Endotoxin-induced liver hypoxia: defective oxygen delivery versus oxygen consumption.

In vivo EPR was used to investigate liver oxygenation in a hemodynamic model of septic shock in mice. Oxygen-sensitive material was introduced either (i) as a slurry of fine particles which localized at the liver sinusoids (pO2 = 44.39 +/- 5.13 mmHg) or (ii) as larger particles implanted directly into liver tissue to measure average pO2 across the lobule (pO2 = 4.56 +/- 1.28 mmHg). Endotoxin caused decreases in pO2 at both sites early (5-15 min) and at late time points (6 h after endotoxin; sinusoid = 11.22 +/- 2.48 mmHg; lobule = 1.16 +/- 0.42 mmHg). The overall pO2 changes observed were similar (74.56% versus 74.72%, respectively). Blood pressures decreased transiently between 5 and 15 min (12.88 +/- 8% decrease) and severely at 6 h (59 +/- 9% decrease) following endotoxin, despite volume replacement with saline. Liver and circulatory nitric oxide was elevated at these times. Liver oxygen extraction decreased from 44% in controls to only 15% following endotoxin, despite severe liver hypoxia. Arterial oxygen saturation, blood flow (hepatic artery), and cardiac output were unaffected. Pretreatment with l-NMMA failed to improve endotoxin-induced oxygen defects at either site, whereas interleukin-13 preserved oxygenation. These site-specific measurements of pO2 provide in vivo evidence that the principal cause of liver hypoxia during hypodynamic sepsis is reduced oxygen supply to the sinusoid and can be alleviated by maintaining sinusoidal perfusion.     

Simultaneous measurement of NO(*) and PO(2) from tissue by in vivo EPR.

We describe a technique that utilizes electron paramagnetic resonance (EPR) to measure NO(*) and pO(2) directly, and non-invasively, from tissue in vivo. Diethyldithiocarbamate (DETC) was injected with iron so as to complex with NO(*) in the tissue. Gloxy (an oxygen-sensitive, paramagnetic material) was also implanted into the tissue of interest (brain or liver). Because the signals arising from gloxy and NO-Fe-(DETC)(2) did not overlap, they could be monitored and measured simultaneously in vivo. The gloxy was not responsive to NO(*) and/or DETC. As model systems we either injected SNP (an NO(*) donor) into animals and monitored NO(*) and pO(2) simultaneously from brain, or endotoxin (lipopolysaccharide; LPS) was injected in order to induce a septic episode and NO(*) and pO(2) measured from liver. We found a close correlation between levels of SNP-derived NO(*) and brain pO(2) in vivo. During sepsis, liver pO(2) decreased dramatically at 300-360 min after endotoxin injection, and this coincided with decreases in mean arterial blood pressure and increased tissue NO(*) detected. These studies demonstrate the potential usefulness of this technique for making direct in vivo measurements of NO(*) and pO(2) simultaneously from tissue.     

A fully coupled porous media and channels flow approach for simulation of blood and bile flow through the liver lobules

Two dimensional, steady state, and incompressible blood and bile flows through the liver lobules are numerically simulated. Two different geometric models A and B are proposed to study the effects of lobule structure on the fluid flow behaviour. In Model A, the lobule tissue is represented as a hexagonal shape porous medium with a set of flow channels at its vertices accounting for the hepatic artery, portal and central veins along with bile ductules. Model B is a channelized porous medium constructed by adding a set of flow channels, representing the bile canaliculies and lobule sinusoids, to Model A. The bile and blood flow through the lobule is simulated by the finite element approach, based on the Darcy/Brinkman equations in the lobule tissue and the Navier-Stokes (or Stokes) equations in the flow channels. In Model B, a transmission factor on the boundaries of the bile canaliculies is introduced to connect the bile and blood flows. First, a single regular lobule is utilized to exhibit the fluid flow pattern through the liver lobule represented by proposed geometric models. Then, the model is extended to a group of liver lobules to demonstrate the flow through a liver slice represented by irregular lobules. Numerical results indicate that the Darcy and Brinkman equations provide nearly the same solutions for Model A and similar solutions with a little difference for Model B. It is shown that the existence of sinusoids and bile canaliculies inside the liver lobules has noticeable effects on its fluid flow pattern, in terms of pressure and velocity fields.     

