The dynamics of cytokine release during 24 hours continuous normothermic machine perfusion liver preservation: An explorative porcine study.

BACKGROUND: Normothermic machine perfusion (NMP) has been proposed to preserve liver grafts in a less pro-inflammatory environment. However, the effect of NMP on liver inflammation remains unclear. Therefore, we aimed at characterizing the inflammatory response during continuous NMP with a comprehensive investigation of cytokine release during perfusion. METHODS: Ten porcine livers underwent either 24 h NMP or whole blood-based NMP (WB-NMP) immediately after procurement. WB-NMP was used as a positive control to mimic early post-reperfusion inflammation. High mobility group box-1 (HMGB1), interleukin 1-beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha), interleukin 6 (IL-6), 8 (IL-8), and 10 (IL-10), transforming growth factor-beta (TGFbeta), aspartate transferase (AST), and hyaluronic acid were measured in the perfusate. The area under the curve (AUC) of their perfusate concentration was compared between groups. Median (IQR) is given. RESULTS: The AUC of HMGB1 and IL-1beta was similar between groups. Compared to WB-NMP, NMP inhibited the release of TNFalpha [NMP: 20275 (18402-32 152), WB-NMP: 242100 (203511-244 238); p = 0.01], IL-6 [NMP: 1206 (338.9-1686), WB-NMP: 8444 (7359-10 087); p = 0.03], and IL-8 [NMP: 1635 (106.90-2130), WB-NMP: 3951 (3090-4116); p = 0.008]. The release of TGFbeta remained unchanged but IL-10 release was lower in NMP [1612 (1313-1916), WB-NMP: 5591 (4312-6421); p = 0.01]. The ratios TGFbeta:TNFalpha and IL-10:TNFalpha were significantly higher in the NMP than in the WB-NMP group. Importantly, the AUC of AST was significantly lower during NMP [1960 (1950-2893)] than WB-NMP [6812 (6370-7916); p = 0.02]. CONCLUSIONS: Continuous NMP leads to the release of detectable levels of cytokines with a slow, linear increase over time and a shift toward anti-inflammatory signaling.

The Distinct Innate Immune Response of Warm Ischemic Injured Livers during Continuous Normothermic Machine Perfusion.

Although normothermic machine perfusion (NMP) provides superior preservation of liver grafts compared to static cold storage and allows for viability testing of high-risk grafts, its effect on the liver immune compartment remains unclear. We investigated the innate immune response during 6 h of continuous NMP (cNMP) of livers that were directly procured (DP, n = 5) or procured after 60 min warm ischemia (WI, n = 5), followed by 12 h of whole blood (WB) reperfusion. WI livers showed elevated transaminase levels during cNMP but not after WB reperfusion. Perfusate concentrations of TNF-α were lower in WI livers during cNMP and WB reperfusion, whereas IL-8 concentrations did not differ significantly. TGF-ß concentrations were higher in WI livers during NMP but not after WB reperfusion, whereas IL-10 concentrations were similar. Endoplasmic stress and apoptotic signaling were increased in WI livers during cNMP but not after WB reperfusion. Additionally, neutrophil mobilization increased to a significantly lesser extent in WI livers at the end of NMP. In conclusion, WI livers exhibit a distinct innate immune response during cNMP compared to DP livers. The cytokine profile shifted towards an anti-inflammatory phenotype during cNMP and WB reperfusion, and pro-apoptotic signaling was stronger during cNMP. During WB reperfusion, livers exhibited a blunted cytokine release, regardless of ischemic damage, supporting the potential reconditioning effect of cNMP.

Porcine Normothermic Isolated Liver Perfusion.

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost. In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 °C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.

Porcine Liver Normothermic Machine Perfusion: Methodological Framework and Potential Pitfalls.

