| The plastic surgeons have tried for the first time to preform skin transplantation from one burned soldier to another during World War II. Most of the allografts were rejected as a result of poor understanding of major histocompatibility complex(MHC) and graft rejection. Peter Medawar, a zoologist from Oxford University, observed this phenomenon and wondered whether the epidermis can survive as an allograft. He persisted in his research after World War II, publishing a series of momentous papers in 1944 and subsequent years; he demonstrated the accelerated rejection of a second-set skin allograft, thus suggesting that allograft rejection was caused by an immunological response to the foreign transplant. Later, he developed the mice skin transplantation model and induced skin transplantation tolerance in mice. The skin transplantation model and availability of genetically defined strains of mice has led to the understanding that complete MHC mismatched or even single class I or class II MHC mismatched skin allografts are quickly recognized by recipient and rejected. The perceived high allogenicity of the skin is considered as one of the major obstacles to widespread clinical applications of skin contained allotransplantation. However, recent clinical applications of composite tissue allotransplantation(CTA) such as hand, face, abdominal wall, penis and other organs have signified an emergence of a new era in reparative and reconstructive surgery. Allogeneic skin components are thought to be highly antigenic and require long-term and intense immunosuppression for the prevention of rejection. However, the associated mechanisms of rejection and tolerance have not been well understood. And a large part of our understanding of allogeneic skin rejection has derived from animal studies using non-vascularized skin allografting. However, clinical CTAs primarily involve vascularized allogeneic skin tissues. Therefore, the insight gained through vascularized skin allotransplantation model is more relevant to and required by mechanistic studies on CTA rejection and appropriate tolerance protocol is needed.Over one million cells die every second in our body, and the dead cells are cleared by the phagocytic cells of the innate immune system including macrophages, dendritic cells and monocytes. These cells are capable of discriminating between self and nonself components via pathogen-associated molecular patterns(PAMPs). Apoptotic cells exert an active and potent immunosuppressive effect on monocytes, promoting secretion of interleukin(IL)-10 and decreasing release of the pro-inflammatory cytokines such as Tumor Necrosis Factor(TNF)-?, IL-1? and IL-12. Based on this specialty of apoptotic cells, many researchers induced bone marrow, heart and islet transplants tolerance through intravenous infusion apoptotic cells. Among these protocols, ECDI fixed donor splenocytes(SP) show superiority over other protocols. In order to develop a reliable method to induce skin allotransplantation tolerance, we tried to prolong skin graft survival by intravenous infusion ECDI fixed SP(ECDI-SP).Objective: 1. The authors propose vascular anastomosis through cuff technique during allotransplantation of mouse donor free groin skin flaps to either recipient inguinal or cervical site and evaluate, on this mouse model, the success rate, extent of allograft rejection, and donor tissue ischemia time, and the time spent for vascular anastomosis as well as for whole surgical procedure. 2. To investigate the effect of ECDI-SPs on the survival of a fullthickness skin or vascularized skin flap allograft and its mechanism.Methods: 1. Free groin skin flaps from BALB/c or C57BL/6 donor mice were transplanted to the groin(inguinal vascularized skin transplantation, IVST) or to the neck of recipient sites(cervical vascularized skin transplantation, CVST) of C57BL/6 mice. A non-suture cuff technique was utilized to anastomose the donor vessels with either femoral vessels in IVST recipients or common carotid arteries and external jugular veins in CVST mice. Immunosuppressant drugs were used in the allogeneic skin group. 2. Different concentration of ECDI were used in vitro to detect the best concentration which could induce donor SPs apoptosis. IL-10 and TGF-? in cell supernatant were tested by elisa when ECDI-SP were co-cultured with SPs of recipients. 3. Full-thickness skin transplantation was established and mice were divided into 4 groups. RAPA group: skin allograft with 3 mg/kg rapamycin for 28 days; ECDI-SP~+RAPA group: ECDI-treated splenocytes were injected i.v. on day 7 before(-7) and 1 day after(~+1) skin transplantation with 3 mg/kg rapamycin for 28 days; SP~+RAPA group: Untreated splenocytes were injected the same way and time as ECDI-SP~+SP group with 3 mg/kg rapamycin for 28 days; C3 H group: ECDI-treated splenocytes were injected i.v. on day 7 before(-7) and 1 day after(~+1) skin transplantation with 3mg/kg rapamycin for 28 days. The donor skin graft came from C3 H, another strain of mice. There are three groups for vascularized skin flap allotransplantation. Control group: allotransplantation were performed without immunosuppressive; RAPA group: allotransplantation were performed with a 28-day rapamycin; ECDI-SP~+RAPA group: ECDI-treated splenocytes were injected i.v. on day 7 before(-7) and 1 day after(~+1) skin transplantation with 3 mg/kg rapamycin for 28 days. Mixed leukocyte reaction and detection of regulatory T cells were performed 10 days after skin transplantation.Results: 1. The overall success rate was higher in the CVST(88.5%) when compared with the IVST(78.9%). Total operation time in CVST mice lasted 96~+3 min that was shorter than that for IVST mice(136~+8 min, P < 0.01). Complications, such as hind limb necrosis and self-mutilation, were observed in IVST mice. 2. Rapamycin(3 mg/kg, daily) significantly prolonged vascularized skin allografts survival with mean survival time(MST) of more than 80 days but did not prolong nonvascularized skin graft. All syngeneic grafts survived for more than 80 days. 3. The donor SPs became apoptotic quickly after treated by ECDI(30 mg/ml). And the ECDI-SP increased the level of IL-10 produced by SPs of recepients in vitro. Infusions of donor splenocytes treated with ECDI prolonged both vascularized and nonvascularized skin allograft survival significantly(P < 0.01), and the SP~+RAPA group and C3 H group didn't show any effect on skin graft survival(P > 0.05). The proportion of regulatory T cells increased in ECDI-SP~+RAPA group than RAPA group(P < 0.05). The ECDI-SP~+RAPA group showed lower reactivity than RAPA group in mixed leukocyte reaction.Conclusions: 1. With the assistance of cuff technique, we demonstrate that the transplantation of free skin flaps to either inguinal or cervical recipient sites and reestablishment of the graft blood supply in mouse model is technically less challenging. It takes 48-72 hours before a nonvascularized skin graft becomes revascularized. During this 48-72 hours period, the skin graft is vulnerable to ischemia injury. Severe ischemia would result in tissue damage and accelerate the immune rejection to the skin allograft. 2. The ECDI of 30 mg/ml can best induce SPs apoptosis and control the rate of necrosis of SPs. ECDI-SP could increased the level of IL-10 produced by SPs of recepients in vitro. 3. Preand post-transplant infusions of splenocytes treated with ECDI prolonged skin allograft survival probably by up-regulation of regulatory T cells in spleen. |
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