脂多糖干预对大鼠周围神经损伤后瓦勒变性的影响

Effect of lipopolysaccharide on Wallerian degeneration after peripheral nerve injury in rats

目的探讨脂多糖对于大鼠坐骨神经损伤瓦勒变性早期髓鞘碎片清除的影响。方法将50只Wistar大鼠随机分成假手术组(10只),模型组(20只)和脂多糖LPS组(20只),LPS组及模型组横断大鼠右侧坐骨神经后,行神经外膜端端吻合;假手术组仅游离出坐骨神经,然后关闭切口。LPS组大鼠在神经断端显微注射LPS(2 g/L)1μL,模型组及假手术组大鼠注射同等体积生理盐水。于术后1.5、24 h和7 d取术侧坐骨神经。实时定量PCR(qRT-PCR)检测坐骨神经中白介素1β(IL-1β)mRNA、单核细胞趋化蛋白-1(MCP-1)mRNA水平;免疫荧光法检测坐骨神经中CD68+巨噬细胞的表达;HE染色观察坐骨神经的病理变化;油红O染色观察坐骨神经脱髓鞘程度;LFB染色观察坐骨神经髓鞘变化;坐骨神经功能指数(SFI)评价大鼠运动功能的恢复情况。结果实时定量PCR显示,与假手术组相比,术后1.5 h模型组IL-1βmRNA和MCP-1 mRNA的表达均明显升高(P<0.001,P<0.001),与模型组相比,术后1.5 h LPS组IL-1βmRNA和MCP-1mRNA的表达明显升高(P<0.001,P<0.001)。术后24 h模型组IL-1βmRNA和MCP-1m RNA的表达均明显升高(P<0.001,P<0.001),与模型组相比,术后24h LPS组IL-1βmRNA和MCP-1 mRNA的表达明显升高(P<0.01,P<0.01)。免疫荧光可见,与模型组相比,术后7 d LPS组中CD68+细胞表达显著上调(P<0.05)。术后7 d坐骨神经HE染色可见,LPS组坐骨神经断端较多炎性细胞浸润,许旺细胞增殖活跃,模型组神经断端炎性细胞和许旺细胞较少。术后7 d坐骨神经ORO染色可见,与模型组相比,LPS组断端远侧脱髓鞘程度较高。术后7 d坐骨神经LFB染色可见,模型组和LPS组坐骨神经断端均出现脱髓鞘反应,但与模型组相比,LPS组神经断端残余髓鞘碎片明显减少(P<0.05)。SFI显示,与模型组相比,LPS组大鼠在术后10、20、30、40和50 d分别不同程度升高,术后20 d明显增高,差异有显著性(P<0.05)。结论脂多糖通过激活固有免疫系统加快大鼠坐骨周围神经损伤后瓦勒变性早期髓鞘碎片的清除。

Objective To investigate the effects of lipopolysaccharide（LPS）on myelin phagocytosis during Wallerian degeneration after early peripheral nerve injury in rats.MethodsFifty male Wistar rats were recruited and randomly divided into LPS group（n=20）,model group（n=20）and sham group（n=10）.The right sciatic nerves of rats in the LPS and model groups were cut and sutured end-to-end,while the sciatic nerve of sham group rats were only exposed.Immediately after surgery,the rats in LPS group were given microinjections of LPS（2 g/L）into the surgical site in a final volume of 1μL,and the rats in other two groups were injected with the same volume of saline.The sciatic nerves were taken at 1.5 h,24 h and 7d after surgery.Real-time quantitative PCR（qRT-PCR）was applied to detect the dynamic expressions of IL-1βmRNA and MCP-1 mRNA.Immunofluorescence staining was used to test the expression of CD68＋macrophages in sciatic nerves.HE staining was used to observe the pathological alterations of sciatic nerves tissue.ORO staining was used to observe sciatic nerves demyelination.LFB staining was used to detect the sciatic nerves myelin.Sciatic function index was used to evaluate the recovery of motor function in rats.Results Compared with the model group,qRT-PCR indicated that the expression of IL-1βand MCP-1 from LPS group were increased at 1.5 h and 24 h after surgery（P〈0.001,P〈0.001）,respectively.Compared with the model group,the expression of CD68＋cells was increased significantly at 7th day after surgery（P〈0.05）.Histological examination showed that compared with the model group,a lot of inflammatory cells and Schwann cells were found at sciatic nerve stump in the LPS group at 7th day after operation.ORO staining showed that the degree of demyelination in the LPS group was higher than that in the model group.LFB staining showed that the sciatic nerve stump demyelination appeared in both model group and the LPS group at 7th day after operation,but compared with the model group,myelin debris clearance in the LPS group was significantly accelerated（P〈0.05）.Finally,compared with the model group,the SFI in the LPS group was increased significantly at 20 d after surgery（P〈0.05）.Conclusions The results confirm that LPS is possible to manipulate the innate immune response to accelerate myelin clearance during Wallerian degeneration after early peripheral nerve injury in rats.