Normothermic Preservation Research Articles

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic <em>Ex Vivo</em> Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods. Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes. In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic and hypothermic machine perfusion preservation versus static cold storage for deceased donor kidney transplantation

Normothermic and hypothermic machine perfusion preservation versus static cold storage for deceased donor kidney transplantation

Antibiotic Prophylaxis in (Sub)Normothermic Organ Preservation

Bacterial contamination during cold organ preservation occurs without major complications. However, with organ preservation steering toward (sub)normothermic temperatures, bacterial contamination may be detrimental with limited evidence to support the choice of antibiotic. This study aimed to determine the effective antibiotic prophylaxis for (sub)normothermic preservation by investigating whether Staphylococcus epidermidis was capable of growing in a subnormothermia-compatible preservation solution Polysol (PS) and in solutions designed for cold preservation (University of Wisconsin solution, histidine-tryptophan-ketoglutarate solution, and Belzer-machine perfusion solution). Various S. epidermidis and Staphylococcus aureus strains were exposed to ceftriaxone and cefazolin at concentrations from 0 to 1000 μg/mL under subnormothermic and normothermic conditions in PS. To mimic procedural conditions, the effect of cefazolin was determined after exposure of bacteria to 20-hr incubation at 28°C in the presence of cefazolin and subsequent incubation at 37°C in the absence of cefazolin. The toxicity of cefazolin was assessed by cell viability and caspase activation assays in porcine kidney endothelial cells. Without antibiotics, PS sustained bacterial growth under sub(normothermic) conditions, whereas growth was absent in cold preservation solutions. Cefazolin exhibited greater bactericidal effect on S. epidermidis than ceftriaxone. However, after inoculating PS with 10 colony-forming units/mL, only a cefazolin concentration of 1000 μg/mL was able to exert a complete bactericidal effect on S. epidermidis and S. aureus strains and maintain sterility after removal of cefazolin. Finally, 1000 μg/mL cefazolin showed no adverse effects on porcine kidney endothelial cells. Based on these findings, we recommend that high-dose cefazolin be used for prophylaxis in (sub)normothermic organ preservation with PS.

Anesthesia Literature Review

Anesthesia Literature Review

The role of normothermic extracorporeal perfusion in minimizing ischemia reperfusion injury

The primary objective of organ preservation is to deliver a viable graft with minimal risk of impaired postoperative graft function. In current clinical practice, preservation of transplanted organs is based on hypothermia. Organs are flushed and stored using specific preservation solutions to reduce cellular metabolism and prevent cell swelling. However, the ongoing organ donor shortage and consequent expansion of donor criteria to include the use of grafts that would once have been discarded as unsuitable have underlined the need for a technique that prevents any further damage during the preservation period. The principle of normothermic machine perfusion preservation is the maintenance of cellular metabolism in a physiological environment throughout the preservation period. Normothermic preservation, at least in theory, thereby overcomes the 3 major weaknesses inherent in traditional static cold storage by (1) avoiding ischemia/reperfusion injury, (2) avoiding cold injury, and (3) allowing viability assessment. Furthermore, normothermic machine perfusion might transpire to be the ideal vehicle to deliver other therapeutic interventions during preservation to modulate and optimize the graft before transplantation. By restoring function in marginal donor organs and enabling the clinician to appraise its viability, the donor pool might be greatly expanded.

Impact of Normothermic Preservation with Extracellular Type Solution Containing Trehalose on Rat Kidney Grafting from a Cardiac Death Donor

BackgroundThe aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx).MethodsPost-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer's (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2).ResultsUsing the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion.ConclusionsNormothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.

