End Of Perfusion Research Articles

Preclinical validation of a customized circuit for ex situ uninterrupted cold-to-warm prolonged perfusion of the liver.

Clinical adoption of ex situ liver perfusion is growing. While hypothermic perfusion protects against ischemia-reperfusion injury in marginal grafts, normothermic perfusion enables organ viability assessment and therefore selection of borderline grafts. The combination of hypothermic and normothermic perfusion, known as "cold-to-warm," may be the optimal sequence for organ preservation, but is difficult to achieve with most commercial perfusion systems. We developed an adaptable customized circuit allowing uninterrupted "cold-to-warm" perfusion and conducted preclinical studies on healthy porcine livers and discarded human livers to demonstrate the circuit's efficacy. In collaboration with bioengineers, we developed a customized circuit that adapts to extracorporeal circulation consoles used in cardiovascular surgery and includes a proprietary reservoir enabling easy perfusate change without interrupting perfusion. This preclinical study was conducted on porcine and human livers. Perfusion parameters (pressures, flows, oxygenation) and organ viability were monitored. The customized circuit was adapted to a LivaNova S5® console, and the perfusions were flow-driven with real-time pressure monitoring. Ten porcine liver and 12 discarded human liver perfusions were performed during 14 to 18 h and 7 to 25 h, respectively. No hyperpressure was observed (porcine and human portal pressure 2-6 and 2-8 mm Hg; arterial pressure 10-65 and 20-65 mm Hg, respectively). No severe histological tissue injury was observed (Suzuki score ≤ 3 at the end of perfusion). Seven (70%) porcine livers and five (42%) human livers met the UK viability criteria. The customized circuit and system design enables smooth uninterrupted "cold-to-warm" perfusion not present in current commercial perfusion systems.

Ex vivo normothermic preservation of a kidney graft from uncontrolled donation after circulatory death over 73 hours.

The transplant community is focused on prolonging the ex vivo preservation time of kidney grafts to allow for long-distance kidney graft transportation, assess the viability of marginal grafts, and optimize a platform for the translation of innovative therapeutics to clinical practice, especially those focused on cell and vector delivery to organ conditioning and reprogramming. We describe the first case of feasible preservation of a kidney from a donor after uncontrolled circulatory death over a 73-h period using normothermic perfusion and analyze hemodynamic, biochemical, histological, and transcriptomic parameters for inflammation and kidney injury. The mean pressure and flow values were 71.24 ± 9.62mmHg and 99.65 ± 18.54mL/min, respectively. The temperature range was 36.7°C-37.2°C. The renal resistance index was 0.75 ± 0.15mmHg/mL/min. The mean pH was 7.29 ± 0.15. The lactate concentration peak increased until 213mg/dL at 6h, reaching normal values after 34h of perfusion (8.92mg/dL). The total urine output at the end of perfusion was 1.185mL. Histological analysis revealed no significant increase in acute tubular necrosis (ATN) severity as perfusion progressed. The expression of KIM-1, VEGF, and TGFβ decreased after 6-18h of perfusion until 60h in which the expression of these genes increased again together with the expression of β-catenin, Ki67, and TIMP1. We show that normothermic perfusion can maintain a kidney graft viable ex vivo for 3days, thus allowing a rapid translation of pre-clinical therapeutics to clinical practice.

Microbial Contamination During Long-term Ex Vivo Normothermic Machine Perfusion of Human Livers.

