一氧化氮在周围神经冷损伤中作用的研究

目的 探讨一氧化氮(NO)在周围神经冷损伤病理过程中的作用及其可能的作用机制.方法 建立大鼠坐骨神经冷损伤模型,分别给予持续或间断4℃低温2 h,于低温完全结束后即刻、4 h、1、3 d时处死取材,同时设立诱导型一氧化氮合酶(iNOS)特异性抑制剂氨基胍给药组,测定各组坐骨神经实验侧、对照侧和血清中NO含量、神经节iNOS免疫组化阳性表达的面积和累积光密度(IOD)值,并对给药组和未给药组的神经组织在半薄光镜及超薄电镜下的病理形态学变化进行对比观察.结果 持续低温组即刻、4 h、3 d时间点和间断低温组即刻、4 h、1 d时间点实验侧坐骨神经NO含量[(0.146±0.047),(0.216±0.048),(0.137±0.035),(0.154±0.027),(0.260±0.027),(0.218±0.042)μmoL/g]均不同程度高于各自对照侧[(0.098±0.022),(0.158±0.030),(0.085±0.020),(0.127±0.016),(0.172±0.027),(0.174±0.026)μmol/g,P<0.01,P<0.05],且间断低温组4 h、1、3 d时间点实验侧NO含量不同程度高于持续低温组(P<0.01,P<0.05);间断低温组即刻、1 d时间点血清NO含量[(4.98±1.33)μmoL/L,(4.02±0.68)μmol/L]高于持续低温组[(2.47±0.36)μmol/L,(3.00±0.67)μmol/L,P<0.01];两组实验侧iNOS阳性表达面积及IOD值均不同程度大于对照侧(P<0.01,P<0.05,P>0.05),且间断低温组即刻时间点[(131 686±24 549)像素单位,(4.10±0.13)×109]明显大于持续低温组[(78 558±34 849)像素单位,(2.10±0.93)×109,P<0.05];间断低温给药组实验侧神经NO含量[(0.178±0.030)μmol/g]明显低于间断低温组1 d时间点[(0.218±0.042)μmol/g,P<0.05],持续低温给药组和间断低温给药组大鼠血清NO含量、实验侧iNOS阳性表达的面积、IOD值较相应时间点未给药组均有不同程度的减少(P<0.05),以间断低温给药组的变化较显著(P<0.01,P<0.05);光镜及电镜下,间断低温后1 d神经组织病理损伤程度重于持续低温后1 d,给予氨基胍干预后神经组织病理损伤程度减轻,以间断低温给药组的变化较为明显.结论 NO参与了周围神经冷损伤的病理过程,且在间断低温冷损伤的过程中尤为明显;NO主要来源于iNOS的激活表达,生成大量自由基继而造成相应的毒性损伤可能是其主要的作用机制.

Role of nitric oxide in pathogenesis of cold nerve injury

Objective To study the role of nitric oxide (NO) in the pathogenesis of cold nerve injury and to explore its mechanism. Methods Cold nerve injury model was established in rat's sciatic nerve. Consistent and intermittent cooling of 4℃ for 2 hours were respectively applied to the sciatic nerves. Nerve samples were taken at different time points: immediately post-cooling and 4 h, 1 d & 3 d post-cooling. The group of aminoguanidine (AG) intervention was also established. Then NO levels in the nerves and blood serum were detected. Induced nitric oxide synthase (iNOS) in ganglion was determined by immunohistochemistry. Morphology of cooled sciatic nerves in the AG groups (consistent & intermittent cooling AG groups) and control groups (1 d time point of consistent & intermittent cooling groups) was observed by light microscope and electron microscope. Results At the time points of immediacy, 4 h, 3 d in the consistent cooling groups and the time points of immediacy, 4 h, 1 d in the intermittent cooling groups, different incremental degrees of NO levels in the cooled nerves (0. 146±0. 047), (0. 216± 0. 048), (0. 137±0. 035), (0. 154 ±0. 027), (0. 260±0. 027), (0. 218±0. 042) μmol/g as compared with those of controls (0. 098±0. 022), (0. 158 ±0. 030), (0. 085 ±0. 020), (0. 127 ±0. 016), (0. 172± 0. 027), (0. 174 ± 0. 026) μmol/g, P < 0. 01, P < 0. 05). And NO levels in the cooled nerves at the time points of 4 h, 1 d, 3 d in the intermittent cooling groups were higher than those in the consistent cooling groups (P < 0. 01, P < 0. 05). NO levels in blood serum at the time points of immediacy and 1 d in the intermittent cooling groups (4. 98 ± 1.33) μmol/L, (4. 02 ±0. 68) μmol/L were higher than those in the consistent cooling groups (2.47±0. 36) μmol/L, (3.00 ±0. 67) μmoL/L, P <0. 01). Both in consistent cooling and intermittent cooling groups, different incremental degrees of areas and integrated optical density (IOD) of iNOS positive staining in ganglions were found in cooled side as compared with the control side at different time points (P < 0. 01, P < 0. 05, P > 0. 05), while those at the time point of immediacy in the intermittent cooling groups (131 686 ±24 549), (4. 1 ±0. 13) ×109 were higher than those in the consistent cooling groups (78 558 ± 34 849), (2. 1 ± 0. 93) ×109, P < 0. 05. NO level of cooled nerves in the intermittent cooling AG group (0. 178 ±0. 030) μmol/g was lower than those at the time point of 1 d in intermittent cooling groups (0. 218 ±0. 042) μmol/g, P <0. 05). Also a reduction of NO levels of blood serum, areas and IOD of iNOS positive staining was found in the AG groups as compared with the control groups (P < 0. 05) while the reduction was more significantly found in the intermittent cooling AG group (P <0. 01, P <0. 05). Through light microscope and electron microscope, more severe pathological injury of cooled nerve was seen in the intermittent cooling group as compared with 1 d time point of the consistent cooling groups, and a marked reduction of pathological injury was found when AG was administered. It was also more significant in the intermittent cooling AG group. Conclusion NO plays a causative role in the pathogenesis of cold nerve injury, especially in intermittent cold nerve injury. The expression of iNOS is the main source of NO. Production of free radicals and the resulting toxic injury may be its main mechanism.