Hepatic Microvascular Perfusion Research Articles

Increased Hepatic Microvascular Density, Oxygenation, and VEGF in the Hypertrophic Lobe following Portal Vein Embolization in Rabbits

Introduction: The microvascular events following portal vein embolization (PVE) are poorly understood despite the pivotal role of the microcirculation in liver regeneration and tumor progression. We aimed to assess the changes in hepatic microvascular perfusion and neo-angiogenesis after experimental PVE. Methods: PVE of the cranial liver lobes was performed in 12 New Zealand White rabbits divided into 2 groups of permanent (P-PVE) and reversible PVE (R-PVE), respectively. Hepatobiliary scintigraphy and CT were used to evaluate hepatic function and volume. Hepatic microcirculation was assessed using a handheld vital microscope (Cytocam) to measure microvascular density (total vessel density; TVD) before PVE, right after PVE, and 20 min after PVE, as well as at 14 days (D14 post-PVE) and 35 days (D35 post-PVE). Additionally, on D35, microvascular PO₂ and liver parenchymal VEGF were assessed. Results: Eleven rabbits were included after PVE (R-PVE, n = 5; P-PVE, n = 6). TVD in the nonembo­lized (hypertrophic) lobes was higher than in the embolized (atrophic) lobes of the P-PVE group at D35 post-PVE (36.7 ± 7.2 vs. 23.4 ± 4.9 mm/mm²; p < 0.05). In the R-PVE group, TVD in the nonembolized lobes was not increased at D35. Function and volume were increased in the nonembolized lobes of the P-PVE group compared to the embolized lobes, but not in the R-PVE group. Likewise, the mmicrovascular PO₂ and VEGF staining rate were higher in the nonembolized lobes of the P-PVE group at D35 post-PVE. Discussion/Conclusion: Successful volumetric and functional hypertrophy of the nonembolized lobe was accompanied by microvascular alterations featuring increased neo-angiogenesis, microvascular density, and microvascular oxygen pressure following P-PVE.

Interpatient heterogeneity in hepatic microvascular blood flow during vascular inflow occlusion (Pringle manoeuvre).

Vascular inflow occlusion (VIO) during liver resections (Pringle manoeuvre) can be applied to reduce blood loss, however may at the same time, give rise to ischemia-reperfusion injury (IRI). The aim of this study was to assess the characteristics of hepatic microvascular perfusion during VIO in patients undergoing major liver resection. Assessment of hepatic microcirculation was performed using a handheld vital microscope (HVM) at the beginning of surgery, end of VIO (20 minutes) and during reperfusion after the termination of VIO. The microcirculatory parameters assessed were: functional capillary density (FCD), microvascular flow index (MFI) and sinusoidal diameter (SinD). A total of 15 patients underwent VIO; 8 patients showed hepatic microvascular perfusion despite VIO (partial responders) and 7 patients showed complete cessation of hepatic microvascular perfusion (full responders). Functional microvascular parameters and blood flow levels were significantly higher in the partial responders when compared to the full responders during VIO (FCD: 0.84±0.88 vs. 0.00±0.00 mm/mm2, P<0.03, respectively, and MFI: 0.69-0.22 vs. 0.00±0.00, P<0.01, respectively). An interpatient heterogeneous response in hepatic microvascular blood flow was observed upon VIO. This may explain why clinical strategies to protect the liver against IRI lacked consistency.

Contrast-enhanced ultrasonography is a feasible technique for quantifying hepatic microvascular perfusion in dogs with extrahepatic congenital portosystemic shunts.

