Experimental Heart Transplantation Research Articles

Treatment with RNase alleviates brain injury but not neuroinflammation in neonatal hypoxia/ischemia.

There is a need for new treatments to reduce brain injuries derived from neonatal hypoxia/ischemia. The only viable option used in the clinic today in infants born at term is therapeutic hypothermia, which has a limited efficacy. Treatments with exogenous RNase have shown great promise in a range of different adult animal models including stroke, ischemia/reperfusion injury, or experimental heart transplantation, often by conferring vascular protective and anti-inflammatory effects. However, any neuroprotective function of RNase treatment in the neonate remains unknown. Using a well-established model of neonatal hypoxic/ischemic brain injury, we evaluated the influence of RNase treatment on RNase activity, gray and white matter tissue loss, blood-brain barrier function, as well as levels and expression of inflammatory cytokines in the brain up to 6 h after the injury using multiplex immunoassay and RT-PCR. Intraperitoneal treatment with RNase increased RNase activity in both plasma and cerebropinal fluids. The RNase treatment resulted in a reduction of brain tissue loss but did not affect the blood-brain barrier function and had only a minor modulatory effect on the inflammatory response. It is concluded that RNase treatment may be promising as a neuroprotective regimen, whereas the mechanistic effects of this treatment appear to be different in the neonate compared to the adult and need further investigation.

PHENOMENON OF DEMIKHOV. Heart transplantation in experiment and clinical cases in the USSR and abroad (1968–1972). The Second in the USSR heart transplantation in a Clinic (Solovyev G.M., June 10, 1971)

The article presents materials of Soviet and foreign medical literature of 1968–1972 devoted to heart transplantation in experiment and clinical practice. It is shown that in the USSR after unsuccessful heart transplantation performed by A.A. Vishnevsky on November 4, 1968, experimental studies on isolated heart preservation in order to preserve its viability were conducted; models of orthotopic and heterotopic heart transplantation on large and small animals were created; morphological, physiological, biochemical and immunological changes in the transplanted heart were studied. The second heart transplantation in this country was performed by G.M. Solovyev on June 10, 1971, but it was also unsuccessful. It’s remarkable that the 20-year experience of V.P. Demikhov in experimental heart transplantation was used only partially: a number of surgeons applied the cardiopulmonary complex isolated according to V.P. Demikhov's technique for biological heart preservation in experimental animals. At the same time after the successful heart transplantation performed by C. Barnard on December 3, 1967, the world boom of clinical transplantation began. In 1969, 101 such operations were performed. For example, D. Cooley performed them on 21 patients and on another one performed two surgeries. There were observations that patients operated on in 1968 lived 800–900 days or more. The best results were shown by R. Lower, M. DeBakey and N. Shumway. However, the vast majority of heart transplants resulted in lethal outcomes in the immediate or distant postoperative period. This led to the fact that the euphoria of successful transplants gradually began to diminish: in 1969 only 47 operations were performed, and in 1970–1971 only 17 surgeries per year.

Recipient sex and estradiol levels affect transplant outcomes in an age-specific fashion

Sex-specific influences have been shown for a variety of diseases. Whether donor or recipient sex and sex hormone levels impact alloimmune responses remains unclear. In unifactorial and multifactorial analyses of more than 400 000 SRTR listed kidney transplant patients, we found that younger female recipients had an inferior death-censored graft survival that was independent of donor sex. In contrast, graft survival was superior in older female recipients, suggesting the impact of recipient sex hormones over chromosomal sex mismatches. Those clinical changes were delineated in experimental skin and heart transplant models showing a prolongation of graft survival in ovariectomized young female recipients. In contrast, graft survival was comparable in ovariectomized and naïve old female recipients. Young ovariectomized mice showed reduced amounts and a compromised T cell proliferation. Deprivation of female hormones dampened the production of interferon (IFN)-γ and interleukin (IL)-17+ by CD4+ T cells while augmenting systemic counts of Tregs. Increasing estradiol concentrations in vitro promoted the switch of naïve CD4+ T cells into Th1 cells; high physiological estradiol concentrations dampening Th1 responses, promoted Tregs, and prolonged graft survival. Thus, clinical observations demonstrate age-specific graft survival patterns in female recipients. Estrogen levels, in turn, impact the fate of T cell subsets, providing relevant and novel information on age- and sex-specific alloimmunity.

