| Objective: The inability to reconstruct form and function of extensive tracheal defects is a clinical dilemma for many years. With the development of study on allotransplantation, tracheal allotransplantation is gradually regarded as an ideal method for long-segment tracheal allografts. However, two tough problems remain unsolved for modern tracheal graft. One is the immunological rejection of host to the homo trachea, and the other is the revascularization of the tracheal allografts. We used modified Courtman method to remove epithelia of tracheal mucosa and programmable cryopreserved respectively to dispose homo trachea and orthotopicly transplant the tracheal segment, and then observe the effect. We are looking for an effective way to reduce the immunogenicity of tracheal allotransplantation and provide new thoughts for reconstruction of form and function of extensive tracheal defects.Method: Fifteen dogs were randomly assigned to 3 groups. Group A: fresh tracheal grafts (n=5). Group B: cryopreserved tracheal allografts (n=5). Group C: allografts treated with modified Courtman to remove epithelium of tracheal mucosa (n=5). Group A: Fresh tracheal grafts were directly transplanted orthotopicly without any disposal. Group B: The grafts were stored in liquid nitrogen (-196 degrees C) for six weeks and then rewarmed and transplanted orthotopicly. Group C: The grafts were treated with non-ionic surfactants (NIS) (Triton X-100 and enzyme preparation) to remove epithelium of mucosa and then transplanted orthotopicly. The index and method of observation: (1) Observed microscopic structure change of mucosa and cartilage with different treatment after histological section and HE staining. (2) Observed canine living condition after orthotopic transplantation, and made a record of when they cough and breathe shortly as well as the changing. Took X-ray photographs to observe the stenotic condition of trachea. (3) After orthotopic transplantation, drew blood of canine peripheral vein and examine lymph cell subset by flow cytometer to analyze the change of CD3, CD4 and CD8 cells. (4) After the dogs died, dislodged the trachea grafts and observed gross change, tracheal stenosis degree and structure change of tracheal mucosa and cartilage after histological section and HE staining and.Results: (1) The microscopic structure change of tracheal grafts in each group after treatment but before transplantation. After HE staining, pseudostratified columnar ciliated epithelium lined up in order in fresh trachea, and mucosa, matrix cell of submucosa, gland cell and vascular endothelial cell were well-preserved. Cartilaginous part had compact texture, and chondrocyte grew in cartilage lacuna alone or in cluster. Ciliated columlar epithelia of cryopreserved tracheal allograft mucosa were partly exfoliated. The structures of mucosa and submucosa were well-preserved. The chondrocytes remained viable. In the specimen of allografts which were treated with modified Courtman, cell in mucosa and submucosa were removed completely, and red-dyed acellular matrix left. The chondrocytes remained viable. (2) Observe canine living condition after orthotopic transplantation. Group A: dogs began to cough about 7 days after the transplantation, and dyspnea appeared 14 days after transplantation. Degree of stenosis overtopped 50% and they died within 3 weeks. Group B: dogs began to cough about 2 weeks after transplantation and they breathed a little fast. Four dogs died within 2 months, and they got different degree of stenosis. Group C: dogs breathed stably after transplantation. They endured short breath to some degree 2 months after transplantation. Two dogs showed no clinical symptom for a long time after transplantation. They showed no symptom of the respiratory tract such as cough and short breath. They all survived after 3 months and no obviously stenoses were examined.(3) Drew blood of canine peripheral vein each group and examine lymph cell subset by flow cytometer to analyze the change of CD3, CD4 and CD8 cells. Group A: CD3, CD4 T lymph cell and the ratio of CD4+/CD8+ increased slightly 7 days after transplantation(P>0.05)(no statistical difference), but increased noticeably 14 days after transplantation( P<0. 05) which meant statistical difference. Group B: CD3, CD4 T lymph cell and the ratio of CD4+/CD8+ increased slightly 7 days after transplantation(P>0. 05) (no statistical difference), but increased noticeably 14 days after transplantation( P<0.05) which meant statistical difference. Group C: No obviously changes were observed after transplantation.(4) Microscopic structure change of tracheal grafts after transplantation. Group A: Gross appearance showed tracheal chondromalacia and some annulus cartilaginous fell into the lumina. Degree of lumina stenosis overtopped 50%. Histological appearance showed no epithelium and no complete cartilage. Some cells appeared squamous differentiation. There were a lot of granulation tissue in the lumina as well as many lymph cells. Interstitial substance bleeded severely. Group B: Gross appearance also showed tracheal chondromalacia. Degree of lumina stenosis was approximately 50%. Histological appearances showed no epithelium. There were a lot of granulation tissue and many lymph cells. Group C: Gross appearance showed slightly stenosis. There were little granulation tissue in the lumina and few lymph cells. Epithelia proliferate and neovascularization occured under cartilage and little calcification also occurs there.Conclusion: Both modified Courtman method and cryopreserved method can remove the antigenicity of tracheal allografts and reduce immunological rejection to tracheal allografts. However, we got better effect with modified Courtman method. The life time is longer and live quality is higher in dogs using allografts treated with modified Courtman method. There' s much hope that tracheal allgrafts treated with modified Courtman to remove epithelia will be a new choice of reconstructing form and function of extensive tracheal defects.
|
PDF Full Text Request