Recent observations provide evidence that complement is implicated as an important factor in the pathophysiology of ischemia/reperfusion injury (IRI). Here, we assessed the effects of complement inhibition on hepatic microcirculation by in vivo microscopy (IVM) using a rat model of warm hepatic ischemia clamping the left pedicle for 70 min. Ten animals received the physiological complement regulator soluble complement receptor type 1 (sCR1) intravenously 1 min prior to reperfusion. Controls were given an equal amount of Ringer's solution (n = 10). Microvascular perfusion and leukocyte adhesion were studied 30 to 100 min after reperfusion by IVM. Microvascular perfusion in hepatic sinusoids was significantly improved in the sCR1 group (80.6 ± 0.6% of all observed sinusoids were perfused [sCR1] vs 67.3 ± 1.2% [controls]). The number of adherent leukocytes was reduced in sinusoids (49.9 ± 3.4 [sCR1] vs 312.3 ± 14.2 in controls {adherent leukocytes per square millimeter of liver surface}; P < 0.001) as well as in postsinusoidal venules after sCR1 treatment (230.9 ± 21.7 [sCR1] vs 1906.5 ± 93.5 [controls] {adherent leukocytes per square millimeter of endothelial surface}; P < 0.001). Reflecting reduced hepatocyte injury, liver transaminases were decreased significantly upon sCR1 treatment compared to controls. Our results provide further evidence that complement plays a decisive role in warm hepatic IRI. Therefore, we conclude that complement inhibition by sCR1 is effective as a therapeutical approach to reduce microcirculatory disorders after reperfusion following warm organ ischemia.

Complement plays a decisive role in postischemic tissue injury, a process responsible for severe damage after organ ischemia. Several pathophysiologic mechanisms initiated upon reperfusion are mediated by complement inducing micro-circulatory disturbances. Here, we demonstrate the effects of complement inhibition using C1-esterase inhibitor (C1-INH) on microcirculation after liver ischemia by invivo microscopy (IVM). In rats, the left liver lobe was clamped for 70 min. C1-INH was given 1 min prior to reperfusion. Controls received Ringer's solution. IVM was performed 30-100 min after reperfusion. Non-perfused acini decreased and sinusoidal perfusion increased substantially after treatment. Leukocyte adherence to sinusoidal and venular endothelium was markedly reduced by C1-INH. Transaminases were significantly decreased by C1-INH. Our data obtained by IVM suggest that complement activation is an early key event of ischemia/reperfusion injury. These observations demonstrate for the first time that reperfusion related microcirculatory disorders can be minimized by C1-INH. This compound should be evaluated in clinical application.

Der postischämische Reperfusionsschaden ist wesentlich für die postoperative Maloder Nonfunktion von Lebern nach warmer Ischämie im Rahmen von ausgedehnter Resektionen oder aber Lebertransplantationen verantwortlich. Es konnte nachgewiesen werden, daß Mikrozirkulationsstörungen die postischämische Leberfunktion ganz entscheidend beeinflussen [1]. Im Rahmen des pathophysiologischen Prozesses der Reperfusion eines ischämischen Organes kommt es zur Auslösung inflammatorischer Prozesse, welche durch Entzündungsmediatoren initiiert and gesteuert werden. Das Komplementsystem steht dabei als der wichtigste Mediator im Mittelpunkt dieses Geschehens, wobei eine Aktivierung sowohl über den klassischen als auch den alternativen Weg erfolgt [2]. Die Anaphylatoaxine C3a and C5a, als potente Entzündungsmediatoren, initieren in der Leber pathophysiologisch bedeutsame Prozesse, wie die Aktivierung und Akkumulation von Leukozyten [3], die Kontraktion von glatter Muskulatur [2], die Steigerung der Permeabilität der Gefäßwände, sowie die Aktivierung von Kupffer Zellen [4]. Komplement initiiert die Aktivierung von Endothelzellen, was zu Fibrinanlagerung, Thrombozytenaggregation und anlagerung sowie zur Adhäsion von Leukozyten führt. Diese durch Komplementaktivierung hervorgerufenen pathophysiologischen Prozesse werden für einen verminderten, eventuell bis zum Stillstand reduzierten mikrozirkulatorischen Blutfluß in der postischämischen Reperfusionsphase der Leber verantwortlich gemacht [1]. Mikrozirkulationsstörungen sind ja wie beschrieben eine wesenfliche Determinante der postischämischen Leberfunktion [1].

