of morphine tolerant rats. MethodsTwelve Sprague-Dawley rats were randomly divided into two groups with 6 rats each: control group (C) were intrathecally administrated 0.9% NaCl 10μl and morphine group(M) received 10μg morphine (i.t.). Drugs were administrated twice daily for 7 consecutive days. Tail flick latency (TFL) in the hot water immersion test was used to evaluate changes of thermal hyperalgesia latency of each group before and 30min after administration every morning. Rats were killed the day after last administration and L4～5 segment of the spinal cord was removed for determination the expression of NR2A by immunofluorescence method. ResultsTFL of group M was decreased gradually after chronic administration of morphine intrathecally. There was no significant difference between group M[(3.25±0.93)s] and group C[(2.66±0.27)s] on the 7th day (P＞0.05). A morphine tolerant model was established successfully. NR2A was distributed throughout the rat spinal cord. The expression of NR2A in group M(OD:9617±1233) was increased compared with group C(OD:2.66±0.93) (t=3.133,P＜0.05).ConclusionThe expression of NR2A was upregulated after repeated administration of morphine intrathecally in the superficial dorsal horn of spinal cord of morphine tolerant rats,which may be part of the mechanisms of morphine tolerance.

Objective To investigate the interaction between NMDA receptor subunit 2B (NR2B) and metabotropic glutamate receptor 5 (mGluR5) in the spinal cord of morphine-tolerant rats. Methods Sixteen male SD rats weighing 220-280 g were randomly divided into 2 groups with 8 rats in each group: control group (C) and morphine group (M). In group M morphine 10μg was administered intrathecally (IT) twice a day for 7 consecutive days. In group C equal volume of normal saline was given instead of morphine. Tail flick latency (TFL) (a segment of tail 4-5 cm long was immersed in 52-53℃ hot water) was used to measure the antinocicepitve effect of morphine before and 30 min after morphine administration every morning. MPE (percentage of maximal possible effect) was calculated (pain threshold after drug administration - baseline pain threshold)/(10- baseline pain threshold)× 100% . The animals were killed at the next day after last IT administration and the dorsal half of L4-5 spinal cord was isolated and homogenized for determination of NR2B and mGluR5 protein expression by Western blot and CO-IP. Results MPE was significantly increased at 1-5 d of drug administration and returned to baseline at 7 d in group M as compared with group C. The mGluR5 protein expression was significantly up-regulated in group M as compared with group C but there was no significant difference in NR2B protein expression between the 2 groups. Conclusion There is interaction between NK2B and mGluR5 in the spinal cord of morphine-tolerant rats.

Objective To establish a LC-MS method of cisatracurium assay in human plasma for clinical therapeutic drug monitoring. Method Propafenone Hydrochloride was used as the internal standard. The plasma samples were treated with 2% formic acid aqueous solution and acetonitrile containing the internal standard to precipitate protein. Agilent SB-C₁₈ column was used for gradient elution with the mobile phase of 0.1% formic acid-water and 0.1% formic acid-acetonitrile solution at 35 ℃ and 0.3 ml/min flow rate. The degradation products of cisatracurium m/z 464.6-358.4 and propafenone hydrochloride m/z 342.2-116.2 were identified by ESI positive-ion detection. Results There was a linear rage of cisatracurium in 2-500 ng/ml (r=0.996 5) with a detection limit of 2 ng/ml. The intra-day coefficients of variation (CVs) were less than 16.00%, and the inter-day CVs were less than 6.00%. The mean recoveries were in the range of 97.63%-111.93%. The plasma samples were stable for 4 hours at room temperature, 14 days at -80 ℃ and 24 hours after pretreated. Conclusion This method was simple, accurate, fast and repeatable for the cisatracurium assay in human plasma.