Extrahepatic-congenital portosystemic shunt is a vascular anomaly that connects the portal vein to the systemic circulation and leads to a change in hepatic microvascular perfusion. However, an assessment of hepatic microvascular perfusion is limited by conventional diagnostic modalities. The aim of this prospective, exploratory study was to assess hepatic microvascular perfusion in dogs with extrahepatic-congenital portosystemic shunt using contrast-enhanced ultrasonography (CEUS) using perfluorobutane (Sonazoid® ). A total of 17 dogs were included, eight healthy dogs and nine with extrahepatic-congenital portosystemic shunt. The time-to-peak (TTP), rising time (RT), and rising rate (RR) in the hepatic artery, portal vein, and hepatic parenchyma, as well as the portal vein-to-hepatic parenchyma transit time (ΔHP-PV) measured from time-intensity curve on CEUS were compared between healthy and extrahepatic-congenital portosystemic shunt dogs. The RT of the hepatic artery in extrahepatic-congenital portosystemic shunt dogs was significantly earlier than in healthy dogs (P=0.0153). The TTP and RT of the hepatic parenchyma were significantly earlier in extrahepatic-congenital portosystemic shunt dogs than in healthy dogs (P=0.0018 and P=0.0024, respectively). ΔHP-PV was significantly shorter in extrahepatic-congenital portosystemic shunt dogs than in healthy dogs (P=0.0018). CEUS effectively revealed changes in hepatic microvascular perfusion including hepatic artery, portal vein, and hepatic parenchyma simultaneously in extrahepatic-congenital portosystemic shunt dogs. Rapid hepatic artery and hepatic parenchyma enhancements may reflect a compensatory increase in hepatic artery blood flow (arterialization) caused by a decrease in portal vein blood flow and may be used as an additional diagnostic test to distinguish extrahepatic-congenital portosystemic shunt dogs from healthy dogs.

Quantitative Evaluation of Hepatic Microvascular Perfusion after Ischemia–Reperfusion Injury in Rabbits by Contrast-Enhanced Ultrasound Perfusion Imaging

The aim of this study was to evaluate microvascular perfusion after liver ischemia–reperfusion injury (IRI) in rabbits using the “flash-replenishment” method of contrast-enhanced ultrasound (CEUS) perfusion imaging. Twenty-eight rabbits underwent either 30, 60 or 90 min of ischemia and 120 min of reperfusion. CEUS perfusion imaging was performed using the “flash-replenishment” model, and hepatic microvascular perfusion parameters, including peak intensity (PI), area under the curve (AUC), and hepatic artery-to-vein transit time (HA-HVTT), were calculated. Prolonged ischemia upregulated intracellular adhesion molecule-1 (ICAM-1), alanine transaminase (ALT) and aspartate transaminase (AST) levels. Longer ischemia decreased PI and AUC, but increased HA-HVTT. The perfusion parameters were significantly correlated with Suzuki's pathology scores and ALT and AST levels. The “flash-replenishment” method of CEUS perfusion imaging is an accurate and non-invasive method for evaluating hepatic microvascular perfusion and provides a valuable experimental basis for early prediction of liver IRI damage after liver transplantation or liver resection.

Repeatability of the "flash-replenishment" method in contrast-enhanced ultrasound for the quantitative assessment of hepatic microvascular perfusion.

This study aimed to evaluate the feasibility and repeatability of the flash-replenishment method in contrast-enhanced ultrasound (CEUS) perfusion imaging and assess quantitatively microvascular perfusion in the liver. Twenty healthy New Zealand rabbits were submitted to CEUS perfusion imaging with continuous intravenous infusion. Using flash-replenishment kinetics, the dynamic process of depletion and refilling of microbubble contrast agent was recorded. The hepatic microvascular perfusion parameters were calculated, including region of interest, peak intensity (PI), area under the curve (AUC), and hepatic artery to vein transit time (HA-HVTT). A consistency test was performed for multiple measurements by the same operator and blind measurements by two different operators. The hepatic perfusion imaging of 3×108 bubbles/min had minimal error and the best imaging effect and repeatability. The variability of the perfusion parameter measured at 3 cm depth under the liver capsule was at a minimum with coefficient of variation of 3.9%. The interclass correlation coefficient (ICC) of measurements taken by the same operator was 0.985, (95% confidence interval, CI=0.927-0.998). Measurements taken by two operators had good consistency and reliability, with the ICC of 0.948 (95%CI=0.853-0.982). The PI and AUC of liver parenchyma after reperfusion were lower than before blocking; and HA-HVTT was significantly longer than before blocking (P<0.05). The flash-replenishment method in CEUS perfusion imaging showed good stability and repeatability, which provide a valuable experimental basis for the quantitative assessment of hepatic microvascular perfusion in clinical practice.

