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PubMed1993

Glia cells of the monkey retina. I. Astrocytes.

Morphology and distribution of retinal astrocytes have been studied in macaque monkeys by immunocytochemical localization of glial fibrillary acidic protein (GFAP). With the exception of the fovea and the far periphery, astrocytes are ubiquitous in the nerve fiber layer (NFL) and the ganglion cell layer (GCL) of the monkey retina. The morphology of NFL astrocytes changes gradually, from star-shaped in the periphery to bipolar close to the optic disc. By contrast, GCL astrocytes maintain their star-shaped appearance throughout the retina. Astrocytes are unevenly distributed in the monkey retina, showing the highest concentration around the optic disc, and particularly low densities in the perifoveal region and the far periphery. The fovea proper is devoid of astrocytes. Employing high-resolution confocal microscopy, we could demonstrate that astrocytes form manifold contacts to blood vessels. In addition, bundles of NFL astrocyte processes are co-localized with axon bundles, individual astrocytes forming contacts to several axon bundles. In contrast, a similar affinity of astrocytes to ganglion cell somata was never observed. Thus, our data confirm and extend the current knowledge of morphology and putative function of astrocytes in mammalian and especially the primate retina.

Animals↗
PubMed2002

Astrocyte-derived factors modulate the inhibitory effect of ethanol on dendritic development.

Numerous studies in vivo and in vitro have demonstrated that ethanol disrupts neuromorphogenesis. However, it has not been determined what role, if any, is played by non-neuronal cells in mediating this effect. We recently reported that ethanol inhibits dendritic development in low-density cultures of fetal rat hippocampal pyramidal neurons (Yanni and Lindsley, 2000: Dev Brain Res 120:233-243). In this culture system, cortical astrocytes precondition neuronal culture media for 2 days before the addition of neurons, which then develop on a separate substrate in coculture with the astrocytes. To determine whether astrocyte response to ethanol mediates the effects of ethanol on neurons, the present study compared dendritic development of neurons after 6 days in medium containing 400 mg/dl ethanol in coculture with live astrocytes and in conditioned medium from astrocytes that were never exposed to ethanol. The same experiment was also performed with and without ethanol present during astrocyte preconditioning of the medium. The effects of ethanol differed depending on when it was added to the cultures relative to addition of newly dissociated neurons. However, the effects of ethanol were not related to whether neurons were cocultured with live astrocytes. When astrocytes preconditioned the medium normally, ethanol added at plating inhibited dendritic development of neurons regardless of whether they were maintained in coculture with live astrocytes or in conditioned medium. In surprising contrast, the presence of ethanol during astrocyte preconditioning of the media had a growth promoting effect on subsequent dendrite development despite the continued presence of ethanol in the medium. Thus, astrocytes release soluble factors in response to ethanol that can protect neurons from the inhibitory effects of ethanol on dendritic growth, but the timing of neuronal exposure to these factors, or their concentration, may influence their activity.

Alcohol-Induced Disorders, Nervous System↗
PubMed2002

Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors.

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.

Amino Acid Sequence↗
PubMed2001

Astrocytes attenuate oligodendrocyte death in vitro through an alpha(6) integrin-laminin-dependent mechanism.

Oligodendrocyte (OL) death occurs in many disorders of the CNS, including multiple sclerosis and brain trauma. Factors reported to induce OL death include deprivation of growth factors, elevation of cytokines, oxidative stress, and glutamate excitotoxicity. Because astrocytes produce a large amount of growth factors and antioxidants and are a major source of glutamate uptake, we tested the hypothesis that astrocytes may have a protective role for OL survival. We report that when OLs from the adult mouse brain were initiated into tissue culture, DNA fragmentation and chromatin condensation resulted, indicative of apoptosis. OL death was significantly reduced in coculture with astrocytes, but not with fibroblasts, which provided a similar monolayer of cells as astrocytes. The protection of OL demise by astrocytes was not reproduced by its conditioned medium and was not accounted for by several neurotrophic factors. In contrast, interference with the alpha(6) integrin subunit, but not the alpha(1), alpha(2), alpha(3), alpha(4), alpha(5), or alpha(v) integrin chains, negated astrocyte protection of OLs. Furthermore, a function-blocking antibody to alpha(6)beta(1) integrin reduced the ability of astrocytes to promote OL survival. The extracellular matrix ligand for alpha(6)beta(1) is laminin, which is expressed by astrocytes. Significantly, neutralizing antibodies to laminin-2 and laminin-5 inhibited the astrocyte mediation of OL survival. These results implicate astrocytes in promoting OL survival through a mechanism involving the interaction of alpha(6)beta(1) integrin on OLs with laminin on astrocytes. Enhancing this interaction may provide for OL survival in neurological injury.

