Radial glial cell development and transformation are disturbed in reeler forebrain

Radial glia are among the earliest cell types to differentiate in the developing mammalian forebrain. Glial fibers span the early cortical wall, forming a dense scaffold; this persists throughout corticogenesis, providing a cellular substrate which supports and directs the migration of young neurons. Although the mechanisms regulating radial glial cell development are poorly understood, a secreted cortical radial glial differentiation signal was recently identified in the embryonic mouse forebrain. This signal is abundant at the time radial glia function to support neuronal migration, and down-regulated perinatally, when radial glia are known to undergo transformation into astrocytes. Therefore, it seems that this signal functions as a radial glial maintenance factor, the availability of which regulates the phenotype of cortical astroglia. Here the differentiation signal is further characterized as RF60, a protein with a molecular weight of approximately 60 kD. In addition, the neurologic mutant mouse reeler provides a genetic model for analysis of RF60 function. Radial glia in reeler cortex are shown to be poorly differentiated and the radial scaffold is shown to be maintained for a shorter time than normal. Furthermore, although astroglial cells from normal cortex are induced to elaborate a radial phenotype by RF60, reeler astroglia show an impaired differentiation response to this. These findings suggest that an intrinsic defect in glial differentiation contributes to the phenotype of abnormal cortical lamination seen in reeler mouse, and indicate that RF60 may play a critical role in normal cortical patterning. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 459–472, 1997     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.     

Two forms of cerebellar glial cells interact differently with neurons in vitro

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.     

Granule cell induction of 9-O-acetyl gangliosides on cerebellar glia in microcultures

In previous studies we have shown that the expression of acetylated gangliosides recognized by the JONES monoclonal antibody is correlated with regions of cell migration in the developing rat nervous system. In this study we have investigated the expression of these gangliosides in two different types of cultures prepared from dissociated postnatal rat cerebella. In the first type, cells are plated after dissociation under conditions where most of the glial cells develop a stellate morphology that anchors neurons but does not support their migration. In the second type of culture, cells are plated in a ratio of four neurons to one glial cell and under these conditions the predominant form of astroglia is an elongate form that supports the migration of granule neurons. Granule neurons express JONES antigens in dissociated cell suspensions and in cultures in which cells are plated either after dissociation or in a 4:1 neuron:glia ratio. On the other hand, glial cells grown in the absence of neurons are JONES negative. In addition, the expression of JONES gangliosides by glial cells is different in the two types of culture. In cultures where the astroglial cells display the stellate morphology only a small proportion show JONES staining. Cultures in which the glial cells assume the elongate morphology have a significantly higher number of JONES-positive astroglia.     

Cytoprotective and anti-inflammatory effects of PAL31 overexpression in glial cells

Background

Acute spinal cord injury (SCI) leads to a series of reactive changes and causes severe neurological deficits. A pronounced inflammation contributes to secondary pathology after SCI. Astroglia respond to SCI by proliferating, migrating, and altering phenotype. The impact of reactive gliosis on the pathogenesis of SCI is not fully understood. Our previous study has identified an inflammatory modulating protein, proliferation related acidic leucine-rich protein (PAL31) which is upregulated in the microglia/macrophage of injured cords. Because PAL31 participates in cell cycle progression and reactive astroglia often appears in the injured cord, we aim to examine whether PAL31 is involved in glial modulation after injury.

Results

Enhanced PAL31 expression was shown not only in microglia/macrophages but also in spinal astroglia after SCI. Cell culture study reveal that overexpression of PAL31 in mixed glial cells or in C6 astroglia significantly reduced LPS/IFNγ stimulation. Further, enhanced PAL31 expression in C6 astroglia protected cells from H₂O₂ toxicity; however, this did not affect its proliferative activity. The inhibiting effect of PAL31 on LPS/IFNγ stimulation was observed in glia or C6 after co-culture with neuronal cells. The results demonstrated that the overexpressed PAL31 in glial cells protected neuronal damages through inhibiting NF-kB signaling and iNOS.

Conclusions

Our data suggest that PAL31upregulation might be beneficial after spinal cord injury. Reactive gliosis might become a good target for future therapeutic interventions.     

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development

The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.     

