Toll-like receptor 4 antagonist protects against Wallerian degeneration after peripheral nerve injury

doi:10.3969/j.issn.2095-4344.2016.42.012

Abstract

Abstract:

BACKGROUND: The mechanism underlying Wallerian degeneration following peripheral nerve injury is complex. Immune regulation on Wallerian degeneration is beneficial for early repair of perpheral nerve injury.
OBJECTIVE: To investigate the effects of Toll-like receptor 4 (TLR4) antagonist on Wallerian degeneration and axonal regeneration after early peripheral nerve injury in rats.
METHODS: Fifty male Wistar rats were recruited and randomly divided into treatment group (n=20), model group (n=20) and sham group (n=10). The right sciatic nerves of rats in treatment and model groups were cut and sutured end-to-end, while the sciatic nerves of rats in sham group were only exposed. In the treatment group rats were intravenously injected with 0.15 mg/kg TAK-242 via tail vein 1 hour preoperatively and 7 days postoperatively, and the rats in the other two groups were given intravenous injection of the same volume of normal saline. The sciatic nerves were removed at 24 hours, 3, 4 and 7 days after surgery.
RESULTS AND CONCLUSION: Real-time PCR indicated that the mRNA expressions of interleukin-1β and monocyte chemoattractant-1 were significantly increased in the model group compared with the sham group at 24 hours after surgery (both P < 0.001), while the expressions were significantly decreased after TAK-242 injection (both P < 0.001). Immunofluorescence showed that compared with the model group, down-regulated expression of CD68+ and iba1+ cells appeared in the treatment group at 3 days after surgery (P < 0.01, P < 0.05). Luxol fast blue staining revealed that demyelination at the sciatic nerve stump appeared in both model and treatment groups at postoperative 7 days, but myelin debris clearance in the treatment group was significantly reduced compared with the model group (P < 0.05). Hematoxylin-eosin staining showed that a lot of inflammatory cells, Schwann cells and regenerated nerve fibers at the sciatic nerve stump were found in the model group, while there were few inflammatory cells, Schwann cells and regenerated nerve fibers in the treatment group at 7 days after surgery. Immunohistochemistry found that the expression of growth-associated protein-43 in the treatment group was significantly lower than that in the model group at 4 days postoperatively (P < 0.05). Besides, compared with the model group, a significantly decreased sciatic functional index was found in the treatment group at 20, 30 and 40 days after surgery (P < 0.05). These results show that TLR4 antagonists delay early nerve regeneration in rats after sciatic nerve injury probably by inhibiting the TLR4 signaling pathway.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Peripheral Nerves, Toll-Like Receptor 4, Myelin Sheath, Tissue Engineering

CLC Number:

R318

Xiong Le1, Zhang Bei1, Shen Ruo-wu2, Ji Ai-yu3, Sun Guang-qiang1, Bian Hong-lin3, Zhang Feng-yu1, Wang Yi1, Huang Heng4, Li Hua-qiao1, Zhou Shan-yu5, Shen Zhao-kang6, Wang Zhong1 . Toll-like receptor 4 antagonist protects against Wallerian degeneration after peripheral nerve injury[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(42): 6308-6316.

