Selective extravascular escape of albumin into the cerebral cortex of the diabetic rat.

The extravasation of plasma proteins (albumin, IgG, and complement C3) into the cerebral cortex was studied in streptozotocin-induced diabetic rats using immunohistochemical techniques. The results indicate that albumin, but not IgG or complement C3, selectively enters the cerebral cortex within 2 wk after induction of diabetes. It is suggested that albumin may be an oncotic substance that contributes to diabetic cerebral microangiopathy, astrocytic swelling, and the cerebral edema that occasionally is seen during fluid and insulin therapy of juvenile ketoacidotic diabetes.

Deficient Kupffer cell phagocytosis and lysosomal enzymes in the endotoxin-low-responsive C3H/HeJ mouse.

Various substances, including lysosomal enzymes, are produced by Kupffer cells and other macrophages; their release has been implicated in the toxic response to endotoxins. C3H/HeJ mice exhibit little or no response to doses of endotoxin that are lethal in syngeneic C3HeB/FeJ mice. To explore the nature of this deficient response, the Kupffer cells of these mice were studied using in vivo microscopic as well as histochemical and electron microscopical methods. In vivo, the rate of phagocytosis of single 0.8 micron latex particles was measured in individual Kupffer cells as was the number of phagocytic cells per microscopic field. Frozen sections of livers were stained for a variety of lysosomal enzymes and liver specimens also were processed for electron microscopy. In comparison to the endotoxin-sensitive C3HeB/FeJ mice, the livers of the C3H/HeJ mice contained 60% fewer Kupffer cells that phagocytosed latex. However, the rate of phagocytosis by these cells was not statistically different and ranged from 19-26 sec. The volume density of acid-phosphatase-positive Kupffer cells was 40% less in the C3H/HeJ mice. Similar differences were observed with other lysosomal enzymes including cathepsins B and H and dipeptidyl peptidases I and II. However, light and electron microscopy revealed a relatively normal number of Kupffer cells in livers stained for peroxidase, a nonlysosomal enzyme. The results suggest that the insensitivity of C3H/HeJ mice to endotoxin may be related in part to a lysosomal enzyme deficiency and a paucity of phagocytic Kupffer cells in these animals.

Studies of the hemopoietic microenvironment. IV. In vivo microscopic and histochemical study of allografts of bone marrow in the hamster cheek pouch chamber.

Within a transparent chamber enclosing subcutaneous tissue of the hamster cheek pouch, allogeneic femoral marrow was grafted. This permitted in vivo microscopic study of the grafts for 14 days. Five to seven days after grafting, blood flow was established within sinusoids arranged in interconnecting polygonal networks, a pattern characteristic of marrow. All vessels appeared to have a complete and continuous endothelial lining. The graft contained foci of erythropoiesis, granulopoiesis, and megakaryocytopoiesis, but no osteogenic activity. Degeneration of the grafts began to occur between days 9 and 13 after implantation. Generally, this was preceded by infection within the chamber, fibroblastic proliferation, and an accumulation of acid mucopolysaccharide in the stroma. The concomitant loss of hemopoiesis and accumulation of acid mucopolysaccharides was consistent with our hypothesis reported previously that excessive concentrations of acid mucopolysaccharide in the hemopoietic microenvironment are not conducive to supporting hemopoiesis, especially erythropoiesis. Degeneration of the grafts probably was the result of infection since there was no evidence of an immunologic response to the graft, and in chambers not containing grafts, similar infections and changes in the connective tissue were observed. The technique of grafting marrow into the hamster cheek pouch chamber provides a model for long-term, in vivo microscopic study of bone marrow. However, methodologic improvements, especially in the control of infections, are needed.

Hematopoiesis on cellulose-ester membranes (CEM). VI. Histochemical evaluation of stromal-CEM interactions after stromal enrichment.

