Rat Heart Transplantation Model Research Articles

Non-invasive diagnosis of allograft rejection with positron emission tomography in a heterotopic rat heart transplantation model

Objectives: Allograft rejection is a major complication after heart transplantation. Endomyocardial biopsy is the gold standard in diagnosis of rejection; however, this technique is invasive and is associated with complications. Therefore, we investigated a non-invasive method for the detection of allograft rejection reaction with positron emission tomography (PET). 68-Ga-DOTA-TATE, which binds to the somatostatin-receptors of lymphocytes, was used to detect a cellular infiltration of the graft.

Mesenchymal stem cells together with mycophenolate mofetil inhibit antigen presenting cell and T cell infiltration into allogeneic heart grafts

Donor-derived mesenchymal stem cells (MSC) can induce long-term acceptance in a rat heart transplantation model when injected prior to transplantation in combination with mycophenolate mofetil (MMF). In contrast, MSC alone cause accelerated graft rejection. To better understand these conflicting data we studied the effects of MSC and MMF on lymphocyte populations in heart allografts and secondary lymphatic organs. Allogeneic MSC injected prior to transplantation are immunogenic in this model because activated CD4 + and CD8 + cells emerged earlier in secondary lymphatic organs of MSC- and MSC/MMF-treated animals, compared to animals not treated with MSC. Consequently T cells infiltrated the grafts of MSC-only treated animals promptly causing accelerated graft rejection. However, few T cells or antigen-presenting cells (APC) infiltrated the grafts of animals treated with MSC and MMF. Consistent with this finding, intercellular adhesion molecule 1 (ICAM-1) and E-selectin was down-regulated exclusively in MSC/MMF-treated grafts, indicating that MSC together with MMF interfere with endothelial activation. Additionally, the presence of interferon-gamma (IFN-γ) enhanced MSC capabilities to suppress T cell proliferation in vitro. Interestingly, MMF did not influence serum IFN-γ levels in vivo. Together, our data indicate that MSC pre-activate T cells, but co-treatment with MMF eliminates these T cells, decreases intragraft APC and T cell trafficking by inhibiting endothelial activation, and allows IFN-γ stimulation of suppressive MSC.

Mechanism Analysis of Long-Term Graft Survival by Monocarboxylate Transporter-1 Inhibition

Monocarboxylate transporter (MCT)-1, a member of a family of molecules, transports monocarboxylates such as lactate. Inhibiting MCT-1 leads to long-term graft survival in rodent heart transplantation and induces tolerance. We evaluated an MCT-1 inhibitor, AS2495674, in a rat heart transplant model and analyzed its underlying mechanism. AS2495674 was tested on rat lymphocytes to determine its effect on lactate accumulation, proliferation, and immunoglobulin production. The effect of AS2495674 on graft survival was tested on the Brown Norway to Lewis rat strain combination with a second heart transplantation to test donor-specific suppression. Histology and ex vivo analyses were done to examine the AS2495674 effects on the immune response. In vitro, AS2495674 resulted in lactate accumulation, inhibited lymphocyte proliferation, and prevented immunoglobulin production. AS2495674 induced long-term allograft survival with little evidence of chronic rejection and induced donor-specific suppression. Evaluation of the allograft and peripheral T lymphocytes from the AS2495674 group compared with that of vehicle showed (1) decreased donor-specific T lymphocyte response, (2) more forkhead box P3+ (Foxp3+) and CD45RA+ cells in the allograft, (3) higher gene expression of chemokines and chemokine receptors in the allograft, and (4) preferential inhibition of Foxp3(-) cells with little or no effect on Foxp3+ cells. AS2495674 prevents acute rejection, reduces features of chronic rejection, and induces tolerance. Our data suggest that the mechanism of AS2495674 involves generating a tolerogenic graft environment by preferentially targeting T effector cells while sparing the generation of T regulatory cells.

