Histologic organization of the myocardium of the rat during hypokinesia (stereologic study)

For 30 days Wistar rats have been kept under conditions of hypokinesia. Using stereological methods, the volume and surface density of muscle fibers, microcirculatory bed and connective tissue components have been defined, and volumetric and surface--volume ratios of parenchymal and stromal structures have been calculated. Intracellular stereological parameters of the cardiac myocytes have been studied. Three-dimensional parameters in the parenchymatous-stromal interrelations of the atrophying myocardium accompanied with a deep rearrangement of the intracellular architectonics have been determined, as well as certain destructive changes of the cardiac myocyte ultrastructures. Such a treatment as a decreased functional loading is demonstrated to be the cause of cardiomyocytic lesions.     

Ultrastructural changes in the components of the filtration barrier in the glomerular capillaries of a remaining kidney following its temporary ischemia

The components of the filtration barrier of the glomerular capillaries of the rat remaining kidney were studied ultrastructurally 3, 7, 14, 30, 60, 180 and 360 days after exposure to 30 minutes and to 1-2 hours of ischemia. Submicroscopic changes found in the glomerular capillaries and in compensation -adaptive processes occurring in the remaining kidney were ascertained to be dependent on the duration of ischemia and the time elapsed since recirculation in the kidney. After 30-minute ischemia experienced by the remaining kidney the structural alterations in the glomerular capillaries were not remarkable, disappearing 14 days following recirculation, with the emergence by that time of the signs of hyperplasia and hypertrophy of intracellular structures. After raising the time of temporary ischemia of the remaining kidney up to 60 min followed by recirculation, appreciable ultrastructural postischemic disorders were recorded in the components of the filtration barrier of the glomerular capillaries. In addition, the compensation-adaptive processes in the kidney remained suppressed for a longer period of time. All these disorders were particularly demonstrable as a result of 2-hour ischemia. It was also discovered that destructive processes dominated over reparative ones thereby leading to the animals' death at early times of experiment.     

Ultrastructural changes in the microcirculatory bed and filtration-reabsorption barrier of the kidney during temporary ischemia after unilateral nephrectomy

Ultrastructure of cellular elements of the microcirculatory bed and filtration-reabsorption barrier has been studied in 150 mature white rats, in which vascular fasciculus of the left kidney has been compressed for 30 min, 1-2 h with a subsequent restoration of the blood stream in the organ undergone ischemia on the 3rd, 7th, 14th, 30th, 60th, 180th, 360th days under conditions of the preliminarily right kidney nephrectomy. On the 3rd day after ischemia of the remained kidney for 30 min, structural components of the walls of the glomerular arterioles and those of the filtration-reabsorption barrier undergo certain ultrastructural changes, that with time elapsed (7, 14 days) gradually pass away, and amount of cells with hypertrophic processes increases. Ischemia for 1 h in the remained kidney with subsequent restoration of the blood stream on the 3rd, 7th days produces in the structures mentioned more pronounced destructive changes. During subsequent compensatory hypertrophy (the 30th, 60th days) of the remained kidney after its ischemia, in the microcirculatory bed elements and in the convoluted canal epitheliocytes intracellular regenerative and hyperplastic processes develop. However, ischemia for 2 h in the remained kidney produces severe destructive-necrotic phenomena in ultrastructure of the microcirculatory bed and of the filtration-reabsorption barrier.     

Arterial repair after microvascular anastomosis. Scanning and transmission electron microscopy study

In order to study the morphological aspects of endothelial regeneration and vascular wall reaction after microvascular anastomosis, rat femoral arteries were sectioned and successively sutured (end-to-end anastomosis) with microsurgical techniques. Control arteries and anastomosed vessels (recovered after 1, 4, 7, 14, 21, 30, 60, 120, 180 and 360 days) were studied by means of scanning (SEM) and transmission electron microscopy (TEM). The reendothelialization phenomena started after 7 days and were mainly evident at 21 days. Areas of subendothelial connective tissue with fibrin deposition remained exposed to the blood stream up to 21-30 days. Thrombus formations or post-anastomotic stenosis have been occasionally observed. Regenerating endothelium showed evident morphological differences from the control. These changes mainly consisted of shortened cell length, absence of pinocytotic vesicles, presence of cytoplasmic prolongations, and microvillous proliferations. The arterial wall showed subintimal thickening. The anastomotic site appeared completely covered by new endothelium after 30-60 days. Subintimal vascular wall changes (thickening of the media) as well as slight alterations of endothelial cells (shortened length, reduced number of pinocytotic vesicles) were evident in 60-day vessels. Lumen reduction, due to the protruding of endothelial-covered sutures, was occasionally observed in 60- to 120-day arteries. Endothelial cell morphology normalized after 60-120 days. However, thickening of the media and occasional lumen reduction were observed also after 180-360 days. Although the endothelial regeneration phenomena were clearly evident after 2 weeks, nevertheless the reestablishment of arterial wall took longer time.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of chronic alloxan- and streptozotocin-induced diabetes on isolated rat heart performance

