【作者】 罗永春；

【导师】 朱刚；

【作者基本信息】 第三军医大学 ， 外科学， 2004， 硕士

【摘要】 周围神经损伤后,伤口处和远侧段全长神经纤维发生瓦勒变性,由于部分神经元凋亡,局部疤痕增生,吻合口处感觉、运动神经纤维轴突易相互错长,长期失神经支配后终末器官萎缩、变性等有害因素,导致治疗后功能恢复多数并不理想。因此神经损伤后的形态学变化和各种促进神经再生及功能恢复的方法成为近年来研究的热点。目的:为了研究不同程度瓦勒变性对大鼠坐骨神经再生与功能恢复的影响及白细胞介素-1β对神经再生的影响机制,以及神经损伤后变性与再生过程的形态学变化规律,探索周围神经再生过程的可视化研究方法,为周围神经的治疗提供理论依据。材料与方法:本课题第一部分研究在大鼠坐骨神经切割伤模型中分别利用白细胞介素-1β(IL-1β)和二氯亚甲基二磷酸盐脂质体(Cl2MDP)干预神经变性过程,损伤后1、3、5、7、15、30、60、90天用组织学方法、免疫组化方法检测损伤后不同时间点坐骨神经断端巨噬细胞的聚集与瓦勒变性程度,结合坐骨神经功能指数(SFI)、神经电生理等指标的恢复情况,评估不同程度的瓦勒变性对神经再生的影响。课题第二部分利用免疫组化方法检测损伤局部神经生长因子及胶质细胞源性神经营养因子的动态表达变化,结合组织学方法及轴突图像分析,探索白细胞介素-1β在神经损伤与再生中的作用机制。课题第三部分对不同时间点、不同部位的坐骨神经进行连续切片,经过改良三色染色法染色后光学显微镜观察并摄像后输入微机进行图像配准和二维处理,再利用图形工作站对坐骨神经损伤后变性、再生过程中典型的显微形态结构进行三维重建和显示,探讨坐骨神经再生过程的形态特征及量化规律。结果:1. 伤后3天、7天硅胶管内可见淡红色胶冻样物质,组织易碎;伤后15天白细胞介素-1β处理组可见纤细浅红色条索状物把坐骨神经两断端连接起来;伤后30～90天各组硅胶管内再生神经逐渐变粗、变韧,颜色呈亮白色,但是白细胞介素-1β处理组再生神经直径明显大于Cl2MDP处理组和对照组,而后两组没有显著差异。2. 光镜及电镜结果显示,IL-1β处理组神经损伤后巨噬细胞聚集的时间及轴突断裂、髓鞘崩解的时间均较早,变性组织清除也相应较快,增生的胶原纤维较少;再生神经轴突直径、数量及髓鞘厚度均优于对照组。Cl2MDP处理组早期很少有巨噬细胞出现于神经断端,变性组织清除较慢,再生神经分布密度也较小。 <WP=10>3.坐骨神经功能指数测定结果显示,白细胞介素-1β处理组坐骨神经功能指数恢复较快而且较好,Cl2MDP处理组早期恢复不如对照组,但后期与对照组差异不显著。4.神经电生理结果显示,白细胞介素-1β处理组神经传导速度及复合肌肉动作电位波幅的恢复明显优于其余两组(P<0.01);Cl2MDP处理组复合肌肉动作电位波幅较对照组差(P<0.01),神经传导速度与对照组相比早期较差(P<0.01),晚期则无明显差异(P>0.05)。5.神经损伤早期,白细胞介素-1β处理组损伤局部神经生长因子及胶质细胞源性神经营养因子的表达明显高于同一时期的对照组(P<0.01),神经再生晚期无明显差异性。6.再生神经轴突图像分析结果显示,白细胞介素-1β处理组再生神经的有髓神经纤维的数目和轴突直径、髓鞘厚度均明显优于对照组(P<0.01)。7.利用改良三色法对正常及再生神经进行染色并于光镜下摄像,输入微机进行图像配准和二维处理,在图形工作站中进行三维重建和显示,三维图像显示神经变性和再生过程较为典型的病理结构,如Bungner带的形成,郎飞氏节的结构等。结论:1. 白细胞介素-1β可加剧神经损伤后瓦勒变性,使其变性组织清除加快,神经再生的程度及功能恢复较好。二氯亚甲基二磷酸盐脂质体耗竭了大鼠的单核细胞后,致使神经损伤后的变性组织清除延迟,早期神经再生较差,但对晚期神经功能恢复的影响较小。2. 白细胞介素-1β能增加损伤早期局部的神经营养因子(NGF及GDNF)的合成与分泌,抑制胶质瘢痕的增生,对周围神经再生有一定的促进作用,再生小室内用药结合局部注射白细胞介素-1β可以作为治疗周围神经损伤的一种方法与手段。3. 利用计算机图像重建技术,采用三维图像的形式对外周神经再生过程和再生规律进行可视化研究,是一种有效的方法。更多还原

