Role of microcirculatory derangements in manifestation of portal triad cross-clamping-induced hepatic reperfusion injury.

Postischemic liver dysfunction following portal triad cross-clamping (PTC) predisposes patients for the development of multiple system organ failure (MSOF) and is associated with increased mortality of MSOF. The deterioration of the hepatic microcirculation may play a pivotal role in the pathophysiologic sequelae of PTC-induced reperfusion injury. We quantitatively analyzed in vivo the role of microcirculatory derangements in the manifestation of hepatic reperfusion injury following ischemia by PTC. Sprague-Dawley rats were subjected to 20 min PTC followed by 60 min reperfusion (PTC, n = 18). Sham-operated animals without ischemia served as controls (sham, n = 15). Within 45-60 min of reperfusion, hepatic microcirculation (sinusoidal perfusion, leukocyte-endothelial cell interaction) was analyzed by means of intravital fluorescence microscopy; tissue oxygenation was assessed using a platinum multiwire surface electrode. Liver enzymes and bile flow were determined as indicators of hepatocellular integrity and liver function. In vivo analysis of postischemic hepatic microcirculation revealed the induction of leukocyte-endothelial cell interaction within sinusoids and postsinusoidal venules and concomitant sinusoidal perfusion deficits associated with tissue hypoxia. In addition, PTC resulted in increased serum levels of liver enzymes and a marked reduction of bile flow. Regression analyses demonstrated significant correlations between hepatocellular desintegration/liver dysfunction and PTC-induced microcirculatory disorders. These results underline the predominant role of microcirculatory disturbances in the development of PTC-induced hepatic reperfusion injury and support the concept that normalization of postischemic microcirculation may be a most effective therapeutic regimen to prevent hepatocellular damage and liver dysfunction.

Leukocytes contribute to hepatic ischemia/reperfusion injury via intercellular adhesion molecule-1-mediated venular adherence.

BACKGROUND: Leukocytes are suggested to modulate ischemia/reperfusion injury via membrane receptor-controlled interaction with the microvascular endothelium. METHODS: With the use of intravital fluorescence microscopy we investigated the role of the intercellular adhesion molecule-1 (ICAM-1) in a rat model of hepatic reperfusion injury with a neutralizing monoclonal antibody (anti-ICAM-1). RESULTS: Sixty minutes of left lobar ischemia and reperfusion (isotype-matched immunoglobulin G1 control antibody) caused leukostasis in sinusoids (240 +/- 15 cells per liver lobule), leukocyte adherence in postsinusoidal venules (679 +/- 76 cells per mm2 endothelial surface of postsinusoidal venules), nutritive perfusion failure (15% +/- 2% nonperfused sinusoids), excretory dysfunction (bile flow, 1.2 +/- 0.3 microliters.min-1.gm-1), and loss of hepatocellular integrity (serum aspartate aminotransferase, 1353 +/- 317 units.L-1; serum alanine aminotransferase, 1055 +/- 265 units.L-1). Anti-ICAM-1 did not affect sinusoidal leukostasis; however, it effectively inhibited postischemic leukocyte adherence to the venular endothelial lining (217 +/- 38 cells/mm2, p < 0.01). Concomitantly, hepatic reperfusion injury, including sinusoidal perfusion (6% +/- 1% nonperfused sinusoids, p < 0.01), excretory function (bile flow, 1.8 +/- 0.1 microliters.min-1.gm-1, p < 0.05), and hepatocellular integrity (aspartate aminotransferase, 480 +/- 108 units.L-1; alanine aminotransferase, 447 +/- 80 units.L-1, p < 0.05), was significantly ameliorated by anti-ICAM-1. CONCLUSIONS: These findings prove in vivo the pivotal role of ICAM-1 in leukocyte-dependent manifestation of postischemic liver damage.

Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm ischemia-reperfusion.

