Analysis Of Microglia Research Articles

A rotenone organotypic whole hemisphere slice model of mitochondrial abnormalities in the neonatal brain.

Mitochondrial abnormalities underscore a variety of neurologic injuries and diseases and are well-studied in adult populations. Clinical studies identify critical roles of mitochondria in a wide range of developmental brain injuries, but models that capture mitochondrial abnormalities in systems representative of the neonatal brain environment are lacking. Here, we develop an organotypic whole-hemisphere (OWH) brain slice model of mitochondrial dysfunction in the neonatal brain. We extended the utility of complex I inhibitor rotenone (ROT), canonically used in models of adult neurodegenerative diseases, to inflict mitochondrial damage in OWH slices from term-equivalent rats. We quantified whole-slice health over 6days of exposure for a range of doses represented in ROT literature. We identified 50nM ROT as a suitable exposure level for OWH slices to inflict injury without compromising viability. At the selected exposure level, we confirmed exposure- and time-dependent mitochondrial responses showing differences in mitochondrial fluorescence and nuclear localization using MitoTracker imaging in live OWH slices and dysregulated mitochondrial markers via RT-qPCR screening. We leveraged the regional structures present in OWH slices to quantify cell density and cell death in the cortex and the midbrain regions, observing higher susceptibilities to damage in the midbrain as a function of exposure and culture time. We supplemented these findings with analysis of microglia and mature neurons showing time-, region-, and exposure-dependent differences in microglial responses. We demonstrated changes in tissue microstructure as a function of region, culture time, and exposure level using live-video epifluorescence microscopy of extracellularly diffusing nanoparticle probes in live OWH slices. Our results highlight severity-, time-, and region-dependent responses and establish a complimentary model system of mitochondrial abnormalities for high-throughput or live-tissue experimental needs.

Prenatal drivers of microglia vulnerability in the adult.

Environmental insults during early development heavily affect brain trajectories. Among these, maternal infections, high-fat diet regimens, and sleep disturbances pose a significant risk for neurodevelopmental derangements in the offspring. Notably, scattered evidence is starting to emerge that also paternal lifestyle habits may impact the offspring development. Given their key role in controlling neurogenesis, synaptogenesis and shaping neuronal circuits, microglia represent the most likely suspects of mediating the detrimental effects of prenatal insults. For some of these environmental triggers, like maternal infections, ample literature evidence demonstrates the central role of microglia, also delineating the specific transcriptomic and proteomic profiles induced by these insults. In other contexts, the analysis of microglia is still in its infancy. Fostering these studies is needed to define microglia as potential therapeutic target in the frame of disorders consequent to maternal immune activation.

High-Dimensional Methods of Single-Cell Microglial Profiling to Enhance Understanding of Neuropathological Disease.

Microglia are the innate myeloid cells of the central nervous system (CNS) parenchyma, functionally implicated in almost every defined neuroinflammatory and neurodegenerative disorder. Current understanding of disease pathogenesis for many neuropathologies is limited and/or lacks reliable diagnostic markers, vaccines, and treatments. With the increasing aging of society and rise in neurogenerative diseases, improving our understanding of their pathogenesis is essential. Analysis of microglia from murine disease models provides an investigative tool to unravel disease processes. In many neuropathologies, bone-marrow-derived monocytes are recruited to the CNS, adopting a phenotype similar to that of microglia. This significantly confounds the accurate identification of cell-type-specific functions and downstream therapeutic targeting. The increased capacity to analyze more phenotypic markers using spectral-cytometry-based technologies allows improved separation of microglia from monocyte-derived cells. Full-spectrum profiling enables enhanced marker resolution, time-efficient analysis of >40 fluorescence parameters, and extraction of cellular autofluorescence parameters. Coupling this system with additional cytometric technologies, including cell sorting and high-parameter imaging, can improve the understanding of microglial phenotypes in disease. To this end, we provide detailed, step-by-step protocols for the analysis of murine brain tissue by high-parameter ex vivo cytometric analysis using the Aurora spectral cytometer (Cytek), including best practices for unmixing and autofluorescence extraction, cell sorting for single-cell RNA analysis, and imaging mass cytometry. Together, this provides a toolkit for researchers to comprehensively investigate microglial disease processes at protein, RNA, and spatial levels for the identification of therapeutic targets in neuropathology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Processing the mouse brain into a single-cell suspension for microglia isolation Basic Protocol 2: Staining single-cell mouse brain suspensions for microglial phenotyping by spectral cytometry Basic Protocol 3: Flow cytometric sorting of mouse microglia for ex vivo analysis Basic Protocol 4: Processing the mouse brain for imaging mass cytometry for spatial microglia analysis.

