Andrea Ferrigno

Subnormothermic machine perfusion protects steatotic livers against preservation injury: A potential for donor pool increase?†

We tested whether rat liver preservation performed by machine perfusion (MP) at 20°C can enhance the functional integrity of steatotic livers versus simple cold storage. We also compared MP at 20°C with hypothermic MP at 8°C, and 4°C. Obese and lean male Zucker rats were used as liver donors. MP was performed for 6 hours with a glucose and N‐acetylcysteine–supplemented Krebs‐Henseleit solution. Both MP and cold storage preserved livers were reperfused with Krebs‐Henseleit solution (2 hours at 37°C). MP at 4°C and 8°C reduced the fatty liver necrosis compared with cold storage but we further protected the organs using MP at 20°C. Necrosis did not differ in livers from lean animals submitted to the different procedures; the enzymes released in steatotic livers preserved by MP at 20°C were similar to those showed in nonsteatotic organs. The adenosine triphosphate/adenosine diphosphate ratio and bile production were higher and the oxidative stress and biliary enzymes were lower in steatotic livers preserved by MP at 20°C as compared with cold storage. In livers from lean rats, the adenosine triphosphate/adenosine diphosphate ratio appears better conserved by MP at 20°C as compared with cold storage. In steatotic livers preserved by cold storage, a 2‐fold increase in tumor necrosis factor‐alpha levels and caspase‐3 activity was observed as compared with organs preserved by MP at 20°C. These data are substantiated by better morphology, higher glycogen content, and lower reactive oxygen species production by sinusoidal cells in steatotic liver submitted to MP at 20°C versus cold storage. MP at 20°C improves cell survival and leads to a marked improvement in hepatic preservation of steatotic livers as compared with cold storage. Liver Transpl 15:20–29, 2009.

Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions

The contribution of endogenous fluorophores - such as proteins, bound and free NAD(P)H, flavins, vitamin A, arachidonic acid - to the liver autofluorescence was studied on tissue homogenate extracts and on isolated hepatocytes by means of spectrofluorometric analysis. Autofluorescence spectral analysis was then applied to investigate the response of single living hepatocytes to experimental conditions resembling the various phases of the organ transplantation. The following conditions were considered: 1 h after cells isolation (reference condition); cold hypoxia; rewarming-reoxygenation after cold preservation. The main alterations occurred for NAD(P)H and flavins, the coenzymes strictly involved in energetic metabolism. During cold hypoxia NAD(P)H, mainly the bound form, showed an increase followed by a slow decrease, in agreement with the inability of the respiratory chain to reoxidize the coenzyme, and a subsequent NADH reoxidation through alternative anaerobic metabolic pathways. Both bound/free NAD(P)H and total NAD(P)H/flavin ratio values were altered during cold hypoxia, but approached the reference condition values after rewarming-reoxygenation, indicating the cells capability to restore the basal redox equilibrium. A decrease of arachidonic acid and vitamin A contributions occurred after cold hypoxia: in the former case it may depend on the balance between deacylation and reacylation of fatty acids, in the latter it might be related to the vitamin A antioxidant role. An influence of physico-chemical status and microenvironment on the fluorescence efficiency of these fluorophores cannot be excluded. In general, all the changes observed for cell autofluorescence properties were consistent with the complex metabolic pathways providing for energy supply.

Machine perfusion at 20 °C reduces preservation damage to livers from non-heart beating donors

