INTRODUCTION

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ISCHEMIA and subsequent reperfusion lead to substantial damage to cells and organs, depending on the ischemic time. Although reperfusion supplies O₂ to ischemic tissues, it may trigger a complex series of pathophysiological events including free radical formation (4, 8, 15, 17, 29), and little information is available regarding tissue blood flow during reperfusion (7, 18, 28). Although transient hyperemia may be observed after short-term ischemia (3, 20), the final tissue blood flow after ischemia-reperfusion does not reach preischemic levels. Some investigators have reported that no reflow or decreased tissue perfusion after ischemia causes substantial damage after reperfusion (28). These phenomena may exacerbate cellular and organ damage after ischemia-reperfusion.

Hepatic blood circulation is unique in that the supply comes from both venous blood from the portal vein and arterial blood from the hepatic artery. The venous blood contributes 70-80% of the total hepatic blood flow, and the arterial blood contributes 20-30% (9). It is well known that portal blood flow is inversely correlated with hepatic arterial blood flow, a phenomenon known as the hepatic arterial buffer response (9). Although tissues with a single blood supply show transient hyperemic or decreased blood flow after ischemia (3, 20), no detailed information is available regarding hepatic microcirculation and the recovery of hepatic arterial and portal venous blood flow. It is therefore important to evaluate separately the recoveries of hepatic arterial and portal blood flows after transient ischemia and their roles in O₂ transport to hepatocytes during reperfusion.

In the present study, we analyzed the recovery of O₂ transport and hepatic microcirculation during postischemic reperfusion using 1) a laser-Doppler flowmeter for measuring tissue blood flow, 2) an ultrasonic transit-time volume flowmeter for separate measurements of portal venous and hepatic arterial blood flow, 3) phosphorescence imaging for monitoring microvascular O₂ pressure, and 4) a dual-spot microspectroscope for determining the O₂ transport parameters in single sinusoids. The results showed different recovery patterns of blood flow from the hepatic artery and portal vein and biphasic recovery for hepatic tissue blood flow, with the first peak predominantly from the portal vein and the second peak from the hepatic artery.

	MATERIALS AND METHODS

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Reagents. Vinblastine (1.6 mg/kg body wt) purchased from Sigma Chemical (St. Louis, MO) was intraperitoneally administered to the rats at 5 and 2 days before the experiments. Allopurinol (50 mg/kg body wt, Sigma Chemical) was intraperitoneally administered 1 h before the experiments. All other chemicals were of analytical grade.

Animals. Male Lewis rats weighing 200-250 g were deprived of food and had free access to water for 12 h before the experiments. All animal experiments were conducted in accordance with our institutional guidelines for the care and use of laboratory animals. Rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip) and allowed to breathe spontaneously. Catheters were inserted into the femoral artery and a tributary of the superior mesenteric vein for monitoring systemic arterial and portal venous pressures, respectively. Physiological saline (3 ml · h¹ · kg body wt¹) was infused with an infusion pump (model 11, Harvard Apparatus, South Natick, MA) from a catheter inserted in the femoral vein throughout the experiments. After a transverse upper abdominal laparotomy, the liver was mobilized and isolated by dividing all of its attachments; negligible bleeding occurred.

Measurement of hepatic blood flow. A laser-Doppler flowmeter (ALF 21, Advance, Tokyo, Japan) was used for monitoring tissue blood flow. After a baseline recording of hepatic blood flow for 10 min, the ischemic procedures were performed for the indicated times. Postischemic hepatic blood flow was monitored for 30 min after release of the microsurgical clip. Hepatic blood flow monitoring was conducted in the same area of the left lateral lobe.

Microspectroscopic measurements of erythrocyte velocity and hemoglobin oxygenation. The apparatus and the analytical method were essentially the same as those reported previously (19, 25). Three units were combined with a microscope: 1) a computer-controlled scanning spectrophotometer for measuring the visible spectra of tissues and erythrocytes to obtain the O₂ saturation (SO₂) of hemoglobin flowing in single sinusoids; 2) two photomultipliers for measuring the erythrocyte velocity (v) according to the dual-spot cross-correlation method, and 3) a charge-coupled device camera and an image analyzer for measuring the sinusoid diameter and length of the sinusoid.