Characterization of a mouse model of chronic hydrocephalus induced by partial occlusion of the aqueduct of Sylvius in the adult brain

BackgroundHydrocephalus is a neurologic disturbance produced by the abnormal production, circulation, and absorption of cerebrospinal fluid (CSF). Late-onset idiopathic aqueductal stenosis induces normal pressure hydrocephalus (NPH) in adults. To date, no animal model replicating chronic NPH is available to study the pathophysiological changes observed in these subjects. New MethodWe performed and characterized a model that induces chronic hydrocephalus in the adult mouse brain by producing a pre-aqueductal semiobstruction using an acetate lamina inserted into the atrium of the aqueduct of Sylvius. After surgical procedure, we analyzed the hydrocephalus development on days 60 and 120 and sham-operated animals were used as controls. We included an additional group of hydrocephalus resolution in which we removed the obstruction and analyzed the morphological changes in the brain. ResultsThe hydrocephalus was fully established on day 60 after the obstruction and remained stable for 120 days. In all animals, the intracranial pressure remained ~4.08 mmHg and we did not find statistically significant differences between the hydrocephalus groups and controls. We did not find motor impairments and anxiety-like behaviors among groups and the analysis of microglia and astrogliosis revealed mild glial reactivity. Comparison with Existing MethodsThis model generates a long-term ventricular enlargement with normal intracranial pressure and moderate glial reactivity. Importantly, this model allows the reversibility of ventricular enlargement after the removal of the obstructive film from the brain. Conclusions. This mouse model may be useful to study the long-term cerebral alterations that occur during NPH or after its surgical resolution.

Proteomic analysis of microglia during LPS‐induced acute neuroinflammation

AbstractBackgroundDysregulation of microglia function is associated with neuroinflammation. A major limitation in understanding microglial contribution to cellular processes and their role in disease has been the lack of tools to specifically distinguish these cells from other myeloid cells.MethodWe generated and validated a rat monoclonal antibody against a highly selective microglia marker P2RY12. We utilized LPS administration as a model for neuroinflammation to assess alterations in P2RY12 expression, a homeostatic protein. To gain better understanding of microglial function, we assessed their proteomic profile using BioLegend’s proprietary technology, TotalSeq™, which allows simultaneous analysis of both the transcriptome and proteome at the single‐cell level.ResultMice were injected with either PBS, low or high dosages of LPS daily for 4 days after which microglia were harvested from the brain. Isolated cells were co‐stained with CD11b, CX3CR1, and P2RY12 as general markers for microglia. Flow cytometric quantification using live cells showed a significant increase in CD11b+ expression in both low and high dose LPS treated mice compared to the control untreated group. Low and high dose LPS injections led to a significant reduction in P2RY12 and CX3CR1 expression in these cells. These data are consistent with a decrease in P2RY12 expression under inflammatory conditions. We utilized a panel of 200 antibody‐oligonucleotide conjugates from our TotalSeq™ platform, including P2RY12, CD11b, CD45, and CX3CR1, to analyze differentially expressed proteins under normal and LPS‐induced inflammation in mice.ConclusionThese studies help elucidate the diversity and physiology of microglia, and potentially aid in the discovery of novel markers in response to neuroinflammation. Our future goal is to extend our studies and use our platform to assess alternations in microglial response in neurodegeneration.

JAM-A functions as a female microglial tumor suppressor in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary brain tumor and has a dismal prognosis. Previously, we identified that junctional adhesion molecule A (JAM-A), a cell adhesion molecule, is highly elevated in human GBM cancer stem cells and predicts poor patient prognosis. While JAM-A is also highly expressed in other cells in the tumor microenvironment, specifically microglia and macrophages, how JAM-A expression in these cells affects tumor growth has yet to be determined. The goal of this study was to understand the role of microenvironmental JAM-A in mediating GBM growth. Male and female wild-type (WT) and JAM-A-deficient mice were transplanted intracranially with the syngeneic glioma cell lines GL261 and SB28 and were assessed for differences in survival and microglial activation in tumors and in vitro. RNA-sequencing was performed to identify differentially regulated genes among all genotypes, and differences were validated in vitro and in vivo. We found that JAM-A-deficient female mice succumbed to GBM more quickly compared with WT females and JAM-A-deficient and male WT mice. Analysis of microglia in the tumors revealed that female JAM-A-deficient microglia were more activated, and RNA-sequencing identified elevated expression of Fizz1 and Ifi202b specifically in JAM-A-deficient female microglia. Our findings suggest that JAM-A functions to suppress pathogenic microglial activation in the female tumor microenvironment, highlighting an emerging role for sex differences in the GBM microenvironment and suggesting that sex differences extend beyond previously reported tumor cell-intrinsic differences.

