Astroglia Development Research Articles

MicroRNA Expression Profiling of Propofol-Treated Developing Rat Hippocampal Astrocytes.

Although propofol exerts toxic effects on the developing central nervous system (CNS), it remains a first-choice anesthetic in the pediatric population. Astrocytes represent a major glial cell population whose role in CNS development is widely appreciated and that has been recently shown to be mediated in large part by microRNAs (miRNAs). In contrast, relatively little is known about the roles of miRNAs in developing astrocytes during propofol treatment. Here, miRNA microarray was used to profile fluctuations in miRNA expression in immature hippocampal astrocytes in response to propofol treatment, and results were subsequently validated using quantitative real-time polymerase chain reaction. Predictive analysis of genes targeted by propofol-regulated miRNAs indicated enrichment of genes in the gene ontology (GO) nervous system development and differentiation category, and in the Kyoto encyclopedia of genes and genomes (KEGG) apoptotic pathway category. A total of 24 (10 short-term dosage and 14 long-term dosage) miRNAs were significantly regulated, one of which was rno-miR-665. Ectopic overexpression and silencing of rno-miR-665 demonstrated its role in the neurotoxic effects of propofol on hippocampal immature astrocytes. We present evidence that the role of rno-miR-665 in anesthesia-induced disturbances in astroglia development may involve direct downregulation of the anti-apoptotic gene Bcl2l1, and subsequent increased caspase-3-mediated apoptosis. Our results shed light on the anesthetic mechanism of propofol and have implications for its use in the clinical setting.

Development of astroglia heterogeneously expressing Pax2, vimentin and GFAP during the ontogeny of the optic pathway of the lizard (Gallotia galloti): an immunohistochemical and ultrastructural study

The successful regrowth of retinal ganglion cell (RGC) axons after optic nerve (ON) axotomy in Gallotia galloti indicates a permissive role of the glial environment. We have characterised the astroglial lineage of the lizard optic pathway throughout its ontogeny (embryonic stage 30 [E30] to adults) by using electron microscopy and immunohistochemistry to detect the proliferation marker PCNA (proliferating cell nuclear antigen), the transcription factor Pax2 and the gliofilament proteins vimentin (Vim) and GFAP (glial fibrillary acidic protein). PCNA(+) cells were abundant until E39, with GFAP(+)/PCNA(+) astrocytes being observed between E37 and hatching. Proliferation diminished markedly afterwards, being undetectable in the adult optic pathway. Müller glia of the central retina expressed Pax2 from E37 and their endfeet accumulated Vim from E33 and GFAP from E37 onwards. Astrocytes were absent in the avascular lizard retina, whereas abundant Pax2(+) astrocytes were observed in the ON from E30. A major subpopulation of these astrocytes coexpressed Vim from E35 and also GFAP from E37 onwards; thus the majority of mature astrocytes coexpressed Pax2/Vim/GFAP. The astrocytes were ultrastructurally identified by their gliofilaments, microtubules, dense bodies, desmosomes and glycogen granules, which preferentially accumulated in cell processes. Astrocytes in the adult ON coexpressed both gliofilaments and presented desmosomes indicating a reinforcement of the ON structure; this is physiologically necessary for local adaptation to mechanical forces linked to eye movement. We suggest that astrocytes forming this structural scaffold facilitate the regrowth of RGCs after ON transection.

Isoflurane Impairs Immature Astroglia Development In Vitro: The Role of Actin Cytoskeleton

General anesthetics, either alone or in combination, can be detrimental to the developing mammalian brain and induce extensive apoptotic degeneration of immature neurons when they are administered at the peak of synaptogenesis. Because neuron development and normal functions depend on the integrity of astroglia, we sought to determine whether general anesthesia also causes disturbances in the early development of astroglia. Using isoflurane, an inhaled anesthetic that is highly toxic to immature neurons, we studied primary astroglia cultures, focusing on very early development (Day-In-Vitro 4 treatment). Exposure to 3% isoflurane for 24 hours delayed morphological differentiation and impaired the growth of immature astrocytes. The timing of delayed astroglia maturation and growth coincided with a major disturbance in actin cytoskeleton sculpting that was manifest as impaired actin stress fiber formation and cytoskeletal organization and downregulation of the focal adhesion protein, paxillin. Isoflurane-induced actin cytoskeletal changes were accompanied by a significant decrease in protein levels of the endogenous GTPase RhoA that regulates the phosphorylation of myosin light chain protein, suggesting that isoflurane-induced impairment in glial growth and morphological development is, in part, mediated by the RhoA/myosin light chain protein signaling pathway.

