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The expression of tyrosine-hydroxylase (TH) gene was analysed in tissue sections of bovine adrenal glands, by in situ hybridization using a single-stranded cDNA probe. Tissue fixation and hybridization conditions were found that led to a specific and sensitive detection of TH.
Eighty-four male white leghorn chickens were killed by CO2 gas to determine the type, rate, and sequence of postmortem microscopic changes in the kidneys of dry and wet intact carcasses. They were held at 29 or 18 C with 50% relative humidity for different times postmortem. Microscopic postmortem changes in the different segments of the nephron underwent a different rate and sequence of cellular changes. Cellular changes occurred earlier at 29 C than at 18 C and earlier in chickens not wetted with detergent solution. The decrease in body temperature of wetted chickens over dry chickens was significant (P less than 0.05). The proximal convoluted tubule (PCT) underwent the earliest postmortem changes, followed by the distal convoluted tubule (DCT), collecting tubule (CT), medullary loop (ML), medullary collecting duct (MCD), and glomerulus. The PCT, DCT, and thin and thick segments of the ML underwent a sequential nuclear change of chromatin margination, progressive shrinkage, pyknosis, karyorrhexis, and karyolysis. Nuclei were pyknotic if cytoplasmic changes were severe. Primary karyorrhexis was the predominant feature of collecting tubules and ducts. As early as one hour after death, some PCT cells of all kidney sections were pyknotic, emphasizing that immediate tissue fixation was necessary for critical evaluation. By 9 and 18 hr postmortem, PCT of dry and wet chickens, respectively, held at 29 C had pyknotic and karyorrhectic nuclei with slight karyolysis and moderate to marked cytoplasmolysis that extended until 36 hr. At this time, DCT were hardly distinguishable because of loss of basophilia. Karyorrhectic nuclei were already evident in collecting tubules and ducts. At 48 hr postmortem, all tubular cells were non-nucleated with homogeneous, acidophilic cytoplasm. Basement membranes no longer stained with periodic acid-Schiff (PAS). Erythrocytes were pyknotic with unstained cytoplasm. Pyknotic glomeruli were first observed at 9 hr postmortem in dry chickens and 12 hr in wet chickens. Histologic appearance of dry chickens at 9 hr and wet chickens at 18 hr when held at 29 C was similar to that of dry chickens at 12 hr and wet chickens at 24 hr when held at 18 C, with minor differences in some tubular changes. At 18 C, pyknotic glomeruli appeared by 6 hr in dry chickens and 24 hr in wet chickens. Widespread bacterial invasion was noted at 72 hr in dry chickens and at 96 hr in wet chickens.(ABSTRACT TRUNCATED AT 400 WORDS)
It has been shown that between living and surviving receptors of a central and local origin there is similarity in a number of morphological, reactive and tinctorial properties as well as in their response to tissue fixation. At the same time these structures possess a number of specific properties making them different from one another.
It has been postulated that the selective accumulation of circulating desialylated cells in the mammalian liver results from the binding of desialylated glycoproteins on surfaces of the cells to asialoglycoprotein receptors in the liver. Since circulating cells in the liver are in contact predominantly with sinusoidal lining cells (Kupffer cells and endothelial cells), this postulate requires the presence of asialoglycoprotein receptors on the luminal surface of the sinusoidal lining cells. Whether the receptor is present on these cells, however, remains controversial. To clarify this issue, we used an indirect immunoelectron microscopic method to determine the distribution of the receptor on the surfaces of hepatic cells accessible to the circulation. F(ab')2 fragments of antireceptor antibodies were perfused in situ via the portal vein prior to tissue fixation. After perfusion fixation, sections were reacted with peroxidase-labeled antibodies to the antireceptor F(ab')2. The plasma membranes of Kupffer cells, endothelial cells, and fat-storing cells were devoid of the asialoglycoprotein receptor. In contrast, the receptor was associated with hepatocytes, where it was present diffusely on the sinusoidal plasma membranes and concentrated within coated pits. We conclude that it is unlikely that circulating desialylated cells bind to the asialoglycoprotein receptor in the liver unless a breach in the continuity of sinusoidal lining cells exists.
