Principles of organization of tissue mosaics of simple squamous epithelia

With the aim to study general principles on organization of tissue mosaics of various types of simple squamous epithelii, a quantitative topological analysis has been performed in the frog mesothelial mosaics, in the posterior epithelium of the rabbit cornea and in the rat aortal endothelium after Lewis method. To all the types of simple squamous epithelii studied the histological law of Lewis can be applied. As demonstrates the qualitative analysis of the mosaics, the mesothelium and the posterior epithelium of the cornea are typical isotropic mosaics, while the mosaic of the aortal endothelium is nearer to the polystichous one. The possibilities of qualitative and quantitative topological analysis of the biological mosaics after Lewis method for studying their organization, degree of regularity, histogenesis and proliferative properties are discussed.     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

Ultrastructure of the inner surface of the aorta of mature and old animals

In the experiments, performed on 12 white rats and 8 rabbits, by means of scanning electron microscopy of the native preparations and in a number of cases with use of silver nitrate impregnation, the internal surface structure has been studied in the aortal membrane of mature and old animals. At ageing the integrity and continuity of the endothelial monolayer is preserved, on the surface local intimal pits, craters and microdefects appear, adhesiveness of endothelium to leucocytes increases. Orientation of the intimal folds is disturbed. The type of the senescent remodeling in the endothelial layer revealed predisposes to development of atherosclerosis.     

Adaptation to short-term stress exposure increases the activity ATP-sensitive potassium channels in the smooth muscle cells of coronary blood vessels

The study was aimed at the effect of prior adaptation to short-term stress exposure on changes in K(ATP)-channel activity induced by severe stress and the dependence of the changes on the state of endothelium which plays important role in autoregulation of the coronary flow and myocardial contractility. Experiments were conducted on isolation hearts of female rats. At the first step of experiment, the heart was perfused by Krebs-Henseleit solution; at the second step, the heart was perfused with the same solution in which glibenclamide (1 microM), glibenclamide with saponin or N(omega)-nitro-L-arginine (60 microM) methyl ether was added. During the experiment, the perfusion pressure was stepwise elevated from 40 to 120 mm Hg with 20 mm intervals (coronary autoregulation). Adaptation to short-term stress prevented development of stress-specific myocardial hyperperfusion (increased volumetric velocity of coronary flow against the background of decreased myocardial contractility) and the reduction of coronary dilation reserve. In coronary vessels of adapted rats, as distinct from control rats, basal glibenclamide-sensitive functional activity of K(ATP)-channels depended on presence and functional activity of endotheliocytes; it was reduced in presence of endothelium and increased after de-endothelization or NO synthase inhibition. In all experimental groups, the increase in glibenclamide-sensitive functional activity of K(ATP)-channels induced by NO synthase inhibition more than twice as great as after the endothelium denudation. In adapted animals, stress did not decrease the functional activity of K(ATP)-channels and their activity slightly depended on presence of endotheliocytes. In addition, the elevation of their functional activity characteristic of adaptation and evident after endothelium removal has vanished. Therefore adaptation to short-term stress exposure is associated with a potential increase in basal activity of K(ATP)-channels which enhances the potency of vascular dilation system and may apparently reduce the risk of high vascular tone when such important local regulatory system as the NO system is damaged.     

Mitotic activity of pial microvascular endothelium rat in postnatal ontogenesis

Mitotic activity of endothelium in pial microvessels has been studied with the aid of intra-vessel autoradiography in 4-, 12- and 30-day old rats using these parameters: number of labeled nuclei per vessel or per 100 mcm of its length, or per 1 mm2 endothelial surface area, as well as number of vessels with labeled endothelium cells. The first parameter was independent of vessel diameter. The other parameters had highest value in the pericapillary vessels in all rats. These values decrease with rat age. The highest rate of brain growth was revealed after reaching the greatest mitotic endothelium activity in pial bed.     

