Perivascular Astrocytic Endfeet Research Articles

Astrocyte-endothelial interactions and blood-brain barrier permeability.

The blood-brain barrier (BBB) is formed by brain endothelial cells lining the cerebral microvasculature, and is an important mechanism for protecting the brain from fluctuations in plasma composition, and from circulating agents such as neurotransmitters and xenobiotics capable of disturbing neural function. The barrier also plays an important role in the homeostatic regulation of the brain microenvironment necessary for the stable and co-ordinated activity of neurones. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of more complex tight junctions than in other capillary endothelia, and a number of specific transport and enzyme systems which regulate molecular traffic across the endothelial cells. Transporters characteristic of the BBB phenotype include both uptake mechanisms (e.g. GLUT-1 glucose carrier, L1 amino acid transporter) and efflux transporters (e.g. P-glycoprotein). In addition to a role in long-term barrier induction and maintenance, astrocytes and other cells can release chemical factors that modulate endothelial permeability over a time-scale of seconds to minutes. Cell culture models, both primary and cell lines, have been used to investigate aspects of barrier induction and modulation. Conditioned medium taken from growing glial cells can reproduce some of the inductive effects, evidence for involvement of diffusible factors. However, for some features of endothelial differentiation and induction, the extracellular matrix plays an important role. Several candidate molecules have been identified, capable of mimicking aspects of glial-mediated barrier induction of brain endothelium; these include TGFbeta, GDNF, bFGF, IL-6 and steroids. In addition, factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) have been shown to induce astrocytic differentiation. Thus endothelium and astrocytes are involved in two-way induction. Short-term modulation of brain endothelial permeability has been shown for a number of small chemical mediators produced by astrocytes and other nearby cell types. It is clear that endothelial cells are involved in both long- and short-term chemical communication with neighbouring cells, with the perivascular end feet of astrocytes being of particular importance. The role of barrier induction and modulation in normal physiology and in pathology is discussed.

Astrocyte–endothelial interactions and blood–brain barrier permeability

The blood–brain barrier (BBB) is formed by brain endothelial cells lining the cerebral microvasculature, and is an important mechanism for protecting the brain from fluctuations in plasma composition, and from circulating agents such as neurotransmitters and xenobiotics capable of disturbing neural function. The barrier also plays an important role in the homeostatic regulation of the brain microenvironment necessary for the stable and coordinated activity of neurons. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of more complex tight junctions than in other capillary endothelia, and a number of specific transport and enzyme systems which regulate molecular traffic across the endothelial cells (Abbott & Romero, Mol. Medical Today2, 1996). Transporters characteristic of the BBB phenotype include both uptake mechanisms (e.g. GLUT‐1 glucose carrier, L1 amino acid transporter) and efflux transporters (e.g. P‐glycoprotein, MRP). In addition to their role in long‐term barrier induction and maintenance, astrocytes and other cells can release chemical factors that modulate endothelial permeability over a timescale of seconds to minutes.Cell culture models, both primary and cell lines, have been used to investigate aspects of barrier induction and modulation. Conditioned medium taken from growing glial cells can reproduce some of the inductive effects, evidence for involvement of diffusible factors. However for some features of endothelial differentiation and induction the extracellular matrix plays an important role. Several candidate molecules have been identified capable of mimicking aspects of glial–mediated barrier induction of brain endothelium; these include TGF(GDNF, bFGF, IL‐6 and steroids. In addition factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) have been shown to induce astrocytic differentiation. Thus endothelium and astrocytes are involved in 2‐way mutual induction. Short‐term modulation of brain endothelial permeability has been shown for a number of small chemical mediators produced by astrocytes and other nearby cell types. Thus endothelial cells are involved in both long and short‐term chemical communication with neighbouring cells, with the perivascular end feet of astrocytes being of particular importance. The role of barrier induction and modulation in normal physiology and in pathology will be discussed.

