rhNGF-mediated pathway activation and gene expression in adult zebrafish during retinal regeneration. (A, B) Western blot analysis of ERK during retinal regeneration. (A) Total ERK protein levels in non-injected and rhNGF-injected adult zebrafish eyes at 0 h post injury (hpi) and at 36 hpi. Ctr-0 hpi (N: 15 eyes); 36 hpi rhNGF − (N: nine eyes); 36 hpi rhNGF + (N: 9 eyes) (3 eyes × time point × condition). (B) Phosphorylated ERK protein levels in the same samples analyzed in A. (C) Bar plot of pERK/ERK ratio as normalized fluorescence intensity relative to protein levels. Data are shown as means and standard error of the mean (SEM). The statistical analysis test used was one-way ANOVA followed by a Dunnett’s multiple comparisons test; 0 hpi vs. 36 hpi rhNGF +: Adjusted p-value: 0.0078; 36 hpi un-injected N: nine eyes; 36 hpi rhNGF − vs. 36 hpi rhNGF +: Adjusted p-value: 0.0347. (D–F). Expression levels of Müller glia-specific genes gfap, vim, and drgal1-L2 during retinal regeneration (N three eyes time point condition). Data are shown as mean ± SEM. ***p < 0.001; **p < 0.01; *p < 0.05 (two-way ANOVA test followed by a Sidak’s multiple comparisons test). gfap: 21 dpi un-injected vs. 21 dpi rhNGF-injected. Adjusted p-value: 0.0134; vim: 21 dpi un-injected vs. 21 dpi rhNGF-injected: Adjusted p-value: 0.0052. drgaI: 72 hpi un-injected vs. 72 hpi rhNGF-injected: Adjusted p-value: <0.001. rhNGF: recombinant human nerve growth factor.
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