The morphology and histochemistry of adult rats neurocytes after BCNU administration

Studies were performed on adult rats of Wistar strain given four 7-days-spaced intraperitoneal doses of BCNU with single dose resembling to used in clinical practice. The animals were sacrificed at the seventh day after the last dose of the drug. Morphological alterations were evaluated in H + E or cresyl violet stained sections. In frozen microtome sections histoenzymatic reactions were performed to detect enzymatic activity of some phosphatases and esterases. Karyo- and cytophotometric measurements of pyramidal cell nuclei in frontal and parietal cortex and of motor neurons in trigeminal nerve nucleus were performed in sections subjected to Feulgen reaction, using automatic microscopic image analyzer "Morphoquant" (VEB Carl Zeiss, Jena). The performed studies showed that administration of multiple therapeutic doses of BCNU lead to primary injury of vascular wall as oedema and proliferation of endothelium and small perivascular haemorrhages. The cytostatic drug induced a decrease in NsE and AlkP enzymatic activities, increased activity of AChE, ChE, AcP and ATPase and topographically variable changes in intensity of TPPase enzymatic reaction. Several karyo- and cytophotometric alterations were observed also in neurocyte cell nuclei which became elongated and acquired a more round shape. This was associated with a decrease in relative DNA content, loosening of nuclear chromatin structure and with shifting chromatin lumps toward periphery of cell nuclei.     

Functional interrelationships of the sympathetic nervous system and the hypothalamo-hypophyseo-adrenal complex]

Adrenal and hypothalamic structure has been studied in mice at functional inhibition of the sympathetic region of the vegetative nerve system by means of antibodies against the nerve growth factor. Routine histological, histochemical, morphometric, electron microscopic and radioautographic methods have been applied. In the experiment the hypothalamic region is in the state of a continuous functional stress which is evident from enlargement of neurons and their nuclei in the macrocellular neurosecretory nuclei of the hypothalamus. The method of radioautography proves increasing activity in synthetic processes taking place in supraoptic, paraventricular and ventromedial nuclei of the hypothalamus and in the medial habenular nucleus of the epithalamus. In 30-day-old mice certain structural changes have been revealed in the adrenals demonstrating an increasing activity in their cortex: blood vessels are dilated, cholesterine, lipid and ascorbic acid granules are not evenly distributed, enzymatic activity in cytoplasm of adrenocorticocytes is increased. Ultramicroscopic destructive and regenerative alterations have no definite zonal specificity, nevertheless they are more pronounced in the fascicular zone. Radioautographic and morphometric investigations demonstrate an increasing functional activity in glomerular and fascicular adrenal zones. Taking into account morphologic demonstration of functional stress in hypophysial adenocytes in the same animals (Molostov O. K., 1974), it is possible to conclude that lesions in the sympathetic system center result in adaptive reaction of the hypothalamo-hypophysial system which, in its turn, produces reactions in the adrenals and in the vascular complex. This interconnection is accompanied by the reaction depending on breaking off direct sympathetic effects in the adrenals and vessels.     

Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation

Granulocytes are large, stiff viscoelastic cells that adhere naturally to the vascular endothelium. On their passage through the capillary network they have to be deformed, and recent evidence indicates that they may impose a significant hemodynamic resistance. The entry time of granulocytes into capillaries is about three orders of magnitude longer than that for red cells. Inside the capillary the granulocytes move with a lower velocity than red cells. Under conditions when the capillary perfusion pressure is reduced and/or elevated levels of inflammatory products are present that increase the adhesion stress to the endothelium, granulocytes may become stuck in the capillary. In such a situation, the granulocytes form a large contact area with the capillary endothelium, they obstruct the lumen, and they may initiate tissue injury. After the restoration of the perfusion pressure the granulocytes may not be removed from the capillary owing to the adhesion to the endothelium. Capillary plugging by granulocytes appears to be the mechanism responsible for the no-reflow phenomenon, and together with oxygen free radical formation and lysosomal enzyme activity may constitute the origin for ischemic injury as well as other microvascular occlusive diseases.     

