Focus on Ex situ/Ex vivo Perfusion
Facilitators and Barriers of Ex Situ Heart Perfusion (ESHP) in Pediatric Donation (20 minutes presentation + 10 minutes Q&A)
Purpose: Despite medical advances, pediatric heart transplant (HTx) candidates are at significant risk for morbidity and mortality. Ex Situ Heart Perfusion (ESHP) is a method for continuous perfusion of the donor heart, allowing for extended out-of-body time and functional assessment of the organ. Currently available devices are targeted to adults and have been used in the context of neurological and circulatory determination of death. Pediatric-specific devices are in development, but before adoption, knowledge of stakeholder attitudes towards ESHP is needed. The aim of this study was to explore pediatric HTx stakeholders’ understanding and perspectives of ESHP.
Methods: A virtual focus group study was conducted international pediatric HTx stakeholders. Data was analyzed using qualitative content analysis.
Results: There were 17 participants from 12 institutions representing 3 countries. Findings were organized under the topics of: general knowledge, indications, risks, adoption/barriers, and pediatric-specific considerations. Knowledge ranged from limited grasp of the technology to first-hand experience in clinical practice. Discussions encircled the current use of ESHP in adult-sized HTx candidates, and potential future uses in infants and young children. In all focus groups, it was discussed that risks need to be considered regarding ESHP itself, including the potential of damaging the donor heart, subjecting recipients to unquantified risks, and potential adverse impacts on pediatric HTx programs if poor outcomes occur. Concerns surrounding the material costs and human resources associated with ESHP were also discussed. While all discussions highlighted the need for pediatric ESHP devices, participants emphasized the need for rigorous research and implementation oversight of ESHP to navigate ethical issues, program administration, and support best practices in the context of technological innovation.
Conclusion: For stakeholders to become adopters of ESHP, they must understand the goals, benefits, and risks. This project represents the first step in the adoption of new technology, and the knowledge gained will form the basis for future education, clinical trial design, and rollout of pediatric ESHP technologies.
24-hour negative pressure ventilation ex-situ lung perfusion with a porcine transplantation model (10 minutes presentation + 5 minutes Q&A)
Background: Lung transplantation has a wait-list mortality of 30% due to a shortage of high-quality donor lungs. Ex-situ lung perfusion (ESLP) allows for improved preservation and reconditioning of marginal quality donor lungs to expand the donor pool, increasing donor utilization rates in some centres by 20%. To date, the longest pre-clinical preservation period with favourable transplantation data is 24-hours using positive pressure ventilation ESLP. Negative pressure ventilation has been shown to result in reduced lung injury and inflammation, yet reliable 24-hour preservation has yet to be achieved.
Objectives: The aim of this study was to refine our protocol of negative pressure ventilation (NPV)-ESLP to achieve reliable 24-hour preservation and assessment with acceptable in-vivo transplantation data.
Methods: Twelve double-lung blocks from 45-55kg pigs were preserved on normothermic NPV-ESLP for 24-hours using a cellular solution with autologous packed red blood cells. Lung protective ventilation and perfusion strategies were employed throughout preservation. Six left lungs were transplanted into recipients post-ESLP and reperfused for 4-hours to evaluate the impact on in-vivo post-transplant lung function. Final assessment of the transplanted left lung was performed with the right lung clamped. Lung oxygenation capacity (PF ratio = PaO2/FiO2), dynamic lung compliance (Cdyn), pulmonary artery pressure (PAP), pulmonary vascular resistance (PVR), and lung weight-gain were monitored.
Results: All twelve lung blocks demonstrated stable lung function during 24-hours of NPV-ESLP. At 24-hours, PF ratios were 473.9 ± 29.5 mmHg, Cdyn was 20.2 ± 2.8 mL/cmH20, PAP was 15.0 ± 1.1 mmHg, PVR was 570.7 ± 46.9 dynes/s/cm5, percentage weight-gain after ESLP was 54.66 ± 8.0. Acceptable lung oxygenation was demonstrated in all six transplanted left lungs with a mean PF ratio >300 mmHg (329.5 ± 21.7), equivalent to primary graft dysfunction (PGD) grade 0. Isolated left lung weight gain (%) post-transplant was 15.3 ± 39.02.
Conclusions: Although limited by small sample size, this study illustrates that reliable 24-hours of normothermic NPV-ESLP with a cellular perfusate is achievable, resulting in acceptable post-transplant oxygenation capacity. Prolonged preservation will allow for the treatment more advanced lung pathology and the elimination of geographic barriers for transplant. Future studies will strive to achieve 36-hours of NPV-ESLP with favourable transplantation data.
Ninjurin1 Contributes to Lytic Cell Death in Hepatic Ischemia-Reperfusion Injury (20 minutes presentation + 10 minutes Q&A)
Background: Liver transplantation is the gold standard therapy for many terminal liver diseases but the field remains limited by the availability of donor organs. Ischemia reperfusion injury (IRI) is an inherent part of organ transplantation, and is particularly problematic when utilizing high-risk organs, a necessary step for expanding the donor pool. Mechanistically, IRI leads to hepatocellular damage, lytic cell death and allograft dysfunction. Recently, ninjurin1 (NINJ1) has been implicated in macrophages as the common executioner of multiple lytic cell death pathways seen in IRI. Here we investigate the contribution of NINJ1 to hepatic IRI, including its role in mediating lytic cell death in hepatocytes. We focus on the pyroptosis cell death pathway, as whether hepatocytes are capable of undergoing pyroptosis has been hotly debated
Methods: For in vivo liver IRI, mixed-sex cohorts of Ninj1 knockout or wildtype littermate control mice were subjected to segmental warm liver injury consisting of 1 hour of ischemia and 6 hours of reperfusion. Liver injury and inflammation were evaluated by histopathology, serum AST/ALT levels and flow cytometric analysis of inflammatory infiltrate. For in vitro experiments, primary mouse hepatocytes and Kupffer cells were stimulated to undergo lytic cell death in normoxic or hypoxic conditions and membrane rupture assayed by the release of lactate dehydrogenase.
