Hypothermic Reconditioning Research Articles

Role of Pulsatility in Hypothermic Reconditioning of Porcine Kidney Grafts by Machine Perfusion After Cold Storage

Brief in-house machine perfusion after cold storage (CS) (hypothermic reconditioning) has been proposed as a convenient tool to improve kidney graft function. The present study aimed to investigate the mechanistic role of vascular pulsatility in this context. Kidney function after cold preservation (4°C, 18 hr) and subsequent reconditioning by 90 min of pulsatile machine perfusion (PP) (30/20 mm Hg) or nonpulsatile machine perfusion (NPP) (30 mm Hg) was studied in an isolated kidney perfusion model in pigs (n=6 for both) and compared with simply CS grafts. Compared with CS, PP but not NPP significantly improved renal perfusate flow and urine production and significantly increased the reduction of perfusate levels of creatinine and urea during reperfusion. Perfusate levels of fatty acid binding protein, a marker of tubular cell injury, were dramatically reduced by PP but not NPP. PP and NPP lowered fractional excretion of sodium, but significance was only reached for PP. Molecular effects of PP comprised a significant (vs. CS) mRNA elevation of the endothelial anti-inflammatory transcription factor Krüppel-like factor 2 as well as endothelial nitric oxide synthase, along with significantly higher perfusate levels of the endogenous vasodilator nitric oxide. Functional efficiency of PP over CS was confirmed in additional porcine transplant experiments (n=5 for both) by, for example, up to threefold improved clearance of creatinine during the first days after transplantation. PP of 90 min shortly before transplantation seems to be an efficient mechanism to reduce proinflammatory endothelial phenotype and improve functional outcome of kidney grafts even after preceding static storage.

Hypothermic reconditioning in organ transplantation

To summarize the recent understanding of postpreservation reconditioning of organ grafts. Notable parts of ischemia-associated tissue alterations during static storage do not represent irreversible tissue damage, but rather biochemical and molecular disturbances of cellular homeostasis. Manifestation of irreversible organ injury upon warm reperfusion, triggered by these ischemic cellular disorders, could in large part be circumvented by a brief period (1-2 h) of hypothermic oxygenation between cold storage and transplantation. The procedure called 'hypothermic reconditioning' has first been found to be effective in the liver using simple gaseous oxygen persufflation, but was seen equally effective using hypothermic machine perfusion. Under certain circumstances, anoxic, but pulsatile vascular perfusion may suffice to alleviate ischemia-induced proinflammatory phenotype on vascular endothelium. Vascular and parenchymal tissue homeostasis can be restored to a notable extent in cold-preserved organ grafts by hypothermic reconditioning: a short period of preimplantation treatment allows for better graft function after transplantation in livers and kidneys. Hypothermic reconditioning represents a promising and easily applicable tool for post hoc enhancement of organ viability in marginal grafts and should be validated in randomized controlled trials.

Hypothermic Reconditioning of Porcine Kidney Grafts by Short-Term Preimplantation Machine Perfusion

Clinical trial data suggest that continuous hypothermic machine perfusion (HMP) during the entire preservation period reduces the incidence of delayed graft function and improves graft survival. This study evaluates whether short-term MP after cold storage (CS) is also effective. Kidney function after cold preservation (4°C, 21 hr) and transplantation was studied in an autotransplant model using Landrace pigs (25-30 kg; n=5 per group) with 1 week follow-up. Preservation was performed by conventional CS or HMP with a modified Lifeport Kidney Transporter either continuously during the entire preservation period or only for 2 hr of hypothermic reconditioning (HR) subsequent to conventional CS. HMP and HR similarly improved cortical microcirculation and significantly reduced maximal serum creatinine levels and recovery of creatinine clearance to normal values compared with CS. Fractional excretion of Na+ was unaltered after HMP and HR but significantly increased until postoperative day 5 on CS. On a molecular level, HR reduced innate immunoreactivity (toll-like receptor 4 expression and high mobility group protein B1 [HMGB-1] release) and normalized antiinflammatory tissue expression of von Kruppel-like Factor-2. Short-term reconditioning after CS proves to be as effective as continuous MP during the whole storage time. Because of its logistical convenience, the concept of an a posteriori treatment recommends itself to be evaluated in clinical trials.

