Journal: Journal of neurochemistry

Article Title: Neuroinflammatory and behavioural changes in the Atp7B mutant mouse model of Wilson's disease.

doi: 10.1111/j.1471-4159.2011.07278.x

Figure Lengend Snippet: Fig. 2 Copper accumulation in liver and brain of toxic milk mice. (a, b) Copper content (a) and fold change (b) in copper in liver and brain areas of toxic milk mice in comparison to aged-matched controls. The number of livers was three per group and five brain tissues of different animals were pooled. Toxic milk mice and controls were matched for age and gender. Note differential change in different brain areas. Tx, toxic. Fig. 1 Livers of toxic milk mice show distinct histopathological chan- ges and inflammatory infiltrates. (a, b) Macroscopic pathology in livers (a) and HE stained liver sections (b) of toxic milk mice compared to age-matched controls. (c) Immunofluorescence or CD11b in liver sections of toxic milk mice and age-matched controls, demonstrating infiltrating lymphocytes. Scale bars in (b) and (c) are 100 lm. (d) Fold change in cytokine mRNA production in livers of toxic milk mice in comparison to controls. (e) Cytokine levels in the circulation of toxic milk mice compared to controls. Number of animals was five per group (three females, two males). Bars represent means + SEM. Asterisks indicate significant differences (*p < 0.05, ***p < 0.01). Tx, toxic.

Article Snippet: The following primary antibodies were used with respective concentrations: rabbit polyclonal anti-glial fibrillary acetic protein (GFAP) (1 : 1000, Dako, Glostrup, Denmark), rat monoclonal anti-mouse CD11b (1 : 200, Serotec, Oxford, UK) and mouse monoclonal against neuronal nuclei (NeuN) (1 : 500, Millipore, Temecula, CA, USA).

Techniques: Comparison, Staining

Journal: Journal of neurochemistry

Article Title: Neuroinflammatory and behavioural changes in the Atp7B mutant mouse model of Wilson's disease.

doi: 10.1111/j.1471-4159.2011.07278.x

Figure Lengend Snippet: Fig. 4 Inflammation in brains of toxic milk mice. (a–d) Astroglial (a) and microglial (c) reactivity in different brain regions of toxic milk and control mice as assessed by im- munohistochemistry for GFAP and CD11b, respectively, and quantification thereof (b, d). Scale bars in (a) are 100 lm, in (b) upper panels 200 lm and lower panels 500 lm. (e, f) Fold difference in cytokine, chemokine and inflammatory enzyme mRNAs in striatum (e) and hippocampus (f) of toxic milk and control mice. Number of animals was five per group (three females and two males per group). Significant dif- ferences are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Str, striatum; Cc, corpus callosum; Tx, toxic.

Article Snippet: The following primary antibodies were used with respective concentrations: rabbit polyclonal anti-glial fibrillary acetic protein (GFAP) (1 : 1000, Dako, Glostrup, Denmark), rat monoclonal anti-mouse CD11b (1 : 200, Serotec, Oxford, UK) and mouse monoclonal against neuronal nuclei (NeuN) (1 : 500, Millipore, Temecula, CA, USA).

