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Background: Although there is a wide application of the rat extended groin flap (epigastric skin flap) in studying different clinical issues, inconsistency arises between studies because many parameters of the extended groin flap have not been well defined., Materials and Methods: The flap is based on the superficial inferior epigastric vessels, which give into a lateral and a medial branch distally. Herein, three steps were taken to redesign this model: First, the ventral vascular anatomy was visualized through an imaging study to determine the flap borders. Second, different ischemic durations were induced on five groups of Lewis rats (n = 5 in each group) by clamping the femoral artery; group 1 (sham group) received no ischemic insult after elevation and was immediately repositioned, and groups 2, 3, 4, and 5 received 12-, 14-, 16-, and 18-hour ischemia, respectively. Percentage of necrosis area was measured after 5 days. Third, the redesigned groin flap model was tested with the ischemic postconditioning for validation., Results: The flap borders were determined such that both branches of the superficial inferior epigastric vessels were always included to ensure blood supply consistency. As the 14-hour ischemia induced the least variation in necrotic area on rats, it was chosen for further studies. In addition, ischemic postconditioning after 14-hr ischemia resulted in significant reduction of necrosis in this model., Conclusions: We have redesigned the extended groin flap model with better-defined borders and consistent vascular anatomy. The ischemia duration was calibrated with predictable necrosis pattern and the practicality was demonstrated. With this model, precise assessment of treatment efficacies on ischemia-reperfusion injury could be achieved in future studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Cotreatment with regulatory T cells (T(reg)) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether T(reg) treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats., Methods: Recipient Lewis rats treated (day 0) with or without naturally sorted T(reg) (3 × 10(6)) from Lewis rat spleen and lymph nodes received costimulation blockade (anti-CD154 monoclonal antibody, days 0 and 1 and CTLA-4 immunoglobin, days 2, 4, and 6), rapamycin (days -1, 0, and 2), and concurrent transplantation of fully mismatched allogeneic donor VCAs (day 0) from the Brown Norway rat hindlimb containing VBMT. The mixed chimerism level was assessed monthly using flow cytometry. Survival of VCAs and occurrence of graft-versus-host disease were assessed clinically and histologically., Results: The combination of T(reg) and VBMT treatment led to long-term multilineage hematopoietic mixed chimerism (12-18%) and long-term donor-specific tolerance to VCAs (89% acceptance rate). Neither stable mixed chimerism nor VCA acceptance was observed in recipients without T(reg) treatment. Graft-versus-host disease did not occur in the VBMT recipients., Conclusions: Cotreatment with T(reg) and VBMT created stable mixed chimerism and induced long-term donor-specific tolerance to VCAs without requiring cytoreductive conditioning. This noncytoreductive T(reg)-VBMT protocol has potential for clinical application in VCAs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Mixed chimerism with long-term composite tissue allotransplant (CTA) acceptance can be achieved through allogeneic bone marrow transplantation (BMT). The present study investigated the optimal chimerism level by giving different irradiation dosages to recipients to induce tolerance to CTA., Methods: Chimera were prepared using Brown-Norway and Lewis rats with strong major histocompatibility complex incompatibility. The Lewis rats received 5 mg antilymphocyte globulin (day -1 and 10) and 16 mg/kg cyclosporine (day 0-10) and were separated into groups 1, 2, 3, 4, and 5 according to the day -1 irradiation dosage: 0, 200, 400, 600, and 950 cGy, respectively. The Lewis rats were then reconstituted with 100 × 10(6) T-cell-depleted Brown-Norway bone marrow cells (day 0) and received vascularized Brown-Norway-CTA on day 28. Chimerism was assessed monthly by flow cytometry starting on day 28 after BMT. Graft-versus-host disease (GVHD) was assessed clinically and histologically., Results: Chimerism, 4 weeks after BMT, averaged 0.2%, 9.2%, 30.7%, 58%, and 99.3% in groups 1 to 5, respectively. GVHD occurred as follows: groups 1 and 2, none; group 3, 1 case of GVHD; group 4, 7 cases of GVHD (of which 3 died); and group 5, 10 cases of GVHD (of which 6 died). The percentage of long-term CTA acceptance was 0%, 0%, 90%, 70%, and 40% in groups 1 to 5, respectively. The percentage of regulatory T cells was significantly lower in high-chimerism (≥ 20%, n = 15) than in low-chimerism (<20%, n = 5) rats that accepted CTA long-term ., Conclusions: The chimerism level correlated positively with GVHD occurrence and long-term CTA acceptance but correlated negatively with regulatory T-cell levels. Optimal chimerism for CTA acceptance through pre-CTA BMT and irradiation occurs at 20-50% at day 28 after BMT in the rat model., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa, which represents cell-mediated autoimmune diseases. Pathological study demonstrated that abundant T lymphocytes infiltrated the oral mucosa, in which