Isolation of Microglia and Immune Infiltrates from Mouse and Primate Central Nervous System

Galatro, Thais Fernanda de Almeida; Vainchtein, Ilia D.; Brouwer, Nieske; Boddeke, Erik; Eggen, Bart J. L.

doi:10.1007/978-1-4939-6786-5_23

Cited by 42 publications

(42 citation statements)

References 14 publications

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“…10A-D). We used CD45 and CD11b expression to identify inflammatory cells and sort them into three populations: microglia (CD11b high, CD45 intermediate); myeloid cells, including macrophages and neutrophils (CD11b high, CD45 high); and lymphoid cells, including B and T lymphocytes (CD11b low, CD45 high; Galatro et al, 2017). We found no effect of genotype on the proportion of cells of each lineage isolated from cerebral cortex ( Figure 10E-G; microglia t test: t (27) ϭ 1.61, p ϭ 0.1189; myeloid cells t test: t (27) ϭ 1.72, p ϭ 0.0969; lymphoid cells t test: t (27) ϭ 0.9363, p ϭ 0.3574).…”

Section: Resultsmentioning

confidence: 99%

“…Samples were stained with SYTOX blue (Thermo Fisher catalog #S348857) at a final concentration of 1 M per the manufacturer's instructions. Cells were analyzed on a FACScan 2 flow cytometer (BD Biosciences) using a gating strategy similar to one described previously to isolate microglia, myeloid, and lymphoid cells (Galatro et al, 2017). All gating strategies incorporated size and doublet discrimination based on forward and side scatter parameters.…”

Section: Methodsmentioning

confidence: 99%

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Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion ofCtcfinCamk2a-Cre-Expressing Neurons

McGill

Barve

Maloney³

et al. 2017

J. Neurosci.

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CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying 3D chromatin structure. Human mutations in cause intellectual disability and autistic features. Knocking out in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out postnatally in glutamatergic forebrain neurons under the control of ; ( CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor coordination, and decreased sociability by 4 months of age. CKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA from CKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically from CKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia in CKO hippocampus. Finally, we found that microglia in CKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm that KO in postnatal neurons causes a neurobehavioral phenotype in mice and provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction. CCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations in cause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lacking in -expressing neurons ( CKO mice) have spatial learning/memory deficits, impaired fine motor skills, subtly altered social interactions, and decreased dendritic spine density. We demonstrate that CKO mice overexpress inflammation-related genes in the brain and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron-microglia interactions are factors in the pathology of CTCF deficiency.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion ofCtcfinCamk2a-Cre-Expressing Neurons

McGill

Barve

Maloney³

et al. 2017

J. Neurosci.

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“…Microglia were isolated from adult mouse brain using the protocol as described before (Galatro, Vainchtein, et al, ). Briefly, the brains were isolated and triturated using a tissue homogenizer.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional profiling of microglia; current state of the art and future perspectives

Gerrits

Heng

Boddeke

et al. 2019

Glia

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Microglia are the tissue macrophages of the central nervous system (CNS) and the first to respond to CNS dysfunction and disease. Gene expression profiling of microglia during development, under homeostatic conditions, and in the diseased CNS provided insight in microglia functions and changes thereof. Single‐cell sequencing studies further contributed to our understanding of microglia heterogeneity in relation to age, sex, and CNS disease. Recently, single nucleus gene expression profiling was performed on (frozen) CNS tissue. Transcriptomic profiling of CNS tissues by (single) nucleus RNA‐sequencing has the advantage that it can be applied to archived and well‐stratified frozen specimens. Here, we give an overview of the significant advances recently made in microglia transcriptional profiling. In addition, we present matched cellular and nuclear microglia RNA‐seq datasets we generated from mouse and human CNS tissue to compare cellular versus nuclear transcriptomes from fresh and frozen samples. We demonstrate that microglia can be similarly profiled with cell and nucleus profiling, and importantly also with nuclei isolated from frozen tissue. Nuclear microglia transcriptomes are a reliable proxy for cellular transcriptomes. Importantly, lipopolysaccharide‐induced changes in gene expression were conserved in the nuclear transcriptome. In addition, heterogeneity in microglia observed in fresh samples was similarly detected in frozen nuclei of the same donor. Together, these results show that microglia nuclear RNAs obtained from frozen CNS tissue are a reliable proxy for microglia gene expression and cellular heterogeneity and may prove an effective strategy to study of the role of microglia in neuropathology.

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“…Microglia were isolated as described previously (Galatro, Vainchtein, Brouwer, Boddeke, & Eggen, 2017b). Briefly, mice were perfused using PBS (Lonza, BE17-512F) and brain and spinal cord were isolated in HBSS (Gibco, 14170-088) containing 15 mM HEPES (Lonza, BE17-737E) and 0.6% glucose (Sigma-Aldrich, G8769) (=Medium A).…”

Section: Acute Microglia Isolationmentioning

confidence: 99%

VISTA expression by microglia decreases during inflammation and is differentially regulated in CNS diseases

Borggrewe

Grit

Dunnen

et al. 2018

Glia

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V‐type immunoglobulin domain‐containing suppressor of T‐cell activation (VISTA) is a negative checkpoint regulator (NCR) involved in inhibition of T cell‐mediated immunity. Expression changes of other NCRs (PD‐1, PD‐L1/L2, CTLA‐4) during inflammation of the central nervous system (CNS) were previously demonstrated, but VISTA expression in the CNS has not yet been explored. Here, we report that in the human and mouse CNS, VISTA is most abundantly expressed by microglia, and to lower levels by endothelial cells. Upon TLR stimulation, VISTA expression was reduced in primary neonatal mouse and adult rhesus macaque microglia in vitro. In mice, microglial VISTA expression was reduced after lipopolysaccharide (LPS) injection, during experimental autoimmune encephalomyelitis (EAE), and in the accelerated aging Ercc1 Δ/− mouse model. After LPS injection, decreased VISTA expression in mouse microglia was accompanied by decreased acetylation of lysine residue 27 in histone 3 in both its promoter and enhancer region. ATAC‐sequencing indicated a potential regulation of VISTA expression by Pu.1 and Mafb, two transcription factors crucial for microglia function. Finally, our data suggested that VISTA expression was decreased in microglia in multiple sclerosis lesion tissue, whereas it was increased in Alzheimer's disease patients. This study is the first to demonstrate that in the CNS, VISTA is expressed by microglia, and that VISTA is differentially expressed in CNS pathologies.

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Isolation of Microglia and Immune Infiltrates from Mouse and Primate Central Nervous System

Cited by 42 publications

References 14 publications

Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion ofCtcfinCamk2a-Cre-Expressing Neurons

Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion ofCtcfinCamk2a-Cre-Expressing Neurons

Transcriptional profiling of microglia; current state of the art and future perspectives

VISTA expression by microglia decreases during inflammation and is differentially regulated in CNS diseases

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