BACKGROUND： It has been reported that activation and/or translocation of protein kinase C （PKC） is related to hyperalgesia, and changes in PKC expression in the dorsal horn of spinal cord take place during inflamma...BACKGROUND： It has been reported that activation and/or translocation of protein kinase C （PKC） is related to hyperalgesia, and changes in PKC expression in the dorsal horn of spinal cord take place during inflammatory pain. OBJECTIVE： To observe PKC changes in the dorsal horn of spinal cord using immunohistochemistry and to measure the time-course during persistent pain produced by chemical stimulation with a right hind-paw injection of formalin. DESIGN： Randomized controlled animal experiment. SETTING： Institute of Basic Medical Science, Hebei Medical University MATERIALS： The present experiment was performed at the Department of Pathophysiology, Institute of Basic Medical Science, Hebei Medical University between September 2000 and June 2002. Forty-two Sprague-Dawley rats, weighing 260-280 g, irrespective of gender, were provided by the Center of Animal Experimentation at Hebei Medical University. PKC antibody was provided by Sigma, USA. Immunohistochemistry kits were purchased from Zhongshan Biotechnology Company, Beijing. HPIAS-1000 definition multicolor system was provided by Qianping Wuxiang Project Company of Tongji Medical University. Animal use during experimentation was consistent with the standards of Animal Ethics Committee. METHODS： Sprague-Dawley rats were divided randomly into control （n = 6） and experimental groups （n = 36）. Experimental rats were given an intracutaneous injection of 5% formalin into the planta surface of the right hind-paw. Animals with inflammatory pain were anesthetized and sacrificed to obtain the L5 spinal region at 1, 3, 12 hours, 1, 3, and 7 days after formalin treatment, with 6 rats in each time group. The spinal cords at the L5 region were collected from the control group following sodium chloride injections into the planta surface of the right hind-paw, identical to the experimental group. MAIN OUTCOME MEASURES： Pain reaction of experimental rats after formalin treatment. PKC-positive neurons, and distribution of PKC-immunoreactive particles, in the ipsi- and contralateral dorsal horn were investigated during different stages of inflammatory pain using immunohistochemistry. RESULTS： All 42 rats were included in the final analysis, without any loss. Pain reaction： consistent with previous findings, it was determined that a unilateral injection of formalin into the hind-paw resulted in significant edema and induced a series of nociceptive responses, such as licking, biting, or shaking the injected paw. The maximal inflammation change was observed 1 day after formalin injection and changes did not disappear until the day 7. Number of the PKC positive neurons： results demonstrated that the number of PKC immunoreactive neurons in the dorsal horn increased slightly after formalin injection at 1 hour, compared with the control group. PKC immunoreactivity was up-regulated at day 1, reduced at day 3, and appeared to recover at day 7. The number of PKC-positive neurons in the contralateral side was less than the ipsilateral side at each time sampled. Distribution of PKC immunoparticles over the neurons： PKC immunoreactivity was observed in the nucleus and cytoplasm, as well as on or near the membrane of neurons and synaptosomes in the spinal cord of the control group. PKC activated and translocated from nucleus to the membrane-associated site