Perivascular Glial Processes Research Articles

Actin cytoskeleton remodeling governs aquaporin‐4 localization in astrocytes

Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4.

Increased expression of aquaporin-1 in the anterior temporal neocortex of patients with intractable epilepsy

Objective: Aquaporin-1 (AQP1), the osmotic water channel, is located in choroidal plexus, which facilitates the cerebrospinal fluid formation in central nervous system (CNS). AQP1 has been speculated to maintain the homeostasis of intracellular and extracellular water in the brain, while the intractable epilepsy (IE) may be related to the imbalance in water and ion homeostasis.Methods: To investigate the role of AQP1 in pathophysiology of IE, we studied the expression of AQP1 in surgical samples of the anterior temporal neocortex of patients with IE and the age-matched controls samples.Results: Using immunohistochemistry, it was shown that AQP1 expression increased in astrocytes, but not in neurons or oligodendrocytes. Double-label immunofluorescence and confocal microscopy disclosed AQP1 immunoreactivity at the astrocyte membranes, where the most abundant expression was in the perivascular glial processes, which were recognized with antiglial fibrillary acidic protein antibodies (anti-GFAP).Conclusion: For the first time, we showed high expression of AQP1 water channels in IE cases and suggest two mechanisms to explain this finding. Increased AQP1 expression of astrocytes may be a cause or a consequence of IE. Thus, additional works are needed to elucidate the true mechanism underlying their relationship.

Blood-brain barrier disruption highly induces aquaporin-4 mRNA and protein in perivascular and parenchymal astrocytes: Protective effect by estradiol treatment in ovariectomized animals

Strong evidence involves aquaporin-4 (AQP4) in the physiopathology of brain edema. Two major points remain unsolved: (1) the capacity of perivascular glial cells to regulate AQP4 in response to disruption of the blood-brain barrier (BBB); and (2) the potential beneficial role of AQP4 in the clearance of brain edema. We used intraparenchymal injection of lipopolysaccharide (LPS) as an efficient model to induce BBB disruption. This was monitored by IgG extravasation and AQP4 was studied at the mRNA and protein level. The first signs of BBB disruption coincided with strong induction of AQP4 mRNA in perivascular glial cells. At the early phase, estradiol treatment highly prevented the LPS-induced disruption of the BBB and the induction of AQP4. Efficient clearance of vasogenic edema is supposed to occur once BBB is restored. This phase coincided with high induction of AQP4 mRNA in parenchymal reactive astrocytes and perivascular glial processes. High levels of AQP4 mRNA may be beneficial under these conditions. Our data may clarify why estradiol treatment reduces mortality in conditions typically associated with edema formation, like stroke.

Involvement of metalloprotease-2 in the development of human brain microvessels.

The involvement of the metalloprotease-2 (MMP-2) in vessel development was investigated in the human telencephalon by double immunoreactions with antibodies to the enzyme, latent (proMMP-2) and active (aMMP-2) forms, and an antibody against collagen type IV, a constitutive component of the extracellular matrix (ECM) of the vessel basal lamina. MMP-2 is expressed in both 12- and 18-week telencephalic vessels, the proenzyme being mainly localised in endothelial cells and the active form prevailing in alpha-actin-reactive periendothelial cells identified as pericytes. Endothelial cells intensely positive for aMMP-2 were revealed in some microvessels and appeared locally associated with discontinuities of the collagen basal lamina. No detectable expression of MMP-2 was observed in perivascular glial processes revealed by vimentin/glial fibrillary acidic protein immunostainings. Double immunoreactions performed to further investigate telencephalon angiogenesis have demonstrated that both the endothelial cells and pericytes strongly express vascular endothelial growth factor (VEGF). Taken together, the results indicate that MMP-2 is largely involved in human brain angiogenesis and suggest that endothelial cells and pericytes tightly interplay in both angiogenesis mechanisms, by ECM proteolysis, and angiogenesis regulation, by local (autocrine/juxtacrine) VEGF action.

Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice.

In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.

GABA immunoreactivity in the human cerebellar cortex: a light and electron microscopical study.