Normothermic machine perfusion permits the ex vivo preservation of human livers before transplantation. Long-term perfusion for days-to-weeks provides the opportunity for enhanced pretransplant assessment and potential regeneration of organs. However, this risks microbial contamination and infection of the recipient if the organ is transplanted. An understanding of perfusate microbial contamination is required to inform infection control procedures and antimicrobial prophylaxis for this technology. We modified a liver perfusion machine for long-term use by adding long-term oxygenators and a dialysis filter. Human livers that were not suitable for transplantation were perfused using a red-cell-based perfusate under aseptic and normothermic conditions (36 °C) with a goal of 14 d. Cephazolin was added to the perfusate for antimicrobial prophylaxis. Perfusate and bile were sampled every 72 h for microbial culture. Eighteen partial human livers (9 left lateral segment grafts and 9 extended right grafts) were perfused using our perfusion system. The median survival was 7.2 d. All organs surviving longer than 7 d (9/18) had negative perfusate cultures at 24 and 48 h. Half of the grafts (9/18) became culture-positive by the end of perfusion. Microbial contaminants included Gram-negative (Pseudomonas species, Proteus mirabilis, Stenotrophomonas maltophilia) and Gram-positive bacteria (Staphylococcus epidermidis, Enterococcus faecalis, and Bacillus species) as well as yeast (Candida albicans). Microbial contamination of perfusate is common during long-term perfusion of human livers with both exogenous and endogenous sources. Enhanced infection control practices and review of targeted antimicrobial prophylaxis are likely to be necessary for translation into the clinical arena.

Ex vivo lung perfusion moderates gene expression differences between cardiac death and brain death donor lungs

Donation after cardiac death (DCD) donor lungs have been shown to express less pro-inflammatory genes than donation after brain death (DBD) lungs, likely due to the absence of brain-death related inflammatory physiology. However, it is unclear whether this difference is clinically significant following reperfusion. To avoid confounding by the recipient immune system and activation state, we utilized ex vivo lung perfusion (EVLP) as a reperfusion-like event and examined the effect of EVLP on the transcriptome of DCD (n=39) and DBD (n=49) lungs. To validate our RNA results, banked EVLP perfusates from a separate cohort of DCD (n=24) and DBD (n=24) cases were assayed for IL-6, IL-8, IL-10, IL-1β, sTNFR1, and sTREM1 protein levels at 15 min intervals for three hours. While DCD lungs demonstrated lower levels of pro-inflammatory transcripts and perfusate cytokine protein levels than DBD lungs prior to EVLP, after EVLP there were no significant gene expression differences or cytokine protein levels between groups. Therefore, while DCD and DBD lungs differ by the amounts of pro-inflammatory cytokines following procurement, the propagation of inflammation becomes limited during EVLP, and DBD and DCD lungs reach a similar plateau of transcript expression, including pro-inflammatory cytokines at the end of perfusion. EVLP may therefore play a pre-conditioning role by dampening the pro-inflammatory state prior to transplant reperfusion.

Interleukin-6 Levels During Normothermic Machine Perfusion Impact Postreperfusion Hemodynamics of Liver Graft Recipients: A Prospective Single-center Observational Study.

Hemodynamic instability after liver graft reperfusion increases recipient morbidity after liver transplantation. The etiologies of hemodynamic disturbances appear to be multifactorial and are poorly understood. Normothermic machine perfusion (NMP) provides an opportunity to analyze graft quality prior to transplantation. In the present study, we aim to investigate the influence of interleukin-6 (IL-6) levels during NMP on postreperfusion hemodynamics of the recipient. Consecutive NMP-liver transplants at a single-center were prospectively analyzed. Perfusate samples were collected at the beginning, after 6 h, and at the end of perfusion and analyzed for IL-6 levels. Mean arterial pressure (MAP) and catecholamine consumption during surgery were recorded. IL-6 levels at the end of NMP were correlated to donor and perfusion characteristics as well as changes in MAP and catecholamine requirements during the anhepatic and reperfusion phase. IL-6 perfusate measurements were assessed in 77 livers undergoing NMP and transplantation. Donor age, sex, cold ischemic time, and NMP time did not correlate with IL-6 levels. Perfusates of donation after circulatory death grafts showed higher IL-6 levels at the end of NMP than donation after brain death grafts. However, IL-6 levels at the end of NMP correlated with catecholamine requirements and MAP in the reperfusion phase. Per log10 increase in IL-6 levels, an increase of 42% points in administered catecholamine dose was observed, despite MAP being decreased by 3.6% points compared to baseline values. IL-6 levels may be a predictor for recipient hemodynamic instability during liver reperfusion. Larger studies are needed to confirm this finding.

48h Normothermic Machine Perfusion With Urine Recirculation for Discarded Human Kidney Grafts.