Neurons from radial glia: the consequences of asymmetric inheritance

Recent work suggests that radial glial cells represent many, if not most, of the neuronal progenitors in the developing cortex. Asymmetric cell division of radial glia results in the self-renewal of the radial glial cell and the birth of a neuron. Among the proteins that direct cell fate in Drosophila melanogaster that have known mammalian homologs, Numb is the best candidate to have a similar function in radial glia. During asymmetric divisions of radial glial cells, the basal cell may inherit the radial glial fibre, while the apical cell sequesters the majority of the Numb protein. We suggest two models that make opposite predictions as to whether the radial glia or nascent neuron inherit the radial glial fiber or the majority of the Numb protein.     

Basement membrane attachment is dispensable for radial glial cell fate and for proliferation, but affects positioning of neuronal subtypes

Radial glial cells have been shown to act as neuronal precursors in the developing cortex and to maintain their radial processes attached to the basement membrane (BM) during cell division. Here, we examined a potential role of direct signalling from the BM to radial glial cells in three mouse mutants where radial glia attachment to the BM is disrupted. This is the case if the nidogen-binding site of the laminin gamma1 chain is mutated, in the absence of alpha6 integrin or of perlecan, an essential BM component. Surprisingly, cortical radial glial cells lacking contact to the BM were not affected in their proliferation, interkinetic nuclear migration, orientation of cell division and neurogenesis. Only a small subset of precursors was located ectopically within the cortical parenchyma. Notably, however, neuronal subtype composition was severely disturbed at late developmental stages (E18) in the cortex of the laminin gamma1III4-/- mice. Thus, although BM attachment seems dispensable for precursor cells, an intact BM is required for adequate neuronal composition of the cerebral cortex.     

Administration of Hepatocyte Growth Factor Increases Reelin and Disabled 1 Expression in the Mouse Cerebral Cortex: An In Vivo Study

Hepatocyte growth factor (HGF) and its receptor, c-Met, are widely expressed in the developing brain. HGF also known as scatter factor enhances cell proliferation and cell growth, and stimulates cell migration and motility. Neurons and glia produced in the neuroepithelium migrate along radial glial fibers into the cortical plate. Reelin, a glycoprotein which is produced by Cajal–Retzius cells in the marginal zone directs neuronal migration indirectly via the radial glial cells. It has been demonstrated that Disabled 1 functions downstream of reelin in a tyrosin kinase signal transduction pathway that controls appropriate cell positioning in the developing brain. In this study, administration of HGF on reelin and Disabled 1 expression in the cerebral cortex has been studied. Using Western blot, it was shown that the expression of reelin and Disabled 1 is increased in response to infusion of HGF when compared to control group. It is concluded that HGF is essential for reelin and Disabled 1 expression in the cerebral cortex of the newborn mouse. Moreover, this method may be applied to the other factors, allowing identification of molecules involved in neural cell migration.     

Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex

The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.     

Bergmann Glia Function in Granule Cell Migration During Cerebellum Development

Granule cell migration influences the laminar structure of the cerebellum and thereby affects cerebellum function. Bergmann glia are derived from radial glial cells and aid in granule cell radial migration by providing a scaffold for migration and by mediating interactions between Bergmann glia and granule cells. In this review, we summarize Bergmann glia characteristics and the mechanisms underlying the effect of Bergmann glia on the radial migration of granule neurons in the cerebellum. Furthermore, we will focus our discussion on the important factors involved in glia-mediated radial migration so that we may elucidate the possible mechanistic pathways used by Bergmann glia to influence granule cell migration during cerebellum development.     

Adrenergic and cholinergic stimulation of arachidonate and phosphatidate metabolism in cultured astroglial cells

Primary cultures of neonatal rat brain polygonal astroglia, or cells of the C62B glioma line, were incubated with [1-¹⁴C]arachidonic acid (AA) in culture for 18 hr. In both culture systems, more than 80% of the added [1-¹⁴C]AA was taken up into cellular glycerolipids; less than 1% of the radioactivity in the cells was present in an unesterified form. When prelabeled C62B cells were stimulated with acetylcholine (ACh), there was a rapid accumulation of arachidonyl-phosphatidic acid (PA) accompanied by a liberation of [1-¹⁴C]AA. A variety of other neurotransmitters failed to activate this response in C62B glioma cells, primary astroglia generated PA and liberated [1-¹⁴C]AA in response to several neurotransmitters (i.e., ACh, norepinephrine, glutamate, and histamine) Treatment of astroglia with a combination of norephrine, ACh, and histamine resulted in a greater production of PA and free [1-¹⁴C]AA than did treatment with any one of these neurotransmitters alone. The results suggest that cultures of astroglia can respond to several different neurotransmitters with specific changes in AA and PA metabolism. Thus, a variety of neurotransmitters initiate cascades of lipid metabolism which may be of physiological significance in glial function.Special Issue dedicated to Prof. Eduardo De Robertis.     

