Morphological Study On The Change Of Ventral Horn Motor Neurons In The New Spinal Cord Center And The Regeneration Of Axon After The Artificial Reflex Arc Established

Bladder dysfunction after spinal cord injury (SCI), also called neurogenic bladder. At early stage it is the main barrier that affects the paraplegia patients' recovery, later it always causes uninary system infection and kidney damages etc., it is also the main fact that caused the long-term paraplegia patients' death. In recent centuries, both domestic and foreign medical workers have been trying very hard to explore the way to solve this problem.Today, there are many methods can be used to treat the neurogenic voiding dysfunction after SCI, including urethral catheterization such as suprapubic cystotomy, persistence catheterization and clean intermittent catheterization (CIC); compression voiding such as Crede maneuver and abdominal presser; pharmacological voiding to promotes voiding by drugs including cholinergic and anticholinergic agents acting on the detrusor,α- orβ- adrenal arousal (or block) agent targeting on the urethral sphincter; surgical voiding such as reinnervating of the bladder and rebuilding of bladder and urethra; voiding by sacral anterior root stimulation (SARS) to evok voiding by electric stimulation after sacral deafferentation (SDAF). Although all the techniques above have been applied in some medical organizations for many years, they still haven't been widely used either because the clinic effect was uncertain or the operation was complicated. Xiao CG, et al. proposed an assumption in 1990:using the useless nerves below the plane of spinal cord injury to set up an artificial somatic- autonomic reflex arc, so as to establish a new nerve reflex pathway to realize controllable voiding which can be controlled by skin. Later, this assumption has proved to be successful both in animal and clinic experiments. The artificial somatic- autonomic reflex arc can be surgically established to provide a novel method of controlling bladder function for complete suprasacral SCI patients with hyperreflexic bladder and detrusor external sphincter dyssynergia (DESD). Although the artificial reflex arc can be successfully established both in animal and clinic experiments, the mechanism is not fully understood, especially the mechanism involves ventral horn motor neurons and the regeneration of axon in the new spinal cord center after the artificial reflex arc established. Therefore, we designed the following experiments to afford more theory support and experiment proof for the artificial reflex arc assumption. Part ILocating the spinal cord center after the artificial reflex arcestablishedObjective: To examine whether the artificial somatic-autonomic nerve reflexarc was smooth or not and locate the segments in the new spinal cord center after the rats models were established, so as to lay solid foundation for the coming experiment.Methods: Four months after the operation of artificial somatic-autonomic nerve arc was done on the SD male rats models, injected 5% DiI-DMSO into the left major pelvic ganglia by using the retrograde neural tracing method. Two weeks later, sectioned L2-S2 spinal cord with frozen section method and observed DiI labeled neuron under a Confocal laser scanning microscope.Result: DiI labeled neuron was found in locus of the left anterior ventral horn from L3 caudal to L5 rostral, (mainly at the L4 segment of spinal cord).Conclusion: Four months after the artificial somatic-autonomic nerve anastomosis was set up, the new somatic motor axons regenerated into major pelvic ganglia and replaced autonomic pre-ganglionic nerve fibers, there was no obstruction in the newly built artificial reflex arc, which had formed a morphological basis of controllable voiding. Part IIMorphological study on the change of ventral horn motor neurons inthe new spinal cord center and the regeneration of axon after theartificial reflex arc establishedObjective: Study on the change of ventral horn motor neurons in the new artificial reflex arc spinal cord center in different period of time and the regeneration of axon in the anastomosis areas after the artificial somatic-autonomic nerve anastomosis was set up.Methods: Forty-eight SD male rats were randomly divided into three groups: normal control group(n=8), model group(n=20) and sham group(n=20). Kept the normal control group unchanging, made artificial somatic-autonomic nerve anastomosis operation on the model group and only cut the somatic nerve and autonomic nerve of the sham group without making anastomosis. At 1 week, 4 weeks and four months, using the traditional HE dyeing method to dye the L4 segment of spinal cord and the nerve of anastomosis areas respectively, observed the morphological change of ventral horn motor neurons at the L4 segment of spinal cord and the passing situation of the regeneration axon in the anastomosis areas, as well as counted the number of ventral horn motor neurons.Result: The sham and the model group were observed at 1 week, 4 weeks and 4 months, the ventral horn motor neurons of the former group reduced 34.1%, 57.7% and 72.5% respectively and which of the latter group reduced 32.9%, 43.7% and 51.3% respectively. After statistics processes, significant differences were found in the survival rate of the ventral horn motor neurons at the L4 segment of spinal cord in the sham and model group. The anastomosis areas situation of model group at 1 week: the passing of the nerve fibers through the anastomosis areas could not be observed. The anastomosis areas situation of model group at 4 weeks: the far end of the regenerated nerve fibers was sparse and disordered. The anastomosis areas situation of model group at 4 months: there were full of regenerated nerve fibers at the far end, the order of which was compact and regular.Conclusion: After somatic autonomic nerve anastomosis was established, part of the ventral horn motor neurons in the spinal cord center could survive, the survival neurons were the material basis of the regenerated axon, which in turn could accelerate the survival of the neurons. With the joint function of the regenerated axon and the survival neurons, the satisfactory artificial reflex arc could be established successfully. Part IIIThe neural tracing study on the regenerated nerve fibers after theartificial somatic-autonomic nerve anastomosis was establishedObjective: Study on the terminals of the regenerated nerve fibers ending after the artificial somatic-autonomic nerve anastomosis was establishedMethods: Four months after the artificial somatic-autonomic nerve anastomosis operation was done on the SD male rats, using neural tracing method to inject DiI-DMSO into the left ventral horn at the L4 segment of spinal cord. Two weeks later, put the left major pelvic ganglia (MPG), the external urethral sphincter (EUS ) and the bladder under the microscope for frozen section and observed the regenerated nerve fiber terminals under the fluorescent microscope.Result: The result of the neural tracing method showed that: after DiI was injected into the ventral horn at the L4 segment of spinal cord , DiI positive axon terminals were found in the left major pelvic ganglia (MPG) and the external urethral sphincter (EUS) not in the bladder detrusor.Conclusion: After somatic-autonomic nerve anastomosis was established, the new somatic motor nerve fibers could regenerate into and replace autonomic pre-ganglionic nerve fibers, which also could successfully reach the target organ innervated by autonomic nerve. The artificial reflex arc provided a morphological basis with controllable voiding. However, how did the regenerated nerve fibers and the target organ get connected and how did they function remain to be studied and proved.