The distribution of gamma-aminobutyric acid (GABA) in surgical samples of human cerebellar cortex was studied by light and electron microscope immunocytochemistry using a polyclonal antibody generated in rabbit against GABA coupled to bovine serum albumin with glutaraldehyde. Observations by light microscopy revealed immunostained neuronal bodies and processes as well as axon terminals in all layers of the cerebellar cortex. Perikarya of stellate, basket and Golgi neurons showed evident GABA immunoreactivity. In contrast, perikarya of Purkinje neurons appeared to be negative or weakly positive. Immunoreactive tracts of longitudinally- or obliquely-sectioned neuronal processes and punctate elements, corresponding to axon terminals or cross-sectioned neuronal processes, showed a layer-specific pattern of distribution and were seen on the surface of neuronal bodies, in the neuropil and at microvessel walls. Electron microscope observations mainly focussed on the analysis of GABA-labelled axon terminals and of their relationships with neurons and microvessels. GABA-labelled terminals contained gold particles associated with pleomorphic vesicles and mitochondria and established symmetric synapses with neuronal bodies and dendrites in all cortex layers. GABA-labelled terminals associated with capillaries were seen to contact the perivascular glial processes, basal lamina and endothelial cells and to establish synapses with subendothelial unlabelled axons.

Immunohistochemical and Ultrastructural Characterization of Cortical Plate Microvasculature in the Human Fetus Telencephalon

The blood–brain barrier (BBB) differentiation was investigated by immunohistochemistry and electron microscopy in the radial microvasculature of the telencephalon cortical plate (CP) of 12- and 18-week human fetuses. The BBB-specific glucose transporter isoform 1 (GLUT1) is expressed in both stages, with a main localization on the ablumenal and lateral plasma membranes of the endothelial cells. The endothelial cells are welded by short junctions with fusion points of the plasma membranes at 12 weeks and by extensive tight junctions at 18 weeks. The basal lamina is discontinuous beneath the endothelium–pericyte layer at 12 weeks and splits into two continuous layers circumscribing the pericytes in the later stage. The expression of laminin, a basal lamina glycoprotein, is continuous already at 12 weeks. The CP microvessels are tightly surrounded by processes of glial cells. Immunodetection of the cytoskeletal filament proteins, vimentin (VIM), and glial fibrillary acidic protein (GFAP), demonstrates that at 12 weeks the perivascular glial processes are mostly represented by VIM-stained fibers of the radial glia. At 18 weeks, GFAP-stained radial glia fibers, processes of VIM-stained astroblasts, and GFAP-positive astrocytes also build the perivascular envelopes. The results indicate that the vessel differentiation is already under way in the human CP at the midgestational age and entails the establishment of some barrier devices. The early relationship between perivascular glia coverage formation and endothelial barrier maturation suggests that also immature astroglial cells are involved in the setting up of the BBB.

Antisera to glutathione: characterization and immunocytochemical application to the rat cerebellum.

Rabbits were immunized with reduced glutathione (gamma-glutamyl-cysteinyl-glycine) coupled to bovine serum albumin by glutaraldehyde or a mixture of glutaraldehyde and formaldehyde. The antisera that were formed were tested qualitatively, by screening them against more than 50 amino acids and peptide conjugates that had been immobilized on cellulose discs (spot test), and quantitatively, by immunogold analysis of test conjugates that had been embedded in an epoxy resin. It was shown that the antisera selectively recognized the reduced and oxidized forms of glutathione and that they did not exhibit any significant crossreactivity with glutamate, cysteine, glycine, gamma-glutamyl-cysteine or cysteinyl-glycine. Immunocytochemistry of Vibratome sections of rat cerebellum suggested that glutathione occurs in glial cells as well as in neurons. This was confirmed by electron microscopic, immunogold cytochemistry of tissue from rat cerebellum that had been freeze-substituted and embedded in Lowicryl under low temperature. Gold particles were concentrated over Golgi epithelial cells and perivascular glial processes, but also occurred over several types of neuronal profile including Purkinje and granule cell bodies, and mossy fibre terminals. At the subcellular level, glutathione-like immunoreactivity was found in the cytoplasmic matrix, mitochondria and nuclei. The immunolabelling intensity was strongly reduced in animals that had been pretreated with buthionine sulphoximine, which is known to depress the level of glutathione by inhibiting gamma-glutamyl-cysteine synthetase. The availability of antisera to glutathione is likely to further our understanding of the physiological and pathophysiological roles of this tripeptide.

Freeze-fracture evidence for a novel restricting junction at the blood-brain barrier of the cuttlefish Sepia officinalis.

