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To clarify the in vivo relevance of leukocyte-endothelial cell interactions in the manifestation of hepatic ischemia-reperfusion (I/R) injury, we studied leukocyte flow behavior in sinusoids and postsinusoidal venules of postischemic hepatic tissue in rats using intravital microscopy. Reperfusion following either 20 min (n = 9) or 60 min (n = 9) of left hepatic lobar ischemia resulted in a significant increase of the number of stagnant leukocytes in sinusoids and adherent cells in postsinusoidal venules compared with sham-operated controls (n = 10). Transmission electron microscopy revealed the extravasation of leukocytes from both sinusoids (into the space of Disse) and postsinusoidal venules. In parallel, hepatic I/R was associated with increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and reduced bile flow. Linear regression estimates revealed significant (P < 0.01) correlations between serum ALT (r = 0.76) and AST (r = 0.65) activities, bile flow (r = -0.62), and the number of adherent leukocytes in postsinusoidal venules. In contrast, parameters of hepatocellular integrity and function did not directly correlate with the number of stagnant leukocytes in liver sinusoids. We conclude that hepatic I/R induces accumulation, adherence, and extravasation of leukocytes in both hepatic sinusoids and postsinusoidal venules. However, the adherence of leukocytes to the endothelial lining of venules, rather than of sinusoids, may determine the manifestation of hepatocellular damage and liver dysfunction.

Growing evidence supports a pathophysiological role for platelets during the manifestation of postischemic reperfusion injury; in the current study, we investigated the nature and the molecular determinants of platelet-endothelial cell interactions induced by ischemia/reperfusion (I/R). Platelet-endothelium and leukocyte-endothelium interactions after 1 hour of ischemia were monitored in vivo within mouse small intestine. By intravital fluorescence microscopy, we observed that platelets, like leukocytes, roll along or firmly adhere to postischemic microvascular endothelial cells. In contrast, few leukocyte-endothelial cell interactions were detected in sham-operated controls. Monoclonal antibodies against P-selectin significantly attenuated platelet rolling and adherence in response to I/R. To identify whether platelet or endothelial P-selectin plays the major role in mediating postischemic platelet-endothelial cell interactions, P-selectin-deficient or wild-type platelets were transfused into wild-type or P-selectin-deficient mice, respectively. Whereas platelets lacking P-selectin rolled along or adhered to postischemic wild-type endothelium, interactions between wild-type platelets with mutant endothelium were nearly absent, indicating that I/R-induced platelet-endothelium interactions are dependent on the expression of P-selectin by endothelial cells. Concomitantly, P-selectin expression in the intestinal microvasculature was enhanced in response to I/R, whereas no upregulation of P-selectin was observed on circulating platelets. In summary, we provide first in vivo evidence that platelets accumulate in the postischemic microvasculature early after reperfusion via P-selectin-ligand interactions. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of I/R injury.

Following ischemia-reperfusion (I/R), platelet adhesion is thought to represent the initial event leading to remodeling and reocclusion of the vasculature. The mechanisms underlying platelet adhesion to the endothelium have not been completely established. Endothelial cells rendered ischemic acquire a procoagulant phenotype, characterized by fibrinogen accumulation. Therefore, we evaluated whether fibrinogen deposition during I/R mediates platelet adhesion. Using fluorescence microscopy, fibrinogen deposition and the accumulation of platelets were assessed in vivo in a model of intestinal I/R (1.5 hours/60 minutes). Fibrinogen accumulated in arterioles and venules early after the onset of reperfusion. The deposition of fibrinogen colocalized with large numbers of adherent platelets (520 ± 65 and 347 ± 81 platelets/mm2 in arterioles and venules). Pretreatment with an antifibrinogen antibody attenuated platelet adhesion. Intracellular adhesion molecule (ICAM)-1 served as a major receptor for fibrinogen, since fibrinogen deposition and platelet adhesion to the endothelial cell surface were markedly decreased in ICAM-1–deficient mice. The platelet IIb/β3 integrin plays a key role in fibrinogen-dependent platelet accumulation, because (1) platelet adhesion involved RGD-recognition sequences, and (2) platelets isolated from a patient with Glanzmann’s disease showed decreased interaction with the postischemic endothelium. Since platelets are demonstrated here to induce tyrosine phosphorylation in endothelial cells, platelet recruitment might contribute to the development of an inflammatory reaction during I/R.

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