| ObjectiveSince plerosis of peripheral nerve injury is one of trauma surgery problems, screening ideal graft of accelerating neural regeneration is becoming the key of solution. Various kinds of nature and artificial synthetic materials have been adopted in experiments and some progress had been made for many years. But there are still more difficulties hard to overcome. In recent years, with the development of constitution engineering, a better and broader prospect has been expected for the research and developing of artificial neural graft. In my study, acellular method of constitution engineering was used to prepare Acel-lular Rat Sciatic Nerve (ARSN ). HE (Hematoxylin and eosin) staining , PAGE (polyacrylamidedel electrophoresis) and immunohisto-chemical method were used to analysis its components. In order to observe the histocompatibility we embedded ARSN in muscle tissue and finally provided experimental basis of screening a nature, easy to obtain and satisfactory ECM (extracellular matrix) cage of peripheral nerve.Methods1. Preparation of ARSN and histological observationAfter resected bilateral sciatic nerve of Wistar rat and fixed, put them in Tris - HCL buffer. Through action of low permeating, cells such as Schwann cell, epineurium and nerve fascicles membrane would distend and break. According to certain steps and time and under appropriate temperature and pH, acellular treatment of sciatic nerve of Wistar rat with Triton X-100, protease inhibitor (0.1μg/ml aprotinin,0. 5μLg/ml leupeptin,0. 6μg/ml pepstatinA ) , DNase and RNase.Compared acellular with fresh sciatic nerve the difference of form and structure through optical microscope after routine steps of desiccation and clearing, dipping wax and embedding, sectioning before HE staining.2. Compare acellular with fresh sciatic nerve the difference of protein components using supernate by PAGE after ultrasound homoge-nated and centrifugated.3. Laminin ( LN) and fibronectin ( FN) were examined by immu-nohistochemical method.4. Wistar rats were divided into two groups; The rats of controlled group were embedded with fresh sciatic nerve while the rats of experimented group were embedded with acellular sciatic nerve. Histological observation was on the 4th and the 7th day.Results1. Histo - morphological observation of ARSNObservation by optical microscope we found axis-cylinder and myelin sheath disappeared on the transverse section of ARSN and only left areole basement membrane of Sehwann cells. On the longitudinal section, basement membrane of Sehwann cells appeared typical long dummy tube shape as the axis - cylinder, myelin sheath and nuclei of Sehwann cells disappeared. There were no cell structures of epineuri-um or nerve fascicles.2. Compared protein components of ARSN with fresh sciatic nerve by using PAGEIn the area of 30kDa, there was a significant change. It nearly vanished in ARSN group verifying that most components of ARSN were removed; There was a small quantity reduce in the area of 43kDa while no evidence change in the area of 65kDa, these indicated that the growth associated protein GAP - 43 and 65kDa protein which can induce the growth of axis - cylinder were reserved in the ARSN.3. Observation of LN and FN examined by immunohistochemical method.Yellow and brown reaction of basement membrane was seen under the optical microscope verifying components of LN and FN were saved with basement membrane after acellular treatment. But compared with the normal sciatic nerve, the reaction was feeble, it showed that some of the ECM were taken of during the procedure of removing cell.4. Histological observation of ARSN after embedded in muscle. ARSN embedded in quadriceps femoris of rats were stained byHE and observed after 4 and 7 days respectively. On the 4th in the ARSN group, the absorption phenomenon of remained basement membrane structure was more distinctly than that in fresh group. Lots of phlogistic cell, palingenetic blood capillary and fibroblast formed a cricoid... |
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