Sowing "seeds of trust": How trust in normothermic regional perfusion is built in a continuum of care.

Normothermic regional perfusion (NRP) is a promising technology to improve organ transplantation outcomes by reversing ischemic injury caused by controlled donation after circulatory determination of death. However, it has not yet been implemented in Canada due to ethical questions. These issues must be resolved to preserve public trust in organ donation and transplantation. This qualitative, constructivist grounded theory study sought to understand how those most impacted by NRP perceived the ethical implications. We interviewed 29 participants across stakeholder groups of donor families, organ recipients, donation and transplantation system leaders, and care providers. The interview protocol included a short presentation about the purpose of NRP and procedures in abdomen versus chest and abdomen NRP, followed by questions probing potential violations of the dead donor rule and concerns regarding brain reperfusion. The results present a grounded theory placing NRP within a trust-building continuum of care for the donor, their family, and organ recipients. Stakeholders consistently described both forms of NRP as an ethical intervention, but their rationales were predicated on assumptions that neurologic criteria for death had been met following circulatory death determination. Empirical validation of these assumptions will help ground the implementation of NRP in a trust-preserving way.

Circular RNA MAP2K2-modified immunosuppressive dendritic cells for preventing alloimmune rejection in organ transplantation.

Long-term patient and graft survival has been achieved in organ transplantation but at the expense of toxic side effects that are associated with long-term use of nonspecific immunosuppressive drugs. Discovering new regulators of dendritic cells is the key for development of an ideal treatment to prevent immune rejection. We hypothesized that knockdown of circMAP2K2 induces immunosuppressive DCs and that treatment with circMAP2K2 silenced-DCs can prevent alloimmune rejection. DCs were cultured and transfected with siRNA for circMAP2K2. circMAP2K2 levels were measured by qRT-PCR. DC's maturation and immune function were assessed by flow cytometry and mixed lymphocyte reactions. The function of circMAP2K2 was illustrated by a series of RIP and IP. The therapeutics of engineered DCs was tested in a mouse heart transplantation model. We found that circMAP2K2 was highly expressed in mature DCs. Knockdown of circMAP2K2 reduced expression of MHCII, CD40 and CD80, attenuated the ability of DCs to activate allogeneic naïve T cells, and enhanced CD4+CD25+FOXP3+ regulatory T cells (Treg). circMAP2K2-induced immunosuppressive DCs by interacting with SENP3. Treatment with circMAP2K2-knockdown DCs attenuated alloimmune rejection and prolonged allograft survival in a murine heart transplantation model. The immune suppression induced in vivo was donor-antigen specific. In conclusion, knockdown of circMAP2K2 can induce immunosuppressive DCs which are able to inhibit overactive immune response, highlighting a new promising therapeutic approach for immune disorder diseases.

Survival benefit of neoadjuvant FOLFIRINOX for patients with borderline resectable pancreatic cancer.

Backgrounds/Aims: While patients with borderline resectable pancreatic cancer (BRPC) are a target population for neoadjuvant chemotherapy (NAC), formal guidelines for neoadjuvant therapy are lacking. We assessed the perioperative and oncological outcomes in patients with BRPC undergoing NAC with FOLFIRINOX for patients undergoing upfront surgery (US). Methods: The AHPBA criteria for borderline resectability and/or a CA19-9 level > 100 µ/mL defined borderline resectable tumors retrieved from a prospectively populated institutional registry from 2007 to 2020. The primary outcome was overall survival (OS) at 1 and 3 years. A Cox Proportional Hazard model based on intention to treat was used. A receiver-operator characteristics (ROC) curve was constructed to assess the discriminatory capability of the use of CA19-9 > 100 µ/mL to predict resectability and mortality. Results: Forty BRPC patients underwent NAC, while 46 underwent US. The median OS with NAC was 19.8 months (interquartile range [IQR], 10.3-44.24) vs. 10.6 months (IQR, 6.37-17.6) with US. At 1 year, 70% of the NAC group and 41.3% of the US group survived (p = 0.008). At 3 years, 42.5 % of the NAC group and 10.9% of the US group survived (p = 0.001). NAC significantly reduced the hazard of death (adjusted hazard ratio, 0.20; 95% confidence interval, 0.07-0.54; p = 0.001). CA19-9 > 100 µ/mL showed poor discrimination in predicting mortality, but was a moderate predictor of resectability. Conclusions: We found a survival benefit of NAC with FOLFIRINOX for BRPC. Greater pre-treatment of CA19-9 and multivessel involvement on initial imaging were associated with progression of the disease following NAC.