We previously reported that machine perfusion (MP) performed at 20°C enhanced the preservation of steatotic rat livers. Here, we tested whether rat livers retrieved 30 min after cardiac arrest (NHBDs) were better protected by MP at 20°C than with cold storage. We compared the recovery of livers from NHBDs with organs obtained from heart beating donors (HBDs) preserved by cold storage. MP technique: livers were perfused for 6h with UW-G modified at 20°C. Cold storage: livers were perfused in situ and preserved with UW solution at 4°C for 6h. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2h at 37°C). AST and LDH release and mitochondrial glutamate dehydrogenase (GDH) levels were evaluated. Parameters assessed included: bile production and biliary enzymes; tissue ATP; reduced and oxidized glutathione (GSH/GSSG); protein-SH group concentration. Livers preserved by MP at 20°C showed significantly lower hepatic damage at the end of reperfusion compared with cold storage. GDH release was significantly reduced and bile production, ATP levels, GSH/GSSG and protein-SH groups were higher in livers preserved by MP at 20°C than with cold storage. The best preserved morphology and high glycogen content was obtained with livers submitted to MP at 20°C. Liver recovery using MP at 20°C was comparable to recovery with HBDs. MP at 20°C improves cell survival and gives a better-quality of preservation for livers obtained from NHBDs and may provide a new method for the successful utilization of marginal livers.

Exogenous melatonin enhances bile flow and ATP levels after cold storage and reperfusion in rat liver: Implications for liver transplantation

Abstract:   Although the use of melatonin in the transplantation field has been suggested, it has not been previously tested in a liver cold‐storage model. We used a rat liver model to study (a) the dose‐dependent effect of melatonin on bile production, and (b) the potential of melatonin to improve liver function after cold‐storage. Male Wistar rats were perfused with Krebs–Henseleit bicarbonate buffer (KHB) at 37°C without or with 25, 50, 100 and 200 μm melatonin. Each dose of melatonin stimulated bile production. For cold‐storage studies, livers were flushed with either University of Wisconsin (UW) or Celsior solution and stored for 20 hr at 4°C. Reperfusion (120 min) was performed with KHB at 37°C. In subsequent studies, 100 μm melatonin were added to the perfusate during the reperfusion period. ATP and melatonin levels in the tissue were measured. Bile analysis was performed by measuring melatonin, bilirubin and gamma‐glutamyl transpeptidase (γ‐GT) levels in the fluid. A dose‐dependent increase in bile secretion, associated with an enhanced melatonin and bilirubin levels in the bile were observed. Also, tissue levels of melatonin increased in a dose‐dependent manner. When melatonin was added during the reperfusion period, bile production and bile bilirubin levels increased both with UW and Celsior solutions. The analysis of γ‐GT in the bile showed an increase in the Celsior‐preserved liver and the addition of melatonin to the perfusate reduced this effect. Tissue ATP levels were higher when melatonin was added to the perfusion medium. Higher levels of melatonin in bile than in tissue were found. In conclusion, we demonstrate that melatonin improves significantly the restoration of liver function after cold‐storage and reperfusion.

Correlation between the liver temperature employed during machine perfusion and reperfusion damage: Role of Ca2+

This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, the role of Ca2+ levels in the MP medium was evaluated. For MP, rat livers were perfused for 6 hours with Krebs‐Henseleit (KH) solution (with 1.25 or 2.5 mM CaCl2) at 4°C, 10°C, 20°C, 25°C, 30°C, or 37°C. For cold storage, livers were perfused in situ and preserved with Celsior solution at 4°C for 6 hours. The reperfusion period (2 hours at 37°C) was performed under the same conditions used for MP‐preserved and cold storage–preserved livers. Hepatic enzyme release, bile production, adenosine triphosphate (ATP) levels, and morphology were evaluated during MP and reperfusion. MP at 37°C caused marked enzyme release; the same findings were obtained during reperfusion. By contrast, MP temperature lowering induced a significant decrease in liver damage. High levels of biliary gamma‐glutamyltransferase and lactate dehydrogenase were found with MP at 4°C and 10°C but not with MP at 20°C. When a KH–1.25 mM CaCl2 solution was used during MP at 20°C, very low enzyme release was observed and significantly lower hepatic damage was present at the end of the reperfusion period in comparison with cold storage. The same results were obtained when ruthenium red, a calcium uniporter blocker, was added to KH–2.5 mM CaCl2. ATP levels were higher and morphology was better in liver preserved with KH–1.25 mM CaCl2. MP at 20°C with KH–1.25 mM CaCl2 resulted in better quality liver preservation, improving hepatocyte and endothelial biliary cell survival, in comparison with cold storage. This raises the need to reconsider the temperature and calcium levels to be used during liver MP. Liver Transpl 14:494–503, 2008.

