[Carbon dioxide embolization: is it a complication of laparoscopic cholecystectomy? ]. / Embolia gassosa da biossido di carbonio: è una complicanza della colecistectomia laparoscopica?

The occurrence of pulmonary gas embolism in patients undergoing laparoscopic cholecystectomy is reported in the medical literature. Severe intraoperative complications or the patient's death were correlated to gas embolism during laparoscopic procedures. However, the careful retrospective study or the autoptic exam of such casualties have always showed an erroneus direct puncture of vessels or the straight insertion of the Veress needle into a parenchymal organ. It is obvious that the direct gas injection into a vein or into parenchymal organs is a primary cause of gas embolism, as well as the high flow insufflation of gas into the peritoneal cavity in concomitance with the lesion of major abdominal vessel's wall. Gas embolism may occur each time the vein internal pressure is lower than the external pressure and not only during a laparoscopic procedure when carbon dioxyde is inflated into the peritoneal cavity, but also during open surgery such as major liver resections, neurosurgery, vascular or cardiac surgery. The review of large series of laparoscopic cholecystectomies reported in the international literature, as well as our own clinical experience in this field, together with the results of laboratory animal studies based on the experimental insufflation or injection of carbon dioxyde, show that gas embolism must not be considered as a complication of laparoscopic surgery. Due to the above mentioned risks with the use of the Veress needle, the surgeon should revalue alternative means in creating the pneumoperitoneum.

Retinoic acid maintains differentiated cell morphology and functions in long-term cultured porcine hepatocytes: obtaining functional cells for prolonged treatments with bioartificial liver.

Porcine hepatocytes show several immunological characteristics and enzymatic activities of human liver, representing an ideal xenogenic source of cells as biological component of bioartificial liver (BAL). Isolated hepatocytes rapidly lose their specific metabolic activities and their typical morphology when cultured in the presence of serum. Since in BAL porcine hepatocytes are perfused by the patient's plasma, procedures able to minimize de-differentiation of cells could be useful for long-term treatment of acute liver failure (ALF). In this work we found that, in the presence of micromolar concentration of All trans-retinoic acid (ATRA), porcine parenchymal liver cells undergo to a lower extent the de-differentiating effects of long-term culture in the presence of serum. The evaluation of lidocaine metabolism showed that ATRA-treated cells retain specific hepatocyte function for a significantly longer time when compared to control hepatocytes. A tyrosine phosphorylation of PLC-gamma1 was observed in concomitance with the ATRA-induced maximal functional activity. An increased expression of PLC-beta3 and PKC-alpha and -beta2 was also evidentiated at the longer time points explored, when the effects of ATRA in preservation of the differentiated morphology were maximal. These results provide the first evidence that ATRA plays a differentiating role in adult porcine hepatocytes cultured under de-differentiating conditions. The administration of ATRA to isolated parenchymal cells from pig liver may provide functional hepatocytes for prolonged treatment with BAL.

Modulation of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) gene expression in isolated porcine hepatocytes perfused within a radial-flow bioreactor after low-temperature storing.

