Expeimental Study On Adenoviral-Mediated Exogenous Genes Expression In Spinal Cords And Peripheral Nerves And Influencing Factor On The Transgene Expression

Injuries of peripheral nerves have long been a common clinical problem. Sophisticated microsurgery skills have made neurorrhaphy precise, however, the functional recovery of injured nerve is unsatisfactory up to the present, for peripheral nerve regeneration involves a complex process of biochemical and cellular events and microenviroment of the regenerating nerves influences its repairing results very much. Recent years, reseachs of delivery of neurotrophic factors encoded by adenoviral vector to spinal cord for protecting the original neurons of injured nerves and promoting peripheral regeneration have become more popular. Successful gene transfer in peripheral and central nervous system is dependent on efficient methods of introducing and expressing a particular transgene in the appropriate population of neural cells effectively. But there have been disadvantages of low transgene expression,complicated operational performance and high inflammation response in vector injected site with the commonly used method of delivery of vetors to nervous system. Moreover the detail course of transgene expression in spinal cord and peripheral nerve has not been understood in detail. Immune response to adenoviral vectors have severely affected the ability of these vetors to induce long-term gene expression and even caused side effects. Since several experimental and clinical articles reported more successful use of end-to-side neurorrhaphy in past ten years, this repair method which has been used at the beginning of last century drew the nerve researcher'considerable attention. The end-to-side neurorrhaphy has several advantages over conventional end-to-end nerve repair in that it does not require nerve grafts if there are long defects, and it may be applicable to injuries in which proximal nerve end is unavailable, such as an avulsion injury of the brachial plexus. Still the end-to-side repair method is controversial and a number of issues remain to be clarified one of which concerns the source of the regenerating axons. In order to resolve the above problems, recombinant replication-defective adenoviral vectors encoding LacZ gene (AdLacZ), green fluorescent protein (GFP) gene (AdGFP),CTLA4Ig gene (AdCTLA4Ig) and adenoviral vector without encoding any exogenous gene (Ad0) are administrated singly or jointly to the proximal stumps of transected peripheral nerves or end-to-side repaired nerves in rats. Through these series experiments we could integrally analysis the characterization and time course of LacZ gene retrograde transfer to spinal cord and anterograde labeling of peripheral nerve by transgene product, explore immune tolerant mechanism induced by CTLA4Ig and trace regenerating axons after end-to-side neurorrhaphy. The experiment is divided into 3 parts. 1 Monitoring retrograde adenoviral transgene expression in spinal cord and anterograde labeling of the peripheral Nerves Objective: AdLacZ and AdGFP administrated to proximal stumps of transected median and tibial nerves in order to explore specific, high effective and safe method of transfect adenoviral vectors to peripheral nerves and its original neurons, analysis integrally the characterization and time course of LacZ gene retrograde transfer to spinal cord and anterograde labeling of peripheral nerve by transgene product. Methods: 192 Wister female rats aged 7 weeks were used. 168 of the rats were administrated with AdLacZ, half of that from median nerves (LacZ-M group) and half of that from tibial nerves (LacZ-T group). The remaining 24 rats were administrated with AdGFP, also half of that from median nerves (GFP-M group) and half of them from tibial nerves (GFP-T group). In median nerve models, at the site 3mm distal to the cross of median and tendon of latissimus dorsi, the median nerve was transected. In the tibial nerve models, at the site 5mm distal to the trifurcation of tibilal, peroneal and sural nerves the tibial nerve was transected. Then 2μl (2×107 pfu) AdLacZ or 2.0μl (2.0×106 pfu) AdGFP was dropped to the proximal stumps of transected median nerves or tibial nerves. The transectednerve was repaired with 10-0 nylon sutures, and then the wound was closed with 4-0 nylon sutures. At 24 different time points within 9 weeks post transfect in LacZ-M group and LacZ-T group, and at 4 days, 7 days post transfect in GFP-M group and GFP-T group, the C5 to Th1 spinal cords attached with their DRGs and brachial plexus or L3 to L6 spinal cords attached with their DRGs and sacral plexus were removed. In LacZ-M group and LacZ-T 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. In GFP-M group and GFP-T group, the spinal cords, DRGs, nerve roots and peripheral nerves were sectioned and the positive neurons and axons were observed directly under Epi-fluorescence microscope. 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 L4～L6 segments in tibial nerve models. In both of LacZ-M group and LacZ-T 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 of β-gal staining was shortest in motor neurons, then in sensory neurons and longest in peripheral nerves. The initial detected time of β-gal staining was earlier in LacZ-M group compared with that in LacZ-T group. The labeled neurons were more in LacZ-T group than that in LacZ-M group (P<0.01). In both of the LacZ-M group or LacZ-T group, the labeled sensory neurons of DRGs were more than labeled motor neurons of ventral horn (P<0.01). In GFP-M group and GFP-T group, the FITC labeled neurons were observed within corresponding spinal cord segments and DRGs, and FITC labeled axons were observed in corresponding root. But no FITC labeledaxons were observed in nerve trunks around repair site. Conclusion: In this study the method of administrating adenoviral vectors to injured nerves 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. Nerve tissues transfected by AdGFP can be observed directly under Epi-fluorescence microscope without staining. 