Quantitative immunogold study of increased expression of metallothionein-I/II in the brain perivascular areas of diabetic scrapie-infected mice.

Quantitative immunogold procedure was used to study the distribution of metallothionein I/II (MT-I/II) at the ultrastructural level in the perivascular areas, including microvascular endothelial cells (ECs) and astrocytes with their perivascular end-feet, in brains of scrapie-infected hyperglycemic (diabetic) and normoglycemic (non-diabetic) mice. Samples of the fronto-parietal cortex obtained from diabetic and non-diabetic scrapie-infected, as well as from non-infected (control) SJL/J mice, were processed for immunocytochemical examination. In control mice, the labelling of the ECs was of low intensity, restricted to few immunogold particles in the cytoplasm. More intense labelling was present in the cytoplasm of astrocytic perivascular processes and perikarya, where it was associated with endoplasmic reticulum and fibrils. A few immunosignals were also present inside the nuclei of astrocytes. In diabetic mice the labelling of the EC cytoplasm was slightly increased, whereas in the cytoplasm of perivascular processes and pericarya of astrocytes, including their nuclei, there was significant enhancement of labelling. In these cells the density of immunosignals was highest in the areas of cytoplasm containing bundles of fibrils. In non-diabetic, scrapie-infected mice the intensity of immunolabelling was higher than in control mice but slightly lower than in diabetic mice. These results are similar to those in Alzheimer's disease reported by other authors, and suggest that neurodegenerative diseases as well as metabolic stress enhance the metallothionein expression in perivascular regions of brain cerebral cortex, predominantly in astrocytes.

Immunogold study of altered expression of some interendothelial junctional molecules in the brain blood microvessels of diabetic scrapie-infected mice.

Quantitative immunogold procedure was used to study the distribution of molecular components of interendothelial junctions in blood-brain barrier (BBB) microvessels of scrapie infected SJL/J hyperglycemic mice showing obesity and reduced glucose tolerance. Samples of brain (fronto-parietal cerebral cortex and thalamo-hypothalamic region) obtained from hyperglycemic (diabetic) mice and from non- infected, normoglycemic (non-diabetic) SJL/J mice, were processed for immunocytochemical examination. The localization of the following tight junction (TJ)-associated proteins was studied: occludin as an integral membrane (transmembrane) protein, and zonula occludens one (ZO-1) as a peripheral protein. The localization of beta-catenin as a representative of the cadherin/catenin complex that is typical for adherens junctions (AJs) also was studied. Morphometric analysis revealed that the density of immunosignals for occludin, represented by colloidal gold particles (GPs), was significantly lower in the brain microvessels of diabetic than in non-diabetic mice. No significant differences in the density of immunosignals for ZO-1 and beta-catenin between both experimental mouse groups were observed. It indicates that abnormal glucose metabolism affects mostly occludin which is believed to play a fundamental role in the maintenance of the tightness of endothelial lining in brain microvascular network and thereby in the preservation of its barrier function. These results also support the previously expressed opinion that occludin, detected with the applied morphological method, can be considered a sensitive indicator of altered molecular architecture of the interendothelial junctions due to the action of some metabolic or pathological insults.

Immunogold localization of tight junctional proteins in normal and osmotically-affected rat blood-brain barrier.

