去部分背根及电针刺对猫脊髓Ⅱ板层SP表达的影响
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摘要
【目的】采用免疫组织化学技术,探讨SP在正常猫、部分去背根及针刺部分去背根猫脊髓Ⅱ板层(L_1、L_3、L_5、L_7)表达的时空变化,藉以初步了解SP与脊髓可塑性和针刺关系。
【方法】取成年健康雄猫20只。随机分4组:正常对照组、术后14天组、1月和3月组(行双侧部分去背根手术,切除双侧L_1-L_5、L_7-S_2背根节,保留L_6为备用根)术后当天针刺备用根14天组、1月组和3月组的左侧,左侧为针刺侧右侧为非针刺侧(动物行双侧部分去背根术后,针刺备用背根节外周支配区内的两组穴位足三里和悬钟,伏兔和三阴交。针刺频率98次/分,时间为30分钟,每分钟15时交换电极,每天针刺1次,每次针刺一组穴位,两组穴位交替进行。动物于术后分别存活14天、1月和3月后处死)。每组选取五只猫。动物于术后14天、1月和3月分别灌注固定,取正常组、14天组、1月组和3月组的脊髓L_1、L_3、L_5、L_7节段,—20℃恒冷箱切片,片厚20μm,用兔抗SP(1∶5000)抗体行免疫组织化学两步法染色。观察、计数并比较各组脊髓Ⅱ板层(L_1、L_3、L_5、L_7)中SP的分布、含量及时空变化。进行组间多个样本均数比较的单因素方差分析和组内两样本均数比较的配对t检验。
【结果】1.SP阳性膨体主要分布于猫脊髓后角浅层,呈颗粒状。2.部分去背根和针刺后,脊髓L_1、L_3、L_5、L_7的后角Ⅱ板层SP阳性神经膨体数在术后14天明显组下降,1月最低,随后至3个月明显恢复L_1、L_3达14天水平,而L_5、L_7已明显超过14天水平,但仍低于正常水平;且针刺侧L_1、L_3、L_5、L_7脊髓同节段Ⅱ板层SP阳性膨体数量均较非针刺侧明显减少(P＜0.05)。
【结论】部分去背根导致脊髓Ⅱ板层SP免疫阳性神经膨体数呈现一种先减少后增加趋势,提示备用背根来源的SP在脊髓内发生了侧枝出芽,支持备用根猫脊髓可塑性。
针刺进一步导致备用根来源的脊髓Ⅱ板层SP免疫阳性神经膨体数较部分去背根组显著减少,提示针刺镇痛可能与SP下调有关。
[Objective] To explore effects of partial dorsal rhizotomy and electro-acupuncture (EA) on the expression of SP in lumbar II of the spinal cord (L_1,L_3, L_5, L_7) in cats so as to find out the relationship between SP expression and EA promoting spinal cord plasticity.
[Methods] 20 adult healthy male cats were divided randomly into 4 groups (n=5 in each group): normal control group; bilateral partial dorsal root rhizotomy 14~(th) day, 1 month and 3 month groups (bilateral L_1-L_5, L_7-S_2 DRGs were transected; but the L_6 DRG was spared) and received left EA treatment after operation (at acupoints Zusanli and Xuanzhong, Futu and Sanyinjiao, were stimulated alternately at the frequency of 98 HZ for 30 minutes every day. They were allowed to survive 14 day, 1 month and 3 months after operation). The cats in different groups were perfused and L_1,L_3,L_5,L_7 spinal segments were collected and were cut into 20μm frozen sections. Then, the sections were stained with specific SP (1:5000) antibody by the immunohistochemistry two-step method under the same condition. The distribution and the number of SP positive varicosity in lumbar II of the spinal cord (L_1,L_3, L_5, L_7) were observed and counted. All data were analyzed by One-Way ANOVA and Independent-Samples t-test.
[Results] 1. SP immunopositive varicosity mainly dispersed in superficial lamina of dorsal horn in spinal cord with the form of particles. 2. At 14 days after partial removal of dorsal root ganglia and EA, the number of SP immunopositive varicosities decreased in lamina II of L_1, L_3, L_5, L_7 spinal segments and reached the lowest at 1 month. At 3 months after operation, the number of these varicosities increased to different extents: equal to the level of 14 day in L_1 and L_3 spinal segments while surpassed it in L5 and L7 segments, but still less than the normal level. And there were less SP positive staining varicosities in the unilateral spinal cord receiving acupuncture than the other lateral (P<0.05).
[Conclusion] The partial removal of dorsal root ganglia lead to the change in the number of SP positive staining varicosities: first decreased and subsequently increased. This suggested that SP positive varicosities derived from spared dorsal root ganglion sprouted in the spinal cord and promoted spinal cord plasticity in rats subjected to partial dorsal root ganglionectomies.