Due to the scarcity of available human livers, porcine hepatocytes are currently being evaluated as a xenogeneic cell source for extracorporeal bioartificial liver (BAL). Hypothermic storage of isolated porcine hepatocytes could support stocking of cell-loaded bioreactors for BAL use and may provide bioreactors ready to be used at the patient's bedside. For the development of this technology, it is of utmost importance to ensure cell viability and differentiated functions after low-temperature storage and following warm reperfusion. We compared cell viability, functional activity and apoptosis in isolated porcine hepatocytes which were perfused within a radial-flow bioreactor (RFB), stored at 4 degrees C and then reperfused at 37 degrees C. RFBs were loaded with 8 x 10(9), > or = 90% viable hepatocytes at 37 degrees C for 3 h. RFBs were then flushed with 4 degrees C University of Wisconsin solution (UW) and subsequently stored for 24 h or 48 h. RFBs were then reperfused for 8 h with recirculating medium plus serum at 37 degrees C . Cytochrome P450 (CYP) activity was studied before and after cold storage by means of monoethylglycinexylide (MEGX) detection in the effluent medium, after repeated lidocaine injections. After reperfusion experiments, hepatocytes were harvested for total RNA isolation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used in order to amplify specific mRNAs for Bcl-2 and Bax genes, by using appropriate primers; beta-actin primers were used as control. Total RNA was extracted by northern blotting analysis and for Bcl-2, Bax and beta-actin RNA messenger detection, RT-PCR amplification was used. Freshly isolated hepatocytes perfused into the RFB showed a progressive increase of MEGX while a loss in Bax expression was paralleled by an increase in Bcl-2 expression, in comparison to starting hepatocytes. After 4 degrees C storage and warm reperfusion, MEGX production was preserved in 24 h- and 48 h-stored bioreactors as well as a sharp increase of Bcl-2 and a decrease of Bax mRNAs. Our study suggests that refrigeration of hepatocyte-bioreactors is a suitable strategy to maintain both viability and function of isolated hepatocytes, for up to 48 h a time-length that is compatible with long-distance delivery of ready-to-use bioreactors.

Advanced technology for extracorporeal liver support system devices.

Acute Liver Failure (ALF) still presents high mortality rates, and liver transplant is the only treatment with proven efficacy. However transplant is not always possible and systems for Extracorporeal Liver Support (ELS) are being developed which can treat patients with ALF, for whom a transplant is not available, or is delayed. They can also treat patients with chronic liver disease who develop ALF. There are two types of ELS: artificial systems (hemoperfusion, plasmaperfusion, therapeutic plasma exchange, continuous hemodialysis and high volume continuous hemofiltration) and bioartificial systems. These are based on a biological component (animal or human hepatocytes) inserted into a bioreactor, whose main function is to perform the metabolic activity and synthesis that the liver can no longer perform. The results obtained in clinical trials have so far shown that the best results in terms of compensating for lost metabolic function and detoxification are obtained inserting artificial components in the bioartificial circuit.

Biologic liver support: optimal cell source and mass.

Hepatic support is indicated in acute liver failure (ALF) patients to foster liver regeneration, or until a liver becomes available for orthotopic liver transplantation (OLT), in primary non function of the transplanted liver, and hopefully in chronic liver disease patients affected by ALF episodes, in whom OLT is not a therapeutic option. The concept of bioartificial liver (BAL) is based on the assumption that only the hepatocytes can perform the whole spectrum of biotransformation functions, which are needed to prevent hepatic encephalopathy, coma and cerebral edema. Among others, two important issues are related to BAL development: 1) the choice of the cellular component; 2) the cell mass needed to perform an adequate BAL treatment. Primary hepatocytes, of human or animal origin, should be considered the first choice because they express highly differentiated functions. Accordingly, a minimal cell mass corresponding to 10% of a human adult liver, i.e. 150 grams of freshly isolated, > or = 90% viable hepatocytes should be used. When 4 degrees C cold-stored or cryopreserved hepatocytes are used, the cellular mass should be increased because of a drop in cell viability and function. In case of hepatoma-derived cells, cultured cell lines or engineered cells, an adequate functional cell mass should be used, expressing metabolic and biotransformation activities comparable to those of primary hepatocytes. Finally, the use of porcine hepatocytes or other animal cells in BAL devices should be presently directed only to ALF patients as a bridge treatment to OLT, because of potential transmission of animal retrovirus and prions which may potentially cause major pandemics.

Serum bile acids in patients with liver failure supported with a bioartificial liver.