Histochemical procedures were used to elucidate differences in the distribution of glycosaminoglycans (GAGs) and glycoproteins (GPs) in tubes made of cellulose-ester membrane (CEM) in six weeks and 12 months following intraperitoneal (i.p.) implantation. After six weeks, CEM tubes coated on their inner surfaces with bone marrow (BM), bone homogenate (BH), or regenerating medullary mesenchyme (M) contained a highly vascularized loose connective tissue and scattered hematopoietic elements. Numerous mononuclear cells were seen at the stromal-CEM interface and infiltrating the CEM. At the site of infiltration, there were accumulations of GPs and nonsulfated acidic GAGs. The remainder of the CEM contained limited amounts of sulfated and nonsulfated GAGs. By 12 months, both coated and uncoated CEMs were saturated with sulfated and nonsulfated acidic GAGs. However, only coated CEM tubes contained trilineal hematopoiesis surrounded by a shell of bone that was incorporated into the structure of the CEM. Exterior to the bone, the CEM was infiltrated with GPs, some of which were acidic and nonsulfated, possibly sialoglycoproteins. No bone or glycoproteins infiltrated the uncoated CEMs. The stroma supporting trilineal hematopoiesis contained GPs and nonsulfated acidic GAGs, but not sulfated acidic GAGs. However, both sulfated and nonsulfated GAGs were found in the fibrous connective tissue filling the lumens of uncoated CEMs. While CEM tubes accumulate peritoneal GAGs that may be conducive for the retention, differentiation, and proliferation of osteogenic and other stromal cells provided by coatings of BM, BH, or M, it is these cells that produce the bone and glycoproteins that appear to contribute to a microenvironment conducive to trilineal hematopoiesis. However, the relative roles of these substances in this process remain to be elucidated.

The microcirculation during endotoxemia.

The initial responses to endotoxemia are detectable in the microcirculation as a microvascular inflammatory response characterized by activation of the endothelium stimulating these cells from their normal anticoagulant state to a procoagulant state with increased adhesiveness for leukocytes and platelets. Concomitantly, arteriolar tone is lost and reactivity to a variety of agonists is modified. Tissue damage subsequently results not only from reduced perfusion of the exchange vessels, but also from injurious substances released from activated, sequestered leukocytes as well as activated endothelial cells, macrophages, and platelets. This is the result of endotoxins inducing activation and interaction of a number of effector cells, cascades, and acute-phase responses, such as the complement, coagulation, bradykinin/kinin, and hematopoietic systems accompanied by the release of a myriad of mediators. These include eicosanoids, cytokines, chemokines, adhesion molecules, reactive free radicals, platelet-activating factor, and nitric oxide. This paper briefly reviews the microvascular responses to endotoxemia and discusses some of the mechanisms involved.

In vivo microscopic studies of age-related changes in the structure and the reactivity of cerebral microvessels.

To determine if hemodynamic changes in cerebral microvessels could contribute to the age-related changes in the blood-brain barrier (BBB) function, the cerebral microvessels of male Fischer 344 rats at different ages were studied using intravital fluorescence microscopy. Aging in rats was associated with significant arteriovenous shunting in the cerebral microvessels without alterations in blood flow characteristics or changes in vascular permeability to FITC dextran (150 kDa). The basal diameter of terminal arterioles examined in 24- to 27-month-old (aged) rats (28.6 +/- 2.8 microns) was not different from that in 12- to 15-month-old (intermediate age) rats (32.5 +/- 2.5 microns) or in 3- to 6-month-old (young) rats (28.6 +/- 3.0 microns). At 3 s following addition of 5% BaCl2 there was 23.3 +/- 3.47% constriction of arterioles in young rats and 14.8 +/- 5.16% constriction in intermediate age rats, but only a 3.43 +/- 5.69% change in aged rats (P less than 0.03). This initial brief constriction phase was followed by a dilatory response which was similar in all age groups. One minute following suffusion with artificial cerebrospinal fluid, the arteriolar diameter essentially returned to baseline in all rats examined. It is concluded that aging in rats is associated with alterations in cerebral microvascular reactivity in vivo along with arteriovenous shunting. These changes may contribute to age-related alterations in the BBB function.

