Abstract-Although the serine protease, tissue plasminogen activator (tPA), is approved by the US Food and Drug Administration for therapy to combat focal cerebral infarction, the basic concept of thrombolytic tPA therapy for stroke was challenged by recent studies that used genetically manipulated tPA-deficient (tPAϪ/Ϫ) mice, which suggested that tPA mediates ischemic neuronal damage. However, those studies were potentially flawed because the genotypes of tPAϪ/Ϫ and wild-type control mice were not entirely clear, and ischemic neuronal injury was evaluated in isolation of tPA effects on brain thrombosis. Using mice with appropriate genetic backgrounds and a middle cerebral artery occlusion stroke model with nonsiliconized thread, which does lead to microvascular thrombus formation, in the present study we determined the risk for cerebrovascular thrombosis and neuronal injury in tPAϪ/Ϫ and genetically matched tPAϩ/ϩ mice subjected to transient focal ischemia. Cerebrovascular fibrin deposition and the infarction volume were increased by 8.2-and 6.7-fold in tPAϪ/Ϫ versus tPAϩ/ϩ mice, respectively, and these variables were correlated with reduced cerebral blood flow up to 58% (PϽ0.05) and impaired motor neurological score by 70% (PϽ0.05). Our findings indicate that tPA deficiency exacerbates ischemia-induced cerebrovascular thrombosis and that endogenous tPA protects the brain from an ischemic insult, presumably through its thrombolytic action. In addition, our study emphasizes the importance of appropriate genetic controls in murine stroke research. (Arterioscler Thromb Vasc Biol.

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SUMMARY Liver damage was induced in rats by injection of dimethylnitrosamine (DMN) or carbon tetrachloride (CC4). Fibrin clots were observed in the hepatic sinusoids at 12 hours and soluble fibrin monomer complexes were markedly detected at 24 hours only in the rats given DMN. When antithrombin III concentrate was infused at 12 hours there was a dose dependent improvement of the values of serum total bilirubin, SGPT, prothrombin time, peripheral platelet count, and plasma fibrinogen and coagulation factor VIIIC and of the histological degree of liver injury at 24 hours in the DMN group. The CCl4-group showed no such improvement. Intravascular coagulation may complicate the course of certain types of acute liver injury and contribute to its aggravation in rats. Under such circumstances, treatment with antithrombin III concentrate would be beneficial.Both the sinusoidal deposition of fibrin and the increased catabolism of fibrinogen in acute liver failure suggest that intravascular coagulation may contribute to the cell injury in this condition.'`7 The efficacy of early treatment of such intravascular coagulation is, however, still unproven.2`7 It is not clear whether all types of acute liver failure are accompanied by intravascular coagulation and whether in any specific case it contributes to the aggravation of liver damage.Fibrinogen is converted into fibrin by thrombin. Thrombin activity can be inhibited by various plasma inhibitors.' Of the well known three protease

bance induced by microthrombotic occlusion following We investigated the role of anticoagulant in the isischemia/reperfusion. (HEPATOLOGY 1997;25:1136-1140.) chemia/reperfusion injury of the liver, using activated protein C (APC), active human urinary thrombomodulin (UTM), and factor Xa blocked at the active site Endothelial cells play an important regulatory role in the (DEGR-Xa). Liver ischemia was induced in male Wistar coagulation system. Protein C is a vitamin K-dependent rats by occlusion of the portal vein with a microvascuplasma glycoprotein that circulates as an inactive zymogen. lar clip for 30 minutes. Each anticoagulant was injectedAt the endothelial cell surface, thrombin in complex with intravenously 10 minutes before clamping the portal the integral membrane protein, thrombomodulin, converts vein. Serum concentrations of cytokine-induced neuprotein C to its active form by specific cleavage of an activatrophil chemoattractant (CINC) were determined by enzyme-linked immunosorbent assay. The serum levels tion peptide. [1][2][3] The active form of protein C has potent anticoof CINC increased significantly following reperfusion, agulant activity as a feedback regulator of thrombin generareaching a peak in 6 hours, and then decreasing gradu-tion, 4,5 and also has profibrinolytic, 6,7 anti-ischemic, 8 and ally to control levels by 24 hours. CINC levels in rats anti-inflammatory properties. 9 pretreated with APC (500 U/kg), UTM (3,000 TMU/kg), Neutrophil activation has been shown to play an important or DEGR-Xa (10 mg/kg) peaked 3 hours following reper-role in ischemia/reperfusion injury associated with the shock fusion and decreased rapidly to baseline level within process. 10,11 Recently, Jaeschke et al. 12 and Poggeti et al. 13 6 and 12 hours, respectively. These peak values were showed the importance of neutrophils in the development significantly lower than those observed in untreated of liver ischemia/reperfusion injury. When activated in the rats (P õ .01). Expression of CINC transcripts in liver microcirculation, neutrophils release a variety of inflammatissue of untreated rats was evaluated by Northern blot tory mediators capable of producing endothelial cell injury. 14 analysis and peaked 3 hours following reperfusion. Pre-Among these mediators, granulocyte proteases and hydrogen treatment with these anticoagulants significantly de-peroxide have been shown to induce inactivation of thrombocreased the expression of CINC messenger RNA tran-modulin synergistically, as well as damage to the endothelial scripts as compared with untreated animals. cell integrity. 15 Thus, it is likely that microthrombus can be Myeloperoxidase activity and the number of neutro-formed in ischemia/reperfusion as a result of activated leukophils accumulated into the liver 24 hours following cyte-induced endothelial cell injury, which may lead to tissue ischemia/reperfusion were also significantly decreased ischemia. Because production of interleukin (IL)-1 by monoin animals pretreated with these anticoagulants. In ad-cyte...