PHENOMENON OF DEMIKHOV. In the Sklifosovsky Institute (1960–1986). C.N. Barnard and the first clinical heart transplantation (December 3, 1967). V.P. Demikhov and C.N. Barnard: touchpoints

Having studied the available printed, visual, and verbal sources from Russia, South Africa, the USA, and Germany, we have identified and reviewed in the article the facts of face-to-face and correspondence communication between V.P. Demikhov, the "father" of experimental heart transplantation (Moscow, USSR), and C.N. Barnard, a pioneer of clinical heart transplantation (Cape Town, South Africa). We have shown that C.N. Barnard mastered the heart surgery techniques, including those under conditions of artificial circulation, in the USA in 1956-1958, and later improved them in his homeland both in clinic (heart surgery for cardiac defects), and in the experiment (heart transplantation). The main events preceding the first world human heart transplant performed by C.N. Barnard on December 3, 1967, were his trip to the United States in August 1967 to study immunosuppression techniques, and the kidney transplantation he had performed in Cape Town in September, 1967. Prior to that time, C.N. Barnard had visited the USSR only once, in May 1960, as a delegate to the XXVII All-Union Congress of Surgeons. In the Soviet Union, he visited a number of clinics dealing with heart surgery and tissue and organ transplantation, including the N.V. Sklifosovsky Institute for Emergency Medicine, where he met V.P. Demikhov, but C.N. Barnard could neither talk to him personally, nor watch his operations. In December 1967, V.P. Demikhov spoke with C.N. Barnard on the phone, but the conversation was highly professional. This paper has shown different approaches of V.P. Demikhov and C.N. Barnard to the transplantation problem: the Soviet surgeon paid more attention to the transplantation technique, meanwhile, the South African surgeon considered the solution of immunological problems to be the basis of success. Nevertheless, C.N. Barnard knew about V.P. Demikhov's scientific achievements and used some of them in his surgical practice. The authors have substantiated the interaction between V.P. Demikhov and C.N. Barnard as between an ideological mentor and a student (in a broad sense) rather than as a teacher and a student (in a narrow sense). Therefore, in a broad, philosophical sense, the Soviet surgeon can be considered one of the inspirers of the world's first heart transplantation, which, in turn, proved that his ascetic work was not in vain.

Methane supplementation improves graft function in experimental heart transplantation

Maintenance of cell viability during cold storage is a key issue in organ transplantation. Methane (CH4) bioactivity has recently been recognized in ischemia/reperfusion conditions; we therefore hypothesized that cold storage in CH4-enriched preservation solution can provide an increased defense against organ dysfunction during experimental heart transplantation (HTX). The hearts of donor Lewis rats were stored for 60 minutes in cold histidine-tryptophan-ketoglutarate (Custodiol [CS]) or CH4-saturated CS solution (CS-CH4) (n = 12 each). Standard heterotopic HTX was performed, and 60 minutes later, the left ventricular (LV) pressure-volume relationships LV systolic pressure (LVSP), systolic pressure increment (dP/dtmax), diastolic pressure decrement, and coronary blood flow (CBF) were measured. Tissue samples were taken to detect proinflammatory parameters, structural damage (by light microscopy), endoplasmic reticulum (ER) stress, and apoptosis markers (CCAAT/enhancer binding protein [C/EBP] homologous protein, GRP78, glycogen synthase kinase-3β, very low-density lipoprotein receptor, caspase 3 and 9, B-cell lymphoma 2, and bcl-2-like protein 4), whereas mitochondrial functional changes were analyzed by high-resolution respirometry. LVSP and dP/dtmax increased significantly at the largest pre-load volumes in CS-CH4 grafts as compared with the CS group (114.5 ± 16.6 mmHg vs 82.8 ± 4.6 mmHg and 3,133 ± 430 mmHg/s vs 1,739 ± 169 mmHg/s, respectively); the diastolic function and CBF (2.4 ± 0.4 ml/min/g vs 1.3 ± 0.3 ml/min/g) also improved. Mitochondrial oxidative phosphorylation capacity was more preserved (58.5 ± 9.4 pmol/s/ml vs 27.7 ± 6.6 pmol/s/ml), and cytochrome c release was reduced in CS-CH4 storage. Signs of HTX-caused myocardial damage, level of ER stress, and the transcription of proapoptotic proteins were significantly lower in CS-CH4 grafts. The addition of CH4 during 1 hour of cold storage improved early in vitro graft function and reduced mitochondrial dysfunction and activation of inflammation. Evidence shows that CH4 reduced ER stress-linked proapoptotic signaling.