Deferoxamine, the newly developed iron chelator LK-614 and N-α-acetyl-histidine in myocardial protection

During cold storage of donor hearts, reactive oxygen species produced by intracellular redox-active chelatable iron potentially alter myocardial function. To reduce this cold-induced injury we investigated the efficacy of two new modifications of the well established histidine-tryptophan-ketogluterate (HTK) solution (Custodiol) with the addition of N-alpha-acetyl-l-histidine and iron-chelators in a heterotopic rat heart transplantation model. The donor hearts were cardioplegically arrested with 20 ml cardioplegia and stored for 1 h. Then the hearts were anastomosed to the abdominal aorta and vena cava of the recipient (n=30). After 1 h reperfusion, myocardial function and energy charge potential were measured in three groups: HTK-1: addition of l-arginine and N-alpha-acetyl-l-histidine; HTK-2: addition of iron-chelators deferoxamine and LK-614; traditional HTK - control. After 1 h reperfusion, left ventricular systolic pressure (106+/-33 vs. 60+/-39, vs. 67+/-8 mmHg, P<0.05) and dP/dt minimal (-1388+/-627 vs. -660+/-446, vs. 871+/-188 mmHg/s, P<0.05) were significantly higher in the HTK-1 group. Energy charge potentials were not significantly different. This study showed that the novel modified HTK-1 solution improves myocardial contractility and relaxation after heart transplantation. Nevertheless, addition of the iron-chelators deferoxamine and LK-614 diminished these beneficial effects.

Custodiol-N, a novel organ preservation solution reduces ischemia/reperfusion injury after heart transplantation

Custodiol (HTK-solution) is a leading organ preservation solution. Custodilol-N includes amino acids and antioxidative iron chelators to address reperfusion injury. Here, we investigated the effects of Custodiol-N in a rat model of heart transplantation.

103. The effect of supercoolant, Kaempferol 7-o-glucoside, on a heterotopic rat heart transplantation model

103. The effect of supercoolant, Kaempferol 7-o-glucoside, on a heterotopic rat heart transplantation model

Intragraft gene expression profile associated with the induction of tolerance by allochimeric MHC I in the rat heart transplantation model

The MHC class I allochimeric protein containing donor-type epitopes on recipient-type heavy chains induces indefinite survival of heterotopic cardiac allografts in rats. We analyzed gene expression profile of heart allograft tissue. Mutated peptide [alpha1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) was delivered into ACI recipients of WF hearts at the time of transplantation in addition to a 3 days course of oral cyclosporine. Microarray analysis was performed using Affymetrix Rat 230 2.0 Microarray. Allochimeric molecule treatment caused upregulation of genes involved in structural integrity of heart muscle, downregulation of IL-1beta a key modulator of the immune response, and downregulation of partitioning defective six homolog gamma PAR6, which is involved in T cell polarity, motility, and ability to scan dendritic cells (DC). These indicate that the immunosuppressive function of allochimeric molecule and/or the establishment of allograft tolerance depend on the induction of genes responsible for the heart tissue integrity, the suppression of cytokine pathway(s), and possibly the impairment of T cells mobility and their DC scanning ability. These novel findings may have important clinical implications for inhibition of chronic rejection in transplant recipients.

Impact of animal strain on gene expression in a rat model of acute cardiac rejection

BackgroundThe expression levels of many genes show wide natural variation among strains or populations. This study investigated the potential for animal strain-related genotypic differences to confound gene expression profiles in acute cellular rejection (ACR). Using a rat heart transplant model and 2 different rat strains (Dark Agouti, and Brown Norway), microarrays were performed on native hearts, transplanted hearts, and peripheral blood mononuclear cells (PBMC).ResultsIn heart tissue, strain alone affected the expression of only 33 probesets while rejection affected the expression of 1368 probesets (FDR 10% and FC ≥ 3). Only 13 genes were affected by both strain and rejection, which was < 1% (13/1368) of all probesets differentially expressed in ACR. However, for PBMC, strain alone affected 265 probesets (FDR 10% and FC ≥ 3) and the addition of ACR had little further effect. Pathway analysis of these differentially expressed strain effect genes connected them with immune response, cell motility and cell death, functional themes that overlap with those related to ACR. After accounting for animal strain, additional analysis identified 30 PBMC candidate genes potentially associated with ACR.ConclusionIn ACR, genetic background has a large impact on the transcriptome of immune cells, but not heart tissue. Gene expression studies of ACR should avoid study designs that require cross strain comparisons between leukocytes.

Myocardial protection with the use of deferoxamine and L-arginine

Objective: Effective myocardial preservation is an important condition for cardiac surgery especially in heart transplantation with long ischemic times. During ischemia and reperfusion myocardial function is altered by cold-induced ischemic injury. One source of free oxygen radicals are iron-iones. We investigated the efficacy of new modifications with addition of iron-chelators and L-arginine of the well established HTK solution (Custodiol) in a rat heart transplantation model.