Terminal lymphatics: the potential "lethal corner" in the distribution of tissue pO2

BACKGROUND: Terminal lymphatic fluid is the compartment furthest removed from the oxygen supply, and therefore should present the lowest pO(2) in the tissue due to oxygen consumption by the tissue and the lymphatic vessel wall. METHODS AND RESULTS: The distribution of pO(2) was determined in the tissue, the lymphatic microvessels, and arterioles and venules of the hamster chamber window model, which is studied without anesthesia with the tissue isolated from the environment. Lymphatic fluid pO(2) was measured with the phosphorescence oxygen quenching method. Small terminal lymphatic fluid pO(2) was 18.4 +/- 2.6 mmHg, and 18.0 +/- 2.4 mmHg in collecting lymphatics. Tissue pO(2) averaged 24.6 +/- 2.7 mmHg. The significant difference between tissue and intralymphatic pO(2) was due in part to the presence of an oxygen gradient across the lymphatic wall, which ranged from 3.7 +/- 1.3 mmHg for terminal lymphatics, to 6.0 +/- 1.2 mmHg for collecting lymphatics. This gradient is assumed to be due to the oxygen consumption by the cellular component of the lymphatic wall. CONCLUSION: The increased vessels wall gradient found in collecting lymphatics was reconciled by the findings that these microlymphatic vessels tend to be contiguous to the arterioles, whereas the terminal lymphatics are dispersed in the tissue. These findings indicate that terminal lymphatic present the lowest oxygen tension in the tissue, and therefore are the locations at risk to develop anoxia when the microvascular oxygen supply becomes limited.     

Determination of oxygen gradients in engineered tissue using a fluorescent sensor

Nutrient and oxygen supply of cells are crucial to tissue engineering in general. If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely. Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO(2)) in culture media or described the use of invasive techniques with spatially limited resolution. The experimental setup described here allows for continuous, noninvasive, high-resolution pO(2) measurements over the cross-section of cultivated tissues. Applying a recently developed technique for time-resolved pO(2) sensing using optical sensor foils, containing luminescent O(2)-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period. Cylindrical tissue samples were immobilized on top of the sensors. By measuring the luminescence decay time, two-dimensional pO(2) distributions across the tissue section in contact with the foil surface were determined. We applied this technique to cartilage explants and to tissue-engineered cartilage. For both tissue types, changes were detected in monotonously decreasing gradients of pO(2) from the surface with high pO(2) to minimum pO(2) values in the center of the samples. Nearly anoxic conditions were observed in tissue constructs ( approximately 0 Torr) but not in excised cartilage discs ( approximately 20 Torr) after 1 day. Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function. Additionally, histological analysis revealed a maximum depth of approximately 1.3 mm of regular cartilage development in constructs grown under the applied culture conditions. Correlating analytical and histological analysis with the oxygen distributions, we found that pO(2) values below 11 Torr might impair proper tissue development in the center. The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures.     

Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia

Oxygen supply and diffusion into tissues are necessary for survival. The oxygen partial pressure (pO(2)), which is a key component of the physiological state of an organ, results from the balance between oxygen delivery and its consumption. In mammals, oxygen is transported by red blood cells circulating in a well-organized vasculature. Oxygen delivery is dependent on the metabolic requirements and functional status of each organ. Consequently, in a physiological condition, organ and tissue are characterized by their own unique 'tissue normoxia' or 'physioxia' status. Tissue oxygenation is severely disturbed during pathological conditions such as cancer, diabetes, coronary heart disease, stroke, etc., which are associated with decrease in pO(2), i.e. 'hypoxia'. In this review, we present an array of methods currently used for assessing tissue oxygenation. We show that hypoxia is marked during tumour development and has strong consequences for oxygenation and its influence upon chemotherapy efficiency. Then we compare this to physiological pO(2) values of human organs. Finally we evaluate consequences of physioxia on cell activity and its molecular modulations. More importantly we emphasize the discrepancy between in vivo and in vitro tissue and cells oxygen status which can have detrimental effects on experimental outcome. It appears that the values corresponding to the physioxia are ranging between 11% and 1% O(2) whereas current in vitro experimentations are usually performed in 19.95% O(2), an artificial context as far as oxygen balance is concerned. It is important to realize that most of the experiments performed in so-called normoxia might be dangerously misleading.     

