Transfection Of Neural Agrin Gene On The Recovery Of Muscle Function Following The Free Neurovascular Muscle Transfer

[Background] Free muscle transplantation by microneurovascular anastomosis is a valuable clinical option in the reconstruction of functional deficits in the extremities and the face. The diminution in strength of the transferred muscle that invariably follows the procedure is problematic. The failure to regain full function has been attributed to a disturbance in resting muscle tension, the obligatory period of ischemia during the transfer, and the changes that occur in the muscle with denervation and subsequent reinnervation. Despite advances in nerve repair by microsurgical techniques, careful restoration of muscle tension, less period of ischemia during the transfer, clinical observations and experimental studies still document residual force deficits in transferred muscle. Recent studies have found that the weakness of the muscle after free transfer may be associated with the change to a simple morphology and pattern of the neuromuscular junction as well as to the decline in nAChR number at the re-formed neuromuscular junction following free muscle transfer. So it showed the probability of a new method to improve the postoperative muscle function by regulation of nAChR number at regenerated endplate. Much evidence suggest that nerve-derived protein agrin may play a unique role in formation of neuromuscular junction, especially in induction, differentiation, maturation, maintenance and regeneration of postsynaptic apparatus. To study the distribution of neural agrin on the endplate following muscle free transfer and investigate the effects of transfection of agrin gene on the recovery of nAChR number at regenerated endplate and transferred muscle function was important. So far, few researches on the field have been reported yet at present.[Objective] The purpose of this study was to study the quantitative changes of mRNA expression of s and y acetylcholine receptor subunit and distribution of neural agrin at the neuromuscular junction following muscle free transfer. Then to investigate the possibility of a new method to improve the restoration of the motor function of transferred muscle that accomplished by regulation of nAChR number at regeneratedendplate, so as to provide the experimental basis for its clinical application in the future.[Methods](1) In part I: The gracilis muscle was orthotopic transferred in adult rat to establish the animal model. TaqMan real-time fluorescent quantitative RT-PCR was set up by establishing clone vector pMD18-T-e as standard object and evaluated with sensibility, particularity and reiteration. Then the quantitative changes of mRNA expression of e and Y acetylcholine receptor subunit at the neuromuscular junction was tested by this method and the distribution of neural agrin was detected by immunohistology (EnVision method) and Western blot in the different postoperative period following free muscle transfer.(2) In part II: The carbonic end of rat neural agrinY4Zs cDNA was composed by SOE (splicing by overlap extension) transverse polymerase chain reaction from ratembryo RNA. The purified products were ligated with the eukaryotic expression vector pCDNA3. Musculus pectolaris major was isolated from adult rat and primary skeletal myoblasts were cultured by tissue culture method. The cells were counted and observed by light microscopy combined with immunocytochemical identification. The plasmid pCDNA3-AGRIN Y4Zs was established as positive control and pCDNA3 as negative contral. The plasmid pCDNA3-agrinY4Z8, pCDNA3-LacZ, pCDNA3-AGRrNY4Z8 and pCDNA3 were transfected into cultured myoblasts by LipoGen? reagent respectively. The instant expression of LacZ gene was detected two days later. The positive transfectant clons were selected by G418 for 3 weeks. The successful transfectant cells expressing neural agrin were identified by RT-PCR and immunofluorescent method.(3) In part III: The transfection efficiency of pCS2 + -AGRINY4Z8 with different Electric-Pulse parameters in vivo was detected by Western blot. Then the electrical gene transfection with the most favorable Electric-Pulse parameters was performed at once in gracilis muscle of the SD rat following free neurovascular transfer. The first group was treated with pCS2+- AGRINY4Z8 ;the second group with plasmid pCS2 +as the negative control ; the third group with normal saline as the frank control. The muscle function, expression of e and Y acetylcholine receptor subunit mRNA, neural agrin and the junctional nAChR number was measured at different stage postoperatively.