Reconstruction Of Anorectal Function Through End-to-side Neurorrhaphy By Autonomic And Somatic Nerves In Rats

Objective:To investigate the neural regeneration in the efferent pathway of the rectum and the external anal sphincter after end-to-side neurorrhaphy of autonomic nerve and somatic nerve.Methods:Thirty-six adult male SD rats (240-300g) were randomly divided into four groups:End-to-side coaptation group (n=15), the rats were subjected to laminectomy from L3to S1vertebrae through the dorsal median incision. An epineurial window was made on the lateral face of the left L4ventral root (L4VR). The left L6and S1spinal nerves were transected in dura at L3level. Then the distal stump of left L6ventral root (L6VR) was wrapped in helicoid fashion around L4VR, and sutured to the lateral face of the L4VR in an end-to-side fashion. No coaptation group (n=6), rats received the same operation as the end-to-side coaptation group but without coaptation; Control group (n=6), the same operation as end-to-side coaptation group was performed but the L6ventral root was preserved. Normal rats group (n=9), no treatment. At16week,6rats from end-to-side coaptation group, no coaptation group and control group, respectively, were used for nerve regeneration mechanism study. Fluorogold (FG) was injected into the left major pelvic ganglion (MPG),3days after the fast blue (FB) was injected into the left sciatic nerve,7days later, serial coronal sections of spinal cord was made on a cryostat. An ultraviolet fluorescence microscope was used to examine fluorescent labeled neurons.9rats from end-to-side coaptation group and9rats from the normal group were used for efferent pathway study. These rats were divided into three groups. In the first group, HRP was injected in anterior horn of L4spinal cord, in the second group,3%fast blue was injected in MPG, in the third group,4%fluorogoldrectal was injected in the wall of the rectum. Transverse serial sections (10-μm thick) of the EAS were obtained using a cryostat, sections were stained with the method of the HRP-TMB and examined under light microscope. Serial coronal sections of spinal cord were made on a cryostat. An ultraviolet fluorescence microscope was used to examine fluorescent labeled neurons.Results:FG-FB double labeled neurons and FG single labeled neurons were mainly observed in the ventral horn of the L4spinal cord segment, FB single labeled neurons were observed in the left ventral horn of L3to L5, and no FG labeled neurons appeared in that of L6segment in end-to-side coaptation group. In no coaptation group, Only FB-single labeled neurons were found in the cross-sections of L3to L5spinal cord, and no FG-single or FG-FB double labeled neurons appeared in that of L3-5or L6segments. In control group, FG-single labeled and FB-single labeled neurons appeared in the cross-sections of L6and L3-5segments, respectively, no double labeled neurons being observed. After HRP was injected into L4left ventral horn, HRP positive axon terminals were observed in the left external anal sphincter(EAS) of the end-to-side coaptation group, there was no HRP positive axon terminals in the left EAS of the normal rat group. In end-to-side coaptation group and normal rat group after injection FG into the rectum, FG labeled neurons appeared in the MPG. In end-to-side coaptation group and normal rat group after injection FB into the MPG, FB labeled neurons appeared in the L4and L6-S1segments of spinal cord, respectively.Conclusions:There were more FG-FB double-labeled neurons than FG single labeled neurons in the spinal L4segment, which indicated that the main nerve regeneration mechanism was axonal collateral sprouting after end-to-side anastomosis. A small amount of FG single labeled neurons proved that small amount of axonal regeneration was caused by the donor nerve injury in the stitching process. The spinal cord innervations of the rectum and external anal sphincter were transferred from the L6S1spinal segments to the L4spinal segment by the L6-L4end to side neurorrhaphy. Objective:To study the number and ultra-structural features of the regenerated fibers with neural morphological techniquesMethods:Twenty four adult male SD rats (240-300g) were randomly divided into three groups:End-to-side coaptation group (n=8), The left L6and S1spinal nerves were transected and the distal stump of L6ventral root (L6VR) was sutured to L4ventral root (L4VR) through end-to-side neurorrhaphy. No coaptation group (n=8), rats received the same operation as the end-to-side coaptation group but without coaptation; Control group (n=8), the same operation as end-to-side coaptation group was performed but the L6VR was preserved. At16week, the left pelvic parasympathetic nerve (PPN) was found along the the major pelvic ganglion (MPG),5mm long nerve segments