Hepatic Steatosis and Normothermic Perfusion—Preliminary Experiments in a Porcine Model

Steatotic livers are increasingly common in the donor population. Cold storage of steatotic livers exacerbates ischemia-reperfuson injury and risks primary nonfunction and recipient death. Normothermic preservation avoids prolonged cooling of the organ and may be well suited to the preservation and resuscitation of damaged livers. By ex vivo normothermic perfusion, it may be possible to preserve and improve steatotic livers, so that transplantation is a viable option. In a porcine model, streptozotocin was used to induce a hyperglycemic, ketotic state that, together with a high fat diet, resulted in mild hepatic steatosis at 5 weeks. A blood-based oxygenated ex vivo normothermic preservation system was then used to compare extended preservation of normal and mildly steatotic porcine livers at physiological pressures and flows. Serial liver biopsies were stained with Oil Red O, a specialist triglyceride stain, and were analyzed using custom-designed image analysis to quantify the degree of lipid deposition. Steatotic livers were capable of correcting the perfusate base excess and maintaining factor V and bile production and showed markers of liver injury comparable with normal livers. Steatotic livers had a significantly higher urea production and required no glucose support. Preliminary results suggest that prolonged normothermic perfusion results in a reduction in steatosis. This study suggests that steatotic livers can be successfully preserved using normothermic preservation for prolonged periods and that normothermic preservation facilitates a reduction in hepatic steatosis. Further studies are now needed including transplantation of steatotic livers after normothermic preservation.

Normothermic kidney preservation

Kidneys from marginal or donation after cardiac death (DCD) donors are particularly susceptible to injury during hypothermic preservation and may benefit from alternative methods of preservation. Normothermic preservation can be adapted to improve the quality of kidneys for transplantation by a variety of techniques. Extracorporeal membrane support to maintain circulation before cooling and organ retrieval has been used to improve the condition of DCD donor kidneys, with lower rates of delayed graft function (DGF) compared with standard retrieval conditions. Experimentally, normothermic perfusion has been used in conjunction with hypothermic techniques as a resuscitation technique to improve graft outcome. An ex-vivo porcine kidney model showed that energy levels could be replenished to improve tissue perfusion during reperfusion. This technique was translated into a porcine transplant model demonstrating that it was a feasible and safe method of preservation. Normothermic preservation techniques have the potential to be adapted into an improved method of retaining tissue viability compared with hypothermic techniques. Furthermore, they may be used as a device to enhance and assess the condition of the kidney which would be particularly beneficial for kidneys from DCD donors.

Modification de l’épidémiologie des donneurs d’organes et nouvelles stratégies de préservation des greffons rénaux

Changes in the epidemiology of brain death are leading the transplant community to use more marginal grafts from so-called extended criteria donors. Current preservation techniques, while simple, are no longer adequate for these organs, which harbor chronic lesions making them very sensitive to ischemia-reperfusion injury. New preservation solutions are needed, along with machine perfusion techniques (dynamic preservation) and normothermic preservation phases.

A Pilot Study Assessing the Feasibility of a Short Period of Normothermic Preservation in an Experimental Model of Non Heart Beating Donor Kidneys

Restoring metabolism to an organ after hypothermic storage and before transplantation could reverse some of the detrimental effects of ischemic injury. This may be particularly beneficial for kidneys from non-heart-beating (NHBD) donors that sustain significant periods of warm and cold ischemic injury. This pilot study assessed the feasibility of a short period of normothermic preservation (NP) in a porcine autotransplant model. Kidneys were subjected to 30 min of warm ischemia, then preserved by hypothermic machine perfusion (HMP) for 22 h or 20 h HMP followed by 2 h of NP using autologous blood. Kidneys were then re-implanted, a contralateral nephrectomy performed, and renal function measured over 10 d. Post-transplant, 4/6 animals survived in the NP group compared with 5/6 in the HMP group (P = 1.00). Creatinine levels fell below 250 μmol/L in all four of the surviving animals in the NP group compared with 2/5 in the HMP group (P = 0.608). There was no difference in levels of renal function (peak creatinine, HMP = 1736 ± 866 versus NP = 1553 ± 516 μmol/L; P ≥ 0.990). Levels of lipid peroxidation were significantly lower 60 min post-transplant in the NP group (NP = 477 ± 118.0 versus HMP = 671 ± 99.4 pg/mL; P = 0.026). A period of NP at the end of the renal preservation period in NHB kidneys is a feasible method of kidney preservation. NP could prove to be a useful technique to predetermine graft function and allow pre-transplant modification of organs.

