Thomas Minor

Protective effect of heat shock pretreatment with heat shock protein induction before hepatic warm ischemic injury caused by Pringle's maneuver

BACKGROUNDnInduction of heat shock proteins is thought to have a > cytoprotective effect against environmental stress and to result in a better ischemic tolerance. The protective ability of heat exposure and heat shock protein 72 (HSP 72) induction before warm ischemia caused by Pringles maneuver was evaluated in rats.nnnMETHODSnHeat exposed rats (HS) were compared with control animals (C). The gene expression (messenger RNA) of HSP 72 and HSP 72 were detected by Northern and Western blot analyses. During 40 minutes of in situ reperfusion, liver energy metabolism and levels of standard liver enzymes were evaluated. The survival rate was determined after postoperative day 7.nnnRESULTSnAfter heat exposure and recovery, messenger RNA of HSP 72 and HSP 72 can be detected strongly in HS group but not in C group. During reperfusion HS group exhibited a significantly (p < 0.01) improved energy metabolism, and the release of liver enzymes was significantly (p < 0.001) reduced compared with C group. Seven-day survival rate was 100% in HS group but at 50% was significantly impaired (p < 0.05) in C group.nnnCONCLUSIONSnHeat exposure associated with HSP induction has a significant protective effect against warm ischemic liver injury, which results in a relevant improvement of survival rate.

The effects of allopurinol and SOD on lipid peroxidation and energy metabolism in the liver after ischemia in an aerobic/anaerobic persufflation

This study was aimed at examining the vulnerability of the liver to oxygen-free radicals upon reoxygenation after prolonged ischemia. Livers from male Wistar rats were first flushed with Ringers and Euro-Collins solutions. After ischemic storage in Krebs-Henseleit solution at 37°C for 60 min and in Euro-Collins solution at 4°C for another 60 min, they were then persufflated with either gaseous O2 or N2 for 30 min at 37°C, and rinsed again with Ringers solution. Enzyme concentrations and calcium ion activities were measured in the effluent rinsing solution after passage through the liver. Treatment with superoxide dismutase (SOD) or allopurinol resulted in a significant reduction of tissue injury, determined by the enzyme loss, calcium uptake, and lipid peroxidation upon persufflation with O2. Allopurinol also improved the tissue levels of ATP and the sum of adenine nucleotides after aerobic persufflation, whereas SOD did not. Notwithstanding, neither treatment had any effect on anoxic persufflation with N2. Thus, we conclude that the postischemic liver is susceptible to oxygen-induced free radical injury and that allopurinol and SOD promote specific antioxidative protection of the liver, with the exclusion of side effects related to substrates or perfusion modalities.

A Correlation of Intravital Microscopically Assessed NADH Fluorescence, Tissue Oxygenation, and Organ Function during Shock and Resuscitation of the Rat Liver

Multiple organ failure following severe hemorrhagic shock and trauma continues to be a major problem and accounts for a high mortality rate in surgical intensive care units. Persisting depression of micro vascular blood flow and, thus, tissue hypoxia have been suggested to promote multiple organ failure after hemorrhagic shock. Through modulation of the host response to shock and trauma the liver plays a central role for the maintenance of metabolic homeostasis, protein synthesis, as well as clearance and elimination of bacteria and endotoxins.

Polyethylene glycol 4000 attenuates adhesion formation in rats by suppression of peritoneal inflammation and collagen incorporation.

BACKGROUNDnPostoperative separation of the peritoneal surfaces is a promising approach for the prevention of adhesion formation. Instillation of polyethylene glycol 4000 (PEG) was recommended as an appropriate method.nnnMETHODSnIn a time-course study in rats, efficacy and mode of action of PEG was investigated. Adhesions were induced by crushing of the cecum. Sixty animals were randomly allocated to three control groups with intraperitoneal instillation of 5 mL 0.9% NaCl and to three therapy groups treated with 5 mL 20% PEG. After 3, 7, and 21 days, adhesion grade, intraperitoneal leukocytes, and collagen incorporation into the adhesion strands were evaluated.nnnRESULTSnAdministration of PEG resulted in a decrease of leukocytes by 43% (days 3 and 7), a reduction of the adhesion score by 85% (day 3), 77% (day 7), and 79% (day 21), and an impairment of collagen deposition by 54% (day 7) and 44% (day 21).nnnCONCLUSIONSnCoating the peritoneal surfaces with PEG is a highly effective measure to suppress the fatal sequence of posttraumatic inflammation, formation of initial adhesions, and their subsequent organization to strong permanent adhesion strands.