[Results](1) Part I : TaqMan real-time fluorescent quantitative RT-PCR was set up by establishing clone vector pMD18-T-e as standard object. The dynamic range of the assay varied from 102 to 107 copies. Both the copies of mRNA expression of y and 8 acetylcholine receptor subunit increased and then decreased after transfer . At 3 weeks after transfer ,the copies of mRNA expression of e acetylcholine receptor subunit reduced to the lowest level ,and then increased gradually. At 30 weeks the mRNA expression of y acetylcholine receptor subunit was still detectable and the expression of eacetylcholine receptor subunit remained lower than the control in the reinnervated muscle (P<0.05 ). There was a little anti-neural agrin staining on the endplate in normal gracilis muscle and then a greatly overall decrease during the early period of muscle transfer but not entirely absent. At 10 weeks, the expression of neural agrin recovered gradually, but failed to return to the presurgical state tested by Western blot method even at 30 weeks after muscle transfer.(2) Part If: The plasmid pCDN A3-agrin Y4Z8 was successfully established. Cultured for 2 weeks by tissue culture method, the number of skeletal myoblasts reached 8X10 per milliliter. Immunocytochemical analysis showed that over 90% cells were stained positive for myogenin, a factor specific for skeletal myocytes after 8 weeks of cell culture. Detected by RT-PCR and immunofluorescent method, the myoblasts transfected with neural agrin gene pCDN A3-AGRIN Y4Z8 expressed mRNA and active protein of neural agrin.(3) Part III: At the first stage of transfection efficiency experiment, the expression of 95KD agrin protein in 200V group and 140V group were obviously higher than the 100V and 0V groups. Western blot results documented increased expression of agrinprotein at 4 weeks following the operation but decreased remarkably at 15 weeks. Then after the electrical transfection of pCS2+-AGRINY4Z8invivo with MOV and 40 ms parameters the pCS2+-AGRIN group was significantly better than the control groups in muscle function(P<0.05) at 4,5 and 10 weeks postoperatively. In experimental group, anti-neural agrin staining increased markedly at 1 week postoperatively but decreased gradually to the level of control groups at 30 weeks. Following the transfection of neural agrin, it was found that fluorescent a-bungarotoxin staining at the neuromuscular junction region changed greatly from denervation to reinnervation. At the first week after transfection, the clear outline of the junction with the fluorescent stain disappeared, and the neuromuscular junction appeared as some irregular spots or speckles, the borders of which were out of focus. Then the fluorescent spot number of each reinnervated endplate area decreased but with increased intensity and size following continued reinnervation. At 30 weeks postoperation, the reinnervated neuromuscular junction of experimental group differentiated to form a compact structure with a crisp, regular outline both in two and three dimensions similar to those of normal junction. Furthermore, the reinnervated endplate outline of pCS2+-AGRIN group looked more smooth and showed a regular structure compared with the controls. The experimental group was significantly more than the control groups but less than normal muscle in junctional nAChR number at each period postoperatively.[Conclusion](1) The detection of mRNA expression of e and y acetylcholine receptor subunit by TaqMan real-time fluorescent quantitative RT-PCR is more credible, accurate, sensitive, specific and time-saved. The decline in AChR number and persistent weakness of muscle following free transfer may be attributed to these quantitative changes of mRNA expression of e and y acetylcholine receptor subunit at the neuromuscular junction .(2) The decline in AChR number, a simpler morphology and delay reconstruction of endplate and persistent weakness of muscle following free transfer may be attributed tothese changes of distribution of neural agrin.(3) The plasmid pCDN A3 -agrin Y4Z8 was successfully established and the tissue culture method for skeletal myoblast was effective. These results provide the basis for future studies.(4) Primary culture of rat skeletal myoblasts were transfected with agrin gene but only the cells transfected by positive control pCDN A3-AGRIN Y4Z8 could express active neural agrin protein.(5) The transfection of pCS2 +-AGRINY4Z8 in vivo was feasible and electrical transfection of this gene with optimized parameters could get higher transfection efficiency and longer expression period.(6) Transfection of agrin gene in the transfer muscle increased the early(4,5,10weeks) recovery of muscle function by accelerating the regeneration of endplate following the free neurovascular muscle transfer.In conclusion, our study elucidates that transfection of agrin gene in the transferred muscle can increase the early recovery of muscle function in rat. But the clinical usefulness of this procedure awaits further investigations in clinical trial and need more practice and follow-ups to confirm the feasibility in human.