Populations of radial glial cells respond differently to reelin and neuregulin1 in a ferret model of cortical dysplasia

Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not.     

Neuronal inhibition of astroglial cell proliferation is membrane mediated

Previously we have used a microwell tissue culture assay to show that early postnatal mouse cerebellar astroglia have a flattened morphology and proliferate rapidly when they are cultured in the absence of neurons, but develop specific cell-cell contacts and undergo morphological differentiation when they are co-cultured with purified granule neurons (Hatten, M. E., 1985, J. Cell Biol., 100:384-396). In these studies of cell binding between neurons and astroglia, measurement with light and fluorescence microscopy or with [35S]methionine-labeled cells indicated that the kinetics of the binding of the neurons to astroglial cells are rapid, occurring within 10 min of the addition of the neurons to the growing glia. 6 h after neuronal attachment, astroglial DNA synthesis decreases, as shown by a two- to fivefold decrease in [3H]thymidine incorporation, and glial growth ceases. No effects on astroglial cell growth were seen after adding medium conditioned by purified cerebellar neurons cultured in the absence of astroglia, by astroglia cultured in the absence of neurons, or by a mixed population of cerebellar cells. This result was unchanged when any of these media were concentrated up to 50-fold, or when neurons and astroglia were cultured in separate chambers with confluent medium. Two groups of experiments suggest that membrane-membrane interactions between granule neurons and astroglia control astroglial cell growth. First, neurons fixed with dilute amounts of paraformaldehyde (0.5%) bound to the astroglia with the same kinetics as did living cells, inhibited DNA synthesis, and arrested glial growth within hours. Second, a cell membrane preparation of highly purified granule neurons also bound rapidly to the glia, decreased [3H]thymidine incorporation two- to fivefold and inhibited astroglial cell growth. The rate of the decrease in glial growth depended on the concentration of the granule neural membrane preparation added. A similar membrane preparation from purified cerebellar astroglial cells, PC12 cells, 3T3 mouse fibroblasts, or PTK rat epithelial cells did not decrease astroglial cell growth rates. Living neurons were the only preparation that both inhibited glial DNA synthesis and induced the astroglial cells to transform from the flat, epithelial shapes they have when they are cultured without neurons to highly differentiated forms that resemble Bergmann glia or astrocytes seen in vivo. These results suggest that membrane-membrane interactions between neurons and astroglia inhibit astroglial proliferation in vitro, and raise the possibility that membrane elements involved in glial growth regulation include neuron-glial interaction molecules.     

Heparin affin regulatory peptide/pleiotrophin negatively affects diverse biological activities in C6 glioma cells

Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be involved in the progression of several tumors of diverse origin. In this study, we tried to determine the role of HARP in rat C6 glioma cells by using an antisense strategy for inhibition of HARP expression. Decrease of the expression of endogenous HARP in C6 cells (AS-C6 cells) significantly increased proliferation, migration, and anchorage-independent growth of cells. Implantation of AS-C6 cells onto chicken embryo chorioallantoic membranes resulted in a significant increase of tumor-induced angiogenesis compared with that induced by non-transfected or C6 cells transfected with the plasmid alone (PC-C6 cells). In the same line, conditioned medium from AS-C6 cells significantly increased endothelial cell proliferation, migration, and tube formation in vitro compared with the effect of conditioned medium from C6 or PC-C6 cells. Interestingly, vascular endothelial growth factor (VEGF) induced C6 cell proliferation and migration, and SU1496, a selective inhibitor of VEGF receptor 2 (VEGFR2), blocked increased glioma cell growth, migration, and angiogenicity observed in AS-C6 cell cultures. The above results seem to be due to a direct interaction between HARP and VEGF in the culture medium of C6 and PC-C6 cells, while AS-C6 cells secreted comparable amounts of VEGF that do not interact with HARP. Collectively, these data suggest that HARP negatively affects diverse biological activities in C6 glioma cells, mainly due to binding of HARP to VEGF, which may sequester secreted VEGF from signalling through VEGFR2.     