Effect of 17-β estradiol and epidermal growth factor on DNA and RNA labeling in astroglial cells during development, maturation and differentiation in culture

Growth factors stimulate astroglial and neuronal proliferation and differentiation in culture. Estrogens markedly influence astroglia, and are key factors participating in neurodegeneration. The aim of the present study was to investigate interactions between estradiol (E 2) and epidermal growth factor (EGF) during astroglia development, maturation and differentiation in culture. DNA or RNA labeling in 16 or 40 or 60 days in vitro (DIV) astrocyte cultures treated for 24 or 48 h with EGF and/or E 2 was evaluated. A significant increase in DNA labeling in 16 DIV astrocyte cultures treated for 24 h with EGF (5 ng/ml) and E 2 (1 nM) was found. EGF (5 or 10 ng/ml) addition in the last 24 h in 48 h E 2 (1 or 5 nM)-treated astrocyte cultures at 16 DIV caused a slight, but significant increase in DNA labeling. No differences in RNA labeling were observed in 16 DIV astrocyte cultures treated for 24 or 48 h with EGF (5 or 10 ng/ml) in the presence of E 2 (1 or 5 nM). A significant stimulation in DNA labeling was shown in 40 DIV astrocyte cultures treated for 48 h with E 2 (1 or 5 nM) in the presence of EGF (5 or 10 ng/ml) added in the last 24 h. In well differentiated astroglial cell cultures (60 DIV), DNA labeling was remarkably increased after 24 h treatment with 1 nM E 2 or 5 ng/ml EGF. Co-addition of 1 nM E 2 and 5 ng/ml EGF for 24 h reduced [methyl- 3H]thymidine incorporation, when data are compared to E 2- or EGF-treated cultures. Addition of EGF in the presence of E 2 for 48 h or only in the last 24 h caused a significant decrease of [methyl- 3H]thymidine incorporation in comparison with EGF-treated cultures at 60 DIV or with untreated cultures. Treatment of cultures for 24 h with EGF (5 or 10 ng/ml) alone or in combination with E 2 (1 or 5 nM) induced a strong increase of RNA labeling in 60 DIV astrocyte cultures. Addition for 48 h of E 2 (1 or 5 nM) or EGF (5 or 10 ng/ml) alone or in association stimulated significantly RNA labeling in astrocyte cultures at 60 DIV. When 60 DIV astrocyte cultures were treated for 48 h with E 2 (1 or 5 nM) in the presence of EGF (5 or 10 ng/ml) added only in the last 24 h, a potentiating effect of RNA labeling was observed. The above results suggest that interaction between growth factors and estrogens may contribute to regulate astroglia development, maturation and differentiation.

Neuroanatomical and neurophysiological mechanisms involved in central nervous system dysfunctions induced by prenatal alcohol exposure.

One of the most severe consequences of maternal ethanol consumption is the damage to the developing central nervous system, which is manifested by long-term cognitive and behavioral deficits in the offspring. Prenatal exposure to ethanol affects many crucial neurochemical and cellular components of the developing brain. Ethanol interferes with all of the stages of brain development, and the severity of the damage depends on the amount of ethanol intake and level of exposure. Experimental observations also indicate that the toxic effects of ethanol are not uniform: some brain regions are more affected than others and, even within a given region, some cell populations are more vulnerable than others. The neocortex, the hippocampus, and the cerebellum are the regions in which the neurotoxic effects of ethanol have been associated with the behavioral deficits. At the cellular level, ethanol disrupts basic developmental processes, including interference with division and proliferation, cell growth, and differentiation and the migration of maturing cells. Alterations in astroglia development and in neuronal-glial interactions may also influence the development of the nervous system. An impairment of several neurotransmitter systems and/or their receptors, as well as changes in the endocrine environment during brain development, are also important factors involved in the behavioral dysfunctions observed after prenatal ethanol exposure. Finally, some molecular mechanisms of ethanol-induced behavioral dysfunctions will be discussed.

A rapid replacement of vimentin-containing radial glia by glial fibrillary acidic protein-containing astrocytes in transplanted telencephalon.

