Hours Of Cold Ischemia Research Articles

Amputated Digit Replantations: Critical Digit Ischemia Timing, Temperature, and Other Predictors of Survival.

A common consideration for replantation success is the ischemia time following injury and the preservation temperature. A classic principle within the hand surgery community describes 12 hours of warm ischemia and 24 hours of cold ischemia as the upper limits for digit replantation; however, these limits are largely anecdotal and based on older studies. We aimed to compare survival data from the large body of literature to aid surgeons and all those involved in the replantation process in hopes of optimizing success rates. The PubMed database was queried on April 4th, 2023, for articles that included data on digit replantation survival in terms of temperature of preservation and ischemia time. All primary outcomes were analyzed with the Mantel-Haenszel method within a random effects model. Secondary outcomes were pooled and analyzed using the chi-square statistic. Statistical analysis and forest plot generation were completed with RevMan 5.4 software with odds ratios calculated within a 95% confidence interval. Our meta-analysis identified that digits preserved in cold ischemia for over 12 hours had significantly higher odds of replantation success than the amputated digits replanted with 0-12 hours of warm ischemia time ( P ≤ 0.05). The odds of survival in the early (0-6 hours) replantation group were around 40% greater than the later (6-12 hours) replantation group ( P ≤ 0.05). Secondary outcomes that were associated with higher survival rates included a clean-cut amputation, increased venous and arterial anastomosis, a repair that did not require a vein graft, and replants performed in nonsmokers ( P ≤ 0.05). Overall, these findings suggest that when predicting digit replantation success, time is of the essence when the digit has yet to be preserved in a cold environment. This benefit, however, is almost completely diminished when the amputated digit is appropriately maintained in a cold environment soon after injury. In conclusion, our results suggest that there is potential for broadening the ischemia time limits for digit replant survival outlined in the literature, particularly for digits that have been stored correctly in cold ischemia.

Mid-term and long-term outcomes following heart transplantation with prolonged cold ischemia

Objective: comparative analysis of long-term outcomes following heart transplantation (HT) with prolonged and short cold ischemia.Materials and methods. We analyzed the data of 29 orthotopic HT with >4 hours of cold ischemia. The transplant surgery was performed at Meshalkin National Medical Research Center between 2013 and the present time. Organs were obtained from donors from other regions. The control group consisted of 29 HTs with cold ischemia <4 hours, performed in the same period. The minimum distance between the transplant center and the donor base was about 250 km (Barnaul); the maximum distance was about 850 km (Krasnoyarsk). Recipient survival and postoperative peculiarities were analyzed.Results. In-hospital survival in the prolonged cold ischemia group was 89.7% (n = 26) with 3 deaths (10.3%). In the second group (<240 min), in-hospital survival was 79.3% (n = 23) with 6 (20.7%) deaths. The Kaplan-Meier survival analysis showed no difference between the groups (Log-Rank Test, P 1/4 0.59). In addition, cold ischemia time did not increase the risk of graft rejection and the risk of transplant coronary artery disease (TCAD).Conclusion. HT with cold ischemia >4 hours did not have worse outcomes than in short graft ischemia. This provides grounds for further accumulation of experience in the use of heart donors from remote locations.

344.3: Extracellular Vesicles Released During Normothermic Machine Perfusion Are Associated With Human Donor Kidney Characteristics