Expression of zebrafish fkd6 in neural crest-derived glia

The zebrafish fkd6 gene is a marker for premigratory neural crest. In this study, we analyze later expression in putative glia of the peripheral nervous system. Prior to neural crest migration, fkd6 expression is downregulated in crest cells. Subsequently, expression appears initially in loose clusters of cells in positions corresponding to cranial ganglia. Double labelling with a neuronal marker shows that fkd6-expressing cells are not differentiated neurones and generally lie peripheral to neurones in ganglia. Later, expression appears associated with the posterior lateral line and other cranial nerves. For the posterior lateral line nerve, we show that fkd6-labeling extends caudally along this nerve in tight correlation with lateral line primordium migration and axon elongation. Expression in colourless mutant embryos is consistent with these cells being satellite glia and Schwann cells.     

Formation and preservation of cortical layers in slice cultures.

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984-985; Berry and Rogers, 1965, J. Anat. 99:691-709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodeoxyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells continued to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slice, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61-84; Hatten and Mason, 1990, Experientia 46:907-916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cultures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells.     

Astroglial c-Myc overexpression predisposes mice to primary malignant gliomas

Malignant astrocytomas are common human primary brain tumors that result from neoplastic transformation of astroglia or their progenitors. Here we show that deregulation of the c-Myc pathway in developing astroglia predisposes mice to malignant astrocytomas within 2-3 weeks of age. The genetically engineered murine (GEM) gliomas harbor a molecular signature resembling that of human primary glioblastoma multiforme, including up-regulation of epidermal growth factor receptor and Mdm2. The GEM gliomas seem to originate in an abnormal population of glial fibrillary acidic protein-expressing cells in the ventricular zone and, analogous to human glioblastomas, exhibit molecular and morphological heterogeneity. Levels of connexin 43 in the majority of the tumors are unaltered from normal tissue, indicating that GEM tumors have retained the capacity to establish syncytial networks. In line with this, individual glioma foci are composed of a mixture of actively proliferating cells expressing c-Myc and proliferating cell nuclear antigen and less dividing bystander cells that express glial fibrillary acidic protein and the broad complex tramtrack bric-a-brac/poxvirus and zinc finger domain protein HOF. A subset of the transgenic mice harbored, in addition to brain tumors, vestigial cerebellums in which granule cell migration and radial Bergman glial cell differentiation were disturbed. These observations argue for a window of vulnerability during astrocyte development where c-Myc overexpression is sufficient to trigger the neoplastic process, presumably by inducing the sustained growth of early astroglial cells. This is in contrast to most other transgenic studies in which c-Myc overexpression requires co-operating transgenes for rapid tumor induction.     

Effect of astroglial cells on hypoxia-induced permeability in PBMEC cells

An in vitro model of the blood-brain barrier (BBB),consisting of porcine brain-derived microvascular endothelial cells(PBMEC), was used to evaluate the effect of astrocytes in theBBB disruption during hypoxia. Hypoxia-induced hyperpermeability wasdecreased significantly in a coculture model of astroglia cells, either astrocytes or C6 glioma cells, with PBMEC and, to the same extent, whenglia cell-conditioned medium was used. Corresponding to effects onhypoxia-induced hyperpermeability, astrocyte- and C6 cell-conditioned medium diminished hypoxia-induced vascular endothelial growth factor(VEGF) mRNA and protein expression, which recently was shown to beresponsible for hypoxia-induced permeability changes in vitro. Theeffect on hypoxia-induced hyperpermeability and VEGF expression wasspecific for astroglia cells because conditioned medium from bovinesmooth muscle cells (BSMC) did not show any effect. Immunocytochemistryrevealed that 24 h of hypoxia disrupted the continuity of thetight junction protein, zonula occludens-1 (ZO-1), which lines thecytoplasmic face of intact tight junctions. These changes wereprevented when hypoxia was performed in glia cell-conditioned medium.Results suggest that astrocytes protect the BBB from hypoxia-inducedparacellular permeability changes by decreasing hypoxia-induced VEGFexpression in microvascular endothelial cells.