Immunoglobulin M, C-reactive protein and complement activation in rat hepatic ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) models have shown that C-reactive protein (CRP) and immunoglobulin M (IgM) are involved in complement activation. Binding of CRP and IgM to damaged cell membranes initiates complement activation and aggravates I/R injury in various organs. However, the time course of CRP- and IgM-mediated complement activation and the relation to hepatocellular injury and inflammation in liver I/R are unknown. AIM: To evaluate the time course of IgM- and CRP-related complement activation and the relation to hepatocellular injury and inflammation in a hepatic I/R rat model. METHODS: Male Wistar rats were allocated to (1) five groups of animals exposed to 60 min of partial ischemia (70%) induced via clamping of the left segmental portal triad, followed by 0, 3, 6, 12 or 24 h of reperfusion (n = 6 in each group); (2) five groups of sham-operated animals with corresponding reperfusion times (n = 5), and (3) a control group sacrificed before ischemia (n = 5). Hepatocellular injury, inflammatory response, rat plasma CRP and IgM levels and immunohistochemical depositions of CRP, IgM and C3 were assessed for each group. RESULTS: Histopathological injury scores of hematoxylin and eosin sections of ischemic liver lobes demonstrated increasing values throughout the reperfusion time with a peak at 12 h. Plasma aminotransferases (alanine aminotransferase and aspartate aminotransferase) significantly increased after 3 h of reperfusion, peaking at 6 h (3,100 ± 800 U/l; p < 0.05). Hepatic neutrophil influx significantly increased from 3 to 6 h of reperfusion (p < 0.05) and demonstrated the highest value at 12 h (1.1 ± 0.2 U/mg of protein). Plasma IL-6 levels in the ischemia groups showed peak values after 6 h of reperfusion, decreasing significantly thereafter (p < 0.05). Plasma CRP values reached highest levels after 3 h of reperfusion (mean 91 ± 5% of control pool), decreasing significantly thereafter. Rat IgM concentrations in plasma did not significantly change throughout the reperfusion time. Immunohistochemical depositions of IgM, CRP and C3 in ischemic lobes demonstrated a similar pattern in time, reaching maximum values at 12 h of reperfusion. The percentages of depositions of CRP and IgM were significantly correlated [r(S) = 0.569; p < 0.001; Spearman test]. The time course of C3 and CRP depositions throughout reperfusion and C3 and IgM staining were significantly similar [r(S) = 0.797 and r(S) = 0.656, respectively; p < 0.0001; ANOVA]. CONCLUSIONS: CRP and IgM depositions demonstrate a parallel time course throughout the reperfusion to hepatocellular damage, inflammatory response and activated complement deposition in this rat hepatic I/R model. Furthermore, the time course of CRP and IgM depositions was significantly similar to that of activated complement depositions.

Hypothermic in situ perfusion of the porcine liver using Celsior or Ringer-lactate solution.

BACKGROUND: Hypothermic perfusion (HP) of the liver is applied during total vascular exclusion (TVE) to reduce ischemic injury during liver resection. No studies have been performed comparing different perfusion solutions for HP. The aim of this experimental study was to compare Ringer-lactate solution (RL) with Celsior solution (Cs) for HP in a pig model of 60-min TVE. METHOD: Twenty pigs underwent 60-min TVE of the liver. Groups were TVE without HP (no-HP, n = 9), TVE with HP using RL (n = 6), and TVE with HP using Cs (n = 5). Blood and liver tissue samples were taken before TVE and during 24-h reperfusion. RESULTS: In the no-HP group, plasma aspartate aminotransferase values were significantly increased during reperfusion (p < 0.05), while liver tissue pO(2) levels (p < 0.01) were decreased when compared to the HP groups. After 24-h reperfusion, bile production and liver tissue glutathione content were significantly higher (p < 0.05) in the Cs group (42.0 +/- 1.7 mL/h and 44.9 +/- 2.2 nmol/mg, respectively) as compared to the RL group (31.5 +/- 3.5 mL/h and 19.6 +/- 1.8 nmol/mg, respectively). CONCLUSION: The protective effect of HP during TVE was confirmed in this study. HP with Cs was more effective in reducing ischemic injury as compared to HP with RL.