Porcine models of liver normothermic machine perfusion (NMP) are increasingly used in transplant research, although known to be challenging because of their complex methodology and their scarcely documented operational aspects. Here, we aimed to provide a methodological framework for researchers looking to adopt NMP technology in research setting by giving an in-detail account of the implementation of a previously validated porcine liver NMP model. We subjected groups of 3-5 porcine livers to 24 h NMP and, using a trial-and-error principle, introduced stepwise changes in the NMP setting with the objective to obtain stable preservation of liver function and histology for 24 h. Female porcine livers were procured, and packed red-blood-cell perfusate was prepared. Perfusate oxygenation, hemodynamics, markers of hepatic injury (aspartate transaminase [AST]), function (lactate, perfusate pH, bile production), and histology were analyzed. Intermediate analysis was performed within groups and a minimum of 3 (out of 5) failed experiments prompted methodological reevaluation. Overall, 13 liver NMP experiments were needed in 3 phases. In phase 1, loss of oxygenator performance occurred from 6 h onward in 3 consecutive experiments because of perfusate leakage. In phase 2, a plasma-tight hollow fiber oxygenator ensured adequate perfusate oxygenation in 5 experiments. However, portal vein resistance increased during all liver NMP, associated with high perfusate AST levels (range, 106-322 IU/L/100 g) and pan-lobular sinusoidal dilation and hemorrhage, suggesting liver outflow impairment. In phase 3, an improved inferior vena cava cannulation technique avoided liver outflow impairment, resulting in lower AST release (range, 29-101 IU/L/100 g), improved lactate clearance, preserved biliary excretion, and normal histology in 5 experiments. This study underscores the critical importance of auditing all equipment and operational components of NMP circuits to obtain successful and reproducible perfusion setup and advocates for in-detail reporting of methodological aspects and potential pitfalls.

Hepatocellular uptake of cyclodextrin-complexed curcumin during liver preservation: A feasibility study.

The increasing demand for donor organs and the decreasing organ quality is prompting research toward new methods to reduce ischemia reperfusion injury (IRI). Several strategies have been proposed to protect preserved organs from this injury. Before curcumin/dextrin complex (CDC), a potent antioxidant and anti-inflammatory agent, can be used clinically we need to better understand the intracellular uptake under hypothermic conditions on a rat model of liver donation after circulatory death (DCD) and brain death (DBD). To be able to use the fluorescence of CDC for quantification the stability of CDC in different preservation solutions at 4°C or 37°C was investigated. Livers from Wistar rats were procured after being flushed-out through the portal vein using CDC-enriched preservation solutions and stored at 4°C for variable periods. The CDC signal was stable in different preservation solutions over a period of 4 h and allowed the rapid and lasting uptake of curcumin into cells. After 4 h of preservation, CDC was no longer visible microscopically, and HPLC analysis showed very low to non-detectable tissue levels of CDC, proving metabolization during preservation. However, the distribution of CDC was not affected by warm ischemia damage (p = 0.278) nor by flushing the livers before or after 4 h of cold storage and without a warm preflush. Finally, curcumin reduced oxidative stress, lowered histological injury and did not change gene expression after WI/cold storage. Therefore, the use of CDC flush solution for the initial organ flush can offer a promising approach to the enhancement of liver preservation and the maintenance of its quality.

Plasmatic Villin 1 Is a Novel In Vivo Marker of Proximal Tubular Cell Injury During Renal Ischemia-Reperfusion.

BACKGROUND: Early detection of acute kidney injury (AKI), a common condition with a high mortality risk, can be facilitated by specific and reliable biomarkers. Villin 1, a protein typically found in the brush borders of proximal tubular cells, has been detected in urine of patients with AKI, but its possible release in plasma remains unexplored. METHODS: We measured the presence of villin 1 by immunohistochemistry on kidney sections and by Western blotting in plasma samples from rats subjected to renal ischemia-reperfusion injury, pigs subjected to renal transplantation and liver transplantation patients that developed AKI. Moreover, rats were treated with necrostatin-1, an inhibitor of programmed necrosis (necroptosis), which occurs in renal tubular cells during AKI. Villin 1 levels were compared with other renal injury markers (creatinine, aspartate transaminase, and heart-type fatty acid binding protein). RESULTS: During AKI, plasmatic villin 1 levels corresponded with the severity of kidney injury and dysfunction. Its detection in plasma was associated with a redistribution in the kidney tissue. Unlike the levels of other markers, plasmatic villin 1 decreased already after a short (3 hours) treatment with necrostatin-1 during renal ischemia-reperfusion injury. The presence of plasmatic villin 1 was confirmed in patients who experienced AKI after liver transplantation. CONCLUSIONS: Villin 1 is released in plasma during AKI and shows potential as an early marker for proximal tubular injury/necrosis and warrants further investigation.

