【作者】 马彦；

【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】 第一部分躯体神经-内脏神经吻合后神经再生研究目的：应用神经追踪技术及形态学方法研究躯体神经-内脏神经显微吻合后神经的再生情况。方法：取成年雄性SD大鼠（250～300g）33只，随机分为3组：假手术组、神经切断组、神经吻合组（每组11只）。假手术组，打开椎管后只分离出左侧腰4前根（L4VR）、双侧腰6前根（L6VR）及骶1前根（S1VR）而不切断。神经切断组，分离出左侧L4VR、双侧L6VR及S1VR并切断。神经吻合组，分离出左侧L4VR、双侧L6VR及S1VR切断后，左侧L4VR与左侧L6VR行端端显微吻合。术后16周，假手术组、神经切断组、神经吻合组各取6只大鼠行逆行追踪研究，左侧盆神经节内注射荧光金（FG），大鼠再存活一周后取脊髓切冰冻切片，在荧光显微镜下观察FG阳性神经元数目与分布。3组再各取剩余5只大鼠，分离出5mm L6VR神经段，行半薄切片甲苯胺蓝染色及透射电镜超微结构观察。结果：逆行神经追踪结果显示，假手术组，大鼠脊髓左侧L6及S1脊髓节段的中间外侧柱（SPN）可以看到FG阳性神经元。神经切断组，大鼠各个脊髓节段均未见FG阳性标记神经元。神经吻合组，左侧L4脊髓前角可以见到FG阳性神经元。假手术组L6及S1脊髓SPN内FG阳性神经元数目分别为13.2±1.3和4.8±0.5。神经吻合组L4脊髓前角FG阳性神经元为7.9±1.1。神经吻合组FG阳性神经元数目显著多于神经切断组（p <0.05）。半薄切片甲苯胺蓝染色显示，假手术组可见大量正常的有髓神经纤维，有髓神经纤维数量为784±45。神经切断组轴突崩解、变性，未见到正常神经纤维。神经吻合组可见大量有髓神经纤维再生，有髓神经纤维数量为510±53。假手术组及神经吻合组有髓神经纤维数量均显著多于神经切断组（p <0.05）。神经超微结构示，假手术组大鼠L6VR内可见大量发育成熟的有髓轴突，轴突髓鞘较厚。神经切断组未见到正常形态的有髓神经纤维。神经吻合组大鼠L6VR可见大量的有髓神经纤维再生，轴突髓鞘较厚，致密，轴浆丰富。结论：逆行神经追踪、半薄切片甲苯胺蓝染色及电镜超微结构研究证明躯体神经—内脏神经通过端端吻合可以实现神经再生，再生的神经纤维可以长入并取代内脏神经（L6VR）。第二部分躯体神经-内脏神经吻合对大鼠勃起功能影响研究目的：验证躯体神经-内脏神经端端吻合后再生的神经能否发挥功能，引发阴茎勃起。方法：取雄性SD大鼠（体重250～300g）30只，随机分为3组（每组10只）：假手术组，只分离出神经而不切断；神经切断组，双侧L6VR、S1VR及左侧L4VR切断；神经吻合组，双侧L6VR、S1VR及左侧L4VR切断后，左侧L4VR与左侧L6VR行端端显微吻合。术后16周，假手术组、神经切断组、神经吻合组大鼠行海绵体内压力（ICP）测定，三组分别电刺激L6VR、L6VR断端及L4VR，同时记录阴茎海绵体内压力及动脉压力。结果：电刺激时，假手术组达到的最大海绵体压力（ICPmax）为72.7±2.5mmHg，海绵体压升高值（ICP increase）为60.5±3.3mmHg，海绵体压力升高值与平均动脉压比值（ICP/MAP）为0.68±0.03。神经切断组ICP max为17.1±0.9mmHg，ICP increase为2.7±0.5mmHg，ICP/MAP为0.03±0.01。神经吻合组ICP max为39.4±2.7mmHg，ICP increase为26.7±3.0mmHg，ICP/MAP为0.31±0.04。假手术组和神经吻合组ICPmax、ICP increase及ICP/MAP均显著高于神经切断组（p <0.05）。结论：大鼠躯体神经—内脏神经反射通路建立后，刺激L4VR可以导致海绵体内压力的升高，证实躯体神经不但能够长入并取代内脏神经，再生的神经还能发挥功能，引发阴茎勃起。第三部分躯体神经-内脏神经吻合重建大鼠勃起功能后阴茎NOS表达及组织学改变目的：探讨躯体神经—内脏神经反射通路建立后，大鼠阴茎NOS表达情况及组织学改变。方法：假手术组、神经切断组、神经吻合组大鼠（各10只）行海绵体测压后剪刀取下阴茎组织，取一部分用4%多聚甲醛固定，石蜡包埋，切片行Masson染色，观察阴茎平滑肌与胶原纤维的变化；另一部分用4%多聚甲醛固定后置入30%蔗糖脱水，冰冻切片行NADPH-黄递酶染色及nNOS免疫荧光染色，检测阴茎组织中NOS阳性神经纤维表达量。结果：阴茎组织Masson染色后平滑肌和胶原纤维分别呈红色和蓝色。假手术组大鼠平滑肌与胶原纤维比值为0.109±0.008，神经切断组为0.043±0.005，神经吻合组为0.089±0.007。假手术组和神经吻合组大鼠平滑肌与胶原纤维比值显著高于神经切断组（P<0.05）。假手术组大鼠阴茎背神经中可见大量NADPH阳性神经纤维表达。神经切断组NADPH阳性神经纤维表达数量显著下降。神经吻合组NADPH阳性神经纤维表达量显著高于神经切断组（P<0.05）。假手术组阴茎背神经中nNOS阳性神经纤维所占比例为0.014±0.002。与假手术组相比，神经切断组阴茎背神经中nNOS阳性神经纤维所占比例明显降低（p <0.05）。神经吻合组阴茎背神经中nNOS阳性神经纤维所占比例为0.009±0.001，显著高于神经切断组（p <0.05）结论：躯体神经—内脏神经反射通路的建立显著改善大鼠阴茎组织内平滑肌与胶原纤维比值，抑制阴茎的纤维化，增加阴茎组织中NOS阳性神经纤维的表达。更多还原

【Abstract】 PartⅠ Neural regeneration research after anastomosis ofautonomic nerve and somatic nerveObjective: To investigate the neural regeneration through retrograde tracing andmorphological studies after anastomosis of autonomic nerve and somatic nerve.Methods: Thirty-three adult male SD rats (250~300g) were randomly divided into threegroups: sham group, neurotomy group and anastomosis group. In sham group, left L4ventral root (VR) and bilateral L6-S1VR were detached and left intact. In neurotomy group,the left L4VR and bilateral L6-S1VR were detached and then transected. In anastomosisgroup, the left L4VR and bilateral L6-S1VR were transected, and the proximal stump ofthe left L4VR was anastomosed to the distal stump of the left L6VR.16weeks afteroperation,6rats from sham group, neurotomy group and anastomosis