PD-1 and PD-L1 inhibitors in cold colorectal cancer: challenges and strategies.

Colorectal cancer (CRC) is the second most common cause of cancer mortality, with mismatch repair proficient (pMMR) and/or microsatellite stable (MSS) CRC making up more than 80% of metastatic CRC. Programmed death-ligand 1 (PD-L1) and programmed death 1 (PD-1) immune checkpoint inhibitors (ICIs) are approved as monotherapy in many cancers including a subset of advanced or metastatic colorectal cancer (CRC) with deficiency in mismatch repair (dMMR) and/or high microsatellite instability (MSI-H). However, proficient mismatch repair and microsatellite stable (pMMR/MSS) cold CRCs have not shown clinical response to ICIs alone. To potentiate the anti-tumor response of PD-L1/PD-1 inhibitors in patients with MSS cold cancer, combination strategies currently being investigated include dual ICI, and PD-L1/PD-1 inhibitors in combination with chemotherapy, radiotherapy, vascular endothelial growth factor (VEGF) /VEGF receptor (VEGFR) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, and signal transducer and activation of transcription 3 (STAT3) inhibitors. This paper will review the mechanisms of PD-1/PD-L1 ICI resistance in pMMR/MSS CRC and potential combination strategies to overcome this resistance, summarize the published clinical experience with different combination therapies, and make recommendations for future avenues of research.

Liver transplantation with donation after medical assistance in dying: Case series and systematic review of the literature.

Medical assistance in dying (MAiD) has been a legally approved practice in Canada since 2016. Only recently have patients undergoing MAiD also been considered as donors for liver transplantation (LT). This study aimed to evaluate a case series of LT outcomes for recipients with MAiD donors and was paired with a systematic literature review of studies assessing the efficacy of MAiD-associated liver donation. A retrospective chart review of patients registered within the LT Registry at London Health Sciences Centre (LHSC) in London, Ontario, Canada, that had received MAiD donor LT was conducted to develop a case series. Descriptive statistics were produced based on available patient outcomes information. The systematic review included euthanasia due to MAiD being a term exclusive to Canada. Case series had a 100% 1-year graft survival rate, with 50% of patients experiencing early allograft dysfunction but having no significant clinical outcome. A single case of postoperative biliary complication was reported. Median warm ischemic time ranged from 7.8-13 minutes among case series and literature reviews. Utilization of donation after circulatory death allografts procured after MAiD appears to be promising. Mechanisms associated with potential impact in postoperative outcomes include relatively lower warm ischemic time relative to donation after circulatory death Maastricht III graft recipients.

Reducing length of stay in patients following liver transplantation using the model for continuous improvement.

Length of stay (LOS) is a significant contributor to overall patient outcomes for patients undergoing liver transplantation. This study documents a quality improvement project aiming to reduce the median post-transplant LOS for liver transplant patients. We instituted five Plan-Do-Study-Act cycles with the goal of reducing LOS by 3 days from a baseline median of 18.4 days over 1 year. Balancing measures such as readmission rates ensured any decrease in stay was not associated with significantly increased patient complications. Over the 28-month intervention period and 24-month follow-up period, there were 193 patients discharged from hospital with a median LOS of 9 days. The changes appreciated during quality improvement interventions carried over to sustained improvements, with no significant variability in LOS postintervention. Discharge within 10 days increased from 18.4% to 60% over the study period, with intensive care unit stay decreasing from a median of 3.4-1.9 days. Thus, the development of a multidisciplinary care pathway, with patient engagement, led to improved and sustained discharge rates with no significant differences in readmission rates.

Protocol for a qualitative pilot study to explore ethical issues and stakeholder trust in the use of normothermic regional perfusion in organ donation in Canada.