Cilostazol improves hepatic blood perfusion, microcirculation, and liver regeneration after major hepatectomy in rats.

Major hepatectomy or small-for-size liver transplantation may result in postoperative liver failure. So far, no treatment is available to improve liver regeneration. Herein, we studied whether cilostazol, a selective phosphodiesterase III inhibitor, is capable of improving liver regeneration after major hepatectomy. Sprague-Dawley rats (n = 74) were treated with cilostazol (5 mg/kg daily) or a glucose solution and underwent either 70% liver resection or a sham operation. Before and after surgery, hepatic arterial and portal venous blood flow and hepatic microvascular perfusion were analyzed. Liver morphology, function, and regeneration were studied with histology, immunohistochemistry, western blotting, and bile excretion analysis. Cilostazol significantly increased hepatic blood flow and microcirculation before and after hepatectomy in comparison with sham-operated controls. This was associated with an elevation of hepatic vascular endothelial growth factor expression, an increase of hepatocellular proliferation, and an acceleration of liver regeneration. Furthermore, cilostazol protected the tissue of the remnant liver as indicated by an attenuation of hepatocellular disintegration. In conclusion, cilostazol increases hepatic blood perfusion, microcirculation, and liver regeneration after a major hepatectomy. Thus, cilostazol may represent a novel strategy to reduce the rate of liver failure after both extended hepatectomy and small-for-size liver transplantation.

Hepatic ischemic preconditioning increases portal vein flow in experimental liver ischemia reperfusion injury

Ischemic preconditioning (IPC) has been shown to decrease liver injury and to increase hepatic microvascular perfusion after liver ischemia reperfusion. This study aimed to evaluate the effects of IPC on hemodynamics of the portal venous system. Thirty-two rats were randomized into two groups: IPC group and control group. The rats of the IPC group underwent IPC by 10 minutes of liver ischemia followed by 10 minutes of reperfusion before liver ischemia, and the rats of the control group were subjected to 60 minutes of partial liver ischemia. Non-ischemic lobes were resected immediately after reperfusion. The animals were studied at 4 hours and 12 hours after reperfusion. Mean arterial pressure, heart rate, portal vein flow and pressure were analyzed. Blood was collected for the determination of the levels of aspartate aminotransferase, alanine aminotransferase, calcium, lactate, pH, bicarbonate, and base excess. IPC increased the mean portal vein flow at 4 hours and 12 hours after reperfusion. IPC recovered 78% of the mean portal vein flow at 12 hours after reperfusion. IPC decreased the levels of aspartate aminotransferase, alanine aminotransferase and lactate, and increased the levels of ionized calcium, bicarbonate and base excess at 12 hours after reperfusion. This study demonstrated that IPC increases portal vein flow and enhances hepatoprotective effects in liver ischemia reperfusion. The better recovery of portal vein flow after IPC may be correlated with the lower levels of transaminases and with the better metabolic profile.

Endoglin and activin receptor-like kinase 1 heterozygous mice have a distinct pulmonary and hepatic angiogenic profile and response to anti-VEGF treatment

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia associated with dysregulated angiogenesis and arteriovascular malformations. The disease is caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase 1 (ALK1; HHT2) genes, coding for transforming growth factor β (TGF-β) superfamily receptors. Vascular endothelial growth factor (VEGF) has been implicated in HHT and beneficial effects of anti-VEGF treatment were recently reported in HHT patients. To investigate the systemic angiogenic phenotype of Endoglin and Alk1 mutant mice and their response to anti-VEGF therapy, we assessed microvessel density (MVD) in multiple organs after treatment with an antibody to mouse VEGF or vehicle. Lungs were the only organ showing an angiogenic defect, with reduced peripheral MVD and secondary right ventricular hypertrophy (RVH), yet distinctly associated with a fourfold increase in thrombospondin-1 (TSP-1) in Eng (+/-) versus a rise in angiopoietin-2 (Ang-2) in Alk1 (+/-) mice. Anti-VEGF treatment did reduce lung VEGF levels but interestingly, led to an increase in peripheral pulmonary MVD and attenuation of RVH; it also normalized TSP-1 and Ang-2 expression. Hepatic MVD, unaffected in mutant mice, was reduced by anti-VEGF therapy in heterozygous and wild type mice, indicating a liver-specific effect of treatment. Contrast-enhanced micro-ultrasound demonstrated a reduction in hepatic microvascular perfusion after anti-VEGF treatment only in Eng (+/-) mice. Our findings indicate that the mechanisms responsible for the angiogenic imbalance and the response to anti-VEGF therapy differ between Eng and Alk1 heterozygous mice and raise the need for systemic monitoring of anti-angiogenic therapy effects in HHT patients.