Comparing with the operated group, the number of SP positive varicosities derived from spared dorsal root ganglion significantly decreased after EA. This suggested that EA releasing pain might have a relation with decrease of SP.
【方法】取成年健康雄猫20只。随机分4组:正常对照组、术后14天组、1月和3月组(行双侧部分去背根手术,切除双侧L_1-L_5、L_7-S_2背根节,保留L_6为备用根)术后当天针刺备用根14天组、1月组和3月组的左侧,左侧为针刺侧右侧为非针刺侧(动物行双侧部分去背根术后,针刺备用背根节外周支配区内的两组穴位足三里和悬钟,伏兔和三阴交。针刺频率98次/分,时间为30分钟,每分钟15时交换电极,每天针刺1次,每次针刺一组穴位,两组穴位交替进行。动物于术后分别存活14天、1月和3月后处死)。每组选取五只猫。动物于术后14天、1月和3月分别灌注固定,取正常组、14天组、1月组和3月组的脊髓L_1、L_3、L_5、L_7节段,—20℃恒冷箱切片,片厚20μm,用兔抗SP(1∶5000)抗体行免疫组织化学两步法染色。观察、计数并比较各组脊髓Ⅱ板层(L_1、L_3、L_5、L_7)中SP的分布、含量及时空变化。进行组间多个样本均数比较的单因素方差分析和组内两样本均数比较的配对t检验。
【结果】1.SP阳性膨体主要分布于猫脊髓后角浅层,呈颗粒状。2.部分去背根和针刺后,脊髓L_1、L_3、L_5、L_7的后角Ⅱ板层SP阳性神经膨体数在术后14天明显组下降,1月最低,随后至3个月明显恢复L_1、L_3达14天水平,而L_5、L_7已明显超过14天水平,但仍低于正常水平;且针刺侧L_1、L_3、L_5、L_7脊髓同节段Ⅱ板层SP阳性膨体数量均较非针刺侧明显减少(P＜0.05)。
【结论】部分去背根导致脊髓Ⅱ板层SP免疫阳性神经膨体数呈现一种先减少后增加趋势,提示备用背根来源的SP在脊髓内发生了侧枝出芽,支持备用根猫脊髓可塑性。
针刺进一步导致备用根来源的脊髓Ⅱ板层SP免疫阳性神经膨体数较部分去背根组显著减少,提示针刺镇痛可能与SP下调有关。
[Objective] To explore effects of partial dorsal rhizotomy and electro-acupuncture (EA) on the expression of SP in lumbar II of the spinal cord (L_1,L_3, L_5, L_7) in cats so as to find out the relationship between SP expression and EA promoting spinal cord plasticity.
[Methods] 20 adult healthy male cats were divided randomly into 4 groups (n=5 in each group): normal control group; bilateral partial dorsal root rhizotomy 14~(th) day, 1 month and 3 month groups (bilateral L_1-L_5, L_7-S_2 DRGs were transected; but the L_6 DRG was spared) and received left EA treatment after operation (at acupoints Zusanli and Xuanzhong, Futu and Sanyinjiao, were stimulated alternately at the frequency of 98 HZ for 30 minutes every day. They were allowed to survive 14 day, 1 month and 3 months after operation). The cats in different groups were perfused and L_1,L_3,L_5,L_7 spinal segments were collected and were cut into 20μm frozen sections. Then, the sections were stained with specific SP (1:5000) antibody by the immunohistochemistry two-step method under the same condition. The distribution and the number of SP positive varicosity in lumbar II of the spinal cord (L_1,L_3, L_5, L_7) were observed and counted. All data were analyzed by One-Way ANOVA and Independent-Samples t-test.
[Results] 1. SP immunopositive varicosity mainly dispersed in superficial lamina of dorsal horn in spinal cord with the form of particles. 2. At 14 days after partial removal of dorsal root ganglia and EA, the number of SP immunopositive varicosities decreased in lamina II of L_1, L_3, L_5, L_7 spinal segments and reached the lowest at 1 month. At 3 months after operation, the number of these varicosities increased to different extents: equal to the level of 14 day in L_1 and L_3 spinal segments while surpassed it in L5 and L7 segments, but still less than the normal level. And there were less SP positive staining varicosities in the unilateral spinal cord receiving acupuncture than the other lateral (P<0.05).
[Conclusion] The partial removal of dorsal root ganglia lead to the change in the number of SP positive staining varicosities: first decreased and subsequently increased. This suggested that SP positive varicosities derived from spared dorsal root ganglion sprouted in the spinal cord and promoted spinal cord plasticity in rats subjected to partial dorsal root ganglionectomies.
Comparing with the operated group, the number of SP positive varicosities derived from spared dorsal root ganglion significantly decreased after EA. This suggested that EA releasing pain might have a relation with decrease of SP.
引文
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