In vivo single microglial cell isolation after intracerebral hemorrhage in mice.

Failure to translate promising potential therapeutics for intracerebral hemorrhage (ICH) partially results from limited understanding of cellular mechanisms underlying brain injury and repair. Understanding neural repair mechanisms after brain injury requires intricate comprehension of microglial behavior; however, studying individual microglial cell behavior is challenging. Further single cell isolation techniques may be an excellent means to expand known differences in male and female microglial cell response to ICH. In this study, 24 h after intrastriatal collagenase injection, one male and one female CX3CR1-GFP mouse underwent ex vivo microglial cell isolation via micropipette from perihematomal regions and equivalent location of contralateral striata. After cell collection, individual and grouped cell samples underwent reverse transcription and analyses for gene expression using Fluidigm RT-PCR technology. Data were analyzed by t-tests and visualized as a heatmap of the log2 Ct values. Gene expression assays were chosen for target-specific amplification, including markers of M1 pro-inflammatory microglial phenotype (i.e., Tnf, Il6, Fcgr3/CD16), M2 anti-inflammatory markers (i.e., Mrc1/CD206, Arg1, Tgfb1), and genes involved in the toll-like receptor pathway (i.e., Tlr2, Tlr4 and Myd88). Greater number of individual microglia cells expressed Mcr1, Tlr2, and Arg1 in perihematomal tissue than in contralateral hemispheres. Additionally, more male microglia expressed Myd88, Tlr2, Il6, and Arg1 than did female microglia. Single cell microglial isolation is feasible after in vivo rodent ICH. Differential gene expression can be detected between individual cells from different brain regions and experimental conditions. Cell-specific analyses will contribute to improved understanding of microglial roles in both post-ICH pathogenesis and recovery.

Wildfires and extracellular vesicles: Exosomal MicroRNAs as mediators of cross-tissue cardiopulmonary responses to biomass smoke.

INTRODUCTION: Wildfires are a threat to public health world-wide that are growing in intensity and prevalence. The biological mechanisms that elicit wildfire-associated toxicity remain largely unknown. The potential involvement of cross-tissue communication via extracellular vesicles (EVs) is a new mechanism that has yet to be evaluated. METHODS: Female CD-1 mice were exposed to smoke condensate samples collected from the following biomass burn scenarios: flaming peat; smoldering peat; flaming red oak; and smoldering red oak, representing lab-based simulations of wildfire scenarios. Lung tissue, bronchoalveolar lavage fluid (BALF) samples, peripheral blood, and heart tissues were collected 4 and 24 h post-exposure. Exosome-enriched EVs were isolated from plasma, physically characterized, and profiled for microRNA (miRNA) expression. Pathway-level responses in the lung and heart were evaluated through RNA sequencing and pathway analyses. RESULTS: Markers of cardiopulmonary tissue injury and inflammation from BALF samples were significantly altered in response to exposures, with the greatest changes occurring from flaming biomass conditions. Plasma EV miRNAs relevant to cardiovascular disease showed exposure-induced expression alterations, including miR-150, miR-183, miR-223-3p, miR-30b, and miR-378a. Lung and heart mRNAs were identified with differential expression enriched for hypoxia and cell stress-related pathways. Flaming red oak exposure induced the greatest transcriptional response in the heart, a large portion of which were predicted as regulated by plasma EV miRNAs, including miRNAs known to regulate hypoxia-induced cardiovascular injury. Many of these miRNAs had published evidence supporting their transfer across tissues. A follow-up analysis of miR-30b showed that it was increased in expression in the heart of exposed mice in the absence of changes to its precursor molecular, pri-miR-30b, suggesting potential transfer from external sources (e.g., plasma). DISCUSSION: This study posits a potential mechanism through which wildfire exposures induce cardiopulmonary responses, highlighting the role of circulating plasma EVs in intercellular and systems-level communication between tissues.