Th. Minor

Resuscitation of cadaveric livers from non-heart-beating donors after warm ischemic insult: a novel technique tested in the rat

Clinical liver transplantation has become the therapy of choice in end-stage liver disease, but the limited availability of suitable donor organs still impedes its widespread application. In order to increase the availability of donor organs for liver transplantation, it would be advantageous if ischemically damaged livers could be resuscitated from cadavers in which the heart has stopped beating. A method for doing this has been developed in a rat model. Compared to livers excised from rats in which the heart is still beating, severe deteriorations of tissue integrity and functional performance were evident in predamaged livers after cold preservation without supplementary treatment. A treatment of those livers which included an antioxidant rinse with superoxide dismutase, and venous vascular insufflation of gaseous oxygen during preservation, completely prevented tissue alterations upon reperfusion, and promoted a functional recovery of the livers, making them comparable to organs harvested from heart-beating donors.

Evaluation of antioxidant treatment with superoxide dismutase in rat liver transplantation after warm ischemia.

In order to investigate the effects of the exogenously administered radical scavenger superoxide dismutase (SOD) on the orthotopic liver graft, livers from male Wistar rats were transplanted after subjection to 40 min of warm ischemia and 30 min of storage at 4 degrees C. SOD was given at the onset of ischemia and before reperfusion as a supplement (6,000 IU) to the washout solutions. 30,000 IU were infused into the recipient. SOD reduced tissue levels of thiobarbituric acid-reacting substances at the end of ischemia (737 vs. 956 nmol/g; p < 0.01) and 60 min after the onset of reperfusion (629 vs. 947 nmol/g; p < 0.001) and preserved total adenine nucleotides after reperfusion (11.69 vs. 10.40 mumol/g; p < 0.01). Survival 2 weeks after transplantation was 18% (2/11) in the SOD group versus 10% (1/10; nonsignificant) in untreated animals. It is concluded that SOD protects the ischemically altered liver from radical mediated peroxidation and preserves hepatic energy stores upon reperfusion. However, in our model no major improvement in organ viability could by achieved.

Role of the Hepatovasculature in Free Radical Mediated Reperfusion Damage of the Liver

This study was undertaken in order to assess the role of purely circulation-related effects upon free-radical-mediated reperfusion injury in the liver by comparing the respective effects of the oxygen free-radical scavenger superoxide dismutase (SOD) and the vasodilative action of papaverine in an ischemia/reperfusion model of the liver. Livers from male Wistar rats were rinsed blood free via the portal vein and stored ischemically (60 min at 37 degrees C in Krebs-Henseleit solution and 60 min at 4 degrees C in Euro-Collins solution). Reperfusion was carried out at a constant flow of 30 ml/min for 45 min at 37 degrees C in a nonrecirculating manner. Warm ischemic damage was evident in untreated livers compared to control livers, submitted solely to cold ischemia for 2 h at 4 degrees C, by increased vascular resistance upon reperfusion, enhanced enzyme leakage from the parenchyma (glutamate pyruvate transaminase, glutamate dehydrogenase) and from the endothelium (purine-nucleoside phosphorylase), reduced tissue content of ATP and enhanced lipid peroxidation. Preischemic treatment with SOD or papaverine (the latter also given during reperfusion) significantly reduced hepatic vascular resistance and parenchymal enzyme loss in a comparable manner. Both drugs resulted in a significant increase of hepatic tissue content of ATP at the end of reperfusion. SOD, but not papaverine, prevented the leakage of purine-nucleoside phosphorylase and significantly reduced the tissue levels of lipid peroxides.(ABSTRACT TRUNCATED AT 250 WORDS)

Endischemic oxygen persufflation to improve viability of marginally preserved donor livers

Abstract The quality of colD‐stored donor livers slowly declines beyond approximately 12 h, although these organs may still be used for clinical transplantation. The aim of the present study was to improve the energetic status and viability of long‐term‐preserved livers by short‐term gaseous oxygen insufflation prior to implantation of the organ using a technique that has already been shown to promote aerobic energy metabolism during hypothermia. Livers from ten male Wistar rats were isolated, rinsed blooD‐free. Five livers (group 1) were stored for 48 h at 4°C in UW preservation solution, and five livers (group 2) were isolated and stored in the same manner for 47 h, and were then, during the last 60 min of the preservation period, connected to a persufflation device and gaseous oxygen was introduced into the organ via the inferior caval vein, with the liver still immersed in cold UW solution. This technique of endischemic gaseous oxygenation resulted in a significant normalization of vascular resistance upon isolated reperfusion in vitro and a reduction in hepatic efflux of alanine aminotransferase as well as glutamate dehydrogenase, which led to improved recovery of the reperfused grafts of group 2 as evidenced by an elevated energy charge potential at the end of the reperfusion period. In conclusion, the technique described seemed effective in enhancing the preoperative viability of marginal donor grafts.