Systemic TLR2 tolerance enhances central nervous system remyelination

BackgroundMultiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by both inflammatory demyelination and impaired remyelination. Studies indicate that Toll-like receptor 2 (TLR2) signaling contributes to both the inflammatory component and the defective remyelination in MS. While most MS therapeutics target adaptive immunity, we recently reported that reducing TLR2 signaling in innate immune cells by inducing TLR2 tolerance attenuates adoptively transferred experimental autoimmune encephalomyelitis. Given that previous reports suggest TLR2 signaling also inhibits myelin repair, the objective of this study was to assess how reducing TLR2 signaling through TLR2 tolerance induction affects CNS myelin repair.MethodsChow containing 0.2% cuprizone was fed to male and female wild-type (WT) C57BL/6 mice or TLR2-deficient (TLR2−/−) mice for 5 weeks to induce demyelination. During a 2-week remyelination period following discontinuation of cuprizone, WT mice received either low dose TLR2 ligands to induce systemic TLR2 tolerance or vehicle control (VC). Remyelination was evaluated via electron microscopy and immunohistochemical analysis of microglia and oligodendrocytes in the corpus callosum. Statistical tests included 2-way ANOVA and Mann-Whitney U analyses.ResultsInducing TLR2 tolerance in WT mice during remyelination significantly enhanced myelin recovery, restoring unmyelinated axon frequency and myelin thickness to baseline levels compared to VC-treated mice. Mechanistically, enhanced remyelination in TLR2 tolerized mice was associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. This result was confirmed in vitro by inducing TLR2 tolerance in WT microglia cultures. TLR2−/− mice, without TLR2 tolerance induction, also significantly enhanced myelin recovery compared to WT mice, adding confirmation that reduced TLR2 signaling is associated with enhanced remyelination.DiscussionOur results suggest that reducing TLR2 signaling in vivo by inducing TLR2 tolerance significantly enhances myelin repair. Furthermore, the enhanced remyelination resulting from TLR2 tolerance induction is associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. While deletion of TLR2 would be an impractical approach in vivo, reducing innate immune signaling through TLR2 tolerance induction may represent a novel, two-pronged approach for treating both inflammatory and myelin repair components of MS.

Abstract TP141: Mesenchymal Stem Cell Derived Exosomes as a Modulator of Neuroinflammation Following Cortical Injury

In this study, we tested Mesenchymal Stem Cell derived exosomes as a therapeutic to enhance recovery in our established nonhuman primate model of cortical injury. After pretraining monkeys on fine motor hand task,10 aged female monkeys were randomly assigned to an exosome treatment group (n=5) or a vehicle control (PBS) group (n=5). Then, in a single surgery, monkeys received a lesion in the hand representation of M1. One day and 14 days after injury, monkeys were given 4x10 11 exosomes/kg or PBS intravenously. Fifteen days after surgery, motor assessments were resumed for 12 weeks. The five treated monkeys all returned to pre-operative latencies to retrieve on motor tasks while none of the control monkeys returned to pre-operative latencies. To assess recovery mechanisms, cerebrospinal fluid (CSF) collected across the post-injury recovery period and terminal brain tissue were analyzed for markers of neuroinflammation. Preliminary ELISA results in both groups showed a trend towards increased Myelin Basic Protein (MBP) in CSF 24 hours after cortical injury followed by a gradual decline over 6 weeks. Interestingly, MBP levels declined more rapidly in exosome-treated animals, suggesting a potential reduction in damage. Scholl analysis of microglia branching in perilesional grey matter showed that exosomes increased ramification length, branching complexity, and convex hull 3D coverage volume. These findings suggest an increase in the surveying capacity of microglia in treated monkeys that may serve to promote repair after injury. Further, optical density analyses of microglial markers revealed a lower expression in treated monkeys of IBA1/P2RY12 and the MHCII antigen-presenting marker, LN3. This is consistent with decreased microglial activation associated with exosome treatment. These results provide evidence for the translational potential of exosomes as a treatment for human stroke.