【Abstract】 The peripheral nerve injury is always accompanied by Wallerian degeneration in the wound and distal regions of the nerve and apoptosis of some related neurons.But some harmful factors such as wrong growth each other of motor and sensory neuritis in regeneration regions, the fibroplasias in nerve gap and atrophy of axon terminals after a long time of denervation always result the nerval function recovery is always dissatisfactory.So, it has become focus in clinical and basic research to investigate morphological changes of the injured nerve and measures of promoting peripheral nerve regeneration.Objective 1) To study the effects of different extent of the wallerian degeneration on injured sciatic nerve regeneration and recovery of neural function in rat .2) To investigate the mechanisms and effects of Interleukin-1 beta on nerve regeneration.3) To research morphological changes of degenerating and regenerating sciatic nerve following injury, and grope for medical image visualization methods for peripheral nerve regeneration.The findings of these researches may provide theoretical basis for clinical therapy to peripheral nerve injury.MethodsIn the part one of the research, the rats’ sciatic nerve was transected and then bridge-connected with silicone tube, The wallerian degeneration of transected sciatic nerve were intervened with recombinative human Interleukin-1 beta(IL-1β) and dichloromethylene diphosphonate-containing liposome(Cl2MDP) in respectively. Macrophage congregation in the local and the extent of wallerian degeneration were observed by pathological and immunohistochemical means on the 1、3、5、7、15、30、<WP=6>60、90 days after injury, and the outcome of sciatic nerve function index(SFI)and Electrophysiolgical assessmentwere observed, by which the effects of different extent of wallerian degeneration on the nerve regeneration was evaluated.In the part two of the research, the dynamic expression of the nerve growth factor (NGF)and Glial cell line-derived neurotrophic factor(GDNF) in the local were inspected with immunohistochemical technique, associating with pathology and morphometric analysis, the mechanisms and effects of Interleukin-1 beta were explored on injured peripheral nerve in rats. In the part three of the research, The images of serial slices which were stained with trichrome stain was taken by micro-photograph system and inputted into computer by which the image matching and 2-D integrate processing were completed.The 3-D reconstruction and display of typical morphological structure of the injured nerve was accomplished in SGI workstation, and then we can explore morphological character of sciatic nerve regeneration and quantization method for nerve regeneration process.Result1.Soft red crisp colloid was seen in the silicone tube for 3 and 7 days after injury.15 days later it was observed that a soft red strip has connected two sides of transected sciatic nerve in the IL-1β group.After 30 to 90 days all of the newborn nerve became thicker , more tenacious, and its color became brighter than before, whereas, the diameter of the rebirth sciatic nerve in the IL-1β group were bigger than that of Cl2MDP and control groups.But the difference between the Cl2MDP group and control group has been inapparent .2.The findings in the light microscope and electron microscope indicated that the time of macrophage assemble in the local region of injury and the time of axon rupture and myelin collapse in the IL-1β group was earlier than that of other groups, and the cleanness of degenerative tissue was more faster in the early stage in the IL-1β group.There was fewer hyperplastic collagen fiber in the IL-1β group.The indexes of regeneration nerve were superior to that of control group.On the contrary, there is fewer macrophage which invade into the local region in the early stage in the Cl2MDP group, and the elimination of degenerative tissue was also slow.The distributing density of the newborn nerve also was inferior. <WP=7>3.The recovery rate of sciatic nerve functional inde更多还原