Journal: Nature Communications

Article Title: Schwann cell TRPA1 mediates neuroinflammation that sustains macrophage-dependent neuropathic pain in mice

doi: 10.1038/s41467-017-01739-2

Figure Lengend Snippet: TRPA1 mediates pSNL-evoked allodynia and neuroinflammation. a Drawing representing the pSNL surgery in mice. b – e Time-dependent (3–20 days, d) mechanical allodynia ( b ), number and representative images of macrophages (F4/80 + cells) ( c , e ) and H 2 O 2 content ( d ) in the sciatic nerve trunk induced by pSNL in C57BL/6 compared to sham mice ( n = 6, * P < 0.005, ** P < 0.01 *** P < 0.001 pSNL vs. Sham; two-way ANOVA followed by Bonferroni post hoc analyses and unpaired two-tailed Student’s t-test). f Time-dependent (3–20 d) mechanical allodynia in sham/pSNL Trpa1 +/+ / Trpa1 −/− mice ( n = 8, *** P < 0.001 pSNL +/+ vs. Sham +/+ ; n = 6, § P < 0.05 and §§§ P < 0.001 pSNL −/− vs. pSNL +/+ ; two-way ANOVA followed by Bonferroni post hoc analyses). g Mechanical allodynia (at day 10 after surgery) in sham/pSNL mice after HC-030031 (HC03, 100 mg kg −1 , i.p.), A-967079 (A96, 100 mg/kg, i.p.) and α-lipoic acid (αLA, 100 mg kg −1 , i.p.) or respective vehicles (veh, 4% DMSO and 4% tween 80 in isotonic saline) ( n = 6, *** P < 0.001 pSNL veh vs. Sham veh; §§§ P < 0.001 pSNL-HC03, A96 or αLA vs. pSNL-veh; two-way ANOVA followed by Bonferroni post hoc analyses). h – l Representative images, number of F4/80 + cells, and H 2 O 2 content in the sciatic nerve of sham/pSNL Trpa1 +/+ / Trpa1 −/− and C57BL/6 mice, before (BL) and 1–6 h after HC03, A96, αLA (all, 100 mg kg −1 , i.p.) or respective vehicles (veh, 4% DMSO and 4% tween 80 in isotonic saline) ( n = 6, ** P < 0.01 and *** P < 0.001 pSNL Trpa1 +/+ vs. Sham- Trpa1 +/+ and pSNL veh vs. Sham veh; §§ P < 0.01 and §§§ P < 0.001 pSNL Trpa1 −/− vs. pSNL- Trpa1 +/+ and pSNL HC03, A96 or αLA vs. pSNL-veh; two-way and one-way ANOVA followed by Bonferroni post hoc analyses). (Scale bars: 50 μm; ( e , h ) dashed lines, perineurium). Data are represented as mean ± s.e.m

Article Snippet: Mouse thioglycollate-elicited peritoneal macrophages were harvested (up to 250 × 10 6 cells per ml) and incubated for 15 min at RT with VivoTrack 680 (PerkinElmer, Inc., Waltham, USA), dissolved in sterile PBS, washed, centrifuged (400 × g, 10 min) and diluted to a final concentration of 5 × 10 6 cells 40 μl −1 .

Techniques: Two Tailed Test, Saline

Journal: Nature Communications

Article Title: Schwann cell TRPA1 mediates neuroinflammation that sustains macrophage-dependent neuropathic pain in mice

doi: 10.1038/s41467-017-01739-2

Figure Lengend Snippet: Oxidative stress from Schwann cell TRPA1 recruits macrophages and signal pain in C57BL/6 mice. a , f Schematic representation of perineural/intrathecal injection of TRPA1 antisense/mismatch oligonucleotides (AS/MM-ODN). b , g TRPA1 immunofluorescence (mean gray value) and TRPA1 mRNA relative expression in DRGs and acute nociception after perineural AITC (20 nmol 10 µl −1 ) or capsaicin (CPS, 1 nmol 10 µl −1 ) following perineural (10 nmol 10 µl −1 ) ( b ) or intrathecal (5 nmol 5 µl −1 ) ( g ) TRPA1 AS/MM-ODN treatment (once/day for 4 consecutive days) in C57BL/6 ( n = 6, *** P < 0.001 MM/AS AITC, CPS vs. MM/AS veh; §§§ P < 0.001 AS AITC vs. MM AITC; one-way ANOVA followed by Bonferroni post hoc analyses, Scale bars: 20 µm). c , h Representative images (Scale bars: 50 μm; dashed lines, perineurium), ( j ) colocalization value (Rcoloc) of S100/TRPA1 and mRNA-TRPA1 expression in sciatic nerve after perineural ( c ) and intrathecal ( h ) AS/MM-ODN ( n = 6, * P < 0.05; *** P < 0.001 AS vs MM; unpaired two-tailed Student’s t-test). d , i Mechanical allodynia, and ( e , j ) representative images, F4/80 + -cells, and H 2 O 2 -content (at day 10 after surgery) in sham/pSNL mice after perineural ( d , e ) and intratechal ( i , j ) AS/MM-ODN ( n = 8, *** P < 0.001 pSNL-MM-ODN vs. sham-MM-ODN; § P < 0.05 and §§§ P < 0.001 pSNL-AS-ODN vs. pSNL-MM-ODN; ( d , i ) two-way ANOVA followed by Bonferroni post hoc analyses and ( e , j ) one-way ANOVA followed by Bonferroni post hoc analyses) (Scale bars: 50 μm; dashed lines, perineurium). Data are represented as mean ± s.e.m

Article Snippet: Mouse thioglycollate-elicited peritoneal macrophages were harvested (up to 250 × 10 6 cells per ml) and incubated for 15 min at RT with VivoTrack 680 (PerkinElmer, Inc., Waltham, USA), dissolved in sterile PBS, washed, centrifuged (400 × g, 10 min) and diluted to a final concentration of 5 × 10 6 cells 40 μl −1 .