The pancreatic tissue damage was investigated histologically in rats kept on liquid diet containing 6% ethanol for 18 months. Rats kept on a liquid diet without ethanol served as controls. The daily ethanol intake was 1.2 g/100 g b.w. The daily intake of liquid food was 26 ml/100 g b.w. (26 cal/100 g b.w.) (108 Joule/100 g b.w.) without ethanol and about the same (24 ml/100 g b.w.) with ethanol. Mortality rate was 75% in the group fed with ethanol and 30% in the control group. Ethanol decreased both body weight and pancreatic weight but did not effect the relative pancreatic weight. In histological sections no difference in character, intensity or extent of the damage was observed between the ethanol fed rats and controls. In both groups there were protein plugs in the pancreatic ducts and ductules. The noncalcified intraductal protein plugs are thought to have been induced by the protein precipitant effect of the tissue fixative applied in histology.
The results obtained with some types of bioprostheses together with the present economical situation of the country pointed out that the manufacture of another type of bioprostheses was necessary. After a careful analysis of the results that had been reported in the literature with the diverse types of bioprostheses had been made, it was concluded that pericardial valves were the best choice for our specific requirements. The first problem that had to be solved was the identification of the adequate fraction of purified glutaraldehyde that would provide for adequate tissue fixation. This was determined using several types of biophysical and biochemical techniques. The next step was to find the adequate manufacturing techniques that would provide us with optimal function valves. This was achieved by testing the functional characteristics of these valves on a cardiac cycle simulator. The first group of 132 patients that received this type of valve includes 9 multiple valve replacements, 24 aortic, 98 mitral and 1 pulmonary valve replacements. All the surviving patients have shown a clear improvement and they all show adequate function of their bioprostheses. The function of the valves has not been involved in any of the patient deaths. Since there has not been any evidence of disfunction, these results show that the manufacturing techniques and the design of these bioprostheses is adequate. Naturally to long-term durability of these valves will have to wait before it can be valorated.
Early culture supernatants from hybridomas that were obtained through fusions of mouse myeloma cells with lymphocytes of melanoma-immunized mice were screened for their reactivity with a paraffin-embedded cell block of a melanoma cell line, using a biotin:avidin immunoperoxidase procedure. Eleven monoclonal antibodies were derived that define several new melanoma-associated antigens. The antigens include a neutral glycolipid, gangliosides, membrane-associated proteins, cytosolic proteins, and strongly secreted proteins. These antibodies, which detect antigens that withstand tissue fixation and embedding procedures, were tested for reactivity in fixed cell lines, as well as in melanoma biopsies. These antibodies may provide powerful tools in diagnostic studies of human malignant melanoma biopsy material.
The electrophysiological effects of intravenous bepridil were studied at doses of 2 mg/kg and 3 mg/kg. The drug was tested on 25 patients of both sexes who were undergoing electrophysiological investigation for other reasons (11 normal ECGs, 11 pathological ECGs, 3 WPW syndromes). Electrophysiological parameters before and after bepridil were compared. The following changes were noted: - the refractory period was significantly prolonged at all conducting levels; - the sinus cycle was significantly lengthened in a practically constant fashion (+9,2 p. 100); - conduction in the sinoatrial and atrioventricular nodes was significantly prolonged (+15 p. 100 and + 11 p. 100); - the Luciani-Wenckebach point was significantly decreased in the anterograde, and even more so in the retrograde, directions (-20 and -23 p. 100 respectively); - however, infrahisian conduction was not affected either in patients with normal or pathological ECGs. The drug was effective in blocking the accessory pathway in two of the three patients with the WPW syndrome. A study of drug plasma levels did not show a dose-effect relationship. The plasma levels were extremely variable from one subject to another, suggesting tissue fixation of the active part of the molecule. These results demonstrate the valuable electrophysiological effects of bepridil which, by its mode of action, is very similar to amiodarone.
The horizontal and vertical distributions of immunoglobulin (Ig) A cells were determined in canine intestines by immunoperoxidase assay on formalin-fixed, paraffin-embedded tissues. With this technique, excellent preservation of tissue morphology was obtained, and special tissue fixation and handling were not required. Immunoglobulin A cells were counted in 2-different ways at different magnifications. It was seen that (i) the duodenum usually had the highest cell counts at all levels of the lamina propria, (ii) IgA cells in the zone closest to the intestinal lumen became less numerous caudad from the duodenum to the colon, (iii) the zone closest to the submucosa (zone D) had the fewest IgA cells in the small intestines, and (iv) the IgA cell counts in zone D tended to be lowest in the jejunum and ileum and highest in the duodenum and colon.
