| I . Background and PurposeIt is frequent to see in clinical about large peripheral nerve defect,degeneration, necrosis caused by trauma, tumor, etc. The way of treatment often need the transplanting thing which is used in bridging,pomoting and homing nerve regeneration. Since all the time, self nerve transplanting is the idealest operation method in restoring peripheral nerve defect . It is being extensive used in clinical, and it is as a "gold standard " to judge various nerve bridging material. But self nerve transplanting often result in the loss of sense and function in supply area , and limited sources of autogenous nerve. Allogeneic nerve graft can solve these problems, But how to reduce or eliminate the immune rejection and to accelerate nerve growth is the problem to the clinical workers and the basis researchers . This experiment for both of the above-mentioned problems, With RGD (arginine-glycine- aspartic acid Arg-Gly-Asp) Polymer Materials and add FK506 (immunosuppressant), NGF (nerve growth factor) release synthetic outer membrane around the nerve for transplantation, investigate the effect of promoting nerve regeneration. II .methods1. Select eight healthy adult SD male rats, weighing 180～200g, under the microscope isolated and cut bilateral sciatic nerve, made of 10mm nerve saved in dry sterile test-tube Placed in -196℃liquid nitrogen for 3 weeks ,then taken out for rewarming before operation.2. Select 40 healthy adult SD male rats , weighing 180～200g, as receptors. They were randomly divided into 4 groups of 10 per set. Group A: cold preservation of nerve allograft; Group B: cold preservation of nerve allograft with FK506/RGD slow-releasing film; GroupC: autograft; GroupD: cold preservation of nerve allograft with FK506/NGF/RGD slow-releasing film. With concentration of 0.4% pentobarbital sodium (40mg/kg) injected the rat abdominal cavity to anesthetized rats. After the anesthesia satisfaction ,the rats were fixed in surgery board in prone position. Then shaving except the hair of rat's left lateral thigh skin, operation area disinfects Jay the sterile surgical drape. Along the left thigh for longitudinal posterolateral incision, the skin and subcutaneous tissue incision. Blunt separation of biceps femoris muscle and semitendinosus, semimembranosus muscle, fully exposed the sciatic nerve. From the lower edge of the piriformis 5mm. Department under the sciatic nerve cut and removal of 10mm, then transplantation for treatment of nerve. B group cold preservation of nerve allograft with FK506/RGD slow-releasing film. D group cold preservation of nerve allograft with FK506/NGF/RGD slow-releasing film, fixed at both ends of up in the epineurium. After operation closing the rat in the cage ,and feeding for 12 weeks.3. After each group of rats were generally observed, in the first nerve electrophysiological testing 12 weeks after operation, and then take three bilateral calf muscle recovery rate measured separately ,at the last the nerve' organization and ultrastructural morphology was observed in light and electron microscope . III. Results1. General observation: The rats of each group acted difficulty, the spirit was dejected, eat small food, every group animal's operation cut had not had infection.Two weeks pastoperation ,the rat's leg and foot occurred swelling and ulcer in varying degrees. Eight weeks pastoperation , the swelling and ulcer was subside. Four weeks pastoperation, the muscle of the surgery side legs of rats appeared vary degree atrophy. Eight weeks pastoperation, the musle of surgery side of rats in B.C.D groups start to restorate , the rats in D groups most obvious. 12 weeks pastoperation, when the nerve of transplatation was exposed , except the nerve of the A group had adhesion with around organization , the nerve of the rest of the group have limpid boundary with around organization , the continuity was fine , quality of work equation , the texture was soft. Under the microscope could be clearly observed the rich fresh blood vessels in nerve outside film. Sustained-release film had been a marked brittle, thinning.2. Electrophysiologial examination: the measuring results of motor nerve conduction velocity showed that: four sets of statistical difference between the groups(F = 26.190, P = 0.000), D group is better than A, B, C group (P <0.05, difference statistical significance); B, C group was better than the A group (P <0.05, statistically significant difference); B, C groups had no significant difference (P = 0.348> 0.05, the difference was not statistically significant).3. Rat triceps weight resumption rate: 12 weeks pastoperation, the muscle of the surgery side legs of all rats appeared vary degree atrophy .The rat triceps weight resumption rate measurements by the statistical analysis showed that: four sets of statistical difference between the groups (F = 19.132, P = 0.000), D group is better than A, B, C group (P <0.05, statistically significant difference); B, C group was better than the A group (P <0.05, the difference was statistically significant); B, C groups had no significant difference (P = 0.351> 0.05, the difference was not statistically significant).4. HE staining: 12 weeks pastoperation, the nerve of transplatation was stained with HE, the result showed that: The nerve perineurium of A group was rather ambiguous, a relatively small number of regenerative nerve fiber was seen, the axons of nerve distributed in disorder; The nerve perineurium of B, C group was well-defined perineurium, a relatively large number of regenerative nerve fibers were seen, the axons of nerve distributed in order; The nerve perineurium of D group was clear, the largest number of regenerative nerve fibers were seen, and clearly visible, the axons of nerve arranged in rules, distributed evenly towards consensus.5. Methyelin staining: 12 weeks pastoperation, the nerve of transplatation was stained with methyelin , the result showed that: The density of nerve fibers in A group was sparse, non-uniform size, myelin thickness, various shapes; The nerve fiber of B, C group was density, the size of nerve was relative uniformity, the number of myelinated nerve was large; The nerve fiber of D group was the greatest density, there are more myelinated nerve, myelin sheath thickness, mostly circular.6. Image-analysis: 12 weeks pastoperation, image analysis showed that: of The differences of the number of myelinated nerve fibers, axon diameter, myelin thickness among the four groups were significant difference (F = 194.918, P = 0.000; F = 54.342 , P = 0.000; F = 109.294, P = 0.000;), the number of myelinated nerve fiber, axon diameter, myelin thickness of D group was greater than that of A, B, C group (P <0.05, the difference was statistically significant); the number of myelinated nerve fibers, axon diameter, myelin thickness of B, C group was greater than that of A group (P <0.05, statistically significant difference); the number of myelinated nerve fibers, axon diameter, myelin thickness of B, C group compared to each other have no significant difference (P = 0.058> 0.05; P = 0.412> 0.05 P = 0.219> 0.05 no significant difference).7. S-100 immunohistochemical staining: 12 weeks pastoperation, the nerve of transplatation was stained with S-100 immunohistochemical,the result showed that: after S-100 immunohistochemical staining , the regenerative nerve of A , B, C, D group were showing positive response. Group D in which the most positive, B group, C group a strong positive, A group the weakest positive.8. The ultrastructure of electron microscope: 12 weeks pastoperation, the nerve of transplatation was counterstained with uran-lead, the result showed that: Much amorphous connective organization structure mixed with degeneration of nerve fibers could be seen in A group picture vision, it's myelin sheath was vary thickness, in which a few of Schwann cells and myelinated axons could be seen, some regeneration axon myelin-sheath was variable; In B, C group picture vision we could see more thickened nerve fibers myelin and rich Schwann cells, and normal mature nerve fibers with degeneration of nerve fibers co-exist, the ratio was quite; In D group picture vision we could see a large number of thickened nerve fibers myelin and the most rich Schwann cells, and most of the normal maturation of nerve fibers.V. ConclussionThrough the partial using of nerve transplants with immunosuppressive agents (FK506), nerve growth factor (NGF), RGD peptide release the film, not only be able to play against the effects of immune rejection, but also a large extent, improve the nerve growth speed, its test results significantly better than the other experimental group, even more than autograft group.
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