Background: Recent observations provide evidence that the complement system is involved in the pathophysiology of ischemia/reperfusion injury. Methods: In this study we assessed the impact of complement inhibition on hepatic microcirculation by in vivo microscopy (IVM) using a rat model of warm hepatic ischemia. Therefore the left liver lobe was clamped for 70 min. 12 animals received the physiological complement regulator C1-Esterase-Inhibitor (C1-Inh) intravenously 1 min prior to reperfusion, This component blocks the classical pathway of the complement cascade. First clinical trials showed a beneficial effect in cases of capillary leak syndromes, especially angioedema. Controls received an equal amount of Ringer's solution (n = 12). Microvascular perfusion and leukocyte-endothelial cell interaction were studied 30 to 100 min after reperfusion by IVM. Results: Microvascular perfusion in hepatic sinusoids was improved in the C1-Inh group (90% +/- 2.2 vs. 71.9% +/- 3.3; p < 0.001). The number of adherent leukocytes was reduced in sinusoids (96.5 +/- 13.7 vs. 198.8 +/- 20 [adherent leukocytes per mm(2) liver surface]; p < 0.001) as well as in postsinusoidal venules after sCR1 treatment (129.4 +/- 12.2 vs. 366.1 +/- 31.9 [adherent leukocytes per mm2 endothelial surface]; p < 0.001). Plasmatic liver enzyme activity was decreased significantly upon C1-Inh treatment, indicating reduced parenchymal cell injury. Conclusion: Our results provide further evidence that the complement system plays a decisive role in hepatic ischemia/reperfusion injury. Based on our results we conclude that complement inhibition by C1-Inh represents an effective treatment to prevent reperfusion injury in warm organ ischemia not restricted to the liver.

Two groups of male and female Sprague-Dawley rats (50 animals/group per sex) were treated with either 15.37 or 46.77 μmole of 1,1,2-TCE in DMSO/rat for 2 years. The animals were treated once a week by s.c. injection of test compound in the skin of neck. Two groups of controls received either DMSO or no treatment at all. The incidence of benign mesenchymal and epithelial tumors was not significant when compared with either DMSO-treated or untreated controls. The animals treated with 46.77 μmole 1,1,2-TCE significantly developed sarcomas when compared with the untreated controls. In a further experiment, either 40 μmole or 160 μmole 1,1,2-TCE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in 24-h urine samples. Comparative studies were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. Analysis of the metabolites showed that no detectable HEMA was excreted in urine after treatment of rats with 1,1,2-TCE or chloroethanol. TdGA was excreted in urine much more among chloroacetaldehyde-treated animals than among 1,1,2-TCE- or chloroethanol-treated rats.

Either 40 μmole or 160 μmole 2,2′-DDE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in the 24-h urine specimens. Comparative investigations were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. In a further step, inhalation experiments were performed to determine urinary excretion of the two metabolites after an 8-h exposure of male Wistar rats to 10, 50, 100, and 500 ppm 2,2′-DDE and to 50, 200, und 1000 ppm vinyl chloride.

Since the early studies of Durston and Ames in 1974, the Salmonella oxygenase test (Ames et al. 1973 b) and the bacterial fluctuation tests (Gatehouse and Delow 1979; Green et al. 1977) have been extended for the detection of mutagenic metabolites of the environmental carcinogens in urine of man and laboratory animals (Aeschbacher and Ruch 1982; Commoner et al. 1974; Durston and Ames 1974; Falck et al. 1980; Gibson et al. 1983; Kriebel et al. 1983; Legator et al. 1975; McCann and Ames 1975; Norpoth 1984). Histidine-requiring mutants of Salmonella typhimurium which revert to prototrophy by a variety of mutagens are used to detect mutagenic activity in the urine of patients receiving chemotherapeutic agents and workers exposed to various mutagens.

Several male Wistar rats were individually placed in a chamber resembling a room provided with minimal air flow. They were exposed separately to the main- and sidestream smoke of a commerical brand of cigarettes smoked by a smoking machine. Exposure to both sidestream and mainstream smoke of at least two cigarettes resulted in significant excretions of frameshift mutagens in urine within 24h, detected by the bacterial microtiter fluctuation test with Salmonella typhimurium TA1 538. Doubling exposure to the mainstream smoke resulted in similar quantitative mutagenic activities. Doubling exposure to the side-stream smoke resulted in reduced water intake by the animals and thus toxic effects of the urine concentrates on the test bacteria.

Fifteen workers employed in a PVC producing plant were investigated concerning their individual vinyl chloride (VCM) exposure and the urinary excretion of the VCM metabolite thiodiglycolic acid (TdGA). The urine concentrations found were in the range 0.94–20.4 μg/ml. These could be compared with exposure data calculated from VCM air analyses performed by personal air sampling and corrected with respect to the exposure times of the workers. The amounts of TdGA excreted within 24 h were correlated with the effective VCM body concentrations calculated from the exposure data as mean values for 12 h periods (Spearman coefficient P=α<0.005). This correlation resembles a function of the Michaelis-Menten type. It could be shown that during short exposure periods of less than 5 min, the metabolite formation in relation to the exposure data was lower than during longer periods of exposure although, as would be expected, there were some fluctuations of the exposure level. Therefore, the VCM body concentrations could not normally reach steady state values.