IgA nephropathy, as Berger defined it, is characterized by mesangial deposits of IgA, which are easily visualized by immunofluorescence on kidney biopsies. The structure (mono- or dimeric) of these IgA has not been clearly defined so far. Fifteen renal biopsies were studied to find out whether these IgA are serum monomers, or are polymers from a different origin. This was done by tissue fixation in vitro of free secretory component, which was then visualized by immunofluorescence (IF). In all 15 cases, the IgA deposits were shown to lack bound secretory component, but were able to bind, specifically, with the free secretory component. The presence of J chain in these deposits was also evidenced by indirect IF. These findings favour the hypothesis that these immunoglobulins are polymeric.
The technique of in situ hybridization (ISH) is presented with special reference to its use in the practice of pathology. Methodological and theoretical aspects of ISH are first considered with regard to tissue fixation, probe selection and preparation, various types of labeling reactions and aspects of hybridization conditions as they relate to hybridization protocols. This is followed by a discussion about the interpretation of the results of ISH with regard to sensitivity and specificity and the importance of, and various types of, controls. Lastly, specific applications of ISH in pathology are discussed, concluding with possible future applications for ISH in research and diagnosis.
Bromodeoxyuridine (BrdUrd), a thymidine analogue incorporated into DNA, can be quantified by fluorescent or chromophoric quenching of dyes bound to DNA or with antibodies to BrdUrd. These technologies have been used since the 1970s as tools for measuring DNA synthesis in isolated chromosomes and in cells and tissues. This paper is Part I of a three-part comprehensive review of the literature over the last 20 years (to the end of 1993) describing the histochemical methods for measuring BrdUrd in cells and tissues. Fixation, denaturation and staining procedures are compared for quantifying BrdUrd for microscopy and flow cytometry. Non-immunochemical methods related to the quenching of fluorescent DNA stains by BrdUrd are also described. Methods are described for the comparative assay of cell kinetic parameters by tritiated thymidine and bromodeoxyuridine. The multivariate BrdUrd/DNA assay of Ts and Tc, and a comparison of recent methods based on the single biopsy bivariate analysis of Tpot, is presented. Recent developments in the use of double halopyrimidine label to determine kinetic parameters are also reviewed.
In an experimental study, endothelial cell seeding on glutaraldehyde-fixed and detoxified bioprosthetic tissue, suitable for valve fabrication, was investigated in vitro. These findings were compared to spontaneous endothelial cell ingrowth on vascular grafts fabricated from the same materials. Special consideration was given to the quality of cell attachment with regards to improved shear stress resistance in the endothelial layer covering the bioprosthetic surface. On glutaraldehyde detoxified bovine pericardium, in vitro endothelial cell seeding resulted in uninhibited cell proliferation, but the cells were loosely bound to the underlying tissue. In vivo, endothelial cells grew spontaneously over the surface of vascular implants in direct contact with the bioprosthetic material. In contrast to standard fixed bovine pericardium, a significant decrease in thrombotic appositions could be observed. Cells exhibited intensive production of extracellular matrix, which renders the method of spontaneous in vivo cell ingrowth as the most promising approach for further research.
Reinecke-salt (ammonium tetracyanato diamine chromate) is known as amine precipitating reagens in applied chemistry. Its amine precipitates can be demonstrated in electron micrographes. The amine reineckates are unable to diffuse and difficult to dissolve. Depending from that they are localized in the cells. From our findings received in the boundary layer between adrenal cortex and adrenal medulla, we conclude that there are no non-specific precipitations with other cell components caused by Reinecke-salt. It could be determined that the catecholamines are accumulated in the cells of the adrenal medulla in granular shape in a typical manner. In the adrenal medulla cells of Acomys cahirinus first investigated by us we found amine containing vesicles with a diameter of 200 to 250 nm. In the neighbouring cells of the adrenal cortex no precipitates were visable. In the adrenal cortex we noticed the mitochondria from the tubulus typ specific for this region. The assumption that Reinecke-salt is a specific amine precipitating substance able of forming localized amin precipitates was confirmed by these investigations. So the Reineck-salt precipitation of biogenic amines is a new method beside the known ones for the ultrahistochemical amine demonstration. In our method the precipitation of amines by Reinecke-salt is the first step followed later by the tissue fixation with glutaraldehyde.