Results: Our data demonstrate that, like macrophages and Kupffer cells, primary hepatocytes are capable of undergoing pyroptosis. Furthermore, membrane rupture across cell types is abrogated in Ninj1 knockout cells as compared to wildtype. In vivo, Ninj1 genetic deficiency reduces hepatic IRI as compared to wildtype littermate control animals.
Conclusion: This work demonstrates that hepatocytes can directly undergo pyroptosis. Furthermore, it offers the first evidence that targeting membrane disruption through NINJ1 inhibition reduces hepatic IRI in vivo, thereby demonstrating a new potential therapeutic target for prevention of allograft injury during liver transplantation.
Normothermic ex vivo kidney perfusion preserves ATP generation and graft function after warm ischemia, which is further enhanced with pharmacologic mitochondrial protection (10 minutes presentation + 5 minutes Q&A)
Background: Normothermic Ex-vivo kidney machine perfusion (NEVKP) is a novel preservation technique. We recently determined that NEVKP preserves the expression of proteins involved in mitochondrial biogenesis in kidneys. We hypothesize that ex vivo machine perfusion will replenish energy levels in mitochondria, thereby restoring mitochondrial function and reducing injury in kidney grafts. AP39, a mitochondria-targeted hydrogen sulfide donor, has been shown to stimulate mitochondrial electron transport and improve cellular bioenergetic function. Here, we investigated whether administering AP39 during NEVKP protects mitochondrial function and protects renal grafts from ischemia reperfusion injury.
Methods: Porcine kidneys were subjected to 60 minutes of warm ischemia (WI) followed by 5 hours of static cold storage (SCS) or NEVKP. The warm ischemia grafts were subsequently divided into three groups: SCS group, NEVKP group, and a group in which AP39 was additionally administered during NEVKP (NEVKP + AP39). After contralateral nephrectomy, grafts were auto-transplanted and animals were followed for 3 days. Renal function and ATP levels were assessed.
Results: All animals (n=5-6 in each group) survived the follow-up period. Grafts preserved with NEVKP had lower serum creatinine (SrCr) on postoperative day 3 compared to the SCS group. Treatment with NEVKP + AP39 further reduced SrCr when compared to the NEVKP group (SCS vs NEVKP vs NEVKP + AP39: 12.7±1.1 vs 7.6±2.8 vs 3.5±1.1 mg/dl, mean±SD). We measured ATP in biopsy-derived cell suspensions from grafts stored using SCS, NEVKP, and NEVKP + AP39. ATP levels were increased in the NEVKP group compared with SCS group at the time of pre-implantation and this level was further increased when AP39 was administered. (1.9±11.4 vs 73.6±9.3 vs 121.6±36.3 nM/10000cells).
Conclusions: For grafts subjected to warm ischemia, NEVKP has the potential to preserve ATP generation and graft function, which is further enhanced with mitochondria-targeted hydrogen sulfide donor, AP39.
Quantitation of mitochondrial damage-associated molecular patterns in perfusate and bile for analysis of donor liver quality during ex-vivo NMP (10 minutes presentation + 5 minutes Q&A)
Background: Tissue damage during liver transplantation (LT) results in expulsion of mitochondrial damage-associated molecular patterns (mitoDAMPs), including mitochondrial DNA (mtDNA), into the extracellular space. High mtDNA levels positively correlate with negative clinical outcomes, and mtDNA is emerging as a predictive biomarker for a variety of disease processes. Normothermic machine perfusion (NMP) has emerged as an attractive alternative to traditional static cold storage (SCS), yet there remains an unmet need for an agile and universal marker of graft function during NMP. We hypothesized that mtDNA could be quantitated in perfusate and bile collected during ex vivo NMP of donor livers prior to transplant and would correlate with other metrics of graft quality.
Methods: DNA was isolated from perfusate (n=29) and bile (n=18) collected from donor livers undergoing NMP. MtDNA levels were measured via quantitative polymerase chain reaction (qPCR) using primers specific to mtDNA gene targets (COXI, CytB, ND1, ND6) and standard curves generated by commercially available synthetic oligonucleotides of primer-specific amplicons.
Results: MtDNA can be quantitated in both perfusate and bile samples collected during NMP. MtDNA quantitation via qPCR with an amplicon-specific standard curve accurately reproduced values measured by droplet digital PCR (ddPCR). Concentration of mtDNA in perfusate was positively correlated with warm ischemia time (COXI, p=0.042; CytB, p=0.041) and donor lactate (CytB, p=0.012; ND1, p=0.040). Donor international normalized ratio (INR) showed significant positive association with mtDNA in both perfusate (CytB, p=0.003; ND1, p=0.048) and bile (CytB, p=0.048; ND1, p=0.049; ND6, p=0.032).
Conclusions: Here, we demonstrate the feasibility and reproducibility of mtDNA quantitation in both perfusate and bile via qPCR and show association with clinical parameters. We anticipate that mtDNA will serve as a stable and inexpensive marker for liver function during NMP, which may inform the expected course of disease and illuminate opportunities for precision medicine in patients undergoing LT.