A Mechanism Study On Regeneration Of Peripheral Nerve Promoted By FG-NGF Membranes Embedded At Anastomotic Site

Background:Peripheral nerve injury is very common in orthopedic trauma, accounting for 12.5% of all lesions involved in extremities, in which half of cases require surgical treatment when nerves are transected or severely contused. Direct nerve anastomosis, nerve graft and nerve bridging are main treatment alternatives for peripheral nerve transection at present. The former includes the epineurium suture, perineurium suture, joint epi-&perineurium suture, adhesive bonding nerve, laser repair and so on. The latter includes autologous nerve graft, allogeneic nerve grafting, nerve transfer surgery, end-to-side anastomosis, biological graft bridging (skeletal muscle, biological membrane tube, basal membrane tube, chitin tube, etc.) and non-biological graft bridging (silicone tube, carbon fiber tube) and so on.For nerve transplantation and bridging anastomosis, autologous nerve grafting sacrifices the nerve function of donor nerve and nerve transfer surgery causes immunological rejection to allogenic nerve graft, non-biological graft needs the second surgery to remove bridging graft. While biological grafts are still limited at the stage of animal testing, hence such operations remain to use selectively for neurological defects or a large segment contusion. For most cases of peripheral nerve transection, the direct nerve anastomosis is still the main operative method. Lu Yu-Pu et al considered that when the nerve defect is less than 2cm, direct anastomosis can be achieved by dissociate the both end of injured nerve which could make nerve stretch out by 15% to 25% of the original nerve length. However, the result of direct anastomotic method remains unsatisfactory because of the following shortcomings and deficiencies:(1) The anastomotic site is prone to adhesion with the surrounding tissue that will form a scar and lead to local pressure to affect recovery of neurological function. (2) The regenerative nerve fibers growing into the distal part through the anastomosis could be hampered by scar resulting from invasion of fibrous tissue into the anastomotic site. (3) The "regeneration chamber" effect which is benefit to the nerve fiber regeneration is hard to form because of the lack of the micro-environment rich of a variety of growth factors. (4) The growth-blocked regenerative nerve fiber forms neurofibroma locally. Thus, how to find a simple and useful way for peripheral nerve repairing to avoid or reduce these shortcomings is an urgent need to address the important clinical orthopedic issues. Injured nerve repair is a complicated process involving comprehensive factors. Studies have shown that the environment surrounding the anastomosis is the determinant for the peripheral nerve regeneration and growth. A number of cytokines play an important role in the nerve regeneration process. Studies have demonstrated that the factors promoting nerve regeneration include:transforming growth factor (TGF-β), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), tumor necrosis factor (TNF), glial cell-derived neurotrophic factor (GDNF) and so on.As the dose of endogenous factors are insufficient to meet the needs of nerve regeneration, it is necessary to add exogenous factors to promote the nerve growth. Experiments have revealed that the nerve regeneration can be significantly promoted by administratering these factors in which nerve growth factor (NGF) is the first discovered and most useful cytokines. Gao Yan-ming et al studied the effects of local and continuous administration of NGF on regeneration and growth of injured peripheral nerves and the results revealed that the administration could significantly improve the renovatation and regeneration of the injured peripheral nerve, and local continuous administration of NGF had a better result than that of localized single dose. However, these factors are biologically active peptides or proteins, so they are vulnerable in the body and susceptible to degeneration and inactivation. It is intriguing how to remain active under the controlled release. In the previous study we have added the nerve growth factor into the fibrin glue and made the factor integrated into the preparation process of fibrin glue. Since both of them are biologically active molecules which have the same living environment, without chemical reaction between them, the fibrin glue including nerve growth factor was processed. The fibrin glue containing NGF was applied for embedding sciatic nerve anastomosis in rat and:observed functional recovery of the injured extremities and the sciatic functional index; neurophysiological detection of sciatic nerve conduction velocity and potential peak of calf muscle action; morphological observation of anastomotic adhesions and generation of neurofibromas; pathological examination of regeneration the number of nerve fibers of sections from anastomotic site by light microscopy and electron microscopy; the passing ratio of regenerative nerve fibers and fibrous tissue invasion; the diameter and architect of regenerative axon and myelin sheath thickness. The results shown that parameters mentioned above in the experimental group were significantly better than that of the control group. The authors have surgically treated peripheral nerve transection in patients with more than 20 patients and achieved good therapeutic effect with this method in previous three years. However, the method currently used for treatment of peripheral nerve injury is still lack of theoretical evidence, and it is necessary to explore its underlying mechanisms. The authors designed this research to address this problem.Objective:To discuss the mechanism of promoted peripheral nerve growth at anastomotic site embedded in FG-NGF membranes by immunohistochemistry、RT-PCR and electronic microscope etc. at cellular、molecule and ultrastructure level study, which provide theoretical evidence for the efficiency of this treatment。