Induction Of Carbon Monoxide In Recipients Ameliorates Transplant-induced Ischemia/Reperfusion Injury And Allotransplant Rejection In A Murine Heart Transplantation Model

PartⅠImprovements of mouse cervical heterotopic heart transplantation model using the tail-cuff techniqueObjective The model of mouse cervical heart transplantation is an ideal medical research tool for study of transplant-induced ischemia/reperfusion injury and immunological rejection. However, technical problems have limited the widespread use of mouse cervical vascularized heart transplantation. The aim of this study is to describe an improved method for performing cervical heterotopic heart transplantation by the tail-cuff technique.Methods The kunming mice were used for preliminary experiment. And for formal experiment, inbred male C57BL/6 mice and Balb/c mice were used for isogeneic transplantation from BALB/c to BALB/c mice and allogeneic transplantation from C57BL/6 to BALB/c mice. The right common carotid artery and the external jugular vein of the recipient were equipped with a tail cuff made from 24G and 22G intravenous catheter, and everted over the cuff, and then connected with the aorta and the pulonary artery of donor heart, respectively. Results For preliminary experiment, the successful rate was 72.2%(13/18) and the the total surgical procedures took 56.2±8.9min. For formal experiment, the total surgical procedures took 49.6±7.4 min, the average time for evert and reconnection of both vessels was 3.6±1.4min and 4.1±1.1 min, and the successful rate was 100%(36/36).Conclusions This improved cuff technique shows its superiority, and can serve as an ideal method for the establishment of mouse cervical heterotopic heart transplantation model.Part IIInduction of carbon monoxide in recipients ameliorates cold ischemia/reperfusion injury in a murine heart transplantation modelObjective Carbon monoxide (CO), a product of heme degradation by heme oxygenase, induces cytoprotection against ischemia/reperfusion (I/R) injury in a variety of organs such as the liver, lung, kidney, and small intestine. The aim of this study is to examine whether induction of carbon monoxide (CO) with methylene chloride (MC) in recipients would protect heart grafts against cold I/R injury associated with transplantation and explore the possible mechanisam in a murine heart transplantation model..Methods Inbred male Balb/c mice were used as donors and recipients to establish transplant-induced cold I/R injury model with 2 hours cold preservation. Recipients were treated with either methylene chloride (MC) (100mg/kg or 500mg/kg, per os)(Group MC100mg, n=10; Group MC 500mg, n=12) or olive oil(0.15ml, per os, Group IR, n=10) 3h prior to anesthesia. Age-matched normal mice served as controls(Group N, n=5). The serum COHb and the CO content of myocardial tissue were measured at Oh, 1h,3h,6h,12h, 24h after oral MC administration. Half of Recipients mice were killed at 3h and 24h after transplantation for serum or cardiac graft samples(n=5-6 per time point). Recipients mice were killed at 3h and 24h after transplantation for serum or cardiac graft samples. The serum cTnI levels, the mRNA levels of TNF-α,IL-10,Bcl-2,Bax, the protein of NF-κB and the ultrastructure of myocardium were measured, respectively.Results The serum COHb and tissue CO increased significantly and peaked within 3h in Group MC100mg and Group MC 500mg(P<0.01 vs Olive oil). Compared with Group IR, The serum cTnI levels in Group MC100mg and Group 500mg were significantly decreased(P<0.01), especially in Group 500mg. Induction of CO in recipients in Group MC100mg and Group MC 500mg significantly inhibited the pro-inflammatory gene expression of TNF-a mRNA and the pro-apoptotic gene expression of Bax mRNA(P<0.01), and increased the anti-apoptotic gene expression of Bcl-2 mRNA(P<0.01), but did not increase the anti-inflammatory gene expression of IL-10 mRNA(P>0.05) in cardiac grafts. Compared with Group N, the myocardial NF-κB activation increased significantly in Group Olive, Group MC100mg and Group MC500mg(P<0.01), but did not differ between the later three groups at the same point(P>0.05).The myocardial ultrastructure was also improved significantly in Group MC100mg and Group MC500mg, especially in Group MC500mg.Conclusions Induction of CO in recipients with MC suppresses pro-inflammatory and pro-apoptotic gene expression and efficiently ameliorates transplant-induced heart cold I/R injury. The possible mechanism does not seem to be associated with down-regulation of the NF-κB signaling pathway. Part IIIInduction of carbon monoxide in recipient mice inhibits cold ischemia/reperfusion-induced apoptosis of cardiac grafts via PI3K/Akt signal pathwayObjective Carbon monoxide (CO) is emerging as an important gaseous molecule. And exogenous low-dose CO protects against vascular injury, transplant rejection and acute lung injury. However, little is known regarding the precise signaling mechanisms of CO. The aim of this study is to observe whether induction of carbon monoxide in recipients could