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Fig. 4 Brain neuroinflammation and neurodegeneration in galcb knockout zebrafish. (A and B) RT-qPCR analysis of the expression of neuroinflammation-related genes interleukin-1b (il1b), il8, tumor necrosis factor-a (tnfa), apolipoprotein Eb (apoeb), macrophage expressed 1 (mpeg1) and glial fibrillary acidic protein (gfap) in the brain of wild-type (WT), galcb+/− and galcb−/− zebrafish at 4 months post fertilization (mpf). For each gene, the data were normalized to its expression in one WT animal. Data are the mean ± standard deviation of 5–7 animals per group. (C) Immunofluorescence analysis shows the presence of numerous L-plastin-positive cells in the brain of galcb−/− zebrafish at 4 mpf, absent in WT and galcb+/−siblings. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Right: Quantification of L-plastin signal intensity in ≥7 microscopic fields/group. (D) Periodic acid–Schiff (PAS) staining shows the presence of globoid cells (arrows) in the periventricular grey zone (PGZ) of the brain of galcb−/− zebrafish at 4 mpf, absent in WT and galcb+/−siblings. The number of PAS-positive globoid cells were counted in ≥4 microscopic sections/group. (E) Immunohistochemical analysis shows the increase of cleaved caspase3-positive cells in the brain of galcb−/− zebrafish at 4 mpf compared with WT siblings. The number of cleaved caspase3-positive cells were counted in at least 14 microscopic fields/group. (F) RT-qPCR analysis of the expression of neuronal marker genes nestin, neuronal differentiation 1 (neurod1) and RNA binding fox-1 homolog 3a (neun) in the brain of WT, galcb+/− and galcb−/− zebrafish at 4 mpf. For each gene, the data were normalized to its expression in one WT animal (n > 4 animals/group). Data are the mean ± standard deviation. One-way ANOVA (Tukey's multiple comparison test): *P < 0.05, **P < 0.01, ***P < 0.001. RT-qPCR = reverse transcriptase-quantitative PCR.