【作者】 汪隆旺；

【导师】 肖传国； 李兵；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 第一部分神经源性膀胱模型制作及人工体神经-内脏神反射弧手术后再生神经形态学研究目的：观察人工体神经-内脏神反射弧手术后再生神经纤维的形态学特点和膀胱输尿管形态。方法：清洁级雌性SD大鼠60只(120～160)随机分为三组：端端吻合组(n=20),在腰4水平切断两侧]AVR、L6VR和SIVR神经,将L6VR与L4VR行端端吻合。损伤未吻合组(n=20),切断两侧L6VR和S1VR神经,不进行神经端端吻合术。对照组(n=20),切断两侧S1VR神经。手术16周后,沿大鼠背侧原切口找到L4VR-L6VR神经端端吻合口,在体视显微镜下取下距吻合口10-20mm处取10mm长再生L6神经段行1μm半薄切片甲苯胺蓝染色后统计再生神经纤维数量及电镜观察超微结构形态;肉眼观察三组大鼠膀胱形态并测量膀胱残余尿量,在体视显微镜下观察三组大鼠输尿管形态。结果：超微病理显示在端端吻合组再生L6神经前根可见大量的有髓神经纤维和雪旺氏细胞；半薄切片甲苯胺蓝染色显示再生L6VR中有髓神经纤维平均数量为700.4±50.7。肉眼观察可见端端吻合组大鼠膀胱形态正常残余尿0.7±0.9ml,输尿管无扩张,而未吻合组大鼠可见膀胱增大,残余尿可达14.2±5.6ml,输尿管明显扩张；对照组大鼠膀胱形态正常有少许残余尿平均约为0.3±0.2ml无输尿管扩张。结论：我们成功构建了神经源性膀胱和人工体神经-内脏神反射弧手术大鼠模型,L4VR和L6VR行端端吻合后L4VR运动神经元轴突可以长入L6VR,再生的L6VR可见大量的再生有髓神经纤维和雪旺氏细胞。第二部分人工体神经-内脏神反射弧手术重建膀胱神经支配的逆行神经追踪和膀胱测压研究目的：应用逆行神经追踪技术研究人工体神经-内脏神经反射弧手术后的神经再生并利用膀胱测压技术研究膀胱能否重新获得有功能的神经支配。方法：清洁级雌性SD大鼠60只(120-160g)随机分为三组：端端吻合组(n=20),切断两侧LAVR、L6VR和S1VR神经,将L6VR与L4VR行端端吻合。损伤未吻合组(n=20),切断两侧L6VR和S1VR神经,不进行神经端端吻合术。对照组(n=20),切断两侧S1VR神经。手术16周后,取端端吻合组、损伤未吻合组和对照组各10只,将荧光金(Fluoro gold, FG)1μl注射至两侧盆神经节,大鼠存活7天后4%多聚甲醛灌注固定,取L4、L6和S1脊髓节段行冰冻切片并在荧光显微镜下观察被荧光标记的神经元。另取端端吻合组、损伤组及对照组各10只分别手术显露L4VR-L6VR吻合口、L6VR残端及正常L6后将自制膀胱测压管置于三组大鼠膀胱内分别连接微量灌注泵和BL-410生物机能采集系统,电刺激已游离的神经后测量膀胱压力。结果：逆行追踪结果显示在端端吻合组L4脊髓节段切片两侧可见大量FG标记神经元,在L6和Sl脊髓节段中均未见FG标记神经元；在损伤未吻合组中LA、L6和S1脊髓节段中均未见FG标记神经元；在对照组中在L6脊髓节段中可见少量双侧FG标记神经元,在L4及S1脊髓节段中均未见FG标记神经元。膀胱测压结果显示：端端吻合组电刺激供体神经L4VR可以观察到膀胱压力先迅速升高到峰值,然后伴随着尿液排出,压力快速下降到基线水平,与对照组相类似；而刺激L6VR见膀胱压力随着持续的膀胱灌注缓慢升高并保持在基线水平,而无明显的波幅形成结论：人工体神经-内脏神反射弧手术后L4VR可再生长入L6VR并重新支配盆神经节并且膀胱可获得有功能的神经支配。第三部分神经源性膀胱行人T体神经—内脏神经反射弧手术后膀胱尿路渗透性屏障的功能研究目的：本实验利用普通HE染色、免疫荧光、电镜及实时荧光定量PCR研究大鼠膀胱尿路上皮渗透性屏障功能方法：清洁级雌性SD大鼠60只(120-160g)随机分为三组：A端端吻合组(n=20),切断两侧L4、L6和S1神经前根,将L6VR与L4VR行端端吻合。B损伤未吻合组(n=20),切断两侧L6和S1神经前根,不进行神经端端吻合术。C对照组(n=20),切断两侧S1神经前根。手术16周后,取膀胱分别行普通HE染色；uroplakinⅢ、AQP3、ZO-1免疫荧光染色,尿路上皮电镜研究及实时荧光定量PCR检测膀胱炎症因子TNF-α、IL-6和IL-1β的变化。结果：HE染色显示端端吻合组和对照组大鼠膀胱尿路上皮结构层次清晰,未吻合组大鼠膀胱尿路上皮普遍存在死亡伞细胞。免疫荧光染色显示三组中ZO-1表达没有显著性差异,在端端吻合组和对照组大鼠膀胱尿路上皮AQP-3主要表达于尿路上皮基底膜,而在未吻合组中AQP3表达于尿路上皮全层,uroplakinⅢ染色中显示尿路上皮伞细胞层被破坏。尿路上皮电镜研究显示：端端吻合组和对照组大鼠膀胱尿路上皮中没有中性粒细胞浸润,紧密联结结构清晰,伞细胞顶壁含有大量梭状囊泡,而未吻合组大鼠膀胱尿路上皮中出现中性粒细胞浸润,紧密联结结构模糊,含有大圆盘状囊泡。实时荧光定量PCR结果显示未吻合组大鼠膀胱中炎症因子TNF-α、IL-6和IL-1p的表达明显高于端端吻合组和对照组,差异具有统计学意义。结论：人工体神经-内脏神经吻合术在重新支配膀胱的同时能够修复神经源性膀胱尿路上皮的渗透性屏障,降低尿路感染的发生率。更多还原

【Abstract】 PartⅠTo make the model of neurogenic bladder and investigate the morphology of the regeneration nerve after artificial somatic-autonomic reflex pathway procedureObjective:To investigate the number and ultra-structural features of the regenerated fibers with neural morphological techniques, the morphology of the bladder and ureterMethods:Sixty female SD rats (120-160g) were randomly divided into three groups: End-to-end anastomosis group (n=20), the bilateral lumbar4ventral root (L4VR), lumbar6ventral root (L6VR) and sacral1ventral root (S1VR) were transected at lumbar4level and the distal stump of L6VR was sutured to L4VR through end-to-end anastomosis. No coaptation group (n=20), the bilateral L6VR and S1VR were transected but without neurorrhaphy; Control group (n=20), the bilateral SI VR were transected only. At16weeks, the bilateral L4VR-L6VR nerve anastomosis were found along the dorsal original incision.10mm long nerve segments of the regenerated L6VR and nerve anastomosis were harvested under operating microscope. Regenerated L6VR was cut into1-μm thick sections