Techniques: Injection, Immunofluorescence, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Schwann cell TRPA1 mediates neuroinflammation that sustains macrophage-dependent neuropathic pain in mice

doi: 10.1038/s41467-017-01739-2

Figure Lengend Snippet: TRPA1 blockade and antioxidant reduced the number of fluorescent macrophages accumulated at the site of pSNL. a In vivo imaging and quantitative data (NIR area/total ROI) of NIR labeled macrophages (at day 10 after surgery) in sham/pSNL mice at baseline (BL), 1 and 3 h after HC-030031 (HC03, 100 mg kg −1 , i.p.) ( n = 4, *** P < 0.001 pSNL HC03 vs. pSNL Veh HC03; two-way ANOVA followed by Bonferroni post hoc analyses). b Representative images and F4/80 + -cell number surrounding the injured nerve trunk (at day 10 after surgery) of sham/pSNL-mice at BL and 1, 3 and 6 h after HC03 or alpha-lipoic acid (αLA) (both, 100 mg kg −1 , i.p.). ( n = 4, *** P < 0.001 pSNL vs sham; §§§ P < 0.001 pSNL HC03, αLA vs. pSNL veh; one-way ANOVA followed by Bonferroni post hoc analyses) (Scale bars, 200 µm; inside (in) and outside (out) sciatic nerves). Data are represented as mean ± s.e.m

Article Snippet: Mouse thioglycollate-elicited peritoneal macrophages were harvested (up to 250 × 10 6 cells per ml) and incubated for 15 min at RT with VivoTrack 680 (PerkinElmer, Inc., Waltham, USA), dissolved in sterile PBS, washed, centrifuged (400 × g, 10 min) and diluted to a final concentration of 5 × 10 6 cells 40 μl −1 .

Techniques: In Vivo Imaging, Labeling

Journal: Nature Communications

Article Title: Schwann cell TRPA1 mediates neuroinflammation that sustains macrophage-dependent neuropathic pain in mice

doi: 10.1038/s41467-017-01739-2

Figure Lengend Snippet: Cellular and molecular events contributing to TRPA1-mediated mechanical allodynia and neuroinflammation in a neuropathic pain model. Partial sciatic nerve ligation (pSNL) by releasing CCL2 ( a ) promotes the extravasation of hematogenous monocytes ( b ) that, via their rapid NOX2-dependent oxidative burst (red dots) target the TRPA1 channel localized in Schwann cells ( c ). TRPA1 activation in Schwann cells evokes a Ca 2+ -dependent, NOX1-mediated ( d ) prolonged H 2 O 2 (green dots) generation ( e ) with a dual function. The outward H 2 O 2 release ( f ) produces a space-scaled gradient that determines the final macrophage influx to the injured nerve trunk, whereas the inward H 2 O 2 release ( g ) targets nociceptor TRPA1 to produce mechanical allodynia ( h ). ROS reactive oxygen species

Article Snippet: Mouse thioglycollate-elicited peritoneal macrophages were harvested (up to 250 × 10 6 cells per ml) and incubated for 15 min at RT with VivoTrack 680 (PerkinElmer, Inc., Waltham, USA), dissolved in sterile PBS, washed, centrifuged (400 × g, 10 min) and diluted to a final concentration of 5 × 10 6 cells 40 μl −1 .

Techniques: Ligation, Activation Assay

Journal: Biomedicines

Article Title: Selenium-Containing (Hetero)Aryl Hybrids as Potential Antileishmanial Drug Candidates: In Vitro Screening against L. amazonensis

doi: 10.3390/biomedicines12010213

Figure Lengend Snippet: Antileishmanial activity of selenium compounds.

Article Snippet: Murine peritoneal macrophages (1 × 10 6 cells/well), NIH/3T3 cells (ATCC CRL-1658, mouse fibroblasts lineage), and J774A.1 (ATCC, mouse macrophages lineage) were cultured in 96-well plates (1 × 10 5 cells/well) in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% FBS (Cultilab, Campinas, Brazil), 10 IU.mL −1 of penicillin, and 100 μg·mL −1 of streptomycin (Sigma-Aldrich, USA) and adhered overnight at 37 °C and 5% CO 2 .

Techniques: Activity Assay

Journal: Biomedicines

Article Title: Selenium-Containing (Hetero)Aryl Hybrids as Potential Antileishmanial Drug Candidates: In Vitro Screening against L. amazonensis

doi: 10.3390/biomedicines12010213

Figure Lengend Snippet: Cytotoxicity of synthesized selenium compounds MRK-105 , MRK-106 , MRK-107 , MRK-108 , and MRK-113 against NIH/3T3 fibroblasts ( A – E , upper graphs), J774A.1 macrophages ( F – J , middle graphs) and murine peritoneal macrophages ( K – O , lower graphs). *** mean p < 0.001 (ANOVA and Tukey´s post-test).

Article Snippet: Murine peritoneal macrophages (1 × 10 6 cells/well), NIH/3T3 cells (ATCC CRL-1658, mouse fibroblasts lineage), and J774A.1 (ATCC, mouse macrophages lineage) were cultured in 96-well plates (1 × 10 5 cells/well) in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% FBS (Cultilab, Campinas, Brazil), 10 IU.mL −1 of penicillin, and 100 μg·mL −1 of streptomycin (Sigma-Aldrich, USA) and adhered overnight at 37 °C and 5% CO 2 .

Techniques: Synthesized