Hypothermic reconditioning by gaseous oxygen persufflation after cold storage of porcine kidneys

BackgroundDelayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by hypothermic reconditioning using retrograde oxygen persufflation (ROP) immediately prior to reperfusion. MethodsKidneys from female German Landrace pigs were flushed with Histidine–Tryptophan–Ketoglutarate (HTK) solution and cold-stored for 18h (control).Some grafts were subsequently subjected to 90min of retrograde oxygen persufflation (ROP) via the renal vein during cold preservation. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n=6, resp.). ResultsRenal function upon reperfusion was significantly enhanced by ROP with an approximately twofold increase in renal clearances of creatinine and urea. ROP also led to higher renal vascular flow rates, enhanced urine output and mitigated histological alterations. ConclusionIt is concluded that initial graft function can be improved by 90min of hypothermic gaseous oxygenation after arrival of the preserved organ in the transplantation clinic.

Hypothermic Reconditioning by Gaseous Oxygen Improves Survival After Liver Transplantation in the Pig

The quality of cold-stored livers declines with the extension of ischemic time and the risk of primary dys- or nonfunction increases. Here, we provide in vivo evidence for the efficacy of the previously developed end-ischemic gaseous oxygen persufflation technique to resuscitate liver grafts after extended storage times. Porcine livers were recovered according to standard multiorgan procurement protocol. Control livers were cold stored in histidine tryptophan ketoglutarate solution for 10 h (cold storage [CS]; n = 6) at 4°C. In the treatment group (n = 6), livers were additionally subjected to hypothermic reconditioning (HR) by gaseous oxygen persufflation via the caval vein for 2 h before transplantation. Viability was assessed by orthotopic liver transplantation and 1 week follow-up. HR significantly improved pretransplant energy charge and initial graft function after transplantation. One week survival after CS was 0% whereas five of six pigs (83%) survived in the HR group. At that time, coagulation parameters were in the normal range and histological analysis disclosed healthy liver tissue with normal trabecular architecture in the treated grafts. Molecular analyses identify the prevention of ischemia-induced decline of cellular autophagy and mitigation of innate immune machinery (high-mobility group protein B1, interferon-β) as operative mechanisms among the protective effects provided by HR.

Oxygen persufflation as adjunct in liver preservation (OPAL): Study protocol for a randomized controlled trial

BackgroundEarly graft dysfunction due to preservation/reperfusion injury represents a dramatic event after liver transplantation. Enhancement of donor organ criteria, in order to cope with the ever increasing donor shortage, further increases graft susceptibility to ischemic alterations.Major parts of post-preservation injury, however, occur at the time of warm reperfusion but not during ischemic storage; successful reperfusion of ischemic tissue in turn depends on an adequate redox and intracellular signal homeostasis. The latter has been shown experimentally to be favorably influenced by oxygen persufflation within short time spans. Thus viability of marginally preserved liver grafts could still be augmented by transient hypothermic reconditioning even after normal procurement and static cold storage. The present study is aimed to confirm the conceptual expectations, that hypothermic reconditioning by gaseous oxygen persufflation is a useful method to suppress injurious cellular activation cascades and to improve post-ischemic recovery of marginally preserved liver grafts.Methods/DesignOPAL is a prospective single center randomized proof of concept study, including two parallel groups in a total of 116 liver transplant patients. The effect of an in hospital treatment of the isolated liver graft by 2 hours of oxygen persufflation immediately prior to transplantation will be assesses as compared to standard procedure (cold storage without further intervention). The primary endpoint is the peak transaminase serum level (AST) during the first three days after transplantation as a surrogate readout for parenchymal liver injury. Other outcomes comprise patient and graft survival, time of intensive care requirement, hepatic tissue perfusion 1h after revascularisation, early onset of graft dysfunction based on coagulation parameters, as well as the use of a refined scoring-system for initial graft function based on a multi-parameter (AST, ALT, Quick and bilirubin) score. Furthermore, the effect of OPAL on molecular pathways of autophagy and inflammatory cell activation will be evaluated. Final analysis will be based on all participants as randomized (intention to treat).Trial RegistrationCurrent Controlled Trials ISRCTN00167887

Optimal Time for Hypothermic Reconditioning of Liver Grafts by Venous Systemic Oxygen Persufflation in a Large Animal Model