Abstract PR15: The oncometabolite R-2-Hydroxyglutarate suppresses the innate immune microenvironment of IDH1-mutated gliomas via aryl hydrocarbon receptor signaling

Abstract Background: IDH1-mutated gliomas are associated with less abundant and phenotypically skewed innate and adaptive immune cell infiltrates compared to IDH1 wild-type tumors. Despite this, the most frequent mutation—IDH1R132H—represents a clonal shared neoantigen and mutations in IDH are associated with a more favorable prognosis. While the tumor cell-intrinsic consequences of the oncometabolite R-2-hydroxyglutarate (R-2-HG) accumulating in IDH1-mutated gliomas as a result of a neomorphic enzymatic function are well-characterized, potential direct paracrine effects of R-2-HG influencing the glioma immune microenvironment remain incompletely understood. Aim: This study aimed at characterizing the impact of the oncometabolite R-2-HG on the innate immune microenvironment of IDH1-mutated gliomas. Methods and Results: By means of comprehensive analyses of expression datasets from human gliomas and syngeneic murine tumor models as well as transporter studies, we demonstrate that R-2-HG is imported by both microglia and macrophages via SLC family transporters and suppresses their function in a paracrine manner. Functional analyses of microglia and macrophages indicate an R-2-HG-driven induction of tolerogenicity as evidenced by accumulation of IL10 and TGFβ and suppression of MHC-II expression, which results in impaired activation of antigen-specific T-cells and activation of immune checkpoint molecules. Multilevel signature profiling of human tumor-infiltrating as well as primary immune cells was complemented by reporter gene assays and pathway analyses and revealed that R-2-HG activates the cytosolic transcription factor aryl hydrocarbon receptor (AHR), a key immunomodulatory target of immunosuppressive tryptophan metabolism. By means of knockout models, the observed immunosuppressive phenotype was shown to be AHR-dependent. Functional relevance of R-2-HG-mediated, AHR-driven impairment of myeloid cell immunity was demonstrated in vivo by pharmacologic AHR inhibition, increasing the efficacy of checkpoint blockade. Conclusion: R-2-HG impairs antitumor immunity in IDH1-mutated gliomas by activating the AHR in innate immune cells, thus suppressing the innate immune microenvironment by compromising antigen presentation and activation of antigen-specific T-cells. This, together with recent findings on inhibitory effects on T-cell immunity, represents a novel mechanism of immune evasion of an immunogenic driver mutation and opens a novel therapeutic approach to IDH1-mutated gliomas. Citation Format: Mirco Friedrich, Lukas Bunse, Theresa Bunse, Edward Green, Tobias Kessler, Stefan Pusch, Katrin Deumelandt, Rafael Carretero, Andreas von Deimling, Francisco J Quintana, Wolfgang Wick, Michael Platten. The oncometabolite R-2-Hydroxyglutarate suppresses the innate immune microenvironment of IDH1-mutated gliomas via aryl hydrocarbon receptor signaling [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR15.

Dissecting functional phenotypes of microglia and macrophages in the rat brain after transient cerebral ischemia.

Ischemic brain injury causes local inflammation, which involves activation of resident microglia, leukocyte, and monocyte infiltration. Involvement of peripheral immune cells in ischemia-induced damage and repair is debatable. Using flow cytometry, gene expression profiling, and immunocytochemistry, we show that microglia predominate in the ischemic brain and express inflammation mediators at Day 1 after transient middle cerebral artery occlusion (MCAo) in rats. At Day 3, both resident microglia and bone marrow (BM)-derived macrophages are detected in the ischemic hemispheres and display unique transcriptomic profiles. Functional groups enriched in BM-macrophages are indicative of the pro-regenerative, immunosuppressive phenotype. Transient depletion of peripheral macrophages with clodronate-filled liposomes reduced the number of Arg1+ Iba1+ expressing cells in the ischemic brain. The analysis of microglia and macrophage signature genes shows that each cell type maintains the expression of their identity genes, even if gene expression is modified in a response to environmental clues. At Day 7, infiltrating BM-macrophages exhibit the reduced expression of Arg1, the elevated expression of iNos and many inflammatory genes, as shown by RNA sequencing. This is consistent with their switch toward a pro-inflammatory phenotype. We propose that BM-macrophages recruited to the injured brain early after ischemia could contribute to functional recovery after stroke, but they switch toward a pro-inflammatory phenotype in the ischemic parenchyma. Our results point to the detrimental role of microglia in an ischemic brain and the primarily pro-regenerative role of infiltrating BM-macrophages.