Neutrophils mediate microvascular and parenchymal cell dysfunction in a variety of organs after ischemia-reperfusion. 1,2 Reperfusion of ischemic tissue results in the formation of proinflammatory mediators that promote the adherence and emigration of neutrophils in postcapillary venules. 3 The activated neutrophils subsequently injure tissue by releasing oxidants and proteases. 4,5 This hypothesis is based largely on studies demonstrating that tissue injury induced by ischemia-reperfusion is attenuated by rendering the animals neutropenic with antineutrophil serum. 6,7 Additional support for this view is derived from several studies showing that monoclonal antibodies against either the CD11a or CD11b ␣ subunits of the CD11/CD18 on neutrophils 8,9 or intercellular adhesion molecule-1 (ICAM-1) on endothelial cells 10 afford significant protection against ischemia-reperfusion injury. Thus, ICAM-1 is important for the development of a neutrophil-dependent injury mechanism.Alterations in the endothelial cell in response to cytokines may be of critical importance in interactions between circulating leukocytes and the vessel wall, because changes in the expression of adhesion receptors may regulate the number and type of leukocytes emigrating from the blood into inflamed tissues. 11 Tumor necrosis factor ␣, interleukin-1, and interferon gamma have been reported to induce the production of ICAM-1 message and protein in isolated murine hepatocytes, 12 human hepatocytic cell lines, 13 and endothelial cells. 14,15 These observations suggest that ICAM-1 expression may be induced by other proinflammatory cytokines.Monocyte chemoattractant protein 1 (MCP-1), originally described as the JE gene, is a 148-amino acid chemotactic cytokine [16][17][18] that is expressed by a number of cells including monocytes, leukocytes, fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, and pulmonary epithelial cells. [16][17][18][19][20] Jiang et al. 21 and Vaddi et al. 22 demonstrated that MCP-1 induced expression of CD11c (p150,95, ␣-subunit) and CD11b (Mac-1 ␣-subunit), suggesting that MCP-1 is not only a chemoattractant, but also a cytokine with the capacity to regulate monocyte function. Thus, MCP-1 may modulate leukocyte-endothelial cell interactions, specifically by affecting ICAM-1 expression. Kupffer cells generate a wide range of inflammatory mediators including cytokines, eicosanoids, reactive oxygen intermediates, and other factors. The production of several cytokines following Kupffer cell activation may be important in the pathophysiology of liver ischemiareperfusion injury. Therefore, the present study was undertaken to investigate the effects of MCP-1 produced by Kupffer cells on ICAM-1 expression after ischemia-reperfusion of the rat liver. MATERIALS AND METHODSAnimals. Male Wistar rats were obtained from a commercial supplier (Kyudo, Inc., Kumamoto, Japan). All animals were maintained under standard conditions and were fed water and rodent chow ad libitum. The animals weighed between 225 and 250 g.Preparatio...

When a liver perfusion with nitro blue tetrazolium (NBT) and phorbol myristate acetate (PMA) was performed in carbon tetrachloride (CCl4)-intoxicated rats, formazan deposition was remarkable in macrophages in the necrotic areas of the liver, its intensity varying with the extent of injury. The deposits almost disappeared after addition of Cu(Lys)2, a scavenger of intra- and extracellular superoxide, but were not affected by superoxide dismutase (SOD), which acts extracellularly. The formazan content after incubation with NBT and PMA was higher in macrophages isolated from CCl4-intoxicated liver than in those from normal liver, though their PMA-induced chemiluminescence did not differ. In Corynebacterium parvum-treated liver, both Cu(Lys)2 and SOD reduced the deposits. This method can estimate in situ the ability of hepatic macrophages to produce superoxide and the cellular sites of its production.