Tract Tracing Of Nerves Of Upper Extremity Using Adenoviral Vectors Containing LacZ Gene

Objective: Adenoviral vectors containing LacZ gene (AdLacZ) is a effective Neural tracer. The researching of tracct pathways of nerve fibers has become one of the important technologies. In this study, through its specific and efficient transgene expression, the AdLacZ gene and transgenic producttion can trace the tract of nerves and its motor and sensory neurons in the spinal cord and DRG. We can analysis the scope of the nerve axons sources. Ulnar nevers or median nerves and their targeted neurons was traced by remote delivery of adenoviral vectors containing LacZ gene (AdLacZ). Successful gene transfer in peripheral system is dependent on efficient methods of introducing and expressing a particular transgene in the appropriate population of neural cells effectively. The process is researched in the median nerve axons and their target motor and sensory neurons or in ulnar nerve. The rules and characteristics are mastered that the AdLacZ transgenic expression in the peripheral nervous system.Methods: 240 Wistar femal rats aged 7 weeks were andomly divided into 2 groups: group A and group B. group A is adminnistrated with AdLacZ from median nerve(LacZ-M), group A is adminnistrated with AdLacZ from ulnar nerve(LacZ- U). After anesthetized with ketamine and xylazine(75-100mg±5mg/kg), In median nerve or ulnar nerve models, at the site 3 mm distal to the cross of median nerve or ulnar nerve and tendon of latissimus dorsi, the median nerve or ulnar nerve was transected. Then 2μl (2×10~7pfu) AdLacZ was dropped to the proximal stumps of transected median nerves or ulnar nerves. The transected nerve was repaired with 10-0 nylon sutures, and then the wound was closed with 4-0 nylon sutures. At 24 different time points within 7 weeks post transfect in LacZ-M group and LacZ-U group, the C4 to Th1 spinal cords attached with their DRGs and brachial plexus were removed. In LacZ-M group and LacZ-U group, the sections of spinal cord and DRGs were processed with X-gal staining and immunohistochemical staining, and the whole specimens of brachial plexus and sacral plexus were processed with X-gal staining and then were sectioned transversely. Then positive neurons and axons were counted and the initial detected time of retrograde labeling, peak time and persisting period of gene expression in DRG sensory neurons, spinal cord motor neurons and peripheral nerves were studied.Results: The gene transfer was specifically targeted to the particular segments of spinal cord and DRGs, and transgene expression was strictly unilaterally corresponding to the infected nerves. The transgene expression was within C6～Th1 segments in median nerve models and within C7～Th1 segments in ulnar nerve models. In both of LacZ-M group and LacZ-U group, the initial detected time of gene expression was earliest in DRGs neurons, then in the motor neurons and latest in peripheral nerves and the persisting duration ofβ-gal staining was shortest in motor neurons, then in sensory neurons and longest in peripheral nerves. The initial detected time ofβ-gal staining was earlier in LacZ-U group compared with that in LacZ-M group. The labeled neurons were more in LacZ-M group than that in LacZ-U group (P<0.01). In both of the LacZ-M group or LacZ-U group, the labeled sensory neurons of DRGs were more than labeled motor neurons of ventral horn (P<0.01). The motor neurons of ventral horns of spinal cords and sensory neurons of DRGs were labeled by transgene productβ-gal in C7～Th1 segments transfected from ulnar nerve and C6～Th1 segments transfected from median nerves. The corresponding neural roots and nerve trunks of transfected nerves were also labeled. The number of labeled neurons of ulnar nerves was different from that of median nerves. The labeled neurons and axons could not be observed after several weeks post transfection.Under the microscope, it is observed that the DRGs, root silk, before and after the root of spinal nerve, neural stem and nerve near the distal anastomotic virus post transfection in the near and far from the order marked. The average distance from adminnistrating position in neural stem to the roots is 26 mm, the average distance to the DRGs is 23 mm. 3d post transfection it has been seen that a small number of axons from root to wireline marker of anastomotic site, 3d post transfection it can be seen anastomotic distal axonal regeneration is marked, 6w post transfection the blue color of neural stem disappeared. In medain nerve,the stained motor neurons, sensory neurons and axons are originated from C6～Th1 segments. The staining of axons in C7 and C8 roots is dense. In Ulnar nerve, the stained motor neurons, sensory neurons and axons are originated from C7～Th1 segments. The staining of axons in C8 and Th1 roots is dense.Conclusion: In this study the method of administrating advLacZ to injured median nerves or nular nerve and then repairing the nerves simulated the familar injurying and repairing process of peripheral nerves and didn't cause the mechanical injuries to spinal cord and DRGs. This method can make targeted and effective trangene expression in neurons and excellent labeling of neural processes and peripheral nerves. The time course of anterograde transport of AdLacZ to neurons and retrograde transport of transgene product along nerves was observed in detail which will renders this system particularly attractive for gene therapy of peripheral nerve injury and neuroanatomical tracing studies.LacZ gene mediated by Ad could specially target the motor and sensory neurons anterogradlly and then label the axons of the ulnar and median nerves retrogradelly. This gene delivery method renders this system particularly attractive for neuroanatomical tracing studies of brachial plexus nerves and might offer potentialities for gene therapy of might offer periph- eral nerve injury.