TRPM7 inhibition protects mice from increased neutrophil-endothelial interaction (NEI) during endotoxemia. A NEI in mice treated with LPS 3 mg/kg i.p. and or carvacrol 80 mg/kg i.p. (1 h post-endotoxemia) were inspected using a scanning laser ophthalmoscope (SLO) to obtain confocal images of the fundus from the left eye. B-E Representative SLO images obtained from a real-time movie depicting NEI count at 3 h (B) and 12 h (C) post-endotoxemia. Representative SLO images obtained from a real-time movie depicting NEI count at 3 h (D) and 12 h (E) post-endotoxemia in mice subjected to injection of carvacrol 1 h after endotoxemia. F Serial quantification of SLO captures from endotoxic mice in the absence or presence carvacrol at 0,3, 6, 12, 24, 48 and 72 h post-endotoxemia. Grey: reflectance with 638 nm laser. Red: rat anti-Ly6G. Arrowheads: neutrophils labelled with an anti-Ly6G IgG. v: Venules. a: Arterioles. n: Optic nerve head. Scale bar = 100 μm. (N = 5 per group). Statistical differences were assessed by two-way ANOVA followed by Dunnett's post hoc test. **p < 0.01, ****p < 0.0001 comparing endotoxemic (red) vs endotoxemic/ carvacrol (green) conditions. Results showed as mean ± SEM

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... evaluate the participation of TRPM7 in neutrophils rolling on blood vessels in an in situ model, intravital imaging experiments were performed in mice subjected to endotoxemia by i.p. administration of 3 mg/kg endotoxin in the presence or absence of 80 mg/kg carvacrol 1 h after endotoxemia induction (Fig. 3A). Because TRPM7 knockout in mice leads to early embryonic lethality [58], pharmacological modulation of TRM7 is the preferred approach. Confocal retinal reflectance imaging revealed the anatomical features that are characteristic of the retina: optic nerve head (n), arterioles (a), and venules (v) (Fig. 3B), which provide a ...

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... 1 h after endotoxemia induction (Fig. 3A). Because TRPM7 knockout in mice leads to early embryonic lethality [58], pharmacological modulation of TRM7 is the preferred approach. Confocal retinal reflectance imaging revealed the anatomical features that are characteristic of the retina: optic nerve head (n), arterioles (a), and venules (v) (Fig. 3B), which provide a morphological context to aid in following the trajectory and period of interaction of fluorophore-labeled neutrophils in ...

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... (NEI), which increased starting at 6 h after endotoxemia . Results were normalized against vehicle-exposed cells (control condition). Statistical differences were assessed by a two-way analysis of variance (ANOVA) followed by Tukey post hoc test. ****p < 0.0001, compared with the saline-treated condition. Results showed as mean ± SEM induction (Fig. 3F), reached a peak at 12 h ( Fig. 3C and F; Additional file 3: Movie S2) compared with 3 h ( Fig. 3B and F; Additional file 2: Movie S1), and returned to baseline at 72 h (Fig. 3F). However, carvacrol administration 1 h after endotoxemia induction significantly attenuated the rise in NEI at the peak ( Fig. 3E and F; Additional file 4: ...

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... 6 h after endotoxemia . Results were normalized against vehicle-exposed cells (control condition). Statistical differences were assessed by a two-way analysis of variance (ANOVA) followed by Tukey post hoc test. ****p < 0.0001, compared with the saline-treated condition. Results showed as mean ± SEM induction (Fig. 3F), reached a peak at 12 h ( Fig. 3C and F; Additional file 3: Movie S2) compared with 3 h ( Fig. 3B and F; Additional file 2: Movie S1), and returned to baseline at 72 h (Fig. 3F). However, carvacrol administration 1 h after endotoxemia induction significantly attenuated the rise in NEI at the peak ( Fig. 3E and F; Additional file 4: Movie S4) compared with 3 h ( Fig. 3D ...