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

Transcriptome analysis of microglia in a mouse model of Rett syndrome: differential expression of genes associated with microglia/macrophage activation and cellular stress

BackgroundRett syndrome (RTT) is a severe, neurodevelopmental disorder primarily affecting girls, characterized by progressive loss of cognitive, social, and motor skills after a relatively brief period of typical development. It is usually due to de novo loss of function mutations in the X-linked gene, MeCP2, which codes for the gene expression and chromatin regulator, methyl-CpG binding protein 2. Although the behavioral phenotype appears to be primarily due to neuronal Mecp2 deficiency in mice, other cell types, including astrocytes and oligodendrocytes, also appear to contribute to some aspects of the RTT phenotype. In addition, microglia may also play a role. However, the effect of Mecp2 deficiency in microglia on RTT pathogenesis is controversial.MethodsIn the current study, we applied whole transcriptome analysis using RNA-seq to gain insight into molecular pathways in microglia that might be dysregulated during the transition, in female mice heterozygous for an Mecp2-null allele (Mecp2+/−; Het), from the pre-phenotypic (5 weeks) to the phenotypic phases (24 weeks).ResultsWe found a significant overlap in differentially expressed genes (DEGs) with genes involved in regulating the extracellular matrix, and those that are activated or inhibited when macrophages and microglia are stimulated towards the M1 and M2 activation states. However, the M1- and M2-associated genes were different in the 5- and 24-week samples. In addition, a substantial decrease in the expression of nine members of the heat shock protein (HSP) family was found in the 5-week samples, but not at 24 weeks.ConclusionsThese findings suggest that microglia from pre-phenotypic and phenotypic female mice are activated in a manner different from controls and that pre-phenotypic female mice may have alterations in their capacity to response to heat stress and other stressors that function through the HSP pathway.

Tomita-Seylaz法による慢性頭窓作製後のマウス大脳皮質ミクログリアの長期反復<i>in vivo</i>リアルタイム解析

Tomita-Seylaz法による慢性頭窓作製後のマウス大脳皮質ミクログリアの長期反復<i>in vivo</i>リアルタイム解析

High-resolution transcriptome analysis reveals neuropathic pain gene-expression signatures in spinal microglia after nerve injury.

Microglial cells, the resident immune cells of the spinal cord, become activated in response to peripheral nerve injury. Microglia activation contributes to the development of neuropathic pain. Here we employed microarray analysis of individually collected pools of 10 spinal microglia cells to identify changes of levels and cell-to-cell expression variance of microglial genes during their activation after peripheral nerve injury. The analysis of microglia on postoperative day 1 (POD1) identified miR-29c as a critical factor for microglial activation and the development of neuropathic pain. Early POD1 microglia exhibited a very distinct expression profile compared to late POD7 microglia, possibly leading to the transition from initiation to maintenance of neuropathic pain. We found sample variance patterns that were consistent with the hypothesis that microglia were highly heterogeneous at the level of individual cells, and variation analysis identified 56 microglial genes potentially linked to the maintenance of neuropathic pain which included Gria1. This study provides insights into spinal microglial biology and reveals novel microglial targets for the treatment of neuropathic pain.

C4-P-09Three-dimensional analysis of microglia and synapses with large-volume optical reconstruction

C4-P-09Three-dimensional analysis of microglia and synapses with large-volume optical reconstruction

Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice

Although microglial activation is associated with all CNS disorders, many of which are sexually dimorphic or age-dependent, little is known about whether microglial basal gene expression is altered with age in the healthy CNS or whether it is sex dependent. Analysis of microglia from the brains of 3-day (P3)- to 12-month-old male and female C57Bl/6 mice revealed distinct gene expression profiles during postnatal development that differ significantly from those in adulthood. Microglia at P3 are characterized by relatively high iNOS, TNFα and arginase-I mRNA levels, whereas P21 microglia have increased expression of CD11b, TLR4, and FcRγI. Adult microglia (2-4 months) are characterized by low proinflammatory cytokine expression, which increases by 12 months of age. Age-dependent differences in gene expression suggest that microglia likely undergo phenotypic changes during ontogenesis, although in the healthy brain they did not express exclusively either M1 or M2 phenotypic markers at any time. Interestingly, microglia were sexually dimorphic only at P3, when females had higher expression of inflammatory cytokines than males, although there were no sex differences in estrogen receptor expression at this or any other time evaluated here. Compared with microglia in vivo, primary microglia prepared from P3 mice had considerably altered gene expression, with higher levels of TNFα, CD11b, arginase-I, and VEGF, suggesting that culturing may significantly alter microglial properties. In conclusion, age- and sex-specific variances in basal gene expression may allow differential microglial responses to the same stimulus at different ages, perhaps contributing to altered CNS vulnerabilities and/or disease courses.