Cardiac disease is a common secondary complication appearing in chronic diabetics. Isolated perfused working hearts obtained from both acute and chronic diabetic rats have also been shown to exhibit cardiac functional abnormalities when exposed to high work loads. We studied cardiac performance at various time points after induction of diabetes in rats to determine exactly when functional alterations appeared and whether these alterations progressed with the disease state. Female Wistar rats were made diabetic by a single i.v. injection of either alloxan (65 mg/kg) or streptozotocin (STZ 60 mg/kg). Cardiac performance was assessed at 7, 30, 100, 180, 240, and 360 days after induction of diabetes using the isolated perfused working heart technique. No changes were observed in the positive and negative dP/dt development at various atrial filling pressures in the diabetic hearts 7 days after treatment. Alloxan diabetic rat hearts exhibited depressed left ventricular pressure and positive and negative dP/dt development when perfused at high atrial filling pressures, at 30, 100, and 240 days after treatment. STZ diabetic rat hearts exhibited depressed cardiac performance at high atrial filling pressures, at 100, 180, and 360 days after treatment, but not at 30 days after treatment. Control hearts exhibited slight but significant depressions in cardiac function with age. These results suggest that cardiac functional alterations appear in diabetic rats about 30 days after induction and progress with the disease. These alterations may indicate the development of a cardiomyopathy.     

Immunolocalization of matricial components during the early stages of chick embryonic liver development

In the chick embryo, the first liver primordium is observed at the end of the second day of incubation. At 3 and 4 days, ultrastructural analysis of the primitive vascular spaces showed that the endothelial limiting plate was constituted by one or several cell layers. At the vascular pole of the hepatoblasts, mesenchymal cells and connective matrix, present as fibrillar and non fibrillar components, were closely associated. At 5 days, some vascular spaces were limited by a simple endothelial layer. The limiting plate was fenestrated and the connective matrix was reduced to rare collagen fibrils and fibers. Collagen types I, III, IV, procollagen type III, fibronectin and laminin were visualized in the perivascular spaces using immunoperoxidase labeling methods. These components were also detected in the endoplasmic reticulum of hepatoblastic, endothelial and mesenchymal cells. All these appeared to be involved in connective matrix synthesis. Comparing 4 and 5 days, we demonstrated that the number of cells showing intracellular labelling of matricial components dropped dramatically at 5 days, indicating a possible decrease of connective matrix synthesis. Quantification of parenchymal and vascular surfaces was carried out using a semi-automatic image analyzer on consecutive parasagittal sections chosen in the axial part of the embryonic liver. These measurements were performed in order to quantitate the vascular distribution pattern during early development of the liver. These combined immunomorphological studies and morphometrical analyses suggest that during embryogenesis of the liver the synthesis of connective matrix precedes and possibly initiates the vascular differentiation.     

Mechanism of the protective immunity against murine typhoid: persistence of salmonella L forms in the liver after immunization with live-cell vaccines

Abstract Live-cell vaccines of Salmonella typhimurium , either a sub-lethal dose of a wild-type (strain LT2) or a high dose of its two-heptose Rd₁ mutant (strain SL1004), induced acquired resistance to murine typhoid, which remained 180 days after immunozation. Growth of S. typhimurium as a bacillary form ceased between days 30 and 60 of immunization, but L forms of this bacterium colonized the liver (the mean number of L forms in the liver: 600 L-forming units) even at 180 days post-immunization. In contrast, a high inoculum of either a Ra mutant (strain TV148) of strain LT2 or S. schottmülleri 8006 sharing the same O antigenic components with those of S. typhimurium induced only a short-lived protection in proportion to the number of L forms in the liver, and the protective immunity was lost before day 180. However, there was no significant difference in the salmonella-specific T-cell responses among groups of immunized mice on day 180 of immunization. A lethal infection with strain LT2 in mice which had been immunized 75 days previously with living cells of strain SL1004 resulted in a rapid clearance of the challenge inoculum, together with a rapid elevation of anti- S. typhimurium antibody responses. Thus, the present data suggest that the long-lived immunity conferred upon live S. typhimurium vaccines is attributable to the colonization of this bacterium in the liver as L forms and the ability to colonize the liver as L forms is independent of the brain length of salmonella O-antigens.     

