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. 2023 May 26;22(7):e13832. doi: 10.1111/acel.13832

FIGURE 1.

FIGURE 1

GCV and ABT263 treatment successfully deplete senescent endothelial cells from the brains of PTX‐treated p16‐3MR mice. (a) Schematic representation of the experimental design. PTX: paclitaxel, GCV: ganciclovir, NVC: neurovascular coupling. (b–f) Flow cytometric detection of RFP+/CD‐31+ senescent endothelial cells, RFP−/CD‐31+ non‐senescent endothelial cells and RFP+/CD‐31− senescent non‐endothelial cells in single‐cell suspensions obtained from the brains of control and PTX treated p16‐3MR mice that received vehicle, GCV or the senolytic drug ABT263. Brains were analyzed 6 months after PTX. (b) shows a representative dot plot of RFP‐Booster Atto647N fluorescence (which correlates with p16‐3MR expression) versus CD‐31 staining (583/26 nm), depicting the percentage of senescent endothelial cells with bright fluorescence in the brain of a PTX treated mouse. Summary data for all senescent cells are shown in (e). Data are mean ± SEM (n = 4 for each data point). ***p < 0.001 versus control. (d) Representative density plots of RFP‐Booster Atto647N fluorescence versus autofluorescence obtained from the FACS sorting of single cell brain suspension of a PTX treated mouse. Note the distinct population of RFP+ senescent cells (purple arrow). (e) Flow cytometric detection of RFP+/CD‐31+ senescent endothelial cells and RFP+/CD‐31− senescent non‐endothelial cells in single‐cell suspension obtained from the brain of a PTX treated p16‐3MR mouse, which was enriched for RFP+ cells by FACS. Dot plot of RFP‐Booster Atto647N fluorescence versus CD‐31 staining depicts the percentages of senescent endothelial cells with bright fluorescence in both channels and that of RFP+/CD‐31− senescent non‐endothelial cells. (f) Pie charts in the upper row show the ratio of senescent endothelial cells as percentage of all endothelial cells. Note that PTX treatment significantly increases the presence of senescent endothelial cells in the mouse brain, which is reversed by both GCV and ABT263 treatment. Pie charts in the bottom row show the ratio of senescent endothelial cells as percentage of all senescent cells. (g) Identification of cerebromicrovascular endothelial cells based on differentially expressed marker genes (scRNAseq data). Shown is two‐dimensional UMAP plot based on differentially expressed marker genes, colored by cluster. Cluster identity was assigned based on previously reported differentially expressed genes listed in Table S1. (h) Cells with high expression of senescence markers overlaid on UMAP plots for brains of PTX treated mice. (i) Bar charts showing percentage of senescent endothelial cells, microglia, pericytes, vascular smooth muscle cells (VSMCs) and oligodendrocytes in brains of PTX treated mice. (j) Principal component analysis of RNA‐Seq data generated from brain endothelial cells derived from PTX treated mice. Capillary‐, venous‐, and arterial endothelial cells were identified based on their specific gene expression signatures. Shown is visualization of endothelial cell subclusters, color coded for the identified endothelial phenotypes. Marker genes of each cluster are provided in Table S3. Senescent endothelial cells identified based on high expression of senescence markers are highlighted (red) in (k). (l) Spatially‐resolved mRNA expression of the senescence marker gene Cdkn2a (green ST spots) in brains of control and PTX treated p16‐3MR mice that received vehicle, GCV or ABT263. Representative H&E stained coronal sections of brains are shown. (m) Spatial distribution of Cdkn2a positive spots, expressed as a percentage of all spots in the different anatomical regions in brains of each group of mice. GCV and ABT263 treatment successfully depleted senescent cells from the isocortex of PTX treated p16‐3MR mice.