BACKGROUND: Serum bile acids are increased in liver failure, but the composition of the bile acid pool in this condition has not been studied in detail. This information is of interest because of dihydroxy bile acid toxicity. METHODS: We measured serum bile acids by gas chromatography-mass spectrometry in 13 patients with fulminant liver failure and five patients with acute-on-chronic liver failure. Furthermore, serum bile acids were analysed in the same patients after 6 h of treatment with a bioartificial liver, consisting of a hollow-fibre cartridge with microcarrier-attached porcine hepatocytes and a charcoal column. RESULTS: Pre-bioartificial liver serum bile acids demonstrated a high dihydroxy/trihydroxy ratio and were higher in patients with acute-on-chronic liver failure than in those with fulminant liver failure (452.8 +/- 98.6 vs. 182.1 +/- 39.7 micro mol/L; P < 0.05). Bioartificial liver treatment decreased significantly serum bile acids in patients with fulminant liver failure (-38.8%) and acute-on-chronic liver failure (-35.8%), with a decreased dihydroxy/trihydroxy ratio. In vitro, porcine hepatocytes in the bioreactor cleared most conjugated bile acid species from pooled patient plasma. CONCLUSIONS: Acute liver failure is associated with very high serum levels of toxic bile acids that could contribute to the pathogenesis of the syndrome. Bioartificial liver treatment reduces both serum bile acid concentrations and the hydrophobicity of the bile acid pool.

Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients.

Orthotopic liver transplantation (OLT) is the only effective therapeutic modality in severe acute hepatic failure (AHF). The scarcity of organs for transplantation leads to an urgent necessity for temporary liver support treatments in AHF patients. A hepatocyte-based bioartificial liver (BAL) is under investigation with the main purpose to serve as bridging treatment until a liver becomes available for OLT, or to promote spontaneous liver regeneration. We developed a novel radial-flow bioreactor (RFB) for three-dimensional, high-density hepatocyte culture and an integrated pumping apparatus in which, after plasmapheresis, the patient's plasma is recirculated through the hepatocyte-filled RFB. Two hundred thirty grams of freshly isolated porcine hepatocytes were loaded into the RFB for clinical liver support treatment. The BAL system was used 8 times in supporting 7 AHF patients in grade III-IV coma, all waiting for an urgent OLT Three patients with no history of previous liver diseases were affected by fulminant hepatic failure (FHF) due to hepatitis B virus, 3 by primary non-function (PNF) of the transplanted liver, and one by AHF due to previous abdominal trauma and liver surgery. Six out of 7 patients underwent OLT following BAL treatment(s), which lasted 6-24 hours. All patients tolerated the procedures well, as shown by an improvement in the level of encephalopathy, a decrease in serum ammonia, transaminases and an amelioration of the prothrombin time, with full neurological recovery after OLT Our initial clinical experience confirms the safety of this BAL configuration and suggests its clinical efficacy as a temporary liver support system in AHF patients.

Long-term expression of highly differentiated functions by isolated porcine hepatocytes perfused in a radial-flow bioreactor.

To overcome the limitations of standard hollow-fiber module in ensuring efficient cell perfusion and long-term expression of highly differentiated hepatocyte functions, we developed a novel bioreactor in which a three-dimensional hepatocyte culture system was perfused in radial-flow geometry. Isolated porcine hepatocytes were cultured for 2 weeks in recirculating serum-free tissue culture medium, in which NH4Cl and lidocaine were repeatedly added, and ammonia removal, urea synthesis, monoethylglycinexylide (MEGX) production, albumin secretion, Po2, Pco2, O2 consumption, and pH were measured thereafter. During the whole duration of the study, ammonia removal was paralleled by urea production, while MEGX concentration was constantly increased. Our results indicated that hepatocytes remained differentiated and metabolically active throughout the duration of the study. The radial-flow bioreactor allowed physiological contact between recirculating fluid and cells by equalizing the concentration of the perfusing components, including O2, throughout the module, suggesting a potential use of this configuration for extracorporeal liver support.

[Fulminant liver failure: etiopathogenesis and therapy]. / Insufficienza epatica fulminante: eziopatogenesi e terapia.