A light and electron microscopic study of oedematous human cerebral cortex in two patients with post-traumatic seizures

Primary objective : Brain cortical biopsies of two patients with clinical diagnosis of complicated brain trauma who had seizures, were studied by means of light and electron microscopes in order to correlate structural alterations with seizure activity. Methods and procedures : Biopsy samples of left frontal cortex and right parietal cortex were processed by current techniques for light and transmission electron microscopy. Results : The tissue showed severe vasogenic oedema with perivascular and intraparenchymatous haemorrhages. At the capillary wall, increased vesicular and vacuolar transendothelial transport, open endothelial junctions, thickened basement membrane and swollen perivascular astrocytic end-feet were observed. Some pyramidal and non-pyramidal nerve cells appeared dense and shrunken and others exhibited marked intraneuronal enlargement of membrane compartment. The myelinated axons displayed signs of degeneration and a process of axonal sprouting. Numerous swollen asymmetrical axo-dendritic synaptic contacts were observed in the neuropil, which exhibited mostly closely aggregated spheroidal synaptic vesicles toward the presynaptic membrane and numerous exocytotic vesicles sites. The perisynaptic astrocytic ensheathment appeared retracted or absent, whereas the extracellular space appeared notably dilated. Synaptic disassembly was also observed. Conclusion : The findings demonstrate, in two patients with post-traumatic seizure activity, brain brarrier dysfunction, vasogenic oedema, anoxic-ischaemic neurons, axonal sprouting, numerous altered excitatory synapses and synaptic disassembly. Some considerations on clinical and research applications are discussed.

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein.

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, beta-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking alpha-syntrophin (alpha-Syn(-/-)). The total level of AQP4 expression appears normal in brains of alpha-Syn(-/-) mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of alpha-Syn(-/-) mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in alpha-Syn(-/-) mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse-chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires alpha-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

Presence of aquaporin-4 and muscarinic receptors in astrocytes and ependymal cells in rat brain: a clue to a common function?

Using combined double immunofluorescence and laser confocal microscopy, we studied the common cellular localization of cholinergic muscarinic receptors (mAChRs) and aquaporin-4 water channels (AQP4) in the cortex, the corpus callosum and in ependymal cells of the rat brain. In the cortex, AQP4 staining was restricted to the perivascular end-feet of astrocytes. It was more widely distributed on the astrocytes of the corpus callosum. On astrocytes, mAChRs were often present in regions immunoreactive to AQP4. Ependymal cells bordering the third ventricle were also stained by both antibodies. The double staining of mAChRs with AQP4 on two different cell-types might indicate that further interactions exist which may be important in the regulation of water and electrolyte movements in the brain.

Do Glia Have Heart? Expression and Functional Role forEther-A-Go-GoCurrents in Hippocampal Astrocytes

Potassium homeostasis plays an important role in the control of neuronal excitability, and diminished buffering of extracellular K results in neuronal Hyperexcitability and abnormal synchronization. Astrocytes are the cellular elements primarily involved in this process. Potassium uptake into astrocytes occurs, at least in part, through voltage-dependent channels, but the exact mechanisms involved are not fully understood. Although most glial recordings reveal expression of inward rectifier currents (K(IR)), it is not clear how spatial buffering consisting of accumulation and release of potassium may be mediated by exclusively inward potassium fluxes. We hypothesized that a combination of inward and outward rectifiers cooperate in the process of spatial buffering. Given the pharmacological properties of potassium homeostasis (sensitivity to Cs(+)), members of the ether-a-go-go (ERG) channel family widely expressed in the nervous system could underlie part of the process. We used electrophysiological recordings and pharmacological manipulations to demonstrate the expression of ERG-type currents in cultured and in situ hippocampal astrocytes. Specific ERG blockers (dofetilide and E 4031) inhibited hyperpolarization- and depolarization-activated glial currents, and ERG blockade impaired clearance of extracellular potassium with little direct effect on hippocampal neuron excitability. Immunocytochemical analysis revealed ERG protein mostly confined to astrocytes; ERG immunoreactivity was absent in presynaptic and postsynaptic elements, but pronounced in glia surrounding the synaptic cleft. Oligodendroglia did not reveal ERG immunoreactivity. Intense immunoreactivity was also found in perivascular astrocytic end feet at the blood-brain barrier. cDNA amplification showed that cortical astrocytes selectively express HERG1, but not HERG2-3 genes. This study provides insight into a possible physiological role of hippocampal ERG channels and links activation of ERG to control of potassium homeostasis.