following formalin treatment. Significant changes were observed at 1 hour and 1 day. The intensity of staining was stronger in the ipsilateral side than the contralateral side at all time points following formalin injection （P 〈 0.01）, whereas the expression patterns of PKC immunoreactivity in the nuclei were very similar in the right and left hemispheres. CONCLUSION： PKC expression in the dorsal horn of the spinal cord peaked at 1 hour and 24 hours, and was very obvious at 24 hours. Protein kinase C expression in the spinal cord increased bilaterally, although it was greater in the ipsilateral hemisphere. In addition, PKC expression at the neuronal membrane and synaptosome was significantly increased. These results indicate that PKC expression is activated in the dorsal horn of the spinal cord during hyperalgesia.展开更多
目的:探讨大鼠背根神经节持续受压(chronic compression of the dorsal root ganglion,CCD)后,脊髓背角星型胶质细胞的激活情况,以及PKCε是否可以通过调节星型胶质细胞的活性而参与CCD后神经病理性疼痛。方法:将大鼠随机分为6组,每组大...目的:探讨大鼠背根神经节持续受压(chronic compression of the dorsal root ganglion,CCD)后,脊髓背角星型胶质细胞的激活情况,以及PKCε是否可以通过调节星型胶质细胞的活性而参与CCD后神经病理性疼痛。方法:将大鼠随机分为6组,每组大鼠8只:假手术组、CCD7天组、CCD14天组、CCD7天+BIM I组、CCD+DMSO组、CCD+PDBu组,分别通过鞘内注射不同药物,或对正常大鼠鞘注DMSO/PDBu后,测量大鼠机械刺激缩爪阈值(paw withdrawal mechanical threshold,PWMT)的变化,利用Western Blot技术检测脊髓背角PKCε和GFAP蛋白表达的变化,利用免疫荧光技术检测脊髓背角星型胶质细胞激活情况。结果:CCD术后第4天,鞘内注射PKCε的激动剂PDBu 1—4h,明显降低CCD大鼠PWMT(P<0.05),而给予BIM I 1—4h,可升高CCD大鼠PWMT(P<0.05)。从CCD术后第4天起,连续3天鞘注BIM I,可明显缓解大鼠的机械痛敏(4天,P<0.05),但从停止注射后镇痛作用消失(P>0.05)。与假手术组比较,CCD后7天和14天,手术侧背角PKCε和GFAP表达升高,差异有显著性意义(P<0.05),星型胶质细胞激活增加,而注射BIM I可明显抑制PKCε和GFAP表达(P<0.05)和星型胶质细胞激活。PDBu可导致正常大鼠PWMT明显降低(P<0.05),GFAP蛋白质表达量增加(P<0.05),促进星型胶质细胞激活。结论:大鼠背根神经节持续受压后,机械痛阈下降的同时,手术侧脊髓背角内PKCε和GFAP蛋白表达上调,星型胶质细胞激活增加。PKCε可能通过调节星型胶质细胞激活参与CCD后病理性神经痛的中枢敏化机制。展开更多
目的：观察豆腐果苷（helicid）对坐骨神经慢性压迫伤（CCI）后大鼠热痛觉过敏的影响及对脊髓背角磷酸化cAMP反应元件结合蛋白（phosphorylated cAMP response element binding protein,p-CREB）表达的影响。方法：32只180～220 g Wista...目的：观察豆腐果苷（helicid）对坐骨神经慢性压迫伤（CCI）后大鼠热痛觉过敏的影响及对脊髓背角磷酸化cAMP反应元件结合蛋白（phosphorylated cAMP response element binding protein,p-CREB）表达的影响。方法：32只180～220 g Wistar雄性大鼠随机分为4组（n=8）;分别为Sham组（假手术组）、CCI组（CCI模型组）、H1组（CCI 10天后豆腐果苷,25 mg·kg^-1·d^-1×10 d灌胃）和H2组（CCI 10天后豆腐果苷,50 mg·kg^-1·d^-1×10d灌胃）。4组分别于CCI手术前1天、CCI手术后1、3、5、7、9、11、12、13、14、15、16、17、18、19、20天进行热板实验、测后肢回缩时间（PWL）。CCI后20天取L4、5脊髓,免疫组织化学染色,计数p-CREB免疫反应阳性（p-CREB-IR）神经元数量。结果：第一部分：CCI手术后第9天,Sham组、CCI组、H1组、H2组分别与术前PWL基础值比较,Sham组无差异（P〉0.05）,CCI组、H1组、H2组均有差异（P〈0.05）,且下降达30（以上;CCI手术后第9天,CCI组、H1组、H2组分别做PWL值组间两两比较均无差异（P〉0.05）,证实CCI模型制作成功。给药后第1～10天,H1组、H2组分别与CCI组做组间比较,PWL值增高,H2组第3天已有差异,H1组第5天开始有差异,两组PWL值增高至第7天达峰值,后一直保持稳定,H2组较H1组保持较高水平。第二部分：脊髓背角p-CREB免疫反应阳性神经元细胞计数比较：与Sham组比较,CCI组、H1组和H2组计数明显升高,有差异（P〈0.01）;H1组和H2组分别与CCI组比较,数量较之为少（P〈0.01）;H1组计数稍大于H2组,但无差异;即CCI〉H1〉H2〉Sham组。结论：口服豆腐果苷能剂量依赖性提高CCI大鼠热痛阈值,降低热痛觉过敏,并与影响脊髓背角p-CREB的表达有关。展开更多
文摘BACKGROUND： It has been reported that activation and/or translocation of protein kinase C （PKC） is related to hyperalgesia, and changes in PKC expression in the dorsal horn of spinal cord take place during inflammatory pain. OBJECTIVE： To observe PKC changes in the dorsal horn of spinal cord using immunohistochemistry and to measure the time-course during persistent pain produced by chemical stimulation with a right hind-paw injection of formalin. DESIGN： Randomized controlled animal experiment. SETTING： Institute of Basic Medical Science, Hebei Medical University MATERIALS： The present experiment was performed at the Department of Pathophysiology, Institute of Basic Medical Science, Hebei Medical University between September 2000 and June 2002. Forty-two