The present study follows the early events in the development of astroglia in rat embryonic (El5) tissue grafted into the cortex of adult rats. Astroglial elements (radial glia and astrocytes) were studied by glial fibrillary acidic protein (GFAP) and vimentin immunohistochemistry on post-transplantation (PT) days 7, 11, 14, 17, and 21. At PT7, GFAP-immunopositive elements were only scarce fibers in the transplants. At PTll, a dense network of long, GFAP-immunopositive fibers enmeshed the entire transplant, and astrocytes were already recognized. The fibers also showed vimentin immunoreactivity. By PT14, astrocytes became the predominant GFAP-labeled elements, although a few long fibers persisted. When compared with in situ development, the grafts showed earlier GFAP-immunoreactivity and earlier appearance of astrocytes, as well as a more rapid transition from the immature to the mature form of the glial system.

Immuno-electron microscopical localization of vimentin and glial fibrillary acidic protein in mouse astrocytes and their precursor cells in culture.

Immuno-electron microscopy was used to localize the distribution of vimentin and glial fibrillary acidic protein (GFAP) in mouse astrocytes and their precursor cells in primary cultures. In astroblasts and astrocytes, vimentin and GFAP form intermediate filaments (IF), which are heteropolymers, as previously observed in gliomas. Astrocytes and their precursor cells may have IF composed of GFAP-vimentin heteropolymer or vimentin alone, but IF composed of GFAP only were not seen. It seems that the formation of IF that are GFAP-vimentin heteropolymers is a feature of normal astroglia development and that the ratio of GFAP to vimentin in these IF reflects the degree of differentiation and functional state of the cell.

Actin and actin‐binding proteins in differentiating astroglia in tissue culture

In this paper we have described the organization of F-actin and actin-binding proteins (ABP): alpha-actinin, myosin, tropomyosin, caldesmon, vinculin, talin, and spectrin, in differentiating astroglia in colony cultures. We observed that the microfilament (MF) network arrangements differ at various stages of astroglia development, but the composition of MF bundles and stress fibers is the same at all developmental stages. F-actin is closely colocalized with myosin, tropomyosin, caldesmon, and alpha-actinin. The striated pattern of myosin, tropomyosin, and caldesmon are superimposable. Tropomyosin and caldesmon extend along F-actin but are interrupted for short periods, whereas myosin is interrupted for longer periods. alpha-actinin colocalizes with tropomyosin and caldesmon but not with myosin. In astroglia at different stages of development spectrin is arranged in the form of fine networks spreading through the cell and does not follow the arrangement of MF bundles. Only F-actin, alpha-actinin, and vinculin can be detected at cell-cell junctions. In the areas of the focal contacts, F-actin, alpha-actinin, vinculin, and talin are present. They overlap each other, although talin and vinculin extend toward the cell membrane beyond F-actin and alpha-actinin. Astroglia undergo well-defined states of nonmotility, motility, and nonmotility again during differentiation. The changes in motility are paralleled by changes in the organization of F-actin and ABP: as GFAP-containing intermediate filaments increase in differentiating astroglia, the F-actin and ABP are down-regulated, leading to non motility.

Effects of thyroid state and undernutrition on S100 protein and astroglia development in rat cerebellum.

The developmental pattern of S100 protein, a specific marker of astroglia, was studied by radioimmunoassay in the cerebellum of thyroid-deficient, thyroxine-treated and undernourished rats during development. In the control animals, the S100 protein content of the cerebellum increased maximally after the 3rd postnatal week, i.e., after cell multiplication had stopped and when the cerebellum had acquired more than 70% of its adult weight and protein content. This developmental pattern of S100 protein reflected essentially the maturation of astroglia. In the thyroxine-treated rats the total amount and the concentration of S100 protein were higher than in controls during the first 3 weeks of postnatal life and returned to normal values thereafter. In the thyroid-deficient rats both the amount and concentration were lower than in controls throughout development. In the undernourished animals the amount of S100 protein per organ was also lower than in controls during the whole experimental period studied; the S100 protein concentration was higher during the first 2 postnatal weeks and became lower thereafter. These results are discussed taking into consideration previous histological and ultrastructural observations on the effects of altered thyroid state and undernutrition on the formation and maturation of cerebellar astrocytes.