The Feasibility Study Of Reconstruction Of Erectile Function Through Anastomosis Of Somatic Nerve And Autonomic Nerve In Rats

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.