The anthracycline antibiotics constitute a major series of anti-cancer drugs, the best known and most widely used being doxorubicin. Among hundreds of analogues, only a few have reached routine clinical use. Their main metabolic feature is the reduction of a ketone group to an hydroxyl group, giving an -ol derivative generally less active than the parent compound. Anthracyclines are characterized by a rapid distribution phase and a slow elimination phase. The successive half-lives of doxorubicin in plasma are about 5 minutes, 1 hour and 30 hours. Its total plasma clearance is about 30 l/hr/m2, and its total volume of distribution at steady state is approximately 15 l/kg. Anthracyclines are excreted mostly through bile, and special care must be taken with their use in patients with hepatic dysfunction. The new anthracyclines of clinical interest in solid tumours (epirubicin, pirarubicin) are more lipophilic than doxorubicin and have a higher volume of distribution and an increased total plasma clearance. Idarubicin is active in leukaemia rather than against solid tumours, and an oral form is available. Because of their high tissue fixation, these new anthracyclines are of particular interest for locoregional therapy, especially through the hepatic artery. Myelosuppression is the dose-limiting toxicity of anthracyclines and is related to drug exposure, so that pharmacokinetic-pharmacodynamic relationships have been clearly established for these drugs. A new subfamily, characterized by a morpholino group, presents very original features such as direct covalent linking to DNA after cytochrome P450 activation. These molecules are active at 100-fold lower concentrations than the conventional anthracyclines and are currently in clinical trials.
PURPOSE: To examine the in vitro properties of gap junctions on the isolated rabbit ciliary epithelium. METHODS: Intracellular potential was measured and lucifer yellow (5% in 1 M LiCl) was iontophoretically injected into a ciliary epithelial cell. Fixated tissue was examined with a laser confocal microscope. RESULTS: Dye spread was observed throughout both layers (horizontally and vertically) of the ciliary epithelium adjacent to the injected cell that was more intensely labeled. Dye reflux did not occur at the site of microimpalement. Microiontophoretic dye injection in a bathing solution with a high Ca2+ (10 mM) or an acidic pH (6.3) completely inhibited cell-cell dye coupling in the rabbit ciliary epithelial cells. CONCLUSIONS: Laser confocal microscopy of intracellularly injected lucifer yellow dye demonstrates the physiologic presence of gap junctions between both ciliary epithelial layers. Extracellular acidosis and high concentration of extracellular Ca2+ cause loss of cell-cell coupling in the rabbit ciliary epithelium.
Ninety fluid specimens (30 each of urine, ascitic and pleural fluid) were studied by preparing three comparable smears. One was air dried for Giemsa stain, one wet fixed in 95% ethanol and one dried on a hot plate at 37 degrees C, rehydrated in normal saline for 30 seconds and fixed in ethanol. The latter two were stained with Papanicolaou stain, and a comparison was made of the (1) retention of red blood cells, (2) retention of epithelial or mesothelial cells, and (3) cytologic preservation. The Giemsa-stained smear was used as a control for (1) and (2). Complete lysis of red blood cells was noted in the rehydration smears as compared with 70% red blood cell retention in the wet-fixed smears (P < .01). The rehydration smears retained 78% urothelial cells as compared with 55% in the wet-fixed smears (P < .01). For ascitic and pleural fluid the cell retention did not differ significantly. The wet-fixed smears scored better for overall cytologic preservation, but the difference was not significant. The rehydration smears showed a decrease in the chromaticity of staining, more flattened cell clusters and slight cell enlargement. The rehydration method was beneficial for urine and blood-stained body cavity fluids.
Mac-2, a 30-35-kDa galactose-binding protein, is synthesized at similar levels in murine peritoneal exudate macrophages whether recruited in response to an intraperitoneal pathogen Mycobacterium microti, to sterile inflammatory stimuli such as thioglycollate broth, or to concanavalin A. In elicited or activated macrophages up to 30% of Mac-2 is constitutively secreted, and secretion is stimulated markedly by calcium ionophore A23187. Only thioglycollate-elicited macrophages express cell surface Mac-2, and binding is mostly (> 80%) a result of affinity for cell surface carbohydrate structures. Mac-2 surface expression is markedly reduced upon further activation of thioglycollate-elicited macrophages with bacterial lipopolysaccharide in vitro. Polylactosamine structures are present on all macrophage populations examined as determined by binding of Lycopersicon esculentum lectin, whereas alpha-galactosyl residues detected by Griffonia simplicifolia isolectin B4 are expressed only on the thioglycollate-elicited macrophages, indicating that these residues are the major determinants responsible for Mac-2 surface expression. Chemical cross-linking experiments have identified binding of endogenous cell-surface Mac-2 to three glycoproteins of molecular masses of 92, 125, and 180 kDa containing alpha-galactosyl and polylactosamine structures on thioglycollate-elicited macrophages. The restricted cell surface distribution of Mac-2 on thioglycollate-elicited peritoneal macrophages, a population of recently recruited monocytes, suggests a role(s) in early events of macrophage infiltration and tissue fixation such as extravasion and cell-matrix interactions.