Imaging of plasma PO₂ in hepatic microcirculation. The PO₂ in hepatic sinusoids was measured and visualized using a Wild Macrozoom microscope with an epifluorescence attachment (OXYMAP, Medical Systems, Green Valley, NY) (24, 30). Optical filters were used to measure phosphorescence (excitation window of 45 nm centered at 545 nm and emission at 645 nm with a cutoff filter). Pd-meso-tetra(4-carboxyphenyl)porphine (20 mg/kg body wt) solubilized in physiological saline (pH 7.5), containing 60 mg/ml bovine serum albumin was injected from the femoral vein before the measurements. In a dark room, phosphorescence was collected and imaged as follows: after eight flashes, the phosphorescence intensity was measured at 20, 40, 60, 80, 100, 160, 300, 600, 700, 800, and 2,500 µs. The phosphorescence-measurable area of the liver was 3 cm × 3 cm in this series, and the phosphorescence decay was fit by a single exponential function. A quenching constant of 331 Torr¹ · s¹ and a lifetime of 586 µs in a zero-oxygen environment were used as the calibration values (26). The PO₂ was calculated using the Stern-Volmer relationship and then mapped (24, 30).

Statistical analysis. Results are expressed as means ± SD unless otherwise indicated. Data were analyzed with the Welch t-test and one-way ANOVA with post hoc test of the Bonferroni multiple-comparison test. P values <0.05 were considered significant.

	RESULTS

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Hepatic tissue circulation after temporary portal triad clamping. Figure 1 shows typical examples of hepatic tissue blood flow and systemic arterial blood pressure during and after temporary portal triad clamping for 5 min. Immediately after total clamping of the hepatoduodenal ligament (PTC model), hepatic blood flow stopped and arterial blood pressure decreased slightly, whereas portal venous pressure increased from 16 ± 2 (mean ± SD) to 53 ± 6 mmHg after clamping. After release of clamping, systemic blood pressure was restored to the preclamping level before recovery of hepatic blood flow, and portal venous pressure returned to the preclamping level within 5 s. Postischemic hepatic blood flow showed a biphasic increase; two peaks of hepatic blood flow appeared at ~4 and 18 min after the initiation of hepatic tissue perfusion (Fig. 1). This biphasic profile was confirmed in separate experiments using portal-systemic shunt rats and partial clamping model rats, in which there was no significant increase in portal venous pressure (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Changes in hemodynamics induced by temporary portal triad clamp and subsequent reperfusion. In rats under pentobarbital anesthesia (40 mg/kg), portal vein and hepatic artery were occluded for 5 min, followed by reperfusion with declamping (PTC model). Hepatic blood flow (A) and systemic arterial blood pressure (B) were monitored as described in MATERIALS AND METHODS. Data from 4 similar experiments represent a typical trace (see Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Typical traces of postischemic hepatic blood flow after varying periods of ischemia. Traces denote hepatic blood flow after 5 (), 15 (), and 30 () min of ischemia (PTC model), respectively. Each point shows 1 typical profile from 4 separate experiments (total of 12 rats, see Table 1). Hepatic blood flow was obtained and traced every 36 s.

Different recovery patterns of hepatic arterial and portal venous blood flow after temporary portal triad clamping. Figure 3 shows simultaneous measurements of postischemic hepatic tissue blood flow and blood flow of afferent vessels (portal vein and hepatic artery) after 5-min ischemia (PTC model). The recovery of hepatic blood flow showed two distinct peaks at ~4 and 22 min (Fig. 3A). Simultaneous measurements of portal venous and hepatic arterial blood flow indicated that the first peak of hepatic blood flow coincided with the peak of portal blood flow and the second peak coincided with that of hepatic arterial blood flow (Fig. 3B). Similar results were obtained for the other ischemic periods.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Simultaneous measurements of postischemic blood flow of liver and its afferent vessels. Under pentobarbital anesthesia (40 mg/kg), portal vein and hepatic artery were occluded for 5 min, followed by reperfusion (PTC model). Hepatic blood flow was measured with laser-Doppler flowmetry (A), and portal venous blood flow () and hepatic arterial blood flow () were separately measured with an ultrasonic transit time volume flowmeter (B) as described in MATERIALS AND METHODS. Each blood flow was obtained and traced at 36-s intervals. Similar patterns of blood flow recovery were obtained for each ischemic period (5, 15, and 30 min).