Embryonic development of the bovine pineal gland (Bos taurus) during prenatal life (30 to 135 days of gestation)

The ontogenesis of the pineal gland of 30 bovine embryos (Bos taurus) has been analysed from 30 until 135 days of gestation by means of optical microscopy and immunohistochemical techniques. For this study, the specimens were grouped into three stages in accordance with the most relevant histological characteristics: Stage 1 (30 to 64 days of prenatal development); Stage 2 (70 to 90 days) and Stage 3 (106 to 135 days). In the cow, it is from 30 days of gestation that the first glandular outline becomes differentiated from the diencephalic ependyma of the third ventricle. This differentiation includes the phenomena of proliferation and multiplication of the ependymal cells that form the epithelium of the pineal outline in development. At 82 days of intrauterine life, in the interior of the pineal parenchyma, we witnessed some incipient pseudoglandular structures that at 135 days were well differentiated. The pineal parenchyma displays a cytology made up of two cellular types of structurally distinct characteristics: pinealoblasts and interstitial cells. Both cellular types begin differentiation at 70 days of embryonic development, the pinealoblasts being greater in number than the interstitial cells. The glandular stroma is formed from the capsular, trabecular and the perivascular connective tissue, filling the interparenchymal space. A dense network of capillaries, which drive across the trabecular connective tissue towards the central glandular zone where their density increases and their calibre is reduced, complete the glandular structure. GFAP positive cells were observed in the embryonic pineal parenchyma in stage 3. At 135 days of gestation, NPY positive fibers entered the pineal gland through the pineal capsule occupying a perivascular localization. Morphological studies of this nature are vital for future use as parameters, indicative of the functional activity of the bovine pineal gland during embryonic development.     

Late effects of ionizing radiation on the microvascular networks in normal tissue

Damage to the microvascular networks constitutes one of the most important components of ionizing radiation damage to normal tissue. Previously, we have reported the early (3, 7 and 30 days postirradiation) effects of ionizing radiation on the structure and function of normal tissue microvascular networks. Here we report on the late effects of ionizing radiation on the structural and functional changes in microvascular networks in locally irradiated (single 10-Gy dose) hamster cremaster muscles observed 60, 120 and 180 days postirradiation; age-matched animals were used as controls. As in the previous study, intravital microscopy was used to measure structural and functional parameters in complete microvascular networks in vivo. A factorial design was used to examine the effects of radiation status, time postirradiation, and network vessel type on the structure and function of microvascular networks. Our results indicate that the progression of radiation-induced microvascular damage continues during the late times but that there is partial recovery from radiation damage within 6 months postirradiation. Red blood cell flux, red blood cell velocity, and capillary blood flow in irradiated networks at 180 days postirradiation were significantly greater than control levels. As at the early times, all vessel types were not damaged equally by radiation at every time.     

The effect of high energy electron irradiation on blood-brain barrier permeability to haloperidol and stobadin in rats

Summary The heads of rats were irradiated by 4 MeV electrons in doses 90, 180, and 360 Gy. The observed times of deaths ranged 120–600, 60–420, and 150–370 min after 90, 180, and 360 Gy, respectively. A dose dependent decrease of the brain uptake index of haloperidol was observed 1 and 3 h post radiation. On the other hand an increased brain uptake index was found for stobadin after head irradiation with doses of 180 and 360 Gy. Regional cerebral blood flow, blood pressure, and heart rate were not significantly altered in the period following irradiation with 180 Gy. The observed changes in blood-brain barrier (BBB) permeability seem to be the result of the damaged function of morphological structures forming the BBB rather than altered regional blood flow.     

The capacity for regeneration of the myocardium in rat fetuses

A reproducible model of heart injury in 16-days old rat fetuses has been developed. 30 days after the injury a 4-fold increase of the damaged area was observed. Decrease of reparative capacity of myocardium in fetuses as compared with the adults is due to the functional immaturity of the connective tissue cells.     

