| Neurogenic bladder,which can be caused by sacral roots injury,pelvic nerves injury and spinal cord injury(SCI),often leads to severe urinary voiding dysfunction.Previous studies have demonstrated that complications of urinary tract infection and renal failure have contributed the most to the mortality of SCI patients.For decades,various therapeutics,including drugs,rehabilitation exercises,bladder stoma and multiple surgeries,etc.were employed to improve urination function for these patients.Among,reinnervation,stitching the proximal end of lower extremity nerves to the distal end of the nerves which innervate the bladder,was first applied in dog models more than a century ago to restore urinary bladder function.Based on this theory,a novel technique of artificial skin-CNS-bladder reflex was first proposed by Xiao and its effects have been demonstrated in both animals and humans.This new pathway was mainly established by intradural anastomosis of the L5 ventral root(VR)to the S2 dorsal root(DR).However,failure of this operation was not rare.Regarding the reasons,we concluded as follow:(1)Identification between the ventral root and dorsal root was difficult;accurate suture of VR-VR intradurally cannot be guaranteed.(2)Intradural anastomosis was an extensively destructive surgery which was associated with many clinical complications such as cerebrospinal fluid leakage and infection,etc.Given above,fast and accurate identification of the nerve bundles is very important for successful nerve anastomosis,but still remains a worldwide problem.So far,kinds of methods have been proposed to rapidly identify the nerve bundles,including anatomic,electrophysiological,radio isotopic,immunohistochemical,near-infrared diffuse reflectance spectroscopy methods and Raman spectroscopy.Some of these methods achieved quick identification,however poor selectivity,low specificity and sensitivity,and expensive equipment blocked the widely application.In recent years,advances in bioengineering have promoted the development of trace detection methods for kinds of biologically active substances,for example,proteins and receptor-ligand compounds.Piezoelectric immunosensor,based on the quartz crystal microbalance(QCM),is explicitly made quartz plates.The mass change on the surface can alter the resonance frequency of the crystal and correlates with the frequency change of the crystal electrode linearly.In the present study,we tried to detect the differences of acetylcholinesterase(AchE)content between VRs and DRs using the piezoelectric immunosensor,thus distinguish them.Detailly,the AchE antibody was immobilized on the crystal surface and reacted with the AchE.Formation of AchE-AchE antibody compounds increased the mass of the crystal which induced the frequency change of the crystal electrode.The VRs and DRs were cut into 1-2mm long segments and dissolved in the PBS(250?l,0.01M).After centrifugation at 12000 X g for 3 min,the supernatant(about 200?l)was collected.The reaction between AchE and AchE antibody in the QCM device was carried at a flow of 70?l/min after immobilization of AchE antibody on the surface of the crystal electrode.The frequency shift(AF)was recorded by real time observation and the procedure only cost 10 min.The more AchE reacted with AchE antibody,the more AF was recorded on the real time observation.Therefore,piezoelectric immunosensor based on quartz crystal microbalance can be developed as a quick,sensitive and convenient tool for identification of spinal VRs and DRs.However "artificial bladder reflex" reconstruction via intradural approach has made breakthrough in recovering the bladder function,we found that complications,such as extensive destruction,high rate of cerebral spinal leakage and infection,cannot be ignored.So we further explored the feasibility of spinal root micro anastomosis without opening the dural sac.A total of 10 beagle dogs were used to investigate the following anatomical indicators:(1)The length of the extradural segment of the nerve root;(2)upper nerve root outlet(the point at which it emerges from the spinal dura mater)to S2(dS2),(3)the S3(dS3)nerve root outlet distance;(4)the diameters of the extradural spinal roots,(5)the numbers of nerve fibers from L6 to S3 ventral roots,which were calculated using immunohistochemical staining.Results showed that the extradural spinal roots could be divided into ventral root(VR)and dorsal root(DR)before the ganglionic enlargement of the dorsal root,and the extradural motor nerve roots located ventrally to their corresponding sensory nerve roots.The extradural nerve root lengths of S1 and parts of L7 were longer than the corresponding dS2.The numbers of nerve and motor nerve fibers,and the diameters of extradural nerve roots,were gradually descending from L6 to S3.So the S1 VRs and parts of the L7 VRs can be extradurally anastomosed to the S2 nerves without tension,but a nerve graft was needed for extradural anastomosis of L6 VRs and parts of L7 VRs to S2 VRs.Therefore,our study demonstrated the feasibility of extradural spinal nerve anastomosis for treating neurogenic bladder in canines.Based on our previous studies,16 "artificial bladder reflex" beagle dog models were established using extradural micro anastomosis of S1 ventral root to S2 ventral root without cutting S1 and S2 dorsal roots.Twelve months after the operation,spinal cord at L1 to L2 was cut to establish the model of super spinal cord injury(SCI).Among,3 dogs died after SCI and 5 failed in the second surgical procedures.Totally,8 beagle models of "artificial bladder reflex" were established successfully.Although detrusor pressure,bladder compliance and residual volume were less than the normal,we found that the "artificial bladder reflex" of extradural S1 ventral root to S2 ventral root micro anastomosis can improve the bladder function of the dogs with SCI. |
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