INTRODUCTION: The process of controlled organ donation after circulatory determination of death (cDCDD) results in ischaemic injury to organs and leads to poorer outcomes in organ recipients. Although not yet used in Canada, normothermic regional perfusion (NRP) is a perfusion technology used postmortem with cDCDD donors to selectively restore perfusion of oxygenated blood to target organs in situ, reversing ischaemic injury and improving organ viability and post-transplant outcomes. However, NRP poses significant ethical challenges. To preserve trust in deceased donation, these ethical challenges must be addressed to the satisfaction of Canadian stakeholders before NRP's implementation. This study will identify ethical issues pertaining to NRP and explore perspectives of NRP among key stakeholders. By developing an explanatory framework delineating how stakeholder perceptions of NRP's ethical implications impact trust in Canada's donation and transplantation systems, this study will inform the development of responsible policy on NRP's use in Canada. METHODS AND ANALYSIS: This study includes two workstreams. Workstream 1 is a scoping review of medical and bioethical literature to identify ethical issues stemming from NRP. We will apply a common search string across Medline, PubMed (other than Medline) and Embase to identify relevant articles. We will identify grey literature through Google searches, websites of organ donation organisations and consultation with our research network. No date limits will be applied. All peer-reviewed publications, commentaries, editorials or documents that engage with ethical issues in NRP (or conceptual and empirical issues as they relate to these ethical issues) will be included. News articles, conference abstracts and publications not in English will be excluded. Workstream 2 consists of interviews with healthcare providers, institutional stakeholders, organ recipients and deceased donors' family members (n=24-36), as well as focus groups with healthcare providers involved in deceased donation and transplantation (n=20-32). Constructivist grounded theory methodology will guide data collection and analysis in workstream 2. ETHICS AND DISSEMINATION: This study was approved by Western University's research ethics committee (Western REM; ID: 120001). All participants will be asked to provide written informed consent to participate. Findings will be shared with Canadian organ donation and transplantation organisations, presented at national conferences and published in medical journals.

Tranexamic acid versus placebo to reduce perioperative blood transfusion in patients undergoing liver resection: protocol for the haemorrhage during liver resection tranexamic acid (HeLiX) randomised controlled trial.

INTRODUCTION: Despite use of operative and non-operative interventions to reduce blood loss during liver resection, 20%-40% of patients receive a perioperative blood transfusion. Extensive intraoperative blood loss is a major risk factor for postoperative morbidity and mortality and receipt of blood transfusion is associated with serious risks including an association with long-term cancer recurrence and overall survival. In addition, blood products are scarce and associated with appreciable expense; decreasing blood transfusion requirements would therefore have health system benefits. Tranexamic acid (TXA), an antifibrinolytic, has been shown to reduce the probability of receiving a blood transfusion by one-third for patients undergoing cardiac or orthopaedic surgery. However, its applicability in liver resection has not been widely researched. METHODS AND ANALYSIS: This protocol describes a prospective, blinded, randomised controlled trial being conducted at 10 sites in Canada and 1 in the USA. 1230 eligible and consenting participants will be randomised to one of two parallel groups: experimental (2 g of intravenous TXA) or placebo (saline) administered intraoperatively. The primary endpoint is receipt of blood transfusion within 7 days of surgery. Secondary outcomes include blood loss, postoperative complications, quality of life and 5-year disease-free and overall survival. ETHICS AND DISSEMINATION: This trial has been approved by the research ethics boards at participating centres and Health Canada (parent control number 177992) and is currently enrolling participants. All participants will provide written informed consent. Results will be distributed widely through local and international meetings, presentation, publication and ClinicalTrials.gov. TRIAL REGISTRATION NUMBER: NCT02261415.

Hepatitis C positive organ transplantation to negative recipients at a multiorgan Canadian transplant centre: ready for prime time.

