角叉菜胶炎性痛及痛过敏诱导大鼠脊髓后角神经元一氧化氮合酶变化的时间特征

Time characteristics of changes of nitric oxide synthase activity in neurons of spinal cord posterior horn in rats with carrageenan-induced inflammatory pain and hyperalgesia

目的:观察角叉菜胶炎性痛及痛过敏中脊髓后角一氧化氮合酶的变化,特别是其时间特征。方法:实验于2004-05/08在河北工程学院医学院生理学实验室完成。选用清洁级健康雄性Wistar大鼠40只,随机分为5组,每组8只:对照组:不加任何处理。注射角叉菜胶12h组,注射角叉菜胶24h组,注射角叉菜胶48h组,注射角叉菜胶72h组,大鼠右后掌足跖部皮下注射30g/L角叉菜胶0.1mL建立炎性痛模型,分别在注射角叉菜胶后12,24,48,72h取材。应用尼克酰胺腺嘌呤二核苷酸-黄递酶组织化学法测定脊髓一氧化氮合酶表达。应用光学显微镜下观察并计数脊髓后角Ⅰ～Ⅱ板层内尼克酰胺腺嘌呤二核苷酸-黄递酶阳性细胞数目;应用HPLAS-1000高清晰度彩色病理图文分析系统软件测定尼克酰胺腺嘌呤二核苷酸-黄递酶阳性细胞的灰度值,用于定量分析染色深度,灰度值与染色深度呈反比。结果:纳入动物40只,均进入结果分析。①L5脊髓后角Ⅰ～Ⅱ板层尼克酰胺腺嘌呤二核苷酸-黄递酶阳性细胞数量的变化:对照组可见少量尼克酰胺腺嘌呤二核苷酸-黄递酶阳性细胞,注射角叉菜胶后12,24,48h,无论是同侧还是对侧的脊髓后角Ⅰ～Ⅱ板层内,阳性细胞数目均较对照组增加,24h达到高峰,以后逐渐减少,至72h,两侧均恢复至对照水平。此外,两侧脊髓后角比较,在注射角叉菜胶后各时间点,注射侧的阳性细胞数目均比对侧多。②L5脊髓后角Ⅰ～Ⅱ板层尼克酰胺腺嘌呤二核苷酸-黄递酶阳性细胞染色深度的变化:注射角叉菜胶后,双侧脊髓后角尼克酰胺腺嘌呤二核苷酸-黄递酶阳性细胞染色均逐渐加深,至24h达高峰,以后逐渐降低。统计结果表明,12,24,48h组阳性细胞染色深度均较对照组明显加深(即灰度值降低),其中24h加深最显著;至72h时,注射部位对侧脊髓后角Ⅰ～Ⅱ板层内的阳性细胞染色已恢复至对照组水平,而同侧尚未降至对照组水平。两侧比较,发现注射角叉菜胶后各时间点,注射同侧均较对侧阳性细胞染色加深。结论:角叉菜胶炎性痛及痛过敏中,脊髓后角一氧化氮合酶活性增强,且这种增强有一定的时间变化特征。

AIM： To observe the changes of nitric oxide synthase （NOS） activity in the spinal＇ cord posterior horn during carrageenan inflammatory pain and hyperalgesia, especially the time characteristics. METHODS： The experiment was performed in the Laboratory of Physiology, Medical College of Hebei Engineering University from May to August 2004. Forty healthy male Wistar rats of clean grade were chosen and randomly divided into 5 groups with 8 rats in each group： Control group： Rats received no treatment. Carrageenan-injection 12-hour group, carrageenan-injection 24-hour group, carrageenan-injection 48-hour group and carrageenan-injection 72-hour group, voix pedis of right hind paw in rats were hypodermicly injected with 30 g/L carrageenan 0.1 mL to establish inflammatory-pain models. Materials were drawn respectively in the 12^th, 24^th, 48^th and 72^nd hours after injection of carrageenan. NOS expression of spinal cord was determinated with nicotinamide adenine dinucleotide （NADPH） diaphorase （NADPH-d） histochemical stain. NADPH-d positive cells in laminae Ⅰ - Ⅱ of spinal cord posterior horn were enumerated and observed with light microscope; The grey degree of NADPH-d positive cells was determined with HPLAS-1000 color pathological image analysis system to quantitatively analyze the staining intensity. Value of. grey degree was inversely proportional to staining intensity. RESULTS： Forty included animals were involved in the analysis of results. ①Changes of the number of NADPH-d positive cells in laminae Ⅰ - Ⅱ of L5： Some NADPH-d positive cells were detected in the control group while the number of positive cells in laminae I - lI of spinal cord posterior horn in either same lateral or contralateral sides 12, 24, 48 hours after injection of carrageenan were increased and reached the peak in the 24^th hour and gradually decreased 24 hours after the injection of carrageena and that in bilateral sides recovered to the control level in the 72^nd hour. In addition, number of NADPH-d positive cells in the ipsilateral side was more than that in the contralateral side in each time-point after injected with carrageenan. ②Changes in the staining degree of NADPH-d positive cells in laminae Ⅰ -Ⅱ of the L5： After injection of carrageenan, that in bilateral spinal cord posterior horns gradually became deeper after injection of carrageenan and reached the peak in the 24^th hour and began to attenuate 24 hours after the injection of carrageenan. Statistical result showed that those in the 12 hour, 24 hour and 48 hour groups were remarkably deeper than that in the control group （ i.e. the value of grey degree decreased）, among which the staining degree was the deepest in the 24^th hour of injection of carrageenan. At the 72^nd hour, the staining degree of NADPH-d positive cells in laminae Ⅰ - Ⅱ of the contralateral spinal cord posterior horn recovered to control level, while neither did those in the bilateral sides. It was found in comparison between bilateral sides that the staining intensity of NADPH-d positive cells in ipsilateral side was deeper than that in the contralateral side at each time-ppoint after the injection of carrageenan. CONCLUSION： During the process of carrageenan-induced inflammatory pain and hyperalgesia, NOS activity in spinal cord posterior horn is reinforced with a certain changing characteristics in time.