神经移位术重建高位颈脊髓损伤大鼠膈肌功能及神经通路重塑的研究

发布时间：2018-04-29 13:00

本文选题：脊髓损伤 + 膈肌功能　；参考：《第二军医大学》2015年硕士论文

【摘要】：[目的]脊髓膈神经元胞体所在核团主要位于颈3-颈5水平,高位颈脊髓损伤病人,若损伤累及颈3-颈5水平,随之则出现完全性或不完全性的膈肌瘫痪。受损的呼吸系统相关肌肉严重限制了患者的活动、生活质量及发声,因而在慢性进展阶段不得不采用机械通气以辅助患者呼吸。然而伴随机械通气所带来的并发症逐渐增加,如呼吸机相关性肺炎、张力性气胸及纵膈气肿等等。长期机械通气引起的呼吸道相关并发症是导致高位脊髓损伤患者高死亡率高主要原因。本课题组发现可以将大鼠副神经作为动力神经,移位吻合至膈神经以重建高位颈髓横断伤后膈肌运动功能。本课题拟在其基础上建立单侧颈3脊髓半切动物模型,进一步探讨神经移位术后大鼠膈肌功能恢复的程度,并通过神经逆行示踪的方法,研究呼吸相关神经通路重塑的的情况。[方法]健康成年雄性S-D大鼠(100-150克)48只,平均随机分配到A、B、C三组,每组16只。A组为健康大鼠组,B组为左侧膈神经、副神经在锁骨水平离断组,C组为左侧副神经斜方肌支锁骨水平移位吻合膈神经干组。饲养6个月后,每组随机选取8只大鼠行胸片、肺功能、动脉血气分析,之后将这部分大鼠处死。剩余每组8只大鼠行颈3脊髓左侧半切损伤,饲养1周后,再次将每组剩余8只大鼠,行胸片、肺功能、动脉血气分析。再次选取健康成年雄性S-D大鼠(100-150克)24只,随机平均分配至D、E、F三组,每组8只。其中D、E组为健康大鼠组,F组为神经移位组,正常饲养6月后,E、F组大鼠行颈3脊髓左侧半切。左侧膈肌行绿色荧光蛋白基因重组的伪狂犬病毒定点注射,顺时针选取4点,每点注射5微升。正常饲养4天后行心脏灌注、取脑。连续冰冻切片后于荧光显微镜观察,并同大鼠脑解剖图谱进行比对。选取带有荧光的切片行免疫组织化学染色。使用NIS-Elements2.1软件获取图像,使用Image-Pro Plus 6.0行绿色荧光显色阳性神经元胞体计数。统计学分析采用SPSS 20.0软件,采用单因素方差分析进行组间比较。组间两两比较采用Bonferroni法(方差齐时)或Mann-Whitney法(方差不齐时),P=0.05。[结果]颈3左侧脊髓半切前,B、C两组大鼠肺功能相关指标均低于A组,但C组优于B组。颈3左侧脊髓半切后,A组大鼠各项肺功能指标约下降50%,B、C两组指标同脊髓半切前基本持平,略有下降,此时C组大鼠各项指标均优于A、B两组。E组大鼠未观察到荧光显像的神经元细胞,D、F组大鼠在延髓内检测到绿色荧光,免疫组织化学染色证实为绿色荧光蛋白基因重组伪狂犬病毒转染的神经元细胞核团。[结论]副神经斜方肌支在锁骨水平移位吻合膈神经主干的方法可以部分重建颈3至颈5水平颈脊髓损伤大鼠的膈肌功能,同时可以一定程度上改善大鼠的呼吸功能。通过逆行示踪的方式发现神经移位术重新建立了膈肌同延髓之间的联系,此神经通路在颈脊髓损伤大鼠膈肌功能恢复中起到了重要的作用。
[Abstract]:[objective] the nucleus of the phrenic neuron is located mainly at the level of cervical 3 neck 5. If the injury involves the level of 3 neck 5 of the cervical cord, complete or incomplete diaphragmatic paralysis will occur in the patients with high cervical spinal cord injury. Damaged respiratory muscles severely limit the patient's activity, quality of life, and voice, so mechanical ventilation has to be used to assist the patient in the chronic stage of progression. However, complications associated with mechanical ventilation, such as ventilator-associated pneumonia, tension pneumothorax and mediastinal emphysema, are increasing. Respiratory tract related complications caused by long-term mechanical ventilation are the main cause of high mortality in patients with high spinal cord injury. Our team found that the accessory nerve can be used as the dynamic nerve and anastomosed to the phrenic nerve to reconstruct the diaphragm motor function after high cervical spinal cord transection. Based on the model of unilateral cervical 3 spinal cord hemisectomy, this study aims to explore the degree of recovery of diaphragm function in rats after nerve transposition, and to study the remodeling of respiratory related nerve pathway by retrograde tracing of nerve. [methods] Forty-eight healthy adult male S-D rats were randomly assigned to group A (n = 16), group B (n = 16) and group B (n = 16), with left phrenic nerve in group B (n = 8). The accessory nerve was transfered horizontally from the clavicular branch of the left trapezius muscle to the phrenic trunk group. After 6 months of feeding, 8 rats in each group were randomly selected for chest radiography, pulmonary function and arterial blood gas analysis. The remaining 8 rats in each group were treated with left side hemisection injury of the cervical 3 spinal cord. After feeding for 1 week, the remaining 8 rats in each group were again given chest radiographs, pulmonary function and arterial blood gas analysis. Twenty-four healthy adult male S-D rats were randomly divided into three groups (8 rats in each group). Among them, DU E group was healthy rat group and F group was nerve transposition group. After 6 months of normal feeding, the left side of the cervical 3 spinal cord was cut in the EOF group. The left diaphragm was injected with pseudorabies virus with green fluorescent protein gene recombination, 4 points clockwise, 5 microliters per point. After normal feeding for 4 days, the heart was perfused and brain was taken. Continuous frozen sections were observed by fluorescence microscope and compared with the anatomical map of rat brain. Sections with fluorescence were selected for immunohistochemical staining. NIS-Elements2.1 software was used to obtain images and Image-Pro Plus 6.0 was used to count the cell bodies of green fluorescent positive neurons. Statistical analysis was carried out with SPSS 20.0 software and single factor analysis of variance (ANOVA). The two groups were compared with each other by Bonferroni method (homogeneity of variance) or Mann-Whitney method (P0. 05 when variance was not equal). [results] the relative indexes of lung function in group C were lower than those in group A, but group C was superior to group B. The indexes of lung function in group A decreased about 50% after hemisection of the left side of the cervical 3 spinal cord, and the indexes in group C were almost the same as those in group C before spinal cord hemisection, and decreased slightly. At this time, the indexes of group C were better than those of group A B. Group E. the rats in group D and F had detected green fluorescence in medulla oblongata. It was confirmed by immunohistochemical staining that it was a neuronal nuclear mass transfected by recombinant pseudorabies virus (PRV) with green fluorescent protein gene (GFP). [conclusion] the method of horizontal transposition of trapezius branch of accessory nerve and anastomosis of phrenic nerve trunk can partly reconstruct the diaphragm function of the rats with cervical spinal cord injury from 3 to 5 levels, and improve the respiratory function of the rats to some extent. It is found by retrograde tracing that the connection between diaphragm and medulla oblongata is re-established by nerve transposition, which plays an important role in the recovery of diaphragmatic function in rats with cervical spinal cord injury.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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