BACKGROUND: Transplantation offers the best survival for patients with end stage organ disease. Transplant of hepatitis C virus (HCV) nucleic acid test (NAT) positive organs into negative recipients is a novel strategy that can expand the donor pool. We aim to evaluate our centre's experience. METHODS: We preformed a retrospective review of anti-HCV NAT positive and negative organs into negative recipients transplanted over 27 months. Primary outcome was the success rate of eradication of HCV post-transplant. Secondary outcomes were rate of transmission of HCV, treatment adverse events, and graft failure. RESULTS: 33 anti-HCV positive organs were transplanted into negative recipients. 22 (66.7%) were NAT positive. Median recipients age was 49 years (interquartile range [IQR] 44.5-62.0) with the majority being males (57.6%). NAT positive organ transplantations included 16 kidneys, 3 livers, 1 kidney-pancreas, 1 liver-kidney, and 1 heart. The most common HCV genotype was 1a (59.1%). The median time to initiating therapy was 41.5 days. SVR12 was 100% in patients who finished therapy. There were no adverse events with therapy and no graft failure. CONCLUSIONS: Anti-HCV NAT positive organ transplantation into negative recipients is safe with excellent eradication rates and no significant adverse events or graft failure. This would expand donor pool to close the gap between supply and demand.

PATCH-DP: a single-arm phase II trial of intra-operative application of HEMOPATCH™ to the pancreatic stump to prevent post-operative pancreatic fistula following distal pancreatectomy.

BACKGROUND: Post-operative pancreatic fistula (POPF) is the most significant cause of morbidity following distal pancreatectomy. Hemopatch™ is a thin, bovine collagen-based hemostatic sealant. We hypothesized that application of Hemopatch™ to the pancreatic stump following distal pancreatectomy would decrease the incidence of clinically-significant POPF. METHODS: We conducted a prospective, single-arm, multicentre phase II study of application of Hemopatch™ to the pancreatic stump following distal pancreatectomy. The primary outcome was clinically-significant POPF within 90 days of surgery. A sample size of 52 patients was required to demonstrate a 50% relative reduction in Grade B/C POPF from a baseline incidence of 20%, with a type I error of 0.2 and power of 0.75. Secondary outcomes included incidence of POPF (all grades), 90-day mortality, 90-day morbidity, re-interventions, and length of stay. RESULTS: Adequate fixation Hemopatch™ to the pancreatic stump was successful in all cases. The rate of grade B/C POPF was 25% (95%CI: 14.0-39.0%). There was no significant difference in the incidence of grade B/C POPF compared to the historical baseline (p = 0.46). The 90-day incidence of Clavien-Dindo grade ≥3 complications was 26.9% (95%CI: 15.6-41.0%). CONCLUSION: The use of Hemopatch™ was not associated with a decreased incidence of clinically-significant POPF compared to historical rates. (NCT03410914).

Kidney transplant outcomes after medical assistance in dying.

INTRODUCTION: After nearly four years of Canadian experience with medical assistance in dying (MAID), the clinical volume of organ transplantation following MAID remains low. This is the first Canadian report evaluating recipient outcomes from kidney transplantation following MAID. METHODS: This was a retrospective review of the first nine cases of kidney transplants following MAID at a Canadian transplant center. RESULTS: Nine patients underwent MAID followed by kidney retrieval during the study period. Their diagnoses were largely neuromuscular diseases. The mean warm ischemic time was 20 minutes (standard deviation [SD] 7). The nine recipients had a mean age of 60 (SD 19.7). The mean cold ischemic time was 525 minutes (SD 126). Delayed graft function occurred in only one patient out of nine. The mean 30-day creatinine was 124 umol/L (SD 52). The mean three-month creatinine was 115 umol/L (SD 29). CONCLUSIONS: We report nine cases of kidney transplantation following MAID. The process minimized warm ischemia, resulting in low delayed graft function rates, and acceptable post-transplant outcomes. Further large-scale research is necessary to optimize processes and outcomes in this novel clinical pathway.

Paving the Road for the Adoption of Normothermic Regional Perfusion in Canada.

Donation after circulatory determination of death has increased the number of organs available but can result in worse recipient outcomes than organs recovered from donors after neurologic death. Normothermic regional perfusion is a novel tool that can circumvent the shortcomings of donation after circulatory determination of death. However, its implementation may pose a threat to existing laws surrounding death declaration. Here, we propose a research agenda that will allow this technology to be introduced within current Canadian organ donation frameworks.

Recurrent Hepatocellular Carcinoma After Liver Transplantation: Validation of a Pathologic Risk Score on Explanted Livers to Predict Recurrence.

