| 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. |
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