Effects of temporary portal triad clamping on PO₂ of hepatic microcirculation. The changes in plasma PO₂ in the hepatic microcirculation were examined before and after hepatic ischemia using the PTC model. Before temporary portal triad clamping, the PO₂ level measured by the OXYMAP system was heterogeneous over the liver from 35 to 55 mmHg. During reperfusion, the PO₂ level of the liver heterogeneously increased to 60-70 mmHg in some parts at 4 min after declamping and then decreased (Fig. 4). This increase corresponded to the first peak of hepatic tissue blood flow (Figs. 1, 3, and 4). A second increase in the PO₂ level appeared at 16 min after declamping and spread more widely over the liver than did the first increase (Fig. 4). The peak of PO₂ reached 80 mmHg and then declined to below the preclamping level. This second increase in the PO₂ level coincided with the second peak of hepatic tissue blood flow (Figs. 1, 3, and 4).

View larger version (97K): [in this window] [in a new window]   Fig. 4.   Changes in postischemic PO2 in hepatic microvasculature during reperfusion. In rats under pentobarbital anesthesia (40 mg/kg), portal vein and hepatic artery were occluded for 5 min followed by reperfusion with declamping (PTC model) as described in MATERIALS AND METHODS. Pd-meso-tetra-(4-carboxyphenyl)porphine (20 mg/kg body wt) was injected from femoral vein before measurement. After release of clamp on hepatoduodenal ligament, phosphorescence intensity was measured at 2- to 3-min intervals and calculated PO2 pressure was mapped. Maps of PO2 are presented in pseudocolor with color scale on right. There were 2 increases in plasma PO2; 1 appeared at 4 min after declamping and the other at 16 min. Photographs before clamping and representative photographs for first and second increases are shown. Similar patterns of blood flow recovery were obtained for each ischemic period (5, 15, and 30 min).

Hepatic hemodynamics after temporary portal triad clamping in rats with hepatic artery ligation. The hypothesis that the first peak of hepatic tissue blood flow after reperfusion corresponds to the peak of portal blood flow and the second peak to that of hepatic arterial blood flow was confirmed using a hepatic artery ligation model (HAL model). After ligation of the hepatic artery, hepatic tissue blood flow decreased to 80 ± 6% (mean ± SD) of the initial values, but systemic blood pressure was unchanged (data not shown). Figure 5 shows a typical example of the hepatic hemodynamics and systemic blood pressure during ischemia and reperfusion in the HAL model. Hepatic tissue blood flow showed uniphasic changes, with a maximal peak flow at 3 min after the declamping. Hepatic tissue blood flow in the HAL model lacked the second peak observed in the PTC model (Table 1). The recovery pattern of hepatic tissue blood flow in the HAL model was independent of the ischemic period (Table 1) and was similar to that of portal venous blood flow presented in Fig. 3B.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effects of hepatic artery ligation on microvascular hemodynamics under ischemia and subsequent reperfusion. After ligation of hepatic artery (HAL model), portal vein was occluded for 5 min followed by reperfusion as described in MATERIALS AND METHODS, and hepatic blood flow (A) and systemic arterial blood pressure (B) were monitored as described. Data show a typical trace from 4 different experiments (see Table 1).