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. 2 Targeted adenoviral vector-mediated CTLA4Ig gene delivery to the spinal cord and its effect on exogenous gene expression in nervous system Objective: Immune response to adenoviral vectors has severely affected the ability of these vetors to induce long-term gene expression and impeded the effect of gene therapy. In this study, AdLacZ was transferred to spinal cords jointly with Ad0 or with AdCTLA4Ig through median nerves of rats in order to investigate the effect of immune tolerant induced by CTLA4Ig and its mechanism. Methods: 144 Wister female rats aged 7 weeks were randomly divided into 2 groups: group A, the AdlacZ+Ad0 transfer group and group B, the AdlacZ+AdCTLA4Ig transfer group. At the site 3mm distal to the cross of median and tendon of latissimus dorsi, the median nerve was transected. 1.5μl (1.5×107 pfu) AdlacZ and 1.5μl (7.5×106 pfu) Ad0 were dropped to the proximal stumps of median nerves in group A and 1.5μl (1.5×107pfu) AdlacZ and 1.5μl (7.5×106 pfu) AdCTLA4Ig were dropped to the proximal stumps of median nerves in group B. The transected nerve was repaired with 10-0 nylon sutures, and then the wound was closed with 4-0 nylon sutures. At 10 different time points within 8 weeks post transfect 2ml blood was got from caudal vein of each rat and then the C5～Th1 spinal cords attached with their DRGs and brachial plexus were removed. The spinal cord and DRGs were sectioned andprocessed with β-gal and CTLA4Ig double immunohistochemical staining to observe positive neurons of transgene expression. Sections of spinal cord, DRGs and brachial plexus were processed with X-gal staining and positive neurons and axons were counted in both two groups. The title of serum anti-adenoviral antibodies and neutralizing antibodies were assayed. Results: The LacZ gene and CTLA4Ig gene transfer was specifically targeted to the particular (C6～Th1) segments of spinal cord and DRGs, and transgene expression was strictly unilaterally corresponding to the infected nerves. In group A only FITC labeled neurons (expressing β-gal) were observed in spinal cord sections of immunohistochemical staining. While FITC labeled neurons (expressing β-gal) or Rho labeled neurons (expressing CTLA4Ig) or double labeled neurons (expressing both β-gal and CTLA4Ig) were observed in group B. There was no significant difference of the number of X-gal stained neurons in spinal cord or DRGs sections between two groups (P>0.05). There was also no significant difference of the number of X-gal stained axons in median nerves between two groups (P>0.05). Mononuclear cell infiltration was observed obviously in spinal cord sections 6 days post transfect in both two groups, disappeared mostly in group B, but still observed obviously in group A 21 days post transfect. Some mononuclear cell infiltration still could be observed in group A 42 day post transfect. The title of serum anti-adenoviral antibodies was detected from 2 days, and increased similarly within 8 days post transfect, but increased persistently until 8 weeks post transfect in group A. The title of neutralizing antibodies was ≤1:4 in each time point post transfect. Conclusion: AdCTLA4Ig and AdLacZ can retrograde transfer to spinal cords and DRGs singlely and jointly from injured peripheral nerves and express transgene product of CTLA4Ig andβ-gal. CTLA4Ig can inhibit cellular immune response and anti-adenoviral antibody production. Neutralizing antibodies can not be detected after AdLacZ being transferred with Ad0 or with AdCTLA4Ig to spinal cords and DRGs from injured peripheral nerves. Although transfecting jointly with AdCTLA4Ig to spinal cords and DRGS can inhibit cellular immune response to AdLacZ, it does notaffect AdLacZ transgene expression in nervous system. 3 Tracing the origin of regenerating axons after end-to-side neurorrhaphy using adenoviral vectors containing LacZ gene. Objective: The source of the regenerating axons was investigate by administrating AdLacZ to nerves of brachial plexus nerves after end-to-side neurorrhaphy and tracing the range of transgene expressing neurons in spinal cord and DRGS and axons in brachial plexus nerves. Methods: 40 Wister female rats aged 7 weeks were randomly divided into 2 groups: the normal control group (n=12) and the experimental group (n=28). AdLacZ was administrated to median nerves (group M, n=4), ulnar nerves (group U, n=4) and musculocutaneous nerves (group Mc, n=4) respectively in control group. At the site 3mm distal to the cross of median and tendon of latissimus dorsi, the median nerve was transected in group M and the ulnar nerve was transected in group U in the same level. In group Mc the musculocutaneous nerve was transected at site 5mm after it divided from lateral branch. Then 2μl (2×107 pfu) AdLacZ was dropped to the proximal stumps of transected nerves. The transected nerve was repaired with 10-0 nylon sutures, and then the wound was closed with 4-0 nylon sutures. The rats were divided into two sub-groups in experimental group. Mc-M group (n=14): musculocutaneous nerve was transected as recipient nerve coaptated end-to-side to median nerve which was as donor nerve. Mc-U group (n=14): musculocutaneous nerve was transected as recipient nerve coaptated end-to-side to ulnar nerve which was as donor nerve. AdLacZ was transferred from two sites in each group 6 weeks post operation. In Mc-M1 group (n=8), AdLacZ was transferred from musculocutaneous nerve 5mm distal to coaptation site and in Mc-M2 group (n=6), AdLacZ was transferred from median nerve 5mm distal to coaptation site. In Mc-U1 group (n=8), AdLacZ was transferred from musculocutaneous nerve 5mm distal to coaptation site and in Mc-U2 group (n=6), AdLacZ was transferred from ulnar nerve 5mm distal to coaptation site. Then the transected nerve was repaired with 10-0 nylon sutures, and the wound was closed with 4-0 nylon sutures. The C5～Th1 spinal cord attached with their DRGs and...