Aquaporin-4 is absent at the sarcolemma and at perivascular astrocyte endfeet in α1-syntrophin knockout mice

α1-Syntrophin, a member of dystrophin-associated proteins, is expressed at the sarcolemma and at perivascular astrocytes, and participates in protein-protein interactions through its PDZ domain. Aquaporin-4 (AQP4) is the predominant water channel protein in the brain, and also expressed at the sarcolemma of fast-twitch muscle fibers. AQP4 is concentrated in orthogonal array particles (OAPs), and its expression has been reported to be decreased at the sarcolemma of dystrophin-deficient mdx mice. We examined whether α1-syntrophin targets AQP4 at the sarcolemma. Immunohistochemistry showed that AQP4 is absent at the sarcolemma in α1-syntrophin knockout mice and that its expression is also lost from the perivascular astrocyte endfeet. On the other hand, expression of AQP4 is not decreased at the sarcolemma of the knockout mice in the neonatal stage. Moreover, AQP4 is expressed in lung, stomach, and kidney of wild-type and α1-syntrophin null mice. Our results show that α1-syntrophin is a key molecule to localize AQP4 to the sarcolemma of mature fast myofibers and astrocyte endfeet, but AQP4 is targeted to the plasma membrane by different molecules in lung, stomach, and kidney.

Heterotetrameric composition of aquaporin-4 water channels.

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.

Connexin30 in rodent, cat and human brain: selective expression in gray matter astrocytes, co-localization with connexin43 at gap junctions and late developmental appearance

We previously presented evidence (Nagy et al., Neuroscience, 1997, 78, 533–548) that, in addition to their ubiquitous expression of connexin43, astrocytes produce a second connexin suggested to be connexin30, a recently discovered member of the family of gap junction proteins. A connexin30 specific antibody was subsequently developed and utilized here to confirm and extend our earlier observations. On Western blots, this antibody detected a 30,000 mol. wt protein in rat, mouse, cat and human brain, and exhibited no cross-reaction with connexin43, connexin26 or any other known connexins expressed in brain. Immunohistochemically, connexin30 was localized in astrocytes, at gap junctions between these cells and on the astrocyte side of gap junctions between astrocytes and oligodendrocytes. Double labelling revealed co-localization of connexin30 and connexin43 at astrocytic gap junctions. Punctate immunolabelling patterns for both connexins were qualitatively similar, but differences were evident. In contrast to regional connexin43 expression, diencephalic and hindbrain areas exhibited considerably greater expression than forebrain areas, subcortical perivascular astrocytic endfeet were more heavily labelled for connexin30, white matter tracts such as corpus callosum, internal capsule and anterior commissure were devoid of connexin30, and appreciable levels of connexin30 during development were not seen until about postnatal day 15. These results indicate that connexin30 is expressed by gray, but not white matter astrocytes, its distribution is highly heterogeneous in gray matter, it is co-localized with connexin43 at astrocytic gap junctions where it forms homotypic or heterotypic junctions, and its emergence is delayed until relatively late during brain maturation. Taken together, these results suggest that astrocytic connexin30 expression at both regional and cellular levels is subject to regulation in adult brain as well as during brain development.