Direct, real-time, simultaneous monitoring of intravitreal nitric oxide and oxygen in endotoxin-induced uveitis in rabbits

Nitric oxide (NO) plays a critical role in the pathogenesis of endotoxin-induced uveitis (EIU). Since NO is a labile free radical, it is difficult to examine the dynamics of NO directly in vivo. In this study, we established a system for direct monitoring of the dynamics of NO and partial pressure of oxygen (pO(2)) in EIU in rabbits. The currents (calculated concentrations) of NO and pO(2) in the vitreous were monitored after the intravitreal injection of lipopolysaccharide (LPS). In addition, the protein concentrations and nitrite levels in the aqueous humor were analyzed. The eyes were enucleated, and a histologic study was performed on their posterior segments. The tissue slices were also immunostained with anti-nitrotyrosine as a marker of peroxinitrite and/or nitrogen-related oxidants. The NO level decreased temporarily after LPS injection and then increased from 1 to 7 h. pO(2) increased temporarily for about 30 min after LPS injection. The change in NO current was inversely proportional to pO(2) after LPS injection and vice versa. The protein concentration and nitrite level after LPS injection increased significantly. These changes were suppressed by pretreatment with N(G)-nitro-l-arginine-methyl-ester. Immunohistochemical study showed enhanced immunoreactivity of nitrotyrosine in the inflamed retina. Since nitrotyrosine was detected, it appears that NO readily reacts with oxygen to produce cytotoxic species, peroxynitrite, and/or nitrogen-related oxidants. This process may be related to the retinal injury in EIU. This monitoring system can provide useful information on dynamic changes in intravitreal NO and pO(2) for understanding EIU.     

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.     

Simulating Microdosimetry in a Virtual Hepatic Lobule

The liver plays a key role in removing harmful chemicals from the body and is therefore often the first tissue to suffer potentially adverse consequences. To protect public health it is necessary to quantitatively estimate the risk of long-term low dose exposure to environmental pollutants. Animal testing is the primary tool for extrapolating human risk but it is fraught with uncertainty, necessitating novel alternative approaches. Our goal is to integrate in vitro liver experiments with agent-based cellular models to simulate a spatially extended hepatic lobule. Here we describe a graphical model of the sinusoidal network that efficiently simulates portal to centrilobular mass transfer in the hepatic lobule. We analyzed the effects of vascular topology and metabolism on the cell-level distribution following oral exposure to chemicals. The spatial distribution of metabolically inactive chemicals was similar across different vascular networks and a baseline well-mixed compartment. When chemicals were rapidly metabolized, concentration heterogeneity of the parent compound increased across the vascular network. As a result, our spatially extended lobule generated greater variability in dose-dependent cellular responses, in this case apoptosis, than were observed in the classical well-mixed liver or in a parallel tubes model. The mass-balanced graphical approach to modeling the hepatic lobule is computationally efficient for simulating long-term exposure, modular for incorporating complex cellular interactions, and flexible for dealing with evolving tissues.     

Real-time monitoring of nitric oxide in ischemic myocardium using an NO-selective electrode calibrated by electron spin resonance

Using a Langendorff-perfused rat heart preparation and selective electrodes, we determined nitric oxide (NO) and oxygen levels in cardiac tissue. An NO-selective electrode that was calibrated by electron spin resonance (ESR) spectroscopy was inserted into the middle of the myocardium in the left ventricle. Simultaneously, we used an O2-selective electrode to measure the partial pressure of oxygen (pO2) in the perfusate, Krebs-Henseleit (K-H) solution, that was ejected from the heart. After 30 min of aerobic control perfusion, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Under ischemic conditions, with a gradually decreasing pO2, NO detected by an NO-sensitive electrode within the myocardium was gradually increased. The maximum concentration increases in NO and decreases in pO2 during global ischemia were +10.200 +/- 1.223 microM and -58.608 +/- 4.123 mmHg, respectively. NO and pO2 levels both recovered to pre-ischemia baseline values when perfusion was restarted after global ischemia (reperfusion). The presence of Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mM), a NOS inhibitor, prevented ischemia/reperfusion-induced changes in NO. This study shows that an NO-selective electrode that is calibrated by ESR can provide accurate, real-time monitoring of cardiac NO in normal and ischemic myocardium.     

Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice

Cholestasis-induced liver injury during bile duct obstruction causes an acute inflammatory response. To further characterize the mechanisms underlying the neutrophil-induced cell damage in the bile duct ligation (BDL) model, we performed experiments using wild-type (WT) and ICAM-1-deficient mice. After BDL for 3 days, increased ICAM-1 expression was observed along sinusoids, along portal veins, and on hepatocytes in livers of WT animals. Neutrophils accumulated in sinusoids [358 +/- 44 neutrophils/20 high-power fields (HPF)] and >50% extravasated into the parenchymal tissue. Plasma alanine transaminase (ALT) levels increased by 23-fold, and severe liver cell necrosis (47 +/- 11% of total cells) was observed. Chlorotyrosine-protein adducts (a marker for neutrophil-derived hypochlorous acid) and 4-hydroxynonenal adducts (a lipid peroxidation product) were detected in these livers. Neutrophils also accumulated in the portal venules and extravasated into the portal tracts. However, no evidence for chlorotyrosine or 4-hydroxynonenal protein adducts was detected in portal tracts. ICAM-1-deficient mice showed 67% reduction in plasma ALT levels and 83% reduction in necrosis after BDL compared with WT animals. The total number of neutrophils in the liver was reduced (126 +/- 25/20 HPF), and 85% of these leukocytes remained in sinusoids. Moreover, these livers showed minimal staining for chlorotyrosine and 4-hydroxynonenal adducts, indicating a substantially reduced oxidant stress and a diminished cytokine response. Thus neutrophils relevant for the aggravation of acute cholestatic liver injury in BDL mice accumulate in hepatic sinusoids, extravasate into the tissue dependent on ICAM-1, and cause cell damage involving reactive oxygen formation.     

The effects of Efaproxyn (efaproxiral) on subcutaneous RIF-1 tumor oxygenation and enhancement of radiotherapy-mediated inhibition of tumor growth in mice

Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO(2). The purpose of this study was to determine the effect of efaproxiral on tumor oxygenation and growth inhibition of RIF-1 tumors that received X radiation (4 Gy) plus oxygen breathing compared to radiation plus oxygen plus efaproxiral daily for 5 days. Two lithium phthalocyanine (LiPc) deposits were implanted in RIF-1 tumors in C3H mice for tumor pO(2) measurements using EPR oximetry. Efaproxiral significantly increased tumor oxygenation by 8.4 to 43.4 mmHg within 5 days, with maximum increases at 22-31 min after treatment. Oxygen breathing alone did not affect tumor pO(2). Radiation plus oxygen plus efaproxiral produced tumor growth inhibition throughout the treatment duration, and inhibition was significantly different from radiation plus oxygen from day 3 to day 5. The results of this study provide unambiguous quantitative information on the effectiveness of efaproxiral to consistently and reproducibly increase tumor oxygenation over the course of 5 days of treatment, modeling the clinical use of efaproxiral. Also, based on the tumor growth inhibition, the study shows the efaproxiral-enhanced tumor oxygenation was radiobiologically significant. This is the first study to demonstrate the ability of efaproxiral to increase tumor oxygenation and to increase the tumor growth inhibition of radiotherapy over 5 days of treatment.     