of the regenerated left PPN and the left L6VR were harvested under operating microscope. PPN and L6nerve segments were used for electron microscopy to study ultrastructural features. Ultrastructure of regeneration nerve was examined by electron microscopy. The PPN was cut into1-μm thick sections, and stained with Toluidine blue. Total nerve fibers counts of PPN were calculated.Results:In the transmission electron microscopy, both myelinated fibers and unmyelinated fibers were observed in the cross-sections of the left PPN and the distal stump of the L6VR in end-to-side coaptation group. There is no myelinated fiber in the cross-sections of the left pelvic parasympathetic nerves in no coaptation group. The average number of mythlinated axons of the left PPN in end-to-side coaptation group and control group was263±66and579±84, respectively.Conclusion:The motor axons of L4VR can regenerate into L6VR to innervate the rectum and EAS after end-to-side coaptation of L6VR to L4VR. Histomorphological examination revealed that the mean number of regenerated myelinated axons (PPN) reached the45.4%compared with that of the PPN in control group. Objective:To investigate the synergy situation of the external anal sphincter and rectal smooth muscle of rat with anorectal dynamics after end-to-side neurorrhaphy of autonomic nerve and somatic nerve, and to clarify its underlying mechanism and the impact on the donor nerve after end-to-side neurorrhaphy by EMG and morphological techniques.Methods:Twenty four adult male SD rats (240-300g) were randomly divided into three groups:End-to-side coaptation group (n=8), The left L6and S1spinal nerves were transected and the distal stump of L6ventral root (L6VR) was sutured to L4ventral root (L4VR) through end-to-side neurorrhaphy. No coaptation group (n=8), rats received the same operation as the end-to-side coaptation group but without coaptation; Control group (n=8), the same operation as end-to-side coaptation group was performed but the L6ventral root was preserved. At16week, in end-to-side group L4VR was stimulated and the rectal pressure and external anal sphincter(EAS) EMG were recorded. In control group the bipolar stimulatory electrodes were placed on the left L4VR and the left L6VR, respectively. In no coaptation group stimulatory electrodes were placed on the left L4VR. After16weeks, the BL-410biological test system was used to exmine the tibialis anterior muscle and gastrocnemius muscle EMG. The right and left tibialis anterior (TA) muscle were carefully separated and integrity removed from the beginning of the tendon to measure the wet weight. The muscle morphology was evaluated by HE staining. In end-to-side coaptation5mm nerve segments of L4donor nerve distal to anastomosis and proximal to anastomosis were harvested, in control group corresponding parts of the L4nerve was harvested, and cut into8-μm thick sections, and stained with Toluidine blue(TB). Total nerve fibers counts were calculated. Results:Anorectal manometry showed a progressive rise in the anorectal pressure and simultaneously induced electric activity of EAS to decrease or disappear by consistent electric-stimuli to the left L4VR proximal to the anastomosis, and the maximum anorectal pressure reached27.9±4.5cm water in end-to-side coaptation group. In no coaptation group, the anorectal pressure did not rise while the left L4VR was stimulated. In control group, the maximum anal pressure reached was68.4±6.6cm water while the left L6VR was stimulated, and there was no rise in the anorectal pressure when the left L4VR was stimulated with the same parameters. The right and left TA muscle weight were0.688±0.061g and0.694±0.059g, respectively. There was no significant difference between the left TA muscles and the right TA muscles (p>0.1). There was no denervation potentials in EMG of the tibial anterior muscles and the gastrocnemius muscles of two sides. No obvious atrophic muscle fiber or degeneration fiber was identified. The average number of mythlinated axons of the L4donor nerve distal to anastomosis and proximal to anastomosis and L4nerve in control group were875±83,869±64and882±75, respectively, and there was no significant difference between groups.Conclusion:Stimulation on the left L4VR proximal to the anastomosis resulted in increased rectal pressure as well as the decrease or disappearance in the electric activity of EAS, which indicated that this technique successfully realized the synergia between rectum and EAS. There was no apparent adverse effect to the donor nerve function in terms of wet weight, morphology, EMG of TA muscle and the numbers of mythlinated axons of the L4VR. These results demonstrated that the end to side anastomosis can successfully repair the rectal function, with no adverse effect to the donor nerve function.