Impact of Normothermic Perfusion and Protein Supplementation on Human Endothelial Cell Function During Organ Preservation

Hypothermia-induced changes in endothelial cell (EC) morphology and function after organ storage may influence the initial outcome and development of transplant-associated coronary artery disease. Human saphenous vein ECs were incubated with saline (NaCl), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solution, with and without protein additives, at 4 degrees C and 37 degrees C. After 6 hours, ECs were recultivated for 24 and 48 hours with culture medium (reperfusion). Mitochondrial activity, adenosine triphosphate concentration, cell count, and inflammatory responses were analyzed. Cold preservation did not affect the mitochondrial activity of ECs and allowed a complete regeneration of the metabolic turnover after reperfusion. However, under normothermic conditions the metabolism of the cells was influenced by time and type of preservation solution. While both the mitochondrial activity and cell count did not change after treatment with NaCl and culture medium, the metabolic turnover of cells treated with HTK and UW solution significantly increased (twofold) and decreased (twofold, p < 0.05), respectively, after reperfusion. The endothelial reactivity remained unchanged after treatment with NaCl and HTK. The addition of serum proteins significantly improved mitochondrial activity of cells treated with warm NaCl and HTK (p < 0.05). The UW-treated cells burned out through a significant up-regulation of the ATP concentration resulting in a complete metabolic regression after reperfusion and induction of apoptosis. Normothermic preservation in UW prevented regeneration of ECs, while treatment with HKT solution did not irreversibly affect mitochondrial activity of ECs and allowed complete regeneration of metabolism and function. Serum proteins improved the preservation effect of HTK and NaCl.

Invited Commentary

Tissue injury related to hypoxic preservation remains a significant problem in organ transplantation. Ischemic reperfusion injury releases inflammatory mediators and attracts pro-inflammatory and pro-thrombotic cells that predispose to organ failure, early re-transplantation, and death. Cold preservation has been a hallmark for organ storage for many years by slowing cellular metabolism and reducing toxic metabolite formation. However, recent investigation has focused on normothermic preservation, which has been shown to result in reduced organ damage and improved function under certain conditions.

Normothermic Perfusion: A Mini-Review

The quality of organ preservation is critical to the outcome of transplantation: preservation technology is a limiting factor in the further development in the use of marginal donor organs. Normothermic preservation (preservation at normal physiological temperature) enables prolonged preservation, resuscitation after warm ischemia and organ viability assessment before transplantation.

242: Functional, Metabolic, and Morphological Aspects of Continuous, Normothermic Heart Preservation: Effects of Different Preparation and Perfusion Techniques

242: Functional, Metabolic, and Morphological Aspects of Continuous, Normothermic Heart Preservation: Effects of Different Preparation and Perfusion Techniques

Functional, Metabolic, and Morphological Aspects of Continuous, Normothermic Heart Preservation: Effects of Different Preparation and Perfusion Techniques

Functional, Metabolic, and Morphological Aspects of Continuous, Normothermic Heart Preservation: Effects of Different Preparation and Perfusion Techniques

Sequential Cold Storage and Normothermic Perfusion of the Ischemic Rat Liver

Extending transplant criteria to include livers obtained from donors after cardiac death (DCD) could increase the liver donor pool, but conventional simple cold storage of these ischemic organs can lead to poor graft function after transplantation. Experimental normothermic machine perfusion has previously proven to be useful for the recovery and preservation of DCD livers, but it is more complicated than conventional cold storage, and, therefore, is perhaps not practical during the entire preservation period. In clinical situations, the combined use of simple cold storage and normothermic perfusion preservation of DCD livers might be more realistic, but even a brief period of cold storage prior to normothermic preservation has been suggested to have a negative impact on graft viability. In this study we show that rat livers subjected to 45 minutes of ex vivo warm ischemia followed by 2 hours of simple cold storage can be reclaimed by 4 hours of normothermic machine perfusion. These livers could be orthotopically transplanted into syngeneic recipients with 100% survival after 4 weeks (N = 10), similar to the survival of animals that received fresh livers that were stored on ice in University of Wisconsin (UW) solution for 6 hours (N = 6). On the other hand, rats that received ischemic livers preserved on ice in UW solution for 6 hours (N = 6) all died within 12 hours after transplantation. These results suggest that normothermic perfusion can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage.