Ultrastructural organization of connective tissue microfibrils in the posterior chamber of the eye in vivo and in vitro

The ultrastructural organization of connective tissue microfibrils was studied in the mouse eye and also by means of in vitro experiments for reconstituting microfibrils. In the posterior chamber of the eye of the C57BL/6J mouse, 3 nm-wide ribbon-like double-tracked structures were present and were periodically associated on either side with 3.5 nm-wide particulate structures identified as pentosomes, the subunits of amyloid P component (AP). At certain sites, such composite structures were observed in various stages of helical winding, and in these helices, pentosomes were preferentially localized internally. In helices in the final stages of winding, the resulting rods appeared increasingly similar to those of microfibrils. In experiments in vitro, incubation of chondroitin sulfate proteoglycan (CSPG) in TRIS buffer, pH 7.4, at 35°C for 1 h produced random aggregates of 3 nm-wide double-tracked structures similar to those observed in the eye. Co-incubation of CSPG and AP resulted in the formation of rod-like structures arranged parallel to one another in approximately 50 nm-thick sheet-like layers. These rods were ultrastructurally similar to microfibrils and were made up of helically wound, 3 nm-wide double-tracked structures containing pentosomes within their core. The results of in vivo as well as in vitro experiments suggest the possibility that the connective tissue microfibril is composed of helically wound, CSPG-containing, 3 nm-wide double-tracked structures periodically associated with pentosomes which, as the helix becomes progressively tighter, fit with one another at the core of the helix to form successive 8.5 nm-wide disks of AP segments.     

Increased expression of the collagen internalization receptor uPARAP/Endo180 in the stroma of head and neck cancer.

Local growth, invasion, and metastasis of malignancies of the head and neck involve extensive degradation and remodeling of the underlying, collagen-rich connective tissue. Urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 is an endocytic receptor recently shown to play a critical role in the uptake and intracellular degradation of collagen by mesenchymal cells. As a step toward determining the putative function of uPARAP/Endo180 in head and neck cancer progression, we used immunohistochemistry to determine the expression of this collagen internalization receptor in 112 human squamous cell carcinomas and 19 normal or tumor-adjacent head and neck tissue samples from the tongue, gingiva, cheek, tonsils, palate, floor of mouth, larynx, maxillary sinus, upper jaw, nasopharynx/nasal cavity, and lymph nodes. Specificity of detection was verified by staining of serial sections with two different monoclonal antibodies against two non-overlapping epitopes on uPARAP/Endo180 and by the use of isotype-matched non-immune antibodies. uPARAP/Endo180 expression was observed in stromal fibroblast-like, vimentin-positive cells. Furthermore, expression of the collagen internalization receptor was increased in tumor stroma compared with tumor-adjacent connective tissue or normal submucosal connective tissue and was most prominent in poorly differentiated tumors. These data suggest that uPARAP/Endo180 participates in the connective tissue destruction during head and neck squamous cell carcinoma progression by mediating cellular uptake and lysosomal degradation of collagen.     

Ontogenetic Development of the Striatal [3H]Spiperone Binding: Regulation by Sodium and Guanine Nucleotide in Rats

Ontogenetic development of specific [3H]spiperone binding to crude synaptic membranes and its regulation by Na+ and GTP was investigated in the rat striatum. (d)-Butaclamol more effectively inhibited [3H]spiperone binding than (l)-butaclamol. The ratio of inhibitory activity of (d)- and (l)-butaclamol for [3H]spiperone binding was not different between 1-, 7-, and 70-day-old animals but eight- to ninefold lower at 18 days of gestation than during the postnatal period. A Scatchard plot of specific binding indicated the presence of two types of binding: low-affinity (KD = 1.51 nM) and high-affinity (KD = 0.09 nM) binding on day 70. Only one component (KD = 0.075 nM) was observed on days 1 and 7 and both types of binding were found on day 15. Bmax gradually increased with age and reached a peak on day 30, followed by a decline on days 70 and 360. Na+, 100 mM, significantly increased specific binding on days 1, 7, 15, and 70. GTP, 50 microM, completely reversed the Na+-induced decrease in IC50 of apomorphine on both days 15 and 70, but not on day 7. It is suggested that receptors could recognize ligand stereospecificity on day 1. The density in dopamine receptors in the striatum reaches a peak on day 30, followed by a decrease on days 70 and 360. In addition, regulation by Na+ and GTP in agonist binding to dopamine receptors seems to become functional between 1 and 2 weeks after birth.     

