Ultrastructural changes in the cerebral cortex following transcranial micropolarization]

Electron microscopy of the cerebral cortex in cats and monkeys following transcranial micropolarization (TCMP) demonstrated ultrastructural changes whose degree was dependent on the electric current intensity and the stimulation period. In the focus of stimulation the current affected the brain tissue directly, different elements of the cerebral cortex showing unegual sensitivity to various TCMP regimens. The glia was the first to respond, then the neuronal bodies, and the last -- the synaptic structures. In the areas distant from the TCMP focus synaptic components altered first. The ultrastructural changes revealed were not of pathological character.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Ultrastructural analysis of major basement membrane types in rhesus monkey Macaca mulatta acellular renal cortex

Increasing interest in animal models of human nephropathies have led to a number of renal studies in nonhuman primates. In the current investigation, sequential detergent extraction of cellular elements was carried out on renal cortical tissue blocks from rhesus monkey in an effort to demonstrate clearly the morphological features of major basement membrane (BM) types and their associated extracellular matrix (ECM). LM and TEM views of acellular tissue blocks demonstrate planar arrangements of ECM components, while SEM studies provide striking three-dimensional images of their surface characteristics. All major BM types maintain their in vivo histoarchitectures despite the absence of cells. We propose that the intrinsic structural rigidity of tubular (TBM), Bowman's capsule (BCBM) and peritubular capillary BM (PTCBM) may be related to to their close external association with collagenous fibrils, while glomerular BM (GBM) may be internally supported by a network of mesangial matrix (MM) plates and trabeculae which extend onto internal surfaces of peripheral GBM loops. Thicknesses of rhesus monkey renal BMs show that they are similar to those seen in the laboratory rat and, in general, BCBM greater than TBM greater than GBM greater than PTCBM. We conclude that rhesus monkey renal BMs closely resemble those described by us in the human [J. Ultrastruct. Res. 82: 96-110, 1983] and that this species offers an attractive model for studies of renal diseases of BM origin-notably diabetes mellitus.     

Ultrastructural development features of the human glomerular basement membrane

In the human embryo, the basement glomerular membrane appears early, at about ten weeks of age. The study of 36 human embryos aged 8-20 weeks revealed many arguments suggesting the epithelial origin of the basement membrane of the visceral capsula.     

Presence of glycosaminoglycans in retinal capillary basement membrane

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [³⁵S]sulfate and [¹⁴C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase and chondroitinase ABC. Retinal microvessels also incorporated [³⁵S]- and [¹⁴C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinalmicrovessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.     

Ultrastructural orientation of laminin 5 in the epidermal basement membrane: an updated model for basement membrane organization.

Laminin 5 is a trimeric glycoprotein involved in cell adhesion in the epidermal basement membrane. To determine the precise orientation of laminin 5 in adult human skin, we used plural epitope-specific monoclonal antibodies, a polyclonal antiserum, and postembedding immunogold electron microscopy (IEM). Immunogold labeling distances from the basal keratinocyte plasma membrane (PM) were measured for each gold particle (>200 particles) and the mean distance (nm) calculated. Antibodies included BM165 (recognizing the alpha 3-chain first globular domain) that was measured at 35.40 +/- 2.20 nm from the keratinocyte PM, K140 (recognizing a region adjacent to the beta 3-chain globular domain IV) that measured 45.20 +/- 3.60 nm from the PM, and an anti-laminin 5 polyclonal antiserum that was 43.43 +/- 6.28 nm from the PM. The laminin 5 gamma 2-chain short arm hinge domain was previously localized to the lower lamina densa (LD) at approximately 56.30 +/- 1.65 nm from the keratinocyte PM. Taken together with previous gamma 2-chain data and the distribution of the polyclonal antisera, we estimate that the long axis of laminin 5 is oriented at an angle of approximately 27 degrees from the horizontal lamina lucida (LL)/LD border and propose that the gamma 2-chain lies farthest from the PM. This novel orientation, with the majority of the laminin 5 molecule lying obliquely along the LL/LD border and not perpendicularly, as was first thought, sheds new light on the organization of the basement membrane and likely molecular interactions.     

Ultrastructural demonstration of dehydrogenases in rat cerebral cortex

Summary Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retension of LDH and NADH-diaphorase was obtained by perfusion with a mixture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase; DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100; DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and intermembrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.     

Ultrastructural immunoperoxidase demonstration of autologous albumin in the alveolar capillary membrane and in the alveolar lining material in normal rats

The location of autologous serum albumin within the alveolar-capillary membrane was studied in the rat under physiological conditions using antialbumin antibodies labeled with peroxidase. Albumin was detected in the lung interstitium, and in numerous pinocytic vesicles within endothelial cells and type I alveolar epithelial cells. The immunoreaction was also positive at the level of plasmalemmal membranes of both cell types and in the alveolar lining material.     

Ultrastructural demonstration of glucose 6-phosphatase in cerebral cortex

Summary A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.     

Ultrastructural demonstration of dehydrogenases in rat cerebral cortex.

Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retention of LDH and NADH-diphorase was obtained by perfusion with a misture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase: DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100: DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and inter-membrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.     

Ultrastructural demonstration of glucose 6-phosphatase in cerebral cortex

A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.     

Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach

Rat kidneys were perfused with fixative solutions containing either a) a polycationic dye (Alcian blue 8 GX, Astra blue 6 GLL, cuprolinic blue, ruthenium red), b) a monocationic dye (safranine 0), or c) Alcian blue in the presence of a 0.3 M MgCl2 concentration. Whereas solutions of a revealed the glomerular basement membrane proteoglycans as particles or threads 60 nm apart and arranged in a reticular pattern, solutions of b and c demonstrated new morphological aspects of these molecules. They appeared as tiny filamentous structures, about 100 to 160 nm long, ordered in a network-like pattern with a mesh of about 60-nm width. The filaments displayed lateral branches about 20 nm apart and about 25 nm long, projecting within the meshes. We suggest that the filamentous structures are the protein core, and the branches are the glycosaminoglycans of proteoglycan molecules. Because of this arrangement the negatively charged sites of the glomerular basement membrane would lie closer to each other than previously assumed.     

Age-related characteristics of intraorganic vascular structures of the cerebral cortex, heart, kidneys and spleen in rats in the normal state and in arterial hypertension

The work has been performed on 40 normotensive and spontaneously hypertensive rats at the age of 6 months-1 year. In all the organs studied a direct dependence between the degree of changes in the intraorganic arterial walls and a relative content of the arterial vessels in the organ tissues has been revealed. The functional changes of the arterial bed of the organs mentioned in spontaneously hypertensive rats during hypertensive phase at the stage of stable hypertension, as a result of a prolonged adaptation transform into organic ones.     

Effects of magnesium and nickel ions on penicillin-induced focal epileptic activity in the cerebral cortex of rats]

In experiments on freely moving male Wistar rats on the model of penicillin-induced focal epileptic activity (EA) (the application onto the sensorimotor cortex of a filter paper soaked with benzylpenicillin sodium salt solution) it was shown that addition of MgSO4 (series 1) and NiCl2 (series 2) into the solution of penicillin significantly weakened EA. The combination of Mg2+ and Ni2+ with penicillin (series 3) produced a more significant suppression of EA as compared with separate application of the above-mentioned ions: the latency period of appearance of interictal discharges (IID) increased, the frequency and amplitude of IID decreased much more, no ictal discharges appeared in any animal, the duration of epileptic foci reduced to a much greater extent. This effect can be explained by the blockade of Ca current by the above-mentioned ions. One can suppose that the amplification of antiepileptic effects of combined action of Mg2+ and Ni2+ was due to an increase in the number of blocked voltage-dependent and NMDA-operated calcium channels.     

Hysteresis of the alveolar capillary membrane in normal subjects

Weibel and associates (Respir. Physiol. 18: 285-308, 1973), using morphometric techniques, demonstrated in the rat that changes in lung volume related to inflation and deflation caused a hysteretic variation in alveolar capillary membrane which is locally pleated at low pulmonary volume, unfolds during inflation but does not immediately refold during deflation, possibly enhancing the CO diffusion throughout the membrane. The present study was conducted to verify the existence of this hysteresis in human lungs in vivo. Single-breath diffusing capacity for CO (DLCO) was measured in five healthy seated subjects before and 0, 0.5, 1, 3, and 7 min after an inflation-deflation maneuver (IDM) in 6 separate days. The value of mean DLCO was 36.4 +/- 3 (SD) before and 42.1 +/- 2.9, 41.6 +/- 3.3, 40.3 +/- 3.3, 39.2 +/- 3.2, and 38.1 +/- 2.7 ml X min-1 X Torr-1 after the IDM. Two mechanisms can explain our findings: an active filling of the capillary bed, or an unfolding of the alveolar capillary membrane. The first mechanism should be accompanied by changes in pulmonary circulation. Therefore, right-heart catheterization was performed in two normal subjects and in four patients examined for a chest pain syndrome. At the end of the IDM, the values for the pulmonary artery pressure and capillary wedge pressure had returned to control levels. This suggests that the capillary bed is not directly involved in the DLCO increase observed from 0.5 to 7 min after the IDM. The unfolding of the alveolar capillary membrane appears to better explain our findings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of lysosomal enzymes in the normal glomerular basement membrane matrix

Summary The question posed in the present study was: are there hydrolytic enzymes, including proteases, present in the extracellular matrix of the glomerular basement membrane? If these enzymes are present they may play a role in the catabolism of the glomerular basement membrane (GBM) and removal of macromolecular debris resulting from ultrafiltration. Enzymes, acid phosphatase - the marker for lysosomal enzymes - β-galactosidase, β-glucuronidase and acid protease (using albumin as substrate) were biochemically assayed in purified basement membrane preparations. It was found that all enzymes were present in significant amounts in the basement membrane. Compared to other enzymes, acid protease activity was present in much higher amounts. The pH optima of these enzymes were variable but all had significant activity at neutral pH. A method was developed to localize the marker enzyme, acid phosphatase, ultrastructurally in the basement membrane in order to substantiate the biochemical findings. Activity was shown by the presence of dense deposits of lead phosphate. Staining for acid phosphatase could also be shown on isolated, purified basement membrane. The demonstration of acid hydrolases in the GBM matrix argues for their role in (i) the extracellular turnover of basement membrane macromolecules, and (ii) clearance of debris of ultrafiltration which tend to clog the membrane pores.