Animals↗
PubMed2005

Astrocytic contributions to bioenergetics of cerebral ischemia.

Astrocytes are multifunctional cells that interact with neurons and other astrocytes in signaling and metabolic functions, and their resistance to pathophysiological conditions can help restrict loss of tissue after an ischemic event provided adequate nutrients are supplied to support their requirements. Astrocytes have substantial oxidative capacity and mechanisms to upregulate glycolytic capability when respiration is impaired. An astrocytic enzyme that synthesizes a powerful activator of glycolysis is not present in neurons, endowing astrocytes with the ability to sustain ATP production under restrictive conditions. The monocarboxylic acid transporter (MCT) isoforms predominating in astrocytes are optimized to facilitate very large increases in lactate flux as lactate concentration increases within (1-3 mM) and above (>3 mM) the normal range. In sharp contrast, the major neuronal MCT serves as a barrier to increased transmembrane transport as lactate rises above 1 mM, restricting both entry and efflux. Lactate can serve as fuel during recovery from ischemia but direct evidence that lactate is oxidized by neurons (vs. astrocytes) to maintain synaptic function is lacking. Astrocytes have critical roles in regulation of ionic homeostasis and control of extracellular glutamate levels, and spreading depression associated with ischemia places high demands on energy supplies in astrocytes and contributes to metabolic exhaustion and demise. Disruption of Ca2+ homeostasis, generation of oxygen free radicals and nitric oxide, and mitochondrial depolarization contribute to astrocyte death during and after a metabolic insult. Novel pharmaceutical agents targeted to astrocytes and hyperoxic therapy that restores penumbral oxygen level during energy failure might improve postischemic outcome.

Animals↗
PubMed2005

CHOP plays a pivotal role in the astrocyte death induced by oxygen and glucose deprivation.

Ischemia has different consequences on the survival of astrocytes and neurons. Thus, astrocytes show a remarkable resistance to short periods of ischemia that are well known to cause neuronal death. We have used a cell culture model of stroke, oxygen, and glucose deprivation (OGD), to clarify the mechanisms responsible for the exclusive resistance of astrocytes to ischemia. The expression of genes implicated in both ischemia-induced astrocyte death and post-ischemic survival was analysed by the RNA differential display technique. Our study revealed that the expression of the CEBP homologous protein (CHOP)-coding gene is promptly an intensely upregulated following astrocyte oxygen and glucose deprivation. CHOP mRNA induction was accompanied by the activation of other genes (grp78, grp95) that, alike CHOP, are involved in the endoplasmic reticulum (ER) stress response. In addition, drugs that cause ER calcium depletion or protein N-glycosylation inhibition mimicked the effects of OGD on astrocyte survival, further supporting the involvement of ER in the astrocyte responses to OGD. Our experiments also demonstrated that upregulation of CHOP during the ER stress response is required for ischemia to cause astrocyte death. Not only the levels of CHOP mRNA and protein correlate perfectly with the degree of OGD-triggered cell injury, but also astrocyte death induced by OGD is significantly overcome by CHOP antisense oligonucleotide treatment. Nevertheless, we observed that astrocytes undergo apoptosis only when CHOP is permanently upregulated, and not when CHOP increases are transient. Finally, we found that the extent of CHOP induction is determined by the length of the ischemic stimulus. Taken together, our results indicate that permanent upregulation of CHOP is decisive for the induction of astrocyte death by OGD.

Animals↗
PubMed1992

Heterogeneity in gap junction expression in astrocytes cultured from different brain regions.

