Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo

The mouse model of liver ischemia and reperfusion injury has proven to be valuable for our understanding of the role that reactive oxygen and nitrogen metabolites play in postischemic tissue injury. This methods paper provides a detailed protocol for inducing partial liver ischemia followed by reperfusion. Liver ischemia is induced in anesthetized mice by cross-clamping the hepatic artery and portal vein for varying lengths of time resulting in deprivation of blood flow to approximately of 70% of the liver. Restoration of blood flow to the ischemic lobes enhances superoxide production concomitant with a rapid and marked decrease in the bioavailability of nitric oxide resulting in alterations in the redox state of the liver in favor of a more oxidative environment. This hepatocellular oxidative stress induces the activation of oxidant-sensitive transcription factors followed by the upregulation of pro-inflammatory cytokines and mediators that ultimately lead to liver injury. This model can be induced in any strain or sex of mouse and requires 1-2 months of practice to become proficient in the surgery and animal manipulation. The role of different reactive metabolites of oxygen and nitrogen may be evaluated using genetically-engineered mice as well as selective molecular, cellular and/or pharmacological agents

Induction of Foxp3-Expressing Regulatory T-Cells by Donor Blood Transfusion Is Required for Tolerance to Rat Liver Allografts

BACKGROUND:Donor-specific blood transfusion (DST) prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx). METHODOLOGY/PRINCIPAL FINDINGS:Tolerance to Dark Agouti (DA; RT1(a)) rat liver allografts was induced by injection (iv) of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1(l)) rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4(+) T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg) transcription factor Foxp3 nor did they suppress alloantigen (DA)-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone) induced the time-dependent formation of CD4(+)Foxp3(+) Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4(+)CD45RC(-) population whereas in which approximately 50% of these Tregs express CD25. CONCLUSIONS/SIGNIFICANCE:We conclude that preoperative DST, in the absence of liver allograft transplantation, induces the formation of CD4(+) T-cells that are not themselves Tregs but give rise directly or indirectly to fully functional CD4(+)CD45RC(-)Foxp3(+)Tregs when transferred into MHC mismatched recipients prior to LTx. These Tregs possess potent suppressive activity and are capable of suppressing acute liver allograft rejection. Understanding the mechanisms by which preoperative DST induces the generation of tolerogenic Tregs in the presence of alloantigens may lead to the development of novel antigen-specific immunological therapies for the treatment of solid organ rejection

An Early Study on the Mechanisms that Allow Tissue-Engineered Vascular Grafts to Resist Intimal Hyperplasia

Intimal hyperplasia is one of the prominent failure mechanisms for arteriovenous fistulas and arteriovenous access grafts. Human tissue-engineered vascular grafts (TEVGs) were implanted as arteriovenous grafts in a novel baboon model. Ultrasound was used to monitor flow rates and vascular diameters throughout the study. Intimal hyperplasia in the outflow vein of TEVGs was assessed at the anastomosis and at juxta-anastomotic regions via histological analysis, and was compared to intimal hyperplasia with polytetrafluoroethylene (PTFE) grafts in the baboon model and in literature reports from other animal models. Less venous intimal hyperplasia was observed in histological sections with arteriovenous TEVGs than with arteriovenous PTFE grafts. TEVGs were associated with a mild, noninflammatory intimal hyperplasia. The extent of intimal tissue that formed with TEVG placement correlated with the rate of blood flow through tissue engineered vascular grafts at 2 weeks postimplant. Outflow vein dilatation was observed with increased flow rate. Both mid-graft flow rates and outflow vein diameters reached a plateau by week 4, which suggested that venous remodeling and intimal hyperplasia largely occurred within the first 4 weeks of implant in the baboon model. Given their compliant and noninflammatory nature, TEVGs appear resistant to triggers for venous intimal hyperplasia that are common for PTFE arteriovenous grafts, including (1) abundant proinflammatory macrophage populations that are associated with PTFE grafts and (2) compliance mismatch between PTFE grafts and the outflow vein. Our findings suggest that arteriovenous TEVGs develop only a mild form of venous intimal hyperplasia, which results from the typical hemodynamic changes that are associated with arteriovenous settings

Randomized Trial of Everolimus-Facilitated Calcineurin Inhibitor Minimization Over 24 Months in Renal Transplantation

TRANSPLANTATION957933-942TRPLAUnited State