Changes in rat liver microcirculation after experimental hepatic arterial embolization: comparison of different embolic agents.

PURPOSE: To evaluate the effects of hepatic arterial embolization on hepatic microcirculation in the rat liver by using different particulate agents. MATERIALS AND METHODS: Polylactic acid microspheres, polyvinyl alcohol particles, and absorbable gelatin powder were injected into the hepatic artery of 50 rats. Saline was used as the control agent. Flow characteristics of hepatic microcirculation were qualitatively assessed on days 0 and 7 after embolization by using in vivo microscopy. Histologic specimens of the rat liver were analyzed. RESULTS: The polylactic acid (1-5 microns) injected into the hepatic artery was seen circulating through the sinusoids into the central venules. The slowing of flow observed with the injection of larger (50-200-micron) particles reflected the arterial occlusion occurring more proximally. After 7 days, all embolic agents caused vascular occlusion that led to necrosis and fibrosis. Networks of irregular, high-speed vessels that resembled arterioles and bypassed the normal sinusoids were observed. CONCLUSION: The necrotic areas observed after experimental distal occlusion of the hepatic arteries in the rats were bypassed by vessels similar to the capillarized sinusoids observed in the cirrhotic liver in humans. These vessels acted as sinusoidal shunts in the embolized territories.

In vivo microscopy of hepatic metastases: dynamic observation of tumor cell invasion and interaction with Kupffer cells.

In vivo microscopy was used in the study of the biological behavior of tumor cells and of the activity of Kupffer cells in hepatic tumors in situ. Three tumor models, Friend erythroleukemia inoculated into Dilute Brown Aguti (DBA)/2 mice, murine colon adenocarcinoma (CT)-26 in Bagg Albino inbred albino (BALB)/c mice, and mammary cancer 13762 NF in Fischer rats, were investigated. Tumor cells showed a strong tendency to adhere to the sinusoidal endothelium, most frequently in the sinusoids near the tumors. Mechanical trapping of tumor cells in the narrow portion of hepatic sinusoids, a phenomenon suggested by previous investigators as a predominant pattern for tumor cells to arrest in the liver, was not confirmed. Our study documented that in tumor-bearing livers, as compared with normal control livers, the population size and the phagocytic capacity of Kupffer cells are increased in nontumorous areas but are significantly decreased inside the tumors. In vivo microscopic images showed that Kupffer cells are not only attracted to tumor cells in the hepatic circulation but also have the ability to phagocytose those tumor cells. In vivo microscopy has been shown to be a useful tool for dynamic studies in tumor biology, pathology, and pharmacology.

Role of Kupffer cells in iodized oil embolization.

RATIONALE AND OBJECTIVES: Iodized oil is a common oily embolic agent used in chemoembolization for treating hepatic tumors. However, how the iodized oil is cleared from the liver has been an unsettled and controversial issue. In this study, the authors attempt to clarify whether Kupffer cells are involved in the clearance of iodized oil and to evaluate the effect of hepatic arterial injection of iodized oil on the functional status of Kupffer cells. METHODS: Iodized oil was injected into the proper hepatic artery in 42 Fischer 344 rats. In vivo microscopy was performed immediately after and 1, 3, 7, 15, 30, and 60 days after injection. Electron microscopy was performed after in vivo microscopy. RESULTS: Kupffer cells actively captured and phagocytosed iodized oil droplets in the hepatic circulation. The number and functional status of Kupffer cells in the liver were significantly increased after the injection of the iodized oil and returned to normal when the liver was cleared of the oil. CONCLUSIONS: Kupffer cells play an important role in clearing iodized oil from the liver. Iodized oil activates the immune defense system in the liver, which may have a synergistic effect in tumor treatment.

An electron microscopy study of Kupffer cells in livers of mice having Friend erythroleukemia hepatic metastases.