Normothermic machine perfusion (NMP) has reshaped organ preservation in recent years. In this preclinical study, prolonged normothermic perfusions of discarded human kidney grafts were performed in order to investigate perfusion dynamics and identify potential quality and assessment indicators. Five human discarded kidney grafts were perfused normothermically (37°C) for 48h using the Kidney Assist device with a red-blood-cell based perfusate with urine recirculation. Perfusion dynamics, perfusate and urine composition as well as injury markers were measured and analyzed. Donor age ranged from 41 to 68years. All but one kidney were from brain dead donors. Perfusions were performed successfully for 48h with all discarded kidneys. Median arterial flow ranged from 405 to 841mL/min. All kidneys excreted urine until the end of perfusion (median 0.43mL/min at the end of perfusion). While sodium levels were consistently lower in urine compared to perfusate samples, this was only seen for chloride and potassium in kidney KTX 2. Lactate, AST, LDH as well as pro-inflammatory cytokines increased over time, especially in kidneys KTX 3 and 4. Ex vivo normothermic perfusion is able to identify patterns of perfusion, biological function, and changes in inflammatory markers in heterogenous discarded kidney grafts.

CARD27: Morphologic and functional assessment of beating heart in normothermic ex-situ preservation

Background: Heart transplantation remains the treatment of choice for patients experiencing end-stage heart failure. The emergence of cardiac ex situ machine perfusion not only facilitates the use of donor heart through the avoidance of potentially damaging ischemia during graft storage, it also opens new horizons for more sensitive monitoring of graft quality in the transplant field, and morphologic-functional assessment. Methods: Twenty-five domestic adult pigs, weighing 93.1±13.1kg were selected for this study. Until cardiectomy, all pigs received Principles of Laboratory Animal Care. The pig heart were cannulated, and then connected to the Portable System for Normothermic Ex-Situ HeartPreservation (our made). In the Heart Preservation System (HPS), oxygenated blood is perfused by a centrifugal pump into the aorta, perfusing the coronary arteries. After the end of experiment, (6-14 hour) pig heart was arrested with normothermic blood cardioplegia and disconnected. The pig isolated heart was conditioned with Levosimendan while in the system and hemofiltration was applied in the HPS in order to protect and improve donor heart function. Ex-Situ Heart Perfusion process was accompanied with morphologic-functional assessment such as echocardiography, contrast-enhanced magnetic resonance, thermodilution technique and biochemistry. Results: Mean (SD) ischemic time was 19.2 (±3.3) min. Mean ex vivo perfusion time was 560 (±143) min. Time of sinus rhythm restoration was 2.3 (5.7) min. All isolated hearts had stable perfusion, biochemical, as well as myocardial functional parameters measured by echocardiography and thermodilution technique, and histological characteristics in the perfusion system. MRI structural images showed no infiltrates alterations in myocardium. Mean venous lactate trend are with normal levels at the end of perfusion 1.6 (±0.6). Conclusion: Normothermic Ex-Situ Heart Perfusion provides near to physiologic, reproducible approach to sustain the isolated heart for long period of time. Multiparametric assessment of organ morphology and function is feasible in Ex-Situ Heart Perfusion, and may improve organ assessment, leading to a better selection for transplant.Figure 1. Simplified schema of the experimental design and protocol.

Exceeding the Limits of Static Cold Storage in Limb Transplantation Using Subnormothermic Machine Perfusion.

For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model. Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days. Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613). To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.

Treprostinil Supplementation Ameliorates Hepatic Ischemia Reperfusion Injury and Regulates Expression of Hepatic Drug Transporters: An Isolated Perfused Rat Liver (IPRL) Study