BACKGROUND: Recurrence of hepatocellular carcinoma (HCC) after liver transplantation is a major cause of morbidity and mortality. To date, there is no widely accepted pathologic assessment tool to predict HCC recurrence. In 2007, we developed a pathologic risk score that stratified patients into low, intermediate, or high risk for recurrence based on explant pathology. The aim of this study was to externally validate this risk score. METHODS: We retrospectively evaluated 124 patients over a 10-year period who underwent liver transplantation for HCC. Using explanted pathology reports, each patient was stratified according to the pathologic risk score and followed over time for HCC recurrence. RESULTS: Recurrence occurred in 15 patients (12%) after a mean follow-up of 25 months. Using the pathologic risk score, 10 (8%), 21 (17%), and 93 (75%) patients were stratified into high, intermediate, and low risk of recurrence, respectively. Among these risk groups, recurrence occurred in 50%, 28.5%, and 4.3% (P < .01) of patients, respectively. Using the optimal cutoff value ≤3.5, our risk score had a sensitivity of 80% and specificity of 79% with an area under the receiver operator characteristic curve of 0.8. Those with lower risk scores had higher recurrence-free survival (P < .0001). CONCLUSIONS: Our pathologic risk score accurately risks stratified patients for HCC recurrence after liver transplant. It can be used to tailor surveillance strategies for those deemed to be at elevated risk for recurrence.

Preventing alloimmune rejection using circular RNA FSCN1-silenced dendritic cells in heart transplantation.

BACKGROUND: While heart transplantation is used as a standard treatment for heart failure, transplant rejection continues to pose a challenge. Recent evidence has shown that circular RNA (circRNA) is a new type of gene regulator in cell development. Our aim was to demonstrate that treatment with tolerogenic dendritic cells (Tol-DCs) generated by circular RNA FSCN1 (circFSCN1) silencing could prevent alloimmune rejection and prolong heart graft survival in heart transplantation. METHODS: Bone marrow-derived DCs were transfected with circFSCN1 siRNA in vitro. The circFSCN1 level was measured by qRT-PCR. DC maturation was determined by flow cytometry. Mixed lymphocyte reactions (MLRs) were conducted to assess the function of DCs to activate T cells and to generate regulatory T cells (Tregs). In situ RNA hybridization and fluorescent microscopy were performed to detect the distribution of circFSCN1 in DCs. A heterotopic allogeneic murine heart transplantation was conducted where recipients were pre-treated with donor derived circFSCN1-silenced Tol-DCs. Heartbeat was monitored to assess immune rejection. RESULTS: Exonic circFSCN1 was highly expressed in the cytoplasm of mature DCs. Knockdown of circFSCN1 using siRNA arrested DCs at an immature state, impaired DC's ability to activate T cells and enhanced Treg generation. Treatment with circFSCN1-silenced Tol-DCs prevented alloimmune rejection, prolonged allograft survival, reduced fibrosis, and induced Tregs in vivo. CONCLUSIONS: Knockdown of circFSCN1 induces Tol-DCs and treatment with these Tol-DCs prevents alloimmune rejection and prolongs allograft survival. This is a promising therapeutic target to combat transplant rejection in heart transplantation and increases our understanding of circRNA in the immune system.

Chronic stress physically spares but functionally impairs innate-like invariant T cells.

The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a "split" inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.

Liver transplantation in adult polycystic liver disease: the Ontario experience.

BACKGROUND: Liver transplantation (LT) remains the curative treatment for symptomatic Polycystic Liver Disease (PCLD) patients and is associated with excellent survival rates. The aim of the study is to review the Ontario experience in LT for PCLD. METHODS: A retrospective study was performed from pre-existing LT databases from the LT Units at Toronto General Hospital and London Health Sciences Center, which are the two LT programs in Ontario, Canada. This database contains demographic, clinical parameters and follow-up of all patients transplanted for PCLD. Data was extracted for patients who underwent LT between January 2000-April 2017 and included follow up until December 31st, 2018. RESULTS: A total of 3560 patients underwent LT, of whom 51 (1.4%) had PCLD and met inclusion criteria. 43 (84%) of these patients were female. The median physiologic Model for End Stage Liver Disease (MELD-Na) score at time of referral was 13 (IQR = 7-22), however all patients required MELD-Na exception points to receive LT. The median age of transplant was 62 years (IQR = 59-64) for male vs. 52 (IQR = 45-56) for female patients. 33 (65%) of our cohort had PCLD while 9 (17.5%) had ADPKD and 9 (17.5%) had both diseases. 39 (76%) had LT due to symptoms of mass effect, while 8 (16%) had portal hypertensive complications. After a median follow-up of 6.3 (IQR = 2.9-12.5) years, the probability of survival was 96% (95% CI: 90%, 100%). Log-rank test, comparing survival analysis between males and females did not show a statistically significant difference (p = 0.26). CONCLUSION: Most patients underwent LT for PCLD due to symptoms of mass effect with women being more likely than men to undergo LT. LT for PCLD had excellent long-term survival.