Laser Doppler Flowmetry of Hepatic Microcirculation during Pringle’s Maneuver: Determination of Spatial and Temporal Liver Tissue Perfusion Heterogeneity

Background: Laser Doppler flowmetry (LDF) is frequently used for non-invasive microvascular perfusion measurements. The aim of the present study was to analyze liver blood flow heterogeneity in detail using LDF devices under normal and low-flow conditions. Materials and Methods: In 5 anesthetized and laparotomized Suabian-Hall strain pigs, systemic hemodynamics and hepatic arterial/portal venous blood flow were constantly recorded. Hepatic microcirculation was assessed by 2 different LDF devices, analyzing microvascular flow and velocity before, during and after inducing a Pringle’s maneuver for hepatic inflow occlusion. Offline data analysis comprised differentiation between the two LDF devices used as well as calculation of temporal and spatial heterogeneity of liver perfusion. Results: Pringle’s maneuver induced complete inflow occlusion, confirmed by hepatic arterial/portal venous blood flow measurement. Laser Doppler signals showed a significant decrease during Pringle’s maneuver. Spatial heterogeneity of flow and velocity increased more than temporal heterogeneity during Pringle’s maneuver. Conclusion: Both LDF devices proved suitable for assessing hepatic microvascular perfusion during normal perfusion and low-flow conditions. Reduced microvascular perfusion induces a significant increase in temporal and spatial perfusion heterogeneity. In particular, the pronounced spatial heterogeneity requires measurements at different places when assessing hepatic microcirculation by LDF during impaired perfusion conditions.

The Rho-kinase inhibitor Y-27632 inhibits cholestasis-induced platelet interactions in the hepatic microcirculation

Bile duct obstruction is associated with hepatic accumulation of leukocytes and liver injury. Emerging data suggest that platelets may play an important role in tissue damage and inflammation. Herein, we characterized the platelet response in cholestatic liver injury and evaluated the role of Rho-kinase signaling. For this purpose, C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 (10 mg/kg) and vehicle before undergoing bile duct ligation (BDL) for 12 h. Platelet rolling and adhesion, formation of platelet aggregates as well as microvascular perfusion in the liver were analyzed using intravital fluorescence microscopy. Liver damage was monitored by measuring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Administration of Y-27632 reduced the BDL-associated increase of ALT and AST by 95% and 89%, respectively. The inhibition of Rho-kinase also reduced cholestasis-induced platelet rolling and adhesion by more than 46% and 73% in postsinusoidal venules and platelet adhesion in sinusoids by 60%. In addition, Y-27632 decreased platelet aggregation in hepatic sinusoids and postsinusoidal venules by 69% and 81%. BDL caused a significant reduction of hepatic microvascular perfusion. Importantly, pretreatment with Y-27632 restored sinusoidal perfusion in cholestatic animals. Our findings demonstrate that Rho-kinase regulates multiple aspects of platelet interaction in the microcirculation of cholestatic animals. Moreover, inhibition of Rho-kinase signaling not only attenuates platelet responses but also maintains microvascular perfusion and protects against hepatocellular injury in cholestasis. Thus, targeting Rho-kinase signaling may be an effective way to protect against platelet-mediated liver injury in obstructive jaundice.