Erythrocyte velocity and hemoglobin oxygenation in single sinusoids after temporary portal triad clamping. Hemoglobin SO₂ and v of erythrocytes flowing in single sinusoids were measured with a dual-spot microspectroscope (Fig. 6). In the rats treated by portal triad clamping (PTC model), the changes in the SO₂ and v values during reperfusion were biphasic; two peaks of the SO₂ and v values appeared at ~5 and 20 min after the initiation of sinusoidal blood flow, which corresponded to the first and second peaks in the hepatic blood flow measured by laser-Doppler flowmetry, respectively (Fig. 6A). The SO₂ values of the second phase were slightly higher than those of the first phase, whereas the v values of the second phase were lower than those of the first phase. Similar biphasic patterns of SO₂ and v were obtained with each ischemic period in the PTC model.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Recovery of hemoglobin (Hb) oxygenation (SO2) and velocity (v) of red blood cells flowing in single sinusoids during reperfusion. Under pentobarbital anesthesia (40 mg/kg), rat livers were placed on a specially designed microscopic stage, and SO2 () and v () of red blood cells in single sinusoids were measured by microspectroscopy as described in MATERIALS AND METHODS. A: PTC model. Portal vein and hepatic artery were occluded for 5 min followed by reperfusion. B: HAL model. After ligation of hepatic artery, portal vein was occluded for 5 min followed by reperfusion. Data presented are mean values obtained from 9 different sinusoids of 3 rats for each preparation and are expressed as percentage of preischemic levels. Preischemic values of SO2 and v in PTC group were 54 ± 4% (mean ± SD) and 0.18 ± 0.05 mm/s, respectively, and those in HAL group were 45 ± 6% and 0.14 ± 0.02 mm/s, respectively. Diameters of measured sinusoids were similar between the 2 groups: 12.6 ± 3.4 µm (18 sinusoids of 6 rats).

Effect of reagents on hemodynamics during postischemic reperfusion. Although the administration of vinblastine markedly decreased the number of white blood cells from 6,800 ± 1,270 (mean ± SD) to 1,780 ± 430 per mm³ (n = 4, P < 0.01 with Welch t-test), two similar peaks of hepatic blood flow (first peak appeared at 3.3 ± 0.5 min after initiation of hepatic blood flow and second peak at 13.0 ± 2.4 min) were observed in the PTC model by laser-Doppler flowmetry, and the recovery patterns of hepatic tissue blood flow after declamping were similar to those without vinblastine. The final flow was also equivalent to that without vinblastine (data not shown). Similar results were obtained with the PTC model using allopurinol, which produced the first peak flow at 5.6 ± 4.4 min and the second at 13.0 ± 2.4 min (Table 1).

	DISCUSSION

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Reperfusion after ischemia causes substantial damage to parenchymal and nonparenchymal cells via oxygen-deprived free radical formation and the activation of the complement cascade, neutrophils, and platelets, which result in cell death and necrosis (10, 16). Recent studies have focused on disturbances in the microcirculation during reperfusion after temporary ischemia (5, 16). Ischemia-reperfusion is reported to activate neutrophils adhering to the endothelial cells, which causes microcirculatory disturbances and tissue injury in various organs (12-14, 17, 21, 27). Ferguson et al. (7) showed that although leukocytes accumulated in ischemic loci were not correlated with local microvascular damage at sites of accumulation, the leukocyte accumulation appears to correlate well with microvascular damage. They concluded that leukocyte-independent factors are likely to be of considerable quantitative importance in microvascular injury during reperfusion after hepatic ischemia. The disturbance in the microcirculation may induce cellular hypoxia even after reperfusion and may aggravate ischemic injury.

Hepatic tissue blood flow has been evaluated by several methods, including those used in the present study (1-3, 20). Laser-Doppler flowmetry is reported to be sensitive to blood flow changes in a substantial area of the liver (1, 2). It is more sensitive to blood flow changes in the hepatic artery and those after venous stasis. In contrast, dual-spot microspectroscopy can accurately measure the erythrocyte velocity in single sinusoids, the sinusoidal diameter, hemoglobin oxygenation in erythrocytes, and the O₂ consumption of regional hepatocytes (11, 19, 25). However, this method cannot be used to evaluate the blood flow in areas such as whole lobules. Another method, ultrasonic transit time volume flowmetry, can separately measure portal venous and hepatic arterial blood flow but cannot be used to evaluate tissue flow. The OXYMAP method more sensitively indicates spatial differences in plasma PO₂ but does not show tissue blood flow. These four methods were used to compensate for each other in the evaluation of hepatic tissue blood flow in the present study. One other issue in this study is anesthesia; anesthetics, e.g., the pentobarbital used in the present study, may affect the vascular response and blood flow after ischemia, which would thus not reflect the physiological response of each vasculature. However, in the clinical setting, ischemia-reperfusion of the liver is usually used in interventional or surgical procedures occurring under anesthesia.