Background: Extracellular Vesicles (EVs) represent stable, tissue specific nano-sized particles that reflect the conditional state of their tissue of origin. Here, the dynamic release and phenotype of kidney EVs was characterized and quantified during Normothermic Machine Perfusion (NMP) of Expanded-Criteria Donor (ECD) kidneys to examine whether EVs could function as a potential biomarker for assessing kidney quality before transplantation. Materials & Methods: Eight discarded ECD kidneys (~13 ± 5 hours of cold ischemia, age 68 ± 7 (mean ± standard deviation), all male) were perfused in a closed system at 37 0C for 6 hours. Perfusates were taken before and at 1, 3 and 6 hours of NMP and examined with Nanoparticle Tracking Analysis (NTA) and Imaging Flow Cytometry (IFCM). For IFCM, perfusates were stained with the tetraspanins CD9, CD63 or CD81 (general EV markers), or a mix of these three markers in combination with CFDA-SE (a non-fluorescent molecule that acquires fluorescent properties after cleavage by intravesicular esterases) to identify, quantify and characterize EVs. Results: Analysis of perfusates with NTA revealed that the majority of nanoparticles present in the perfusates are <300 nm. For CFSE and the mix of tetraspanin double-positive EVs, we observed a ~700/ 740/ 560 fold increase compared to EV levels before perfusion at 1, 3 and 6 hours of NMP, respectively. Analysis of EV concentrations with crude donor characteristics (e.g. age, cold ischemia time (CIT), kidney weight) and NMP viability characteristics (renal flow, renal flow resistance, urine production) revealed that double-positive EV are negatively correlated with CIT whilst positive correlations were found with donor age after the first hour of NMP. Furthermore, tetraspanin CD81 was found to represent the majority (~75%) of the excreted double-positive EV (CD9: ~16%/ CD63 ~8%) (Figure). Conclusion: EVs <300 nm are released by ECD kidneys during NMP with highest excretion levels during the first hour of perfusion. Tetraspanin CD81 is predominantly present on these EVs, and EV concentrations were shown to be correlated with well-established indicators of kidney quality such as donor age and CIT. The characterization of the excreted EVs as well as their correlation with clinical parameters provide a starting point to study their role as potential biomarkers of kidney quality.

Is the Organ Care System (OCS) Still the First Choice With Emerging New Strategies for Donation After Circulatory Death (DCD) in Heart Transplant?

The scarcity of donor hearts continues to be a challenge in transplants for advanced heart failure patients. With an increasing number of patients on the waiting list for a heart transplant, the discrepancy in the number between donors and recipients is gradually increasing and poses a new challenge that plagues the healthcare systems when it comes to the heart. Several technologies have been developed to expand the donor pool in recent years. One such method is the organ care system (OCS). The standard method of organ preservation is the static cold storage (SCS) method which allows up to four hours of safe preservation of the heart. However, beyond four hours of cold ischemia, the incidence of primary graft dysfunction increases significantly. OCS keeps the heart perfused close to the physiological state beyond the four hours with superior results, which allows us to travel further and longer distances, leading to expansion in the donor pool. In this review, we discuss the OCS system, its advantages, and shortcomings.

Luminal preloading with hydrogen‐rich saline ameliorates ischemia‐reperfusion injury following intestinal transplantation in rats

Prolonged intestinal cold storage causes considerable mucosal breakdown, which could bolster bacterial translocation and cause life-threatening infection for the transplant recipient. The intestine has an intraluminal compartment, which could be a target for intervention, but has not yet been fully investigated. Hydrogen gas exerts organ protection and has used been recently in several clinical and basic research studies on topics including intestinal transplantation. In this study, we aimed to investigate the cytoprotective efficacy of intraluminally administered hydrogen-rich saline on cold IR injury in intestinal transplantation. Isogeneic intestinal transplantation with 6hours of cold ischemia was performed on Lewis rats. Hydrogen-rich saline (H2 concentration at 5ppm) or normal saline was intraluminally introduced immediately before preservation. Graft intestine was excised 3hours after reperfusion and analyzed. Histopathological analysis of control grafts revealed blunting of the villi and erosion. These mucosal changes were notably attenuated by intraluminal hydrogen. Intestinal mucosa damage caused by IR injury led to considerable deterioration of gut barrier function 3h post-reperfusion. However, this decline in permeability was critically prevented by hydrogen treatment. IR-induced upregulation of proinflammatory cytokine mRNAs such as IL-6 was mitigated by hydrogen treatment. Western blot revealed that hydrogen treatment regulated loss of the transmembrane protein ZO-1. Hydrogen-rich saline intraluminally administered in the graft intestine modulated IR injury to transplanted intestine in rats. Successful abrogation of intestinal IR injury with a novel strategy using intraluminal hydrogen may be easily clinically applicable and will compellingly improve patient care after transplantation.