with Toluidine blue staining and then total regenerated nerve fibers counts of L6VR were calculated. Ultrastructure and morphology of Regenerated L6VR was examined by transmission electron microscopy. The morphologies of rat bladder and ureter were observed by naked eye and under the operating microscope respectively. Then residual urine of all rats was measured.Results:A large number of mythlinated axons of the regenerated L6VR were observed in End-to-end anastomosis group and The average number was700.4±50.7. A large numbers of myelinated fibers and Schwann cells were observed in the cross-sections under the transmission electron microscopy. The normal bladder morphology without ureteral dilatation were observed in both end-to-end anastomosis group with some residual urine 0.7±0.9ml, but significant expansion of the bladder and ureter was observed in No coaptation group and the average residual urine up to14.2±5.6ml. The normal bladder morphology without ureteral dilatation were observed in No coaptation group with some residual urine0.3±0.2mLConclusion:The rat models of neurogenic bladder and artificial somatic-autonomic reflex pathway procedure were successfully constructed, and The motor axons of L4VR can regenerate into L6VR. A lot of myelinated fibers and Schwann cells were observed in the regenerated L6VR. PartⅢNeural tracking and Cystometry study of reinnervation for bladder through end-to-end anastomosis by artificial somatic-autonomic reflex pathway procedureObjective: To investigate the neural regeneration and innervations in the efferent pathway of the bladder after end-to-end anastomosis by Neural tracking and cystometryMethods:Sixty female SD rats (120-160g) were randomly divided into three groups: End-to-end anastomosis group (n=20), the bilateral lumbar4ventral root (L4VR), lumbar6ventral root (L6VR) and sacral1ventral root (S1VR) were transected at lumbar4level and the distal stump of L6VR was sutured to L4VR through end-to-end anastomosis. No coaptation group (n=20), the bilateral L6VR and S1VR were transected but without neurorrhaphy; Control group (n=20), the bilateral S1VR were transected only. At16weeks,10rats from end-to-end anastomosis group, no coaptation group and control group, respectively, were used for nerve regeneration study. Fluorogold (FG)1μl was injected into the bilateral major pelvic ganglion (MPG).7days later,the rats were per fusion fixed by4%paraformaldehyde and the spinal segments of L4, L6and S1were taken for serial cross-sections(20-μm thick) on a cryostat and then the fluorescent labeled neurons were examined under an ultraviolet fluorescence microscope. Additional10rats from end-to-end anastomosis group, no coaptation group and control group, respectively, were used for cystometry study. the bilateral LAVR-L6VR nerve anastomosis, distal stump of L6VR and normal L6VR were found along the dorsal original incision in the three groups. Homemade cystometry tube were fixed into the bladder and then connected to micro infusion pump and BL-410biological and functional acquisition system respectively. The free nerves were electrical stimulated and bladder pressures were measured at the same time.Results:A large number ofFG labeled neurons were mainly found in the ventral horn of the bilateral L4spinal cord segment in the end-to-end anastomosis group