Taurine Reduces Experimental Liver Injury after Cold Ischemic Preservation and a Period of Rewarming Prior to Reperfusion

Livers of male Wistar rats (250-300 g) were isolated and flushed with 10 ml of Ringers solution and 10 ml of UW preservation solution. Then the organs were stored for 24 h at 4 degrees C in UW solution. Livers of Group 1 were rinsed with 10 ml of Ringers solution and reperfused after hypothermic storage with oxygenated Krebs-Henseleit solution (95% O2; 5% CO2) in a nonrecirculating system at constant pressure (10 mmHg) and 37 degrees C. Livers of Group 2 were incubated for 30 min at 37 degrees C prior to reperfusion, in order to simulate rewarming of the organ upon surgical implantation. Livers of Group 3 were treated like Group 2, but taurine was admixed to the UW solution (1 mM). Livers of Group 1 showed little signs of a preservation/reperfusion injury, with low enzyme activities of the parenchymal ALT and endothelial purine nucleoside phosphorylase (PNP) in the postischemic rinse solution (ALT: 19.9 +/- 12.4; PNP: 3.3 +/- 0.4 U/liter), adequate portal flow values about 3 ml/g/min and high O2 uptake at the end of the experiment (VO2: 3.2 +/- 0.4 ml/100g/min). Livers of Group 2 exhibited nearly tenfold higher enzyme activities in the rinse solution (ALT: 247.0 +/- 94.7*; PNP: 29.5 +/- 17.0* U/l) and disturbed tissue perfusion with significantly reduced flow values of about 2 ml/g/min during the first 10 min of reperfusion. As a result, the recovery of O2 uptake was only 2.2 +/- 0.3 ml/100 g/min*. Addition of taurine (Group 3) resulted in a significant reduction of the enzyme loss (ALT: 96.2 +/- 50.0#; PNP:12.4 +/- 7.0# U/liter) and improved portal flow values and O2 uptake at the end of reperfusion (2.7 +/- 0.3 ml/100 g/min#). The results give evidence for the importance of the rewarming period after hypothermic storage, which is inevitable during implantation of the organ in vivo. Taurine seems to exert a protective effect, affecting both the vascular endothelium and parenchymal tissue (*p < 0.05 vs Group 1; # p < 0.05 vs Group 2).

HYPOXIC CELLULAR DETERIORATION AND ITS PREVENTION BY THE AMINO ACID TAURINE IN A TRANSPLANTATION MODEL WITH RENAL TUBULAR CELLS (LLC-PK1)

Dear Editor: In the field of transplantation medicine one of the determining factors for the outcome of the transplanted organ is the damage due to hypoxia and reoxygenation and the protection against it. Frequently the hypoxic storage of the graft limits the possibility of successful transplantation, and the cell damage due to insufficient organ preservation is a major cause for the loss or the dysfunction of transplanted organs (3,6,7). Some of the determining factors of the extent of hypoxic and posthypoxic deterioration are the energy deficiency (4,23), intracellular calcium overload (24), osmoregulatory dysfunction (21,22), the oxygen paradox and membrane oxidation induced by toxic metabolites, and free oxygen radicals (4,25). Different preservation solutions are conventionally used for the preservation of a kidney graft. One of them is the University of Wisconsin (UW) solution (14-16,18), which is based on components and constituents of the intraand extracellular environment and which was additionally supplemented with agents acting as osmoregulators, antioxidants, antibiotics, radical scavengers, and membrane stabilizers. Because of the unique functions of the endogenous amino acid taurine as modulator of transmembranous calcium transport (10), as osmoregulator (10,21,22), and free radical scavenger (16), one purpose of this study was to investigate in a cell culture model whether UW solution could be improved by the supplementation of taurine and if its addition during hypoxic preservation improves the posthypoxic outcome. Cell cultures have frequently been used to assess hypoxic cell damage and to develop methods of its prevention (1,11,13), but the time course of hypoxic changes was difficult to investigate and often required large-scale and expensive techniques (11,13,26). So another aim of our study was to determine the extent and time course of hypoxic cell damage in renal tubular cells with a simple technique, which should allow one to measure metabolic parameters of the cells. Proximal renal tubular cells (8,9) of the pig (strain LLC PK 1, Kasza pig kidney; ICN Biomedical, Meckenheim, Germany) were cultured as monolayers aerobically (21% 02, 5% CO2, Po 2 140 mmHg, 37 ° C) for 48 h in M199 medium mixed with fetal bovine serum, penicillin, and streptomycin. At the end of the aerobic culturing the M 199 medium was removed and replaced by UW solution, which was formerly equilibrated with nitrogen (Po 2 < 8 mmHg). The cells were preserved hypoxically for 15 or 21 h. After cold hypoxic storage (4 C), the UW solution was removed and replaced by a cristalloid perfusion solution equilibrated with room air (Hanks balanced salt solution, without calcium). Thereafter, the cell cultures were stored aerobically for 30 min (37 ° C, P o 2 140 mmHg). During aerobiosis and hypoxia as well as during reoxygenation the monolayers were placed in modified cell culture flasks. The modification (Fig. 1) allowed microperfusion of the culture chamber and

