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PubMed1990

Dynamics of tracer distribution in radiation induced brain oedema in rats.

The dynamic behaviour of the distribution of Evans blue (EB), sodium fluorescein (SF), Lucifer yellow (LY) and horseradish peroxidase (HRP) was studied using standard light- and fluorescence microscopy following ultraviolet radiation induced brain oedema in the rat model. The cerebral cortex was irradiated after craniotomy (2 x 2 mm) under anaesthesia. The tracers were injected (iv) 30 min prior to radiation. Animals were perfused with glutaraldehyde through the heart at different survival periods ranging from 30 min to 24 h post irradiation. The results showed a remarkable difference in distribution and spread of these tracers in the oedematous brain following radiation. The extravasation of EB was evident in ipsilateral cortex 6 h after radiation which extended to the contralateral side at the end of the 24 h survival period. The HRP reaction product was seen in the necrotic area 3 h after radiation which further extended to the underlying white matter at 24 h survival. The LY stained the ipsilateral micronecrotic area 6 h after radiation, whereas a non-specific diffuse fluorescence of SF was noted at this time period. These results point out a specific selectivity of tracer distribution in oedematous brain following ultraviolet radiation.

Animals↗
PubMed1996

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: effect of tumor necrosis factor-alpha.

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor-alpha (TNF-alpha) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF-alpha inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF-alpha at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-alpha, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF-alpha inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.

Calcification, Physiologic↗
PubMed1986

Canine cortical bone autograft remodeling in two simultaneous skeletal sites.

The morphological and physical aspects of cortical bone autografts implanted in dogs for 1-9 months in two differently located skeletal defects are reported with a twofold aim: to provide a reference system for further comparison with various allografts and to delineate a general pattern of cortical bone graft healing. A 3-cm osteoperiosteal gap was created in the diaphyseal segment of the ulna and fibula of mature dogs. The grafts, freed from periosteum and bone marrow, were then inverted and replaced for the autografts in the left limb bone without internal fixation or external splints. On the right side, different allografts were tested. A group of three animals also had an unfilled segmental resection on the right as control. Dogs were observed for 1, 2, 3, 6, and 9 months and were able to bear weight within 3 days. Twenty-eight ulnae and 27 fibulae were available for this autograft study. Fluorochromes were injected at mid-term and at the end of the observation. All the grafts were assessed morphologically by cross-section microradiographs and ultraviolet light microscopy, and a morphometric analysis for porosity and fluorescence was done. To evaluate the physical aspects of graft healing, the recovered ulnar autografts, when available, were submitted to photon absorptiometry and to torsional loading. Morphologically, resorption was found to invade the cortical bone graft transversely through radial tunnels, and in addition to the host-bone-graft junction, the entire transplant surface provided another way for revascularization. The highest porosity level was achieved 2 months after surgery for both ulna and fibula, while new bone formation, as assessed by fluorochromes, was most important at 3 months. At 9 months, porosity remained above the normal range as determined in a set of five nongrafted dogs. While the lack of correlation for porosity between the two grafts suggests that local factors are more important in graft resorption, the observed correlation for fluorescence indicates that new bone deposition is more dependent upon skeletal metabolic activity. Within each graft, porosity and new bone formation were not well correlated. In the ulna, the bone mineral content (BMC) reflected the graft volumetric variations during the remodeling, with the lowest mean value at 3 months. For each graft, BMC was well correlated with the torsional stiffness. When torsionally loaded, the maximal tangential shear stress at failure of the graft was negatively related to its cortical porosity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Familial disorder of the central and peripheral nervous systems with particular cytoplasmic lamellated inclusions in peripheral nerves, muscle satellite cells, and blood capillaries.

Four siblings of a family of 11 were afflicted with a predominant parkinsonian syndrome, pyramidal signs, intellectual deterioration, and peripheral neuropathy. Symptoms were noticed first when they were aged 8 years; the condition was slowly progressive. All presented similar clinical features of varying severity. Nerve and muscle biopsies of two patients exhibited inclusions of concentric lamellae 2 nm thick with a periodicity of 3.6 nm, in the cytoplasm of perineurial and Schwann cells, endothelial cells and pericytes of blood capillaries, and muscle satellite cells. These inclusions differ in their location and morphological features from other inclusions that have been described in nerve and muscle biopsies of many disorders. The features of these inclusions suggest that they may represent a storage deposit whose nature could not be determined. They may be a distinguishing morphological feature of a multisystem disorder which to our knowledge has not been identified previously.

Adult↗
PubMed1995

Bisbenzimide: a fluorescent counterstain for tissue autoradiography.