Comparison of different gene delivery methods in rat heart transplantation model

Comparison of different gene delivery methods in rat heart transplantation model

Temporal Profile of Calcineurin Phosphatase Activity During Acute Allograft Rejection in the Heterotopic Rat Heart Transplantation Model

Background Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal if it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. Methods The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. Results We have shown that CaN's activity levels are not significantly altered during acute allograft rejection in peripheral blood mononuclear cells and in spleen-isolated mononuclear cells. CaN's intragraft activity decreased with time in both rejectors and controls, and was significantly lower in the allogeneic group. Conclusions CaN failed as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection.

Effects of Selective Phosphodiesterase-5-Inhibition on Myocardial Contractility and Reperfusion Injury After Heart Transplantation

Recently, the infarct reducing and cardioprotective effects of phosphodiesterase-5-inhibitors were described. In this study, we investigated these effects on ischemia/reperfusion injury in a rat model of heart transplantation. Three groups were assigned for our study: a vardenafil preconditioning group, an ischemic control, and a nonischemic control. Hemodynamic parameters were significantly increased in the vardenafil group (Pmax: 82+/-4 vs. 110+/-12 vs. 127+/-13 mm Hg; dP/dtmax: 1740+/-116 vs. 3197+/-599 vs. 4397+/-602 mm Hg/sec; ischemic control vs. vardenafil vs. nonischemic control; P<0.05 vs. ischemic control). Furthermore, we recorded increased ATP levels and significantly less apoptosis in the treatment group after terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (apoptosis index: 27.23%+/-1.54% vs. 16.77%+/-1.42% vs. 18.86%+/-1.07%; ischemic control vs. vardenafil vs. nonischemic control; P<0.05 vs. ischemic control). Our current results support the concept that the cGMP-PKG-pathway plays an important role in ischemia/reperfusion injury. We could show that up-regulating this pathway has a preconditioning-like effect and can effectively reduce ischemia/reperfusion injury.

MITOCHONDRIAL ISCHEMIA-REPERFUSION INJURY OF THE TRANSPLANTED RAT HEART

Cold ischemia time and preservation of organs are limited by I/R injury leading to primary nonfunction of the graft. In a rat heart transplant model, we compared cardioplegic St Thomas (ST) to histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin preservation solutions in terms of contractile function, and mitochondrial respiratory and enzymatic defects after prolonged cold ischemia and reperfusion. Contractile function was scored after transplantation and 24 h of reperfusion. Mitochondrial function was investigated by high-resolution respirometry of permeabilized myocardial fibers. Graft performance in terms of contractile function declined with the duration of cold storage. Recovery was significantly improved after 10 h of cold storage in HTK compared with ST (cardiac scores, 3.3+/-0.5 and 1.8+/-0.8, respectively). Tissue lactate dehydrogenase was better preserved in HTK than ST. Increase of tissue water content (edema) was less pronounced in HTK than ST (3.33+/-0.14 and 3.73+/-0.21 mg/mg dry weight, respectively). Similar cardiac scores (2.6+/-0.9 and 2.9+/-1.2, respectively) and mitochondrial respiratory parameters were obtained after preservation in HTK and University of Wisconsin. Decline in contractile function of individual grafts correlated well with loss of mitochondrial respiratory capacity, whereas citrate synthase activity remained largely preserved, indicating specific damage of respiratory complexes. Our data provide evidence for the superiority of preservation solutions versus a cardioplegic solution for prolonged cold storage of the heart. The correlation of graft performance and mitochondrial function indicates the potential of high-resolution respirometry for quantitative assessment of myocardial injury upon cold I/R, providing a basis for diagnostic approaches and evaluation of improved preservation solutions for heart transplantation.

Targeting of interleukin‐10 is superior to cytotoxic T‐lymphocyte associated antigen 4 with human immunoglobulin G1 for the prevention of chronic allograft deterioration in organ transplantation

