Journal: Neurobiology of aging

Article Title: Old age increases microglial senescence, exacerbates secondary neuroinflammation, and worsens neurological outcomes following acute traumatic brain injury in mice.

doi: 10.1016/j.neurobiolaging.2019.02.010

Figure Lengend Snippet: A representative histogram depicts the relative expression level of NOX2 in brain-resident microglia (A). The MFI of NOX2-positive microglia is shown to the right, demonstrating an effect of age and injury in microglial expression. No change was seen in NOX2 MFI between young and aged CD45hiCD11b+ infiltrating myeloid populations after TBI (B). A representative histogram shows the relative production level of reactive oxygen species, as measured by DHR123 (C). MFI Quantification of DHR123-positive cells reveal a significant effect of injury on microglial-derived ROS production. A comparison of DHR123 MFI in young and old infiltrating myeloid cells is shown (D). A representative histogram illustrates the relative differences in the phagocytic uptake of fluorescent red beads in young and old microglia after TBI (E). To the right, the MFI quantification of red bead-positive microglia in young and old mice demonstrates that the relative level of phagocytosis induced by TBI is reduced in older microglia populations. The relative level phagocytic activity in young and old infiltrating myeloid cells is shown (F). A representative histogram depicts the relative expression level of MMP-9 in brain myeloid cells (G). MFI quantification of MMP-9-positive microglia is shown for young and aged mice at three days after TBI. No difference in relative MMP-9 expression between young and old infiltrating myeloid populations was found (H). A representative histogram shows the relative expression level of TNF (I). MFI Quantification of TNF-positive cells show a significant increase in microglia with both injury and age. An agerelated decrease in TNF production was in the infiltrating myeloid population after TBI (J). A representative histogram illustrates the relative expression level of IL-1β (K). To the right, MFI quantification of IL-1β-positive microglia demonstrates a significant effect of both age and injury. The relative production level of IL-1β is shown for infiltrating myeloid populations (L). For all experiments, N=4 sham and N=6 TBI/group. Statistical comparisons between groups were determined by two-way ANOVA analysis with Tukey’s multiple comparisons test. Significant group effects of age, injury, and interaction between age and injury are shown in each box. Error bars show mean SEM. Abbreviation: FMO fluorescence minus one, MFI mean fluorescence intensity, MMP-9 matrix metalloproteinase-9, NOX2 NADPH oxidase 2, DHR123 dihydrorhodamine 123, TNF tumor necrosis factor, IL-1β interleukin-1 beta, TBI traumatic brain injury, n.s. not significant, SEM standard error of mean. *p<0.05, **p<0.01, and ***p<0.001

Article Snippet: Cells were washed twice in 500 μL Permeabilization/Wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing cytokine antibodies (MMP-9-R-PE, StressMarq Biosciences (SMC-396D); p16 ink4a -APC, StressMarq Biosciences (SPC-1280D); p21 cip1 FITC, StressMarq Biosciences (SPC-1281); H2AX pS139-PE, MACS Miltenyi Biotec (130-107-585); NOX2-AF647, Bioss Antibodies (bs-3889R); TNF-PE-Cy7, eBioscience (MP6-XT22); IL-1β-PerCP-eF710, eBioscience (NJTEN3); and Bcl-2-PE-Cy7, BioLegend (BCL/10C4)) and fixed.

Techniques: Expressing, Derivative Assay, Activity Assay, Fluorescence

Journal: GeroScience

Article Title: Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice

doi: 10.1007/s11357-022-00562-y

Figure Lengend Snippet: Microglial phagocytosis of neurons, expression of autophagy markers, and lipofuscin content is chronically increased with age and injury. Phagocytosis was assessed by intracellular detection of neuronal and myelin antigens. A The percentage of NeuN-positive microglia was significantly increased with age and injury. The mean fluorescence intensity of FluoroMyelin red staining was acutely increased in microglia from both age groups (B) . The presence of vesicular glutamate transporter 1 (vGlut1) (C) and the phagosome marker CD68 (D ) were increased in microglia with both old age and TBI. Representative histograms illustrate the relative abundance of lysosomes and LC3II-positive autophagosomes in microglia as measured by LysoTracker (E) and Cyto-ID Autophagosome dyes (F) . For all histograms, gray = FMO control, blue = young, red = old, sham = no outline/no fill, 48 h TBI = bold outline/no fill, and 12w TBI = bold outline/bold fill. The mean fluorescence intensity of Lamp1 (G) and Sqstm1/p62 (H) for microglia are shown. N = 5–7/group. Data were analyzed using 2-way ANOVA group analysis with Tukey’s test for multiple comparisons. **** p < 0.0001, ** p < 0.01, * p < 0.05