Sprague-Dawley rats, weighing 260-280 g, irrespective of gender, were provided by the Center of Animal Experimentation at Hebei Medical University. PKC antibody was provided by Sigma, USA. Immunohistochemistry kits were purchased from Zhongshan Biotechnology Company, Beijing. HPIAS-1000 definition multicolor system was provided by Qianping Wuxiang Project Company of Tongji Medical University. Animal use during experimentation was consistent with the standards of Animal Ethics Committee. METHODS： Sprague-Dawley rats were divided randomly into control （n = 6） and experimental groups （n = 36）. Experimental rats were given an intracutaneous injection of 5% formalin into the planta surface of the right hind-paw. Animals with inflammatory pain were anesthetized and sacrificed to obtain the L5 spinal region at 1, 3, 12 hours, 1, 3, and 7 days after formalin treatment, with 6 rats in each time group. The spinal cords at the L5 region were collected from the control group following sodium chloride injections into the planta surface of the right hind-paw, identical to the experimental group. MAIN OUTCOME MEASURES： Pain reaction of experimental rats after formalin treatment. PKC-positive neurons, and distribution of PKC-immunoreactive particles, in the ipsi- and contralateral dorsal horn were investigated during different stages of inflammatory pain using immunohistochemistry. RESULTS： All 42 rats were included in the final analysis, without any loss. Pain reaction： consistent with previous findings, it was determined that a unilateral injection of formalin into the hind-paw resulted in significant edema and induced a series of nociceptive responses, such as licking, biting, or shaking the injected paw. The maximal inflammation change was observed 1 day after formalin injection and changes did not disappear until the day 7. Number of the PKC positive neurons： results demonstrated that the number of PKC immunoreactive neurons in the dorsal horn increased slightly after formalin injection at 1 hour, compared with the control group. PKC immunoreactivity was up-regulated at day 1, reduced at day 3, and appeared to recover at day 7. The number of PKC-positive neurons in the contralateral side was less than the ipsilateral side at each time sampled. Distribution of PKC immunoparticles over the neurons： PKC immunoreactivity was observed in the nucleus and cytoplasm, as well as on or near the membrane of neurons and synaptosomes in the spinal cord of the control group. PKC activated and translocated from nucleus to the membrane-associated site following formalin treatment. Significant changes were observed at 1 hour and 1 day. The intensity of staining was stronger in the ipsilateral side than the contralateral side at all time points following formalin injection （P 〈 0.01）, whereas the expression patterns of PKC immunoreactivity in the nuclei were very similar in the right and left hemispheres. CONCLUSION： PKC expression in the dorsal horn of the spinal cord peaked at 1 hour and 24 hours, and was very obvious at 24 hours. Protein kinase C expression in the spinal cord increased bilaterally, although it was greater in the ipsilateral hemisphere. In addition, PKC expression at the neuronal membrane and synaptosome was significantly increased. These results indicate that PKC expression is activated in the dorsal horn of the spinal cord during hyperalgesia.
基金This work was supported by the National Natural Science Foundation of China (No. 30470565) and the Educational Commissionof Fujian Province of China (No. JB04230).