group, respectively,were used for retrograde tracing study.The fluorogold (FG) was injected into the left major pelvic ganglion (MPG) of the rats.7days later, the rats were put to death, and frozen sections of spinal cord were obtained. Thenumbers and distribution of FG-positive neurons were observed using a fluorescencemicroscope.5mm long nerve segments of the L6VR were harvested from the remainingfive rats in each group, and used for semi-thin sections stained with toluidine blue andultrastructural study.Results: FG labeled neurons were mainly observed in the intermediolateral cell columnregion of the L6-S1spinal cord in sham group. However in anastomosis group, FG labeledneurons were observed in the ventral horn of the L4spinal cord segment. No FG-labeledneurons appeared in any segment of the spinal cord in neurotomy group. In sham group, thenumber of FG labeled neurons in L6and S1spinal cord segments was13.2±1.3and4.8±0.5respectively. In anastomosis group, the number of FG labeled neurons in the ventralhorn of the L4spinal cord segment was7.9±1.1. The number of FG labeled neurons in anastomosis group was significantly higher than that in neurotomy group (p <0.05).semi-thin sections of the L6VR in sham group showed a large number of normalmyelinated nerve fibers, and the number of myelinated nerve fibers was784±45. Nonormal nerve fibers were identified in neurotomy group. In anastomosis group, a great dealof myelinated nerve fiber regeneration was observed, and the number of myelinated nervefibers was510±53. The number of myelinated nerve fibers in the sham group andanastomosis group was significantly higher than that in neurotomy group (p <0.05). Inultrastructural study, a large number of mature myelinated axons was observed at L6VR insham group. No normal myelinated nerve fibers was detected in neurotomy group. A greatdeal of myelinated nerve fiber regeneration was observed at L6VR in anastomosis group,and the myelin sheaths of the regenerated nerve fibers were thick and dense.Conclusion: Retrograde tracing, semi-thin sections stained with toluidine blue andultrastructural studies have shown that nerve regeneration can be achieved throughend-to-end anastomosis of autonomic nerve and somatic nerve, and regenerated nervefibers can grow into and replace autonomic nerve (L6VR). Part II The effect of anastomosis of autonomic nerve andsomatic nerve on erectile functionObjective: To investigate whether the regenerated nerve could control penile erection afteranastomosis of autonomic nerve and somatic nerve.Methods: Thirty adult male SD rats (250~300g) were randomly divided into three groups:sham group, sham operation; neurotomy group, transection of left L4VR and bilateralL6-S1VR; anastomosis group, transection of left L4VR and bilateral L6-S1VR andsurgical anastomosis of left L4VR to L6VR.16weeks after operation, intracavernouspressure (ICP) measurement was performed in sham group, neurotomy group andanastomosis group respectively. Electrostimulation was carried out using a bipolar stainlesssteel electrode by hooking intact L6VR in sham group, transected L6VR in neurotomygroup, L4VR in anastomosis group, and the ICP and mean arterial