Light and electron microscopic studies of diet-induced hepatic changes in mice

Adult mice were fed a choline-deficient ethionine enriched (CDE) diet for 24, 48 or 72 h. They were then fasted for 24 or 48 h prior to sacrifice. All tissues were studied by light and electron microscopy. Animals fed the CDE diet for 24 h exhibited cells with vacuolated cytoplasm, and the accumulation of lipid in these cells was clearly abnormal. Animals fed the CDE diet for 24 h and subsequently a regular diet for 48 h displayed normal hepatocytes, suggesting that the alterations at 24 h were reversible. Following 48 or 72 h of feeding the CDE diet, abundant lipid-laden cells were observed in the hepatic lobules, and at the electron microscope level these cells were undergoing frank degeneration. Evidence indicated that changes after 48 or 72 h were irreversible.     

Light and electron microscopic characteristics of signet-ring adenocarcinoma cells in serous effusions and their distinction from mesothelial cells

Signet-ring adenocarcinoma cells in serous fluids have been classically described as possessing vacuolated cytoplasm and eccentrically placed, crescent-shaped nuclei. We studied serous fluids from six patients that contained signet-ring adenocarcinoma cells by light microscopy; one case was also studied by transmission electron microscopy. We found that the adenocarcinoma cells were more often present in a non-signet-ring configuration. The typical crescent-shaped nucleus was rarely displayed in smears and may be seen only in the cell-block preparation. Special stains (PAS, mucicarmine and Diff-Quick) showed globular cytoplasmic positivity in signet-ring adenocarcinoma cells but not in mesothelial cells. Significant characteristic electron microscopic findings in the signet-ring adenocarcinoma cells included (1) cytoplasmic lumens or invaginations or both, (2) cytoplasmic protrusions and (3) mucin granules of various sizes and densities. Singly or in combination, all of the above features were located on one side of the nucleus, which suggests that signet-ring adenocarcinoma cells retain some degree of cellular polarity.     

Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine

Monospecific antisera to purified hepatic fatty acid-binding protein (hFABP) and gut fatty acid-binding protein (gFABP) have been used to localize these two proteins in the small intestine of fed rats at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 4-, 10-, 20-, 22-, and 60-day-old Sprague-Dawley rats. Both cryostat and paraffin sections were studied for the presence of hFABP or gFABP by the avidin-biotin immunoperoxidase method. Slides were graded blind for the intensity of staining. Despite the structural and immunological differences between these two proteins, we showed no major differences between their staining patterns or their staining intensity throughout the intestine during postnatal development. The staining for both fatty acid-binding proteins was cytoplasmic. No brush border staining was found. Staining was more intense in the proximal rather than distal intestine, in the villus rather than crypt cells, and in the apex rather than the base of intestinal cells. Shifts in staining patterns, and staining intensity occurring during development may be related to variations in dietary fat intake, rates of cell proliferation, intestinal anatomy, and mechanisms for fat absorption.     

Glucose turnover in compensated hepatic cirrhosis

Glucose turnover and recycling from glucose derived 3-carbon intermediates were examined in overnight fasted patients with compensated hepatic cirrhosis and in age- and weight-matched normal control subjects. Fasting blood concentrations of glucose, lactate and glycerol were similar in both groups but blood pyruvate (60 +/- 10 vs. 80 +/- mumol/l, P less than 0.05), blood alanine (0.23 +/- 0.02 vs 0.34 +/- 0.02 mmol/l, P less than 0.01) were decreased and serum insulin increased (19 [13-24]v 7 [4-11] mU/l, P less than 0.01) in cirrhotic subjects. Absolute glucose turnover, assessed by analysis of decay of [3H]-3-glucose specific activity was decreased in cirrhotic patients (8.1 +/- 0.6 v 12.1 +/- 0.7 mol/kg-1 min-1). Glucose "recycling", assessed by the difference between absolute glucose turnover and that given by [14C]-1-glucose data, was normal in cirrhotic patients suggesting that Cori cycle (glucose-lactate-glucose) activity was normal. These data support previous findings of decreased peripheral glucose utilisation and insulin resistance in cirrhotic patients.     

Lectins for electron microscopic distinction of eosinophils from other blood cells

Colloidal gold-labeled soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA), and Griffonia simplicifolia lectin (GS-1) were used for electron microscopic observation of blood cells. Colloidal gold-labeled SBA, HPA, and DBA showed marked deposition on eosinophil granules at all stages of maturation. Gold particles were not deposited on basophils, neutrophils, monocytes, lymphocytes, or other blood cells. Only a few colloidal gold-labeled GS-1 were deposited on eosinophil granules. Eosinophil granules are rich in N-acetyl-D-galactosamine compounds, and the colloidal gold-labeled SBA, HPA, and DBA are useful for electron microscopic detection of eosinophil granules.     

