Bertossi M

Expression of P-Glycoprotein in Human Cerebral Cortex Microvessels

P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the blood-brain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.

Ultrastructural and Morphometric Investigation of Human Brain Capillaries in Normal and Peritumoral Tissues

Capillaries of peritumoral and normal brain tissues were ultrastructurally and morphometrically investigated to evaluate the changes in peritumoral capillaries connected with the tumor-associated vasogenic edema. The endothelial cells of peritumoral capillaries showed varying thickness, electron-lucent cytoplasm, and structurally normal tight junctions. The basal lamina was thickened, rarefied, and vacuolated. The pericytes were provided with pinocytotic vesicles and phagocytic bodies. The astrocytic glia appeared empty or swollen, with few glycogen granules and a disarranged cytoskeleton; well-preserved glia was occasionally observed. The brain tissue was slightly edematous. No statistically significant differences were observed between normal and peritumoral capillaries as regards diameter, wall thickness, endothelial thickness, and endothelial vesicle density. Instead, the peritumoral capillaries displayed three times as many endothelial surface-connected vesicles, a markedly thicker basal lamina, and significantly reduced extension of pericytic and glial investments. The kind and severity of the vascular modifications, compared with the slight edematous appearance of the nervous tissue, strengthen the hypothesis that peritumoral capillaries could be involved in the edema resolution process.

Effects of Exogenous Heparin on the Vasculogenesis of the Chorioallantoic Membrane

On the basis of the well-known stimulating role played by heparin on blood vessel neoformation in the chorioallantoic membrane (CAM), demonstrated by means of slow-release polymers soaked in this substance and placed on CAMs, a new technique of introducing heparin directly into the allantoic sac is proposed. According to observations carried out in ovo and after fixation, morphological modifications are demonstrated in the developing vasculature of the heparin-treated CAMs which, compared with the control CAMs, show dilated and sinuous arterial and venous branches, denser and irregular capillary networks, and a high number of vascular primordia. The results, confirming that heparin is involved in angiogenesis, indicate the suitability of the technique and suggest that the effects of this substance could be enhanced by growth factors released by CAM tissues rapidly growing up from the 5th to the 7th incubation day.

Microscopical and ultrastructural investigations on the development of the blood-brain barrier in the chick embryo optic tectum

SummaryThe formation of a blood-brain barrier to horseradish peroxidase was microscopically and ultrastructurally investigated in the tectum opticum of the chick during development of the intraneural blood vessel network from the 6th incubation day to hatching, and in adult specimens.Extravasation of the circulating marker, apparently unimpeded during early stages of vasculogenesis, starts to diminish from the 14th incubation day (i.d.) and is prevented after the 18th i.d. The tracer seems to get out of the vessel lumina through the sites of reciprocal contact between adjacent endothelial cells, and the differentiation of tight junctions there hinders the passage of peroxidase particles. The formation of numerous endothelial vacuoles during early vasculogenesis and the setting of the blood-brain barrier are discussed in connection with the mechanisms of transendothelial transport, and respectively, the processes of moulding of the growing endothelia.

Developmental expression of ZO-1 antigen in the mouse blood-brain barrier.

Tight junction biogenesis during blood-brain barrier development (BBB) in mesencephalon microvessels of mouse embryos of day 9, foetuses of day 15 and 19 and new-born (2-day-old) mice was examined by light and electron microscopy, using monoclonal antibodies recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the light microscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasma membrane leaflets under the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when the endothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout the cytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at the cytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developing brain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation.

REGIONAL DISTRIBUTION AND CELL TYPE-SPECIFIC EXPRESSION OF THE MOUSE F3 AXONAL GLYCOPROTEIN : A DEVELOPMENTAL STUDY

The expression of the mouse axonal adhesive glycoprotein F3 and of its mRNA was studied on sections of mouse cerebellar cortex, cerebral cortex, hippocampus, and olfactory bulb from postnatal days 0 (P0) to 30 (P30). In cerebellar cortex, a differential expression of F3 in granule versus Purkinje neurons was observed. F3 was highly expressed during migration of and initial axonal growth from cerebellar granule cells. The molecule was then downregulated on cell bodies and remained expressed, although at low levels, on their axonal extensions. On Purkinje cells, F3 was strongly expressed on cell bodies and processes at the beginning of the second postnatal week; by P16 it was restricted to neurites of Purkinje cells subpopulations. In the cerebral cortex, the molecule was highly expressed on migrating neurons at P0; by P16, it was found essentially within the neuropil with a diffuse pattern. In the hippocampal formation, where F3 was expressed on both pyramidal and granule neurons, a clear shift from the cell bodies to neurite extensions was observed on P3. In the olfactory pathway, F3 was expressed mainly on olfactory nerve fibers, mitral cells, and the synaptic glomeruli from P0 to P3, with a sharp decline from P11 to P16. As a whole, the data show that F3 protein expression is regulated at the regional, cellular, and subcellular levels and suggest that, in different regions, it can be proposed as a reliable neuronal differentiation marker. J. Comp. Neurol. 413:357–372, 1999.

