Eleonora Boncompagni

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.

Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury

Abstract:  Chronic organ‐donor shortage has required the acceptance of steatotic livers for transplantation purposes despite the higher risk of graft dysfunction or nonfunction associated with the cold ischemia–reperfusion injury. This study evaluated the use of melatonin as an additive to Institute Georges Lopez (IGL‐1) solution for protecting nonsteatotic and steatotic liver grafts against cold ischemia–reperfusion injury. In the current investigation, we used an ex vivo isolated perfused rat liver model. Steatotic and nonsteatotic livers were preserved for 24 hr (4°C) in University of Wisconsin or IGL‐1 solutions with or without melatonin, as well as in University of Wisconsin solution alone. Thereafter, livers were subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (transaminases) and function [bile production and sulfobromophthalein (BSP) clearance, vascular resistance], as well as other factors potentially implicated in the high vulnerability of steatotic livers against ischemia–reperfusion injury (oxidative stress and related inflammatory mediators including nitric oxide and cytokines). We also evaluated well‐known cytoprotective factors as hemeoxygenase 1 (HO‐1). Fatty livers preserved in IGL‐1 solution enriched with melatonin showed lower transaminase levels and higher bile production and BSP clearance when compared to those obtained for livers maintained in IGL‐1 solution alone. A significant diminution of vascular resistance was also observed when melatonin was added to the IGL‐1 solution. The melatonin benefits correlated with the generation of nitric oxide (through constitutive e‐NOS activation) and the prevention of oxidative stress and inflammatory cytokine release including tumor necrosis factor and adiponectin, respectively. The addition of melatonin to IGL‐1 solution improved nonsteatotic and steatotic liver graft preservation, limiting their risk against cold ischemia–reperfusion injury.

Long-term trends of heron and egret populations in Italy, and the effects of climate, human-induced mortality, and habitat on population dynamics

Factors affecting bird population dynamics include climate, harvesting by humans, and habitat changes. Here, we describe the long-term (1972–2006) population trends of seven heron species in NW Italy, an area holding important European breeding populations of these species. Grey (Ardea cinerea), purple (Ardea purpurea), and squacco (Ardeola ralloides) herons, and little egrets (Egretta garzetta) exhibited a strong logistic increase, leveling off around year 2000 at 3–23 times their initial level. Black-crowned night herons (Nycticorax nycticorax) began by increasing like the former species but then dropped to initial levels. Such trends were found to be influenced by several candidate ecological factors, as assessed by ARIMA models. Specifically, grey herons increased following a decrease in human-induced mortality, as quantified by an index of hunting pressure, and an increase in winter temperatures. Little egrets increased mainly with the increase of the extent of ricefields, whereas squacco herons increased with increasing rainfall in the African wintering range. Black-crowned night herons were also positively affected by increasing African rainfall, but only during 1972–1988, whereas in later years competition with other herons could have affected the species’ decline. The improved protection of colony sites by special reserves was unlikely to be the primary trigger of the observed increase, although obviously important for the long-term population persistence. In conclusion, our study shows that heron populations of southern Europe are sensitive to environmental and climatic changes, as well as to temporal variation in human disturbance and changes in foraging habitats, though the importance of the different factors differs among species.

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.

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.

Human liver autofluorescence: an intrinsic tissue parameter discriminating normal and diseased conditions.

Autofluorescence (AF) emission is an intrinsic parameter that can provide real‐time information on morpho‐functional properties of biological tissue, being strictly related with their biochemical composition and structural organization. The diagnostic potentials of AF‐based techniques have been investigated on normal, fibrotic, and steatotic liver tissues, in reference to histological features as evidenced by specific histochemical stainings.

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.

Exposure to heptachlor: evaluation of the effects on the larval and adult epidermis of Rana kl. esculenta.

Widely used in the past against termites and soil insects, the chlorinated insecticide heptachlor (H) is a toxic contaminant which represents a risk for both terrestrial and aquatic organisms. Like many organochlorine pesticides, heptachlor and heptachlor epoxide (HE), with oxidation products synthesized by many plant and animal species, degrade slowly since many of the derived compounds are persistent. This increases the status of heptachlor as a hazardous pollutant. In the present experimental study we exposed specimens of Rana kl. esculenta, from the tadpole stage through to their complete metamorphosis, to three different concentrations of heptachlor (4, 40 and 400 ppb). Mortality and HE bioaccumulation were evaluated on all the experimental groups. Since amphibian integument directly interacts with the environmental constituents (water, air and soil), we investigated the toxic effects on the ventral epidermis of both tadpole and adult samples by employing such histo-cytopathological biomarkers as ultrastructural morphology, certain enzyme activities (acid and alkaline phosphatases, AcPase, and AlkPase; succinic dehydrogenase, SDH; alpha-naphtyl butyrate esterase, ANBE; nitric oxide synthase/NADPH diaphorase, NOS/NADPHd). Also, the levels of reactive oxygen species (ROS) in the different conditions were evaluated. The results obtained were of ecological relevance, in particular as regards the effects of this environmental toxicant on the samples of tadpole epidermis. Severe morphological alterations were observed in the larval epidermal cells (apical and skein cells), whereas the cell epidermis (keratinocytes and mitochondria-rich cells) of the adult survivors showed changes in enzyme activities, particularly those involved in the protective response to xenobiotic injury. In general, morpho-histochemical studies, analysis of HE bioaccumulation and mortality showed a relation to the H doses employed.

The use of a reversible proteasome inhibitor in a model of Reduced-Size Orthotopic Liver transplantation in rats☆

Ischemia/reperfusion injury (IRI), inherent in liver transplantation (LT), is the main cause of initial deficiencies and primary non-function of liver allografts. Living-related LT was developed to alleviate the mortality resulting from the scarcity of suitable deceased grafts. The main problem in using living-related LT for adults is graft size disparity. In this study we propose for the first time that the use of a proteasome inhibitor (Bortezomib) treatment could improve liver regeneration and reduce IRI after Reduced-Size Orthotopic Liver transplantation (ROLT). Rat liver grafts were reduced by removing the left lateral lobe and the two caudate lobes and preserved in UW or IGL-1 preservation solution for 1h liver and then subjected to ROLT with or without Bortezomib treatment. Our results show that Bortezomib reduces IRI after LT and is correlated with a reduction in mitochondrial damage, oxidative stress and endoplasmic reticulum stress. Furthermore, Bortezomib also increased liver regeneration after reduced-size LT and increased the expression of well-known ischemia/reperfusion protective proteins such as nitric oxide synthase, heme oxigenase 1 (HO-1) and Heat Shock Protein 70. Our results open new possibilities for the study of alternative therapeutic strategies aimed at reducing IRI and increasing liver regeneration after LT. It is hoped that the results of our study will contribute towards improving the understanding of the molecular processes involved in IRI and liver regeneration, and therefore help to improve the outcome of this type of LT in the future.