Pre-operative stenting and complications following pancreatoduodenectomy for pancreatic cancer: an analysis of the ACS-NSQIP registry.

BACKGROUND: Historically, pre-operative biliary stenting has been associated with higher infectious complication rates following pancreatoduodenectomy. However, alleviation of biliary obstruction is necessary for consideration of pre-operative chemotherapy, which may improve disease-free survival, or for mitigation of symptoms while awaiting surgery. Our aim is to compare contemporary post-operative complication risk among patients with pre-operative endoscopic retrograde cholangiopancreatography (ERCP) stenting compared to those without. METHODS: Patients who underwent a pancreatoduodenectomy for pancreatic cancer with biliary obstruction within the ACS-NSQIP registry from 2014 to 2017 were identified. The primary outcome was to compare the risk of 30-day complication (composite outcome) between patients with and without pre-operative ERCP stenting. Propensity score matching was used to ensure balanced baseline characteristics and log-binomial regression models were used to estimate risk ratios for overall perioperative complication between groups. RESULTS: From 6073 patients with obstructive jaundice undergoing pancreatoduodenectomy for pancreatic cancer, 92% (5564) were eligible for the study. After performing a propensity score matching on 20 baseline characteristics, 952 patients without stenting were matched to up to four patients who received pre-operative ERCP stenting (n = 3467) for a matched cohort of 4419. A total of 1901 (55%) patients with pre-operative ERCP stenting experienced a post-operative complication compared to 501 (53%) patients without stenting (risk ratio 1.04, 95% CI 0.97-1.11, p = 0.23). CONCLUSION: Pre-operative ERCP stenting was not associated with an increased risk of post-operative complication in patients undergoing pancreatoduodenectomy with obstructive jaundice. Biliary stenting may be safely considered for symptom relief and to potentially facilitate pre-operative chemotherapy for pancreatic cancer.

An Addition of U0126 Protecting Heart Grafts From Prolonged Cold Ischemia-Reperfusion Injury in Heart Transplantation: A New Preservation Strategy.

BACKGROUND: Ischemia-reperfusion injury (IRI) is the major cause of primary graft dysfunction in organ transplantation. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in cell physiological and pathological processes including IRI. This study aims to investigate whether inhibition of ERK signaling with U0126 can prevent prolonged cold IRI in heart transplantation. METHODS: Rat cardiac cell line H9c2 cells were treated with U0126 before exposure to hypothermic hypoxia/reoxygenation (H/R) conditions. The effect of U0126 on H9c2 cells in response to H/R stress was determined by measuring cell death, reactive oxygen species production, mitochondrial membrane potential, and ERK signaling activation. Mouse syngeneic heterotopic heart transplantation was conducted, where a donor heart was preserved in the University of Wisconsin (UW) solution supplemented with U0126 for 24 hours at 4°C before transplantation. Heart graft function, histopathologic changes, apoptosis, and fibrosis were measured to assess IRI. RESULTS: Phosphorylated ERK was increased in both in vitro H/R-injured H9c2 cells and in vivo heart grafts with IRI. Pretreatment with U0126 inhibited ERK phosphorylation and prevented H9c2 cells from cell death, reactive oxygen species generation, and mitochondrial membrane potential loss in response to H/R. Preservation of donor hearts with U0126-supplemented solution improved graft function and reduced IRI by reductions in cell apoptosis/death, neutrophil infiltration, and fibrosis of the graft. CONCLUSIONS: Addition of U0126 to UW solution reduces ERK signal activation and attenuates prolonged cold IRI in a heart transplantation model. ERK inhibition with U0126 may be a useful strategy to minimize IRI in organ transplantation.