The Role of IL-33 in Experimental Heart Transplantation.

Interleukin-33 (IL-33) is a member of the IL-1 family of proteins that are produced by a variety of cell types in multiple tissues. Under conditions of cell injury or death, IL-33 is passively released from the nucleus and acts as an “alarmin” upon binding to its specific receptor ST2, which leads to proinflammatory or anti-inflammatory effects depending on the pathological environment. To date, numerous studies have investigated the roles of IL-33 in human and murine models of diseases of the nervous system, digestive system, pulmonary system, as well as other organs and systems, including solid organ transplantation. With graft rejection and ischemia-reperfusion injury being the most common causes of grafted organ failure or dysfunction, researchers have begun to investigate the role of IL-33 in the immune-related mechanisms of graft tolerance and rejection using heart transplantation models. In the present review, we summarize the identified roles of IL-33 as well as the corresponding mechanisms by which IL-33 acts within the progression of graft rejection after heart transplantation in animal models.

Knockdown of NEAT1 induces tolerogenic phenotype in dendritic cells by inhibiting activation of NLRP3 inflammasome.

Rationale: Tolerogenic dendritic cells (tol-DCs) play essential roles in immune-related diseases and induce immune tolerance by shaping T-cell responses. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) play important regulatory roles in the immune system. However, the potential roles and underlying mechanisms of lncRNAs in tol-DCs remain unclear.Methods: RNA in-situ hybridization, histochemistry, and qRT-PCR were performed to determine the distribution and expression of NEAT1 in DCs. Flow cytometry was used to analyze the tolerogenic function of DCs. Small sequencing, followed by bioinformatic analysis, was performed to determine the target genes of NEAT1. The mechanism of NEAT1 was explored using a luciferase reporter, chromatin immunoprecipitation assays, and Immunofluorescence. In-vivo experiments were used to investigate the induction of immune tolerance via NEAT1-knockdown DCs.Results: Our results show that lncRNA NEAT1 can induce tolerogenic phenotype in DCs. Mechanistically, small RNA-seq analysis revealed that NEAT1 knockdown preferentially affected the expression of miR-3076-3p. Furthermore, NEAT1 used the NLRP3 inflammasome as a molecular decoy for miR-3076-3p, thus facilitating the expression of tolerogenic phenotype in DCs. Moreover, the transcription factor E2F1 acted as a repressor of NEAT1 transcription via activity of H3K27ac. Our results also indicate that NEAT1 knockdown in DCs can induce immune tolerance in models of experimental autoimmune myocarditis and heart transplantation.Conclusions: Taken together, our study shows the mechanism used by NEAT1 in inducing tol-DCs and highlights the therapeutic potential of targeting NEAT1 for the treatment of immune-related diseases.

Targeting Trained Immunity Promotes Organ Transplant Acceptance.