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.

Biliary obstruction exacerbates the hepatic microvascular inflammatory response to endotoxin.

Gram-negative sepsis is a serious complication for patients with obstructive jaundice. The present study was conducted to elucidate the response of hepatic microcirculation to endotoxin 2 weeks after bile duct ligation (BDL) or sham-operation in rats. Two hours after lipopolysaccharide (LPS) injection (1, 10, or 100 microg/kg, iv.), the hepatic microvasculature was examined using in vivo microscopy. BDL elicited increases in leukocytes adhering to the sinusoidal wall, swelling of sinusoidal endothelial cells as well as phagocytic activity of hepatic macrophages and a decrease in the numbers of perfused sinusoids. LPS (1, 10, 100 microg/kg) further increased leukocyte adhesion and reduced the numbers of perfused sinusoids in a dose-dependent manner. Leukocyte adhesion in response to LPS (1, 10, 100 microg/kg) in BDL rats was increased 6.1-fold, 5.9-fold, and 3.3-fold, respectively when compared with sham-operated rats. The numbers of perfused sinusoids in response to LPS (1, 10, 100 microg/kg) in BDL rats were decreased by 42%, 36%, and 45%. While 1 and 10 microg/kg LPS also elicited an increase in phagocytic activity in BDL rats when compared with sham-operated rats, the response to 100 microg/kg LPS was suppressed. LPS did not affect the numbers of swollen endothelial cell in BDL rats. The present study demonstrated that chronic biliary obstruction enhanced the hepatic microvascular response to low doses of endotoxin. This observation suggests that exaggerated hepatic microcirculatory dysfunction during sepsis contributes to the development of liver injury and a high incidence of morbidity and mortality in biliary obstruction.

Ethanol exacerbates hepatic microvascular dysfunction, endotoxemia, and lethality in septic mice.

The effect of acute ethanol administration on the hepatic microvascular responses to sepsis was studied. Polymicrobial sepsis was induced 30 min after mice had received ethanol (1 g/kg b.w.) or isocaloric maltose-dextrin by gastric gavage. Lethality within 24 h was 91.7% in the ethanol-treated animals and 40.0% in septic controls. Endotoxin levels in ethanol treated animals were 107 pg/ml at 6 hr and 1205 pg/ml at 12 h, compared with 32 pg/ml and 104 pg/ml, respectively in the controls. In vivo microscopy revealed that at 3 h in the ethanol treated septic animals, Kupffer cell phagocytic activity was increased by 41%, whereas the number of sinusoids containing blood flow were reduced by 34% concomitant with a 144% increase in the adherence of leukocytes to the sinusoidal walls when compared with the septic controls. By 6 h, however, Kupffer cell phagocytic activity was reduced by 48% in the ethanol treated animals; this was accompanied by a further deterioration in sinusoidal blood flow. Thus, a small, acute dose of ethanol causes significant impairment of the hepatic microcirculation followed by suppression of Kupffer cell activity. This results in exacerbation of endotoxemia and lethality during polymicrobial sepsis.

Effect of immunoglobulin G on the hepatic microvascular inflammatory response during sepsis.

The effects of intravenous immunoglobulin G (ivIG) on the hepatic microvascular inflammatory response to sepsis were studied in rats by in vivo microscopy. High doses of ivIG (300 mg/kg bw) (Sandoglobulin or rat IgG) significantly improved the 48 h survival of septic rats from 25-66% when ivIG was given before or immediately after cecal ligation and puncture. Circulating endotoxin also was significantly reduced. Eight hours after inducing sepsis, the average number of leukocytes adhering to the sinusoidal endothelium increased 15-fold and the average decrease in the number of perfused sinusoids was 22%. IvIG administration minimized these responses. In both septic and nonseptic animals, ivIG also reduced the phagocytic activity of Kupffer cells. The results suggest that high doses of ivIG not only reduce lethality but also limit hepatic microcirculatory dysfunction during sepsis by minimizing leukocyte-endothelial interactions that may be a result of reducing circulating endotoxin and modifying Kupffer cell function.