Immunogold cytochemistry of the blood–brain barrier glucose transporter GLUT1 and endogenous albumin in the developing human brain

The blood-brain barrier (BBB) glucose transporter, GLUT1, was detected by immunogold electron microscopy on the microvascular compartment of the human foetus telencephalon at the 12th and 18th weeks of gestation. By computerized morphometry, the cellular and subcellular localization of the immunosignal for GLUT1 was quantitatively evaluated. The study showed that the glucose transporter is strongly expressed by endothelial cells while a very low signal is detected on vascular pericytes. The GLUT1 antigenic sites are preferentially associated to the ablumenal and junctional plasma membranes of the endothelial cells and tend to increase significantly with age. A parallel study carried out by the endogenous serum protein albumin demonstrated that already at the 12th week the endothelial routes are hindered to the protein as happens at the blood-endothelium interface of mature brain. The results demonstrate that in the human foetus the brain microvessels express BBB-specific functional activities early.

Process of differentiation of cerebellar Purkinje neurons in the chick embryo.

SummaryThe microscopic and ultrastructural differentiation of Purkinje neurons has been studied in 40 chicken embryo cerebella, from the 10th incubation day to hatching, and the transverse diameter of the cell body measured, for each developmental stage, on 30 electron micrographs of sagittally cut Purkinje cells. The developing Purkinje cell bodies, bipolar, at first, given the presence of two processes emerging from the opposite poles of the oval perikaryon, grow progressively in size. After the 12th incubation day, they develop a branched dendritic tree, and, shortly before hatching time, the cells acquire the characteristic flask or pear-shaped configuration. On the 10th incubation day, microtubules are already detectable together with Golgi complexes and a few vesicles of rough endoplasmic reticulum; on the 14th incubation day, RER cisterns are recognizable in the supranuclear cytoplasm, later extending into the whole perikaryon, and attaining their definitive distribution by the 18th incubation day. Pinocytotic and coated vesicles, as well as subsurface cisterns are seen during the whole embryonic life. In the earliest stages of development, three distinct types of junctional contacts between Purkinje cells and surrounding axons are described, and their functional role in relation to synaptogenetic processes is discussed. Beginning with the 16th incubation day, some Purkinje neurons undergo degenerative changes similar to those described in other types of neurons of the central and peripheral nervous system.

An immunohistochemical and morphometric study on astrocytes and microvas culature in the human cerebral cortex

In this study, astrocytes and microvessels of the human cerebral cortex were analysed morphometrically with the aim of acquiring quantitative information on the glio-vascular relationships, considered to be of great importance in the formation and functioning of the blood--brain barrier. Immunohistochemistry for the astrocytic marker, glial fibrillary acidic protein, was used with a computerized image analysis system. The brain tissue was embedded using the progressive lowering of temperature method, and the image analyser was applied to semithin sections subjected to immunogold--silver staining and viewed by epipolarization microscopy. The results show that, in the human cerebral cortex, astrocytes cover 11.4% of the cortex area and that their perivascular processes are nearly as extensive as the vascular bed (0.8% versus 1.72% of the cortex area). These processes form a virtually continuous sheath around the vascular walls, only 11% of the vessel perimeter lacking this astrocytic glia covering. The present results, compared with previous unpublished data obtained by conventional immunocytochemical procedures on wax sections, indicate that low-temperature methods combined with gold--silver immunolabelling on semithin sections significantly improve the detection of immunoreactivity and the performance of the image analyser.

Perivascular astrocytes and endothelium in the development of the blood-brain barrier in the optic tectum of the chick embryo

The role played by perivascular astrocytes in neural vessel maturation was investigated in microvessels of the chick embryo optic tectum. Three-dimensional reconstructions and quantitative analyses were made, and permeability was studied. On embryonic days 14–16, 12.5% of the microvessel wall is surrounded by astrocyte endfeet which, in most cases (82%), are located under endothelium junctions; the latter, at this stage, partly prevent the extravascular escape of the marker horseradish peroxidase. On days 18–21, the astrocyte processes form a nearly complete perivascular sheath enveloping 96% of the microvessel perimeter; the junctions of the endothelial cells are much wider and impermeable owing to extensive fusion of the endothelial plasma membranes. This investigation suggests a close relationship between the perivascular arrangement of glia and differentiation of the endothelium tight junctions and indicates that the morphofunctional maturation of the latter takes place progressively during the prenatal organogenesis of the chick central nervous system.