Figures/Tables 1

2.1 实验动物数量分析实验选用Wistar大鼠50只，分为3组，实验过程无脱失，全部进入结果分析。 2.2 大体观察各组大鼠均发育良好，无死亡，切口甲级愈合。术后20 d内，假手术组术后与术前基本无变化，无行动困难、足趾挛缩及足踝部红肿的表现，针刺足部反应灵敏；处理组和模型组大鼠术后均出现右下肢功能活动受限，术侧腿部肌肉均有萎缩现象，足趾挛缩不能伸展，呈拖曳步态，并出现不同程度右足踝部皮肤红肿，足趾溃疡，针刺反应消失。术后40 d，处理组和模型组大鼠开始出现溃疡干燥，部分大鼠溃疡愈合。术后50 d，模型组大鼠溃疡全部愈合，针刺反应灵敏；处理组仍有2只大鼠足趾未完全愈合，针刺反应迟钝。 2.3 实时定量PCR测坐骨神经组织白细胞介素1β和单核细胞趋化蛋白1 mRNA的表达熔解曲线分析显示2组基因扩增均呈单峰，退火温度一致，无非特异性扩增。与假手术组相比，在术后24 h，模型组的白细胞介素1β和单核细胞趋化蛋白1 mRNA表达水平均显著升高(P < 0.001)。与模型组相比，处理组的白细胞介素1β和单核细胞趋化蛋白1 mRNA表达水平均差异降低(P < 0.001)。结果见图2。 2.4 免疫荧光法测坐骨神经组织CD68+巨噬细胞细胞和iba1+许旺细胞的表达术后3 d神经组织切片免疫特异性巨噬细胞 CD68 抗体和许旺细胞iba1抗体染色可见，模型组神经断端有大量CD68+巨噬细胞和iba1+许旺细胞浸润，而与模型组相比，处理组CD68+巨噬细胞和iba1+许旺细胞均明显减少。通过定量，分析距离损伤中心2 mm内远端免疫阳性细胞面积所占比值显示，处理组CD68+巨噬细胞明显减少(P < 0.01)，处理组iba1+许旺细胞也明显减少(P < 0.05)。结果见图3。 2.5 大鼠坐骨神经髓鞘固兰染色术后7 d坐骨神经固兰染色可见，髓鞘呈天蓝色，轴突不着色，背景为白色,反映在坐骨神经损伤远端崩解的髓鞘碎片被巨噬细胞清除的情况。假手术组大鼠坐骨神经髓鞘结构完整，排列规则，厚度均一，模型组和处理组神经断端可见髓鞘淡染，均为变性崩解的髓鞘，与模型组相比，处理组残余髓鞘碎片较多(方框内表示两组残余髓鞘差异)，通过定量分析，计算距离损伤中心2 mm内远端被染色髓鞘的积分吸光度平均值，可见与模型组相比，处理组残余髓鞘碎片较多，差异有显著性意义(P < 0.05)，反映了处理组的崩解髓鞘碎片清除速率明显降低模型组。结果见图4。 2.6 大鼠坐骨神经组织苏木精-伊红染色术后7d苏木精-伊红染色可见，假手术组大鼠坐骨神经组织结构完整，纤维排列整齐，无炎性细胞浸润，模型组和处理组轴突完全断裂，髓鞘崩解，模型组较多炎性细胞浸润，许旺细胞增殖活跃，纤维组织增生明显，有神经纤维生长通过吻合口，显示了正常的神经断端修复状态；处理组可见有炎性细胞浸润，少量许旺细胞增生，吻合口间仍有缝隙，神经断端修复较差。结果见图5。 2.7 免疫组织化学法测坐骨神经组织生长相关蛋白43(GAP-43)蛋白的表达用距离神经断端4 mm处坐骨神经横切片的生长相关蛋白43表达水平来衡量轴突再生情况。术后4 d免疫组织化学检测可见，免疫组织化学图片背景洁净，几乎无非特异性染色。生长相关蛋白43免疫阳性产物，均呈深褐色，主要分布于轴突内，生长相关蛋白43主要定位于细胞浆、细胞膜及细胞外基质。通过定量分析，计算免疫阳性细胞的积分吸光度平均值可见，与模型组相比，处理组中生长相关蛋白43蛋白表达均显著降低(P < 0.05) 。结果见图6。 2.8 大鼠坐骨神经功能指数(SFI)测定模型组和处理组大鼠在术后10，20，30，40和50 d，行足迹实验分析坐骨神经功能指数，评价大鼠运动功能的恢复情况。各组大鼠行走时步态稳定，足迹清晰。结果显示，在坐骨神经损伤后10 d，两组大鼠的坐骨神经功能指数均为最低值，之后开始逐步上升。处理组在损伤后20，30和40 d，坐骨神经功能指数均分别明显低于模型组(P < 0.05)。结果见图7。"

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