PurposeIR injury is an unavoidable consequence in deceased donor liver transplantation. Cold preservation and warm reperfusion may change the expression and function of drug transporters in the liver due to vasoconstriction, infiltration of neutrophils and release of cytokines. We hypothesize that vasodilation, anti-platelet aggregation and proinflammatory downregulation activities of treprostinil will diminish the IR injury and its associated effects.MethodsLivers obtained from male SD rats (n = 20) were divided into 1) Control, 2) IR, 3) Treprostinil-1 (preservation only), and 4) Treprostinil-2 (preservation and reperfusion) groups. Control livers were procured and immediately reperfused. Livers in the other groups underwent preservation for 24 h and were reperfused. All the livers were perfused using an Isolated Perfused Rat Liver (IPRL) system. Periodic perfusate, cumulative bile samples and liver tissue at the end of perfusion were collected. Liver injury markers, bile flow rates, m-RNA levels for uptake and efflux transporters (qRT-PCR) were measured.ResultsCold preservation and warm reperfusion significantly increased the release of AST and ALT in untreated livers. Treprostinil supplementation substantially reduced liver injury. Bile flow rate was significantly improved in treprostinil-2 group. m-RNA levels of Slc10a1, Slc22a1, and Slc22a7 in liver were increased and m-RNA levels of Mdr1a were decreased by IR. Treprostinil treatment increased Abcb11 and Abcg2 m-RNA levels and maintained Slc22a1m-RNA similar to control livers.ConclusionsTreprostinil treatment significantly reduced liver injury. IR injury changed expression of both uptake and efflux transporters in rat livers. Treprostinil significantly altered the IR injury mediated changes in m-RNA expression of transporters.

313.9: Perfusate IL-6 Levels During Liver NMP Might Be Predictive For Hemodynamic Response and Catecholamine Demand After Reperfusion in the Recipient

Introduction: Normothermic liver preservation (NMP) has become a clinical routine at several transplant centres. Reperfusion-syndrome occurs less often in recipients of NMP-livers compared to cold stored livers. We hypothesized that perfusate interleukin (IL)-6 during liver NMP correlate with recipient hemodynamics in the post-reperfusion period. Method: Consecutive NMP-liver transplants at a single-centre were prospectively analysed. Perfusate samples were collected at 1 and 6 hours of NMP and at the end of perfusion and analysed for IL-6 levels. Median arterial pressure (MAP) and catecholamine need during surgery were recorded. The anhepatic phase was defined as baseline for MAP and catecholamine requirements. Results: Over a period of 36 months, IL-6 perfusate measurements were assessed in 77 livers undergoing NMP and transplantation; 15/77 (19.5%) were DCD organs. The median donor age was 61 (15-87) years, median recipient age was 60 (19-73) years. Median (IQR) cold ischemia time was 6.2 (2.1) hrs, NMP-time and overall preservation time were 17.6 (10.4) hrs and 23.6 (10.6) hrs. Median (IQR) IL-6 levels (ng/L) after 1, 6 hrs and NMP-end were 52 (175), 278 (674) and 174 (2171). Neither duration of CIT nor NMP correlated with IL-6 levels over time. NMP-livers were stratified for the median of the last IL-6 measurement. Recipients receiving NMP-livers with perfusate IL-6 levels above the median developed significantly lower post-reperfusion MAP (dropping 20% from baseline) and displayed a significant higher demand of catecholamines (increase of 25% from baseline) up to 30 minutes after reperfusion. Perfusate IL-6 did not correlate with the occurrence of early allograft dysfunction.Conclusion: Perfusate IL-6 levels during liver NMP are clinically relevant as they help to predict the post-reperfusion hemodynamics in recipients.

Transplantation of Extended Criteria Donor Livers Following Continuous Normothermic Machine Perfusion Without Recooling.

Background.Traditional liver transplant strategies with cold preservation usually result in ischemia-reperfusion injury (IRI) to the donor liver. Regular normothermic machine perfusion (NMP) donor livers suffer IRI twice. Here, we aimed to introduce a novel technique called continuous NMP without recooling to avoid a second IRI and its application in livers from extended criteria donors.Methods.Seven donor livers transplanted following continuous NMP without recooling, 7 donor livers transplanted following standard NMP, and 14 livers under static cold storage (SCS) were included in this study. Perioperative outcomes were recorded and analyzed between groups.Results.During the NMP without a recooling procedure, all livers cleared lactate quickly to normal levels in a median time of 100 min (interquartile range, 60–180) and remained stable until the end of perfusion. In the NMP without recooling and standard NMP groups, posttransplant peak aspartate aminotransferase and alanine aminotransferase levels were both significantly lower than those in the SCS group (P = 0.0015 and 0.016, respectively). The occurrence rate of early allograft dysfunction was significantly lower in the NMP without recooling group than in the SCS group (P = 0.022), whereas there was no difference in the NMP group with or without recooling (P = 0.462).ConclusionsOur pilot study revealed a novel technique designed to avoid secondary IRI. This novel technique is shown to have at least a comparable effect on the standard NMP, although more data are needed to show its superiority in the future.

