Journal: PLOS One

Article Title: In vivo mouse model of calcific myonecrosis induced by injury

doi: 10.1371/journal.pone.0346816

Figure Lengend Snippet: (a-c) Quantitative RT-PCR analysis of Nlrp3 (a) , Asc (b) , and Caspase-1 (c) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (** P < 0.01, *** P < 0.001). (d) Immunostaining of gastrocnemius muscle sections 3 days after notexin injection using anti-NLRP3, anti-ASC, and anti-Caspase-1 antibodies on serial sections. The upper panels show control muscles, and the lower panels show notexin-treated muscles. Scale bars, 20 μm. Asterisks indicate calcified muscle fibers. (e) Immunofluorescence staining showing the localization of NLRP3 (green), ASC or Caspase-1 (red) around calcified muscle fibers in notexin-injected skeletal muscle. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm. Asterisks indicate calcified muscle fibers. (f, g) Quantitative RT-PCR analysis of Il-1β (f) and Il-18 (g) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (*** P < 0.001). (h) Serum IL-18 levels measured by ELISA in control and notexin-injected mice (n = 6 (control), n = 9 (notexin)). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (i) Quantitative RT-PCR analysis of Runx2 mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (j) Western blot analysis of Runx2 and tubulin (loading control) protein expression in skeletal muscle from control and notexin-injected mice.

Article Snippet: Serum IL-18 concentrations were measured using the Mouse IL-18 DuoSet ELISA Kit (DY7625-05; R&D Systems) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Control, Injection, Immunostaining, Muscles, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Addiction Biology

Article Title: Engaging Gut‐to‐Brain Signalling to Treat Alcohol Use Disorder

doi: 10.1111/adb.70144

Figure Lengend Snippet: Effect of LPS and Nezavist on hippocampal cytokine levels and plasma CRF and corticosterone in mice. (A) Experimental design of the study. (B–G) Hippocampal cytokine levels. Levels of IL‐1 β (B), IL‐6 (C), TNF‐ α (D), IL‐10 (E), IL‐12p70 (F) and IL‐18 (G) in hippocampus collected from all treatment groups on Day 5, as determined by MSD assay, are shown (mean ± SEM). Averaged data from two separate experiments with the same samples are shown. Values are given as pg/g protein. No outlier test was performed. Statistics ( n = 10 per group): One‐way ANOVA followed by Bonferroni's multiple comparisons test (TNF‐ α and IL‐10) or Kruskal–Wallis test followed by Dunn's multiple comparisons test (all other measures). The C57BL/6, LPS, vehicle group served as reference group for pairwise comparisons. * p < 0.05, *** p < 0.001. (H–K) Quantification of Iba1 Immunofluorescence in the C57BL/6 Mouse Hippocampus (HC) after ip Nezavist and LPS. Immunofluorescence of Iba1 was detected with guinea pig monoclonal [Gp311H9] antibody. Iba1 immunosignal was significantly increased in LPS‐only treated animals in all four measured read‐outs when compared to vehicle‐only treated controls. Treatment with Nezavist at both doses led to significantly reduced immunoreactive area (H), density (I) and size (K) values of Iba1‐positive objects. In the case of the high dose animals (100‐mg/kg Nezavist) object intensity values were also significantly reduced (J). Graphs show the means of immunofluorescent signal on five brain sections per mouse [ n = 10]. Data were analysed by one‐way ANOVA and Bonferroni's post hoc test. The C57BL/6, LPS, vehicle group was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM. * p < 0.05, *** p < 0.001. (L–O) Quantification of CD68 Immunofluorescence in the C57BL/6 mouse hippocampus (HC) after ip Nezavist and LPS. Immunofluorescence of CD68 was detected with rat monoclonal [FA‐11] antibody. The immunosignal was significantly increased in the LPS‐only treated animals when compared to vehicle‐only treated controls. Slightly lower mean values of immunoreactive area (L) and object density (M) were found in mice that were treated with Nezavist; however, treatment effects were only significant in the case of the object intensity (N) in the lower dose (50 mg/kg) Nezavist‐treated animals. Graphs show the means of immunofluorescent signal on five brain sections per mouse [ n = 10]. Data were analysed by one‐way ANOVA and Bonferroni's post hoc test (object intensity and object size) or by Kruskal–Wallis test and Dunn's post hoc test (immunoreactive area and object density). The C57BL/6, LPS, vehicle group was defined as reference group for pairwise comparisons. Bar graphs represent group means ± SEM. * p < 0.05, *** p < 0.001. (P–T) Effects of LPS and Nezavist on plasma cytokine levels in C57BL/6 mice. Levels of IL‐1 β (P), IL‐6 (Q), TNF‐ α (R), IL‐10 (S) and IL‐12p70 (T) in terminal plasma samples collected from all treatment groups on Day 5, as determined by MSD assay, (mean ± SEM). Data are given as pg/mL plasma. No outlier test was performed. Statistics ( n = 9–10 per group): One‐way ANOVA followed by Bonferroni's multiple comparisons test (IL‐1 β or IL‐12p70) or Kruskal–Wallis test followed by Dunn's multiple comparisons test (all other measures). The C57BL/6, LPS, vehicle group served as reference group for pairwise comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. LLOQ, lower limit of quantification. (U,V) Effects of ip LPS and Nezavist on plasma CRF and corticosterone in C57BL/6 mice. Levels of CRF (U) and corticosterone (V) in terminal plasma samples collected from all treatment groups on Day 5, as determined by ELISA, are shown (mean ± SEM, pg/mL plasma). No outlier test was performed. Statistics ( n = 10 per group): One‐way ANOVA followed by Bonferroni's multiple comparisons test (CRF) or Kruskal–Wallis test followed by Dunn's multiple comparisons test (corticosterone). The C57BL/6, LPS, vehicle group served as reference group for pairwise comparisons. * p < 0.05 and *** p < 0.001.

Article Snippet: Brain (hippocampus) extracts from all animals ( n = 40 samples) as well as terminal plasma samples ( n = 40 samples) were diluted 1:2 and analysed for cytokines included in an inflammation panel (IL‐1 β , IL‐6, IL‐12p70, IL‐10 and TNF α ) with a U‐PLEX custom Cytokine Assay (K15069L‐1) from Mesoscale Discovery (MSD) and for IL‐18 with the Mouse IL‐18 DuoSet ELISA from R&D Systems (DY7625‐05).

Techniques: Clinical Proteomics, Immunofluorescence, Enzyme-linked Immunosorbent Assay