Troubleshooting and improving the mouse and rat isolated perfused liver preparation.

INTRODUCTION Isolated perfused liver (IPL) model is not only widely performed in rats but is also used in mouse liver, although a detailed description of this procedure is absent. A comparison of the different techniques used on rats and mice will be discussed in this article association with a detailed description of the surgical and technical aspects needed to obtain and maintain the integrity of the livers during the organ isolation and perfusion. METHODS The surgical procedures, the IPL set-up, and the evaluation of hepatic function and damage will be described in relation to both rats and mice. In particular, the heparin dosage and administration, the portal vein cannulation avoiding portal leakage, the use of suprahepatic caval vein output, and the insertion of a cannula for bile collection will be reported. For the settings, the perfusion circuit, the perfusion solution, the temperature and the flow rate will be described, with particular regard to the balance between perfusion pressure and oxygen delivery. The monitoring of liver integrity by measuring oxygen concentration and calculating oxygen delivery rate and oxygen uptake rate, and recommendations for the collection of perfusate and bile samples will be considered. Accurate pH measurement with normalization, and the perfusion portal pressure assay by a calibrated water manometer will be also reported. RESULTS AND DISCUSSION This work analyzes the parameters crucial to performing a correct IPL both in rat and mouse, comparing our experience with the equivalent practice from other laboratories. An updated example of IPL applications in liver toxicology and pharmacology, physiology and pathophysiology, and liver graft preservation will be briefly presented, underlining how this technique provides essential information allowing a more accurate planning of the in vivo studies.

Dipeptidylpeptidase-IV, a key enzyme for the degradation of incretins and neuropeptides: activity and expression in the liver of lean and obese rats

Given the scarcity of donors, moderately fatty livers (FLs) are currently being considered as possible grafts for orthotopic liver transplantation (OLT), notwithstanding their poor tolerance to conventional cold preservation. The behaviour of parenchymal and sinusoidal liver cells during transplantation is being studied worldwide. Much less attention has been paid to the biliary tree, although this is considered the Achilles heel even of normal liver transplantation. To evaluate the response of the biliary compartment of FLs to the various phases of OLT reliable markers are necessary. Previously we demonstrated that Alkaline Phosphatase was scarcely active in bile canaliculi of FLs and thus ruled it out as a marker. As an alternative, dipeptidylpeptidase-IV (DPP-IV), was investigated. This ecto-peptidase plays an important role in glucose metabolism, rapidly inactivating insulin secreting hormones (incretins) that are important regulators of glucose metabolism. DPP-IV inhibitors are indeed used to treat Type II diabetes. Neuropeptides regulating bile transport and composition are further important substrates of DPP-IV in the enterohepatic axis. DPP-IV activity was investigated with an azo-coupling method in the liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. Protein expression was studied by immunofluorescence with the monoclonal antibody (clone 5E8). In Wistar rat liver, DPP-IV activity and expression were high in the whole biliary tree, and moderate in sinusoid endothelial cells, in agreement with the literature. Main substrates of DPP-IV in hepatocytes and cholangiocytes could be incretins GLP-1 and GIP, and neuropeptides such as vasoactive intestinal peptide (VIP) and substance P, suggesting that these substances are inactivated or modified through the biliary route. In lean Zucker rat liver the enzyme reaction and protein expression patterns were similar to those of Wistar rat. In obese rat liver the patterns of DPP-IV activity and expression in hepatocytes reflected the morphological alterations induced by steatosis as lipid-rich hepatocytes had scarce activity, located either in deformed bile canaliculi or in the sinusoidal and lateral domains of the plasma membrane. These findings suggest that bile canaliculi in steatotic cells have an impaired capacity to inactivate incretins and neuropeptides. Incretin and/or neuropeptide deregulation is indeed thought to play important roles in obesity and insulin-resistance. No alteration in enzyme activity and expression was found in the upper segments of the biliary tree of obese respect to lean Zucker and Wistar rats. In conclusion, this research demonstrates that DPP-IV is a promising in situ marker of biliary functionality not only of normal but also of fatty rats. The approach, initially devised to investigate the behaviour of the liver during the various phases of transplantation, appears to have a much higher potentiality as it could be further exploited to investigate any pathological or stressful conditions involving the biliary tract (i.e., metabolic syndrome and cholestasis) and the response of the biliary tract to therapy and/or to surgery.