Protective Effects of a Hydrogen-Rich Preservation Solution in a Canine Lung Transplantation Model

Molecular hydrogen (H2) has protective effects against ischemia-reperfusion injury in various organs. Because they are easier to transport and safer to use than inhaled H2, H2-rich solutions are suitable for organ preservation. In this study, we examined the protective effects of an H2-rich solution for lung preservation in a canine left lung transplantation (LTx) model. Ten beagles underwent orthotopic left LTx after 23 hours of cold ischemia followed by reperfusion for 4 hours. Forty-five minutes after reperfusion, the right main pulmonary artery was clamped to evaluate the function of the implanted graft. The beagles were divided into two groups: control group (n= 5), and H2 group (n= 5). In the control group, the donor lungs were flushed and immersed during cold preservation at 4°C using ET-Kyoto solution, and in the H2 group, these were flushed and immersed using H2-rich ET-Kyoto solution. Physiologic assessments were performed during reperfusion. After reperfusion, the wet-to-dry ratios were determined, and histology examinations were performed. Significantly higher partial pressure of arterial oxygen and significantly lower partial pressure of carbon dioxide were observed in the H2 group than in the control group (P= .045 and P < .001, respectively). The wet-to-dry ratio was significantly lower in the H2 group than in the control group (P= .032). Moreover, in histology examination, less lung injury and fewer apoptotic cells were observed in the H2 group (P < .001 and P < .001, respectively). Our results demonstrated that the H2-rich preservation solution attenuated ischemia-reperfusion injury in a canine left LTx model.

Bronchial-arterial-circulation-sparing Lung Preservation: A New Organ Protection Approach for Lung Transplantation.

Compromised microvasculature resulting from disrupted bronchial arterial circulation appears to trigger chronic lung allograft dysfunction. Maintaining the microvasculature throughout the transplant process could improve the long-term health of transplanted lungs. We recently developed a bronchial-arterial-circulation-sparing (BACS) lung preservation approach and tested whether this approach would decrease microvascular damage and improve allograft function. The lungs of Lewis rats were procured using either the BACS approach, where the bronchial and pulmonary arteries were synchronously perfused; a conventional approach, where only the pulmonary artery was perfused; or a conventional approach with a prostaglandin flush. After 4 hours of cold ischemia, physiologic function and vascular tone of the grafts were evaluated during ex vivo lung perfusion (EVLP), and microvasculature damage was assessed using 2-photon microscopy analysis. Lung function was compared after transplant among the groups. After 4 hours of cold ischemia, the BACS group exhibited significantly higher adenosine triphosphate levels and lower expression of phosphorylated myosin light chain, which is essential for vascular smooth muscle contraction. On EVLP, the BACS and prostaglandin groups showed lower pulmonary vascular resistance and less arterial stiffness. BACS attenuated microvasculature damage in the lung grafts when compared with conventional preservation. After transplantation, the lungs preserved with the BACS approach exhibited significantly better graft function and lower expression of phosphorylated myosin light chain. Our data suggest that BACS lung preservation protects the dual circulation inherent to the lungs, facilitating robust microvasculature in lung grafts after transplantation, leading to better posttransplant outcomes.

Prolonged Cold Ischemia Induces Necroptotic Cell Death in Ischemia-Reperfusion Injury and Contributes to Primary Graft Dysfunction after Lung Transplantation.

Primary graft dysfunction (PGD) is a major cause of morbidity and mortality after lung transplantation. Ischemia-reperfusion injury (IRI) is a key event that contributes to PGD, though complex interactions affect donor lungs status, such as preceding brain death (BD), hemorrhagic shock (HS), and pre-engraftment lung management, the latter recognized as important risk factors for PGD. We hypothesized that a multi-hit isogenic mouse model of lung transplantation is more closely linked to PGD than IRI alone. Left lung transplants were performed between inbred C57BL/6 mice. A one-hit model of IRI was established by inducing cold ischemia (CI) of the donor lungs at 0°C for 1, 72, or 96 hours before engraftment. Multi-hit models were established by inducing 24 hours of HS and/or 3 hours of BD before 24 hours of CI. The recipients were killed at 24 hours after transplant and lung graft samples were analyzed. In the one-hit model of IRI, up to 72-hour CI time resulted in minimal cellular infiltration near small arteries after 24-hour reperfusion. Extension of CI time to 96 hours led to increased cellular infiltration and necroptotic pathway activation, without evidence of apoptosis, after 24-hour reperfusion. In a multi-hit model of PGD, "HS + BD + IRI" demonstrated increased lung injury, cellular infiltration, and activation of necroptotic and apoptotic pathways compared with IRI alone. Treatment with an inhibitor of receptor-interacting protein kinase 1 kinase, necrostatin-1, resulted in a significant decrease of downstream necroptotic pathway activation in both single- and multi-hit models of IRI. Thus, activation of necroptosis is a central event in IRI after prolonged CI, though it may not be sufficient to cause PGD alone. Pathological evaluation of donor lungs after CI-induced IRI, in conjunction with pre-engraftment donor lung factors in our multi-hit model, demonstrated early evidence of lung injury consistent with PGD. Our findings support the premise that pre-existing donor lung status is more important than CI time alone for inflammatory pathway activation in PGD, which may have important clinical implications for donor lung retrieval.