Vastatins have a distinct effect on sterol synthesis and progesterone secretion in human granulosa cells in vitro.

Lovastatin and simvastatin are strong inhibitors of cholesterol synthesis in cultured human granulosa cells, as measured within 6 days after isolation, with IC50-values of respectively 27.0 and 18.2 nM obtained after 3.5 hours of incubation with the drugs. Pravastatin is a much weaker inhibitor of cholesterol synthesis (IC50-value of 977.8 nM) in these cells. Under these conditions inhibition of cholesterol synthesis had no influence on progesterone secretion into the medium which was probably due to the presence of large cholesterol pools in the cells. To deplete these pools, granulosa cells were cultured for 7 days after which the culture medium was changed into medium supplemented with 20% lipoprotein-depleted serum to deprive the cells of exogenous cholesterol. Additionally, 30 mIU of follicle-stimulating hormone and luteinizing hormone per ml were added to stimulate the progesterone production and secretion, thereby decreasing the cholesteryl ester pools. After 48 h of incubation, culture was continued without hormones for another two days. Thereafter, the cells were preincubated for 24 h without or with 1 microM of lovastatin, simvastatin or pravastatin in medium containing lipoprotein-deficient serum and the above-mentioned hormones. This period is followed by incubation for another 24 h in the presence of [14C]acetate after which cells and media were collected for determination of 14C-labelled sterols synthesized and progesterone secreted into the media. Now, lovastatin and simvastatin, which strongly inhibited sterol synthesis, significantly attenuated the secretion of progesterone. One microM of pravastatin had no significant effect on sterol synthesis nor on progesterone secretion. When the latter experiment was performed under conditions in which exogenous cholesterol was provided in the form of human low density lipoproteins, no influence of the vastatins on progesterone secretion was observed. So under conditions in which the cholesterol pools were decreased, lovastatin and simvastatin attenuated the progesterone secretion, whereas pravastatin did not. When pools were filled by exogenous cholesterol, no effect on progesterone secretion by either of the drugs was observed.

Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types.

The three vastatins examined, lovastatin, simvastatin and pravastatin, are equally strong inhibitors of the sterol synthesis in human hepatocytes in culture with IC50-values of 4.1, 8.0 and 2.0 nM, respectively. However, in the human extrahepatic cells: umbilical vascular endothelial cells, retinal pigment epithelial cells, cornea fibroblasts and granulosa cells, pravastatin was much less inhibiting the sterol synthesis than lovastatin or simvastatin. It was observed as well that longer incubation with the vastatins resulted in higher IC50-values. In order to show that the feedback regulation mechanism for 3-hydroxy-3-methylglutaryl-coenzyme A reductase was involved in this phenomena mRNA levels were measured in human vascular endothelial cells after incubation with the vastatins for 3.5 h and for 20 h. Indeed, lovastatin and simvastatin gave rise to higher levels of HMG-CoA reductase mRNA after 20 h than after 3.5 h of incubation. The differences observed in different human cell types can be explained by supposing that pravastatin is transported into the human hepatocyte via a liver-specific transporter. This was supported by the results of uptake experiments with 14C-labelled pravastatin and 14C-labelled simvastatin into human hepatocytes compared to that into human umbilical endothelial cells (as an example of an extrahepatic cell type). [14C]-Simvastatin was associated with both cell types, whereas [14C]-pravastatin was hardly associated with human endothelial cells, but to a similar extent as [14C]-simvastatin with human hepatocytes.

Inhibition of proliferation of human smooth muscle cells by various HMG-CoA reductase inhibitors; comparison with other human cell types.