MO662: Effects of Blood Contact with Haemodialysis Membrane on the Kidney in a Rat Extracorporeal Perfusion Model

Abstract BACKGROUND AND AIMS A microcirculatory disturbance was found to occur during hemodialysis in an organ such as the brain or heart, which was recently considered to lead to functional impairment of these organs. Decrease in blood volume due to water removal during hemodialysis and the reaction of blood vessels with activated leukocytes and platelets that have come in contact with the dialysis membrane may cause the microcirculatory disturbance of the organs. Residual renal function decline after initiation of hemodialysis is well-known, but the mechanism has not yet been fully understood. In the present study, we focused on the effects of leucocytes and platelets activated by contact with the dialysis membrane and aimed to clarify the effects of blood contact with the dialysis membrane on the kidney. METHOD Male Sprague–Dawley rats weighing 397.4 ± 38.6 g were used for the experiment. We compared three experimental groups; the sham group, in which only puncture and infusion of Ringer's lactate solution were performed; the extracorporeal perfusion alone group, in which extracorporeal perfusion was performed only through a blood circuit; and the dialyzer group, in which extracorporeal perfusion was performed through a blood circuit and dialyzer. Blood was withdrawn from the femoral artery and returned to the femoral vein. The blood flow rate was 0.5–1.0 mL/min, the perfusion time was 4-h and no water removal was performed. During the perfusion experiment, arterial pressure and percutaneous arterial oxygen saturation (SpO2) were measured and at the end of perfusion, serum interleukin-6 (IL-6), a marker of inflammation and urinary kidney injury molecule-1 (KIM-1), a marker of kidney injury, were measured. After completing the perfusion experiment, kidneys were removed and morphology was evaluated by a hematoxylin and eosin (HE) staining. RESULTS AND DISCUSSION Arterial pressure during perfusion showed a significant upward trend during the first 2-h in the extracorporeal alone and dialyzer groups, while SpO2 showed a significant downward trend in these groups. Serum IL-6 levels were significantly higher in the extracorporeal perfusion alone group (P < .05, n = 8) and the dialyzer group (P < .01, n = 6) than in the sham group (n = 7). Urinary KIM-1 level in the dialyzer group was significantly higher than that in normal rats (P < .01, n = 7). Glomerular congestion was observed in the kidney only in the dialyzer group from the HE staining. There was a significant correlation between the increase in arterial pressure and the decrease in SpO2, suggesting that peripheral circulatory disturbance due to peripheral vasoconstriction occurred in the extracorporeal perfusion alone and the dialyzer groups. In addition, extracorporeal perfusion through the dialyzer caused high levels of IL-6 in serum and KIM-1 in urine, suggesting that systemic inflammatory and kidney injury have occurred. These reactions may contribute to the progression of residual renal dysfunction due to dialysis treatment, especially for the patients after the initiation of hemodialysis. CONCLUSION Extracorporeal perfusion and blood contact with a dialyzer caused systemic inflammation and the microcirculatory disturbance of kidneys, resulting in glomerular congestion and kidney injury in a rat extracorporeal perfusion model.

The mitochondrial electron transport chain contributes to calpain 1 activation during ischemia-reperfusion

