Primary glial cells and brain fibroblasts: Interactions in culture

Estin, Charles D.; Vernadakis, Antonia

doi:10.1016/0361-9230(86)90144-9

Cited by 30 publications

(14 citation statements)

References 32 publications

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“…Thin arrows point to non-neuronal nuclei that were weakly p65-positive in the unstimulated condition and strongly p65 positive after TNFα stimulation. The shape and large nuclear diameter (15–20 μm) of the illustrated responsive cells suggests that they are fibroblasts derived from brain vasculature and meninges (Estin and Vernadakis, 1986). Thick arrows point to neuronal nuclei that were weakly p65-positive in both unstimulated and TNFα-stimulated conditions.…”

Section: Figmentioning

confidence: 99%

Minimal NF-κB activity in neurons

Listwak¹,

Rathore²,

Herkenham

2013

Neuroscience

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NF-κB is a ubiquitous transcription factor that regulates immune and cell-survival signaling pathways. NF-κB has been reported to be present in neurons wherein it reportedly responds to immune and toxic stimuli, glutamate, and synaptic activity. However, because the brain contains many cell types, assays specifically measuring neuronal NF-κB activity are difficult to perform and interpret. To address this, we compared NF-κB activity in cultures of primary neocortical neurons, mixed brain cells, and liver cells, employing Western blot of NF-κB subunits, EMSA of nuclear κB DNA binding, reporter assay of κB DNA binding, immunofluorescence of the NF-κB subunit protein p65, quantitative real-time PCR of NF-κB-regulated gene expression, and ELISA of produced proteins. Assay of p65 showed its constitutive presence in cytoplasm and nucleus of neurons at levels significantly lower than in mixed brain or liver cells. EMSA and reporter assays showed that constitutive NF-κB activity was nearly absent in neurons. Induced activity was minimal—many fold lower than in other cell types, as measured by phosphorylation and degradation of the inhibitor IκBα, nuclear accumulation of p65, binding to κB DNA consensus sites, NF-κB reporting, or induction of NF-κB-responsive genes. The most efficacious activating stimuli for neurons were the proinflammatory cytokines TNFα and IL-β. Neuronal NF-κB was not responsive to glutamate in most assays, and it was also unresponsive to hydrogen peroxide, lipopolysaccharide, norepinephrine, ATP, phorbol ester, and nerve growth factor. The chemokine gene transcripts CCL2, CXCL1, and CXCL10 were strongly induced via NF-κB activation by TNFα in neurons, but many candidate responsive genes were not, including the neuroprotective genes SOD2 and Bcl-xL. Importantly, the level of induced neuronal NF-κB activity in response to TNFα or any other stimulus was lower than the level of constitutive activity in non-neuronal cells, calling into question the functional significance of neuronal NF-κB activity.

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Section: Figmentioning

confidence: 99%

Minimal NF-κB activity in neurons

Listwak¹,

Rathore²,

Herkenham

2013

Neuroscience

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“…(Fig• 1 A). The addition of previously described inhibitors of fibroblast growth (Estin and Vernadakis, 1986;Rutka et al, 1986) prevented any detectable proliferation. Thus, under these experimental conditions, purifed astrocyte cultures were essentially free of proliferating fibroblasts.…”

Section: Resultsmentioning

confidence: 96%

“…To prevent contamination by fibroblast-like cells, only the most intact brain tissue was selected tor processing, and the meninges and vascular tissue were meticulously removed. Two specilic inhibitors of tibroblast growth were added Io lhe cuhure medium: cis-4-hydroxy- L-proliue (Rulka et al, 1986) and D-valine, which replaced I~-valine (Estin and Vernadakis, 1986),…”

Section: Resultsmentioning

confidence: 99%

A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function

Price

Burke

Mayne

1999

In Vitro Cell.Dev.Biol.-Animal

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Studies of brain cell function and physiology are hampered by the limited availability of immortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of gamma-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines' retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.

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“…Some studies revealed that fibroblasts lack D-valine oxidase, and therefore fibroblasts are unable to convert D-valine into L-valine. Without L-valine, fibroblast cannot survive (Estin and Vernadakis 1986). Astrocytes possess appropriate enzymes for oxidizing fatty acids (i.e., glycogen phosphorylase, aldose reductase, and sorbitol dehydrogenase), so they do not require glucose or ketone bodies for respiration.…”

Section: Groupsmentioning

confidence: 99%

Optimized and efficient preparation of astrocyte cultures from rat spinal cord

Yang

Liang

et al. 2006

Cytotechnology

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Astrocytes constitute a major class of glial cells in the CNS, and play crucial roles in physiological functioning, performance and maintenance of the CNS, as well as promotion of neuronal migration and maturation. Astrocytes have also been directly and indirectly implicated in the pathophysiology of various trauma occurrences, development of neurodegenerative diseases and nerve regeneration. To further understand mechanisms by which astrocytes elicit these effects, the first critical step in the study of astrocytes is the preparation of purified astrocytes cultures. Here we describe a simple and convenient procedure for producing rat primary astrocyte cultures of high purity, viability and proliferation. For astrocyte culture, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, seeding density and composition of the culture medium. Using immunofluorescent antibodies against GFAP and OX-42 in combination of Hoechst 33342 fluorescent staining, we found that the purity of the astrocyte cultures was >99%. Astrocytes had high viability as measured by 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, flow cytometric analysis was used to measure and observe variations in the cell cycle after 1-2 passages and proliferation of astrocytes was detected with a high percentage of cells stand in S+G 2 /M phase. Therefore, the method described here is ideal for experiments, which require highly pure astrocyte cultures.

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Primary glial cells and brain fibroblasts: Interactions in culture

Cited by 30 publications

References 32 publications

Minimal NF-κB activity in neurons

Minimal NF-κB activity in neurons

A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function

Optimized and efficient preparation of astrocyte cultures from rat spinal cord

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