Obesity, Insulin Resistance and Hepatic Perfusion

Insulin resistance brings together the collection of hyperglycemia, hyperinsulinemia, and dyslipidemia associated with pathologies such as obesity and type 2 diabetes. As such, it is an important target for the prevention of complications linked with these pathologies. To the extent that insulin resistance involves hyperglycemia and dyslipidemia, there are obvious associations between impaired insulin signaling and microvascular dysfunction. In fact, there is evidence that insulin resistance significantly participates in the microvascular damage that occurs with obesity and type 2 diabetes. Since the liver plays a critical role in glucose homeostasis and lipid metabolism, and the state of the microcirculation reflects its function, the impact of insulin resistance on hepatic microvascular perfusion deficits is important. However, as of yet, no single body of work has addressed the relationships that exist between insulin resistance and the derangements in hepatic microvascular perfusion. Although there is distinct hepatic microvascular dysfunction associated with insulin resistance, a controversy still arises as to whether this dysfunction occurs as a result of reductions to the sinusoidal lumen. Nonetheless, the literature appears to agree that the general effects of obesity and insulin resistance on the hepatic microcirculation are linked to the selective impairment of phosphatidylinositol 3'-kinase signaling, as well as the presence of swollen fatty hepatocytes and sinusoidal "capillarization." This manuscript sets out to review the main aspects of hepatic glucose homeostasis and lipid metabolism, as well as the cellular and molecular mediators regulating hepatic microvascular perfusion deficits during obesity and insulin resistance.

LPS Increases Hepatic HIF-1α Protein and Expression of the HIF-1-Dependent Gene Aldolase A in Rats

Cellular adaptation to hypoxia is mediated in part by the transcription factor hypoxia-inducible factor 1 (HIF-1). Accumulating data suggest that pro-inflammatory mediators can up-regulate HIF-1alpha protein expression and HIF-1 DNA-binding activity in the absence of hypoxia. Accordingly, we investigated HIF-1 mediated signaling in endotoxemic rats. We studied three groups of male Sprague Dawley rats. Controls (N = 5) were injected i.p. with saline. Endotoxemic rats (N = 9) received a sublethal dose of lipopolysaccaride (Escherichia coli; 5 mg/kg, i.p.). A third group of rats (N = 5) received the HIF-1 stabilizing agent CoCl(2) (14 mg/kg, i.p.) at T = 0 h and T = 16 h. At T = 18 h, liver microvascular perfusion was measured using laser Doppler flowmetry and hepatic tissue samples were obtained. RNA was isolated and mRNA levels of the HIF-1 dependent genes aldolase A and vascular endothelial growth factor (VEGF) were determined using quantitative real-time RT-PCR. HIF-1alpha content was estimated by immunoprecipitation followed by Western blotting. HIF-1alpha increased in hepatic tissue after treatment with LPS or CoCl(2). LPS markedly increased hepatic expression of aldolase A, but failed to alter expression of VEGF. CoCl(2) increased aldolase A and VEGF mRNA expression. Although hepatic microvascular perfusion was comparable in saline- and LPS-treated rats, hepatic microvascular blood flow and aldolase A expression were significantly inversely correlated among endotoxemic rats (r = 0.773; P = 0.003). Increased expression of aldolase A in endotoxemic rats is mediated by both hypoxia-dependent and hypoxia-independent mechanisms.

Initial hepatic microcirculation correlates with early graft function in human orthotopic liver transplantation

Microcirculatory disturbances are an initial causative determinant in hepatic ischemia/reperfusion injury. The aim of this study was to assess sinusoidal perfusion during human liver transplantation using orthogonal polarization spectral imaging and to evaluate the significance of intraoperative microcirculation for early postoperative graft function. Hepatic microcirculation was measured in 27 recipients undergoing full-size liver transplantation and compared to a group of 32 healthy living-related liver donors. The microvascular parameters were correlated with postoperative aspartate aminotransferase and bilirubin levels. Hepatic perfusion following liver transplantation was found to be significantly decreased when compared with the control group. Volumetric blood flow within the individual sinusoids increased due to sinusoidal dilatation and enhanced flow velocity. Regression analysis of postoperative aspartate aminotransferase and bilirubin with microvascular parameters revealed significant correlations. The extent of volumetric blood flow increased within the first 30 minutes after reperfusion and showed a significant correlation with postoperative aspartate aminotransferase release and bilirubin elimination. In conclusion, postischemic hepatic microvascular perfusion was analyzed in vivo, demonstrating significant microvascular impairment during liver transplantation. Sinusoidal hyperperfusion appears to confer protection against postischemic liver injury, as given by the correlation with aspartate aminotransferase and bilirubin levels. Thus, these findings may have therapeutic importance with respect to mechanisms mediating postischemic reactive hyperemia.