Genetic manipulation of the allograft is an attractive approach to prevent the graft against chronic deterioration through stable expression of immunomodulatory or protective genes. However, the best strategy for prevention of chronic allograft deterioration remains unclear. The efficacies of adeno-associated viral vector-mediated stable expression of indoleamine 2,3-dioxygenase (IDO), cytotoxic T-lymphocyte associated antigen 4 with human immunoglobulin G(1) (CTLA4Ig) or interleukin-10 (IL-10) in the prevention of chronic allograft deterioration were compared in a rat heart transplantation model. Transduction of grafts with IL-10 significantly prolonged allograft survival, whereas transduction of grafts with IDO did not improve graft survival compared to controls. Analysis of long-term survived heart allografts showed that both CTLA4Ig and IL-10 could significantly reduced the T cells and macrophage infiltration. However, stable expression of CTLA4Ig failed to prevent the development of transplant arteriosclerosis. By contrast, IL-10 suppressed the development of transplant arteriosclerosis effectively. The suppressive effects of IL-10 in preventing the development of chronic allograft deterioration were associated with lower transcript levels of transforming tumor growth factor beta 1 and macrophage migration inhibitory factor in the graft. In addition, higher transcript levels of heme oxygenase-1 were found in IL-10-transduced allograft. Targeting on IL-10 is superior to CTLA4Ig or IDO for the treatment of chronic allograft deterioration.

Mesenchymal stem cells can induce long-term acceptance of solid organ allografts in synergy with low-dose mycophenolate

The induction of tolerance towards allogeneic solid organ grafts is one of the major goals in transplantation medicine. Mesenchymal stem cells (MSC) inhibit the immune response in vitro, and thus are promising candidate cells to promote acceptance of transplanted organs in vivo. Such novel approaches of tolerance induction are needed since, to date, graft acceptance can only be maintained through life-long treatment with unspecific immunosuppressants that are associated with toxic injury, opportunistic infections and malignancies. We demonstrate that donor-derived MSC induce long-term allograft acceptance in a rat heart transplantation model, when concurrently applied with a short course of low-dose mycophenolate. This tolerogenic effect of MSC is at least partially mediated by the expression of indoleamine 2,3-dioxygenase (IDO), demonstrated by the fact that blocking of IDO with 1-methyl tryptophan (1-MT) abrogates graft acceptance. Moreover we hypothesize that MSC interact with dendritic cells (DC) in vivo, because allogeneic MSC are rejected in the long-term but DC acquire a tolerogenic phenotype after applying MSC. In summary, we demonstrate that MSC constitute a promising tool for induction of non-responsiveness in solid organ transplantation that warrants further investigation in clinical trials.

Heterotopic Rat Heart Transplantation (Lewis to F344): Early ICAM-1 Expression After 8 Hours of Cold Ischemia

Primary graft dysfunction is a still poorly understood complication after cardiac transplantation. Ischemia/reperfusion injury contributes to different disorders resulting in impaired graft function. In a heterotopic rat heart transplantation model we extended graft ischemic time up to 8 hours. Using immunohistochemistry we detected an up to 4-fold increase in intracellular adhesion molecule-1 (ICAM-1) expression during 4 hours of reperfusion, independent of ischemic time (30-minute ischemia: 7.65 +/- 2.15 without reperfusion, 19.46 +/- 4.6 after 4-hour reperfusion; 240-minute ischemia: 5.6 +/- 1.99 and 22.3 +/- 3.77; 480-minute ischemia: 3.7 +/- 1.56 and 13.1 +/- 2.2). Eight-hour ischemic allografts had an increase in CD8-positive cells (1.37 +/- 0.5 and 2.3 +/- 0.77) and a significant increase in MHC II expression (11.48 +/- 2.1 and 18.27 +/- 1.34) during 4 hours of reperfusion. We hypothesize that these findings reflect an early inflammatory reaction in the allograft possibly triggered by oxidative stress. During therapeutic interventions, both of these pathways must be considered.

Dietary n‐3 fatty acids attenuate cardiac allograft vasculopathy via activating peroxisome proliferator‐activated receptor‐γ

Recent in vitro data suggested that n-3 fatty acids could inhibit the activation of PPAR gamma. This study was designed to test the hypothesis that fish oil ameliorates CAV development via activating PPAR gamma in an inbred rat model of heart transplantation. Animals were divided into four groups: isograft, control (CsA + vehicle), LFO-treated group (CsA + 0.3% v/w fish oil), and HFO-treated group (CsA + 0.6% v/w fish oil). CsA was administered at 1.5 mg/kg/day for two wk postoperatively. Recipients were treated with fish oil or vehicle daily for eight wk. The histopathological and immunohistochemical examination, activity of NF-kappaB and PPAR gamma, intragraft chemokine levels, and chemokine receptor expression were analyzed. Both LFO and HFO significantly decreased the CAV score, inhibited recruitment of T lymphocytes and macrophages, elevated the activity of PPAR gamma, inhibited the activity of NF-kappaB, reduced levels of intragraft MCP-1 and IP-10 as well as downregulated expression of chemokine receptors CCR2. CXCR3 expression was not affected. Our results demonstrated that fish oil might attenuate CAV development, possibly through activating PPAR gamma and subsequently inhibiting the NF-kappaB activation, the chemokines secretion, as well as the CCR2 expression.

