| Objective ( 1) Take tissue engineering way of hypotonic-chemical detergent to prepare xenogeneic acellular nerve scaffold, which makes an optimal material for the graft of xenogeneic acellular nerve. ( 2) This thesis is intended to explore the feasibility of applying xenogeneic acellular nerve scaffold to nerve graft. ( 3)The experiment is meant to explore the effect of co-application of NGF and GM1 on nerve regeneration and function recovery after xenogeneic acellular nerve scaffold graft.Methods Experiment I: The rabbit tibial were sectioned and then chemically processed with solution of Triton X-100 and sodium deoxycholate in appropriate density and procedure. Fresh and acellular nerve segments were abstracted for morphological observation, for HE (hematoxylineosin) staining. The immunohistochemical staining was conducted to show the presence of laminas which would further dictate that the Schwann cell basal lamina component had been preserved in the nerves after chemical extraction. Experiment II: 48 healthy Sprague-Dawley rats were randomly divided into 4 groups: physiological saline control group, the NGF-treated group, the GM1-treated group and the combined NGF and GM1-treated group. The rats have lived for 4W and 8W respectively with a group of 6 rats living in different time spans. Sciatic nerve defects of the above mentioned four groups of rats were bridge-connected with 10mm long xenogeneic acellular nerve scaffold that have been dipped in NS, NGF, GM1, NGF and GM1 respectively. These rats were injected with 0.1ml of physiological saline fluid, NGF, GM1, and NGF combined with GM1 respectively on the side of operated leg once a day for two weeks. Denervated changes of the rats and morphologic changes of regenerated nerve were observed in each group the fourth and eighth week after operation. A series of measurement were conducted during the experiment such as SFI, recovery rate of the motor nerve conduction velocities (MNCV%), hind limbs recovery rate of the complex muscular action potential (CMAP%), horseradish peroxidase (HRP) retrograde trace, the recovery rate of wet weight of triceps surae muscle. Such activities were also carried out in this period as grafting part and its proximal and distal parts as well as stoma nerve fibers of the proximal part and distal part were stained with hematoxylineosin, lamina with toluidine blue, together with immunohistochemical staining for the presence of nerve fine NF and S-100.Results Experiment I: The xenogeneic acellular nerve scaffold were white long cylinder with well elasticity and ductility. Cells, axons and myelin sheath were removed while basal membrane, endoneurium, perineurium and epineurium was preserved after extraction procedure. Experiment II: In the same time span, the jointly treated group outperformed the two solely treated groups in the following measurements (P<0.05), SFI, recovery rate of the motor nerve conduction velocities (MNCV%), hind limbs recovery rate of the complex muscular action potential (CMAP%), L3-L5 the dorsal root ganglion and the labeled cells HRP in L3-L5 the spinal cord segment motoneurons, the recovery rate of wet weight of triceps, and myelinated nerve fibers in grafting part and in its distal part. Meanwhile, the jointly treated group and the two solely-treated groups outshone the physiological saline-treated group(P<0.05) in those regards. Through morphological observation we could find that nerve graft were in normal size with considerable blood vessels growing around and the fiber slightly connected to peripheral tissue. The jointly treated group once again overshadowed the rest groups in such perspectives as the regenerated nerve fibers, in much more density and order, went smoothly through stoma; Schwann cells proliferated in larger quantity in grafting part; myelinated fiber's density and diameter in the middle and distal part of the grafting part were all greater; connective tissue between microfascicles were much less, close to an ordinary one.Conclusions Experiment I: The chemical extraction procedure with the detergents of Triton X-100 and sodium deoxycholate can remove cells, axons, and myelin sheaths from peripheral nerve of rabbit; while the SC basal lamina tubes, endoneurium, perineurium and epineurium remain intact.Experiment II: ( 1 ) Xenogeneic acellular nerve scaffold got in a hypotonic-chemical tissue engineering way successfully restores peripheral nerve injury. ( 2) NGF or GM1 enhances peripheral nerve regeneration and functional recovery in xenogeneic acellular nerve scaffold graft. ( 3) Co- application of NGF and GM1 outperforms sole application of either one in achieving the effect mentioned above. Therefore, NGF and GM1 are synergistic factors in peripheral nerve injury repair. ( 4) Loca1 application binding with target muscle injection of NGF and GM1 is a good method to treat injured peripheral nerve. |
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