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: The EP2 receptor induces expression of inflammatory enzymes and cytokines in mouse peritoneal macrophages. For all panels: *p < 0.05, **p < 0.01, ***p < 0.001, values are mean ± SEM. A, EP2 immunostaining is detected in wild-type but not EP2−/− C57BL/6 primary peritoneal macrophages costained for Cd11b. Scale bar, 100 μm. B, Peritoneal macrophages were stimulated with LPS (10 ng/ml) for 1 and 6 h. qPCR demonstrates a rapid upregulation of EP2 receptor mRNA by 1 h and subsequent downregulation by 6 h following LPS stimulation (n = 4–6 per group; two-way ANOVA for effect of time ##p < 0.01, and effect of interaction p = 0.02; Bonferroni's multiple-comparison tests comparing mean of 1 h vehicle (veh) and 1 h LPS *p < 0.05). C, Peritoneal macrophages were stimulated with LPS (10 ng/ml) +/− the EP2 agonist butaprost (1 μm) or vehicle. qPCR demonstrates an induction of proinflammatory mediators COX-2, iNOS, and gp91phox with LPS stimulation that is further enhanced by costimulation with butaprost (time points 1 h for COX-2 and 6 h for iNOS and gp91phox; n = 4–6 per group; two-way ANOVA for effect of LPS #p < 0.05, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost *p < 0.05, **p < 0.01). D, LPS-induced macrophage NO release is increased by EP2 receptor activation with butaprost (1 μm), whereas EP2−/− macrophages show reduced NO levels compared with EP2+/+ macrophages (n = 4 per group; Student's tests #p < 0.05, ##p < 0.01 comparing EP2+/+ to EP2−/−). E, qPCR demonstrates induction of IL-6 mRNA at 1 h and IL1β mRNA at 6 h, and a trend of decreased TNFα mRNA at 6 h in LPS-stimulated macrophages with addition of butaprost (n = 4–6 per group; two-way ANOVA for effect of LPS ##p < 0.01, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost **p < 0.01 for IL-6 at 1 h and IL1β at 6 h).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Expressing, Immunostaining, Comparison, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Conditional deletion of the EP2 receptor in macrophages suppresses oxidative enzyme and cytokine gene expression. A, Genomic DNA PCR is shown for EP2+/+, EP2lox/+, and EP2lox/lox C57BL/6 mice. B, Quantitative genomic PCR was assayed for EP2+/+ wild-type, EP2+/−, EP2−/−, Cd11bCre;EP2lox/lox, Cd11bCre;EP2lox/+, and Cd11bCre;EP2+/+; values are relative to wild-type EP2+/+ control DNA. C, Peritoneal macrophages were isolated from adult Cd11bCre;EP2lox/lox and Cd11bCre;EP2+/+ mice and sorted using Cd11b antibody-conjugated magnetic beads before qPCR analysis. Basal levels of EP2 mRNA, assayed by qPCR, are reduced by 91% in Cd11bCre;EP2lox/lox compared with control macrophages (left; Cd11bCre;EP2lox/lox vs Cd11bCre;EP2+/+) and are reduced by 56% in LPS-stimulated Cd11bCre;EP2lox/lox macrophages (right; **p < 0.01; n = 5–6 per group). D, Conditional deletion of EP2 in macrophages reduces LPS-mediated increases in proinflammatory gene expression (two-way ANOVA for effect of LPS treatment is represented by ###p < 0. 001; Bonferroni's multiple-comparisons tests comparing mean of Cd11bCre;EP2+/+/LPS and Cd11bCre;EP2lox/lox/LPS were ***p < 0.001; n = 5–6 per group).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Gene Expression, Control, Isolation, Magnetic Beads

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Examination of levels of monocytic populations in Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice. Splenocytes and peripheral blood immune cells were isolated from 3-month-old mice. A, Representative plots for CD11b+ cells gated on CD115 and Ly6C yielding four populations of cells, including CD115−/Ly6C− macrophages, CD115−/Ly6Cint-hi neutrophils, CD115int/Ly6Cint resident monocytes, and CD115hi-int/Ly6Chi inflammatory monocytes in vehicle and LPS-treated mice. B, Quantification of levels of monocytic populations, including macrophages, resident monocytes, and inflammatory monocytes does not show differences between genotypes in vehicle or LPS-treated mice. Levels of neutrophils are decreased in peripheral blood with LPS, but not vehicle stimulation (*p < 0.05; n = 4 mice per group). C, Quantification of CD11b-positive microglia derived from brains of Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice does not show differences in number (n = 5–7 mice per genotype). D, Comparison of copy number of EP2/copy number of 18S is shown for adult microglia and peritoneal macrophages from Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice (n = 4–6 per group; p < 0.05 unpaired t test). Macrophage expression of EP2 in Cd11bCre;EP2+/+ mice was 28-fold higher; however, the percentage reduction of expression with conditional deletion of EP2 was similar in both microglia and macrophages, and was 62.2 and 62.1%, respectively.

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Isolation, Derivative Assay, Comparison, Expressing