The Key Role of Warm and Cold Ischemia in Uterus Transplantation: A Review.

Introduction: Uterus transplantation (UTx) is a promising treatment for uterine infertility that has resulted in several births since 2014. Ischemia is a key step in organ transplantation because it may lead to changes jeopardizing graft viability. Method: We performed a systematic review of animal and human studies relating to uterine ischemia. Results: We retained 64 studies published since 2000. There were 35 studies in animals, 24 in humans, and five literature reviews. Modest preliminary results in large animals and humans are limited but encouraging. In small animals, pregnancies have been reported to occur after 24 h of cold ischemia (CI). In ewes, uterine contractions have been detected after 24 h of CI. Furthermore, it has been shown in animals that uterine tolerance to CI and to warm ischemia (WI) can be increased by pharmacological products. In women, mean CI time in studies of births from uteri obtained from live donors was between 2 h 47 min and 6 h 20 min from a deceased donor; with only one birth in this case. Muscle contractions have also been demonstrated in myometrial samples from women, after six or more hours of CI. Conclusion: The uterus seems to be able to tolerate a prolonged period of CI, of at least six hours. Studies of the ischemia tolerance of the uterus and ways to improve it are essential for the development of UTx, particularly for procedures using grafts from deceased donors.

Abstract 17: Increasing Ischemia Time Diminishes Chimerism After Vascularized Bone Marrow Allotransplantation

PURPOSE: Vascularized composite allotransplantation (VCA) can include vascularized bone marrow (VBM) to potentially generate chimerism and subsequent tolerance in the recipient. While increasing ischemia time is known to be detrimental to free flaps and solid organ transplants, it is unknown whether increasing VCA ischemia time may diminish VBM’s capacity to achieve chimerism and tolerance. This study determines whether increasing cold ischemia time results in decreased peripheral chimerism levels, increasing loss of the donor bone marrow compartment, and increased skin paddle rejection in a murine VCA model. METHODS: Twenty-three adult Lewis rats received osteomyocutaneous hind limb allografts from 3 groups of Brown-Norway rats. The groups underwent 0, 6, and 12 hours of cold ischemia at 4°C (n=8, 6, and 9 respectively). Immunosuppressive conditioning consisted of anti-lymphocyte serum 1cc 3 days pre-operatively and on post-op day (POD) 1, tacrolimus 2mg/kg/day for PODs 1–7, and then concluded with rapamycin 3mg/kg/day for PODs 8–28. Peripheral chimerism levels were drawn after POD 30. Skin paddle was observed for 30 days after immunosuppression cessation for rejection or tolerance. Allograft was collected at POD 60 for flow cytometry and histologic analysis. RESULTS: Peripheral chimerism levels in both myeloid and lymphoid lines were decreased in the 12 & 6 hours groups versus 0 hours (p<0.01). At POD 60, the amount of donor cells in the allograft bone marrow was significantly decreased at 12 & 6 hours versus the 0 hours group (p<0.05). At 12 hours, hematoxylin & eosin histology demonstrated significantly increased reactive lymphocytes, increased stroma, significantly decreased hematopoetic cells, and significant myelofibrosis. At 6 hours, moderate stromal increases, moderately decreased hematopoetic cells, and moderate myelofibrosis was observed. Trichrome staining revealed viable bone at all time points though with there were increasing amounts of collagen deposition with increasing ischemia and osteoslerosis was noted at 12 hours. Microvasculature was preserved at all time points. By POD 60, 0% of skin paddles survived in the 12 hours group, 17% at 6 hours, and 73% at 0 hours. CONCLUSION: With increasing ischemia time prior to VCA re-vascularization, there is decreased peripheral chimerism levels, increased loss of donor hematopoetic cells in the donor bone marrow, and increased skin paddle rejection. These findings indicate that ischemia time may significantly limit the capacity for VBM to facilitate chimerism and subsequent VCA tolerance and thus strategies using VBM to induce tolerance may need to consider the additional obstacle of time. Prolonged ischemia time may be a contributing factor to the absence of stable chimerism and tolerance in human VBM-bearing VCA recipients.