High doses of intravenous immunoglobulin G enhance Kupffer cell phagocytic function during the late phase of sepsis and endotoxemia in rats.

The effect of intravenous immunoglobuln G (ivIG) on the hepatic microvascular inflammatory response during the late phase of sepsis and endotoxemia in rats was studied by using in vivo microscopy. One hour after administration of a clinically relevant dose of ivIG (0.5 g/kg body weight, Sandoglobulin), rats were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP) or were injected intravenously with lipopolysaccharide (LPS, 0.1 mg/kg body weight). Twenty-four hours after CLP or LPS, the number of leukocytes adhering to the sinusoidal wall was increased 11.0-fold in CLP-treated animals and 5.6-fold in LPS-treated animals, respectively, compared with the controls. Concomitantly, the numbers of swollen sinusoidal endothelial cells were increased 4.2-fold and 3.2-fold. The number of perfused sinusoids was decreased by 35% and by 24%. These responses were minimized by pretreatment with high doses of ivIG. Kupffer cell phagocytic activity in the periportal sinusoids in CLP-treated animals was decreased by 41%, whereas that in the centrilobular sinusoids in LPS-treated animals was increased by 72%. IvIG significantly elevated this activity in both CLP- and LPS-treated animals and the number of ED2-positive Kupffer cells in tissue sections. The results suggest that ivIG limits the hepatic microvascular inflammatory response during the late phase of sepsis and endotoxemia by affecting Kupffer cell function.

Effect of intravenous immunoglobulin G on the TNFalpha-mediated hepatic microvascular inflammatory response.

The effect of intravenous immunoglobulin G (ivIG) on the hepatic microvascular inflammatory response elicited by tumor necrosis factor alpha (TNFalpha) in rats was studied by means of in vivo microscopy and histological examination. One hour after the portal infusion of TNFalpha, the average number of leukocytes adhering to the sinusoidal endothelium was increased sevenfold, and the average number of the perfused sinusoids was decreased by 15% when compared with controls. Concomitantly, the expression of intercellular adhesion molecule-1 (ICAM-1) on the hepatic sinusoidal endothelium and that of the central vein was increased. The phagocytic activity of Kupffer cells in centrilobular sinusoids was increased by 54%, as were the number of ED2-positive Kupffer cells in tissue sections. Pretreatment with a clinically relevant high dose of ivIG (1 g/kg body weight, Sandoglobulin) minimized these responses by reducing leukocyte-endothelial interactions and Kupffer cell phagocytic function. The results suggest that high doses of ivIG limit the hepatic microvascular inflammatory response by inhibiting the action of the proinflammatory cytokine TNFalpha.

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.

In vivo microscopy of the exocrine pancreas.

Light microscopic studies of the living acinar pancreas, although limited in number, have revealed valuable information concerning dynamic aspects of microvascular and parenchymal structure and function. For example, it has been found that: 1) the living organ in anesthetized animals can be imaged with a resolution approaching the limit of the light microscope; 2) blood flow through individual capillaries in the exocrine pancreas is intermittent; 3) blood flow through these capillaries is regulated locally by smooth muscle precapillary sphincters and within individual capillaries by endothelial cells which are spontaneously contractile as well as responsive to vasoactive substances; and 4) the formation and release of zymogen granules occurs within 45-90 minutes in acinar cells stimulated with pancreozymin. This paper reviews these studies and some of the methods used to obtain them.

Sphincters of canine hepatic sublobular veins respond to endothelin-1 and 3.