Myeloid cell functional reprogramming represents a compelling therapeutic approach to induce immunological tolerance, but remains unexplored clinically. We developed a short-term nanoimmunotherapy, based on mammalian target of rapamycin (mTOR) involving drug-loaded high-density lipoprotein (HDL) nanoparticles that target macrophages and prevent epigenetic modifications associated with trained immunity. Using an experimental heart transplantation mouse model, we found that inhibition of aerobic glycolysis in myeloid cells through systemic administration of mTOR-specific nanoimmunotherapy (mTORi-HDL) prevents macrophages’ trained immunity phenotype and induces accumulation of regulatory macrophages. Remarkably, short-term mTORi-HDL treatment in combination with an inhibitory CD40- TRAF6 specific nanoimmunotherapy (TRAF6i-HDL) resulted in indefinite allograft survival. Together, these findings demonstrate that HDL-based nanoimmunotherapy provides an innovative approach to control macrophage function in vivo and promote immunological tolerance.

T2 Mapping for Noninvasive Assessment of Interstitial Edema in Acute Cardiac Allograft Rejection in a Mouse Model of Heterotopic Heart Transplantation.

Heart transplantation (HTX) in mice is used to characterize gene-deficient mice and to test new treatment strategies. The purpose was to establish noninvasive magnetic resonance imaging techniques in mice to monitor pathophysiological changes of the allograft during rejection. Magnetic resonance imaging was performed at baseline and days 1 and 6 after isogenic (n = 10, C57BL/6) and allogenic (n = 12, C57BL/6 to BALB/c) heterotopic HTX on a 7 T small animal scanner. Respiratory- and electrocardiogram-gated multislice multi-echo spin echo sequences were acquired, and parameter maps of T2 relaxation time were generated. T2 times in septal, anterior, lateral, and posterior myocardial segments as well as global T2 times were calculated and compared between groups. At day 7 animals were sacrificed and graft pathology was assessed by semiquantitative regional analysis and correlated with magnetic resonance imaging results. Myocardial T2 relaxation time was significantly increased in allogenic (33.4 ± 0.1 ms) and isogenic cardiac grafts (31.8 ± 1.8 ms) on day 1 after HTX compared with healthy donor hearts at baseline (23.1 ± 0.3 ms, P < 0.001). Until day 6 after HTX, myocardial T2 further increased markedly in allografts but not in isografts (43.4 ± 1.9 vs 31.2 ± 1.1 ms, P < 0.001). Mean segmental T2 values as well as mean global T2 values in allogenic compared with isogenic cardiac grafts on day 6 were significantly higher (P < 0.01). Histologically, isogenic grafts were almost normal besides small focal leukocyte infiltrates and signs of interstitial edema, most likely due to ischemia reperfusion injury (histological sum score, 0.9 ± 0.4). In allogenic HTX, histology revealed severe inflammation and tissue edema representing allograft rejection with increased histological scores (5.3 ± 0.7, P < 0.001). Higher histological scores of rejection were significantly associated with increased T2 times on a segmental and a global level. We could show that T2 mapping is a suitable noninvasive imaging method to monitor global and regional HTX pathologies in experimental heart transplantation in mice. Progressive prolongation of T2 time was significantly associated with pathological signs of rejection.

Disturbances in iron homeostasis result in accelerated rejection after experimental heart transplantation

Clinical data suggest that iron disturbances deleteriously affect graft survival after heart transplantation (HTx), but immunological mechanisms underlying this phenomenon have not yet been elucidated. To identify the mechanistic influence of iron in a murine model of HTx, fully allogeneic BALB/c donor organs were transplanted into iron-overloaded or iron-deficient C57BL/6 mice, and recipients were analyzed for functional and immunological parameters. After HTx, iron overload accelerated acute rejection as observed by shortened graft survival (HTx vs HTx + iron; p = 0.01), elevated rejection score (p < 0.01), and induction of troponin T (p < 0.01). Compared with controls, allografts and recipient spleens derived from iron-overloaded recipients were characterized by a pronounced graft infiltration of CD4+ T cells (p < 0.01), CD3-NKp46+ natural killer cells (p < 0.05), and reduced frequencies of regulatory T cells (p < 0.01). This was accompanied by lower mRNA expression levels of anti-inflammatory cytokines, including interleukin-10, transforming graft factor-β, and Foxp3. Cardiac allograft survival was further tested under co-stimulation blockade (CTLA4-Ig) showing that naïve grafts transplanted into iron-overloaded recipients illustrated restricted graft outcome compared with wild types (p = 0.0051), which was rescued after treatment with the iron chelator deferoxamine. Iron deficiency (ID) also resulted in enhanced intragraft infiltration of inflammatory cells and accelerated rejection in the acute setting (HTx vs HTx + ID; p = 0.02) and after co-stimulation blockade (p = 0.0059). We provide novel insights into the understanding of disturbances in iron homeostasis and their consequences after HTX, allowing novel insights regarding improvements in personalized immunosuppression to prolong allograft survival.