Caffeine targets TOR complex I and provides evidence for a regulatory link between the FRB and kinase domains of Tor1p

The target of rapamycin (TOR) kinase is an important regulator of growth in eukaryotic cells. In budding yeast, Tor1p and Tor2p function as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is specifically inhibited by the antibiotic rapamycin. Significant insight into TORC1 function has been obtained using rapamycin as a specific small molecule inhibitor of TOR activity. Here we show that caffeine acts as a distinct and novel small molecule inhibitor of TORC1: (i) deleting components specific to TORC1 but not TORC2 renders cells hypersensitive to caffeine; (ii) rapamycin and caffeine display remarkably similar effects on global gene expression; and (iii) mutations were isolated in Tor1p, a component specific to TORC1, that confers significant caffeine resistance both in vivo and in vitro. Strongest resistance requires two simultaneous mutations in TOR1, the first at either one of two highly conserved positions within the FRB (rapamycin binding) domain and a second at a highly conserved position within the ATP binding pocket of the kinase domain. Biochemical and genetic analyses of these mutant forms of Tor1p support a model wherein functional interactions between the FRB and kinase domains, as well as between the FRB domain and the TORC1 component Kog1p, regulate TOR activity as well as contribute to the mechanism of caffeine resistance.     

Effect of a constant magnetic field and gamma radiation on the hereditary structure of somatic cells. Effect of the combination of a constant magnetic field and ionizing radiation on human lymphocytes in vitro

Samples of human whole blood were exposed in CMF (field induction, 0.3 T) for 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300 or 360 min. 15 min following exposure, the samples were gamma-irradiated in a dose of 0.0516 C/kg (137Cs) at a dose rate of 1.95 A/kg. The following chromosome aberrations were scored: deletions dicentrics, rings, and symmetrical exchanges. Exposure of the blood in CMF for 15 to 360 min decreased radiation damage to cells as compared with unexposed irradiated samples. The extention of time from 15 to 180 min increases the effect the smallest amount of chromosome damages being scored at 150-180 min. A 2.8 - fold, 3 - fold and 3.5 - fold decrease was registered in the number of aberrant cells, deletions and dicentrics, respectively. With increasing time of exposure (240 min), the radiomodifying effect started decreasing, and with 300-360 min exposure it was the same as that observed at 15-45 min.     

Role of the arterial smooth muscle cell in the pathogenesis of atherosclerosis

The development of an atherosclerotic lesion is characterised by a proliferation of arterial smooth muscle cells and an accumulation of cholesterol, cholesteryl esters and connective tissue. The main connective tissue components of an atherosclerotic lesion, i.e. acidic glycosaminoglycans and collagen, are synthesized by the smooth muscle cells. Cholesterol is chiefly derived from plasma lipoproteins, but there is an enhanced intracellular esterification of cholesterol in the cells of the lesions. The important role of the arterial smooth muscle cell in the development of atherosclerotic lesions has resulted in cultures of these cells being used as experimental models to study the pathogenesis of atherosclerosis. Such studies have revealed many blood-derived and other substances affecting proliferation, as well as lipid and connective tissue metabolism of arterial smooth muscle cells. In this way certain risk factors for cardiovascular disease have turned out to be associated with the metabolic disturbances of atherogenesis at the cellular level. Studies with cultured arterial smooth muscle cells have also demonstrated other factors for example one derived from aggregating platelets that may significantly contribute to the development of atherosclerotic lesions. On the other hand, certain inherent features of the smooth muscle cells of the lesions, such as enhanced proliferation and synthesis of glycosaminoglycans, may also contribute to the pathological changes.     

Changes in germ cell population in young and adult female mice from two inbred strains: CBA/Kw and KE

Female mice from two inbred strains CBA/Kw and KE differ markedly in fertility. The gametes of females from KE strain are of poorer quality than those of CBA/Kw. We analyzed the number of oocytes per ovary in KE and CBA/Kw mice aged 5, 25, 90, 180 and 360 days. The ovaries were dissected and processed according to the routine histological methods. In case of five-day-old females we used a modified distributed point counting method while in order to examine the gonads of older females, the nucleoli counting method was applied. In general, we observed gradual decrease in germ cell number throughout the whole life of females from both strains. The noticeable wave of oocyte loss occurs between 5th and 25th days of life. The mice from KE inbred strain on day 25th (1650 +/- 322 vs. 1140 +/- 210) and 90(th) (1040 +/- 211 vs. 692 +/- 89) days have significantly (p<0.005) more germ cells than the females from CBA/Kw strain. In older females the differences were not statistically significant. Interestingly, CBA/Kw females were found to have more rapid loss of primordial follicles throughout their lives. This can explain their shortened reproductive lifespan which was observed earlier.