pressure (MAP) weremeasured at the same time.Results: When the left L6VR was stimulated in sham group, the ICP max, ICP increaseand ICP/MAP ratio was72.7±2.5mmHg,60.5±3.3mmHg and0.68±0.03respectively.However in neurotomy group, no obvious ICP change was observed. When the left L4VRwas stimulated in anastomosis group, the ICP max, ICP increase and ICP/MAP ratio was39.4±2.7mmHg,26.7±3.0mmHg and0.31±0.04respectively. The the ICP max, ICPincrease and ICP/MAP ratio in anastomosis group presented a significantly increasecompared to neurotomy group (P <0.05), but was obviously lower than that of sham group(P＜0.05).Conclusion: Stimulation on the left L4VR proximal to the anastomosis resulted inincreased ICP after the establishment of the somatic-autonomic reflex pathways, whichindicated that not only the somatic nerve was able to regenerate and grow into theautonomic nerve, but also regenerated nerve can lead to penile erection. Part III NOS expression and histological changes of penis inrats after reconstruction of erectile function throughanastomosis of autonomic nerve and somatic nerveObjective: To investigate NOS expression and histological changes of penis in rats afteranastomosis of autonomic nerve and somatic nerve.Methods: The penile tissues were removed from the rats in sham group, neurotomy groupand anastomosis group respectively after evaluation of erectile function. Following routinedehydration, one part of penile tissues were cut into paraffin sections, and stained byMasson staining that differentially stains smooth muscle cells (red staining) and collagenfibers (green staining). Another part of the penile tissues were fixed in4%formalin, anddehydrated in30%sucrose. The frozen sections were stained by NADPH diaphorase andimmunofluorescence, and the expression of NOS positive nerve fibers were detected in thepenile tissues.Results: Following Masson staining, smooth museles were stained with red, and collagenwith blue. The ratio of smooth museles to collagen fibers was0.109±0.008in sham group,0.043±0.005in neurotomy group and0.089±0.007in anastomosis group respectively.Which was much lower than that in sham group The ratio of smooth museles to collagenfibers in sham group and anastomosis group were much higher than that in sham group (P<0.05). A large number of NADPH-positive nerve fibers was observed in the penile dorsalnerves in sham group. The number of NADPH-positive nerve fibers decreased significantlyin neurotomy group. The number of NADPH-positive nerve fibers in anastomosis groupwas significantly higher than that in neurotomy group (P <0.05). The nNOS positive nervefibers/dorsal nerve was0.014±0.002in sham group. The nNOS positive nervefibers/dorsal nerve was significantly lower in neurotomy group than that in sham group (p<0.05). The nNOS positive nerve fibers/dorsal nerve in anastomosis group was0.009± 0.001, which was much higher than that in neurotomy group (p <0.05).Conclusion: The ratio of smooth museles to collagen fibers in the penile tissues of the ratswas significantly improved, and the expression of NOS positive nerve fibers increased afterthe establishment of the somatic-autonomic reflex pathways.更多还原