the activated T cells that trigger apoptosis of oral epithelial cells is an important mechanism for OLP. However, to date the molecular mechanisms underlying the T lymphocytes infiltration and accumulation in OLP remain unclear. In this paper, we found that the levels of plasma OPN were elevated and were associated with the up-regulated expressions of CD44 in OLP patients. In vitro, the addition of exogenous OPN can suppress the apoptosis of activated CD8(+) T cells via CD44, and this T cell resistance to apoptosis may be attributed to the reduction of endogenous mature granzyme B. Our results suggested that the abnormally elevated levels of OPN may contribute to the abnormal infiltration and accumulation of the activated T cells by up-regulating CD44 in OLP., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: A composite tissue consists of tissues derived from ectoderm and mesoderm typically containing skin, fat, muscle, nerves, lymph nodes, bone, bone marrow or any combination of these. Solid organ transplants possessing larger allograft mass were reported to survive better. As for the vascularized composite tissues however, thus far no study has comparatively studied the survival of grafts possessing different tissue burdens. The purpose of this study was to explore the effect of transplanted tissue burden and tissue type on survival of skin element of composite tissues., Materials and Methods: Forty-five transplantations were performed using four different vascularized composite tissue allotransplantation models. The survival periods and rejection severity of the skin parts of the transplants were compared by histological, immunological, and macroscopic evaluation: (a) under no immunosuppressive treatment (control group) (n = 21); (b) after 1 week of Cyclosporine (CsA) treatment (16 mg/kg) (experimental group) (n = 24). Total rejection was defined as necrosis of >90% of the skin flaps. Histopathologic evaluation and flow cytometric analysis to detect chimerism rates in the blood was performed in the CsA treated animals on day 18., Results: The differences of mean survival times between and within the experimental and control groups were statistically significant (P < 0.05). Histopathological outcomes showed lower rejection grades in skin allografts transplanted with a higher tissue burden. Total CD4+ and CD8+ T-cell chimerism rates were less than 1% in isolated skin transplant groups and ranged from 6.1 to 33.5% in skin flaps transplanted with the entire hindlimb or a part of it., Conclusion: The transplanted tissue burden as well as the tissue type can be an important factor for the skin transplant outcome., (Copyright 2010. Published by Elsevier Inc.)

Background: In vitro experimental studies showed that A2a receptor agonists reduce allostimulatory functions of the dendritic cells through modulation of surface expression of the costimulatory molecules and down-regulation of cytokines. Similar suppressive effects on dendritic cells have also been observed with Cyclosporine A (CsA). In this study we sought to explore if combined use of these two drugs in vivo could prolong the survival of composite tissue allografts across MHC barriers., Materials and Methods: A total of 24 hindlimb transplantations were performed across a major MHC barrier from Brown Norway rats (BN; RT1n) to Lewis rats (Lew; RT1l) In the control group, either isografts (group 1, n = 2) or allografts (group 2, n = 3) were performed and no treatment was given. In the experimental group three kinds of treatment protocols were used: (1) A2a adenosine receptor agonist alone (group 3, n = 6); (2) CsA alone (group 4, n = 7); and (3) A2a adenosine receptor agonist + CsA combined treatment (group 5, n = 6). Mean survival times of each group as well as cytokine levels including IL-2, IL-4, IL-10, INF-gamma, and TNF-alpha were analyzed by ELISA., Results: Isografts survived indefinitely. The mean survival times for allograft groups (group 2 to 5) were 9.8 +/- 1.3, 10.5 +/- 1.0, 29.8 +/- 1.7, and 22 +/- 1.4 days, respectively. Statistically, there was no difference between the allograft control group and the A2a adenosine receptor agonist treated group (P = 0.35). However, survival of the allografts in the CsA-treated group was significantly higher than the A2a adenosine receptor agonist treated (P < 00001) and combined CsA + A2a adenosine receptor agonist treated group (P < 0.0001). In vivo A2a adenosine receptor agonist treatment alone increased the levels of IL-2, INF-gamma, and TNF-alpha, which are important cytokines for induction of allotransplant rejection. However A2a adenosine receptor agonist in combination with CsA significantly reduced the levels of suppressor cytokines IL-4 and IL-10., Conclusion: As opposed to the previous in vitro studies, the results from this in vivo study showed that A2a adenosine receptor agonist treatment does not prolong composite tissue allograft survival. Compared to CsA treatment alone, the allotransplant survival was even shorter with the combined treatment. Treatment with A2a adenosine receptor agonist possibly promotes the differentiation of alloreactive CD4 cells predominantly into T-helper 1 phenotype. As a result, the levels of stimulatory cytokines contributing to allograft rejection are increased and suppressor cytokines are reduced, leading to accelerated allograft rejection.