Interpretation of the data from experiments using autoradiography (e.g. using in situ hybridization histochemistry, receptor binding, neuronal tract-tracing etc.) is aided when the autoradiographic grains can be seen in the context of cellular boundaries. Studies making use of autoradiography in the central nervous system have sometimes used tinctorial stains, such as cresyl violet, as counterstains to visualize the labeling. Tinctorial stains are excellent Nissl stains however, under bright-field illumination such dyes tend to obscure autoradiographic grains. In addition, dark-field illumination provides a common means of visualizing autoradiographic grains but tictorial stains are not optimally visible under these conditions. In an effort to find a counterstain that would be compatible with dark-field illumination, we have investigated the use of fluorescent dyes. Of the fluorescent dyes tested, bisbenzimide (Hoechst 33258) in pH 2.0 buffer was found to be optimal. Bisbenzimide counterstaining gave good resolution of cellular boundaries and appeared not to interfere with the ability to visualize autoradiographic grains. Furthermore, the illumination of bisbenzimide and of the autoradiographic grains could be controlled independently, making it easy to visualize or photograph the bisbenzimide Nissl staining and the autoradiographic grains simultaneously. Thus, bisbenzimide is well suited for use as a fluorescent counterstain in autoradiographic studies.

Animals↗
PubMed1978

[The influence of capillary permeability on the extravascular protein quantities as measured in situ (author's transl)].

The extravascular protein contents in the perivascular connective tissue of a rat's mesenterial plate was measured ultramicrospectrophotometrically in situ and in vivo after changing the hydrostatic and colloidosmotic pressures of the blood. We analized the perivascular area of the different microcirculatory vessels, arterioles, capillaries and venules. The perivascular protein contents, which corresponds besides arterioles to 40% and besides venules to 59% in comparison to the intravascular blood plasma, decreased in a comparison group under the osmotic influence of the perfusion liquid on the mesenterial plate. An injection of an isotonic saline solution is followed only by small fluctuations of the perivascular protein contents during one hour. After blood loss a transitional increase of extravascular protein at the perivenular area was observed indicating the mobilisation of protein depots. Around arterioles the extravascular protein contents did not change significantly. An intravenous injection of albumin solution was followed by a short-termed increase of tissue protein around the arterioles. Around the venules after 50 minutes the extravascular protein contents increased significantly. Within one hour after the changes of the permeability conditions the maximal induceable protein movements in the perivascular space were calculated as +/- 1.35 g% plasma protein concentration corresponding to a maximun protein exchange of +/- 3.1 mg ml(-1) tissue.

Animals↗
PubMedDate not supplied

Morphometric analysis of collagen network and plasma perfused capillary bed in the myocardium of rats during evolution of cardiac hypertrophy.

In order to investigate the consequences of different types of cardiac hypertrophy on myocardial capillary and fibrosis density in rats we describe here, in the same hearts, the pattern of capillary bed density visualized by fluorescein isothiocyanate dextran (FITC-dextran) and the pattern of fibrosis density as determined by automated image analysis. Pressure overload was induced by clipping one renal artery in rats (one-clip, two-kidney Goldblatt hypertension, RHV). Volume overload was induced by creation of an arteriovenous shunt between the abdominal aorta and the vena cava (aorto-caval fistula model ACF). Animals were sacrified at 1, 3 and 6 months following surgical procedure. Immediately prior to sacrifice, FITC-dextran (MW 150,000) was injected with the animal under ether anesthesia. Five minutes later, cardiac diastolic arrest was induced by the i.v. injection of potassium chloride. The heart was rapidly excised and placed in a formaldehyde solution. The degree of cardiac hypertrophy was calculated after measurement of cardiac weight. Left ventricular wall thickness and cavity area were measured by microscopic methods. Capillary density and geometry were determined by morphometric methods, under ultraviolet light microscopy, using a graphic tablet connected to a microcomputer. The degree of myocardial fibrosis, visualized with Sirius Red, was estimated by the use of automated image analysis using light microscopy. In renovascular hypertension, cardiac hypertrophy was maximum at one month (36%) and persisted through the six months of the study. This increase in cardiac mass was concentric, due to a significant increase in ventricular wall thickness and was associated with a marked increase in fibrosis and a significant decrease in subendocardial capillary density. These effects existed already one month and did not change with time. In the aorto-caval fistula model, cardiac hypertrophy was also maximum at one month (+56%), but this eccentric increase in cardiac mass was associated with no significant change in left ventricular wall thickness, but rather with a significant increase in the surface area of the left ventricular cavity. This volume overload hypertrophy was associated with a decrease in subendocardial capillary density which was negatively correlated with time. In contrast to concentric hypertrophy there was no increase in the fibrosis density compared to the sham-operated groups. Despite the identical degrees of hypertrophy, pressure and volume cardiac overload differed in a significant manner in both left ventricular wall thickness and cavity surface area.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Retinal pigment epithelial cell proliferation: potentiation by monocytes and serum.

