Figure 1
Figure 1. DMOG delays resolution of neutrophilic inflammation. (A-B) Fluorescent neutrophil numbers in the mpx:GFP line were counted at 6, 24, and 48 hpi in anesthetized embryos. Six hpi is the time point of maximal neutrophil recruitment. By 24 hpi, neutrophilic inflammation has resolved in wild-type embryos. At 4 hpi, fish were treated with 100μM DMOG (B) or with DMSO as vehicle control (A). Data shown are individual embryos for each line, n = 18 performed as 3 independent experiments. (C) Fluorescence photomicrographs of 8 zebrafish tails from control (DMSO; top panel) and DMOG-treated (bottom panel) larvae. Imaged at 24 hpi, original magnification ×2 on a TE2000U inverted microscope (Nikon) at constant exposure. (D-E) Overlaid fluorescence and brightfield photomicrographs of injured 3 dpf embryos at 6 and 24 hpi (D) or uninjured 3 and 4 dpf embryos (E) after treatment at 3 dpf with DMSO and DMOG. Imaged at original magnification ×2 on a TE2000U inverted microscope (Nikon) at constant exposure. (F) The 6-hpi time point neutrophil counts, in DMSO- and DMOG-treated zebrafish embryos. Data shown are mean ± SEM, n = 18 performed as 3 independent experiments. (G) Resolution of the cellular component of inflammation is decreased in DMOG-treated embryos, expressed as percentage of change in neutrophil number between 6 and 24 hpi. Data shown are mean ± SEM, n = 18 performed as 3 independent experiments. P values were calculated using 1-way ANOVA and Bonferroni multiple comparison test, *P < .05, **P < .01, and ***P < .001. (H) Photomicrograph of a typical tracking experiment, with arrows indicating the path of neutrophil movement over a 1-hour time lapse during the recruitment phase of inflammation (1-2 hpi). No difference was observed in the speed of neutrophil migration (I) or meandering index (displacement/path length; J) in DMOG-treated larvae compared with DMSO controls. Data shown are mean ± SEM, n = 18 performed as 3 independent experiments. (K) TUNEL and TSA colocalization shows the percentage of neutrophils at the injury site undergoing apoptosis. Data shown are mean ± SEM, n = 3 performed as independent experiments each containing 35 to 40 embryos/treatment group.

DMOG delays resolution of neutrophilic inflammation. (A-B) Fluorescent neutrophil numbers in the mpx:GFP line were counted at 6, 24, and 48 hpi in anesthetized embryos. Six hpi is the time point of maximal neutrophil recruitment. By 24 hpi, neutrophilic inflammation has resolved in wild-type embryos. At 4 hpi, fish were treated with 100μM DMOG (B) or with DMSO as vehicle control (A). Data shown are individual embryos for each line, n = 18 performed as 3 independent experiments. (C) Fluorescence photomicrographs of 8 zebrafish tails from control (DMSO; top panel) and DMOG-treated (bottom panel) larvae. Imaged at 24 hpi, original magnification ×2 on a TE2000U inverted microscope (Nikon) at constant exposure. (D-E) Overlaid fluorescence and brightfield photomicrographs of injured 3 dpf embryos at 6 and 24 hpi (D) or uninjured 3 and 4 dpf embryos (E) after treatment at 3 dpf with DMSO and DMOG. Imaged at original magnification ×2 on a TE2000U inverted microscope (Nikon) at constant exposure. (F) The 6-hpi time point neutrophil counts, in DMSO- and DMOG-treated zebrafish embryos. Data shown are mean ± SEM, n = 18 performed as 3 independent experiments. (G) Resolution of the cellular component of inflammation is decreased in DMOG-treated embryos, expressed as percentage of change in neutrophil number between 6 and 24 hpi. Data shown are mean ± SEM, n = 18 performed as 3 independent experiments. P values were calculated using 1-way ANOVA and Bonferroni multiple comparison test, *P < .05, **P < .01, and ***P < .001. (H) Photomicrograph of a typical tracking experiment, with arrows indicating the path of neutrophil movement over a 1-hour time lapse during the recruitment phase of inflammation (1-2 hpi). No difference was observed in the speed of neutrophil migration (I) or meandering index (displacement/path length; J) in DMOG-treated larvae compared with DMSO controls. Data shown are mean ± SEM, n = 18 performed as 3 independent experiments. (K) TUNEL and TSA colocalization shows the percentage of neutrophils at the injury site undergoing apoptosis. Data shown are mean ± SEM, n = 3 performed as independent experiments each containing 35 to 40 embryos/treatment group.

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