The blood-brain barrier in the cuttlefish Sepia officinalis has been studied with the freeze-fracture technique. Previous thin-section electron microscopy showed that a restricting junction is formed between perivascular glial processes in microvessels and venous vessels, and between pericytes in arterial vessels; the restriction appeared not to be a classical zonula occludens or septate junction. In freeze-fracture replicas from brain optic and vertical lobe, endothelial cells, pericytes and perivascular glia could be recognized by their morphology and relation to the vascular lumen. In microvessels, endothelial and pericyte membranes showed sparse but uniform distribution of P-face intramembranous particles, with no particular particle aggregations. Perivascular glial membranes had a higher density of intramembranous particles but again no particle alignments characteristic of known restricting junctions were seen, although clusterings of intramembranous particles resembling gap junctions were present. In larger venous vessels, the perivascular glial layer showed a multilamellated organization, but again no arrays of intramembranous particles were detected, although this should be a favourable site for visualization of the restricting junctions. The walls of arterial vessels showed collagen deposits and cell processes with apparent intracellular myofilamentous profiles, but no intramembranous junctional particle arrays. It is concluded that the junctional zone observed in thin section electron microscopy is not associated with aligned aggregations of intramembranous particles detectable in freeze-fracture replicas, strengthening the evidence that this is a novel type of restricting junction.

Cytology and organization of reactive astroglia in human cerebellar cortex with severe loss of granule cells: A study on the ataxic form of creutzfeldt-jakob disease

In order to investigate the cellular basis of human astrogliosis, we have selected the cerebellar cortex because it provides a relatively simple and geometrical organization of both neuronal and glial populations. A pathological system with severe and progressive loss of granule cells was studied: the ataxic form of Creutzfeldt-Jakob disease, where the tissue geometry is minimally disturbed. The quantitative study revealed a drastic reduction in the numerical density of granule cells in the Creutzfeldt-Jakob disease cerebellum, and a significant increase in the numerical density of astrocytes. Karyometric analysis showed that the nuclear area was significantly greater in reactive astroglial cells than in normal astroglia. Glial fibrillary acidic protein immunocytochemistry revealed astroglial hypertrophy, but the geometry and spatial domains of astroglial subtypes were strictly preserved. Vimentin expression was detected in Bergmann glia and in certain astrocytes of the granular layer. Ultrastructural analysis showed that reactive astroglia had large nuclei, with expanded interchromatinic regions which contained clusters of interchromatin granules and nuclear bodies, and prominent reticulate nucleoli. In the cytoplasm, hypertrophied bundles of intermediate filaments were observed, some of them associated with the nuclear envelope. Numerous adhering and gap junctions were also found among reactive astroglial cells. Perivascular glial processes showed a terminal web of intermediate filaments and a conspicuous plasmalemmal undercoat. Interendothelial tight junctions were preserved. Our results suggest that the severe loss of granule cells induces a highly ordered astroglial response which tends to preserve the geometry of the astroglial scaffold, the domains of each astroglial subtype, the neuronal microenvironmental conditions and the efficiency of the blood-brain barrier, in order to promote neuron survival.

The fine structure of the area postrema of the sheep.

The ultrastructural features of the area postrema (AP) were investigated in the suckling lamb, weaned lamb and adult sheep. No morphological differences were observed between lambs and sheep. Unciliated ependymal cells, linked by zonulae adherentes-type junctions and gap junctions, cover the AP ventricular surface. Clusters of pyriform neurons, glial cells, and axons are present in the parenchyma. The blood vessels are surrounded by wide perivascular spaces, which present an inner and outer basal lamina. The capillaries are of the fenestrated type. Perivascular glial cells rest on the outer basal lamina of the perivascular space and form a continuous ensheathment with their cell bodies or with flattened interdigitating processes. Along adjacent perivascular glial processes gap junctions are present. From our ultrastructural observations it appears that the overall cellular morphology of AP of the sheep does not differ substantially from that of monogastric mammals.

Early vascularization of the embryonic cerebral cortex: Golgi and electron microscopic studies.

Two developmental aspects in the establishment of the vasculature of the embryonic cerebral cortex have been analyzed: the vascular perforation of the external basal lamina and marginal glia by leptomeningeal capillaries and their early intracortical angiogenesis. Electron microscopic and rapid Golgi preparations have been used in this study. Four phases have been identified in the vascular perforation of the cortex. First, the leptomeningeal capillary approaches and establishes direct contact with the external basal lamina and marginal glia of the cerebral cortex. Second, the leading endothelium of the glia-touching capillary undergoes considerable membrane activity forming many filopodia and pseudopodia. Some filopodia of this activated endothelium perforate the vascular and cortical basal laminae and penetrate into the neural tissue. This filopodial perforation is accompanied by swelling and disintegration of the subadjacent marginal glial endfeet. Third, the original perforation enlarges progressively and allows an entire endothelial cell (or cells) to penetrate into the neural tissue. Fourth, proliferation and progressive canalization of penetrated endothelial cells result in the formation of a new cortical capillary in situ. Its proximal wall becomes surrounded by perivascular glial processes which appear to replace the degenerated ones. Thus a vasculoglial barrier is formed around the growing capillary, isolating it from the neural tissue, while the filopodia at its leading edge are still growing among the neural elements without recognizable basal lamina. The formation of a shallow pial-funnel at the capillary entrance can also be recognized at this time. This funnel contains the fine processes of leptomeningeal cells, a few collagen fibers, and the basal laminae of the penetrating vessel and of the perivascular glia. It represents an early stage in the formation of the Virchow-Robin space. The intracortical embryonic vasculature is characterized by both capillary angiogenesis and regression and by the formation of anastomotic plexuses. While capillary angiogenesis and reabsorption are found everywhere and appear as random phenomena, the location of the anastomotic plexuses is specific and always associated with actively growing cortical regions.