The effects of 6 HMG-CoA reductase inhibitors: pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin were analyzed in cultured human smooth muscle cells, fibroblasts, endothelial cells and myoblasts. In vascular smooth muscle cells, pravastatin was a much weaker inhibitor of cholesterol synthesis than the 5 other drugs which displayed equally strong inhibitory potency. The anti-proliferative effects of these 6 drugs were analyzed by measuring cell number and mitochondrial dehydrogenase activity (MTT assay) after 3 days of incubation. IC25 values for inhibition of proliferation were very similar among the 4 cell types and were in the following order of magnitude: pravastatin << lovastatin = simvastatin = atorvastatin = fluvastatin << cerivastatin. Only in the case of pravastatin was proliferation inhibited at lower concentration in smooth muscle cells than in the other cell types. Proliferation was also assessed by measuring DNA synthesis in these cells. A 3 day-incubation with 1 microM of pravastatin had no effect on this parameter in all 4 cell types. However, 1 microM of simvastatin or lovastatin caused either an inhibition (in smooth muscle cells and endothelial cells) or stimulation (in fibroblasts) of this process. The effects of simvastatin on cell number, mitochondrial dehydrogenase activity and DNA synthesis were counteracted by simultaneous mevalonate addition. Simvastatin treatment was also associated with a change in the post-translational modification of the ras protein in smooth muscle cells, probably by inhibition of its farnesylation. Moreover, simvastatin treatment blocked the PDGF and bFGF-induced DNA synthesis in synchronized smooth muscle cells, whereas it does not affect the fetal calf serum-induced DNA synthesis in synchronized fibroblasts, suggesting that simvastatin blocks various steps of the cell cycle and that this effect depends on the cell type and the growth signalling pathway activated.

Machine perfusion preservation of the non-heart-beating donor rat livers using polysol, a new preservation solution.

AIMS: The increasing shortage of donor organs has led to a focus on extended criteria donors, including the non-heart-beating donor (NHBD). An optimal preservation method is required to facilitate successful transplantation of these ischemically damaged organs. The recent literature has shown clear advantages of hypothermic machine perfusion (MP) over cold storage (CS). For MP, modified University of Wisconsin perfusion solution (UW-G) is often used, which, however, is known to cause microcirculatory obstruction, is difficult to obtain, and is expensive. Therefore, Polysol was developed as a MP preservation solution that contains specific nutrients for the liver, such as amino acids, energy substrates, and vitamins. The aim of this study was to compare Polysol with UW-G in a NHBD rat liver model. METHODS: After 24 hours hypothermic MP of NHBD rat livers using UW-G or Polysol, liver damage and function parameters were assessed during 60 minutes of reperfusion with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours CS in UW. RESULTS: Liver enzyme release was significantly higher among the CS-UW group compared to MP using UW-G or Polysol. Flow during reperfusion was significantly higher when using Polysol compared to UW-G. Bile production and ammonia clearance were highest when using Polysol compared to UW-G. There was less cellular edema after preservation with Polysol compared to UW-G. CONCLUSIONS: MP of NHBD rat livers for 24 hours using UW-G or Polysol resulted in less hepatocellular damage than CS in UW. MP of NHBD livers for 24 hours using Polysol is superior to MP using UW-G.

Optimization of a new preservation solution for machine perfusion of the liver: which is the preferred colloid?

AIMS: Machine perfusion (MP) has proven to be beneficial in experimental preservation of the liver. The modified University of Wisconsin solution (UW-Gluconate or UW-G) is used as the MP preservation solution of choice. We have developed Polysol, an enriched MP preservation solution based on a colloid. We sought to optimize Polysol by substituting the colloid hydroxyethylstarch (HES) with the colloids dextran and polyethylene glycol (PEG). METHODS: In an isolated perfused rat liver model, hepatocellular damage and liver function were assessed during reperfusion with Krebs-Henseleit buffer after 24 hours hypothermic MP using Polysol-HES, Polysol-dextran, or Polysol-PEG. Control livers were preserved by MP using UW-G. RESULTS: Compared to MP-UW-G, MP using Polysol resulted in significantly less damage and improved function during reperfusion. MP using Polysol-dextran or Polysol-PEG resulted in equal or less damage than Polysol-HES. Differences in ammonia clearance and bile production were not significant. Tissue edema was higher after MP using Polysol-HES as compared to Polysol-dextran and Polysol-PEG. CONCLUSIONS: MP of rat livers for 24 hours using UW-G results in more extensive damage and reduced liver function compared to MP using Polysol. MP using Polysol-dextran or Polysol-PEG results in equal or even better preservation compared to Polysol-HES.

Wash-out of the non-heart-beating donor liver: a comparison between ringer lactate, HTK, and polysol.

