A novel histone deacetylase inhibitor‐based approach to eliminate microglia and retain astrocyte properties in glial cell culture
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Abstract
The close association between astrocytes and microglia causes great difficulties to distinguish their individual roles in innate immune responses in central nervous system. Current chemical‐based methods to eliminate microglia in glial cell culture introduce various molecular and functional alterations to astrocytes. Here, we describe a novel two‐step approach to achieve a complete elimination of microglia without affecting the biological properties of co‐cultured astrocytes by temporal treatment of histone deacetylase inhibitor trichostatin A (TSA). We verify TSA as a potent inducer for microglial‐specific apoptotic cell death, which also causes comprehensive gene expression changes in astrocytes. However, withdrawal of TSA not only ensures no microglia repopulation, but also restores all the gene expression changes in terms of astrocyte functions, including neurotrophic factors, glutamate and potassium transporters, and reactive astrocyte subtypes. By contrast, withdrawal of PLX5622, the commonly used colony‐stimulating factor 1 receptor inhibitor neither prevents microglia repopulation nor restores the gene expression changes mentioned above. Using this method, we are able to discriminate differential roles of microglia and astrocytes in the induced expression of antiviral and pro‐inflammatory cytokines upon various pathological stimuli including the spike protein of SARS‐CoV‐2. This simple and efficient method can be customized for the understanding of microglia‐astrocyte interaction and the development of epigenetic therapies that target over‐activated microglia in neuroinflammation‐related diseases.
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SciScore for 10.1101/2021.11.09.467827: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The first antibodies include rabbit anti-Iba1 (Wako), mouse anti-GFAP (Sigma) and rabbit anti-Ki67 (Abcam). anti-Iba1suggested: Noneanti-GFAPsuggested: Noneanti-Ki67suggested: NoneExperimental Models: Cell Lines Sentences Resources Pathological stimulation: For SARS-CoV-2 pseudovirus generation, the plasmid carrying spike protein sequence (Miaoling Plasmid Sharing Platform plasmid #P18156) was co-transfected with psPAX2 (Addgene plasmid #12260) into HEK293T cell line using Lipofectamine … SciScore for 10.1101/2021.11.09.467827: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The first antibodies include rabbit anti-Iba1 (Wako), mouse anti-GFAP (Sigma) and rabbit anti-Ki67 (Abcam). anti-Iba1suggested: Noneanti-GFAPsuggested: Noneanti-Ki67suggested: NoneExperimental Models: Cell Lines Sentences Resources Pathological stimulation: For SARS-CoV-2 pseudovirus generation, the plasmid carrying spike protein sequence (Miaoling Plasmid Sharing Platform plasmid #P18156) was co-transfected with psPAX2 (Addgene plasmid #12260) into HEK293T cell line using Lipofectamine 2000 (Invitrogen) according to manufactor’s instruction for 6 hours and cells were replaced with fresh media containing DMEM supplemented with 10% fetal bovine serum. HEK293Tsuggested: NoneRecombinant DNA Sentences Resources Pathological stimulation: For SARS-CoV-2 pseudovirus generation, the plasmid carrying spike protein sequence (Miaoling Plasmid Sharing Platform plasmid #P18156) was co-transfected with psPAX2 (Addgene plasmid #12260) into HEK293T cell line using Lipofectamine 2000 (Invitrogen) according to manufactor’s instruction for 6 hours and cells were replaced with fresh media containing DMEM supplemented with 10% fetal bovine serum. psPAX2suggested: RRID:Addgene_12260)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 19 and 20. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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