Methods:First, to establish animal models of anastomotic site sciatic nerve injury embedded in FG-NGF membrane in rat. Wistar rat were employed. The medial femoral incision was made and the sciatic nerve was cut at about 0.5cm above the bifurcation of the sciatic nerve, then anastomosed under the microscope, Fibrin glue containing nerve growth factor was prepared, spray on evenly around the anastomosis. The rats were raised for 8 weeks postoperatively. The condition of models was observed at 1,2,4,8-week respectively after models were made and the sciatic nerve anastomosis without embedded in FG-NGF fibril glue was established as control group.Second, the effect of sciatic nerve anastomosis embedded in FG-NGF membrane on spinal cord neurons in rat was evaluated. The spinal cord from lumbar 3 to sacral 2 was harvested preoperatively and 1,2,4,8 weeks post-operatively and fixed with paraformaldehyde.The changes in morphology and number of Nissel bodies of neurons. Anterior horn neurons in the spinal cord were observed under the microscope with HE and Nissl staining.Third, the effect of sciatic nerve anastomosis embedded in FG-NGF membrane in rat on p75 levels in Schwann cells was studied. The Schwann cells in sciatic nerve anastomosis were obtained by combined enzyme digestion, cytarabine treatment with identification of S-100 immunofluorescence. These cells were cultured with ordinary culture medium and in media added with high concentrations of IL-1. The expression of surface nerve growth factor p75 in the Schwann cell was evaluated at different time points.Fourth, the effect of sciatic nerve anastomosis embedded in FG-NGF membrane in rat on the sciatic nerve terminal effector was studied.①The nerve entrance to muscle together with muscle itself was fixed with glutaraldehyde of 2.5% concentration at different time points. The small pieces tissue mass were selected under the light microscope, after routine process including re-fixation, dehydration, embedding, sectioning, staining, and observed under GEM1011 electron microscopy at last.②At different times, the sciatic nerve were disociated to neuromuscular junction, and 0.5×0.5cm fresh muscle tissue connected with nerve branches was obtained and placed in -20℃condition. The frozen slices were sectioned (thickness 5μm) and stained with rabbit anti-rat AchE antibody, and the number of motor end-plate was observed under light microscope. Results:After the models were successfully made, the specimens were obtained at 2,4,8-week postoperatively from both experimental and control groups. At the second week after surgery the nissl staining showed that the neurons in the spinal cord were damaged morphologically in two groups, including staining shallow, nuclear contraction, partly disappearing cell processes, and all these changes were more evident in control group. At the fourth week, neuronal damage was still serious in two groups, but in experimental group glial cells increased, and neuronal morphology recovery more than that in control group, with darker staining. At the eighth week, differences in the morphology and structure of neurons between control and experimental group. The morphology of neurons in the experimental group tended to become regular, and the cell processes tended to grow well, Nissel bodies becoming rich and staining deeper. All neurons became well-arranged.The expression detection of p75 by Schwann cell obtained from anastomosis at the first week and cultured in normal medium and medium with IL-1 showed that at the first week p75 level in experimental and control groups reached the low peak. The difference of peak value in both groups was little, with a longer time to reach peak values, but no significant difference was detected. At the second week, in experimental group the peak value of p75 expressed by Schwann cell increased and time needed to reach peak value became slightly shorter compared with the first week. While in control group, the peak value and time for the value just changed a little compared with the first week. At the fourth and eighth week, the peak level of p75 expression became higher and the time to the level became shorter than that in control group, and the differences between two groups were significant.MEP AchE immunohistochemistry staining:The motor end plates were brown, "strip" structures, under light microscope. At 2nd week, in both experimental and control groups, the number of motor end plates decreased and they were light stained with irregular shape. The number of motor end plates in two groups had no significant difference. At 4th,8th week, the number of motor end plates in experimental group further increased, and they were stained more darkly and distributed regularly along muscular fiber; While in control group, their number increased, but was less than that in experimental group with irregular shape, and they were stained lighter. The differences between two groups were significant.Transmission Electromic Microscopy: at 2nd week, the variability of specimen was severe, and we could not find the neuromuscular junction. Skeletal muscle nuclei, myofibrils and interstitial cells were severe degeneration, part of them were swelling and dissolution, fibroblast proliferation and a lot of fiber were hyperplasia. Experimental group and control group had no significant difference. At 8th week, we saw a small amount of the neuromuscular junction, but the number was so little, we were unable to do statistical research. Skeletal muscle cells and interstitial cells could be seen more clearly, and the nucleus and other organelles had more regular and whole structure and shape. Collagen fibers, Z-line structure and the structure of the sarcomere are more clear and complete. However, the number and shape of skeletal muscle and interstitial cells in the experimental group were obviously better than the control group, the two groups had significant differences.Conclusion:①FG-NGF membranes cound provide nerve growth factor continuously, which could be transferred to the cell bodies of neuron to maintain its survival.②The nerve growth factor can promote Schwann cell proliferation and maturation by combination with the p75 receptor which in schwann cell surface, then induce the growth of the nerve axon and the maturity of the myelin sheath.③The nerve growth factor can increase sciatic nerve terminal effector formation and promote it maturation.