Assessment of oxygen radicals during kidney transplantation — effect of radical scavenger

Abstract  In the present study, levels of free oxygen radicals, generated in the very early period of re‐perfusion during human kidney transplantation, were assessed by determination of malondialdehyde (MDA) levels using a high‐pressure liquid chromatography (HPLC) method. Renal blood samples were obtained during reperfusion by in‐traoperative cannulation of the renal vein. Simultaneously, systemic MDA levels were determined. Furthermore, local and systemic levels of interleukin 6 (IL‐6), tumor necrosis factor (TNF) receptors, p55 and p75, and vitamin E were measured. In a second group of patients, 500 mg of ascorbic acid were given prior to reperfusion. Renal MDA levels in the control group were always higher compared to systemic levels. IL‐6 showed a marked increase shortly after reperfusion in the renal blood. In the scavenger group there was a diminution of these effects. TNF receptor levels and vitamin E remained largely unchanged. The results of this pilot study demonstrated clinically the moderate production of reactive oxygen species and the liberation of IL‐6 shortly after reperfusion of human transplanted kidneys. Furthermore, the modulating effect of a radical scavenger on these effects was shown.

Reduction in nonparenchymal cell injury and vascular endothelial dysfunction after cold preservation of the liver by gaseous oxygen.

Abstract  Reintroduction of oxygen to previously anoxic tissue may result in severe cell injury (oxygen paradox) and contribute to the so‐called reperfusion damage of is‐chemic organs. Our study investigated the influence of simple gas eous oxygen supply during ischemia on nonparenchymal cell alterations upon reperfusion of the liver. Livers from male Wistar rats were isolated, rinsed blood‐free and stored for 48 h at 4 °C in UW‐preservation solution (group 1; n= 6). Gaseous oxygen was insufflated into a second group of livers (group 2; n = 6) during the storage period via the inferior caval vein at a pressure limited to 18 mmHg. To simulate the period of slow rewarming of the organ during surgical implantation in vivo, all livers were incubated at 25 °C in saline solution for 30 min prior to reperfusion. Reperfusion was carried out in vitro in a recirculating system with Krebs‐Henseleit buffer. A control group was perfused immediately after harvest. The technique of aero bic storage (group 2) resulted in normal vascular perfusion charac teristics without elevation of portal venous pressure (PVP) above control values, in contrast to group 1 livers which showed a significantly elevated PVP, averaging between 1.5 and 2 times the values of the control. Hepatic efflux of NO (nmol/ml) after 10 min of reperfusion was massively increased in group 1, while only low concentrations were found in group 2 and in control livers. Kupffer cell activation after ischemia was shown by a huge increase in acid phosphate release upon reperfusion compared with the control, with significantly lower values in group 2 after 10 min of reperfusion than in group 1. Thus, aerobic ischemia by gaseous oxygen persuf‐flation seems an appropriate tool for long‐term organ preservation, pre venting vascular and parenchymal dysfunction upon reperfusion.

Allopurinol effects in rat liver transplantation on recovery of energy metabolism and free radical-induced damage

Rat livers were orthotopically transplanted after 90-min cold ischemia (group 1) or after 20-min warm and 70-min cold ischemia without (group 2) or with (group 3) allopurinol treatment (AT) (50 mg/kg i.v. 10 min prior to warm ischemia into the donor, flush perfusates with 1 mmol/l). Recovery processes were followed up for 60 min of reperfusion. Liver tissue levels of ATP and total adenine nucleotides were restored in group 1 to almost preischemic ranges within 15-30 min, remained significantly reduced by 30 and 20%, respectively, in group 2, and recovered with AT within 60 min in group 3 to almost the same extent as in group 1. A massive increase in the tissue malondialdehyde concentration, indicative of lipid peroxidation, occurred in the beginning of reperfusion of warm-ischemically damaged donor livers, which in group 3 with AT tended to be less pronounced than in group 2 without AT. The GSSG/GSH ratio reflecting intracellular oxidant stress averaged 3.3 x 10(-3) in group 1 between 15 and 60 min reperfusion. In group 3 AT resulted in comparably low values averaging 3.8 x 10(-3), while in warm-ischemically damaged livers without AT of group 2 this ratio was significantly and continuously elevated averaging 5.8 x 10(-3).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of intestinal integrity after ischemic preservation by luminal and vascular perfusion in vitro.