Similarity of the carbohydrate moieties of fibronectins derived from blood plasma and synthesised by cultured endothelial cells

Plasma and cellular fibronectins are reported to be very similar but not identical in chemical structure. We have compared bovine plasma fibronectin with fibronectin secreted by bovine aortal endothelial cells in culture. Techniques were chosen to highlight likely structural differences, particularly in the carbohydrate moieties. Both fibronectins were wholly reactive to monospecific antiserum and behaved identically in two-dimensional gel electrophoresis. The oligosaccharide chains were identical in proportion and degree of sialylation by anion-exchange HPLC. Fractionation of the glycopeptides on immobilised lectins and serotonin showed that both fibronectins contained (i)_predominantly biantennary oligosaccharides, (ii) exclusively N-acetylneuraminic acid residues in a non-clustered array and (iii) no L-fucose residues. The overriding structural similarities support the proposal that the endothelium is a site of synthesis of plasma fibronectin in vivo.     

Stress-induced reduction of gastric acid in the rat is not associated with increased tissue somatostatin

In zero, mildly and severely stressed rats, gastric acid secretion, aortal and portal venous gastrin, venous glucagon and somatostatin in gastric, duodenal mucosa and in pancreas were examined. Serum gastrin and gastric acid secretion are reduced markedly by both kinds of stress, whereas plasma glucagon rises steadily with stress. As somatostatin in the tissues of stressed rats is not different from unstressed controls, gastrin and gastric acid reduction may not be attributed to an endocrine or paracrine action of somatostatin.     

Effect of progesterone and estrogens on the state of the hypothalamo-hypophyseal neurosecretory system in pregnancy and labor

Morphofunctional state of the hypothalamo-hypophyseal neurosecretory system was studied in rats at late stages of pregnancy. No special differences both in function of the supraoptical and praventricular nuclei, and in the hypophysis of intact and pregnant rats at administration of progesterone were noted. When estrogenes were administered to pregnant animals, a sharp increase in functioning of the supraoptic and especially of the paraventricular nuclei was noted. The greatest activity of the supraoptic nucleus was observed at delivery that could be resulted from a stress reaction to pain.     

Arteriovenous difference in antithrombin III activity and the antiaggregation properties of the vessel wall

The paper is concerned with the data on the arteriovenous difference in antithrombin III activity and antiaggregation properties of the aortal and inferior vena cava walls in Wistar rats. A correlation between the changes identified and different thromboresistance of the arteries and veins is suggested.     

Role of the hypothalamus in inhibiting the hypophysis-adrenal cortex system through the feedback mechanism

Chronic experiments were made on intact rabbits and rabbits with destroyed paraventricular and ventromedial nuclei of the hypothalamus to explore the hydrocortisone-induced inhibition of the stressor response of the pituitary-adrenocortical system. Intravenous injection of hydrocortisone in a dose of 100 micrograms/kg 5 minutes before immobilization stress led to inhibition of corticosteroid elevation induced by immobilization of the animals. Inhibition of the stressor reaction was maximal in intact animals, less in rabbits with destroyed ventromedial nuclei, and further less in animals with destroyed paraventricular nuclei. The paraventricular and ventromedial nuclei of the hypothalamus are necessary for inhibition of the pituitary-adrenocortical system by the feedback mechanisms.     

Effects of 1-methylnicotinamide and its metabolite N-methyl-2-pyridone-5-carboxamide on streptozotocin-induced toxicity in murine insulinoma MIN6 cell line

1-methylnicotinamide (MNA) is a primary metabolite of nicotinamide. In recent years several activities of MNA have been described, such as anti-inflammatory activity in skin diseases, induction of prostacyclin synthesis via COX-2, aortal endothelium protection in diabetes and hypertriglyceridaemia and increased survival rate of diabetic rats. 1-methylnicotinamide was also suggested to protect pancreatic cells from streptozotocin in vivo. Streptozotocin toxicity is known to be mediated by poly-ADP-ribose polymerase. Nicotinamide and its derivatives have been shown to ameliorate poly-ADP-ribose polymerase-dependent nucleotide pool reduction. We aimed to verify if 1-methylnicotinamide and its metabolite, N-methyl-2-pyridone-5-carboxamide, can protect insulinoma cells from streptozotocin-induced toxicity. We found that N-methyl-2-pyridone-5-carboxamide, but not 1-methylnicotinamide, restores the pool of ATP and NAD+ in streptozotocin-treated cells, but neither compound improved the cell viability. We conclude that inhibition of poly-ADP-ribose polymerase-dependent nucleotide pool reduction may not be sufficient to protect cells from streptozotocin toxicity.     

