条件性损伤的时间与频次对神经根撕脱伤后脊髓前角运动神经元死亡及NOS表达的影响

The time interval and frequency of conditioning lesion affect the motoneuron NOS expression and cell death of anterior horn after spinal roots avulsion

目的 探索条件性损伤 (CL)的时间和频次对神经根撕脱 (TL)后脊髓前角运动神经元一氧化氮合酶 (NOS)表达及细胞死亡的影响。方法 成年SD雌性大鼠 116只 ,体重 2 0 0～ 30 0g ,以钳夹神经干为CL ,分为两个系列。时间系列 :在CL后 1d、3d、1w、2w后进行TL ,频次系列分别在 1w和 2w内行 1次、2次、6次钳夹 ,术后不同时间点处死动物 ,以单纯撕脱臂丛为对照 ,取C5～C8节段脊髓 ,行NADPH d组化染色 ,中性红复染。定量观察前角运动神经元NOS表达及神经元数目。结果 单纯撕脱组术后第 5d脊髓前角运动神经元开始表达NOS ,术后 3wNOS阳性神经元数达到高峰 ,随后逐渐下降并伴有神经元丢失。时间系列 :撕脱后 3d和 2w ,CL与TL间隔 1d组的NOS表达和神经元数目与对照组无显著性差异 ,但 3d、1w、2w组的NOS细胞数及神经元的丢失均较对照组明显 (P <0 0 5P <0 0 1) ,但在撕脱后 4w各CL时间的NOS表达和神经元存活均较对照组减少 (P <0 0 5 )。频次系列 :在 1w和 2w内增加CL次数可使NOS的表达水平和神经细胞的死亡数目有显著增加 ,在撕脱后 2w和 4w等较后的时间点更为明显 ;但在一定时间内 2次与 6次钳夹之间则无显著性差异。结论 条件性损伤与二次损伤之间的间隔影响撕脱后神经元NOS表达和神经元的死亡 。

Objective To investigate the effect of the time interval and the frequency of conditioning lesion (CL) on motoneuron death and NOS expression after spinal roots avulsion (test lesion,TL). Methods One hundred and sixteen female adult SD rats,200～300g, were used. Crushing C 5～C 8 nerve stems of brachial plexus were considered as CL, while avulsing the roots of the same nerve as TL. In the time interval series, TL were performed 1d,3d,1w,2w after CL respectively;In the frequency series, CL once, twice or six times were performed respectively before TL. Rats only received TL were used as control. All rats were killed at 3d,2w or 4w after TL. The C 5～C 8 spinal cord freeze sections 40μm thick were cut for NADPH d histochemistry and counterstained by neural red. The NOS expression level and dying montoneuron number were quantitatively analyzed. Results In the TL group, NOS expression began at the 5th day and reached a maximum by 3 weeks after TL. Then the NOS positive neurons gradually declined accompany with a significant motoneuron loss. In the time interval series, There were not distinct differences in both NOS expression and dying montoneuron number between TL 1d after CL and TL only at 3d or 2w after avulsion. However,the differences appeared between TL only and TL 3d, 1w or 2w after CL. All CL+TL with different time intervals led less NOS positive and survival motoneurons at 4w after avulsion. In the frequency series:the NOS expression level and dying montoneuron number increased distinctively with increasing frequency of CL within 1w or 2w. The difference was more distinguishing at 2w or 4w after avulsion. This effect was not increased in CL six times than in CL twice. Conclusions The time interval between CL and TL can affect the course of montoneuron death and NOS expression. This effect reaches a maximum in one week interval. The frequency change of CL within a certain time span can also affect the montoneuron NOS expression and cell death course, this effect reaches a maximum in CL twice. Our results show that there is the best time to use NGF or other methods inhibiting NOS express and protecting motoneurons after roots avulsion.