Fulminant hepatic failure (FHF) is a clinical syndrome characterized by the development of encephalopathy within eight weeks from the onset of the first symptoms, in the absence of previous hepatic disease. It is an uncommon but not rare disease, often fatal but potentially reversible. This article looks at the diverse aetiologies, clinical features, and current medical management, including orthotopic liver transplantation, and auxiliary orthotopic or eterotopic liver transplantation, that are the most recently adopted surgical procedures. Clinical experience with bio-artificial liver support systems of two of the most active research Groups in this field, concludes the paper.

[Acute and fulminant liver failure: experimental models]. / Insufficienza epatica acuta e fulminante: modelli sperimentali.

Fulminant hepatic failure (FHF) is a complex clinical syndrome, with an invariably high mortality rate, that follows many possible and different infectious, pharmacologic and surgical liver injuries. The appearance of the syndrome is similar whatever the etiology, but the mechanisms which lead to the development of FHF are greatly varied. In order to understand the possible pathways which drive to FHF, experimental animal models have been used for a long time. Six requirements should be fulfilled by any FHF animal model: 1) reversibility; 2) reproducibility; 3) death from liver failure; 4) the presence of a therapeutic window; 5) the need of large laboratory animal; 6) minimal hazard to personnel involved in the study. In the present paper a number of models are reported and described, and advantages and disadvantages are discussed. It is concluded that with respect to the aforementioned criteria, no available experimental model is yet as satisfactory as expected.

[Isolated hepatocytes and their potential therapeutic use]. / Epatociti isolati e loro potenziale uso terapeutico.

Isolated hepatocytes in culture represent an idoneous system for the study of liver physiology and metabolism. Furthermore, they are also widely utilized in pharmacological and toxicological study, in evaluating xenobiotic substance effects on the liver. In this paper, we reviewed the enzymatic methods for liver cell isolation in some mammalian species, as well as the techniques for qualitative and quantitative evaluation of cell number, vitality, purity, morphology and function. Recently, there has been a renewed interest in hepatocyte transplantation and hepatocyte-based liver support systems. From a clinical point of view, isolated hepatocytes could be useful in temporarily substituting an acutely damaged liver, a liver affected by a chronic pathology, or to correct an inherited liver disease carrying a severe metabolic derangement. Early experimental results of allogeneic hepatocyte transplantation, as well as the first clinical trials of bioartificial liver support systems employing xenogeneic hepatocytes are promising and contribute to maintain that interest in liver cell isolation and purification methods.

[In vitro experimentation of a new model of radial flow bioreactor containing isolated hepatocytes]. / Sperimentazione in vitro di un nuovo modello di bioreattore a flusso radiale contenente epatociti isolati.

Hepatocyte based artificial liver support systems are under investigation to support acute liver failure patients. The main purpose of such systems is to serve as a bridge to liver transplantation, or to promote spontaneous liver recovery. Limitation in mass-transfer capacity makes hollow-fiber bioreactors unsuited for long-term functioning of hybrid devices. We developed a novel radial-flow bioreactor in which the fluid perfuses the module from the center to the periphery, after having diffused through a space occupied by a three-dimensional structure filled with the hepatocytes. Five grams of freshly isolated porcine hepatocytes were seeded into uncoated, woven-non woven, hydrophilic polyester fabric, overlaid by two polyethersulfone membranes. Liver cells were perfused with 37 degrees C-warm, oxygenated, serum-free tissue culture medium, in which NH4Cl and Lidocaine were added at the final concentration of 1 mM and 60 micrograms/ml, respectively. Ammonium chloride removal, urea synthesis, monoethylglycinexylide (MEGX), pO2, pCO2, and pH were measured throughout the 14 day duration of the study. In a separate set of experiments, a scaled-up version of the radial flow bioreactor containing 150 grams of cells was perfused for 7 h with recirculating human plasma and MEGX production was monitored. During the 2 weeks of the study, an increasing production of urea was paralleled by constant ammonium removal. MEGX concentration after Lidocaine addition increased throughout the 14 days of perfusion with tissue culture medium, as well as after 7 hour perfusion with human plasma. Under transmission and scanning electron microscopy cells appeared attached to the polyester and one to each other, displaying ultrastructural features typical of functioning hepatocytes. Our study showed that liver cells were metabolically active when perfused into the radial-flow bioreactor. This configuration allowed close contact between media, or plasma, and cells at a physiological flow rate, by equalizing the concentration of the perfusing components, including O2, throughout the module. Our results suggest a potential use of this system for temporary extracorporeal liver support in acute hepatic failure patients.