A mathematical model of tumor oxygen and glucose mass transport and metabolism with complex reaction kinetics

Hypoxia imparts radioresistance to tumors, and various approaches have been developed to enhance oxygenation, thereby improving radiosensitivity. This study explores the influence of kinetic and physical factors on substrate metabolism in a tumor model, based on a Krogh cylinder. In tissue, aerobic metabolism is assumed to depend on glucose and oxygen, represented by the product of Michaelis-Menten expressions. For the base case, an inlet pO(2) of 40 mmHg, a hypoxic limit of 5 mmHg, and a tissue/capillary radius ratio of 10 are used. For purely aerobic metabolism, a hypoxic fraction of 0.16 and volume-average pO(2) of 8 mmHg are calculated. Reducing the maximum oxygen rate constant by 9%, decreasing the tissue cylinder radius by 5%, or increasing the capillary radius by 8% abolishes the hypoxic fraction. When a glycolytic term is added, concentration profiles are similar to the base case. Using a distribution of tissue/capillary radius ratios increases the hypoxic fraction and reduces sensitivity to the oxygen consumption rate, compared to the case with a single tissue/capillary radius ratio. This model demonstrates that hypoxia is quite sensitive to metabolic rate and geometric factors. It also predicts quantitatively the effects of inhibited oxygen metabolism and enhanced mass transfer on tumor oxygenation.     

Mathematical modeling of the circulation in the liver lobule

In this paper, we develop a mathematical model of blood circulation in the liver lobule. We aim to find the pressure and flux distributions within a liver lobule. We also investigate the effects of changes in pressure that occur following a resection of part of the liver, which often leads to high pressure in the portal vein. The liver can be divided into functional units called lobules. Each lobule has a hexagonal cross-section, and we assume that its longitudinal extent is large compared with its width. We consider an infinite lattice of identical lobules and study the two-dimensional flow in the hexagonal cross-sections. We model the sinusoidal space as a porous medium, with blood entering from the portal tracts (located at each of the vertices of the cross-section of the lobule) and exiting via the centrilobular vein (located in the center of the cross-section). We first develop and solve an idealized mathematical model, treating the porous medium as rigid and isotropic and blood as a Newtonian fluid. The pressure drop across the lobule and the flux of blood through the lobule are proportional to one another. In spite of its simplicity, the model gives insight into the real pressure and velocity distribution in the lobule. We then consider three modifications of the model that are designed to make it more realistic. In the first modification, we account for the fact that the sinusoids tend to be preferentially aligned in the direction of the centrilobular vein by considering an anisotropic porous medium. In the second, we account more accurately for the true behavior of the blood by using a shear-thinning model. We show that both these modifications have a small quantitative effect on the behavior but no qualitative effect. The motivation for the final modification is to understand what happens either after a partial resection of the liver or after an implantation of a liver of small size. In these cases, the pressure is observed to rise significantly, which could cause deformation of the tissue. We show that including the effects of tissue compliance in the model means that the total blood flow increases more than linearly as the pressure rises.     

A naphthalocyanine-based EPR probe for localized measurements of tissue oxygenation.

A new electron paramagnetic resonance (EPR) oximetry probe, based on a naphthalocyanine macrocycle, is reported to exhibit high oxygen sensitivity and favorable EPR characteristics for biological applications. The free radical probe, lithium naphthalocyanine (LiNc), is synthesized as fine microcrystalline powder with particle size less than 1 microm and high spin density. It exhibits a single sharp EPR peak, whose width varies linearly with oxygen partial pressure (pO2). The EPR spectrum is nonsaturable at typical microwave power levels (< 25 mW at X-band). These unique characteristics make this probe ideal for measuring oxygen concentration in biological tissues, in vivo. The peak-to-peak width under anoxic conditions is 0.51 G (at X-band), and it increases linearly with increase in oxygen partial pressure and reaches 26.0 G for 100% oxygen (760 mmHg), showing an oxygen sensitivity of 34 mG/mmHg. The probe responds to changes in pO2 quickly and reproducibly, thus enabling dynamic measurements of regional oxygenation in real time. The application of this probe for oximetry is demonstrated in an in vivo biological system. The changes in pO2 were monitored in the leg muscle tissue of a living mouse breathing room air and carbogen (95% oxygen + 5% CO2), alternatively. The mean pO2 measured with this probe in muscle tissues was consistent with values reported previously using other methods. Overall, the probe shows very desirable characteristics for localized measurements of tissue oxygenation.     