BACKGROUND: The development of proliferative vitreoretinopathy (PVR) results often from a breakdown of the blood-retina barrier and the intraocular accumulation of serum proteins and leukocytes, particularly monocytes, that then come into contact with retinal pigment epithelial (RPE) cells. To examine the effect of these two factors on RPE proliferation, which is characteristic of PVR, we used a coculture system of blood monocytes and human RPE cells. METHODS: RPE cells were incubated with a variable number of monocytes at different serum concentrations and assayed for proliferation by [3H]-thymidine incorporation and cell counting. To assess cell-cell communication. RPE cells were labeled with 2', 7' -bis(carboxyethyl)-5(and 6) carboxyfluorescein acetoxy-methyl ester, and the dye transfer to monocytes was analyzed using an UV microscope. RESULTS: Monocytes (P < 0.0004) and serum (P < 0.0001), each on its own, significantly stimulated RPE cell growth, and these two variables were interrelated (P < 0.0001), showing a potentiating synergism. In serum-free medium, monocytes increased proliferation to just above control levels, whereas the same number of monocytes in 5% serum increased the [3H]-thymidine incorporation 3.8 times. This effect was greatly reduced by prevention of direct cell contact by means of placement of a well insert, which also lessened the monocyte-induced proliferation in both serum-free and serum-containing medium. Furthermore, the transfer of the intracellular dye from RPE cells to cocultured monocytes indicates that RPE cells transferred parts of their cytoplasm to monocytes. CONCLUSION: These observations underline the importance of protein leakage through a damaged blood-ocular barrier and of direct contact of monocytes/macrophages with RPE cells, as well as their reciprocal potentiating effect on RPE cell proliferation. Thus, early stabilization of the blood-ocular barrier, which would preclude or reduce protein leakage and invasion of inflammatory cells into the eye, could be a target for pharmacologic prevention of PVR.

Blood Physiological Phenomena↗
PubMed2002

Introgression of self-compatibility from Coffea heterocalyx to the cultivated species Coffea canephora.

Self-compatibility segregation was assessed in two successive backcross progenies originating from an interspecific cross between Coffea canephora (self-incompatible) and Coffea heterocalyx (self-compatible). After self- and cross-pollination, pollen tube behaviour in styles was observed under ultraviolet fluorescence microscopy and fruit-set was determined at harvesting time. Segregation ratios in the two progenies were consistent with monofactorial control of self-compatibility. Self-compatible plants exhibited higher fruit-set than self-incompatible ones in open-pollination conditions. Segregation of AFLP markers was scored in the first backcross progeny. By molecular linkage analysis, the S locus could be mapped to a short linkage group.

Journal Article↗
PubMed2005

Evaluation of rat and rabbit sera lipoproteins in experimentally induced hyperlipidemia by analytical ultracentrifugation.

Animals of various species are widely used as models with which to study atherosclerosis and the lipoprotein metabolism. The objective of this study was to investigate the lipoprotein profiles in Wistar rats and New Zealand white rabbits with experimentally induced hyperlipidemia by means of ultracentrifugation. The Schlieren curves were utilized to compare suckling and adult rat sera to determine whether aging causes alterations in lipoprotein profiles. A striking feature of the data is the high concentration of low-density lipoproteins (LDL), (>5.2 mmol/l cholesterol) in the 2-week old rat serum pool which was greatly decreased in the 3-weeks rat serum pool (<1.3 mmol/l cholesterol). Additional experiments were performed to permit a direct comparison of the amounts of lipoprotein present in rat sera in experimental hyperlipidemia post-Triton WR 1339 administration. Rapid changes in concentrations in very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL) were observed after Triton injection. The administration of Triton WR 1339 to fasted rats resulted in an elevation of serum cholesterol levels. Triton physically alters VLDL, rendering them refractive to the action of lipolytic enzymes in the blood and tissues, preventing or delaying their removal from the blood. Whereas the VLDL concentration was increased markedly, those of LDL and HDL were decreased at 20 h after Triton treatment. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate of LDL aliquots, to prepare radioactive-labeled lipoproteins and to study induced hyperlipidemia in rabbits. Analytical ultracentrifugation was applied to investigate the LDL flotation peaks before and after cholesterol feeding of rabbits. Modified forms of LDL were detected in the plasma of rabbits with experimentally induced atherosclerosis. ApoB-containing particles, migrating as LDL, intermediate density lipoproteins and VLDL were the most abundant lipoproteins. Gamma camera in vivo scintigraphy on rabbits with radiolabeled lipoproteins revealed visible signals corresponding to atherosclerotic plaques of the aorta and carotid arteries.

Age Factors↗
PubMed2006

Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells.

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate-DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfection.

Animals↗