The dog has been used repeatedly as a model in liver transplantation research. The microcirculation and its regulatory mechanisms play a crucial role during ischemia and reperfusion. Little is known about the role of venous sphincters in regulating blood flow in the dog liver. Hence, we performed this study to elucidate their potential role in regulating local blood flow. In 14 dogs mean systemic (MSP) and mean portal venous pressure (MPP) were measured. Light and electron microscopy (scanning and transmission) of tissue sections and vascular corrosion casts were used to elucidate the microvascular morphology. Immunocytochemistry was applied to identify smooth muscle cells and the innervation of venous sphincters. Endothelins 1 and 3 were injected to find whether the hepatic venous sphincters are sensitive to these vasoactive agents. Tufts of smooth muscle cells were found in the sublobular veins (SLV; 100 to 250 microm in diameter), that reduced the luminal diameters of veins by 34%. Nerve endings were not observed close to these venous sphincters. The MSP and MPP were 75.3+/-2.4 mmHg and 8.9+/-0.95 mmHg, respectively. Treatment with 1.0 microg/kg of endothelin-1 (ET-1) significantly increased the MSP, the MPP and the percentage of focal venous sphincter contraction by 39% (105+/-4.7 mmHg), 43% (12.8+/-1.7 mmHg) and 57% (53.5+/-4.7), respectively (P <0.01). Treatment with ET-3 caused a significant (P <0.01) decrease in the MSP, the MPP and the percentage of sphincter contraction by 19% (61.0+/-2.2 mmHg), 39% (5.8+/-2.9 mmHg) and 38% (20.9%+/-3.15). Sinusoids did not contain sphincters. Hepatic arterioles and central veins were not affected by ET-treatment. The contraction of SLV sphincters correlated with increases in MPP (r=0.81, P <0.01) and was related to the MSP (r=0.67, P <0.01). These data show that the smooth muscle sphincters in SLV of the dog liver are involved in the local regulation of blood flow and that these sphincters are stimulated by non-neurogenic mechanisms. These sphincters contract in response to ET-1 and relax in response to ET-3. Since ET-1 is released during and/or causes inflammation, e.g., during ischemia and reperfusion, its antagonists might be of benefit during transplantation reperfusion of liver.

Innervation of the periarteriolar lymphatic sheath of the spleen.

During the course of a neurohistochemical and two independent electron microscopic studies of the mouse spleen, unmyelinated adrenergic nerves containing numerous dense core and lucent vesicles and devoid of neurolemma were observed adjacent to reticular cells and lymphocytes in the white pulp. Some of these nerves formed an intimate relationship with these cells. Since adrenergic substances have been reported to modulate the cell cycle of lymphocytes in vitro, these findings are suggestive of a neural influence on the cell cycle of lymphocytes in vivo.

In vivo and electron microscopic observations of the responses of the hepatic sinusoid to interleukin-1.

Interleukin-1 (IL-1), which is produced by Kupffer cells and sinusoidal endothelial cells, may play an important role in immunological and microvascular responses to a variety of stimuli in the liver. The responses of the hepatic microvasculature including phagocytic activity of sinusoidal lining cells to IL-1 alpha were examined in C57Bl/6 mice in vivo and using electron microscopy. One hour after recombinant mouse IL-1 alpha was injected at doses of 80 U, the low dose group, and 800 U, the high dose group, the phagocytic activity of sinusoidal lining cells showed significant differences between the two treated groups and between the two groups and the controls. In the low dose group, the numbers of sinusoids containing blood flow and of leukocytes adhering to the sinusoidal lining remained unchanged, but the former decreased and the latter increased significantly in the high dose group. Ultrastructurally, Kupffer cells that phagocytosed latex particles appeared to have decreased in number while the sinusoidal endothelial cells became phagocytic. A considerable number of leukocytes were seen adhering to the sinusoidal endothelium. These findings demonstrate that IL-1 alpha not only elicited sticking and plugging by leukocytes in sinusoids but also activated phagocytic functions in the hepatic sinusoidal endothelial cells. These endothelial responses are similar to those seen following FV3 virus infection, chronic administration of ethanol, or a combination of cocaine and ethanol, or during extracorporal perfusion, suggesting that IL-1 may participate in these responses.