Light microscopic immunolocation of thrombospondin in human tissues

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas. Immunostaining was confined to the luminal portions of large blood vessels such as aorta. In large blood vessels that contained lesions of atherosclerosis, immunostaining was observed throughout the lesion area and was especially prominent surrounding some of the lesion cells. These results indicate that thrombospondin is located within the matrix of a variety of human tissues and supports the suggestion that this glycoprotein is an endogenous component of some extracellular matrices.     

Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning

1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.     

Light microscopic histochemistry on plastic sections

As compared with conventional paraffin, celloidin, and frozen sections, semithin plastic sections offer a superior quality of the light microscopic image in terms of better resolution, absence of distortion and shrinkage artifacts, and suitability for calcified tissues. Application of histochemical methods to such sections often encounters, however, serious difficulties resulting from a considerably reduced reactivity of plastic-embedded biological material. Factors involved include a poor penetration of reagents into plastic embedding media due to a steric or hydrophobic hindrance, as well as a blockade of the reactive chemical groups in the sample due to interactions with fixatives and plastics. Embedding in polar (hydrophilic) plastics, such as glycol methacrylate, permits carrying out a large number of histochemical reactions, including the demonstration of enzymatic activities, directly on sections, but is less suitable for combined light/electron microscopic studies because of an imperfect ultrastructural preservation of tissues. Embedding in nonpolar epoxy resins, particularly if combined with a double aldehyde-osmium fixation, results in a high quality ultrastructure but almost fully inhibits the histochemical reactivity of the embedded material. In order to restore this reactivity, i.e. to unmask chemical groups bound by the polymerized resin, semithin epoxy sections require the removal of the embedding matrix by alkoxides prior to the histochemical procedure. Additional steps are also often necessary: treatment of osmium-fixed sections with oxidative agents, e.g., hydrogen peroxide or periodate which reoxidize the bound osmium and remove it from tissue, and a controlled proteolytic digestion, especially useful in immunocytochemical studies, which probably cleaves the bonds between the primary aldehyde fixative, and the reactive sites. This article reviews histochemical methods which have been successfully applied to plastic-embedded material. Using polar methacrylates and/or nonpolar epoxy resins as embedding media, it has been possible to demonstrate proteins and aminoacid residues, carbohydrates, lipids, nucleic acids, biogenic amines, inorganic ions, and some enzymes, although the spectrum of methods found as suitable for plastic-embedded material is far narrower than that available for paraffin or frozen sections.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light microscopic demonstration of myoid material in nuclei.

During the development of configurational staining methods for proteins of the myosin-fibrin group, nuclei showed staining properties similar to those of myofibrils. This dye binding could be attributed to nuclear alpha-helical proteins. More recent chemical and electron microscopic studies demonstrated actomyosins in nuclei of various species. Possible roles of nuclear actomyosin in chromosome movements and condensation and in cell proliferation have been suggested. It seems therefore permissible to assume that the tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN method and similar technics visualize myosin in nuclei. Comparative studies of actomyosins from various sites indicated significant chemical an histochemical differences. It is therefore suggested that, in analogy to the different classes of collagens, there may be several subgroups of myosin which differ in their physico-chemical properties and sensitivity to fixation procedures and pathological conditions.     

Bleeding time in patients with hepatic cirrhosis.

OBJECTIVE--To determine the frequency of an abnormal bleeding time in patients with cirrhosis and to relate this to known factors that affect primary haemostasis and to the severity of liver disease. DESIGN--Prospective clinical and laboratory study in patients admitted for complications or investigations of liver disease. SETTING--Royal Free Hospital hepatobiliary and liver transplantation unit. SUBJECTS--100 Consecutive inpatients aged 17-74 with various forms of cirrhosis, including alcoholic, biliary, autoimmune, viral, and cryptogenic. At least 10 days had elapsed since any episodes of bleeding, resolution of sepsis, or alcohol intake. No patient was taking any drug known to affect primary haemostasis. MAIN OUTCOME MEASURES--Bleeding time as measured with the Simplate double blade template device. A bleeding time longer than 10 minutes was considered abnormal. Other measures were platelet count, prothrombin time, partial thromboplastin time, packed cell volume, and blood urea, serum bilirubin, and serum albumin concentrations, all measured on each subject at the same time by standard laboratory methods. RESULTS--A weak but significant correlation existed between the bleeding time and the platelet count (rs = 0.483; p less than 0.001). There were significantly lower platelet counts, longer prothrombin times, and higher blood urea and serum bilirubin concentrations in the 42 patients with bleeding times of 10 minutes or more compared with the 58 patients with bleeding times less than 10 minutes. Multiple linear regression analysis showed that the bilirubin concentration as well as the platelet count was independently correlated with the bleeding time. The combination of a platelet count greater than 80 x 10(9)/l and a prothrombin time less than 17 seconds (usually taken as safe limits for performing routine liver biopsy) did not predict a normal bleeding time. Ten of 39 patients fulfilling these criteria had a prolonged bleeding time. CONCLUSIONS--Prolonged bleeding time is common in patients with cirrhosis, even in those with prothrombin times and platelet counts within "safe limits" for invasive procedures. The severity of liver disease as assessed by the bilirubin concentration plays an important part in determining the bleeding time in cirrhosis. The bleeding time should be measured when assessing patients for invasive procedures who have a raised bilirubin concentration or poor hepatic function, even if the platelet count and prothrombin time are considered adequate.     