Heterogeneity among astrocytes suggests that their role in the central nervous system is more complex than is commonly recognized. This paper describes just such a functional difference, comparing gap junctions in astrocytes derived from two brain regions. Astrocytes, both in situ and in culture, employ gap junctions as a means of intercellular communication. Recent evidence utilizing cultured rat cortical and striatal astrocytes has shown that these channels consist of subunits of connexin 43, the same protein as that composing cardiac gap junctions. Here we report that astrocytes cultured from neonatal rat hypothalamus contain a greater number of functional channels than astrocytes from the striatum, a difference reflected in both connexin 43 protein and mRNA. Specifically, in hypothalamic astrocytes the level of connexin 43 protein was approximately four times that found in comparable cultures from the striatum, as determined by immunoblotting. Complementary results from immunocytochemical experiments using an antibody specific for connexin 43 reveal significantly greater fluorescence in astrocytes cultured from the hypothalamus as compared to those from the striatum. Northern blot analysis showed that connexin 43 mRNA levels were also approximately 4-fold greater in the hypothalamic cultures, consistent with the difference seen by immunoblotting. Finally, dye coupling studies using confluent cultures consistently showed that within 1 min Lucifer Yellow injected into striatal astrocytes spread to immediately surrounding cells while in hypothalamic astrocytes dye often spread to apparent third or fourth order neighbors within the same time period. Thus, the higher level of connexin 43 expression seen in hypothalamic astrocytes results in cells with greater numbers of functional channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Differences in the disposition and toxicity of 1-methyl-4-phenylpyridinium in cultured rat and mouse astrocytes.

Species difference in the susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was investigated in cultured rat and mouse astrocytes, where 1-methyl-4-phenylpyridinium (MPP+), the toxic mediator of MPTP, is formed. Type A monoamine oxidase (MAO) predominated in both rat and mouse astrocytes, while its activity was slightly higher in mouse cells compared to rat cells; MAO-B activity, on the other hand, was significantly lower in mouse astrocytes than in rat astrocytes. Because both types of MAO have been reported to make similar contributions to MPP+ production in astrocytes, their total activity was examined and results indicated that there was no significant difference between these two species. In addition, MPP+ caused a dose-dependent loss of cell viability as judged by the amount of lactate dehydrogenase released into the incubation medium. The toxicity of MPP+ on astrocytes started to be seen after a 2 day incubation period. Mouse astrocytes were more vulnerable to MPP+ than rat astrocytes. The threshold values for MPP+ toxicity in mouse and rat cultures were 10 microM and 70 microM, respectively. After addition of [3H] MPP+ to the medium, intracellular [3H] MPP+ was found to increase in both cultures. Mouse astrocytes accumulated more MPP+ than rat astrocytes (150 pmol/mg protein vs. 65 pmol/mg protein). When astrocytes were allowed to accumulate [3H] MPP+ and then incubated in fresh medium not containing [3H] MPP+, intracellular levels of [3H] MPP+ in both cells rapidly declined (110 pmol/protein in mouse vs. 40 pmol/mg protein in rat of MPP+ been released).(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2005

Proliferation of cerebellar neurons induced by astrocytes treated with thyroid hormone is mediated by a cooperation between cell contact and soluble factors and involves the epidermal growth factor-protein kinase a pathway.

Cerebellar development is fully dependent on thyroid hormone (T3) levels. We have previously demonstrated a glia-mediated effect of T3 on cerebellar neurons. We have reported that cerebellar astrocytes treated with thyroid hormone secrete epidermal growth factor (EGF), which directly induces neuronal proliferation and, indirectly, by increasing synthesis of extracellular matrix proteins, induces neurite outgrowth in vitro. Here, by using a neuron-astrocyte coculture model, we investigated the involvement of cell contact on neuronal proliferation. Culturing of cerebellar neurons on T3-treated astrocyte carpets or conditioned medium derived from them (T3CM) yielded similar results, revealed by a 60% increase in cell population. However, the absolute number of neurons in coculture assays was greatly enhanced in comparison with that in CM assays (3.5-4-fold). Bromodeoxyuridine (BrdU) incorporation assays revealed that such an increase was due mainly to proliferation of precursors cells. BrdU incorporation was three times higher in cell carpet (31%) than in CM (13%). Treatment of astrocytes by T3 increased neuronal proliferation either by T3CM (2.5 times) or by contact with T3-treated astrocytes (1.5 times). Neuronal death was not affected by T3 treatment of astrocytes as revealed by either trypan blue viability assays or active caspase-3 labeling. Treatment of astrocytes by EGF mimicked T3 effects on neuronal proliferation. Addition of the EGF receptor tyrosine kinase inhibitor genistein and the protein kinase A (PKA) inhibitor KT5720 to cocultutres and T3CM completely reversed neuronal proliferation. Our results implicate EGF and the PKA pathway in the proliferation induced by T3-treated astrocytes. Furthermore, the fact that cocultures potentiate the effect of T3 on neuronal proliferation suggests that neuron-astrocyte contact may cooperate with astrocyte soluble factors to enhance neuronal population. Our data reveal an important role of astrocytes as mediators of T3-induced cerebellar development and partially elucidate the role of cell contact and soluble factors on this process.