Roles of fluid shear stress in physiological regulation of vascular structure and function

The effects of fluid shear stress on the function and structure of the vascular system are outlined, based on the findings obtained in our laboratory or of our colleagues. First, it is pointed out that the adaptive response of the vascular wall to flow changes which we observed in the canine carotid artery shunted with the jugular vein altering the internal diameter to keep the wall shear stress constant, can attain the optimum vascular branching structure as predicted in the minimum work model by Murray. Electronmicroscopic studies of similarly shunted arteries revealing various morphological changes in the endothelial cells have suggested that the shear stress initially affects the endothelium. The in vitro experiments using cultured endothelial cells as well have exhibited that the mitotic activity of the cells significantly increases by applying fluid shear stress. From these findings, it is concluded that the adaptive response of the endothelium to the fluid shear stress is an inherent and key process locally regulating the vascular system to be in the most functional state.     

Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3

Inhibitory effect of 1,25dihydroxycholecalciferol (1,25D₃ = calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D—1,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.     

On the histoenzymology of the human stomach.

By means of histochemical methods, the stomach in the man was studied for some oxidoreducing enzymes and hydrolases. The surface epithelium displays in the fundic area a mean activity which grows intense in the pyloric area. The parietal cells show a very high mitochondrial dehydrogenase activity, while the chief ones display a moderate oxidative activity. The pyloric glands exhibit a positive reaction for oxidoreducing enzymes with great differences of intensity from one cell to another. Around each principal and pyloric gland one can see an intense ATP-ase reaction in the endothelium of vessels and a very intense acid phosphate activity in the histiocytes. The submucosa, muscularis externa and serosa are very active for oxidoreduction and ATP-ase.     

Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3

Inhibitory effect of 1α,25dihydroxycholecalciferol (1,25D₃ = calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D—1α,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.     

Proliferative activity of blood microvascular endothelium of intestinal mesentery during rat postnatal ontogenesis

The study of mitotic activity of mesenteric microvascular endothelium cells (EC) in 4-, 12-, and 30-day old rats has been carried out using following parameters: number of labeled nuclei per vessel, or per 100 microm of vascular length, or per 1 mm2 of endothelial surface area, as well as shares of labeled EC and of vessels with labeled EC, have been estimated. The highest density of labeled nuclei was revealed in the pericapillary vessels in all rats. Its values were significantly higher in 12-day-old rats and were the lowest in 30-day-old ones.     

Participation of ATP-sensitive potassium channel in autoregulation of coronary blood flow under the condition of limited motor activity

The aim of study involved detection of the effect of the K(ATP)-channel blocker glibenclamide on autoregulation of coronary flow, the expression of reactive hyperemia, the value of coronary dilatation reserve, and the myocardial contractile function in isolated rat hearts after a 6-hour immobilization stress. The experiments have been performed on 64 isolated rat hearts (female): into the cavity of left ventricle, a latex balloon connected with electromanometer has been introduced. Every experiment consisted of 2 stages. The heart has been perfused by Krebs-Henseleite solution in the first stage, and in the second stage--by the same solution with glibenclamide (1 mkM) or its combination with verapamile (10(-)6 M) or saponin (44 mcg/ml of coronary flow within 2 minutes) added to it. During the experiment, the perfusion pressure has been elevated step by step from 40 to 120 mm Hg with 20 mm Hg steps (coronary autoregulation). In rats after immobilization, the glibenclamide effect on cardiac vessel tone and expression of maximal hyperemic coronary flow (in contrast to its influence on myocardial contractile function) is lower than in control and depends on endotheliocyte presence which suggests an important role of endothelium in maintaining cardiac vessel smooth cell activity of K(ATP)-channels inhibited under the stress condition. After immobilization stress, the role of endothelium in the reactive hyperemia origin was enhanced, that of the K(ATP)-channels was reduced. The general activity of both mechanisms of tone regulation of cardiac vessels remains the same as in control. This suggests that the K(ATP)-channels as nitric monoxide and eicozanoids are the local system of myogenic tone regulation of the rat cardiac vessels; that the rat immobilization inhibits the activity KATp-channel's smooth cells of coronary vessels and creates a marked dependence of their activity on endothelium presence.