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: A confocal microscopy study

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three-dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S-100β] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S-100β showed numerous well-defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well-defined rosette-like structures lying on the vessel wall. Double immunostaining of GFAP and GLUT1 showed that the endothelial cells were the main targets of these repeated geometrical units formed by the astrocyte endfeet. When three-dimensional images were reconstructed, obvious privileged anatomical relationships were observed between endfeet and individual endothelial cells. These anatomical data provide strong support for the involvement of astrocytes in cerebral metabolic coupling. The finger-like appearance of astrocyte endfeet could allow direct metabolic exchanges between intracerebral vessels and non-glial elements such as nerve terminals. GLIA 23:1–10, 1998. © 1998 Wiley-Liss, Inc.

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: A confocal microscopy study

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three-dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S-100beta] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S-100beta showed numerous well-defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well-defined rosette-like structures lying on the vessel wall. Double immunostaining of GFAP and GLUT1 showed that the endothelial cells were the main targets of these repeated geometrical units formed by the astrocyte endfeet. When three-dimensional images were reconstructed, obvious privileged anatomical relationships were observed between endfeet and individual endothelial cells. These anatomical data provide strong support for the involvement of astrocytes in cerebral metabolic coupling. The finger-like appearance of astrocyte endfeet could allow direct metabolic exchanges between intracerebral vessels and non-glial elements such as nerve terminals.

Morphological astrocytic changes in complicated human brain trauma. A light and electron microscopic study

The astrocytic changes and reactivity in human brain trauma complicated with subdural haematoma or hygroma have been analysed in nine patients. Cortical biopsies of frontal, temporal and parietal regions were examined with light and transmission electron microscopy. At light microscopy level oedematous clear and dense astrocytes, binucleated and multinucleated atrocytes and hypertrophic reactive astrocytes were distinguished in either moderate or severe vasogenic brain oedema. Swollen, clear and dense perineuronal astrocytes were observed compressing and indenting dark, degenerated pyramidal and non pyramidal nerve cells. At electron microscopy level glycogen depleted and glycogen rich astrocytes were found in some patients. Dense and reactive hypertrophic astrocytes exhibited increased amounts of dilated smooth endoplasmic reticulum, microtubules, lipofucsin granules, gliofilaments and alpha type glycogen particles. In severe oedema the astrocytic ensheathment of synaptic contacts is lost, the perivascular astrocyte end feet appeared dissociated from the capillary basement membrane and the interastrocytary gap junctions are disrupted. The posttraumatic neurological deficits and the neurobehavioural disorders of the patient studied are correlated with astrocyte ultrastructural changes and blood brain barrier disruption.

The development of the blood-brain barrier in alcohol-exposed rats

Circulating horseradish peroxidase (HRP) was used as a tracer to determine if the blood-brain barrier to protein was altered by dietary prenatal alcohol exposure. Animals were prepared for light microscopic visualization of HRP after HRP infusion on gestational days 16, 18, 20, 22 and postnatal day 4. There was no consistent evidence of HPR leakage through the BBB in hte alcohol-exposed animals compared to control animals. Capillary endothelial cells and perivascular astrocytic endfeet were morphologically characterized by electron microscopy in rat optic nerve and cerebellum following dietary prenatal and postnatal ethanol exposure. Photomontages of optic nerve capillaries from G20 and P5 animals and cerebellar capillaries from P15 animals were examined for evidences of effects of alcohol on the development of the capillaries and adjacent astroglial endfeet. There was no consistent evidence of any alcohol-induced effects that could indicate a disruption of the vessel, the endothelial tight junctions, the perivascular glial limiting membranes, or the extent of vascular ensheathment by astrocytic endfeet.

Dystroglycan in the cerebellum is a laminin alpha 2-chain binding protein at the glial-vascular interface and is expressed in Purkinje cells.