Animals↗
PubMed1991

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.

Animals↗
PubMed1992

Astrocyte cultures from human embryonic brain: characterization and modulation of surface molecules by inflammatory cytokines.

Astrocyte-enriched cultures were established upon passaging of primary cultures from the myelencephalon and mesencephalon of 7-9-week-old human embryos. Immunocytochemical analysis showed that third-fourth passage cultures were composed of a highly enriched population of proliferating, epithelioid cells, up to 90% of which expressed glial fibrillary acidic protein (GFAP); no macrophages and very few fibroblasts (less than 2%) were present. GFAP expression and proliferation declined upon further culturing in serum-containing medium but could be transiently reinduced by growing the cells in a serum-free chemically defined medium. Large numbers of GFAP+ astrocytes were obtained from each embryo and could be stored frozen and recultured. Using flow cytometric analysis, human astrocyte cultures were examined for basal and cytokine [interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha)]-induced expression of molecules that may be involved in astrocyte-T-lymphocyte interactions. Cultured human astrocytes spontaneously expressed major histocompatibility complex (MHC) class I antigens and variable levels of MHC class II; MHC class I levels were increased upon IFN-gamma and TNF-alpha treatment, whereas MHC class II antigens were induced on most of the astrocytes by IFN-gamma. Among the molecules involved in antigen-independent interactions between T lymphocytes and target cells, lymphocyte function-associated molecule-3 (LFA-3) was spontaneously expressed by most cultured human astrocytes, whereas intercellular adhesion molecule-1 (ICAM-1) was present at variable levels in non-stimulated astrocytes and was greatly induced by IFN-gamma, TNF-alpha, and IL-1 beta. In this study we also show that the above cytokines upregulate astroglial expression of adhesion molecules of the integrin family (VLA-1, VLA-2, and VLA-6) that may be involved in astrocyte-extracellular matrix interaction and play a role in the astrocyte reactive changes occurring at sites of brain injury and inflammation. The human astrocyte cultures developed here represent a useful in vitro model to further investigate mechanisms involved in bidirectional communication between central glia and cells of the immune system.

Astrocytes↗
PubMed1992

Microbeam laser-injured neurons increase in vitro astrocytic gap junctional communication as measured by fluorescence recovery after laser photobleaching.

An important aspect of the neuronal-astrocyte relationship is the interaction of reactive astrocytes with injured and/or dying neurons. Few studies have focused on the signaling of astrocytes by injured neurons or on the possibility that neurons can alter astrocytic gap junctional communication. The purpose of this study was to determine whether the presence of injured neurons could alter astrocytic gap junctional coupling by establishing an in vitro method of microbeam laser neuronal injury and coculturing these neurons with astrocytes. Neurons from two rat neuronal clonal cell lines were injured using a 20-W argon laser operating on the ultraviolet (UV) multiline (351-361 nm) directed through a X40 objective of an inverted microscope. After laser injury, the glass slide with the injured neurons was sandwiched with a slide on which primary rat astrocytes were grown. Although the neurons and astrocytes were bathed in the same medium, they were not in direct contact during the coculture period (24, 48, or 72 hr). Astrocytic gap junctional dye coupling was examined using the fluorescence recovery after laser photobleaching (gap-FRAP) analysis technique. Astrocytes cocultured with the injured neurons for 24 to 48 hr did not show a significant difference in fluorescence recovery when compared to control values. After 72 hr of coculture, there was a significant increase in the gap junctional dye coupling. These results indicate that injured neurons influence in vitro astrocytic gap junctional conductance after 72 hr of coculture as measured by dye coupling.

Animals↗
PubMed2002

Ciliary neurotrophic factor activates spinal cord astrocytes, stimulating their production and release of fibroblast growth factor-2, to increase motor neuron survival.