Ischemia at 4 degrees C: a novel mouse model to investigate the effect of hypothermia on postischemic hepatic microcirculatory injury.

Hypothermia of the ischemic organ at 4 degrees C protects hepatic microcirculation from ischemia-reperfusion (IR) injury. The effect of hypothermia during ischemia was investigated in animal models using liver transplantation and storage of the harvested organ in cold preservation solutions. No investigation of the isolated influence of hypothermia at 4 degrees C of the ischemic organ on hepatic IR injury exists, due to the lack of an appropriate animal model. Therefore, the aim of our present study was to develop such a model using intravital video fluorescence microscopy (IVM). In C57BL/6 mice, a reversible isolated ischemia of the left liver lobe was induced for 90 min, followed by 240 min of reperfusion. The temperature of the ischemic organ was adjusted to either 4 degrees C or 37 degrees C by superfusion with 0.9% NaCl. Sham-operated animals without IR served as controls. The hepatic microcirculation was analyzed using IVM at 30 min and 240 min after reperfusion by quantifying sinusoidal perfusion and leukocyte-endothelial cell interaction in postsinusoidal venules. At the end of the experiment, blood and tissue samples were taken for measurement of liver enzyme activities and light and electron microscopy. Mean arterial pressure and body temperature were kept constant throughout the experiment, while the temperature of the ischemic liver lobe was adjusted to predefined levels. After normothermic ischemia, hepatic microvascular perfusion was significantly impaired compared with sham-operated animals. Perfusion failure was significantly reduced in hypothermic livers and did not differ from livers of the sham-group. Liver enzyme activities in the normotherimic group were significantly higher than in the sham and hypothermic groups. Light and electron microscopy revealed severe histological alterations at 37 degrees C ischemia, whereas at 4 degrees C ischemia only minimal lesions were encountered. Our novel model allows for isolated adjustment of ischemic liver lobe temperature without changing body temperature and systemic macrohemodynamic parameters. Hypothermia at 4 degrees C largely attenuates postischemic microvascular perfusion injury of the liver.

Kupffer cell-initiated remote hepatic injury following bilateral hindlimb ischemia is complement dependent

Intravital fluorescence microscopy was applied to the livers of male Wistar rats to test the hypothesis that complement mobilization stimulates Kupffer cells and subsequently initiates hepatic injury after hindlimb ischemia/reperfusion (I/R). Following 3 h of limb reperfusion, hepatocellular viability (serum levels of alanine transaminase and cell death via propidium iodide labeling) decreased significantly from levels in sham-operated animals. Inhibition of complement mobilization with soluble complement receptor type 1 (20 mg/kg body wt) and interruption of Kupffer cell function with GdCl(3) (1 mg/100g body wt) resulted in significant hepatocellular protection. Although the effects of hindlimb I/R on hepatic microvascular perfusion were manifest as increased heterogeneity, both complement inhibition and suppression of Kupffer cell function resulted in marked improvements. No additional hepatocellular protection and microvascular improvements were provided by combining the interventions. Furthermore, inhibition of complement mobilization significantly depressed Kupffer cell phagocytosis by 42% following limb reperfusion. These results suggest that the stimulation of Kupffer cells via complement mobilization is necessary but is not the only factor contributing to the early pathogenesis of hepatic injury following hindlimb I/R.