Assessing warm ischemic injury of pig livers at hypothermic machine perfusion.

BACKGROUND: Livers originating from donation after circulatory death (DCD) donors are exposed to warm ischemia (WI) before liver transplantation (LTx). Currently, there are no objective tests to evaluate the damage sustained before LTx. This study aims to identify surrogate markers for liver injury that can be assessed during hypothermic machine perfusion (HMP) preservation. In addition, we want to use mathematical equation modeling combining these markers to improve our assessment of DCD livers for transplantation. MATERIALS AND METHODS: Porcine livers were exposed to incremental periods of WI (0-120 min) and subsequently HMP preserved for 4 h. Biochemical and hemodynamic parameters were repeatedly measured in the perfusate during HMP. Subsequently, to mimic LTx, normothermic isolated-liver perfusion was applied for 2 h and the injury assessed using a morphological score. RESULTS: With increasing WI periods, the perfusate became more acidotic, and levels of aspartate aminotransferase (AST), liver fatty acid binding protein, redox-active iron, and arterial vascular resistance increased. A damage index, combining AST and pH (damage index = 2 - 37 × ß(AST) - 257 × ß(pH)) based on multifactorial analysis of the changing pattern of these markers, had increased sensitivity and specificity to reflect WI and reperfusion injury. CONCLUSIONS: This proof of concept study demonstrated the potential role for objective evaluation of DCD porcine livers during HMP and the advantage to use multifactorial analysis on the markers' changing pattern.

Multifactorial biological modulation of warm ischemia reperfusion injury in liver transplantation from non-heart-beating donors eliminates primary nonfunction and reduces bile salt toxicity.

OBJECTIVE: To design a multifactorial biological modulation approach targeting ischemia reperfusion injury to augment viability of porcine liver grafts from non-heart-beating donors (NHBD). BACKGROUND DATA: Liver Transplantation (LTx) from NHBD is associated with an increased risk of primary nonfunction (PNF) and biliary complications. In porcine NHBD-LTx, we previously reported a 50% risk of PNF and toxic bile formation in grafts exposed to > or =30' warm ischemia (WI). METHODS: Porcine livers exposed to 45' WI were cold stored, transplanted and either modulated (n = 6) or not (controls, n = 9). In the modulation group, donor livers were flushed with warm Ringers (avoiding cold-induced vasoconstriction), streptokinase (eliminating stagnating thrombi), and epoprostenol (vasodilator, platelet aggregation inhibitor) prior to cold storage. In recipients, glycine (Kupffer cell stabilizer), alpha1-acid-glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory cytokine generation inhibitor), alpha-tocopherol and glutathione (anti-oxidants), and apotransferrin (iron chelator) were administrated intravenously. PNF, survival, lactate, transaminase, TNF-alpha, redox-active iron, and biliary bile salt-to-phospholipid ratio were monitored. RESULTS: No PNF was observed in modulated versus 55% in control pigs (P = 0.025). Survival was 83% in modulated versus 22% in control pigs (P = 0.02). At 180' postreperfusion, lactate was lower in modulated (5.4 +/- 1.9 mmol/L) versus control pigs (9.4 +/- 2.2 mmol/L; P = 0.011). At 60' postreperfusion, there was a trend for lower AST in modulated versus control pigs at 60' (939 +/- 578 vs. 1683 +/- 873 IU/L; P = 0.089). Postreperfusion, TNF-alpha remained stable in modulated pigs (49 +/- 27 pg/mL at 15' and 85 +/- 26 pg/mL at 180'; P = 0.399) but increased in control pigs (107 +/- 36 pg/mL at 15' and 499 +/- 216 pg/mL at 180'; P = 0.023). At 180' postreperfusion, redox-active iron was higher in control pigs versus modulated pigs (0.21+/-0.18 vs. 0.042+/-0.062 mum; P = 0.038). Biliary bile salt-to-phospholipid ratio post-LTx was lower in modulated versus control pigs (1128 +/- 447 vs. 4836 +/- 4619; P = 0.05). CONCLUSIONS: A multifactorial biological modulation eliminates PNF, improves liver function and increases survival. Biochemically, TNF-alpha and redox-active iron are suppressed and biliary bile salt toxicity is reduced. Translating this strategy clinically may lead to wider and safer use of NHBD.