At focal CNS injury sites, several cytokines accumulate, including ciliary neurotrophic factor (CNTF) and interleukin-1beta (IL-1beta). Additionally, the CNTF alpha receptor is induced on astrocytes, establishing an autocrine/paracrine loop. How astrocyte function is altered as a result of CNTF stimulation remains incompletely characterized. Here, we demonstrate that direct injection of CNTF into the spinal cord increases GFAP expression and astroglial size and that primary cultures of spinal cord astrocytes treated with CNTF, IL-1beta, or leukemia inhibitory factor exhibit nuclear hypertrophy comparable to that observed in vivo. Using a coculture bioassay, we further demonstrate that CNTF treatment of astrocytes increases their ability to support ChAT(+) ventral spinal cord neurons (presumably motor neurons) more than twofold compared with untreated astrocytes. Also, the complexity of neurites was significantly increased in neurons cultured with CNTF-treated astrocytes compared with untreated astrocytes. RT-PCR analysis demonstrated that CNTF increased levels of FGF-2 and nerve growth factor (NGF) mRNA and that IL-1beta increased NGF and hepatocyte growth factor mRNA levels. Furthermore, both CNTF and IL-1beta stimulated the release of FGF-2 from cultured spinal cord astrocytes. These findings demonstrate that cytokine-activated astrocytes better support CNS neuron survival via the production of neurotrophic molecules. We also show that CNTF synergizes with FGF-2, but not epidermal growth factor, to promote DNA synthesis in spinal cord astrocyte cultures. The significance of these findings is discussed by presenting a new model depicting the sequential activation of astrocytes by cytokines and growth factors in the context of CNS injury and repair.

Animals↗
PubMed1990

Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes.

Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP) + 1 microM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP + DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-14C]glutamate was recovered in the medium as radioactive glutamine, 2-3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1979

Assemblies of particles in the cell membranes of developing, mature and reactive astrocytes.

Orthogonal arrays of small intramembranous particles characterize freeze-fractured astrocytic plasma membranes. The normal variation of assemblies in plasma membranes of subpial astrocytic processes in mature and developing rats was established and compared with assemblies in plasma membranes of reactive astrocytes. In mature rats, subpial astrocytic processes had the greatest number of assemblies. As the parenchyma was approached, this number decreased in each successive layer of astrocytic processes. The advent of assemblies within the plasma membrane is a new criterion of astrocytic differentiation. The foot processes of subpial astrocytes in foetal rats began to acquire assemblies between day 19 and 20 and continued to mature postnatally by a constant addition and rearrangement of assemblies. In contrast to the paucity of assemblies in deeper laminae of the normal brain, reactive astrocytes comprising the lower lamellae in glial scars had an increased number of assemblies while the most striking feature of the superficial, astrocytic processes within the scar was a rearrangement of assemblies. Although the function of these intramembranous particles in still unknown, it appears from our measurements that they are localized primarily in the outermost astrocytic foot process and to a progressively lesser degree in the underlying astrocytic lamellae.

Aging↗
PubMed1998

Neurovirulent simian immunodeficiency virus induces calbindin-D-28K in astrocytes.

Astrocyte activation has been postulated to be a major contributor to functional changes in the brain of AIDS patients. We assessed astrocyte activation in the simian immunodeficiency virus (SIV) model. Four groups of macaque brains were examined: uninoculated controls, animals inoculated with virus that did not cause disease, animals inoculated with virus that caused AIDS but did not cause encephalitis, and animals with SIV encephalitis. We examined expression of calbindin-D-28K, a calcium binding protein that is upregulated in astrocytes during excitotoxic events, as well as glial fibrillary acidic protein (GFAP). The presence of calbindin in astrocytes was confirmed by double-labeling using confocal microscopy. Increases in calbindin staining were most apparent in the white matter, but increases in GFAP staining were most apparent in middle layers of the cerebral cortex. Six of the seven animals with SIV encephalitis had calbindin immunoreactive astrocytes in the subcortical white matter, corpus callosum, internal capsule, cerebral peduncle, pontine white matter, and cerebellar white matter. Very rarely, a few, very lightly calbindin-immunoreactive astrocytes were present in the uninoculated control brains. The increase in calbindin expression by astrocytes in SIV encephalitis suggests that these cells are subject to calcium toxicity. In uninoculated control macaques, and in macaques inoculated with virus that did not cause disease, GFAP-immunoreactive astrocytes were present throughout the subcortical white matter and in layer I, but very few were found in layers III-V of the cerebral cortex. Two animals that died of AIDS without encephalitis had somewhat higher numbers of GFAP immunoreactive astrocytes in middle cortical layers. In seven animals that received passaged neurovirulent virus and developed both AIDS and encephalitis, the number of GFAP-immunoreactive astrocytes in middle cortical layers was high, indicating widespread astrocyte activation.