inhibit cold ischemia/reperfusion(I/R)-induced apoptosis of cardiac grafts and to explore the possible anti-apoptotic signaling mechanisms in a murine heart transplantation model.Methods Inbred Balb/c mice were used as donors and recipients to establish transplant-induced cold I/R injury model. Recipients were treated with either methylene chloride (500mg/kg, per os, Group MC, n=12) or olive oil(Group IR, n=12) 3h prior to anesthesia, or treated with MC plus either LY294002 of PI3K inhibitor (40mg/kg, i.p, Group LY, n=10) or DMSO(Group DMSO, n=10) lh prior to reperfusion of cardiac grafts. Recipients mice were killed at 3h and 24h after transplantation for cardiac graft samples(n=5-6 for each time point). The apoptosis index(AI), the protein of phosphorylated, the activities of Caspase-3, the protein of Bcl-2 and Bax were measured, respectively. Age-matched normal mice served as controls(Group N).Results Following MC application serum COHb[(9.82±0.84)%]and tissue CO (2.25±0.08 pmol/mg) peaked within 3h in recipients. Compared with Group IR, induction of CO in recipients decreased significantly the level of AI [3h:(8.65±2.01)%vs. (19.28±4.94)%, P<0.01; 24h:(5.82±2.36)%vs.(10.54±3.66)%, P<0.05], increased the Akt phosphorylation (3h:P<0.01; 24h:P<0.05), inhibited the activities of Caspase-3(3h:2.19±0.18 vs.3.31±0.36, P<0.05)and up-regulated the ratios of Bcl-2/Bax(3h:1.97±0.16 vs.0.46±0.07, P<0.01; 24h:1.89±0.10 vs.0.51±0.04, P<0.01) in cardiac grafts. However, compared with Group MC, recipients pretreated with LY294002 could reverse the anti-apoptotic effects of MC by increasing the level of AI[3h:(17.95±4.92)%, P<0.01; 24h:(9.75±3.14)%, P<0.01], inhibiting the Akt phosphorylation(3h:P<0.01; 24:P<0.05), activating Caspase-3(3h:3.27±0.24, P<0.05) and down-regulating the ratio of Bcl-2/Bax (3h:0.47±0.06, P<0.01; 24h:0.52±0.03, P<0.01). And the DMSO had no impact on the anti-apoptotic effects of MC(P>0.05).Conclusion Induction of carbon monoxide in recipient mice inhibits cold I/R-induced apoptosis of cardiac grafts by regulating caspase-3, Bcl-2 and Bax proteins via the activation of PI3K/Akt signal pathway. Part IVAmelioration of cardiac allograft rejection by induction of carbon monoxide with methylene chloride in recipients is independent of up-regulation of Foxp3 expression in miceObjective Carbon monoxide (CO) is commonly viewed as a poison in high concentrations due to its ability to interfere with oxygen delivery. However, exogenous low-dose carbon monoxide inhibits transplant-induced ischemia/reperfusion injury via its vasodilation, anti-inflammatory and anti-apoptotic effects in various models. The aim of this study is to investigate the protective effects of induction of CO in recipients on amelioration of allograft rejection and prolongation of allograft survival and the possible mechanisms in a mouse heart transplantation model.Methods Inbred male C57BL/6 mice were used as donors and inbred male Balb/c mice were used as recipients to establish allogenic heart transplantation model. Recipients were treated with either methylene chloride (100mg/kg, per os, Group MC) or olive oil(Group Tx, n=6) day 1 prior to transplantation to day 6 posttransplantation (Group MC1w, n=11) or to day 13 posttransplantation (Group MC2w, n=10). The survival of cardiac grafts was recorded. Recipients mice were killed at day 6 and day 10 or the time of non-heartbeating after transplantation for serum, cardiac graft and spleen samples(n=5-6 for each time point). The serum TNF-a and IL-10, the TNF-a mRNA and IL-10 mRNA, the Foxp3 mRNA and Foxp3 protein in cardiac grafts and spleen were measured, respectively. The intercellular adhesion molecule-1(ICAM-1) and Caspase-3 protein in cardiac grafts, the proliferation of collagen fibers and histopathologic changes of cardiac grafts were also recorded. Results Following MC application serum COHb peaked within 3h in recipients[(5.24±0.45)%]. Induction of CO in recipients prolonged significantly the cardiac graft survival(median survival time, MST) [Tx:(6.33±0.56)d; MC1w: (12.14±0.87)d, P<0.01vs.Tx; MC2w:(19.38±0.95)d, P<0.01vs.Tx and MC1w], down-regulated the levels of serum TNF-a(MC1w:P<0.01vs.Tx; MC2w: P<0.01vs.Tx and P<0.05 vs. MC1w)and TNF-a mRNA (MC1w:P<0.01vs.Tx; MC2w: P<0.01vs.Tx and MC1w)of cardiac grafts and spleen in recipient mice, inhibited the protein expression of ICAM-1(MC1w and MC2w: P<0.01vs.Tx) and Caspase-3(MC1w:P<0.01vs.Tx; MC2w:P<0.01vs.Tx and MC1w) of cardiac grafts, and inhibited, especially in Group MC2w, the proliferation of collagen fibers and lymphocyte and monocyte infiltration in cardiac grafts. The levels of serum IL-10, the IL-10 mRNA, the Foxp3 mRNA and the Foxp3 protein in cardiac grafts and spleen of recipients did not differ between the groups(P>0.05).Conclusions Induction of carbon monoxide in recipients could relieve cardiac allograft rejection and prolong cardiac allograft survival via its anti-inflammation and anti-apoptotic effects, not via up-regulation of Foxp3 mRNA and Foxp3 protein in cardiac grafts and spleen in recipient mice.