Decreased apoptosis in fatty livers submitted to subnormothermic machine-perfusion respect to cold storage

Machine perfusion at subnormothermic temperature (20°C), MP20, was developed by Vairetti et al. and showed to afford a better preservation of fatty livers respect to traditional cold storage (CS) in terms of enzyme release into the perfusate, bile production, glycogen stores, energy charge and oxidative stress. Here we investigated whether it also caused decreased cell death by apoptosis. Fatty and lean Zucker rats were submitted to MP20 or CS for 6 h and reperfused normothermically for 2 h. Apoptotic cells were revealed by immunohistochemistry of activated caspase-3 and M30 (new epitope on CK18 degraded by caspase-3) and by the TUNEL assay. Portal pressure was also determined. A statistically significant reduction of hepatocyte apoptosis, but especially of sinusoidal cells was determined for fatty livers submitted to MP20 respect to CS. Portal pressure was significantly lower after MP20 respect to CS. The reduction of sinusoidal cell death by apoptosis without need for anti-apoptotic therapies appears particularly positive since apoptotic sinusoidal cells hinder microcirculation in the sinusoids and are thrombogenic. These results further confirm the potential of MP20 for preserving fatty livers that would be otherwise discarded as grafts, and thus for increasing the donor pool for liver transplantation.

Different susceptibility of liver grafts from lean and obese Zucker rats to preservation injury.

We compared the susceptibility of liver grafts from lean and obese Zucker rats to preservation injury, using two organ-preservation techniques: conventional static preservation (SP) and machine perfusion (MP) preservation. SP: livers preserved by UW solution at 4, 8 or 20 degrees C for 6-h. MP: livers perfused for 6-h with an improved oxygenated Krebs-Henseleit solution (KH) at 4, 8 or 20 degrees C. Reperfusion with KH (2-h) was performed either with the SP or MP preserved livers. Fatty livers tolerate SP poorly at 4, 8 and 20 degrees C as compared with MP at the same temperatures. SP induced a decrease in the ATP/ADP ratio both at 8 and 20 degrees C in obese rats while an increase in energy status was found with MP at 8 and 20 degrees C. Nitrate/nitrite (NOx) concentration was higher and bile flow lower in livers preserved with SP than MP. In lean rats, no differences were observed between MP and SP as regards enzyme release, bile production and NOx levels except for SP at 20 degrees C in which high enzyme release and low bile flow were observed. In lean rats ATP/ADP was higher and NOx was lower with MP at 20 degrees C than with SP at 20 degrees C. To optimize steatotic liver preservation SP should be avoided because it is particularly detrimental as compared with MP.

Bilirubin: an autofluorescence bile biomarker for liver functionality monitoring.

Excitation at 366-465 nm of bilirubin in aqueous solution with solubilizing agents results in emission spectra composed by two main bands. The variation of their relative contributions as shown by changes in the spectral shape are consistent with the bilirubin bichromophore nature. This latter accounts for an exciton-coupling phenomenon, intramolecular interchromophore energy transfer efficiency being affected by microenvironment. Excitation at 366 nm, despite the poor absorption of bilirubin, gives rise to appreciable emission signals from both pure compounds and bile - collected from functionally altered rat livers - favouring the spectral shape response to environment and molecular conformation changes. As compared to the merely bile flow estimation, real-time detection of fluorescence, revealing composition variations, improves near-UV optical-biopsy diagnostic potential in hepatology.