Rescue of Discarded Grafts for Liver Transplantation by Ex Vivo Subnormothermic and Normothermic Oxygenated Machine Perfusion: First Experience in Spain

BackgroundEx vivo machine perfusion (MP) has been reported as a possibly method to rescue discarded organs. The main aim of this study was to report an initial experience in Spain using MP for the rescue of severely marginal discarded liver grafts, and to, secondarily, define markers of viability to test the potential applicability of these devices for the real increase in the organ donor pool. MethodsThe study began in January 2016. Discarded grafts were included in a research protocol that consisted of standard retrieval followed by 10 hours of cold ischemia. Next, either normothermic (NMP) or controlled subnormothermic (subNMP) rewarming was chosen randomly. Continuous measurements of portal-arterial pressure and resistance were screened. Lactate, pH, and bicarbonate were measured every 30 minutes. The perfusion period was 6 hours, after which the graft was discarded and evaluated as potentially usable, but never implanted. Biopsies of the donor and at 2, 4, and 6 hours after ex vivo MP were obtained. ResultsA total of 4 grafts were included in the protocol. The first 2 grafts were perfused by NMP and grafts 3 and 4 by subNMP. The second and third grafts showed a clear trend toward optimal recovery and may have been used. Lactate dropped to levels below 2.5 mmol/L with stable arterial and portal pressure and resistance. Clear biliary output started during MP. Biopsies showed an improvement of liver architecture with reduced inflammation at the end of the perfusion. ConclusionThis preliminary experience has demonstrated the potential of MP devices for the rescue of severely marginal liver grafts. Lactate and biliary output were useful for viability testing of the grafts. The utility of NMP or subNMP protocols requires further research.

Mechanisms of Bone Morphogenetic Protein-7 Protective Effects Against Cold Ischemia-Induced Renal Injury in Rats

Deceased donor kidneys are exposed to cold ischemic insult which makes them particularly susceptible to the effects of cold ischemic injury during hypothermic preservation resulting in high rates of delayed graft function. Bone morphogenetic protein-7 (BMP-7) is a valuable reagent in the field of tissue regeneration and preservation under ischemic conditions. Following these insights, we investigated the effect of recombinant human BMP-7 (rhBMP-7) on graft preservation during cold ischemia.The study was conducted on an experimental model of kidney cold ischemia in rats. Kidneys were perfused with University of Wisconsin (UW) saline solution, rhBMP-7, or rhBMP-7 + UW, and exposed to cold ischemia for 6, 12, and 24 hours.In tubular epithelial cells of kidneys perfused with rhBMP-7 and rhBMP-7+UW solution, the expression of BMP-7 and E-cadherin was observed after 24 hours of cold ischemia. In kidneys not perfused with rhBMP-7, high expression of transforming growth factor-β and α-smooth muscle actin was found. Also, in kidneys perfused with rhBMP-7 solution, statistically higher levels of Smad1, Smad5, and Smad8 messenger RNA expressions were proven.BMP-7 maintains the morphology of kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells.

A New Transport Solution for Parathyroid Allotransplantation: Effects on Cell Viability and Calcium-Sensing Receptors.

Cold ischemia protects organs and tissues by slowing their metabolism, but it also causes ischemic injury. Minimizing cold ischemia has been an important goal in parathyroid auto- and allotransplantation, as well as the transplantation of other major organs. Parathyroid glands are responsible for calcium homeostasis by releasing parathormone (PTH) into the blood circulation. Functionality of a new parathyroid transport solution (NPTS) and effects on cell viability, PTH secretion, and calcium-sensing receptor (CaSR) levels during cold ischemia were evaluated. A NPTS was prepared, and the pH was adjusted to a range of 7.2-7.4 and kept at +4°C until use. Seven patients with parathyroid hyperplasia secondary to chronic renal failure who were scheduled to undergo subtotal parathyroidectomy were enrolled in the study. Glands were cold-preserved in NPTS with different time intervals (0, 6, 12, 18, and 24 hours), and then parathyroid cell viability before and after cryopreservation, PTH secretion, and CaSR levels were determined. The mean cell viability before cryopreservation was 92.7% (range 89.2%-97.2%). There were no significant differences in cell viability rates before and after cryopreservation (p = 0.1168 and p = 0.4085, respectively), and CaSR levels (p = 0.5446) were not significant. NPTS is a solution designed specifically for parathyroid tissue transplantation. This patent pending product can support cellular viability and PTH release, as well as protect CaSR functionality for up to 24 hours of cold ischemia.

