Fig. 5

Figure 5. Retinal spatiotemporal expression pattern of Mmp-2 protein after ONC in adult zebrafish. A: Representative picture showing the presence of active Mmp-2 in adult zebrafish retinal extracts, as confirmed by labeling of both pro (72 kDa) and active (64 kDa) Mmp-2 in zebrafish embryo lysates (left). A,B: Western blotting for Mmp-2 on retinal extracts at different timepoints postinjury indicates a peak in active Mmp-2 expression at 10 dpi during retinotectal regeneration, as compared to sham-operated controls (put at 100%). Data represent at least three retinal samples per timepoint and are shown as mean ± SEM (*P < 0.05). C-F: Immunostainings for Mmp-2 on retinal sections at 4 dpi disclose a decreased expression of Mmp-2 in RGC axons compared to controls, but an increased presence in RGC somata. During axonal regrowth, Mmp-2 expression becomes upregulated in RGC somata and axons, whereafter its expression again confines to RGC axons. G: An additional immunostaining on zebrafish retinal sections at 0 dpi, using a different MMP-2 antibody (EMD Millipore, AB19167), confirms Mmp-2 expression in the nerve fiber layer (NFL). For all immunostainings, DAPI (blue) was used as a nuclear counterstain. ONC, optic nerve crush; dpi, days postinjury; NFL, nerve fiber layer; RGCL, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer. Scale bars = 20 µm.