【作者】 孙丰银；

【导师】 陈敏；

【作者基本信息】 华中科技大学 ， 泌尿外科， 2011， 博士

【摘要】 目的：应用神经电生理学和神经形态学技术,研究大鼠体神经-内脏神经(L4VR-L6VR)吻合术后,新形成的脊髓-盆神经节-直肠、脊髓-肛门外括约肌(EAS)神经反射通路的相关生理学功能,探讨体神经-内脏神经吻合术支配肛管直肠功能的可能性。方法：成年雄性SD大鼠20只,通过端端显微神经吻合技术,人工建立腰4前根(L4VR)-腰6前根(L6VR)异类神经前根。手术12周后存活12只,将模型大鼠随机分为2组(n1=8,n2=4)。取1组8只模型大鼠进行电生理学研究：电刺激L4VR-L6VR异类神经根的吻合口近端,记录再生盆神经节前、节后纤维的诱发电位、直肠压力的变化及肛门外括约肌肌电活动(EMG)变化；电刺激大鼠自身右侧(健侧)L4VR和L6VR作为对照。取2组4只模型大鼠进行神经形态学研究：于左侧盆神经节(MPG)注射荧光金(FG),逆行追踪检测L4VR-L6VR端端吻合术后,异类神经前根的形成及物质转运情况；另取4只正常大鼠作为对照。结果：1、于模型大鼠左侧盆神经节注射荧光金(FG)后,在脊髓L4节段区域可见FG标记神经元,而L6节段未见阳性神经元；正常鼠左侧盆神经节注射FG后,仅在脊髓L6节段可见到FG阳性神经元。2、电刺激L4VR-L6VR神经吻合口近端,于盆神经节前及盆神经节后神经纤维均可记录到诱发电位,并可记录到直肠收缩活动(16.89±9.86mmHg)和肛门外括约肌肌电活动(127.91±28.73 u V)。3、吻合术后再生的传出神经(35.43m/s±12.25m/s)较对照侧(12.33m/s±1.77 m/s)有更快的传导速度(P<0.05)。结论：体神经-内脏神经吻合术后,体神经(L4VR)可以再生长入内脏躯体躯体运动混合神经,再生的L4VR-L6VR异类神经前根能够传递诱发电位而且具有相对独特的电生理特性；再生的轴突具有物质转运功能,支配直肠节前神经元节段及形态发生了改变。从功能学和形态学上证实吻合后神经有功能,可以支配相应的盆腔靶器官—直肠和肛门外括约肌。目的：应用肛肠动力学和神经形态学技术,研究体神经--内脏神经吻合术后,在脊髓损伤(SCI)大鼠的所形成的再生异类神经通路对肛管直肠的支配功能及直肠平滑肌-肛门外括约肌的协同的情况,并阐明其潜在机理。方法：在成年雄性SD大鼠(250-300g)的L4-L6椎管内,将左侧腰4前根(L4VR)与左侧腰6前根(L6VR)分别离断后,通过端端显微吻合技术,人工建立L4VR-L6VR异类神经前根。术后12周行T9-T10椎间脊髓横断,8周后将12只模型大鼠随机分为2组(n1=8,n2=4)。取1组8只模型大鼠进行肛肠动力学研究：分别电刺激L4VR-L6VR吻合口近端及左侧坐骨神经,同步记录直肠压力和肛门外括约肌的肌电活动,以了解直肠平滑肌-肛门括约肌的协同情况；以电刺激自身对侧L6VR及坐骨神经作为对照；取2组4只模型大鼠进行神经形态学研究：于左侧盆神经节(MPG)、左侧肛门外括约肌分别注射荧光金(FG)和四甲基罗丹明葡聚糖(dextran tetramethylrhodamine,TMR),通过神经逆行示踪,了解L4VR-L6VR吻合术后大鼠控制排便的神经通路的变化；以4只正常大鼠作为对照。结果：1.电刺激模型大鼠吻合口近端神经及左侧坐骨神经,在引起直肠持续收缩的同时出现肛门外括约肌的肌电活动大幅减弱；而电刺激自身对照侧L6VR,在引起直肠压力升高的同时出现肛门外括约肌肌电活动明显增强。2.在模型鼠左侧盆神经节(MPG)、肛门外括约肌(EAS)分别注射荧光金(FG)和四甲基罗丹明(TMR)后,主要在脊髓L4节段区域可见少量的FG与TMR双标神经元及FG、四甲基罗丹明单标神经元；正常鼠神经逆行追踪未见双标神经元。结论：体神经-内脏神经吻合术可有效修复脊髓损伤大鼠肛管直肠功能,改善脊髓损伤后的直肠平滑肌-肛门括约肌协同失调情况。在模型大鼠脊髓L4节段新出现的双标神经元同时支配着盆神经节和肛门外括约肌,这可能是体神经-内脏神经吻合术修复模型大鼠排便功能主要神经解剖学基础。更多还原