Innate immune mechanisms in transplant allograft vasculopathy.

Allograft vasculopathy is the leading cause of late allograft loss following solid organ transplantation. Ischemia reperfusion injury and donor-specific antibody-induced complement activation confer heightened risk for allograft vasculopathy via numerous innate immune mechanisms, including MyD88, high-mobility group box 1 (HMGB1), and complement-induced noncanonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The role of MyD88, a signal adaptor downstream of the Toll-like receptors (TLR), has been defined in an experimental heart transplant model, which demonstrated that recipient MyD88 enhanced allograft vasculopathy. Importantly, triggering receptor on myeloid receptor 1, a MyD88 amplifying signal, was present in rejecting human cardiac transplant biopsies and enhanced the development of allograft vasculopathy in mice. HMGB1, a nuclear protein that activates Toll-like receptors, also enhanced the development of allograft vasculopathy. Complement activation elicits assembly of membrane attack complexes on endothelial cells which activate noncanonical NF-κB signaling, a novel complement effector pathway that induces proinflammatory genes and potentiates endothelial cell-mediated alloimmune T-cell activation, processes which enhance allograft vasculopathy. Innate immune mediators, including HMGB1, MyD88, and noncanonical NF-κB signaling via complement activation contribute to allograft vasculopathy. These pathways represent potential therapeutic targets to reduce allograft vasculopathy after solid organ transplantation.

Roles of CXCL9 and CXCL10 in acute rejection after retransplantation in mice

To observe the effects of chemokines CXCL9 and CXCL10 on cardiac allograft acute rejection mediated by alloreactive memory T cells in a retransplantation model. Heart transplantation was performed 6 weeks after skin grafting. The mice were divided into 3 groups of control (direct heterotopic heart transplantation without skin grafting); experimental (heart transplantation after skin grafting) and syngraft (C57BL/6→C57BL/6, heterotopic heart transplantation) (n = 12 each). Graft survival and the pathological changes of cardiac graft were observed. And related gene expression in cardiac grafts and serum concentration of CXCL9/CXCL10 were detected. The mean survival time of control and experimental groups was 7.75 and 3.25 days respectively (P < 0.01).Serum concentrations of CXCL9 and CXCL10 in recipient mice were higher in the experimental group than those in the control group. Compared with the control group, the relative gene expressions of CXCL9 and CXCL10 were higher in the experimental group. According to pathological examinations, the histological rank of cardiac allograft was Grade 2.27 ± 0.25 in the control group versus Grade 4.12 ± 0.03 in the experimental group (P < 0.01). CXCL9 and CXCL10 play critical roles in retransplantation mediated by alloreactive memory T cells. And acute rejection of cardiac allograft is more extensive in retransplantation.

New Immunosuppression in Experimental Heart and Lung Transplantation: Selective JAK1/3 Inhibition Diminishes Acute Cardiac Rejection and Prevents Obliterative Airway Disease

New Immunosuppression in Experimental Heart and Lung Transplantation: Selective JAK1/3 Inhibition Diminishes Acute Cardiac Rejection and Prevents Obliterative Airway Disease

Graft Function After Heterotopic Rat Heart Transplant With an Isolated Reperfused Working Heart: A Methodic Consideration