Activation of calpain1 (CPN1) contributes to mitochondrial dysfunction during cardiac ischemia (ISC) – reperfusion (REP). Blockade of electron transport using amobarbital (AMO) protects mitochondria during ISC-REP, indicating that the electron transport chain (ETC) is a key source of mitochondrial injury. We asked if AMO treatment can decrease CPN1 activation as a potential mechanism of mitochondrial protection during ISC-REP. Buffer-perfused adult rat hearts underwent 25 min global ISC and 30 min REP. AMO (2.5 mM) or vehicle was administered for 1 min before ISC to block electron flow in the ETC. Hearts in the time control group were untreated and buffer perfused without ISC. Hearts were collected at the end of perfusion and used for mitochondrial isolation. ISC-REP increased both the cleavage of spectrin (indicating cytosolic CPN1 activation) in cytosol and the truncation of AIF (apoptosis inducing factor, indicating mitochondrial CPN1 activation) in subsarcolemmal mitochondria compared to time control. Thus, ISC-REP activated both cytosolic and mitochondrial CPN1. AMO treatment prevented the cleavage of spectrin and AIF during ISC-REP, suggesting that the transient blockade of electron transport during ISC decreases CPN1 activation. AMO treatment decreased the activation of PARP [poly(ADP-ribose) polymerase] downstream of AIF that triggers caspase-independent apoptosis. AMO treatment also decreased the release of cytochrome c from mitochondria during ISC-REP that prevented caspase 3 activation. These results support that the damaged ETC activates CPN1 in cytosol and mitochondria during ISC-REP, likely via calcium overload and oxidative stress. Thus, AMO treatment to mitigate mitochondrial-driven cardiac injury can decrease both caspase-dependent and caspase-independent programmed cell death during ISC-REP.

Perfusate Metabolomics Content and Expression of Tubular Transporters During Human Kidney Graft Preservation by Hypothermic Machine Perfusion.

Ischemia-related injury during the preimplantation period impacts kidney graft outcome. Evaluating these lesions by a noninvasive approach before transplantation could help us to understand graft injury mechanisms and identify potential biomarkers predictive of graft outcomes. This study aims to determine the metabolomic content of graft perfusion fluids and its dependence on preservation time and to explore whether tubular transporters are possibly involved in metabolomics variations. Kidneys were stored on hypothermic perfusion machines. We evaluated the metabolomic profiles of perfusion fluids (n = 35) using liquid chromatography coupled with tandem mass spectrometry and studied the transcriptional expression of tubular transporters on preimplantation biopsies (n = 26), both collected at the end of graft perfusion. We used univariate and multivariate analyses to assess the impact of perfusion time on these parameters and their relationship with graft outcome. Seventy-two metabolites were found in preservation fluids at the end of perfusion, of which 40% were already present in the native conservation solution. We observed an increase of 23 metabolites with a longer perfusion time and a decrease of 8. The predictive model for time-dependent variation of metabolomics content showed good performance (R 2 = 76%, Q 2 = 54%, accuracy = 41%, and permutation test significant). Perfusion time did not affect the mRNA expression of transporters. We found no correlation between metabolomics and transporters expression. Neither the metabolomics content nor transporter expression was predictive of graft outcome. Our results call for further studies, focusing on both intra- and extratissue metabolome, to investigate whether transporter alterations can explain the variations observed in the preimplantation period.

Risk of Intraoperative Hyperthermia and Outcomes in Adults Undergoing Cytoreductive Surgery (CRS) with Hyperthermic Intraperitoneal Chemotherapy (HIPEC).

Hyperthermic intraperitoneal chemotherapy (HIPEC) is commonly used to treat peritoneal surface malignancies. We aimed to identify risk factors of intraoperative patient hyperthermia and the postoperative outcome of adults undergoing HIPEC PATIENTS AND METHODS: A retrospective, IRB approved, single center cohort study was conducted. Adults treated with cytoreductive surgery and HIPEC between 2006 and 2021 were included. The primary outcome was bladder hyperthermia during perfusion, stratified by severity and duration. Secondary outcomes were postoperative complications and recurrence-free (RFS) and overall (OS) survival. Multivariable logistic regression models were fit to estimate the effects of important covariates. Out of 214 patients, 114 had mild hyperthermia (≥ 38°C) at any time, and in 73 of these it lasted for ≥ 30min. Independent prognostic factors of mild hyperthermia ≥ 30min were age (OR = 0.958, 95% CI 0.933-0.984), body mas index (BMI; OR = 0.959 95% CI 0.917-1.002), gender (OR = 0.199, 95% CI 0.092-0.431), and type of chemotherapy [cisplatin versus mitomycin (OR = 0.186, 95% CI 0.070-0.491; oxaliplatin versus mitomycin (OR = 0.430, 95% CI 0.163-1.139)]. Prognostic factors of moderate-to-severe hyperthermia (≥ 39°C) at any time were perfusion duration (OR = 1.094, 95% CI 1.018-1.177) and blood transfusion (OR = 5.689, 95% CI 1.784-18.137). Intraoperative hyperthermia was not associated with increased postoperative complications but was associated with better RFS and OS. Our study demonstrates age, gender, BMI, and chemotherapy type to be associated with hyperthermia ≥ 38°C for ≥ 30 min, whereas longer perfusion time and blood transfusion were associated with hyperthermia ≥ 39°C. Mild hyperthermia at the end of perfusion is associated with better RFS and OS.