Dystroglycan is a core component of the dystrophin receptor complex in skeletal muscle which links the extracellular matrix to the muscle cytoskeleton. Dystrophin, dystrophin-related protein (DRP, utrophin) and dystroglycan are present not only in muscles but also in the brain. Dystrophin is expressed in certain neuronal populations while DRP is associated with perivascular astrocytes. To gain insights into the function and molecular interactions of dystroglycan in the brain, we examined the localization of alpha- and beta-dystroglycan at the cellular and subcellular levels in the rat cerebellum. In blood vessels, we find alpha-dystroglycan associated with the laminin alpha 2-chain-rich parenchymal vascular basement membrane and beta-dystroglycan associated with the endfeet of perivascular astrocytes. We also show that alpha-dystroglycan purified from the brain binds alpha 2-chain-containing laminin-2. These observations suggest a dystroglycan-mediated linkage between DRP in perivascular astrocytic endfeet and laminin-2 in the parenchymal basement membrane similar to that described in skeletal muscle. This linkage of the astrocytic endfeet to the vascular basement membrane is likely to be important for blood vessel formation and stabilization and for maintaining the integrity of the blood-brain barrier. In addition to blood vessel labelling, we show that alpha-dystroglycan in the rat cerebellum is associated with the surface of Purkinje cell bodies, dendrites and dendritic spines. Dystrophin has previously been localized to the inner surface of the plasma membrane of Purkinje cells and is enriched at postsynaptic sites. Thus, the present results also support the hypothesis that dystrophin interacts with dystroglycan in cerebellar Purkinje neurons.

Astrocyte-endothelial interaction: physiology and pathology.

The blood-brain barrier of higher vertebrates is formed by the layer of endothelial cells lining the brain microvessels. The close anatomical association between endothelial cells and perivascular astrocytic end feet suggests cooperation between these cell types in forming and maintaining the blood-brain barrier. This review considers evidence from grafting experiments, developmental studies and culture models of the brain endothelium, concerning the inductive influences acting on the endothelium, and from endothelial cells acting on perivascular astrocytes. Examples from pathology and neurotoxicology which may involve breakdown of induction are also considered.

Computerized three-dimensional reconstruction of the developing blood-brain barrier.

Computer-aided techniques for three-dimensional reconstruction of images were employed in a morphological and morphometrical study of blood-brain barrier (BBB) development in the microvasculature of the chick embryo optic tectum. The method proved very useful for understanding the spatial relationships between BBB vascular and perivascular components, and of their modifications during embryonic life. The results indicate that the perivascular astrocytic endfeet, the main periendothelial components of the BBB, appear on the 14th incubation day and form an almost continuous perivascular envelope at hatching time.

Electron microscopic study of capillary wall in human cerebral edema.

Cortical biopsies of 17 patients with diagnoses of congenital malformation, cerebral tumor or brain trauma complicated by subdural hematoma or hygroma were studied with the electron microscope; the alterations of the blood-brain barrier in moderate and severe perifocal cerebral edema were observed. Moderate edema was found associated with central nervous system malformations, while severe edema was seen accompanying brain tumors and serious head injuries. The peripheral cytoplasm of endothelial cells displayed increased formation of microvilli and clear or amorphous electron-dense vacuoles, as well as pinocytotic and coated vesicles. In severe edema, the formation of elongated or chained vacuoles forming transendothelial channels was observed. In moderate edema, endothelial junctions exhibited a tortuous pathway, with a luminal portion sealed by tight junctions and dilated, open, basal portions ending at the basement membrane. In severe edema, the zonulae accludentes partially disappeared and the endothelial junction basal segments became irregularly dilated. The basement membrane showed enlargement, rarefaction, vacuolization, and the presence of collagen fibers. Fine expansion of the basement membrane was distinguished, showing matrix loss and clear, irregularly-dilated channels. Capillaries with reduplicated basement membrane were also observed. The pericytes exhibited edema, vacuolization, and phagocytic activity. In moderate cerebral edema, the clear or dense edematous and vacuolated perivascular end-feet of astrocytes appeared closely applied to the basement membrane, while in severe cerebral edema, they were separated from the basement membrane and showed gradual disappearance of the gap junctions. In all cases, moderately or markedly dilated extracellular spaces were observed in the pericapillary neuropil.