Hepatic ischemia-reperfusion (I/R) is characterized by circulatory and metabolic derangements, liver dysfunction, and tissue damage. However, little is known about the causative role of I/R-induced microcirculatory disturbance on the manifestation of postischemic reperfusion injury. Therefore, the intention of the study was to assess changes of hepatic microvascular perfusion (intravital fluorescence microscopy) as related to hepatic morphology (light/electron microscopy), hepatocellular integrity (serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities), and excretory function (bile flow). Sprague-Dawley rats were subjected to 20 minutes (group B, n = 9) and 60 minutes (group C, n = 9) of left hepatic lobar ischemia followed by 60 minutes of reperfusion. Sham-operated animals without ischemia served as controls (group A, n = 10). Lobar ischemia for 20 minutes followed by reperfusion resulted in a significant reduction of sinusoidal perfusion rate (93.9 +/- 1.4%; P < 0.05) and a decrease in erythrocyte flux (90.0 +/- 5.6%) when compared with controls (99.4 +/- 0.2 and 97.9 +/- 2.7%). This was accompanied by a significant increase of serum AST and ALT activities (P < 0.05) and a reduction of bile flow (P < 0.05). Prolongation of lobar ischemia (group C, 60 minutes) aggravated postischemic reperfusion injury (sinusoidal perfusion rate: 87.4 +/- 2.9%; erythrocyte flux: 62.1 +/- 8.4%) and was paralleled by severed hepatocellular damage. Electron microscopy of postischemic tissue demonstrated alteration of nonparenchymal cells (swelling of sinusoidal lining cells and widening of Disse's space) and substantial parenchymal cell damage (swelling of mitochondria, disarrangement of rough endoplasmatic reticulum, vacuolization, complete cytoplasmic degeneration). Initial postischemic increase in serum AST and ALT activities and reduction of bile flow directly correlated with the extent of microcirculatory failure (P < 0.01), ie, impairment of sinusoidal perfusion and decrease of erythrocyte flux, indicating the decisive role of microvascular perfusion failure for the manifestation of hepatic tissue damage and liver dysfunction.

Impact of leukocyte-endothelial cell interaction in hepatic ischemia-reperfusion injury.

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.

Depressed phagocytic activity of Kupffer cells after warm ischemia-reperfusion of the liver.

Phagocytic activity of Kupffer cells following hepatic ischemia/reperfusion was studied in 39 livers of male Sprague-Dawley rats by in vivo fluorescence microscopy. Animals were subjected to either 20 min (group B, n = 9) and 60 min left hepatic lobar ischemia (group C, n = 9) or to 20 min of global hepatic ischemia (group D, n = 11). Sham-operated animals without ischemia served as controls (group A, n = 10). After 60 min postischemic reperfusion, fluorescent latex beads (3 x 10(8).kg body wt-1; diameter: 1.1 microns) were injected intra-arterially. The zonal distribution and kinetics of adherence of latex beads were quantified by off-line video analysis. After 20 min of left hepatic lobar ischemia, 50%, 38% and 12% of injected latex beads adhered in zones 1, 2 and 3, respectively, and did not significantly differ from control livers (group A: 57%, 32% and 11%). In contrast, after 60 min of left hepatic lobar ischemia (group C) as well as after 20 min of global hepatic ischemia (group D), a more homogeneous distribution of latex beads adherent in zones 1, 2 and 3 was observed (group C: 48%, 36% and 16%; group D: 48%, 36% and 16%). Kinetic analysis of phagocytosis (% adherence of visible latex beads 1 min and 3 min after injection) showed no significant difference between 20 min left hepatic lobar ischemia (group B: 84% and 95%) and control (group A: 81% and 95%).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of leukocytes in the initial hepatic microvascular response to endotoxemia.

INTRODUCTION: Accumulation and adherence of leukocytes within the hepatic microvasculature have been emphasized to play a major role in the pathogenesis of endotoxin/lipopolysaccharide (LPS)-induced liver injury. However, there is no information on their interrelation with hepatic microvascular perfusion failure, hepatocellular damage and liver dysfunction following LPS exposure. AIM AND METHODS: Therefore, we quantitatively assessed the initial LPS-induced hepatic microvascular response, including leukocyte-endothelium interaction and their interrelation with sinusoidal perfusion, hepatocellular integrity (serum AST/ALT activity) and excretory function (bile flow). After infusion of LPS (E. coli 0128:B12; 10 mg.kg-1 i.v.) intravital fluorescence microscopy was applied to livers of Sprague-Dawley rats. RESULTS: 1 h after LPS exposure deterioration of hepatic microcirculation was hallmarked by significant accumulation of leukocytes, stagnant within sinusoids and adherent to the endothelial lining of postsinusoidal venules. This was accompanied by a progressive increase of the number of non-perfused sinusoids (20 +/- 4%). During the 1 h period after LPS exposure, bile flow was found significantly (p < 0.05) reduced, while serum AST/ALT activities remained unchanged. Leukocytes appear to contribute to sinusoidal perfusion failure, since the number of non-perfused sinusoids significantly (p < 0.01) correlated with the number of leukocytes stagnant within the sinusoids. In addition, the inverse correlation (p < 0.01) of bile flow with the number of both, leukocytes stagnant within the sinusoids and non-perfused sinusoids indicates that microvascular injury initiates hepatic dysfunction. CONCLUSION: Inasmuch as LPS exposure initially induces only microcirculatory disturbances without substantial loss of hepatocellular integrity, we propose that therapeutic strategies during early endotoxemia should focus on attenuation of microvascular injury to prevent manifestation of hepatocellular damage.