UNLABELLED: The solution of choice for wash-out of non-heart-beating donor (NHBD) livers is histidine tryptophan ketoglutarate (HTK). This solution has a lower viscosity, due to absence of a colloid, and is less expensive as compared to the University of Wisconsin (UW) solution. A new preservation solution for machine perfusion was developed, named Polysol. In order to apply Polysol clinically in NHBD organ retrieval, the efficacy as a wash-out solution was investigated. METHODS: After a warm ischemic time of 30 minutes, the rat liver was washed out via the portal vein with 50 mL of either ringer lactate (RL), HTK or Polysol. After wash-out and harvesting, the liver was reperfused with Krebs-Henseleit buffer. Samples were taken to assess hepatocellular injury and liver function. RESULTS: Liver damage parameters were elevated in the RL group as compared to the HTK and Polysol groups. Liver/rat weight ratios were significantly lower after wash-out with Polysol. Overall, no differences were seen in ammonia clearance and bile production. In conclusion, wash-out of the NHBD liver with Polysol results in equal to improved reperfusion results as compared to HTK. Polysol is feasible as a wash-out solution in combination with machine perfusion using Polysol.

Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle.

Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 +/- 6 nM and 4.0 +/- 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 +/- 38 nM). Through inhibition of mevalonate production, these compounds have a distinct inhibiting effect on cell proliferation. Because proliferation of myoblasts is important in the repair of damaged skeletal muscle, experiments were performed to investigate the effect of lovastatin, simvastatin, and pravastatin on cell proliferation and cell viability. The more potent inhibitors of sterol synthesis, lovastatin, and simvastatin, were able to inhibit the proliferation of these cells during 3 days of incubation with drug concentrations of 1 microM for lovastatin and 0.1 microM or 1 microM for simvastatin. DNA synthesis was decreased by more than 80% in the presence of 1 microM of lovastatin or simvastatin. In contrast, under these conditions, pravastatin had no influence on cell proliferation or DNA synthesis, which is probably related to the lack of inhibition of sterol synthesis by pravastatin on extended incubation. The three 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors did not disturb cell viability because mitochondrial dehydrogenase activity and ATP content remained proportional to the number of cells in the culture at any concentration used.

Preservation of rat livers by cold storage: a comparison between the University of Wisconsin solution and Hypothermosol.

OBJECTIVES: The University of Wisconsin solution (UW) is the gold standard for cold storage (CS) of donor livers. However, UW contains the colloid Hydroxyethyl starch (HES), which may cause perfusion deficits due to its high viscosity. Recently, a new CS preservation solution, Hypothermosol (HTS), was introduced which contains the less viscous colloid Dextran. The aim of this study was to assess HTS as a cold storage solution for preservation of the liver. METHODS: In an isolated perfused rat liver model, hepatocellular damage was assessed after 24 hours of CS. Liver enzymes were measured during reperfusion with Krebs-Henseleit Buffer. Bile was collected during reperfusion as a parameter of liver function. RESULTS: CS using HTS showed a significant decrease of ALT and LDH levels (as compared to UW) at all time points during reperfusion. For LDH these results where most pronounced at t=10 min (84 +/- 7.09 vs 113 +/- 7.57: p < 0.05) and t=30 min (149.2 +/- 9.68 vs 194 +/-6.52: p< 0.05). Regarding liver function, more bile was produced after 24 hours CS in HTS, but this did not reach statistical significancy. CONCLUSIONS: Cold storage preservation of rat livers using Hypothermosol results in equal or even better preservation as compared to cold storage using UW.

The first disposable perfusion preservation system for kidney and liver grafts.

Machine perfusion systems for continuous hypothermic perfusion preservation are computer controlled and re-usable. The system of perfusion preservation proved beneficial for for storage of kidneys, particularly those harvested from NHBD donors. The first disposable continuous hypothermic perfusion system which can be used for storage of livers is presented.

Biliary drainage attenuates postischemic reperfusion injury in the cholestatic rat liver.