【Abstract】 Objective:To study the physiological functions of the regenerated "spinal cord-major pelvic ganglion (MPG)-rectum" and "spinal cord-external anal sphincter (EAS) neural reflex pathways and to investigate the possible mechanisms underlying the artificial somato-autonomic neuroanastomosis for initiating defecation in the spinal cord injury rats.Methods:In normal male Sprague-Dawley rats, under anesthesia the new regenerated neural pathway was established by intradural microanastomosis of left L4 ventral root(L4VR) to the L6 ventral root (L6VR) while leaving the L4 dorsal root intact to trigger the new the regenerated neural pathway. Then L4VR-L6VR formed the foreign nerve root. Twelve weeks later the model rats were randomly divided into two groups of 8 and 4 respectively (n1=8, n2=4). The rats in the group 1 were used for electrophysiological experiment. Then the L4VR-L6VR foreign nerve root was stimulated by silver electrode and the evoked potentials were recorded on the preganglionic fibers and postganglionic fibers of the major pelvic ganglia (MPG). The rectal pressure and electromyologram of EAS were recorded. The stimulation of the right L4VR and L6VR in contralateral side were used as controls. The rats in the group 2 were used for neural retrograde tracing study. Fluorogold (FG) was injected into the left major pelvic ganglia (MPG). Retrograde tracing technique was used to investigate the material transmitting function of regenerated efferent nerve. Normal rats were used as controls.Results:(1)In the group 1, stimulation of the L4VR proximal end to the anastomosis evoked potentials on the left preganglionic fibers and the postganglionic fibers of the MPG, and change of rectal pressure (16.89±9.86mmHg) and electromyologram of EAS (127.91±28.73μV). Stimulation of the contralateral L4VR failed to evoke potentials on the right preganglionic fibers and the postganglionic fibers of MPG, and can’t induce rectum and EAS to contract. (2) The conduction velocity of the regenerated motor axons was 35.43m/s±12.25m/s, significantly higher than that of the control group(12.33m/s±1.77 m/s). (3)After FG was microinjected into the left major pelvic ganglia (MPG) in the model rats FG labeled neurons were observed in the segment of L4. FG labeled neurons were observed in L6 segment after FG was injected into the left major pelvic ganglia (MPG) in the normal rats.Conclusion:Somatic nerve axons (L4VR) can regenerate to replace the peripheral autonomic and somatic motor nerve fibers through axonal regeneration. And the regenerated nerves can innervate the rectum and EAS to initiate defecating. Objective:A new artifical somatic-autonomic neuroanastomosis has been estabilished in male rat with spinal cord injury (SCI). Anorectal manometry and neural retrograde tracing were conducted in such animal model after estabilishing the regenerated reflex pathway to analyze the mechanisms, and the effects on restoring anorectal function and rectum external sphincter synergetic status.Methods:In normal male Sprague-Dawley rats with weight of 280-300 g, under anesthesia the rats were subjected to limited laminectomy of L4-L6 vertebrae to expose the spinal cord, and the new reflex pathways were estabished by intradural microanastomosis of left L4 ventral root to the L6 ventral root while leaving the L4 dorsal root intact to trigger the new defecation reflex pathway. Then L4VR-L6VR formed the foreign nerve root. Three months later the spinal cord was completely transected at the T9-10 level. Eight weeks later the model rats were randomly divided into two groups of 8 and 4 respectively (n1=8, n2=4). The rats in the group 1 were applied in anorectal manometry. When the left L4VR proximal to the anastomosis and left sciatic nerve were stimulated by silver electrode, the rectal pressure and EMG of external anal sphincter (EAS) were simultaneously recorded. The stimulated L6VR and the right sciatic nerve in contralateral side served as controls. The rats in the group 2 were used for neural retrograde tracing study. Fluorogold (FG) was injected into the left major pelvic ganglia (MPG) and dextran tetramethylrhodamine (TMR) was injected into the left external anal sphincter. Retrograde tracing technique was used to investigate important neuromechanism for defecation of regenerated efferent nerve. The normal rats were used as controls.Result:Stimulation of the L4VR proximal end to the anastomosis and the left sciatic nerve induced rectum to contract and simultaneously electric activity of EAS to become weak or disappearing(indicating synergetic relaxation of EAS). But stimulation of the contralateral L6VR evoked simultaneous contraction of rectum and EAS. After FG was injected into the left major pelvic ganglia (MPG) and TMR was injected into the external anal sphincter (EAS), FG-TMR dual labelled neurons and FG or TMR single labeled neurons were mainly observed in the angulus anterior ofL4 segment in the model rats. FG-TMR positive neurons were not observed in the spinal cord of the normal rats.Conclusion:After established the artifical somato-autonomic neuroanastomosis the regenerated neural pathways are effective to improve the rectum external sphincter synergetic status and restore the anorectal function. Simultaneous reinnervation of MPG and EAS by one group of the dual labelled neurons in L4 may be the key neural anatomy infrastructure for controllable defecation via the somatic-autonomic reflex pathway.更多还原