Journal: The FASEB Journal

Article Title: Stress hormone signaling through β-adrenergic receptors regulates macrophage mechanotype and function

doi: 10.1096/fj.201801429RR

Figure Lengend Snippet: β-AR signaling activation reduces the deformability of macrophages. A) Retention of monocyte (U937) and macrophage (U937+PMA) cells was measured using parallel microfiltration after isoproterenol treatment for 24 h. Retention is measured by the mass of cell suspension that remains in the top well after applying 2 kPa pressure for 30 s relative to the initial mass. Higher retention indicates that cells are more likely to occlude the 5 µm pores. B) Cell size distribution of monocytes and macrophages in vehicle control (Veh) and isoproterenol (Iso)-treated groups. C) Density scatterplot shows data from quantitative deformability cytometry. We measure cell size and transit time, or the timescale for macrophages to pass through the 5 × 10 μm gap of a microfluidic channel (n > 1600). D) Boxplot shows the lower and upper quartiles with median (line). Whiskers show the 10–90th percentiles (P < 0.0001). E) Isoproterenol effects on mouse macrophage (RAW 264.7) cells. F–H) Relative retention measured by PMF following 24 h treatment with Iso (100 nM) and the β-blocker propranolol (Pro) (10 μM) (F). Pro+Iso indicates cotreatment. Adenylyl cyclase activator forskolin (For; 10 μM) (G). Calcium chelator BAPTA-AM [1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis-acetoxymethyl ester] (10 μM) (H). One-way ANOVA test with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant. Results represent data from at least 3 independent experiments (N = 3).

Article Snippet: The adenylyl cyclase activator forskolin (MilliporeSigma) was used at 10 µM for 24 h. To inhibit polymerization of F-actin, we treated cells with 2 μM cytochalasin D (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. To inhibit the activity of Arp2/3 and formins, cells were treated with 50 µM CK666 (MilliporeSigma) or 20 µM SMIFH2 (MilliporeSigma) for 24 h. To chelate intracellular Ca 2+ , cells were treated with 10 μM 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′ -tetraacetic acid tetrakis-acetoxymethyl ester (Thermo Fisher Scientific) for 24 h. Quantitative RT-PCR To measure levels of β-adrenoceptors in U937 cells before and after PMA-differentiation into macrophages, TRIzol reagent (Thermo Fisher Scientific) was used to extract total RNA from cells according to the manufacturer’s protocol. cDNA was obtained using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) starting from 1 µg of RNA.

Techniques: Activation Assay, Cytometry

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on TH striatal levels. ( A ) Representative example of striatal TH immunofluorescence. Scale bar = 1000 μm and ( B ) TH striatal intensity in SHAM and GDX males and SHAM and GDX female mice. Values shown are the means pixel intensity ± SEM of 6-16 mice per group. Two-way ANOVA, Tukey post-hoc test: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques: Immunofluorescence

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on striatal microglial density. ( A ) Representative example of striatal Iba1 immunofluorescence. Scale bar = 20 μm; ( B ) Iba1 positive cells count in the striatum in SHAM and GDX males and SHAM and GDX female mice. Values shown are the mean number of Iba1 positive cells per mm 3 ± SEM of 4 images of 4-6 mice per group and 7 to 25 microglia per image. Two-way ANOVA, Tukey post-hoc test: * p < 0.05 and ** p < 0.01 and ( C ) Correlations between striatal DA contents with striatal microglial density (Iba1 positive cell counts).

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques: Immunofluorescence

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on striatal microglial doublet. ( A ) Representative example of striatal microglial doublet indicated with white arrowheads. Scale bars = 5 μm; ( B ) Microglial doublet counts in the striatum in SHAM and GDX males and SHAM and GDX female mice. Values shown are the mean number of doublets per image ± SEM 4 images of 4-6 mice per group and from 0 to 4 microglial doublet per image. Two-way ANOVA, Tukey post-hoc test: * p < 0.05 and **** p < 0.0001 and ( C ) Correlations between striatal DA contents with striatal microglial doublet counts.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques:

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Three-dimension morphological analysis of striatal microglia schematic and effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on striatal microglial morphology in 3D. ( A ) Number of arborization terminal points representing the number of ramifications terminal points in the whole microglia. Values shown are the sum of terminal points of microglia ± SEM ( B ) Number of arborization branch points representing the number of ramifications branching points in the whole microglia. Values shown are the sum of branch points of microglia ± SEM; ( C ) Number of arborization segments representing the number of ramification segments in the whole microglia. Values shown are the sum of the segment of microglia ± SEM; ( D ) Arborization length defined as the sum of the lengths of all branches within the entire microglia. Values shown are the sum of the length of each branch of each microglia ± SEM; ( E ) Sholl intersections defined as the sum of branches intersections with concentric circles (spheres in 3D) spaced 1 μm each. Values shown are the sum of Sholl intersection of each microglia ± SEM; ( F ) Radius expressed as maximum reach from the nucleus where the concentric circle (spheres in 3D) presenting the intersection of the branches. Values shown are concentric circle numbers having the last branch intersection of microglia. The mean and SEM of 4-6 mice per group and from 1 to 14 microglia per mice is shown. Each point represents one microglia. Two-way ANOVA, Tukey post-hoc test: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques:

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Experimental design. Male and female mice were gonadectomized (GDX) or sham operated (SHAM) and received intraperitoneal injection of dutasteride (DUT) or vehicle and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) or saline.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques: Injection, Saline