Kupffer cells, which are part of the reticuloendothelial system, play an important role in clearing pathogenic substances, including tumor cells, from the liver. The role of Kupffer cells in tumor development is very important as Kupffer cells can be manipulated to a tumoricidal state with biological response modifiers to kill tumor cells and thus to decrease tumor burden and extend survival time. To gain additional information on the role of Kupffer cells and their interaction with tumor cells in hepatic metastases, we studied an established experimental hematogenous metastatic model (Friend erythroleukemia) in mouse livers by light and electron microscopy. Highly activated Kupffer cells were observed in close contact with tumor cells in sinusoids and also in tumor forming foci within the hepatic parenchyma. The Kupffer cells were activated by the presence of the hematogenous tumor cells and were able to lyse and phagocytose them. However, some tumor cells evaded the Kupffer cells as metastases still occurred. Kupffer cells and other macrophages were found to leave the sinusoids and migrate to sites of potential tumor development where they interacted with tumor cells and intimately wrapped their processes around fat storing cells. It is possible that these macrophages which cross biological barriers could be used to deliver drug-loaded microparticles (liposomes and microcapsules) to tumors.

In vivo and electron microscopic studies of rat liver after intravenous injection of polyamino acid microspheres.

In vivo and electron microscopy were used to study the hepatocellular responses of rat livers to intravenously injected polymeric microspheres. Two microsphere preparations with different surface characteristics and degradability were used in this study. In vivo microscopy revealed that both poly(benzyl L-glutamate) (PBLG) and poly(hydroxypropyl L-glutamine) (PHPG) microspheres caused disturbance in the microcirculation of rat liver up to 2 months after injection. The observed changes included stagnant flow and adherence of white blood cells to the endothelial lining of venules an sinusoids. Kupffer cell (KC) activation following phagocytosis of microspheres was evidenced by the enlargement of KCs and increased number of KCs taking up fluorescent latex particles. Electron microscopy of rat livers revealed a wide range of hepatocellular injury associated with the administration of PBLG and PHPG microspheres. These results indicate that a small amount of remaining microspheres is sufficient to induce continuous disturbance to hepatic microcirculation and that particulate drug carriers should be designed to be rapidly degraded so that the return to normal liver function is possible.

In vivo microscopy of hepatic tumors in animal models: a dynamic investigation of blood supply to hepatic metastases.

The dynamics of blood circulation in three experimental animal models of hepatic metastasis were investigated with in vivo microscopy. It was demonstrated that the tumor vasculature communicated with the portal venules and hepatic sinusoids that surrounded the tumors. The hepatic artery was not seen to connect to the tumors directly. However, it was demonstrated that arterial blood entered tumors through the portal venules and that the hepatic arterial flow entered the tumor without resistance, while blood from the portal vein met great resistance at the tumor border, with only small amounts entering the tumor. Interruption of either the hepatic artery or the portal vein did not result in cessation of the blood circulation in hepatic tumors. A reciprocal relationship between the hepatic arterial and portal venous supplies to hepatic tumors was suggested, and it was hypothesized that arterioportal communications play an important role in the arterial and portal venous supply of blood to hepatic tumors. A comprehensive understanding of the blood supply of hepatic tumors is important for improving clinical treatment of hepatic tumors.

Electron microscopic study of the effects of endotoxin on the cells of the hepatic sinusoid in normal and BCG sensitized mice.

Electron microscopic studies were conducted to access ultrastructural alterations in Kupffer cells and other cells lining the hepatic sinusoids at the peak of mediator release two hours after challenge with low doses of endotoxin under various conditions including reticuloendothelial system (RES) expansion and activation with BCG. BCG is known to sensitize animals to endotoxin rendering normally innocuous, low doses of endotoxin lethal. Low non-lethal doses (5 micrograms) of endotoxin activated Kupffer cells as well as caused isolated foci of cellular injury. However, animals which were treated with BCG had a highly activated and expanded RES system as evidenced by enlarged Kupffer cells with many extended cellular processes. Granulomas were prevalent and many reactive cells were present. After two hours marked cellular injury occurred to sinusoid lining and parenchymal cells when BCG treated animals were challenged with these same low doses of endotoxin. Cellular debris, fibrin, and platelets were observed in sinusoids often associated with Kupffer cells. These results suggest that the functional state of Kupffer cells is an important determinant in the host response to endotoxin. While there appears to be an effective clearance of endotoxin; the release of mediators by the highly activated Kupffer cells can be toxic causing hepatocellular injury.

Kupffer cell activity and hepatic microvascular events after acute ethanol ingestion in mice.