Three-dimensional image analysis of hepatic restructuring in liver cirrhosis

OBJECTIVE: To investigate the three-dimensional structure, including the angioarchitecture, of the cirrhotic liver and clarify morphogenesis of the cirrhotic nodule. STUDY DESIGN: The three-dimensional liver structure of nontumor areas in two partially hepatectomized cases of hepatitis C virus-positive liver cirrhosis with hepatocellular carcinoma was examined by computerized reconstruction from serial tissue sections. RESULTS: Our image analysis revealed the following: (1) The parenchyma consisted of two kinds of cirrhotic nodules. The first was the nodule centrifugally formed around the portal veins, and their flows drained into the hepatic veins inside and around the nodule. The second was the nodule derived from the first. The latter was divided into the former by bridging fibrosis-induced intranodular septation. (2) The stroma consisted of the newly formed fibrovascular tissue--i.e., the septum and intranodular inflow and outflow vascular systems and the preexisting one. CONCLUSION: Our computerized reconstruction suggested, from an angioarchitectural point of view, that the first and second kind of cirrhotic nodule might be named the stable and the unstable nodule, respectively, and that the first kind of cirrhotic nodule could be derived from the regenerative nodule appearing in the course of chronic hepatitis.     

Light and electron microscopic studies of crayfish hemocytes

Using light and electron microscopy, three hemocyte types are described in the hemolymph of the crayfish. The coagulocyte comprises 65% of the total hemocyte number and contains medium-sized cytoplasmic granules, abundant dilated rough endoplasmic reticulum, and a highly developed Golgi complex. It rapidly undergoes cytolysis in vitro and participates in coagulation by releasing the contents of its granules to the hemolymph. The granulocyte comprises 31% of the total hemocyte number and is capable of phagocytosis. It contains large, irregularly shaped cytoplasmic granules, a moderately developed Golgi complex, and moderate amounts of non-dilated rough endoplasmic reticulum. During coagulation in vitro, the cell attaches and spreads onto the substratum; this is followed by a slow intracellular granule breakdown and cytolysis. The amebocyte comprises 4% of the total hemocyte number and it is also capable of phagocytosis. It possesses small cytoplasmic granules, many vacuoles, a moderately developed Golgi complex, and large amounts of smooth endoplasmic reticulum. It is distinguished from the other two cell types by being stable and motile in vitro.     

Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections

A procedure is described for light microscopic immunocytochemical localization of catalase and three enzymes of peroxisomal lipid beta-oxidation: acyl-CoA oxidase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase in semi-thin sections of rat liver processed for routine electron microscopy. Satisfactory immunostaining required the removal of the epoxy resin with sodium ethoxide, controlled digestion of deplasticized sections with proteases and, in case of osmiumfixed tissue, bleaching with oxidants. Resin removal was essential for successful immunostaining, and protease treatment enhanced markedly the intensity of the reaction. This study shows that tissues processed for conventional ultrastructural studies can be used for postembedding immunocytochemical demonstration of various peroxisomal enzymes.     

Light and electron microscopic localization of silver in biological tissue

Summary A method is described that visualizes trace amounts of silver in frozen, paraffin and epon sections from biological tissue. After exposure to light, which ensures reduction of silver ions that are not bound to sulphide, histological sections from animals treated with silver compounds are exposed to a photographic developer containing silver ions. Tissue silver acts as a catalyst for the hydroquinone reduction of silver ions to metallic silver which then accumulates at the site of the trace deposit. Light and electron micrographs showing silver in different organs from albino rats treated with silver lactate are presented. Localization of silver in motor neurons of the spinal gray matter and pons indicates a transport of silver over the blood-brain barrier. Silver precipitates in fetal liver suggest that silver ions can penetrate the placental barrier.