Assessment of graft function in experimental cardiac transplant has been underused by insufficient methods (echo-cardiography, magnetic resonance imaging). The isolated reperfused working-heart model is an excellent tool for hemodynamic evaluation of rodent hearts. So far, it has never been used in a cardiac transplant setting. Our study tries to combine the in vivo technique of a rat heart transplant model with the ex vivo method of an isolated working heart. Heterotopic heart transplants have been performed in rats with a nonsuture cuff technique. After 8 hours of cold ischemia and 24 hours of reperfusion, grafts were mounted on the working heart. To assess graft function, cardiac output was measured with increasing levels of afterload pressure. Nontransplanted hearts were mounted directly to the working heart apparatus to serve as a control group. Each heart was assessed subjectively by the Stanford score before being mounted on the working-heart apparatus. The working-heart assessment detected significantly impaired graft function in the transplant group compared with control hearts. In contrast, functional assessment with the score-system could not detect any difference between transplanted and native hearts. The isolated working-heart model is an excellent tool for assessing graft function after experimental heart transplant in rodents.

Melanocortins as potential therapeutic agents in severe hypoxic conditions

Melanocortin peptides with the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) sequences and synthetic analogs have protective and life-saving effects in experimental conditions of circulatory shock, myocardial ischemia, ischemic stroke, traumatic brain injury, respiratory arrest, renal ischemia, intestinal ischemia and testicular ischemia, as well as in experimental heart transplantation. Moreover, melanocortins improve functional recovery and stimulate neurogenesis in experimental models of cerebral ischemia. These beneficial effects of ACTH/MSH-like peptides are mostly mediated by brain melanocortin MC(3)/MC(4) receptors, whose activation triggers protective pathways that counteract the main ischemia/reperfusion-related mechanisms of damage. Induction of signaling pathways and other molecular regulators of neural stem/progenitor cell proliferation, differentiation and integration seems to be the key mechanism of neurogenesis stimulation. Synthesis of stable and highly selective agonists at MC(3) and MC(4) receptors could provide the potential for development of a new class of drugs for a novel approach to management of severe ischemic diseases.

Heart transplantation: Research that led to the first human transplant in 1967

The first experimental heart transplantation was performed by Demikhov in Moscow in the very early 1940s. His experiments were discontinued during World War II but, when they resumed in 1946, he produced a large volume of work devising 24 different methods of heterotopic heart transplantation within the chest. During the 1950s, many research workers transplanted the heart in the orthotopic position and also transplanted the heart and lungs en bloc. They used various methods, including cardiopulmonary bypass (heart- lung machine). During the 1960s, Lower and Shumway established beyond doubt that heart transplantation was a possibility. Christiaan Barnard, using their surgical techniques, performed the first human- to-human heart transplant, in December 1967 in Cape Town. Early transplantation attempts 1,2 He noted the behaviour of autografts, allografts and xenografts, and believed allografts could have a successful outcome. Corneal transplants were carried out as far back as 1872 using xenografts, but were unsuccessful. Allograft corneal grafts, however, were the first successful tissues to be transplanted, the earliest being around 1930. Organ transplantation was first attempted in the early 20th century. Previously, Murphy and Beck had devised vascular suturing methods, and these were used by Carrel in transplanting organs. Carrel observed that kidneys transplanted from one site to another in the same animal had a better survival rate than those from other animals. He believed, however, that the functioning of the transplanted organ depends on the success of the vascular anastomosis. This thinking persisted until the 1940s, with many researchers spending all their time and effort devising better and faster techniques for the anastomoses of vessels. Some human application of these techniques was attempted. Ullman, in 1902, having experimented with kidney allograft transplantation in dogs, transplanted a pig's kidney into the elbow of a uraemic woman. The kidney did not function and he thought this was due to technical problems. At that time, many other research workers reported experimental kidney transplantation, some of whom also attempted transplantation from animals to man. The overall experience was reviewed by Guthrie in 1912, who summed up the situation as follows: 'No one, though many experiments have been reported, has yet succeeded in keeping an animal alive for any great length of time which carried the kidney or kidneys of another animal, after its own kidneys were removed … the outlook is by no means hopeless and the principle of immunity which yields such brilliant results in many other fields would be worthy of being tested in this case'. 1

Partial loading of heterotopic heart transplants results in preserved left ventricular function and morphology

Objectives: Whether partial volume load (VL) in experimental heterotopic heart transplantation (hHTx) is superior to non-loaded (NL) models remains controversial. In this study we demonstrate that an established model of loading transplanted hearts physiologically can preserve left ventricular function and morphology.