Animals↗
PubMed2006

Intracellular calcium-binding protein S100A4 influences injury-induced migration of white matter astrocytes.

Astrocytes play a crucial role in central nervous system (CNS) pathophysiology. White and gray matter astrocytes are regionally specialized, and likely to respond differently to CNS injury and in CNS disease. We previously showed that the calcium-binding protein S100A4 is exclusively expressed in white matter astrocytes and markedly up-regulated after injury. Furthermore, down-regulation of S100A4 in vitro significantly increases the migration capacity of white matter astrocytes, a property, which might influence their function in CNS tissue repair. Here, we performed a localized injury (scratch) in confluent cultures of white matter astrocytes, which strongly express S100A4, and in cultures of white matter astrocytes, in which S100A4 was down-regulated by transfection with short interference (si) S100A4 RNA. We found that S100A4-silenced astrocytes rapidly migrated into the injury gap, whereas S100A4-expressing astrocytes extended hypertrophied processes toward the gap, but without closing it. To explore the involvement of S100A4 in migration of astrocytes in vivo, we induced focal demyelination and transient glial cell elimination in the spinal cord white matter by ethidium bromide injection in S100A4 (-/-) and (+/+) mice. The results show that astrocyte migration into the demyelinated area is promoted in S100A4 (-/-) compared to (+/+) mice, in which a pronounced glial scar was formed. These data indicate that S100A4 reduces the migratory capacity of reactive white matter astrocytes in the injured CNS and is involved in glial scar formation after injury.

Animals↗
PubMed1991

The use of genetically altered astrocytes to provide nerve growth factor to adrenal chromaffin cells grafted into the striatum.

Transplantation of adrenal chromaffin cells into the striatum of Parkinson's disease patients is unlikely to become a reliable therapy unless techniques are devised to improve cell survival. To address this issue, we investigated the use of genetically altered astrocytes that constitutively secrete beta-nerve growth factor (NGF) to provide trophic support for adrenal chromaffin cells grafted into the dopamine-denervated striatum of the rat. Primary rat astrocytes were altered genetically in vitro by infection with a retroviral vector harboring a mouse beta-NGF transgene under constitutive long terminal repeat transcriptional control. Confluent cultures of these genetically altered astrocytes secrete NGF into their culture medium at a rate of approximately 9 pg/10(5) cells/h. This rate of NGF secretion is at least 10-fold higher than that of confluent sister cultures of uninfected astrocytes. The effects of the NGF-secreting astrocytes on the survival and neuronal transformation of dissociated adrenal chromaffin cells were assessed in vitro and following transplantation into the dopamine-denervated striatum of the adult rat. In vitro experiments demonstrated that neuritic outgrowth is stimulated when postnatal day 12 chromaffin cells are grown on a monolayer of the genetically altered astrocytes. When co-grafted with genetically altered astrocytes, young postnatal chromaffin cells displayed extensive neuritic outgrowth within the host brain 2 weeks postimplantation, whereas chromaffin cells grafted alone or with normal astrocytes retain an endocrine-like morphology. Survival of the chromaffin cells is also enhanced 3-6-fold when co-grafted with the genetically altered astrocytes. In addition, the neuronally transformed chromaffin cells appear to lose adrenergic properties as assessed by diminished immunoreactivity to the adrenergic marker, phenylethanolamine-N-methyltransferase. Although their survival is also enhanced approximately 4-fold relative to controls, adult chromaffin cells do not convert to a neuronal morphology when co-grafted with the genetically altered astrocytes. These studies demonstrate that rat astrocytes carrying a mouse NGF transgene provide trophic support for intrastriatal chromaffin cell grafts.

3T3 Cells↗