The effect of exogenous peroxiredoxin 6 on morphofunctional state of isolated rat kidney

Objective: to investigate the role of peroxiredoxin 6 (PRX6) in preserving the morphofunctional state of ischemic isolated kidney during perfusion.Materials and methods. The model of an isolated perfused rat kidney was used. Ischemia time was 5 and 20 minutes, perfusion was 50 minutes. To evaluate the effectiveness of PRX6 at different ischemia times, we used the conventional criteria of kidney function and histological methods.Results. During short warm ischemia times, exogenous PRX6 improves the morphofunctional state of an isolated kidney during perfusion. During this period, the main criteria for functioning of the isolated ischemic kidney reach acceptable values, renal parenchyma is without severe damage. By the end of perfusion, there was an increase in urine flow rate, glomerular filtration rate, fractional glucose reabsorption, urine urea concentration and proportion of primary urine from 1.5 to 2 times compared with the control lesion. At 20-minute ischemia, the isolated kidney can be recognized as non-viable according to the functioning criteria; the positive effect of PRX6 is leveled.Conclusion. The use of recombinant peroxiredoxin 6 for preserving the morphofunctional state of isolated kidneys can be an effective approach in preventing ischemia–reperfusion injury.

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia.

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O2 availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-3H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following 3 weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-3H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH up-regulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase muscle isoenzyme M2 (PKM2) protein content 1.4-fold compared with normoxic hearts. CH also augmented cardiac pentose phosphate pathway (PPP) flux, reflected by higher ribose-5-phosphate (R5P) content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained up-regulation of the glycolytic flux in the chronically hypoxic heart.

O67: PERFUSATE GLUCOSE REFLECTS TISSUE GLYCOGENATION DURING LIVER PERFUSION

Abstract Introduction Normothermic machine perfusion (NMP) is a method of organ preservation that aims to replicate the physiological environment, achieved by perfusing the livers with a blood-based perfusate at physiological inflow pressures and temperature. NMP also permits viability assessment through evaluation of the perfusate flow rates through the portal vein and hepatic artery. In addition to this, biochemical assessment and perfusate gas analysis can be performed to provide insights into the metabolic activity of the liver. Method Discarded human liver grafts (n=6), were perfused for 24 hours. Core biopsies and perfusate samples were taken from each liver at 5 distinct time intervals over 24 hours. Core biopsies were fixed and stained with periodic acid-Schiff and analysed with Leica software to provide a quantitative estimate of the hepatocellular glycogen content. Result Hepatic glycogen concentration rose during the first hour, followed by a steady decline thereafter until the end of perfusion. Contrary to our initial hypothesis that glucose concentration within the circuit would show an inverse relationship to glycogen stores in the liver cells, we found that glucose concentration closely followed the same trend. Conclusion Change in hepatocyte glycogen content provides an important insight into the synthetic function of a liver destined for transplant. Our research suggests that glucose concentration can be used as a surrogate marker for the synthetic function of a liver on NMP and provides valuable information on the glycogen-synthesising capability of the hepatocytes. In future, this could potentially aid the decision-making process with regards to liver graft transplant viability. Take-home message Perfusate glucose concentration could provide an insight into the viability of liver transplants

