中央杏仁核钙/钙调素依赖性蛋白激酶Ⅱα在芬太尼诱发大鼠痛觉过敏中的作用：与mEPSCs的关系

Role of calcium/calmodulin-dependent kinase Ⅱ alpha in central nucleus of amygdale in fentanyl-induced hyperalgesia in rats： the relationship with mEPSCs

目的 评价中央杏仁核钙/钙调素依赖性蛋白激酶Ⅱ（CaMKⅡα）在芬太尼诱发大鼠痛觉过敏中的作用及其与微小自发性兴奋性突触后电流（mEPSCs）的关系.方法 清洁级健康雄性SD大鼠,体重50～ 80 g,中央杏仁核立体定位置管成功的大鼠32只,采用随机数字表法分为4组（n=8）：对照1组（C1组）皮下注射生理盐水,中央杏仁核给予二甲基亚砜;芬太尼诱发痛觉过敏1组（FIH1组）皮下注射芬太尼（每次60 μg/kg,共4次,每次间隔15 min,累积给药240 μg/kg）制备大鼠痛觉过敏模型,中央杏仁核给予二甲基亚砜;KN92组制备模型后中央杏仁核给予CaMKⅡα抑制剂KN92 10 nmol;KN93组制备模型后中央杏仁核给予KN93 10 nmol.于芬太尼或生理盐水注射后6和7h时测定机械痛阈和热痛阈.另取大鼠12只,采用随机数字表法分为2组（n=6）：对照2组（C2组）和芬太尼诱发痛觉过敏2组（FIH2组）分别于颈部皮下注射生理盐水和芬太尼（方法同上）,12h后制备脑片,记录mEPSCs的频率和强度,随后在脑片人工脑脊液中加入KN93 10 nmol,记录mEPSCs的频率和强度.结果 与C1组比较,FIH1组、KN92组和KN93组芬太尼或生理盐水注射后6h时、FIH1组和KN92组芬太尼或生理盐水注射后7h时机械痛阈和热痛阈降低（P＜0.05）;与FIH1组比较,KN93组芬太尼或生理盐水注射后7h时机械痛阈和热痛阈升高（P＜0.05）,KN92组差异无统计学意义（P＞0.05）.与C2组比较,FIH2组给予KN93前mEPSCs频率和强度增加（P＜0.05）,给予KN93后mEPSCs频率和强度差异无统计学意义（P＞0.05）;与给予KN93前比较,C2组给予KN93后mEPSCs频率和强度差异无统计学意义（P＞0.05）,FIH2组给予KN93后mEPSCs频率和强度降低（P＜0.05）.结论 CaMKⅡα激活增强神经元突触兴奋性参与了芬太尼诱发大鼠痛觉过敏的过程.

Objective To evaluate the role of calcium/calmodulin-dependent kinase Ⅱ alpha （CaMK Ⅱα） in the central nucleus of the amygdale （CeA） in fentanyl-induced hyperalgesia in rats and the relationship with miniature excitatory postsynaptic currents （mEPSCs）.Methods Thirty-two male Sprague-Dawley rats,weighing 50-80 g,in which the CeA was successfully cannulated,were randomly divided into 4 groups （n=8 each） using a random number table：control 1 group （group C1）,fentanylinduced hyperalgesia 1 group （group FIH1）,KN92 group,and KN93 group.Normal saline was injected subcutaneously,and dimethyl sulfoxide （DMSO） was given into the amygdale in group C1.In group FIH1,fentanyl was injected subcutaneously （60 μg/kg per time,4 times in total,15-min interval,cumulative dose of 240 μg/kg） to establish the model of hyperalgesia.In KN92 and KN93 groups,KN92 and KN93 10 nmol were given into the CeA after establishing the model.The mechanical paw withdrawal threshold （MWT） and thermal paw withdrawal threshold （TWT） were measured at 6 and 7 h after fentanyl or normal saline injection.Another 12 Sprague-Dawley rats were selected and randomly divided into either control 2 group （group C2） or fentanyl-induced hyperalgesia 2 group （group FIH2） using a random number table with 6 rats in each group.The brains were removed and sliced 12 h later,and the frequency and amplitude of mEPSCs were recorded.KN93 10 nmol was then added to the artificial cerebral spinal fluid,and the frequency and amplitude of mEPSCs were recorded by whole cell patch-clamp technique.Results Compared with group C 1,the MWT and TWT were significantly decreased at 6 h after fentanyl or normal saline injection in FIH1,KN92 and KN93 groups,and at 7 h after fentanyl or normal saline injection in FIH and KN92 groups （P〈0.05）.Compared with group FIH1,the MWT and TWT were significantly increased at 7 h after fentanyl or normal saline injection in group KN93 （P〈0.05）,and no significant change was found in group KN92 （P〉0.05）.Compared with group C2,the frequency and amplitude of mEPSCs were significantly increased before administration of KN93 （P 〈 0.05）,and no significant change was found in the frequency and amplitude of mEPSCs after administration of KN93 in group FIH2 （P〉0.05）.Compared with the value before KN93 administration,no significant change was found in the frequency and amplitude of mEPSCs after administration of KN93 in group C2 （P〉0.05）,and the frequency and amplitude of mEPSCs were significantly decreased after administration of KN93 in group FIH2 （P〈 0.05）.Conclusion Activation of CaMK Ⅱ α in the CeA enhances synaptic excitation in neurons,which is involved in fentanyl-induced hyperalgesia in rats.