| Objective:To investigate the optimal time of making acellular nerve graft using Triton X-100 and sodium deoxycholate as extracting agent. Analyze its components with the methods of Haematoxylin-eosin staining, fast blue staining, Immunohistochemistry staining, transmission electron microscope;Then analyze and arrange this colected data for getting the optimal time of making acellular nerve graft.Then implant the ANG that made with the optimal method in the New Zealand white rabbits' muscle to test it's histocompatibility and offer experimental basis for selecting good ANG to repair the defect of facial nerve.Method:Part1:Preparation of ANG.The bilateral sciatic nerves were obtained from 15 healthy grown New Zealand white rabbits, and removed the adipose tissue and epineurium of the nerve surface under the surgery microscope, then divided these nerves into 66 segments, with each length of 10 mm. The 66 neurons were randomly divided into 11 groups, with 6 neurons in each group. Except the control group, all neurons were placed into Petri dish for 12 hours bathing using distilled water at room temperature, then 5 groups of which were cultured with Triton X-100 for 12, 24, 36, 48, and 60 hours, oscillation at room temperature; the remained 5 groups were cultured with 3% Triton X-100 for 12 hours, followed by 4% sodium deoxycholate for 12 hours, repeated for 1-5 cycles. Got scores from degrees of decellularization,integrality of fiber pipe and degrees of Myelin staining.Part2:Testing of histocompatibility. 3 male and 3 female healthy grown New Zealand white rabbits were selected for this experiment.Made 4 Vertical incision on the bilateral Hind legs of each rabbits,each incision should get to the Muscle layer. Filled the ANG(10mm) in the space,every space was filled 1 nerve. Hierarchically Sutured each incision. Fetched the nerves from each incision in the oder of Top-down at the time points of 1, 2, 3,4weeks.Using the method of Haematoxylin-eosin staining to observe the immunological rejection, the growth of peripheral vascular and the degree of inflammatory cell infiltration.Result:1.Only use Triton X-100 to deal with the nerve of New Zealand white rabbits, even if 60 hours, could not to remove all the cellular components, and the basement membrane of Schwann cells were greatly destroyed. After 2 cycles treatment of Trito X-100 combined with sodium deoxycholate, cellular components and myelin sheath of nerve fibers and axons were removed effectively, and basement membrane of Schwann cell was remained, with epineurium and perineurium could be seen.2.There was no inflammation such as swelling, exudation after implanting the ANG into the muscle.We can see the slightly red color and slight inflammation in the first week.The Neutrophils mainly constituted the inflammatory cells;Two to three weeks later,we can see the ANG adhered to the surrounding tissue closely and there was litte inflammatory cells than the first week.We can also see the growth of fibroblasts and vascular endothelial cells. New vessels began to appear. There was apparently bloody phenomenon when we fetching the ANG from the Muscle tissue after surgry three to four weeks.There are more new vessels than before.And we can not see the inflammatory cells in the Acellular nerve graft.Conclusion:1. Oscillation accompanied by 2 cycle's treatment of TritonX-100 and sodium deoxycholate can obtain acellular nerve graft by removing cellular components completely, and reserving integrated basement membrane of Schwann cells.2.There was no acute inflammatory reaction,the phenomenon of abscess and necrosis.3. After we implanting the ANG into the muscle,we can see the host's fibroblasts and vascular endothelial cells grew into the extracelullar matrix.These cells could consist lots of rich- erythrocytic- vascellum.The ANG could guide the regenerative cell to align oriently.
|
PDF Full Text Request