[Hybrid (bioartificial) systems for liver support. History and current developments]. / Sistemi ibridi (bioartificiali) di supporto epatico. Cenni storici e sviluppi attuali.

Attempts to develop liver support systems for the treatment of acute liver failure patients have ranged in the past, from the use of hemodialysis, or plasma exchange, or activated charcoal particles and synthetic resins, as well as the use of bioreactors loaded with liver tissue. However, no system demonstrated a significant improvement of patient survival, nor has achieved a wide clinical use. Liver transplantation remains the only treatment for severe hepatic failure that can improve patient survival. On the other hands, the chronic scarcity of organs for transplantation, leads to an urgent necessity of liver support systems. In this paper, we reviewed the historical experience and current status of artificial liver support systems, with particular emphasis on the so-called hybrid or bioartificial liver, in which to the traditional artificial components, such as selective membranes, charcoal particles and resins, isolated hepatocytes are used.

Biochemical assessment and clinical evaluation of a non-ionic adsorbent resin in patients with intractable jaundice.

We investigated in vitro and in vivo the ability of a non-ionic adsorbing resin (styrenedivinylbenzene copolymer) to remove bilirubin and bile acids from human plasma. In preliminary experiments, human plasma from healthy donors, enriched in conjugated bile acids and bilirubin, and pooled plasma from jaundiced patients were recirculated through the resin column. The removal of bilirubin and bile acids was evaluated at two different flow rates (200 ml/min and 40 ml/min), and compared to an activated charcoal column. Four patients with severe jaundice were subsequently treated by 4-hour plasmaperfusion through the resin. The in vitro studies showed that after 1 hour the removal of bile acids was almost complete and bilirubin level decreased significantly, reaching a plateau after 4 hours. In the in vivo study, all treatments were well tolerated. After plasmaperfusion, serum bile acid levels decreased by 64.9-94.6% and total bilirubin by 35.3-57.7%. No clinical or biochemical side effects were observed. Our data suggest that plasmaperfusion through this resin is safe and efficient for removal of bilirubin and bile acids in jaundiced patients. Thus, it may serve as a method of artificial liver support in the treatment of cholestatic syndromes.

Morphological and functional evaluation of isolated rat hepatocytes in three dimensional culture systems.

Various three-dimensional configurations, such as polyester tissue and woven-nonwoven, hydrophilic polyester fabric, either collagen-coated or uncoated, were investigated as potential scaffold for hepatocyte culture, in view of their use in bioreactors for hybrid liver support systems. Attachment, morphology and ultrastructure of primary adult rat hepatocytes were evaluated, as well as urea production and ammonium detoxification during a 24h incubation period in serum-free tissue culture medium. As control, hepatocytes were also plated onto collagen-coated dextran microcarriers and on plastic petri dishes, either collagen-coated or uncoated. In all the three-dimensional cultures, hepatocytes appeared morphologically intact without any statistically significant difference in metabolic activity. Collagen-coating did not influence cell attachment to polyester substrates, whereas woven-nonwoven hydrophilic polyester fabric may offer some potential advantages as three-dimensional system for hepatocyte culture in hybrid liver support systems.

Pancreatic beta-cell replication in streptozotocin-diabetic rats: the effect of liver compensatory growth on intraportally engrafted islets.

