Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease.

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-Seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq. Key features • Analysis of microglial phenotypes from murine brain without the need for cell sorting, imaging, or scRNA-seq. • This protocol can distinguish between homeostatic, disease-associated (DAM), lipid-droplet accumulating (LDAM), and interferon response (IRM) microglia from any murine brain region and/or disease model of interest. • This protocol can be modified to incorporate additional markers of interest or dyes when using a cell analyzer capable of multiple color detections.

Phenology of five tick species in the central Great Plains.

The states of Kansas and Oklahoma, in the central Great Plains, lie at the western periphery of the geographic distributions of several tick species. As the focus of most research on ticks and tick-borne diseases has been on Lyme disease which commonly occurs in areas to the north and east, the ticks of this region have seen little research attention. Here, we report on the phenology and activity patterns shown by tick species observed at 10 sites across the two states and explore factors associated with abundance of all and life specific individuals of the dominant species. Ticks were collected in 2020-2022 using dragging, flagging and carbon-dioxide trapping techniques, designed to detect questing ticks. The dominant species was A. americanum (24098, 97%) followed by Dermacentor variabilis (370, 2%), D. albipictus (271, 1%), Ixodes scapularis (91, <1%) and A. maculatum (38, <1%). Amblyomma americanum, A. maculatum and D. variabilis were active in Spring and Summer, while D. albipictus and I. scapularis were active in Fall and Winter. Factors associated with numbers of individuals of A. americanum included day of year, habitat, and latitude. Similar associations were observed when abundance was examined by life-stage. Overall, the picture is one of broadly distributed tick species that shows seasonal limitations in the timing of their questing activity.

Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer's disease.

BACKGROUND: Microglia, the brain's principal immune cells, have been implicated in the pathogenesis of Alzheimer's disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. METHODS: Hippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5-6 month-old [mo]) and old (22-25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings. RESULTS: There were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally and autosomally encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface ( https://neuroepigenomics.omrf.org/ ). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. CONCLUSIONS: These data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.

Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer's disease.

Background: Microglia, the brain's principal immune cells, have been implicated in the pathogenesis of Alzheimer's disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. Methods: Hippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5-6 month-old [mo]) and old (22-25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings. Results: There were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally-and autosomally-encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface ( https://neuroepigenomics.omrf.org/ ). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. Conclusions: These data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.