The Impact of Implantation Time During Liver Transplantation on Outcome: A Eurotransplant Cohort Study.

The liver graft quickly rewarms during transplantation when the vascular anastomoses are being performed, potentially impacting on outcomes. We investigated the relationship between implantation time and outcome in 5223 recipients of deceased-donor livers transplanted in Eurotransplant (2004-2013). Cox regression analyses were corrected for donor, preservation, and recipient variables. Transplant loss represents all-cause graft failure. Median implantation time was 41 minutes (interquartile range, 34-51). Implantation time independently associated with transplant loss (adjusted hazard ratio, 1.04 for every 10-minute increase; 95% confidence interval, 1.01-1.07; P = 0.007). The magnitude of the implantation time effect was comparable to the effect of each additional hour of cold ischemia (adjusted hazard ratio, 1.03; 95% confidence interval, 1.02-1.05; P < 0.001). The effect was most pronounced early posttransplant with no evidence of a significant effect beyond 3 months. A similar detrimental effect of implantation time was seen for graft and patient survivals. The increased risk for transplant loss in livers donated after circulatory determination of death could be attributed to donor warm ischemia time. Implantation time associates with inferior liver transplant outcome in a continuous way. These findings need confirmation and further study of confounding factors is needed so steps toward improving outcomes can be made.

Cold ischemia time is an important risk factor for post-liver transplant prolonged length of stay.

Risk analysis of cold ischemia time (CIT) in liver transplantation has largely focused on patient and graft survival. Posttransplant length of stay is a sensitive marker of morbidity and cost. We hypothesize that CIT is a risk factor for posttransplant prolonged length of stay (PLOS) and aim to conduct an hour-by-hour analysis of CIT and PLOS. We retrospectively reviewed all adult, first-time liver transplants between March 2002 and September 2016 in the United Network for Organ Sharing database. The 67,426 recipients were categorized by hourly CIT increments. Multivariate logistic regression of PLOS (defined as >30 days), CIT groups, and an extensive list of confounding variables was performed. Linear regression between length of stay and CIT as continuous variables was also performed. CIT 1-6 hours was protective against PLOS, whereas CIT >7 hours was associated with increased odds for PLOS. The lowest odds for PLOS were observed with 1-2 hours (odds ratio [OR], 0.65; 95% confidence interval [CI], 0.45-0.92) and 2-3 hours (OR, 0.65; 95% CI, 0.55-0.78) of CIT. OR for PLOS steadily increased with increasing CIT, reaching the greatest odds for PLOS with 13-14 hours (OR, 2.05; 95% CI, 1.57-2.67) and 15-16 hours (OR, 2.06; 95% CI, 1.27-3.33) of CIT. Linear regression revealed a positive correlation between length of stay and CIT with a correlation coefficient of +0.35 (P < 0.001). In conclusion, post-liver transplant length of stay is sensitive to CIT, with a substantial increase in the odds of PLOS observed with nearly every additional hour of cold ischemia. We conclude that CIT should be minimized to protect against the morbidity and cost associated with posttransplant PLOS. Liver Transplantation 24 762-768 2018 AASLD.