Anti-ICAM-1 blockade reduces postsinusoidal WBC adherence following cold ischemia and reperfusion, but does not improve early graft function in rat liver transplantation

Background/Aim: The present in vivo study investigated the impact of a monoclonal antibody directed against the intercellular adhesion molecule-1 (ICAM-1) on initial microvascular reperfusion injury after liver transplantation. Methods: Orthotopic, syngeneic liver transplantation including arterial reconstruction was performed in male Lewis rats after 24 h graft storage in University of Wisconsin (UW) solution at 4°C. Animals received either an anti-ICAM-1 antibody ( n=7), an IgG 1 control antibody ( n=8) or saline only ( n=7). Hepatic microvascular alterations during the initial 90 min of reperfusion were assessed using intravital fluorescence microscopy. Early graft dysfunction was determined by analysis of bile flow. Results: After treatment with anti-ICAM-1 mAb, hepatic microvascular perfusion was found improved when compared with that of IgG 1- and saline-treated controls. In addition, anti-ICAM-1 mAb effectively reduced the number of permanently adherent white blood cells in postsinusoidal venules (284.4±59.1 mm −2 vs IgG 1: 371.9±26.7 mm −2 and saline: 431.8±46.4 mm −2; p<0.05). In contrast, the number of stagnant white blood cells in sinusoids was higher ( p<0.05) in liver grafts with blocked ICAM-1 (320.6±17.2 mm −2) compared with that of IgG 1- (215.2±11.1 mm −2) and saline-treated controls (226.4±14.0 mm −2). Measurement of hepatic uptake of fluorescent-labeled latex particles did not reveal significant differences in phagocytic activity. Finally, bile flow also did not differ between the three groups studied. Conclusion: Together these results indicate that ICAM-1 is involved in the process that mediates white blood cells adherence in postsinusoidal venules, whereas in hepatic sinusoids other mechanisms apart from ICAM-1-mediated white blood cells adherence seem to be fundamental for posttransplant white blood cells accumulation. Our data further suggest that white blood cells adherence in postsinusoidal venules via ICAM-1 does not make a major contribution to the pathogenesis of early cold ischemia/reperfusion injury after liver transplantation.

Prevention of Hepatic Ischemia–Reperfusion Injury by SOD–DIVEMA Conjugate

A protective effect of the SOD (superoxide dismutase)–DIVEMA (divinyl ether and maleic anhydride) conjugate on I–R (ischemia–reperfusion) liver injury was demonstrated. Twenty minutes of normothermic hepatic ischemia was induced by clamping the portal triad of Sprague–Dawley rats. Five minutes before the end of ischemia, SOD, SOD–DIVEMA, or NaCl (0.9%) was given intravenously. Using intravital fluorescence microscopy, hepatic microvascular perfusion was analyzed before ischemia and repeatedly during the 120-min reperfusion period. SOD–DIVEMA significantly restored the sinusoidal perfusion rate (control, 98.0 ± 0.5; NaCl, 65.5 ± 7.7; SOD, 81.5 ± 8.2; SOD–DIVEMA, 95.8 ± 0.7%) and reduced the number of leukocytes stagnant in acini (control, 4.4 ± 0.9; NaCl, 36.6 ± 6.3; SOD, 27.7 ± 6.8; SOD–DIVEMA, 12.3 ± 3.3 cells/lobule) and adherent in postsinusoidal venules (control, 55.0 ± 24; NaCl, 417 ± 63; SOD, 253 ± 58; SOD–DIVEMA, 40.0 ± 14 cells/mm2). In addition, SOD–DIVEMA maintained postischemic hepatocellular integrity. The SOD–DIVEMA-treated group revealed higher serum SOD enzyme activity compared to the SOD group after 120 min of reperfusion (SOD–DIVEMA, 33.0 ± 5.9; SOD, 8.6 ± 3.1 U/ml). The beneficial effect of SOD–DIVEMA was most prominent after 120 min of reperfusion, indicating a longer intravascular half-life of SOD–DIVEMA.

Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity.

Using a murine model, we studied the effect of agonistic anti-CD95 antibodies (aCD95) on sinusoidal lining cells and a potential protection by caspase inhibition. C3H/HeN mice were intravenously administered aCD95 (10 microgram/mouse) or unspecific IgG (control) in the presence or absence of the caspase inhibitor z-VAD-fmk. Analysis of hepatic microcirculation using intravital fluorescence microscopy revealed severe (P<0.01) sinusoidal perfusion failure and reduced (P<0.05) phagocytic activity of Kupffer cells (KC) within 2 h. Transmission electron micrographs demonstrated loss of integrity of sinusoidal endothelial cells as early as 1 h after aCD95 application, whereas histological manifestation of hepatocellular apoptosis and hemorrhagic necrosis was most pronounced at 6 h. Blocking of caspase activity attenuated (P<0.01) both hepatic microvascular perfusion failure and KC dysfunction. Accordingly, full protection of the liver from apoptotic damage and intact microarchitecture was observed in histological sections after z-VAD-fmk treatment. Mortality rate was 40% 6 h after aCD95 administration, whereas all animals survived in the z-VAD-fmk group (P<0.05). The activation of caspases through CD95 may primarily lead to damage of sinusoidal endothelial cells and hepatic microvascular perfusion failure. Moreover, reduced phagocytic capacity of KC may contribute to accumulation of toxic metabolites released by dying cells at the local site of inflammation, further aggravating liver injury.