Article Snippet: Intracellular staining for Ki67-PECy7 (Biolegend, Cat# 652426), PCNA-AF647 (Biolegend, Cat# 307912), CD68-PerCPCy5.5 (Biolegend, Cat# 137010), NeuN-PE (Millipore Sigma, Cat# FCMAB317PE), Vglut1-APC (StressMarq, Cat# SMC-394D-APC), Lamp1-PerCPCy5.5 (Biolegend, Cat# 121626), Lamp2-PE (Biolegend, Cat# 108506), Sqstm1/p62-AF647 (Novus Biologicals, Cat# NBP1-42822AF647), ATG5-AF647 (Biolegend, Cat# 847410), ATG7-AF700 (R&D Systems, Cat# FAB6608N), Ubiquitin-AF647 (Biolegend, Cat# 838710), H3-AF647 (Cell Signaling Technology, Cat# 12230S), Acetylated (Ac) Lysine (Lys)-PECy7 (Biolegend, Cat# 623408), H3-Ac-Lys9-AF488 (Cell Signaling Technology, Cat# 9683S), H3-Ac-Lys18-AF488 (Cell Signaling Technology, Cat# 73508S), H3-Ac-Lys27-AF647 (Cell Signaling Technology, Cat# 39030S), H3-Ac-Lys36-AF647 (Cell Signaling Technology, Cat# 84061S), Phospho(ser149)-H2A.X-PECy7 (Biolegend, Cat# 613420), p16-APC (StressMarq, Cat# SPC-1280D-APC), and p21-AF488 (RND-NBP2-43697AF488) was performed using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Cat# 554714) according to manufacturer’s instructions and as described previously [ ].

Techniques: Expressing, Fluorescence, Staining, Marker

Journal: GeroScience

Article Title: Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice

doi: 10.1007/s11357-022-00562-y

Figure Lengend Snippet: Trehalose treatment reduces long-term microgliosis, lymphocyte infiltration, and phagocytosis of neurons following TBI. A A representative dot plot of leukocyte populations in the brain at 9 weeks after TBI. Quantification of CD45 int CD11b + microglia (B ), CD45 hi CD11b + myeloid cells (C) , and CD45 hi CD11b − putative lymphocyte (D) cell counts are shown. Representative histograms show the relative level of E CD68 protein expression and F LipiBlue-stained lipid bodies in microglia. G The percentage of Vglut1-positive microglia in each treatment group is quantified. H Representative dot plots illustrate the percentage of NeuN-positive microglia in the ipsilateral hemisphere at 8 weeks post-TBI. The mean fluorescence intensity of NeuN immunoreactivity is quantified. For all histograms, gray = FMO control, sucrose (vehicle) treated = blue, trehalose treated = red, sham controls = no fill, and TBI groups = bold fill. N = 9–10/group (A–F) and N = 4–6/group (G–H) . Data were analyzed using 2-way ANOVA group analysis with Tukey’s test for multiple comparisons. **** p < 0.0001, ** p < 0.01, * p < 0.05

Article Snippet: Intracellular staining for Ki67-PECy7 (Biolegend, Cat# 652426), PCNA-AF647 (Biolegend, Cat# 307912), CD68-PerCPCy5.5 (Biolegend, Cat# 137010), NeuN-PE (Millipore Sigma, Cat# FCMAB317PE), Vglut1-APC (StressMarq, Cat# SMC-394D-APC), Lamp1-PerCPCy5.5 (Biolegend, Cat# 121626), Lamp2-PE (Biolegend, Cat# 108506), Sqstm1/p62-AF647 (Novus Biologicals, Cat# NBP1-42822AF647), ATG5-AF647 (Biolegend, Cat# 847410), ATG7-AF700 (R&D Systems, Cat# FAB6608N), Ubiquitin-AF647 (Biolegend, Cat# 838710), H3-AF647 (Cell Signaling Technology, Cat# 12230S), Acetylated (Ac) Lysine (Lys)-PECy7 (Biolegend, Cat# 623408), H3-Ac-Lys9-AF488 (Cell Signaling Technology, Cat# 9683S), H3-Ac-Lys18-AF488 (Cell Signaling Technology, Cat# 73508S), H3-Ac-Lys27-AF647 (Cell Signaling Technology, Cat# 39030S), H3-Ac-Lys36-AF647 (Cell Signaling Technology, Cat# 84061S), Phospho(ser149)-H2A.X-PECy7 (Biolegend, Cat# 613420), p16-APC (StressMarq, Cat# SPC-1280D-APC), and p21-AF488 (RND-NBP2-43697AF488) was performed using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Cat# 554714) according to manufacturer’s instructions and as described previously [ ].