Quality of cold-stored livers declines beyond 12 hr of ischemia, increasing the risk of primary dysfunction. Here we evaluate the potential and optimal treatment interval of gaseous oxygen persufflation for grafts reconditioning after long storage times in an experimental pig liver model. Porcine livers (n=6/group) were cold stored at 4°C for 18 hr in histidine-tryptophan-ketoglutarate solution. Hypothermic reconditioning (HR) was performed in some livers, by insufflation of gaseous oxygen through the caval vein for 1, 2, or 3 hr subsequent to cold storage. Liver integrity was assessed by controlled in vitro reperfusion with autologous blood. HR resulted in a 40% to 50% reduction of serum levels of aspartate aminotransferase, lactate dehydrogenase, and tumor necrosis factor-α with a maximal effect after 2 hr of HR (P<0.05). Functional parameters (bile production, cholinesterase and energetic recovery) were likewise enhanced (P<0.05). Two hours of HR also improved hepatic arterial flow and abrogated the postischemic increase in portal venous perfusion resistance compared with untreated (P<0.05). Gene expression of Toll-like receptor-4 was reduced by 2 hr of HR as was platelet adherence in the reperfused graft (P<0.05), in line with a trend toward lower expression of von Willebrand factor. HR effectively ameliorated graft dysfunction after extended preservation of porcine livers. Two hours of "a posteriori" treatment provide the maximal effect and are recommended for further application.

Hypothermic reconditioning after cold storage improves postischemic graft function in isolated porcine kidneys

Delayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by short-term hypothermic machine perfusion immediately prior to reperfusion. A total of 18 kidneys from female German Landrace pigs was flushed with Histidine-Tryptophan-Ketoglutarate solution and cold-stored for 18 h (control). Some grafts were subsequently subjected to 90 min of hypothermic reconditioning by hypothermic machine perfusion with (HR+O(2)) or without (HR-O(2)) oxygenation of the perfusate. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n = 6, respectively). Renal function upon reperfusion was significantly enhanced by HR+O(2) with more than threefold increase in renal clearances of creatinine and urea. HR+O(2) also led to significantly higher urinary flow rates and abrogated the activation of caspase 3. By contrast, HR-O(2) was far less effective and only resulted in minor differences compared to control. It is derived from the present data that initial graft function can be significantly improved by 2 h of oxygenated machine perfusion after arrival of the preserved organ in the transplantation clinic.

Impaired autophagic clearance after cold preservation of fatty livers correlates with tissue necrosis upon reperfusion and is reversed by hypothermic reconditioning

Fatty livers are particularly susceptible to mitochondrial alterations after cold preservation. We thus aimed to improve graft integrity by brief hypothermic oxygenation prior to warm reperfusion. Macrovesicular steatosis was induced in rat livers by fasting and subsequent feeding of a fat-free diet enriched with carbohydrates. Fatty livers were retrieved and stored ischemically at 4 degrees C for 20 hours in histidine-tryptophan-ketoglutarate solution. Hypothermic reconditioning (HR) was performed in some livers by insufflation of gaseous oxygen via the caval vein during the last 90 minutes of preservation. Viability was assessed upon isolated reperfusion. HR resulted in a significant (approximately 5-fold) reduction of parenchymal (alanine aminotransferase and lactate dehydrogenase) and mitochondrial (glutamate dehydrogenase) enzyme release. Functional recovery (bile production, oxygen consumption, and tissue levels of adenosine triphosphate) was significantly improved by HR. In untreated grafts, cellular autophagy (cleavage of LC3B and protein expression of beclin-1) was significantly impaired (<50% of baseline) after preservation/reperfusion but was restored to normal values by HR. HR also increased cleavage of caspase 9 (P < 0.5) and caspase 3 enzyme activity (by a factor of 1.5). In contrast, histological signs of tissue necrosis were abundant after reperfusion in untreated livers and largely abrogated in reconditioned livers. In conclusion, HR limits mitochondrial defects and restores basal rates of cellular autophagy. This may represent a rescue mechanism for maintaining cellular homeostasis and tissue survival.

Energy charge restoration, mitochondrial protection and reversal of preservation induced liver injury by hypothermic oxygenation prior to reperfusion

Background We investigated the benefit of two different techniques for resuscitating marginally preserved liver grafts, unexpectedly subjected to long storage times. Methods Rat livers were cold-stored for 22 h (CS22). Some grafts were subsequently subjected to 90 min of hypothermic reconditioning by venous systemic oxygen persufflation (VSOP) or oxygenated hypothermic machine perfusion (HMP). Livers stored for only 6 h (CS6) served as reference. Viability of the livers was assessed thereafter by warm reperfusion in vitro. Results VSOP and HMP significantly increased endischemic tissue energy charge, and abrogated cellular enzyme loss upon reperfusion even significantly below control values. Ammonia clearance and bile production were more than 3-fold improved to similar values as CS6. Hypothermic reconditioning by both techniques induced mitochondrial chaperone expression (HSP70 family) and significantly improved early resumption of oxygen utilisation upon reperfusion. Conclusion Viability of long preserved liver grafts can be augmented by transient hypothermic reconditioning using either machine perfusion or gaseous oxygen persufflation, both preventing initial mitochondrial dysfunction and subsequent tissue injury.