Organ reperfusion and preservation

Organ transplantation is one of the medical success stories of the 20th century. Transplantation is, however, a victim of its own success with demand for organs far exceeding supply. The ischemia/reperfusion injury associated with organ transplantation is complex with interlinking cellular pathways and cascades. With increasing use of marginal organs and better understanding of the consequences of ischemia/reperfusion, enhanced organ preservation is required. Traditional static cold preservation cannot prevent ischemia/reperfusion injury, the low temperature itself is damaging and viability testing is limited. Donor preconditioning techniques to enhance organ preservation in advance of retrieval are starting to show convergence on several key pathways (HO-1 and cell apoptosis). Microdialysis and bioimpedence techniques may allow viability assessment during cold storage. Hypothermic machine perfusion has a role to play, particularly in preservation of kidneys from non-heart-beating donors although results of clinical trials are awaited. Normothermic preservation offers benefits over cold storage (at least experimentally) by avoiding damage induced by low temperature, minimising ischemia/reperfusion injury and allowing resuscitation of damaged organs. Normothermic preservation is likely to increase as the average quality of donor organs declines and clinical trials are needed. In the long term, normothermic preservation may be used, not just to resuscitate organs, but facilitate organ immunomodulation.

A Model for Normothermic Preservation of the Rat Liver

Current techniques for the preservation of donor livers typically rely on cold temperatures (approximately 0-4 degrees C) to slow down metabolic processes. Recently, normothermic extracorporeal liver perfusion (NELP) has regained interest as a potentially promising approach for long-term liver preservation. Unlike cold-storage techniques, NELP attempts to maintain the liver in a near physiological environment, thus enabling normal metabolic and tissue repair processes to take place, which may help in the recovery of ischemically damaged and fatty donor livers, both of which represent significant untapped sources of donor livers. However, NELP is technically more complex than cold-storage techniques, and the lack of standardized small animal models limits its development. Here we describe a rat NELP system that is simple and cost-effective to run. We show that rat livers that underwent NELP for 6 h could be routinely transplanted into syngeneic recipient rats with excellent 1-month survival. During perfusion, the release of cytosolic enzymes, bile and urea production, and oxygen uptake rate could be readily monitored, thus providing a comprehensive picture of hepatic function before transplantation. This system will help in the optimization of NELP in several ways, such as for the improvement of perfusion conditions and the development of quantitative metabolic criteria for hepatic viability.

Static normothermic preservation of renal allografts using a novel nonphosphate buffered preservation solution

The aim of this study was to assess the viability and function of renal allografts under normothermic conditions using a novel nonphosphate buffered preservation solution AQIXRS-I. Porcine kidneys were flushed at 30 degrees C with AQIXRS-I at 100 mmHg pressure after 5-10 min warm ischaemic time and stored statically at either 4 degrees C or 30 degrees C for 2 h (n = 6 per group). Assessment of renal function by physiological and biochemical parameters was performed by perfusing the organs with autologous blood at 37 degrees C, with an initial circulating serum creatinine concentration of 1000 mumol/l on an isolated organ perfusion system for 6 h. Although the hypothermic group demonstrated overall superior renal function, the normothermic stored kidneys displayed a statistically comparable acid-base balance (7.37 +/- 0.15 vs. 7.3 +/- 0.09, P = 0.24). Furthermore, renal function was still evident after 6 h perfusion with increasing oxygen consumption, renal blood flow and reduced renal vascular resistance. The effectiveness and versatility of AQIXRS-I as a preservation solution under both normothermic and hypothermic conditions has been demonstrated. Renal viability was maintained after 2 h static normothermic storage. This study provides a foundation for further analysis utilizing normothermic preservation.