BACKGROUND: The combination of hepatic ischemia and cholestasis, both identified as risk factors for oxidative stress, potentially enhances postischemic reperfusion (I/R) injury. Preoperative biliary drainage relieves oxidative stress and therefore seems a worthwhile intervention in cholestatic patients undergoing major liver resection. AIM: To assess the effect of biliary decompression on I/R injury in a reversible bile duct ligation (BDL) model in the rat. METHODS: Male Wistar rats were randomized into 3 groups. The first group underwent 30 minutes of partial liver ischemia after 7 days BDL; the second group underwent internal drainage (ID) after 7 days BDL and after 5 days, were subjected to partial liver ischemia. The last group (control animals) underwent 2 sham laparotomies and subsequent ischemia. Inflammatory response (interleukin [IL]-6, IL-10, GRO/KC, and interferon-gamma), hepatic damage and oxidative stress were assessed during 24 hours of reperfusion. RESULTS: Cholestatic rats, as compared with the ID and control groups, showed significantly increased I/R injury as determined by transaminase release, histologic injury score and neutrophil infiltration. Plasma IL-6, IL-10, and GRO/KC (a CXC chemokine) were significantly increased in the BDL group (P < .05 vs control and ID). Moreover, the hepatic antioxidant capacity was strongly decreased in the BDL group (P < .01 vs control and ID). No significant differences for most parameters were seen in the ID group as compared to the control group. CONCLUSION: The cholestatic rat is more susceptible to postischemic liver injury and these injurious effects were significantly attenuated by biliary decompression.

Mild steatosis impairs functional recovery after liver resection in an experimental model.

BACKGROUND: Mild steatosis has been thought not to affect outcome after liver resection. However, recent studies have reported impaired postoperative recovery of patients with mild steatosis. This study evaluated the recovery of hepatic functional reserve during regeneration in a rat model of mild steatosis and liver resection. METHODS: Male Wistar rats had a standard methione- and choline-deficient diet to induce mild steatosis before 70 per cent liver resection. Evaluation of hepatobiliary function was by (99m)Tc-labelled mebrofenin scintigraphy. Mebrofenin uptake rate, the time for maximum uptake (T peak) and the time required for peak activity to decrease by 50 per cent (T(1/2) peak) were assessed 1, 2, 3 and 7 days after liver resection, along with regeneration of the remnant liver, hepatocellular and sinusoidal damage, and hepatic adenosine 5'-triphosphate (ATP) levels. RESULTS: Liver regeneration and proliferative response in mild steatotic rats were no different from those in controls. However, the mebrofenin uptake rate was lower (P < 0.050) and the recovery of hepatic ATP impaired (P < 0.050) in animals with mild steatosis. Hepatocellular damage was increased (P < 0.050) but sinusoidal endothelial cell function was not affected after liver resection in mildly steatotic rats. CONCLUSION: Mild steatosis impaired functional recovery and increased hepatocellular damage after liver resection.

Alkaline phosphatase reduces hepatic and pulmonary injury in liver ischaemia -- reperfusion combined with partial resection.

BACKGROUND: Lipopolysaccharides mediate inflammation in liver ischaemia-reperfusion (I/R) and partial liver resection (PHX). Bovine intestinal alkaline phosphatase (BIAP) detoxifies lipopolysaccharides by dephosphorylation and reduces inflammation in models of sepsis. This study examined the protective effects of BIAP administration in models of partial (70 per cent) liver I/R with or without partial resection of all non-ischaemic lobes during ischaemia (30 per cent). METHODS: Male Wistar rats were divided into six groups: I/R + BIAP, I/R + saline, I/R + PHX + BIAP and I/R + PHX + saline, PHX only or sham laparotomy only. A single dose of BIAP (0.5 units/g) or vehicle (saline) was administered 5 min before reperfusion. Inflammatory response, and hepatic and pulmonary injury were assessed during 24 h of reperfusion. RESULTS: I/R, with or without PHX, increased all markers of inflammation, and hepatic and pulmonary damage (P < 0.050 versus sham operation). I/R + PHX significantly increased release of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and hepatic neutrophil influx compared with I/R only (P < 0.050). BIAP treatment decreased hepatic wet/dry ratios, neutrophil influx and histopathological damage after I/R with or without PHX (P < 0.050), and also AST, ALT and interleukin (IL)-6 production after I/R + PHX (P < 0.050). BIAP treatment reduced the neutrophil influx after I/R, and pulmonary histopathological injury was decreased after I/R with or without PHX. CONCLUSION: BIAP attenuates hepatic and pulmonary injury after partial liver I/R and PHX.