BACKGROUND In the present study a technique for isolated perfusion of rat intestines in vitro should be tested as an evaluative tool in the assessment of intestinal alterations related to ischemia and reoxygenation. METHODS Segments of upper jejunum (15 cm) were isolated from Wistar rats with vascular pedicle (superior mesenteric artery, SMA and portal vein). The SMA was cannulated with polyethylene tubing and flushed with 10 ml of University of Wisconsin (UW) preservation solution. The intestinal lumen was rinsed with 10-15 ml of UW solution and the organ was stored immersed in UW solution at 4 degrees C for 4 or 18 h. After cold ischemic storage structural and functional integrity of the preparation was tested by biluminal perfusion with artificial buffer via SMA (5 ml/min modified Krebs-Henseleit buffer, 200 mg% glucose, 5% dextran 78, 0.06 mg% dexamethasone, 7 mg% atropine to counteract paralytic hypersecretion) and the intestinal lumen (0.5 ml/min NaCl 0.9% with 200 mg% of galactose). The in vitro model was validated by perfusion of control preparations harvested without ischemic alteration. It was seen that ischemic preservation of 4 h had only a minor impact on the recovery of cellular ATP content and enzyme release (LDH) upon reperfusion, whereas both parameters were significantly changed after 18 h of preservation. Functional parameters like transmucosal carbohydrate absorption and luminal water balance, however, were significantly impaired already after 4 h of ischemic storage of the gut, thus yielding sensitive criteria for the appreciation of the postischemic integrity of the gut. CONCLUSIONS It is concluded that the isolated gut preparation, being an inexpensive and technically feasible model, may be a useful tool in experimental research of intestinal ischemia/reperfusion.

Involvement of oxygen in harvesting injury of the liver: An experimental study including substrate free organ persufflation to evaluate a specific therapeutic approach

SummaryThe present study was undertaken to assess the role of oxygen free radicals relating to cell damage upon reoxygenation of the ischemically altered isolated rat liver. Livers were excised and flushed via the portal vein with Ringers solution and Euro-Collins solution, to which superoxide dismutase (SOD) was added in the experimental group. After warm ischemia at 37°C and cold storage at 4°C, the livers were reperfused via the portal vein with carbogensaturated Krebs-Henseleit solution. Other livers were subjected to a retrograde persufflation via the infrahepatic caval vein with either oxygen or nitrogen and then rinsed with Ringers solution. During reperfusion, SOD-treated livers showed markedly reduced vascular resistance, lower enzyme release and enhanced VO2 accordingly, energy charge at the end of reperfusion was significantly higher in the treated group. With reference to the tissue content of malondialdehyde, SOD-treated livers showed significantly less damage than the controls. Our experiments with substrate-free persufflation provided sound corroboration for these data. Enzyme activities in the eluate were significantly reduced under anoxic conditions as well as in the presence of SOD. We conclude from these data that oxygen free radicals do exert a detrimental impact on the reoxygenated liver, which could be specifically suppressed by application of exogenous SOD.

Determination of plasma activities of purine nucleoside phosphorylase by high-performance liquid chromatography estimates of nonparenchymal cell injury after porcine liver transplantation

An assay is described for measurement of purine nucleoside phosphorylase (PNP) in plasma by high-performance liquid chromatography (HPLC). A plasma sample was incubated with hypoxanthine and ribose-1-phosphate in phosphate-free medium at pH 7.4 to catalyse the production of inosine by plasmatic PNP. The reaction was stopped by addition of perchloric acid to inactivate the enzyme and to precipitate plasma proteins. After centrifugation and neutralization of the supernatant with NaOH the increase in the substrate inosine was determined by HPLC. Plasma activities of PNP averaged 5.0 mU/ml before and 12.3 mU/ml (p < 0.001), 5 min after porcine liver transplantation. At the same time points, the plasma activities of the frequently used liver enzymes lactate dehydrogenase or alanine aminotransferase remained virtually unchanged. Thus, plasmatic activities of PNP may be a suitable and early indicator of ischemic alterations to the graft in vivo.