Mitral Valve Correction Without Usage of Donor Blood

Objective: To study features of mitral valve replacement (MVR) without use of donor blood or its components. Methods: 489 patients (pts) with isolated mitral valve disease were operated during the period from 01/01/2000 till 01/01/2014. All operations were carried out without using donor blood or its components during treatment, and while cardiopulmonary bypass (CPB) haemoconcentrated columns, or cell-savers were not applied. There were 214 males and 275 females. In 372 (76.1%) pts, 468.7 ± 51.4 ml of blood on citrate was taken before the cross-clamping. Diuresis was stimulated on the beginning of operation by 80 mg furosemide and 100 ml mannite. Patient was completed by 6% refortane in a 250-300 ml dose before starting CPB, and the water balance on this stage was in the ranges + 284.4±39.6 ml. Retrograde autological priming (RAP) occurred in 81 (16.6%) pts in doses 588.5±71.2ml. By the time of the end of perfusion, the water balance did not exceed ranges of 700-900.0 ml. After a stop of CPB, its maximum contents were returned into patient, including full evacuation of the CPB`s reservoir. Average time of cross-clamping was 53.4±7.2 min, bloodless – 242.2±34.8 ml. Results: Hospital mortality (HM) among 489 pts was 1.0 % (5 pts died). Duration of stay on artificial lung ventilation was 5.5 ± 0.8 hours, in intensive care unit – 59.5 ± 7.5 hours, average time of the postoperative period was 9.1 ± 0.7 days. At discharge, moderate anemia (reduction of hemoglobin from 145 ± 9.8 g/L to 105 ± 14.3 g/L from initial) was marked. Conclusions: We recommend our method of MVR without use of donor blood and its components. Exceptions for it are significant anemia (hemoglobin less than 120), weight of patients less than 65 kg, arterial hypertension with hypertrophic left ventricle.

Metabolic Profiling of Skeletal Muscle During Ex-Vivo Normothermic Limb Perfusion

Ex vivo normothermic limb perfusion (EVNLP) provides several advantages for the preservation of limbs following amputation: the ability to maintain oxygenation and temperature of the limb close to physiological values, a perfusion solution providing all necessary nutrients at optimal concentrations, and the ability to maintain physiological pH and electrolytes. However, EVNLP cannot preserve the organ viability infinitely. We identified evidence of mitochondrial injury (swelling, elongation, and membrane disruption) after 24 hours of EVNLP of human upper extremities. The goal of this study was to identify metabolic derangements in the skeletal muscle during EVNLP. Fourteen human upper extremities were procured from organ donors after family consent. Seven limbs underwent EVNLP for an average of 41.6 ± 9.4 hours, and seven contralateral limbs were preserved at 4°C for the same amount of time. Muscle biopsies were performed at 24 hours of perfusion, both from the EVNLP and control limbs. Perturbations in the metabolic profiles of the muscle during EVNLP were determined via untargeted liquid chromatography-mass spectrometry (MS) operated in positive and negative electrospray ionization modes, over a mass range of 50 to 750 Da. The data were deconvoluted using the XCMS software and further statistically analyzed using the in-house statistical package, MetaboLyzer. Putative identification of metabolites using exact mass within ±7 ppm mass error and MS/MS spectral matching to the mzCloud spectral library were performed via Compound Discoverer v.2.1 (Thermo Scientific, Fremont, CA, USA). We further validated the identity of candidate metabolites by matching the fragmentation pattern of these metabolites to those of their reference pure chemicals. A nonparametric Mann-Whitney U-test was used to compare EVNLP and control group spectral features. Differences were considered significantly different when P-value < 0.05. We detected over 13,000 spectral features of which 58 met the significance criteria with biologically relevant putative identifications. Furthermore we were able to confirm the identities of the ions taurine (P-value: 0.002) and tryptophan (P-value: 0.002), which were among the most significantly perturbed ions at 24 hours between the experimental and control groups. Metabolites belonging to the following pathways were the most perturbed at 24 hours: neuroactive ligand-receptor interaction (P-values: 0.031 and 0.036) and amino acid metabolism, including tyrosine and tryptophan metabolism (P-values: 0.015, 0.002, and 0.017). Taurine abundance decreased and tryptophan abundance increased at 24 hours. Other metabolites also identified at 24 hours included phenylalanine, xanthosine, and citric acid (P-values: 0.002, 0.002, and 0.0152). This study showed presence of active metabolism during EVNLP and metabolic derangement toward the end of perfusion, which correlated with detection of altered mitochondrial structure, swelling, and elongation.