Sequential Retrograde Tracing For The Assessment Of Reinnervation Accuracy Following Peripheral Nerve Injury And Repair

Part1:Efficacy of retrograde double-labeling of spinal motor neurons by different combinations of tracers in a rat model of tibial nerve tracingAbstractObjective:This study was performed to test the double-labeling efficacy of different combinations of the neural tracers Fluoro-Gold (FG), True Blue (TB) and Fluoro-Ruby (FR).Methods:The left tibial nerve of adult Sprague-Dawley rats was exposed to different tracer combinations, i.e. FG-FR, FG-TB or FR-TB, either by intra-fascicular neural injection or by nerve transection and immediate immersion of the proximal nerve stump into tracer mixture for20min. After survival for5days, the lumbar enlargement of the spinal cord was dissected out and longitudinally cryo-sectioned for laser confocal microscopy and labeled motor neurons counted.Results:The tracing with a combination of FG and TB gave the largest number of labeled motor neurons, among which80%-85%was doubly labeled. The combined use of FG and FR also exhibited the second largest number of labeled motor neurons with over90%double labeling. However, the FR-TB combination group showed poor labeling efficacy in terms of total labeled neurons and double labeling ratio. Exposure to tracers via nerve stump-immersion (20min) was observed to be significantly less efficient, i.e.2/3labeling, than via intra-fascicular neural injection.Conclusion:The combination of either FG with TB or FG with FR seemed suitable for sequential retrograde tracing in assessing nerve regeneration accuracy, with better labeling efficacy for intra-fascicular neural injection. Part2:Functional impairment and nerve degeneration resulted from intra-fascicular injection of fluorescent neuronal tracers in ratsAbstractObjective:In sequential retrograde tracing, which is typically used to evaluate nerve re-innervation accuracy, the first tracer should be of none or low neurotoxicity and lead to minimal functional impairment. In the present study, we sought to determine the alteration of motor, sensory and autonomic nerve fuction of an intact nerve following intra-neural injection of fluorescent neuronal tracers Fluoro-Gold (FG), True Blue (TB) or Fluoro-Ruby (FR).Methods:Sixty adult female Sprague-Dawley rats were randomly divided into6groups. Walking track analysis, plantar tests and laser Doppler perfusion imaging were performed after the application of5%Fluoro-Gold (FG),4%True Blue (TB) or10%Fluoro-Ruby (FR),5μl in volume, to the rat tibial nerve via pressure-injection. Additionally, nerve pathology and labeling efficacy were also observed.Results:The results showed that injection of FG significantly resulted in loss of motor nerve function, lower plantar sensibility and higher neurogenic vasodilatation than control (saline injection); meanwhile, severe nerve degeneration and high retrograde labeling efficacy was observed in FG group. The TB group also showed obvious neurogenic vasodilatation, but less severe loss of motor function and degeneration, and fewer labeled motor neurons were found than in FG group, while no anomalies of nerve function were observed in FR group.Conclusion:This study indicated that FG tracing could lead to significant impairment of motor, sensory and autonomic nerve function, while the function impairment was less severe following TB tracing, and FR injection might not affect nerve function. Part3:A preliminary study on motor re-innervation accuracy following the repair of sciatic nerve defects by chitosan/PLGA artificial nerve grafts in ratsAbstractObjective:The purpose of this study was to determine the re-innervation accuracy within the tibial nerve by regenerated motor axons after the repair of sciatic nerve defects by chitosan/PLGA artificial nerve grafts.Methods:Twenty-four adult female Sprague-Dawley rats were randomly divided into4groups, i.e. chitosan/PLGA artificial nerve grafts for repairing3-mm-long (C/P-3mm) or6-mm-long (C/P-6mm) sciatic nerve defects, autologous nerve repair, and sciatic nerve crush (n=6each). Sciatic nerve injury/repair models were prepared as indicated above, at14days after a tibial nerve tracing by soaking in4%True Blue (TB) for60min. Three months after nerve repair but5days before the sacrifice of animals, the tibial nerve was re-exposed for retrograde tracing by soaking in5%Fluoro-Gold (FG) for60min. The lumbar enlargement of the spinal cord was dissected out and longitudinally cryo-sectioned for laser scanning confocal microscopy and labeled motor neurons counted.Results:The C/P-6mm group gave the lowest number of total labeled motor neurons as compared to other groups while the nerve crush group exhibited the largest number of TB-labeled motor neurons, among which95%was double labeled. However,～84%of TB-labeled motor neurons showed double labeling in C/P-3mm, C/P-6mm groups and autologous nerve repair, but no statistical significance was observed among these three groups. Misdirected motor reinnervation into the tibial nerve, indicated by FG single labeling, was found to be the highest following autologous nerve repair, with a misdirection ratio of～30%which is higher than～20%in repair groups with artificial nerve grafts.Conclusion:As compared to nerve autografting repair, the misdirection ratio of regenerated motor neurons which reinnervated the tibial nerve could be reduced by the repair of the repair of sciatic nerve defects by chitosan/PLGA artificial nerve grafts.