After acute ethanol ingestion in C57Bl/6 mice, phagocytic activity of Kupffer cells and hepatic microcirculation were examined by in vivo and electron microscopy. A ratio of Kupffer cells that phagocytosed 0.8 micron fluorescent latex particles to sinusoids containing blood flow (number of Kupffer cells/number of sinusoids containing blood flow) was used as a measure of Kupffer cell phagocytic activity. Three hours after ingestion of 1 gm/kg ethanol, number of Kupffer cells/number of sinusoids containing blood flow increased in both periportal and centrilobular regions by 62% and 66%, respectively, and blood ethanol was no longer detectable. Ultrastructurally, activation of KC was evidenced by the presence of many pseudopodia and filopodia. Numbers of swollen endothelial cells were increased in both regions by 249% and 174%. Interruption of sinusoidal blood flow by leukocytes was aggravated in both regions by 127% and 167%. Thirty minutes and 3 hr after ingestion of 4 gm/kg ethanol, no significant increase in number of Kupffer cells/number of sinusoids containing blood flow was seen, although number of Kupffer cells were also activated as seen with electron microscopy. An increased number of swollen endothelial cells was observed in both regions by 100% and 71% by 30 min and by 200% and 384% by 3 hr. The interruption of sinusoidal blood flow by leukocytes was also increased (periportal = 161%, centrilobular = 196%) as were sticking or plugging leukocytes in sinusoids (periportal = 320%, centrilobular = 120%) by 3 hr. The diameter of centrilobular sinusoids was decreased by 3 hr. Latex particles and platelets were attached to the sinusoidal wall after both low and high dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Fine structure and function of Kupffer cells.

Kupffer cells are macrophages that are attached to the luminal surface or inserted in the endothelial lining of hepatic sinusoids. In this site, Kupffer cells play a key role in host defense by removing foreign, toxic and infective substances from the portal blood and by releasing beneficial mediators. Under some conditions, toxic and vasoactive substances also are released from Kupffer cells which are thought to play a role in a variety of liver diseases. Many of these activities may be modulated by the levels of gut derived endotoxin normally present in the portal blood. The ultrastructural aspects of Kupffer cell structure function in situ are best studied using perfused-fixed livers. In fixed livers, transmission and scanning electron microscopy reveal Kupffer cells during health to be irregular in shape with their exposed surfaces presenting numerous microvilli, filopodia, and lamellopodia. Long filopodia penetrate endothelial fenestrae to secure Kupffer cells to the sinusoid lining. Specific membrane invaginations known as worm-like bodies or vermiform processes are seen in the cytoplasm of Kupffer cells as are numerous endocytotic vesicles and lysosomes which vary in density, shape and size. Sometimes, annulate lamellae connected to the rough endoplasmic reticulum also are found. The principal endocytic mechanisms of Kupffer cells are phagocytosis of particulates and cells, and bristle-coated micropinocytosis for fluid-phase endocytosis of smaller substances. Many of these events are mediated by specific receptors. In some species, Kupffer cells can be distinguished from other sinusoidal lining cells and monocytes by specific cytoplasmic staining or monoclonal antibodies. Kupffer cells have been shown to be of monocytic origin as well as having the capacity for self-replication.

Relationships between microvascular function and capillary structure in diabetic and nondiabetic human skin.

Despite the commonly held view that abnormalities in capillary morphology, in particular thickening of the capillary basement membrane, are partly responsible for diabetic ischemia, few studies have correlated anatomic and hemodynamic variables in the same diabetic subjects. In a previous study of 24 type II (non-insulin-dependent) diabetic subjects and 24 age-matched control subjects, we showed that a standard finger exercise vasodilated cutaneous forearm vessels nearly equally (51%), but the postarteriolar flow responded differently between groups. Nondiabetic subjects increased flow by recruitment of capillaries, whereas diabetic subjects did so by capillary flow augmentation. Moreover, resting permeability-surface area product (PS) to pentetic acid was 85% higher in diabetic than nondiabetic subjects. In this study, these same subjects had their forearm skin biopsied and examined morphometrically by electron microscopy for capillary radius, basement membrane thickness, endothelial cell density, and a folding index of luminal membrane reduplication. All morphological variables were correlated stepwise in a saturated, analysis of covariance model with the physiological results. The correlations were sparse and specifically excluded basement membrane thickness. The highest r2 value was .432 between resting PS and a ratio of capillary density to endothelial cell number per capillary. These studies show little evidence that diabetic microvascular physiological variables are tightly connected to morphometric changes except for minor permeability changes, which rise with capillary density and decrease with endothelial cell number. Because PS to pentetic acid is increased in diabetic subjects at any level of capillary density, it seems reasonable that permeability may be increased above that of nondiabetic subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental investigation of a new iodinated lipid emulsion for computed tomography of the liver.