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... cells (control condition). Statistical differences were assessed by a two-way analysis of variance (ANOVA) followed by Tukey post hoc test. ****p < 0.0001, compared with the saline-treated condition. Results showed as mean ± SEM induction (Fig. 3F), reached a peak at 12 h ( Fig. 3C and F; Additional file 3: Movie S2) compared with 3 h ( Fig. 3B and F; Additional file 2: Movie S1), and returned to baseline at 72 h (Fig. 3F). However, carvacrol administration 1 h after endotoxemia induction significantly attenuated the rise in NEI at the peak ( Fig. 3E and F; Additional file 4: Movie S4) compared with 3 h ( Fig. 3D and F; Additional file 4: Movie S3) and throughout the course of the ...

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... a two-way analysis of variance (ANOVA) followed by Tukey post hoc test. ****p < 0.0001, compared with the saline-treated condition. Results showed as mean ± SEM induction (Fig. 3F), reached a peak at 12 h ( Fig. 3C and F; Additional file 3: Movie S2) compared with 3 h ( Fig. 3B and F; Additional file 2: Movie S1), and returned to baseline at 72 h (Fig. 3F). However, carvacrol administration 1 h after endotoxemia induction significantly attenuated the rise in NEI at the peak ( Fig. 3E and F; Additional file 4: Movie S4) compared with 3 h ( Fig. 3D and F; Additional file 4: Movie S3) and throughout the course of the experiment time-lapse (Fig. 3F). Neutrophil rolling is the final step in ...

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... Results showed as mean ± SEM induction (Fig. 3F), reached a peak at 12 h ( Fig. 3C and F; Additional file 3: Movie S2) compared with 3 h ( Fig. 3B and F; Additional file 2: Movie S1), and returned to baseline at 72 h (Fig. 3F). However, carvacrol administration 1 h after endotoxemia induction significantly attenuated the rise in NEI at the peak ( Fig. 3E and F; Additional file 4: Movie S4) compared with 3 h ( Fig. 3D and F; Additional file 4: Movie S3) and throughout the course of the experiment time-lapse (Fig. 3F). Neutrophil rolling is the final step in plateletguided interaction with ECs. Thus, NEI is a decisive readout for studying endothelial activation during systemic inflammation, ...

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... at 12 h ( Fig. 3C and F; Additional file 3: Movie S2) compared with 3 h ( Fig. 3B and F; Additional file 2: Movie S1), and returned to baseline at 72 h (Fig. 3F). However, carvacrol administration 1 h after endotoxemia induction significantly attenuated the rise in NEI at the peak ( Fig. 3E and F; Additional file 4: Movie S4) compared with 3 h ( Fig. 3D and F; Additional file 4: Movie S3) and throughout the course of the experiment time-lapse (Fig. 3F). Neutrophil rolling is the final step in plateletguided interaction with ECs. Thus, NEI is a decisive readout for studying endothelial activation during systemic inflammation, regardless of the degree of platelet contribution, yielding an ...

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... file 2: Movie S1), and returned to baseline at 72 h (Fig. 3F). However, carvacrol administration 1 h after endotoxemia induction significantly attenuated the rise in NEI at the peak ( Fig. 3E and F; Additional file 4: Movie S4) compared with 3 h ( Fig. 3D and F; Additional file 4: Movie S3) and throughout the course of the experiment time-lapse (Fig. 3F). Neutrophil rolling is the final step in plateletguided interaction with ECs. Thus, NEI is a decisive readout for studying endothelial activation during systemic inflammation, regardless of the degree of platelet contribution, yielding an approximate readout for detecting platelet interactions with ...

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... results were obtained in zebrafish larvae microinjected with saline solution or endotoxin in the presence or absence of the non-selective TRPM7 inhibitor FTY-720 (Additional file 1: Figure S3 and Additional file 10: Movie 9, Additional file 11: Movie 10, Additional file 12: Movie 11, Additional file 13: Movie 12). ...

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... 30 min. respectively, and platelets and neutrophils adhesion was analyzed (N = 3-4). (G-I) Protein expression quantification of vWF (G), ICAM-1 (H) and P-Sel (I) by densitometric analysis from immunofluorescence experiments in vehicle-and endotoxin-treated ECs in the presence or absence of the TRPM7 α-kinase function inhibitor TG100-115 (N = 3-4). ...