Experimental Study On Targeted Regeneration Guided By Dorsal Root Stump For Spinal Cord Injury Repair

Spinal cord injury often take place in clinic,and with the development of the transportation and the construction,its incidence rate is rising year by year.Spinal cord injury can not only lead to motor and sensory dysfunction,but cause bowel and bladder dysfunction,pathological pain,pressure sore,urinary tract infection,hydronephrosis and other complications,which bring patients with bad physical and psychological health,and bring their families a heavy financial burden.The pathological process of spinal cord injury contains primary injury and secondary injury.The latter includes inflammation,toxicity of excitatory amino acid,apoptosis of neurons and glias,formation of glial scar,and the changes of neuronal electrophysiological properties,which may greatly aggravate spinal cord injury.If the secondary injury is relieved,the degrees of spinal cord injury and disability will be alleviated,which is the chief mechanism of current treatments.However,the environment of the central nervous system is bad for nerve regeneration,which leads to poor effects of current treatments,such as stem cell,peripheral nerve transplant,neuroprotective agents,neurotrophic factors,removal of nerve growth inhibitors,rehabilitation and functional electrical stimulation,etc.Although some nerve tracts,such as corticospinal tract,etc.,can regenerate into the distal spinal cord after injury,the improvement of motor function is limited,which may due to their incorrect reinnervation in the distal spinal cord.Only if reconstruct the correct connections of the supraspinal structures and the neural circuits,such as central pattern generator,etc.,in the distal spinal cord,could recovery of motor function come true after spinal cord injury.Thus,a new repair strategy on spinal cord injury has been put forward after 16 years' research in our laboratory.The sprouted axons of upper intercostal nerve can innervate into the distal spinal cord and reconstruct the connections with the interneurons after intercostal nerve-dorsal root anastomosis.After the cascade of interneurons,signals from the supraspinal structure can be amplificated and transferred to the corresponding ventral roots and peripheral nerves,thereby generate conscious control of the targeted organs.In the preliminary experiments,the anatomic contact of upper intercostal nerve with the lower central pattern generator was reconstructed by this repair strategy,and motor function of hind limbs was partially restored.Therefore,the novel repair strategy is feasible in theory and in practice.However,the neurological mechanism of this repair strategy remains unclear,and the researches on reinnervation of the pelvic vagus nerve after spinal cord injury and improvement of the functions of bowel and bladder by this repair strategy have not been carried out.In the current research,the neurological mechanisms of the repair strategy were elucidated using anterograde and retrograde tracing technique combined with immunofluorescence technique.The results showed that the projection range of L4 primary afferents in the spinal cord was widespread,and the nerve fibers and varicosities from L4 dorsal root ipsilaterally extended to dorsal horn from T12-S4,to Clarke's column from T10-L2 and to anterior horn from L1-L5.The labeled varicosities at L4 segment were very dense,especially in laminae ?-?(2466678/mm3),and decreased gradually in more rostral and caudal segments according to quantitative analysis.In addition,they were predominately distributed in laminae?-?,moderately in laminae ?-?,and sparsely in laminae ?-?.Moreover,direct contacts of L4 afferents with NeuN,ChAT,GABA,GAD65 & 67,GS-positive neurons in gray matter,as well as with the motor neurons in the ventral horn of spinal cord were discovered,forming anatomic contacts similar to synapsis.So,there were extensive connections of the afferents with the various neural circuits,such as CPG,etc.,in spinal cord,and sensory signals from the dorsal root extensively took part in the regulation of the neural circuits and CPG.Furthermore,there were cross distributions of the afferent fibers between L3 and L4 dorsal roots,and between the descending fibers of corticospinal tract or rubrospinal tract and afferents from L4 dorsal root,and some of them contacted with the same neurons which expressed NeuN,ChAT,GABA,GAD65 & 67 or GS at the same time in laminae ?-? in dorsal horn,showing that the neural circuits within the spinal cord was very complicated and the fibers with different origins could regulate different neural circuits in spinal cord together.In addition,we elaborated the neuroanatomy theory of the repair strategy by anterograde and retrograde tracing technique,and the result showed that axons of T11 intercostal nerve could regenerate through the anastomosis site and the central nervous system/ peripheral nervous system transitional zone in dorsal root entry zone guided by L3 dorsal root,and innervated into laminae ? and ? of L3 segment.So,the repair strategy could reconstruct the connections of the upper and the lower spinal cords,which provides the repair strategy a solid neuroanatomy foundation.In addition,we tried to repair the function of pelvic vagal nerve after spinal cord transection by T11 intercostal nerve-S2 dorsal root anastomosis.The results showed that bowel and bladder dysfunction occured after spinal cord injury,and the results of urodynamic indexes of spinal cord injury rats were distinctly worse than that of normal rats(p<0.05).Though there was no significant difference between the repair group and the control group according to urodynamic indexes,such as maximum detrusor pressure and maximum urethral closure pressure(p>0.05),the coordination of bladder detrusor and urethral sphincter,together with the length of urethral function curve in the repair group were better than that in the control group.Moreover,hydronephrosis and ureteral dilation occurred in the control group 4 weeks after injury,but not in the repair group according to pathological examination.Furthermore,T11 intercostal nerve,guided by S2 dorsal root stump,could successfully innervate into S2 segment according to anterograde and retrograde tracing technique.In summary,the repair strategy can not only repair the motor function but also repair the function of pelvic vagal nerve after spinal cord injury.In this study,we quantitatively analyze the distribution of varicosities of single dorsal root in the spinal cord at the first time,and the neurological mechanism on targeted regeneration guided by dorsal root stump for repairing spinal cord injury was preliminarily expounded,which lay the neuroanatomical basis for further experiment and clinical application.The repair strategy has a good prospect for clinical application because of autogenous repairing which do not cause any ethical problem.