Heart allograft acceptance induced by anti-CD3 antibody in high-responder rats: Effect on foxp3 and cytokine expression and graft infiltration

The ability of anti-T cell monoclonal antibody G4.18 and polyclonal anti-lymphocyte serum (ALS) to induce long-term graft survival was examined in a high-responder rat heart transplant model. Heterotopic heart allografts were performed from PVG rat strain donors to high-responder Lewis recipients. Immunosuppressive properties of G4.18 and ALS were investigated by immunohistochemistry and PCR analysis. Untreated graft rejection was 8.5 days while treatment with 1 ml ALS prolonged survival to 11.5 days ( p = 0.01). Treatment with 7 mg/kg G4.18 on days 1 and 3 prolonged survival to > 100 days ( p = 0.002 vs. control and p = 0.002 vs. ALS) but did not induce tolerance. Acceptance was associated with marked inhibition of cellular infiltration and inflammatory cytokine expression and only a brief, slight increase in Foxp3:T cell ratio in the graft and no increase in the spleen. In conclusion, G4.18 treatment led to long-term heart transplant survival associated with marked inhibition of early inflammation. Failure to develop tolerance was associated with a lack of early accumulation of Foxp3 cells in the graft or spleen.

413: The Fibrin Derived Peptide B-beta 15-42 Ameliorates Ischemia-Reperfusion Injury after Heart Transplantation in Rats

Ischemia-reperfusion injury is associated with primary non-function and delayed function of transplanted organs. The inflammtory response after ischemia and reperfusion is caused by leukocytes migrating from the bloodstream into the tissue. B-beta15-42 has prompted to reduce leukocytemigration by inhibiting the transmigration process. The purpose of our study was to investigate the protective effect of the fibrin derived peptide B-beta 15-42 on ischemia reperfusion injury in a rat heart transplant model.

363: Intraluminal Bone Marrow Cells Inhibit Allograft Fibrosis Via the Indoleamine Dioxygenase Pathway

Purpose: Bilirubin is a final byproduct of the heme oxygenase system and is known to be a powerful antioxidant. In this study, we examined whether recipient hyperbilirubinemia would protect cardiac grafts from ischemia/reperfusion (I/R) injury and chronic rejection in a rat heart transplantation (HTx) model. Methods and Materials: Heterotopic HTx was performed using congenitally hyperbilirubinemic GUNN (j/j) and normobilirubinemic GUNN ( / ) rats. Serum bilirubin levels of j/j rats were 4.8 mg/dl, while / rats had bilirubin levels 0.1 mg/dl. To evaluate I/R injury, syngenic grafts from / were transplanted into / or j/j rats with 18 hrs cold storage in UW solution. To evaluate the effect on allograft rejection, fully allogenic BN heart grafts were transplanted into / or j/j rats under a short course of tacrolimus (0.5 mg/kg, d0-6). Results: The / grafts in j/j rats resulted in a significantly lower serum CPK level (5492 IU/L) with smaller infarct area determined by TTC stain, compared to those in / recipients 3 hrs after HTx (9617 IU/L). Graft function evaluated by Langendorf apparatus revealed higher left ventricular developed pressure than those in / at 3 hrs after HTx. Tissue MDA levels, a marker of lipid peroxidation, were significantly lower in j/j recipients (16.3 M/g) compared to those in / recipients (39.5 M/g). Graft survival in j/j recipients improved to 41.7% from 0% in / rats and was associated with reduction of the levels of inflammatory mediator mRNA (TNF and IL-6) and phosphorylation of ERK. The mean allograft survival in j/j recipients was prolonged to a median survival of 150 days from 84 days in / recipients. Prolongation of allograft survival was associated with less inflammatory infiltrates and decrease of intragraft inflammatory cytokine mRNA including IFN , TNF and IL-6 at d50. In vitro T cell proliferation was significantly inhibited in the presence of bilirubin. Conclusions: Recipient hyperbilirubinemia ameliorated cardiac I/R injury, as well as chronic allograft rejection following HTx via regulation of inflammatory responses or T cell proliferation.