Early studies showed that compensatory liver growth after anterior portal branch ligation (aPBL) may restore normoglycemia in streptozotocin (STZ)-diabetic rats, in which a subtherapeutic islet mass was previously transplanted into the liver. We hypothesized that this effect could be related to islet regeneration at the graft site. This study was designed to characterize the proliferative response of the intraportally transplanted islets, shortly after aPBL. Male Wistar-Furth rats were used as syngeneic islet donors and/or recipients. STZ-diabetic rats were divided in four groups: groups 1 and 2 underwent selective 250-islet transplantation (Tx) into the posterior liver lobes, followed by aPBL 10 days later; rats were killed 24 h (n = 9) and 48 h (n = 10) after aPBL, respectively; groups 3 and 4 underwent selective 250-islet Tx into the posterior liver lobes, followed by sham aPBL 10 days later; rats were killed 24 h (n = 3) and 48 h (n = 3) after aPBL, respectively. Two hours before killing, all animals were injected with 5'-bromo-2'-deoxyuridine (BrdU; 50 mg/kg, i.v.). Liver sections were immunostained for insulin and BrdU, and both hepatocyte and islet cell labeling index (LI) were calculated. Islet cell LI was 2.30+/-1.18% in group 1, 2.23+/-1.00% in group 2, 0.43+/-0.29% in group 3, and 0.39+/-0.21% in group 4 (group 1 vs. group 3: p<0.02; group 2 vs. group 4: p<0.01). Hepatocyte LI was 2.50+/-2.14% in group 1, 15.0+/-7.6% in group 2, 0.12 +/-0.04 in group 3, and 0.11+/-0.03% in group 4, respectively (group 1 vs. group 2: p<0.02; group 1 vs. group 3: p<0.001; group 2 vs. group 4: p<0.001). Our study showed that intraportally transplanted islets undergo a concurrent proliferative response after aPBL, although with a lower extent and a different timing when compared with the liver-cell response.

[Extracorporeal liver support systems]. / Sistemi extracorporei di assistenza epatica.

Acute liver failure is very life-threatening since the conventional medical treatments have little effects on the clinical outcome. Artificial liver support systems based on blood detoxification alone have proven ineffective because they cannot correct the severe biochemical disorders. An effective liver support system should be capable of carrying out essential functions such as phase I reaction in which lipid-soluble toxic substance are rendered water-soluble by the enzyme system of the cytochrome P450 and NADPH-cytochrome reductase, and are therefore conjugated by the phase II reaction, before excretion. Liver support systems should be capable of sustaining patients with fulminant liver failure until an organ is available for liver transplantation (bridging treatment), or improving the survival in patients for whom liver transplantation is not a therapeutic option. Recent advances in cell biology and tissue culture techniques have led the way for potential clinical use of isolated hepatocytes so that they are now an important element of bioartificial liver support devices. Some of these systems are currently under clinical investigation in the USA and Europe, and the results of the prospective controlled trials will be soon available.

Haemodynamic and ultrastructural observations on the rat liver after two-thirds partial hepatectomy.

Rat liver ultrastructure was investigated after partial hepatectomy (PH), by scanning and transmission electron microscopy. Portal pressure was monitored before and after PH and, after killing performed at 6, 12, 24, 48 h and 10 d, regenerating livers were fixed by portal vein perfusion under haemodynamic conditions identical to those existing in vivo. An early and persistent increase in portal pressure after PH was found (P < 0.01 for normal vs sham-operated controls). Ultrastructural study showed sinusoid dilatation and disappearance of the sieve-plate arrangement of small endothelial pores, thus leaving the parenchymal liver cell surface directly exposed to portal blood. Widening of sinusoids, endothelial fenestrations, intercellular spaces and spaces of Disse, was accompanied by dilatation of bile canaliculi. At 10 d, liver ultrastructure had returned to normal. Our observations suggest that a rise in portal pressure, as a consequence of PH, may be related to the observed ultrastructural changes in the liver.