Resuscitation with Hypertonic Saline Dextran Reduces Endothelial Cell Swelling and Improves Hepatic Microvascular Perfusion and Function after Hemorrhagic Shock1

Background.Hemorrhagic shock severely compromises hepatic microcirculation and function with tendency to promote hepatic insufficiency and multiple organ failure.Material and methods.The aim of the study was to evaluate the effects of small volume resuscitation on liver microcirculation (intravital fluorescence microscopy and electron microscopy) and function (arterial ketone body ratio (AKBR) and bile flow), in a rat model of traumatic-hemorrhagic shock. One hour after hemorrhage (MAP 40 mm Hg) the rats were resuscitated with HSD (7.2% NaCl/10% dextran 60, 10% of shed blood/2 min,n= 8); DEX (6% dextran 60, 100% of shed blood/5 min,n= 8); or RL (Ringer lactate, 400% of shed blood/20 min,n= 6).Results.HSD yielded a better recovery of sinusoidal perfusion (17.8 ± 0.8% nonperfused sinusoids) than DEX (21.8 ± 0.7%,P< 0.05) and RL (23.9 ± 0.9%,P< 0.01). Hemorrhagic shock produced a moderate increase of mean sinusoidal endothelial cell thickness, which was further enhanced by DEX and RL (P< 0.05 vs baseline), whereas HSD reduced the mean endothelial cell thickness toward baseline (P< 0.05 vs DEX and RL). Both AKBR and bile flow were profoundly reduced after 1 h shock. Resuscitation with DEX and RL produced a weak recovery, still remaining at shock level, while HSD infusion allowed a significant improvement of AKBR and bile flow (P< 0.05 vs shock).Conclusion.Reduction of mean endothelial cell thickness after HSD is very likely the mechanism for the amelioration of sinusoidal perfusion, resulting in a significant improvement of hepatic energetic status and excretory function.

Application of a Novel Method for Subsequent Evaluation of Sinusoids and Postsinusoidal Venules after Ischemia-Reperfusion Injury of Rat Liver

Although several intravital fluorescence microscopic studies demonstrated that microcirculatory derangement is induced during liver ischemia-reperfusion, these data were obtained from randomly selected microvascular areas and microvessels. Repeated observation of the identical microvessels has not been performed yet. Using a specially designed cover glass, it is now possible to relocate desired sites of observation repeatedly over the whole reperfusion time. The aim of this study was to determine the impact of reperfusion time on hepatic microvascular perfusion state. Twenty minutes of ischemia induced a significant decrease in sinusoidal perfusion rate (29.1 ± 10.2%) as compared with baseline values (98.0 ± 0.3%). At 30, 60, and 120 min of reperfusion, the percentage of perfused sinusoids recovered to 62.8 ± 6.6, 67.5 ± 5.7, and 77.2 ± 5.4%. The number of stagnant leukocytes in the same sinusoids was 6.2 ± 1.9/lobule at baseline and increased to 22.3 ± 3.6/lobule at 120 min of reperfusion. The number of leukocytes adhering within postsinusoidal venules was 53.5 ± 12.5/mm² before ischemia and increased to 414.2 ± 62.5/mm² at 120 min of reperfusion. We have demonstrated that during 120 min of reperfusion, there was a steady increase in both sinusoidal and venular leukocyte adhesion along with an attenuation of the initially severely depressed sinusoidal perfusion. A no-reflow phenomenon at an early phase of reperfusion and subsequent reflow were proven.