The no-reflow phenomenon has been reported to play an important role in the recovery of microcirculation (22). Thus tissue blood flow after short-term ischemia shows a transient hyperemic response and then a gradual decrease in many tissues (3, 20). These tissues have a single blood supply from an artery. In contrast, the liver is doubly supplied with blood flow from both the portal vein and the hepatic artery, and the blood flow recovery of these two structures may differ after a temporary portal triad clamp. Chavez-Cartaya et al. (6) previously described a biphasic increase in hepatic tissue blood flow during reperfusion observed by laser-Doppler flowmetry and speculated that free radicals, complement activation, and leukocyte plugging were possible causes. In the present study, the biphasic increase in hepatic tissue blood flow after transient portal triad clamping (PTC model) was confirmed with laser-Doppler flowmetry and dual-spot microspectroscopy, where the maximal and near-maximal blood flows were observed at ~5 min for the first peak and 20 min for the second peak after the initiation of hepatic tissue perfusion (Fig. 2 and Table 1). This phenomenon was independent of the ischemic period (at least up to 30 min) and of portal venous congestion, because it was also observed in rats subjected to a portal-systemic shunt and partial clamping of the liver. This phenomenon was not affected by the administration of vinblastine (which reduces the number of white blood cells) or by allopurinol (which inhibits xanthine oxidase, reported to produce oxygen radicals). These results suggest that mechanisms other than free radical formation and leukocyte plugging may play important roles in the biphasic recovery of postischemic hepatic blood flow.

Simultaneous measurements of hepatic blood flow by laser-Doppler flowmetry and of the blood flows of the hepatic artery and portal vein by ultrasonic transit time volume flowmetry indicated that the first increase in hepatic tissue blood flow consisted mainly of blood flow from the portal vein and the second increase consisted mainly of that from the hepatic artery (Fig. 3). The hepatic artery ligation model (HAL model) did not show the second increase in hepatic tissue blood flow (Fig. 5). The second peak contained highly O₂-saturated blood compared with the first peak (Figs. 4 and 6). These results suggested that the portal venous blood flow mainly constitutes an initial increase in hepatic tissue blood flow at ~5 min after reperfusion, whereas hepatic arterial blood flow forms a second gradual increase in hepatic tissue blood flow at ~20 min.

The precise reasons for the early recovery of portal venous blood flow compared with hepatic arterial blood flow are unknown. Some possibilities regarding the different restoration patterns of arterial and portal blood flow producing the biphasic recovery of postischemic hepatic blood flow are 1) different vascular vulnerability to free radicals, 2) different physical factors of each vessel including blood pressure, elasticity of the vascular walls, and different responses to the physical force of a microvascular clip, 3) increased levels of physiological factors such as vasospastic compounds, and 4) the hepatic arterial buffer response (9). The administration of vinblastine or allopurinol did not alter the biphasic blood recovery, suggesting that free radicals from granulocytes and xanthine oxidase were not responsible for this phenomenon. The clamping times and instruments also did not change the biphasic recovery. Blood pressure was high in the hepatic artery, which displayed slow recovery compared with portal blood flow, suggesting that physical factors including blood pressure and a microvascular clip are also not responsible for this phenomenon. The identification of precise mechanisms underlying the different recovery patterns of portal and hepatic arterial blood flow requires further study. However, hepatic arterial oxygenation is higher than that in the portal vein, and high PO₂ may lead to a predisposition for too much radical formation during ischemia-reperfusion. A predominant recovery of portal venous blood flow in the early phase of reperfusion may be preferable for reducing free radical formation and related cellular injuries.

In summary, we analyzed sinusoidal O₂ transport and hepatic microcirculation during reperfusion after temporary ischemia in rats. The recovery of the hepatic tissue blood flow after portal triad clamping was biphasic; the first increase consisted mainly of portal venous flow, and the second increase consisted mainly of hepatic arterial flow. Although the explanation for preferential recovery of portal blood flow in the early phase of reperfusion compared with hepatic artery blood flow must await further study, this pattern of blood flow recovery may be preferable for attenuating the oxidative injury caused by excessive O₂ inflow from the hepatic artery for a venous blood-dominant organ, the liver.