The distribution of molecular components of interendothelial tight junctions (TJs) was studied in rat blood-brain barrier (BBB) microvessels, using immunogold cytochemistry applied to electron microscopy. Samples of rat brains, both normal (unaffected) and osmotically-affected (1, 5, and 30 min after intracarotid infusion of 1.8 M L(+)arabinose), were processed for immunocytochemical localization of TJ-specific integral membrane (occludin, JAM-1, claudin-5) and peripheral (ZO-1) protein molecules. In unaffected interendothelial junctions of control rats the immunosignals (represented by gold particles) for occludin and ZO-1 were of highest, whereas for claudin-5 and JAM-1 were of lower density. At 1 min after infusion, no discernible changes in distribution of junction-associated molecules were noted. At 5 min, however, changes were most conspicuous, and they consisted of segmental attenuation of the endothelial lining and dilatation (opening) of some junctional clefts accompanied by the diminution of the density of immunosignals for TJ-specific transmembrane and peripheral proteins. It was paralleled by disorganization of the spatial relation of these molecules to the junctional complexes. After 30 min, many interendothelial junctions appeared to be still open, whereas other junctions were partially or totally closed. In the opened interendothelial junctions the expression of TJ-associated molecules was weaker than in closed junctions. Our observations indicate that the localization and expression of TJ-specific proteins, especially occludin, and in lower degree claudin-5 and JAM-1, together with the peripheral ZO-1 molecules, are affected by osmotic shock. Presumably, some of these proteins (e.g., occludin, claudin-5 and ZO-1) could be considered sensitive indicators of normal and also of disturbed functional state of the BBB.

Molecular anatomy of interendothelial junctions in human blood-brain barrier microvessels.

Immunogold cytochemical procedure was used to study the localization at the ultrastructural level of interendothelial junction-associated protein molecules in the human brain blood microvessels, representing the anatomic site of the blood-brain barrier (BBB). Ultrathin sections of Lowicryl K4M-embedded biopsy specimens of human cerebral cortex obtained during surgical procedures were exposed to specific antibodies, followed by colloidal gold-labeled secondary antibodies. All tight junction-specific integral membrane (transmembrane) proteins--occludin, junctional adhesion molecule (JAM-1), and claudin-5--as well as peripheral zonula occludens protein (ZO-1) were highly expressed. Immunoreactivity of the adherens junction-specific transmembrane protein VE-cadherin was of almost similar intensity. Immunolabeling of the adherens junction-associated peripheral proteins--alpha-catenin, beta-catenin, and p120 catenin--although positive, was evidently less intense. The expression of gamma-catenin (plakoglobin) was considered questionable because solitary immunosignals (gold particles) appeared in only a few microvascular profiles. Double labeling of some sections made possible to observe strict colocalization of the junctional molecules, such as occludin and ZO-1 or JAM-1 and VE-cadherin, in the interendothelial junctions. We found that in human brain microvessels, the interendothelial junctional complexes contain molecular components specific for both tight and adherens junctions. It is assumed that the data obtained can help us find the immunodetectable junctional molecules that can serve as sensitive markers of normal or abnormal function of the BBB.

Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist's view.

In this review, we have tried to summarize the current knowledge on the distribution of important molecular components of intercellular junctions-both tight junctions (TJs) and adherens junctions (AJs)-at the level of ultrastructure. For this purpose, immunogold procedure was applied to ultrathin sections of brain samples obtained from mice, rats, and humans and embedded in hydrophilic resin Lowicryl K4M. The results of our observations performed with transmission electron microscopy (EM) are discussed and compared with findings of other authors. Although the main structures responsible for the barrier and fence functions of the blood-brain barrier (BBB) and blood-CSF barrier are TJs present between endothelial cells (ECs) of brain capillaries and epithelial cells of the choroid plexus, their functional characteristics (e.g. tightness of the barrier evaluated by electrical resistance) differ significantly. Therefore, our main attention is focused on the presence and distribution of both intrinsic, i.e. integral membrane (transmembrane), molecules such as occludin, claudins, and junctional adhesion molecule (JAM) in TJs, and cadherins in AJs, as well as peripheral molecules of both types of junctions, e.g. zonula occludens (ZO) proteins and catenins. The latter group of molecules connects transmembrane proteins with the cell cytoskeleton. A close spatial association of the TJ proteins with those of AJs indicates that both junctional types are intermingled in the BBB type of endothelium. One of most important purposes of this work is to find out the junction-associated molecules that can serve as sensitive markers of normal or disturbed function of brain barriers. Understanding the structural-functional relations between molecular components of junctional complexes in physiological and experimental conditions of both barriers can provide important information about the etiology of various pathological conditions of the central nervous system and also help to elaborate new therapeutic approaches.