BASEMENT MEMBRANE SURFACES AND PERIVASCULAR COMPARTMENTS IN NORMAL HUMAN BRAIN AND GLIAL TUMOURS. A SCANNING ELECTRON MICROSCOPE STUDY

The relationship of perivascular tissues to arteries and veins in normal brain and glial tumors were investigated by light microscopy and by scanning and transmission electron microscopy. Vessels and perivascular tissues were separated through various planes by careful tearing of fixed tissue blocks of brain and tumour. Mirror surfaces of torn blocks were examined by scanning electron microscopy and the identity of the vessels and other structure confirmed by transmission electron microscopy. Perivascular glial basement membranes remained adherent to arterial adventitia in both normal brain and in tumour so that the torn surface around the vessel exposed perivascular glial processes attached to the vessel walls. A clear plane of separation of perivascular glial basement membrane from the adventitia of veins was achieved in glial tumours. Mirror surfaces showed the smooth undulating sheet of basement membrane separated from the fine fibrillary connective tissue of the vessel wall. Tears in the basement membrane revealed the perivascular glial processes. The structure of the perivascular basement membrane is discussed in relation to its role as an attachment site for perivascular glial and as an impedence to inflammatory cell migration into brain parenchyma.

Experimental cerebral infarction in dog. (Part IV). Ultrastructural study of microvasculature in recanalization model (author's transl)

Experimental cerebral infarction has been produced in dogs by clipping the middle cerebral artery and lowering the systemic blood pressure to 50mmHg for one hour. In this study, changes in microvasculatures in or near the ischemic area of the experimental animals were examined with light and electron microscope. All the dogs were sacrificed 24 hours after restoration of blood pressure. They were devided into three groups: the clip was permanently placed (Group 1); the clip was removed at the time of sacrifice (Group 2); and the clip was removed 4 hours after restoration (Group 3). Hemorrhage from venules at the boundary zone between the gray and white matters was the common and conspicuous finding in all groups. Venules showed severe ischemic damages including disruption of the limiting membrane of the swollen perivascular glial processes. The enlarged perivascular space was filled with erythrocytes. The capillaries of this boundary zone were occluded by erythrocytes and by the endothelial swelling, but were not ruptured. In the ischemic cortex, capillaries of Group 1 and 2 were packed with erythrocytes and platelets, but were not broken down. In the third group, on the other hand, hemorrhage from necrotic capillaries was one of the characteristic findings; Endothelial flattening and discontinuity and destruction of the basement membrane were observed under electronmicroscope. Marked brain edema was also seen. These results indicated that changes in the fine structure of the microvasculature in the area of focal cerebral ischemia varies according to the location of the area and the degree of ischemia.

Effects of aging on the structural and permeability characteristics of cerebrovasculature in normotensive and hypertensive strains of rats.

This study demonstrates that markedly different patterns of age-related changes in blood pressure and body weight occur among normotensive Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats and spontaneously hypertensive rats (SHR). In addition, a variety of age-related structural alterations occurred in the walls of arterioles, capillaries, and venules of the frontal cortex. These changes include: (1) an increase in the thickness of the vascular wall by deposits of collagen and basal lamina which, in some cases, extended into the surrounding neuropil; (2) the presence of a flocculent material in the adventitia of intracerebral arterioles; (3) vesicular inclusions in perivascular macrophages, pericytes and smooth muscle cells which were labelled with i.v. administered horseradish peroxidase (HRP); (4) fragmentation of smooth muscle cells; and (5) accumulation of lipofuscin-like pigments in perivascular glial processes. The hypertensive rats exhibited these changes, but they were more advanced and more widely distributed throughout the cerebral cortex. The aged hypertensive rats occasionally had large bundles of 10 nm diameter, intermediate filaments in the endothelial cells. Whereas no change in blood-brain barrier permeability to HRP was observed in the aged normotensive rats, all age groups of the hypertensive rats exhibited increased permeability to HRP in the initial segment of penetrating arterioles in laminae I and II of the cerebral cortex.