Enhancement of tumor oxygenation and radiation response by the allosteric effector of hemoglobin, RSR13

Prior studies using pO(2) microelectrodes have shown that RSR13, an allosteric modifier of hemoglobin, increases tissue oxygenation in vivo. Recently, measurements of tissue oxygenation have been performed by many investigators using blood oxygen level-dependent magnetic resonance imaging (BOLD MRI). In this study, we tested the hypothesis that the BOLD MRI signal ratio in tumors will change after administration of RSR13. NCI-H460 human lung carcinoma cells were used as a xenograft in athymic nude mice. Mice with 1-cm(3) tumors in the flank were anesthetized and mounted on the MRI apparatus, and various doses of RSR13 were administered intraperitoneally (i.p.). MR images were then acquired at 10-min intervals for up to 60 min after injection. The effect of RSR13 on tumor response was studied using the same mouse xenograft model with tumor growth delay measurements. RSR13 increased the MRI signal ratio [Intensity(t)/Intensity(t = 0)] in a dose-dependent manner, with maximum increases occurring 30 min after RSR13 was administered. An RSR13 dose of 200 mg/kg proved to be optimum. Since the MRI signal ratio has been shown previously to be linearly related to tissue oxygenation, the changes in the MRI signal ratio can be attributed to changes in tumor oxygen levels. Using a 200-mg/kg dose of RSR13, with a 10-Gy dose of radiation administered to tumors 30 min later, enhancement of radiation-induced tumor growth delay by RSR13 was observed (enhancement factor = 2.8). Thus our MRI results support and verify the previously reported RSR13-induced increase in tumor oxygenation obtained using pO(2) microelectrodes. Based upon these results and other previous studies, the mechanism of enhancement of the effect of radiation by RSR13 probably involves an increase in tumor oxygenation.     

Role of nitric oxide in hepatic microvascular injury elicited by acetaminophen in mice

Nitric oxide (NO) is suggested to play a role in liver injury elicited by acetaminophen (APAP). Hepatic microcirculatory dysfunction also is reported to contribute to the development of the injury. As a result, the role of NO in hepatic microcirculatory alterations in response to APAP was examined in mice by in vivo microscopy. A selective inducible NO synthase (iNOS) inhibitor,l-N6-(1-iminoethyl)-lysine (L-NIL), or a nonselective NOS inhibitor, NG-nitro-l-arginine methyl ester (L-NAME), was intraperitoneally administered to animals 10 min before APAP gavage. L-NIL suppressed raised alanine aminotransferase (ALT) values 6 h after APAP, whereas L-NAME increased those 1.7-fold. Increased ALT levels were associated with hepatic expression of iNOS. L-NIL, but not L-NAME, reduced the expression. APAP caused a reduction (20%) in the numbers of perfused sinusoids. L-NIL restored the sinusoidal perfusion, but L-NAME was ineffective. APAP increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space. L-NIL tended to minimize this infiltration, whereas L-NAME further enhanced it. APAP caused an increase (1.5-fold) in Kupffer cell phagocytic activity. This activity in response to APAP was blunted by L-NIL, whereas L-NAME further elevated it. L-NIL suppressed APAP-induced decreases in hepatic glutathione levels. These results suggest that NO derived from iNOS contributes to APAP-induced parenchymal cell injury and hepatic microcirculatory disturbances. L-NIL exerts preventive effects on the liver injury partly by inhibiting APAP bioactivation. In contrast, NO derived from constitutive isoforms of NOS exerts a protective role in liver microcirculation against APAP intoxication and thereby minimizes liver injury.     

Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats

We previously reported that nitric oxide (NO) derived from endothelial NO synthase (NOS) increased endothelial prostacyclin (PGI(2)) production in rats subjected to hepatic ischemia-reperfusion (I/R). The present study was undertaken to determine whether neutrophil elastase (NE) decreases endothelial production of PGI(2), thereby contributing to the development of I/R-induced liver injury by decreasing hepatic tissue blood flow in rats. Hepatic tissue levels of 6-keto-PGF(1alpha), a stable metabolite of PGI(2), were transiently increased and peaked at 1 h after reperfusion, followed by a gradual decrease until 3 h after reperfusion. Sivelestat sodium hydrochloride and L-658,758, two NE inhibitors, reduced I/R-induced liver injury. These substances inhibited the decreases in hepatic tissue levels of 6-keto-PGF(1alpha) at 2 and 3 h after reperfusion but did not affect the levels at 1 h after reperfusion. These NE inhibitors significantly increased hepatic tissue blood flow from 1 to 3 h after reperfusion. Both hepatic I/R-induced increases in the accumulation of neutrophils and the microvascular permeability were inhibited by these two NE inhibitors. Protective effects induced by the two NE inhibitors were completely reversed by pretreatment with nitro-l-arginine methyl ester, an inhibitor of NOS, or indomethacin. Administration of iloprost, a stable derivative of PGI(2), produced effects similar to those induced by NE inhibitors. These observations strongly suggest that NE might play a critical role in the development of I/R-induced liver injury by decreasing endothelial production of NO and PGI(2), leading to a decrease in hepatic tissue blood flow resulting from inhibition of vasodilation and induction of activated neutrophil-induced microvascular injury.     

Chronic ethanol consumption induces histopathological changes and increases nitric oxide generation in the rat liver

In the present work we evaluated the effect of ethanol consumption in histopathological liver changes and several biochemical biomarkers employed in the detection of hepatic dysfunction. Male Wistar rats were treated with ethanol 20% (vol/vol) for 6 weeks. Histopathological investigation of livers from ethanol-treated animals revealed steatosis. Indices of hepatic function (transaminases) and mitochondrial respiration were not altered in ethanol-treated rats. Chronic ethanol consumption did not alter malondialdehyde (MDA) levels in the liver. Ethanol consumption induced a significant increase on hepatic nitrite and nitrate levels. Treatment with ethanol increased both mRNA expression and immunostaining of iNOS, but not eNOS. Finally, ethanol consumption did not alter hepatic levels of metalloproteinase (MMP)-2 and MMP-9. We conclude that alterations on biochemical biomarkers (nitrite and nitrate levels) and histopathology occurred in ethanol-treated rats, supporting the practice of including both types of evaluation in toxicity studies to detect potential ethanol-related hepatic effects. In our model of ethanol consumption, histopathological liver changes were accompanied by elevation in nitrite and nitrate levels indicating increased nitric oxide (NO) generation. Since iNOS-derived NO contributes to hepatic injury, the increased levels of NO described in our study might contribute to a progressive hepatic damage. Therefore, increases in NO generation may be an early indicator of ethanol-induced liver damage.     

The short-term consumption of a moderately high-fat diet alters nitric oxide bioavailability in lean female Zucker rats

To determine whether short-term consumption of a moderately high-fat diet (MHFD) affects nitric oxide (NO) production, the concentration of stable NO metabolites (NOx) in urine and plasma of rats fed a MHFD (15.6?%g fat) or control diet (4.5?%g fat) was measured weekly for 4?weeks. Plasma and urine NOx levels were significantly depressed in the MHFD group by week 1 and remained so for the duration of the study. Decreased NO bioavailability may result from a decrease in NO production or the scavenging of NO by reactive oxygen species (ROS). Because endothelial NOS (eNOS) is the major contributor to NO production and circulating levels of NOx, eNOS expression was measured in several tissues. At week 1, there was a MHFD-associated decrease in eNOS expression in the liver. Subsequently, eNOS expression declined in the heart and kidney medulla of MHFD-fed rats at weeks 3 and 4, respectively. The expression of eNOS in the kidney cortex and adipose tissue did not change. These results suggest that a MHFD alters eNOS expression in a time-dependent and tissue-specific manner. In the liver, NOS activity and tissue levels of NOx and nitrotyrosine were measured. Nitrotyrosine levels were used as an indirect measure of the NO scavenged by ROS. There was a decrease in NOS activity, suggesting that the low levels of hepatic NOx were due, in part, to a decrease in NO production. In addition, there was a dramatic increase in nitrotyrosine formation, suggesting that the decline in hepatic NOx was also due to an increased interaction of NO with ROS. Tyrosine nitration commonly has detrimental effects on proteins. The decrease in NO and increase in protein nitration could potentially have adverse effects on tissue function.