Microglia Display Distinct Sex‐dependent Gene Expression Profiles in The Postnatal and Adult CNS

Microglial activation is associated with all CNS disorders, many of which are sexually dimorphic or age‐dependent. However, little is known about whether microglia are sexually dimorphic, or how their basal gene expression is altered with age in the healthy CNS. Analysis of microglia from the brains of 3 day‐ to 12 month‐old male and female C57Bl/6 mice revealed distinct gene expression profiles during postnatal development that are significantly different from adulthood. In addition, we found a 2‐fold higher density of microglia/mg tissue in the postnatal brain than in the adult brain. Postnatal microglia are characterized by high iNOS, TNFα and arginase‐1 mRNA levels, whereas adult microglia (2–4 months) are characterized by low pro‐inflammatory cytokine expression that was increased at 12 months of age. Although the microglial phenotype appears to change with age, microglia in the healthy brain were not committed to an M1 or M2 phenotype at any time. We found sexual dimorphisms in microglia in the absence of sex differences in estrogen receptor expression, and only at P3 when females had higher expression of inflammatory cytokines than males. Since basal gene expression may prime microglial responsiveness to physiological or pathological events, the same stimulus may induce different sex‐and age‐dependent microglial activities, and thereby contribute to altered CNS vulnerability at specific ages.

IFN Regulatory Factor 8 Is a Key Constitutive Determinant of the Morphological and Molecular Properties of Microglia in the CNS

IFN regulatory factor (IRF) 8 is a transcription factor that has a key role in the cellular response to IFN-γ and is pivotal in myeloid cell differentiation. Whether IRF8 plays a role in the development and function of microglia, the tissue-resident myeloid cells of the brain, is unknown. Here, we show IRF8 is a constitutively produced nuclear factor in microglia, which suggested that IRF8 might also be a key homeostatic transcriptional determinant of the microglial cell phenotype. In support of this, in mice with a targeted disruption of the IRF8 gene, microglia were increased in number and showed gross alterations in morphology and surface area. In situ analysis of some key myeloid markers revealed that IRF8-deficient microglia had significantly reduced levels of Iba1, but increased levels of CD206 (mannose receptor) and F4/80 as well as increased tomato lectin binding. Analysis of microglia ex vivo revealed IRF8-deficient microglia had significantly increased levels of CD45, CD11b and F4/80, but significantly decreased levels of the chemokine receptors CCR2, CCR5 and CX3CR1. The known involvement of some of these molecular markers in membrane dynamics and phagocytosis led us to examine the phagocytic capacity of cultured IRF8-deficient microglia, however, this was found to be similar to wild type microglia. We conclude IRF8 is a constitutively produced nuclear factor in resident microglia of the CNS being a crucial transcriptional determinant of the phenotype of these cells in the healthy brain.

Subventricular zone microglia possess a unique capacity for massive in vitro expansion

Microglia, the resident immune cells of the brain, have recently been hypothesized to play a role both in neuronal diseases and age-related neurogenic decline, and are theorized to be modulators of adult neurogenesis. Current methods for the isolation of microglia from cultured primary brain tissue result in relatively poor yield, requiring a large tissue sample or multiple specimens to obtain a sufficient number of microglia for cell and molecular analysis. We report here a method for the repetitive isolation of microglia from established glial monolayer cultures from which it is possible to expand the initial population of microglia roughly 10,000-fold. The expanded population expresses appropriate microglial morphology and phenotype markers, and demonstrates functionally normal phagocytosis, thus providing a high-yield assay for the investigation and analysis of microglia from a single initial dissection of primary tissue. Furthermore, this massive expansion is limited to microglia derived from the subventricular zone as the fold expansion of isolatable microglia was found to be up to 20 times greater than cultures from other brain regions, indicating unique properties for this persistently neurogenic region.