Increasing circulating sphingosine-1-phosphate attenuates lung injury during ex vivo lung perfusion

BackgroundSphingosine-1-phosphate regulates endothelial barrier integrity and promotes cell survival and proliferation. We hypothesized that upregulation of sphingosine-1-phosphate during ex vivo lung perfusion would attenuate acute lung injury and improve graft function. MethodsC57BL/6 mice (n = 4-8/group) were euthanized, followed by 1 hour of warm ischemia and 1 hour of cold preservation in a model of donation after cardiac death. Subsequently, mice underwent 1 hour of ex vivo lung perfusion with 1 of 4 different perfusion solutions: Steen solution (Steen, control arm), Steen with added sphingosine-1-phosphate (Steen + sphingosine-1-phosphate), Steen plus a selective sphingosine kinase 2 inhibitor (Steen + sphingosine kinase inhibitor), or Steen plus both additives (Steen + sphingosine-1-phosphate + sphingosine kinase inhibitor). During ex vivo lung perfusion, lung compliance and pulmonary artery pressure were continuously measured. Pulmonary vascular permeability was assessed with injection of Evans Blue dye. ResultsThe combination of 1 hour of warm ischemia, followed by 1 hour of cold ischemia created significant lung injury compared with lungs that were immediately harvested after circulatory death and put on ex vivo lung perfusion. Addition of sphingosine-1-phosphate or sphingosine kinase inhibitor alone did not significantly improve lung function during ex vivo lung perfusion compared with Steen without additives. However, group Steen + sphingosine-1-phosphate + sphingosine kinase inhibitor resulted in significantly increased compliance (110% ± 13.9% vs 57.7% ± 6.6%, P < .0001) and decreased pulmonary vascular permeability (33.1 ± 11.9 μg/g vs 75.8 ± 11.4 μg/g tissue, P = .04) compared with Steen alone. ConclusionsTargeted drug therapy with a combination of sphingosine-1-phosphate + sphingosine kinase inhibitor during ex vivo lung perfusion improves lung function in a murine donation after cardiac death model. Elevation of circulating sphingosine-1-phosphate via specific pharmacologic modalities during ex vivo lung perfusion may provide endothelial protection in marginal donor lungs leading to successful lung rehabilitation for transplantation.

The cardioprotective effect of interleukin-11 against ischemia-reperfusion injury in a heart donor model.

Previously, we have demonstrated the cardioprotective effect of interleukin (IL)-11 in animal models of acute coronary syndrome. In this study, we sought to evaluate its cardioprotective potential during prolonged hypothermic global ischemia and subsequent reperfusion using a rat heart donor model. IL-11 was administered intravenously 10 minutes before harvesting the rat heart. The hearts were preserved in cold (4 °C) Krebs-Henseleit buffer for 6 hours, and then attached to a Langendorff perfusion apparatus and reperfused with an oxygenated Krebs-Henseleit solution containing IL-11. Normal saline was used instead of IL-11 in the control group. Functional recovery of the reperfused heart was observed by using a left ventricular balloon. Myocardial cell injury was quantified by measuring the biomarkers collected from the coronary effluent. Apoptotic cells were identified and counted using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining technique. IL-11 administration improved myocardial function after 6 hours of cold ischemia. Although there were no significant differences in any of the baseline-measured values between the two groups, left ventricular developed pressure (LVDP) and changes in left ventricular pressures (dP/dt) were significantly higher in the IL-11 group at 120-minute reperfusion. The number of TUNEL-labeled cardiomyocytes was also significantly smaller in the IL-11 group. The administration of IL-11 showed a significant recovery of cardiac contractile function after 6 hours of cold ischemia. Our data suggest that it may have significant therapeutic potential for maintaining the functional viability of the heart exposed to prolonged hypothermic global ischemia.

Anti-CD47 monoclonal antibody therapy reduces ischemia-reperfusion injury of renal allografts in a porcine model of donation after cardiac death.

We investigated whether blockade of the CD47 signaling pathway could reduce ischemia-reperfusion injury (IRI) of renal allografts donated after cardiac death (DCD) in a porcine animal model of transplantation. Renal allografts were subjected to 30minutes of warm ischemia, 3.5hours of cold ischemia, and then perfused with a humanized anti-CD47 monoclonal antibody (CD47mAb) in the treatment group or HTK solution in the control group (n=4/group). The animals were euthanized fivedays after transplantation. At the time of reperfusion, indocyanine green-based in vivo imaging showed that CD47mAb-treated organs had greater and more uniform reperfusion. On post-transplant days 3-5, the treatment group had lower values compared to the control for creatinine and blood urea nitrogen. Histological examination of allograft tissues showed a significant decrease of acute tubular injury in the CD47mAb-treated group compared to control. Compared to the control group, CD47mAb treatment significantly decreased genes expression related to oxidative stress (sod-1, gpx-1, and txn), the inflammatory response (il-2, il-6, inf-g, and tgf-b), as well as reduced protein levels of BAX, Caspase-3, MMP2, and MMP9. These data demonstrate that CD47mAb blockade decreases IRI and subsequent tissue injury in DCD renal allografts in a large animal transplant model.