Letter by Deutsch and Kaczmarek Regarding Article, “Donor Pretreatment With Hypertonic Saline Attenuates Primary Allograft Dysfunction: A Pilot Study in a Porcine Model”

To the Editor: We read with great interest the article by Badiwala et al1 describing donor pretreatment with hypertonic saline as being effective in attenuating primary allograft dysfunction in an experimental porcine heart transplant model. The positive influence of hypertonic saline on enhanced ventricular recovery after storage-related ischemia-reperfusion injury and subsequent transplantation was ascribed to an improvement of early hemodynamics and beneficial immunomodulatory and vasculomodulatory effects.1 A work published by Harada and associates2 might explain the early positive hemodynamic effects. Postischemic dysfunction after short-term global ischemia in isolated blood-perfused dog hearts was caused by a decreased responsiveness of the contractile elements to intracellular …

Vitamin E action on oxidative state, endothelial function and morphology in long-term myocardial preservation.

This study assesses the effects of a vitamin E analogue, Trolox, on the oxidative state, endothelial function and morphology in experimental heart transplantation. Heterotopic heart transplantation was carried out in pigs: untreated after 2 and 24 hours of ischemia and treated with Trolox after 24 hours of ischemia. Prolonged preservation of donor hearts was achieved with continuous perfusion and University of Wisconsin solution, in which acid-base balance and enzymes were determined during the procedure. In recipients, hemodynamic and biochemical parameters were determined at baseline and during reperfusion. Trolox diminished the pH of the preservation solution (p<0.01), the left ventricle of the transplanted heart recovered a systolic pressure equaling that of the 2h group and higher than that of the untreated 24h group (p<0.01), the antioxidant levels were not decreased and the glutathione reductase level was maintained throughout the first part of reperfusion. In this group also there was a direct correlation between the concentration of this enzyme and the antioxidant levels (p<0.001). Although the endothelin concentrations increased, the change was less marked in the Trolox group than in the untreated 24h group (p<0.01). Morphologically, mitochondria and myocardial vessels presented a normal structure in the Trolox group, and interstitial edema, inflammatory infiltrate and contraction bands were less prominent than in the untreated group. All these effects indicate that Trolox protected the transplanted heart, at least partially, against ischemia-reperfusion injury.

Perfluorocarbon Improves Post-Transplant Survival and Early Kidney Function following Prolonged Cold Ischemia

Background: The two-layer organ preservation method (TLM) based on oxygenated perfluorocarbon overlaid with University of Wisconsin (UW) solution has been successfully used in clinical islet and experimental heart and intestine transplantation. We tested whether this technique would prevent tissue damage and improve kidney function in a model of syngeneic kidney transplantation with prolonged ischemia time. Methods: Kidneys were stored for 24 h either in UW solution (n = 16), with TLM (n = 16) or transplanted immediately (control group, n = 12). In half of the animals, survival was observed and in the other animals grafts were procured for semiquantitative histological scoring and TUNEL apoptosis assessment 24 h after transplantation. Results: One-month survival rates in the UW, TLM and control groups were 12.5, 62.5 and 100%, respectively (UW vs. TLM, p < 0.01). Median creatinine levels 24 h after transplantation were 381, 299 and 121 µM, respectively (UW vs. TLM, p < 0.02). Histological scoring showed more severe tissue damage in the UW group than in the TLM group (p < 0.05). Apoptosis was more frequent in the UW group than in the TLM group (p < 0.05). Conclusion: We demonstrated for the first time that conservation with TLM significantly improves the outcome of kidney transplantation in a rat model and should therefore be further studied in larger animals.