Techniques: Expressing, Staining, Fluorescence

Journal: Nature Communications

Article Title: Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure

doi: 10.1038/s41467-018-06639-7

Figure Lengend Snippet: Hypertrophy-stage-specific p53 signaling activation. a Bar plot showing the statistical significance of the overlap between modules detected from co-expression analysis with or without hypertrophy-stage cardiomyocytes. The most strongly overlapping module pairs are selected from Supplementary Fig. . Modules are ordered by significance level. The red line indicates the threshold for hypertrophy stage-specific modules. b KEGG pathway enrichment of 12 hypertrophy stage-specific modules identified in a and Supplementary Fig. . c Co-expression network analysis of M7. d Representative images of Cdkn1a mRNA smFISH in the heart from mice at 2 weeks after pressure overload. Wheat germ agglutinin (WGA) and DAPI are used as markers of the plasma membrane and nucleus, respectively. Scale bar, 20 μm. e Bar plots showing the distribution of cells corresponding to the single-cell fluorescent intensity of Cdkn1a mRNA detected by smFISH in the heart after sham and TAC (weeks 2 and 8) operation. f Heatmap showing the enrichment of GO and KEGG pathway terms in 12 hypertrophy stage-specific modules. g Immunohistochemical staining of gH2A.X and p21 in the heart of mice at 2 weeks after pressure overload. WGA and DAPI are used as markers of the plasma membrane and nucleus, respectively. Arrows indicate the nuclei of gH2A.X and p21 double-positive cardiomyocytes. Scale bar, 20 μm

Article Snippet: The sections were incubated with the following antibodies overnight at 4 °C: mouse monoclonal anti-p21 antibody conjugated to Alexa Fluor 647 (1:50; sc6246 AF647; Santa Cruz Biotechnology) and rabbit polyclonal anti-γH2A.X (phospho S139) antibody (1:100; ab2893; Abcam).

Techniques: Activation Assay, Expressing, Clinical Proteomics, Membrane, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure

doi: 10.1038/s41467-018-06639-7

Figure Lengend Snippet: Distinct gene programs and their pathogenicity in human cardiomyocytes. a Co-expression network of human cardiomyocytes. Dot colors indicate module colors, matching the colors in b . b Heatmap showing the enrichment of GO and KEGG pathway terms in each module. c t-SNE visualization of human cardiomyocytes (71 normal and 340 dilated cardiomyopathy (DCM) cardiomyocytes). Cells (dots) are colored according to the cell clusters in Supplementary Fig. . d t-SNE plots of human cardiomyocytes colored by the expression of each module. e Heatmap showing the relative average expression for characteristic genes across the five modules. Representative genes are also indicated. f t-SNE plots of human cardiomyocytes colored by the expression of each gene. g Representative images of CDKN1A mRNA smFISH of the DCM heart. WGA and DAPI are used as markers of the plasma membrane and nucleus, respectively. Scale bar, 20 μm. h Bar plots showing the distribution of cells corresponding to the single-cell fluorescent intensity of CDKN1A mRNA detected by smFISH in the normal and DCM hearts. i Subnetwork analysis of human M1. j Scatter plot showing the relationship between M1 and M2 expression. DCM cardiomyocytes are separated into two groups: responder ( n = 59) and non-responder ( n = 281). k Heatmap showing the significance of gene overlaps between human and mouse modules. The table is colored by −log 10 ( P -value), obtained with Fisher’s exact test, according to the color legend below the table

Article Snippet: The sections were incubated with the following antibodies overnight at 4 °C: mouse monoclonal anti-p21 antibody conjugated to Alexa Fluor 647 (1:50; sc6246 AF647; Santa Cruz Biotechnology) and rabbit polyclonal anti-γH2A.X (phospho S139) antibody (1:100; ab2893; Abcam).

Techniques: Expressing, Clinical Proteomics, Membrane