Comparison of normothermic and hypothermic perfusion in porcine kidneys donated after cardiac death

BackgroundNormothermic machine perfusion (NMP) is an alternative strategy for preserving kidneys donated after cardiac death (DCD). The relative efficacy of prolonged NMP compared to hypothermic machine perfusion (HMP) in DCD kidneys with moderate ischemic injury is undetermined. This study compares NMP and HMP kidney preservation in a porcine DCD model. MethodsTen porcine kidneys underwent NMP or HMP preservation following 45 minutes of warm ischemia and 5 hours of cold ischemia. After 8 hours of machine preservation, hemodynamic stability, renal function, perfusate biomarkers, and histologic integrity were assessed in a simulated reperfusion model. ResultsDuring simulated reperfusion, no differences were observed in oxygen consumption, urine production, creatinine clearance, fractional excretion of sodium, proteinuria, and perfusate levels of lactate dehydrogenase and aspartate aminotransferase. Resistance was no different after 30 minutes of simulated reperfusion. Histologically, NMP kidneys demonstrated increased vacuolization after preservation and greater loss of tubular integrity after simulated reperfusion. Perfusate levels of alkaline phosphatase (AP) and gamma glutamyltransferase (GGT) were higher in NMP kidneys during preservation, but upon simulated reperfusion, AP and GGT levels were higher in HMP-preserved kidneys. Peak AP and GGT during simulated reperfusion of HMP kidneys were over 14 times higher than peak AP and GGT during preservation of NMP kidneys. ConclusionsNMP provided comparable preservation of renal function as HMP and minimized AP and GGT release upon reperfusion.

Anti-inflammatory effects on ischemia/reperfusion-injured lung transplants by the cluster of differentiation 26/dipeptidylpeptidase 4 (CD26/DPP4) inhibitor vildagliptin

ObjectivesWe showed previously that stromal cell−derived factor 1 (SDF-1) is a substrate of cluster of differentiation 26/dipeptidylpeptidase 4 (CD26/DPP4) and exerts regenerative properties on acute lung ischemia-reperfusion injury on CD26/DPP4 inhibition. Here, we extend our studies to test whether an intermediate recovery of lung transplants from ischemia/reperfusion injury by CD26/DPP4 inhibition can be achieved for up to 14 days. MethodsSyngeneic mouse lung transplantation (Tx) was performed in C57BL/6 and in CD26−/− mice by applying 18 hours of cold ischemia. Donor lungs were preconditioned with saline or the CD26/DPP4 inhibitor vildagliptin (1 μg/mL [3 μM]). In vitro, the influence of vildagliptin and SDF-1 on the macrophage cell line RAW 264.7 was tested. Transplants were analyzed up to 14 days after Tx for the expression of SDF-1, tumor necrosis factor-α (TNF-α), interleukin-10, intercellular adhesion molecule-1 (ICAM-1), immune cell infiltration, and oxygenation. ResultsCold ischemic time of 18 hours with vildagliptin preconditioning elevated lung SDF-1 levels (P = .0011) and increased interleukin-10–producing macrophages (P = .0165) compared with the control. SDF-1 reduced macrophage-derived TNF-α (P = .0248) in vitro. Five hours after Tx, vildagliptin significantly reduced macrophages and neutrophils (P = .0306), decreased ICAM-1 expression (P = .002), and improved transplant oxygenation (P = .0181). Seven days after Tx, grafts were preserved on CD26/DPP4-inhibition: perivascular macrophages (P = .0046) and TNF-α (P = .0013) were reduced as well as T and B cells. ICAM-1 was absent in CD26/DPP4-inhibited grafts at all time points. ConclusionsThis study proves an intermediate improvement of ischemia/reperfusion-injured lung transplants by the CD26/DPP4-inhibitor vildagliptin up to 14 days. Enhanced levels of SDF-1 induced an anti-inflammatory effect on a cellular and protein level, and render CD26/DPP4 inhibition preconditioning effective for the protection from lung ischemia/reperfusion injury.