大鼠脊髓压迫性损伤后脱髓鞘病变及硫酸软骨素蛋白多糖的表达变化

- Demyelination and changes in chondrotin sulfate proteoglycan expression after spinal cord compression injury

目的探讨脊髓压迫性损伤（CSCI）后脱髓鞘病变及硫酸软骨素蛋白多糖（CSPG，NG2）的表达变化。方法选取健康成年SD大鼠75只，雌雄不限，采用随机数字表法将其分为正常组、假手术组、CSCI1d组、CSCI3d组和CSCI7d组，每组15只。采用自行设计的大鼠脊髓压迫器制作脊髓压迫模型，应用BBB运动功能评分、锇酸染色及透射电镜观察CSCI后1d、3d及7d时大鼠的运动功能情况及白质有髓神经纤维的病理变化，计算脊髓后索有髓神经纤维的数量及髓鞘厚度与轴突直径的比值即G-ratio值，并采用免疫印迹法检测NG2蛋白的表达变化。结果CSCI后，CSCI1d组、CSCI3d组和CSCI7d组的大鼠后肢BBB评分分别为（1．23±0．45）分、（0．654±0．35）分和（0．00±0．00）分，与正常组（21．004±0．00）分和假手术组（21．00±0．00）分相比，差异有统计学意义（P〈0．05），大鼠的运动功能随受压时间延长而逐渐下降。锇酸染色显示正常组和假手术组大鼠的白质正常，脊髓受压后，CSCI1d组、CSCI3d组和CSCI7d组的有髓神经纤维开始出现水肿、变性、崩解，正常组和假手术组脊髓后索的有髓神经纤维计数分别为（2771±108）根和（2675±199）根，CSCI1d组、CSCI3d组和CSCI7d组的有髓神经纤维计数分别为（2403±161）根、（1708±70）根和（8104-95）根，压迫后有髓神经纤维数目减少，压迫后7d，有髓神经纤维数目减至最低（P〈0．05），与正常组和假手术组比较，差异有统计学意义（P〈0．05）。电镜定量分析结果显示，正常组、假手术组、CSCI1d组、CSCI3d组和CSCI7d组的G—ratio值分别为（18．104±0．4）、（17．704±1．0）、（6．694±0．8）、（5．73±0．4）和（4．954±0．5），CSCI1d组、CSCI3d组和CSCI7d组的G—ratio值均较正常组和假手术组低，且在压迫后7d时降至最低，差异有统计学意义（P〈0．05）。透射电镜结果显示，正常组和假手术组的轴突、髓鞘板层结构正常；脊髓受压后，轴索出现肿胀，轴浆内细胞器变性、坏死、减少；髓鞘折叠、皱缩，出现“洋葱皮”样变，髓鞘崩解；少突胶质细胞的染色质凝聚；巨噬细胞浸润。NG2蛋白免疫印迹结果显示，脊髓受压后，NG2蛋白表达水平在压迫后第1天升至最高（P〈0．05），且表达水平随压迫时间延长而逐渐下调，但均高于正常组和假手术组，差异有统计学意义（P〈0．05）。结论CSCI后，大鼠运动功能随受压时间延长而逐渐下降，有髓神经纤维发生脱髓鞘病变且数量减少，随着压迫时间延长，溃变呈现出进行性加重趋势；NG2细胞与CSCI后髓鞘的变化情况关系密切，可能增殖分化为少突胶质细胞或其它类型细胞，是脊髓髓鞘内源性修复的机制之一。

Objective To investigate the role of demyelination and the alteration of chondrotin sulfate proteoglyean （CSPG,NG2） expression after compression injury of the spinal cord （CSCI）. Methods Seventy-five adult Sprague-Dawley rats were randomly divided into a normal group, a sham-operation group, a CSCI 1 day group, a CSCI 3 day group, and a CSCI 7 day group. There were 15 rats in each group. The injuries in the CSCI groups were inflicted using a technique devised in our laboratory. Basso-Beattie-Bresnahan （BBB） neurological function assess- ment was used to assess the rats＇ motor function, osmic acid staining and transmission electronic microscopy （TEM） were used to observe any pathological changes of myelinated nerve fibers in the white matter at 1, 3 and 7 days after CSCI. The amount of myelinated nerve fibers in the posterior funiculus of the spinal cord and the ratio of myelin sheath thickness to axon diameter （the G-ratio） were calculated. Any alteration in NG2 expression was observed by Western blotting. Results The average neurological function assessment scores in the CSCI groups were （1.23 ± 0.45 ） , （0.65 ± 0.35 ） and （0.00 ± 0.00） respectively. Compared with the normal group （21.00 ± 0.00） and the sham operation group （21.00 ± 0.00 ） , the differences were all statistically significant. The rats＇ motor function deteriorated gradually with time after the CSCI. Osmic acid staining showed that the white matter was intact in the normal and sham groups. After being compressed the myelinated nerve fibers became swollen, degenerated and broke down. The amount of myelinated nerve fibers in the normal group, the sham operation group and the three CSCI groups was （2771 ± 108） , （2675 ± 199）, （2403 ± 161 ） , （ 1708 ±70） and （810 ±95） respectively. The amount of myelinated nerve fibers decreased in the CSCI groups and reached a minimum on the 7th day. The difference was statistically significant. The TEM quantity analysis showed that the G-ratios in the normal, sham operation, and CSCI 1 day, 3 day and7 day groups were （18.10±0.4）, （17.70±1.0）, （6.69±0.8）, （5.73 ±0.4） and （4.95±0.5） respectively. Compared with the normal and sham operation groups, the G-ratios in the 3 CSCI groups were lower and reached their minimum on the 7th day after injury. The difference was statistically significant. TEM observation showed that the axons and myelin sheaths were intact in the normal and sham groups. After CSCI the axons became swollen and cell organelles in the axoplasm degenerated and decreased. The layers of myelin sheath shrank, folded and even wrinkled, which had an onion-like appearance. The oligodendrocytes exhibited chromatin condensation. Macrophages showed infiltration. Western blotting showed that the expression of NG2 in the CSCI groups reached a maximum on the 1st day after injury and then decreased with time. The expression of NG2 in the CSCI groups was higher than in the normal and sham groups, and the difference was statistically significant. Conclusion Demyelination occurs after CSCI-the amount of myelinated nerve fibers decreases and neurological deficits increase with time. The expression of NG2 was associated with changes in the myelin sheaths after CSCI and contributed to recovery of the myelin sheath through proliferation and differentiation to oligodendrocytes and perhaps other kinds of cells.