Iodinated lipid emulsions are highly efficient macrophage imaging agents. Nevertheless, none of them has been accepted for clinical use because of adverse reactions. We have tested a new iodinated lipid emulsion, Intraiodol. The size and surface properties of the particles of this emulsion are similar to those of Intralipid which in turn closely resemble the naturally occurring chylomicrons. Using computed tomography (CT) of the rabbit liver as well as vital microscopy and electron microscopy of the rat liver we found that Intraiodol has low efficiency as a liver-specific contrast medium because its particles are predominantly taken up by the hepatocytes and to a less extent by the Kupffer cells, as is Intralipid. The low efficiency of Intraiodol could be fully compensated by an increase in dosage without any significant effect on sinusoidal blood flow. This in turn suggests that the likelihood of release of toxic mediators (and thereby related adverse reactions from activated macrophages) is reduced. We believe that this new way of delivering iodinated lipid particles to the liver represents an important advance in the search for a non-toxic lipid emulsion for CT of the liver.

Effect of intravenously injected iodinated lipid emulsion on the liver. An experimental study correlating computed tomography findings with in vivo microscopy and electron microscopy findings.

Iodinated lipid emulsions have been shown to have great potential as site specific contrast media for the liver and spleen. Because of unacceptable adverse reactions none of these emulsions has been adopted for clinical use. In an attempt to find an explanation for these adverse reactions we tested three iodinated lipid emulsions, EOE-13, AG 60.99 and AG 66.18. The following models were used: Computed tomography (CT) of the rabbit liver, in vivo microscopy and electron microscopy of the rat liver. The emulsions contained particles of different sizes and were used in varying doses. We found that the larger the emulsion particles, the more likely they were to be taken up by the Kupffer cells and thereby the higher the opacification of the liver achieved at CT. We also observed changes in the microcirculation of the liver when the emulsions were given in doses required to secure satisfactory opacification of the liver at CT. The main changes were 1) a marked increase in the size of the Kupffer cells, and 2) damage to the sinusoidal endothelium, both contributing to sinusoidal congestion. These changes strongly suggest activation of the macrophages and this in turn probably results in the release of toxic mediators. We suspect that the adverse reactions observed in patients when using iodinated lipid emulsions are due to these toxic mediators.

Kupffer cell function in host defense.

High-resolution in vivo microscopic methods have been used to explore the responses to endotoxin of Kupffer cells in the livers of anesthetized mice, rats, hamsters, and guinea pigs under a variety of experimental conditions. These include studies of normal animals as well as of animals sensitized or tolerant to endotoxin, C3H/HeJ mice with a low response to endotoxin, mice rendered septic by cecal ligation and puncture, mice with Kupffer cells selectively destroyed by frog virus 3, and rats with portacaval shunts. The functional state of Kupffer cells was evaluated by measuring both the number of these cells per microscopic field that phagocytosed 1.0-micron latex particles and the rate at which individual Kupffer cells phagocytosed single latex particles. The intrahepatic density and level of activation of Kupffer cells were found to play a role in determining endotoxin sensitivity and to be involved, in conjunction with endotoxin, in the development of tolerance. All of these studies support the concept of a central role for Kupffer cells in host defense mechanisms and of the possible modulation and of this role by gut-derived endotoxins contained in the portal blood.

Normal versus abnormal structure: considerations in morphologic responses of teleosts to pollutants.

Consideration of newer more quantitative morphologic approaches to the study of aquatic pollutants can provide opportunity for collaborative/integrated studies with other subdisciplines in toxicology. Current commonly employed morphologic approaches result largely in subjective findings difficult to analyze statistically and often are directed at levels of structural organization inconsistent with biochemical and physiological approaches. We review some of the methods and approaches available for correlated structure/function studies and present examples from normal and altered skin, gill, and liver of teleosts.