Background: Recent studies have shown that platelets play an important role in the pathogenesis of reperfusion injury. Using an inferior epigastric artery skin flap as a flap ischemia/reperfusion (I/R) injury model, we investigated whether the administration of a nitric oxide (NO) donor, nitrosoglutathione (GSNO), could decrease platelet activation and modulate the NO synthase (NOS) activity of platelets and promote flap survival., Methods: Thirty minutes before flap reperfusion, normal saline (1 mL), nitrosoglutathione (GSNO 0.2, 0.6, 3 mg/kg), or N(G)-nitro-L-arginine-methyl ester (450 mg/kg) was injected intravenously in 10 rats, respectively. The p-selectin (CD62P) expression of platelet activation was detected by a flow cytometry. Immunohistochemical staining was performed to investigate the CD62P deposition on the microvasculature of the flap vessels. NOS isoform expression in the platelets was evaluated by Western blot. Tissue perfusion was monitored by using laser-Doppler flowmetry. Survival areas were assessed at 7 days postoperatively, Results: An optimal dose of GSNO (0.6 mg/kg), significantly decreased in CD62P expression on platelets (P < 0.001) and its deposition on the flap vessels, selectively suppressed iNOS induction of platelet, and significantly improved blood perfusion and the flap survival rate (59.8 +/- 4.9% versus 22.1 +/- 6.1%, P < 0.001). In contrast, the NO synthase inhibitor, N(G)-nitro-l-arginine methyl ester, although inhibiting iNOS expression of platelets, compromised platelet activation, tissue perfusion, and flap survival., Conclusion: This study suggests that GSNO can appropriately donate NO to suppress platelet activation and platelet iNOS induction, resulting in less platelet activation, better blood perfusion, and flap survival after I/R injury.

Background: Evidence has shown that platelets play an important role in the pathogenesis of flap failure. Employing a rat inferior epigastric artery skin flap as a flap reperfusion injury model, we investigated whether platelet activation was involved in the skin flap failure and whether administration of abciximab (ReoPro, chimeric 7E3 Fab) could decrease platelet activation/aggregation and promote flap survival., Methods: Normal saline and abciximab (0.06 mg/kg; 0.2 mg/kg; 1 mg/kg) were injected intravenously into skin flaps 30 min before reperfusion and 1 h after reperfusion (each subgroup n = 6). Platelet activation as demonstrated by P-selectin (CD62P) was analyzed by flow cytometry. P-selectin expression on flap vessels was detected by immunohistochemical staining. Platelet aggregation was induced with adenosine diphosphate (ADP). Laser Doppler flowmetry monitored tissue perfusion. The surviving area was evaluated 7 days postoperatively., Results: CD62P progressively increased after reperfusion. The peak CD62P occurred after reperfusion for 12 h. Immunohistochemical staining showed CD62P significantly deposited on the endothelium after reperfusion. Administration of abciximab (1 mg/kg) effectively improved flap survival rate (P = 0.003), significantly decreased ADP-induced platelet aggregation (P < 0.001), and suppressed CD62P expression on blood platelets (P = 0.002) and its deposition on the flap vessels., Conclusion: Abciximab promotion of skin flap survival is due to blocked platelet activation/aggregation and decreased activated-platelet deposition on the vascular endothelium. Thus, administration of a platelet glycoprotein IIb/IIIa receptor antagonist such as abciximab may save the skin flap from reperfusion injury after a long period of ischemia.