and not found in the L6and S1spinal cord segment. FG labeled neurons in the no coaptation group were not observed in all the spinal cord segment. A small amount of FG labeled neurons only appeared in the L6spinal cord segment. Bladder pressure rapidly increased to peak was the first observed when the the donor nerve L4VR was electrical stimulated, then the pressure was gradually decreased to baseline levels along with the urine excreted in the end-to-end anastomosis group, similar to the control group. The bladder pressure increased slowly with continuous bladder infusion without significant volatility and maintained at baseline levels in the No coaptation group.Conclusions:The motor axons of L4VR can regenerate into L6VR to innervate the bladder and the bladder regained functional innervation. Part IIITo investigate the urothelial Permeability barrier function after end-to-end anastomosis of artificial somatic-autonomic reflex pathway procedureObjective:To investigate the urothelial Permeability barrier function with ordinary HE staining, immunofluorescence, transmission electron microscopy, and real-time PCRMethods:Sixty female SD rats (120-160g) were randomly divided into three groups: End-to-end anastomosis group (n=20), the bilateral lumbar4ventral root (L4VR), lumbar6ventral root (L6VR) and sacral1ventral root (S1VR) were transected at lumbar4level and the distal stump of L6VR was sutured to L4VR through end-to-end anastomosis. No coaptation group (n=20), the bilateral L6VR and S1VR were transected but without neurorrhaphy; Control group (n=20), the bilateral SI VR were transected exclusively. At16weeks,the bladder were used for ordinary HE staining, immune fluorescence staining of uroplakinⅢ、AQP3、ZO-1, transmission electron microscopy of urothelial and real-time fluorescence quantitative PCR for inflammation factor of TNF-α, IL-6and IL-1β。Results:Clear structural levels of urothelial were found in both end-to-end anastomosis group and control group, but a few deaths umbrella cells were observed in the No coaptation group in optical microscope. no significantly difference of the expressions of ZO-1in the urothelial were found among the three groups. urothelial AQP-3was expressed mainly in the urothelial basement layer in both end-to-end anastomosis group and Control group, but in the No coaptation group the AQP-3was expressed all the urothelial layer in a fluorescence microscope. urothelial umbrella cell layer in the staining of uroplakin III was damaged. In the transmission electron microscopy, Neutrophil infiltration, the fuzzy structure of Tight junctions and Multivesicular bodies(MVB) were found in the apical surface of umbrella cells in the No coaptation group, but clear structure of Tight junction and numbers of disco idal/fusiform-shaped vesicles (DFV) appeared in the apical surface of umbrella cells without Neutrophil infiltration in both end-to-end anastomosis group and Control group. The inflammatory cytokines TNF-α, IL-6and IL-1β were higher in the No coaptation group than both end-to-end anastomosis group and Control group.Conclusion:These results demonstrated that the artificial somatic-autonomic reflex pathway procedure can successfully repair the urothelial permeability barrier functions of neurogenic bladder and reduce the incidence of urinary tract infections.更多还原