Phagocytosis, an unrecognized property of murine endothelial liver cells.

Impairment of the phagocytic capacities of Kupffer cells, as is found in Frog Virus 3 hepatitis of mice, allows the endothelial liver cells to take up intravenously inoculated latex particles of 1.0 micron diameter. In vitro experiments with cultivated endothelial cells isolated by collagenase perfusion of the liver and purified by centrifugal elutriation demonstrate that uptake occurs via a typical mechanism of phagocytosis involving pseudopodia. Ingestion of latex is inhibited by incubation of the cells at 4 degrees C and by treatment with cytochalasin B, whereas colchicine has no effect. These results demonstrate that: the Kupffer cells are not the only cells of the hepatic sinusoid capable of phagocytosis; and under conditions where the phagocytosis in Kupffer cells is impaired, the endothelial cells may participate in the clearance of large particles from the blood.

Hepatic microvascular regulatory mechanisms. VIII. Glucogenic responses and morphologic changes following serotonin-induced low flow.

Changes in blood glucose, hepatic glycogen content and distribution, the number of hepatic mast cells, and hepatic morphology were assessed over 30 min in non-fasted and anesthetized Sprague-Dawley rats receiving endoportal or femoral intravenous injections of selected doses of serotonin and/or phentolamine, lodoxamide, or of Ringer's solution (control). Endoportal administration of low-flow producing doses of serotonin (1.0, 10.0, 20.0 micrograms per 100 g b.w.) elevated circulating blood glucose without decreasing hepatic glycogen content when compared to control in unit dry or wet weights. Hyperglycemia was accompanied by centrilobular glycogen depletion and apparent Kupffer cell activation. However, no change in hepatocyte or endothelial cell morphology or in the number of hepatic mast cells was observed following serotonin-induced low flow. The glucotropic response to a nonhypotensive dose of serotonin (1.0 microgram per 100 g b.w.) was modified by phentolamine (100 micrograms per 100 g b.w.) but not lodoxamide (0.1 microgram per 100 g b.w.). These blockers, when given alone, stimulated centrilobular glycogen depletion without producing a net change in blood glucose or hepatic glycogen content. By contrast, injection of serotonin (10.0 micrograms per 100 g b.w.) and/or phentolamine (100 micrograms per 100 g b.w.) into the femoral vein provoked no glucogenesis or systemic hypotension. Given these results, serotonin is suggested to stimulate hyperglycemia by activating alpha-adrenergic receptors. Since centrilobular glycogen depletion proceeds with no detectable change in total hepatic glycogen content, it is postulated that hepatic glycogen catabolism and deposition occur simultaneously and at equivalent rates during conditions of serotonin-induced hyperglycemia and low flow.

Functional units in rainbow trout (Salmo gairdneri) liver: I. Arrangement and histochemical properties of hepatocytes.

The architectural arrangement and selected histochemical properties of hepatocytes in the rainbow trout (Salmo gairdneri Richardson) were examined. Light and transmission electron microscopic (TEM) examination following fixation by portal venous perfusion revealed a tubular arrangement of hepatocytes. Lobules, as defined in the adult mammal, were absent. Biliary epithelial cells associated with bile preductules and ductules were a prominent feature of trout liver. Patterns and location of reaction products for glucose-6-phosphatase (G-6-Pase), glucose-6-phosphate dehydrogenase (G-6-PDH), and magnesium-dependent adenosine triphosphatase (ATPase), enzymes preferentially distributed in mammalian liver, were demonstrated in trout liver. A slightly heavier staining pattern for G-6-Pase was seen around presumptive portal venules but all other enzyme reaction patterns were uniform throughout the liver parenchyma. Following ATPase localization, four sizes of biliary passageways (canaliculi, bile preductules, ductules, and ducts) were visualized. Maximum glycogen retention was achieved with freeze-drying and glycolmethacrylate embedding and with this method intense, uniform glycogen staining was observed in all areas of the liver. Companion TEM examinations revealed large depots of glycogen within hepatocytes. The results are important for interpretation and description of the effects of toxic/carcinogenic alteration on trout liver.