Hiltrud Klauke

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.

Biophysical aspects of liver aeration by vascular persufflation with gaseous oxygen.

BACKGROUND Venous systemic oxygen persufflation of the liver (i.e., gaseous insufflation of oxygen via the venous vascular system) has proven to be an effective tool for preventing anoxic tissue injury during extended time periods of ischemic preservation. It also allows for an improved recovery of the persufflated organ after orthotopic transplantation. METHODS Biophysical aspects of the persufflation technique with regard to persufflation pressure (9 mmHg versus 18 mmHg) and oxygen concentration (pure oxygen versus air) in the persufflation gas were investigated in rat livers, using epi-illumination microscopic detection of autofluorescence of NADH, which accumulates in anoxic tissue. RESULTS We demonstrated that a low-pressure persufflation (9 mmHg) is as sufficient as a higher pressure persufflation (18 mmHg) in oxygenating the ischemic organ. Moreover, oxygenation of the liver was found to be complete and rather homogeneous upon the pure oxygen persufflation, irrespective of the insufflation pressure used. In contrast, insufflation of air instead of pure oxygen resulted in insufficient aeration of the liver, even at the higher persufflation pressure of 18 mmHg. CONCLUSIONS Our results indicate that the oxygen concentration of the persufflation gas rather than the persufflation pressure is a determinant of successful tissue oxygenation during cold storage.

Reduction of proteolysis by venous-systemic oxygen persufflation during rat liver preservation and improved functional outcome after transplantation.

An increase of cytosolic proteolytic activity during ischemic preservation and consecutive tissue degradation have recently been recognized as a major pathogenetic factor for liver injury during ischemia/reperfusion. In the present study, we propose a method for preventing proteolytic tissue disintegration, which results in improved recovery of the liver after transplantation. Livers were harvested from rats and stored for 24 hr at 4 degrees C in University of Wisconsin solution (group A). Others were additionally persufflated with gaseous oxygen via the inferior caval vein during this time (group B). At the end of ischemic preservation, proteolysis was confirmed in group A, with significantly elevated tissue levels of free alanine and free amino groups, whereas proteolysis was prevented in group B. After transplantation, the integrity of the graft was significantly improved in group B, in which there was a 50% reduction of plasma activities of alanine amino-transferase and a twofold increase in hepatic bile production after the onset of reperfusion, as compared with group A. Moreover, venous-systemic oxygen persufflation during cold preservation significantly attenuated the rise in plasma levels of malondialdehyde (MDA) after liver transplantation. In conclusion, venous-systemic oxygen persufflation during ischemic storage prevents tissue proteolysis and reduces parenchymal injury after transplantation in vivo; this technique may, thus, represent a useful adjunct in long-term liver preservation with University of Wisconsin solution.

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.

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.

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).

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.

No evidence for a protective effect of ascorbic acid on free radical generation and liver injury after hemorrhagic shock in rats.

Oxygen free radicals have been shown to be implicated in ischemic tissue injury, and free radical-induced reactions may also play an important role in the pathophysiology of circulatory shock. The present study was designed to investigate the potential use of ascorbic acid as an exogenous antioxidant on the livers recovery from hemorrhagic shock in situ. Rats (fasted overnight) were subjected to 60 min of hemorrhagic shock (HS) (mean arterial pressure = 40 mmHg) under pentobarbital anesthesia, followed by retransfusion of the shed blood. One-half of the animals (n = 6) were injected with 10 mg/kg of ascorbic acid prior to induction of shock, while untreated animals (n = 6) received the same volume of saline solution. In untreated animals, systemic plasma levels of malondialdehyde rose from 1.07 ± .08 during normotension (NT) to 1.36 ± .18* 60 min after resuscitation (RS), documenting oxygen free radical-induced lipid peroxidation. Accordingly, plasma levels of alanine aminotransferase (16.5 ± 2.5; 34.9 ± 12.3*; 105.8 ± 68.7* U/L; NT/HS/RS) and ammonia (127 ± 40; 532 ± 160*; 304 ± 244* μg/dL) rose significantly during the experiment. Hepatic ATP content of the liver fell from 4.8 ± .83 to .56 ± .27* after HS and recovered partially to 2.7 ± 1.6* μmol/g after RS. Leukocyte infiltration in the liver, indicated by tissue levels of myeloperoxidase, remained constant during HS but rose during RS (37.9 ± 18.5; 38.6 ± 16.4; 81.4 ± 30.7*; arbitrary units), thus documenting an inflammatory reaction after HS. In the ascorbic acid group, plasma levels of malondialdehyde were comparable to those of untreated animals after RS, as were enzyme concentrations and ammonia. No differences were observed with regard to the tissue concentrations of ATP or myeloperoxidase. Mean arterial blood pressure as well as liver tissue perfusion, as measured by Laser Doppler flowmetry, did not show significant differences between the groups. It was concluded that, although an effect of oxygen free radicals on liver tissue could be found during and after HS, treatment with ascorbic acid alone, in our model, failed to ameliorate the recovery of the animals upon resuscitation (values are mean ± SD; *,p < .05 vs. NT; one-way ANOVA)

Gaseous Oxygenation of the Ischemic Rat Liver

The lack of aerobic metabolism during ischemic organ preservation plays a pivotal role in the development of tissue alterations during the storage period and favours the manifestation of reper-fusion injuries, deteriorating organ viability upon postischemic resumption of nutritive blood circu-lation. Gaseous insufflation of oxygen via the venous vascular system has proven to be a useful tool to prevent anoxic tissue injury during extended time periods of ischemic preservation of kidneys (Isselhard et al., 1972) or livers (Minor and Isselhard, 1996; Minor et al., 1996) and to allow for an improved recovery of the persufflated organ after transplantation (Fischer et al., 1978; Minor et al., 1997b; Rolles et al., 1989).

Über die Bedeutung von Superoxiddismutase bei der Revitalisierung stark warmischämisch vorgeschädigter Lebern mittels retrograder Sauerstoffpersufflation

Eine warmischamische Vorschadigung der Leber fuhrt zu einem drastischen Anstieg irreversibler Funktionsausfalle des Organs nach Transplantation [1, 2] und ist somit ein limitierender Faktor fur die Verwendung von Lebern zu Transplantationszwecken nach bereits eingetretenem Herz-Kreislaufstillstand des Spenders. In fruheren Studien konnte am Modell der isolierten Rattenleber die Moglichkeit der Revitalisierung von 30 min warmischamisch vorgeschadigten Lebern mittels retrograder Sauerstoffpersufflation wahrend hypothermer Lagerung und Zusatz von Superoxiddismutase (SOD) zu den Freispullosungen aufgezeigt werden [3]. In der vorliegenden Studie sollte am gleichen Modell untersucht werden, erstens, ob eine Verdopplung der Warmischamiedauer auf 60 min mit Hilfe der o.g. Methode noch „reparabel“ ist, und zweitens, ob der Zusatz von SOD zu den Freispullosungen dabei essentiell ist.