Neuroscience
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21633
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Browsing Neuroscience by Author "Hersh, Jessica"
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Item Development of a serum free astrocyte culture method that mimic resting in vivo astrocyte phenotype(2019-03-08) Winters, Ali; Chaudari, Kiran; Hersh, Jessica; Liu, Ran; Yang, Shaohua; Prah, JudePurpose Primary astrocyte cultures have been extensively used for characterization of astrocytes functions in physiological and pathological conditions. The current primary astrocytes are mostly maintained in fetal bovine serum (FBS) containing medium. Although FBS contains growth elements that fulfills many metabolic needs of cultured astrocytes, it alters the genotypic and morphological profiles of primary astrocytes as well as induces astrocyte activation. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state with phenotypes that mimic in vivo astrocytes. Methods Primary astrocytes were isolated from the cerebral cortex of postnatal day 1 C57BL/6 mice and cultured in serum-free astrocyte basal medium containing FGF2 and EGF (ABM-FGF2-EGF). The phenotype of primary astrocytes cultured in ABM-FGF2-EGF were compared with astrocytes cultured in FBS supplemented DMEM medium (MD-10% FBS). Growth assays, immunostaining, Western blot, quantitative polymerase chain reaction, and metabolic assays were used to access the growth rates, metabolic phenotype, mRNA expression profiles and quiescent or reactive states of astrocytes. Results and Conclusions We demonstrated that the novel serum free ABM-FGF2-EGF medium supports astrocytes growth and enhanced glycolytic metabolism with higher glycogen content, lower GFAP and vimentin expression, and increased glutamate transporter mRNA levels as compared to astrocytes cultured in the MD-10% FBS medium. Our study suggests that our serum free culture method produces astrocytes with a biosynthetic phenotype and morphology similar to in vivo resting astrocytes. Additionally ABM- FGF2-EGF cultured primary astrocytes could be activated by various pathological conditions. The developed serum-free and EGF/FGF2-containing astrocyte basal medium will provide a critical tool for defining the precise function of astrocytes under physiological and pathological conditions.Item IMPACT OF T-CELLS ON ASTROCYTES IN VIVO & IN VITRO: IMPLICATIONS POST-ISCHEMIC STROKE(2019-03-05) Xie, Luokun; Li, Wenjun; Liu, Ran; Yang, Shaohua; Hersh, JessicaPurpose: Post-ischemic stroke, T-lymphocytes enter the brain. The role of T-cells in the progression of cerebral infarction or repair mechanisms is unclear. We hypothesized that T-cells interact with astrocytes directly leading to an anti-inflammatory response. Methods: In vivo, ischemic stroke was induced by middle cerebral artery occlusion in young adult C57/B6 male mice. Mice were sacrificed at 3 days or 1-month post-ischemic stroke. Paraffin-embedded brain sections demonstrated co-localization of astrocytes and CD4+ and CD8+ T-cells in the ischemic region 1 month after stroke. T-cells were harvested from the brain by digestion; percoll enriched, and incubated with anti-CD3 and CD25 antibodies. T-cells were sorted via flow cytometry. The cytokine profile of brain infiltrated CD4+ and CD8+ T-cells were compared to spleen T-cells using QT-PCR. In vitro, C8-S murine astrocyte type II clone cell line (ATCC® CRL-2535™), and T-cells extracted from the spleens of 3-month-old C57/B6 female mice were placed in co-culture at a 1:1 for 48 and 72 hours and compared to individual cell cultures. Anti-CTLA-4 antibodies were added to each culture condition as another experimental group. Astrocytes and T-cells were collected separately for QT-PCR analysis. Results: In vivo, the following cytokine gene expressions poststroke, were found to be elevated: IFNγ, IL-10, IL-17, TNFα, and perforin. In vitro, IL-10 gene expression was elevated in astrocytes and T-cells individually harvested from 1:1 co-cultures compared to astrocytes and T-cells alone at 48 and 72 hours respectively. IL-10 was produced primarily by T-cells stimulated by direct contact with astrocytes. Anti-CTLA-4 antibodies blocked the direct cell-to-cell interaction by reducing IL-10 gene expression in both astrocytes and T-cells. Conclusions: Our data suggests that T-cells release pro- and anti- inflammatory cytokines while in close proximity to astrocytes after ischemic stroke. In co-cultures, astrocytes directly interact with T-cells increasing their IL-10 gene expression by 72 h., implying a neuroprotective mechanism exists via astrocyte stimulation of T-cell IL-10 production.