Platelet-activating factor antagonism enhances the liver's recovery from warm ischemia in situ

Hepatic outcome after ischemia is compromised by anoxic injury and by release of oxygen free radicals or other pathological mediators at reperfusion. The platelet-activating factor (PAF) is an endogenous lipid-mediator which plays a key role in catalysing various pro-inflammatory processes. In this study the possible influence of platelet-activating factor antagonism was investigated on the integrity of the vascular endothelium, on free radical-mediated peroxidation and on post-ischemic functional outcome of the liver in the rat. Animals under pentobarbital anesthesia were subjected to 60 min of ischemia of the left and median liver lobes followed by 30 min of reperfusion in vivo. Pre-ischemic injection of the platelet-activating factor antagonist BN 52021 resulted in a significant reduction of endothelial enzyme loss into the plasma (purine nucleoside phosphorylase 56.2 vs. 83.1 U/l), and lowered hepatic lipid peroxidation (malondialdehyde 830 vs 932 nmol/g), leading to a significant improvement of postischemic bile flow and higher tissue levels of ATP. This suggests that the platelet-activating factor may play an important role in hepatic ischemia-reperfusion injury and that PAF antagonists like BN 52021 may be useful for clinical treatment.

Reduction of oxidative tissue injury and endothelial dysfunction by graduated reperfusion after cardioplegic arrest in isolated rat hearts.

The aim of this study was to investigate whether or not a graduated resumption of the perfusion pressure after cardioplegic ischaemic arrest will reduce the impact of oxygen free radicals on myocardium and the cardiovasculature. Langendorff-perfused rat hearts were subjected to cardioplegia and subsequent 40 min of global ischaemia at 25 degrees C. Reperfusion was carried out either abruptly (AR) or gradually (i.e., perfusion pressure stepwise increased from 40 to 75 mmHg within 30 min -GR). GR resulted in a significant improvement of percentage recovery of left ventricular systolic pressure as compared to AR. A marked increase of thiobarbituric acid reactive substances (TBARS) was detected in the effluent during AR, accompanied by an impaired release of the endothelial vasodilator NO and diminished coronary flow rates compared to the baseline values. GR resulted in a significant reduction of TBARS in the effluent and promoted a better recovery of coronary flow as well as endothelial release of NO during the later phase of reperfusion. It is concluded that graduated reperfusion is beneficial in reducing free radical mediated peroxidative tissue injury and endothelial dysfunction upon reoxygenation.

Long-Lasting Hypoxic Preservation of Porcine Kidney Cells

Taurine administered during hypoxia reduced the cell damage due to O2 deficiency markedly. The beneficial effect outlasted the period of reoxygenation. The mechanisms for the improved survival rates are postulated in a reduced osmoregulatory disturbance of cellular integrity, improved Ca2+ homeostasis and induction of accelerated cellular growth processes. We conclude that taurine supplementation of the conventionally used kidney preservation solution (UW) improves this gold standard kidney preservation solution markedly.

Influence of Taurine Supplementation on Ischemic Preservation of the Isolated Rat Kidney

Taurine (2-aminoethanesulfonic acid) is an endogenous amino acid with unique functions as a modulator of transmembraneous calcium transport, an osmoregulator and putatively a free radical scavenger4,7,12.