Fig 1.
Salinomycin (SNC) does not significantly alter the function or structure of the rods and cones of the retina 4 weeks after intravitreal injection.
A) DMSO-treated eyes have similar ERG traces to those treated with SNC for both scotopic (rod function) and photopic (cone function) parameters. B) The average a- and b-wave amplitudes are not significantly different between DMSO (n = 6) and SNC-treated (n = 9) eyes at scotopic and photopic light intensities. Fundus (C) and OCT (D) imaging reveals no differences in retinal appearance. E) H & E staining of the retina 4 weeks after treatment shows no differences in retina layers or appearance. F) The nuclear layer thicknesses for both the inner nuclear layer (INL) and outer nuclear layer (ONL) are not significantly different between DMSO-treated and SNC-treated eyes (n = 5 eyes for each analysis).
Fig 2.
Salinomycin (SNC) is effective at slowing PVR progression over 4 weeks in a mouse model of PVR.
A) A violin plot showing the distribution of PVR grades over 4 weeks for eyes injected with only SNC, RPE/DMSO and RPE/SNC. Mann-Whitney U tests showed significant differences between the distribution of PVR grades at all time-points (*p<0.05, **p<0.01, ***p<0.001). B) Representative weekly fundus images and a 4w OCT image from two representative eyes injected with RPE/DMSO and two representative eyes injected with RPE/SNC. C) H&E staining at 4 weeks post-treatment of the eyes injected with RPE/DMSO and RPE/SNC shown in B.
Table 1.
A summary of the key characteristics that define each grade of the mouse PVR model used in this study [20].
Table 2.
Intravitreal DMSO injection in the mouse PVR model resulted in significantly higher proportions of mice developing severe PVR compared to mice treated with intravitreal SNC injection at 2w, 3w, and 4w post-treatment.
Any eyes in which optical opacities prevented imaging were excluded from analysis. Mann-Whitney U test showed significant differences in average PVR grades at all time-points; all values below p = 0.05 were considered significant.
Fig 3.
Mouse eyes treated with SNC show reduced formation of PVR membranes in the vitreous and along the inner retinal surface.
Immunohistochemistry of markers (stained in brown) of fibrotic cells (A,B), glial cells (C,D), B-cells (E) and T-cells (F) are all expressed in the vitreous and along the retina surface of eyes treated with RPE/DMSO, but show reduced expression in eyes treated with RPE/SNC. Black arrow denotes PVR membranes in the vitreous and black arrowheads are PVR membranes along the retinal surface. PVR grades (PG) assigned to the eyes stained are shown.
Fig 4.
qPCR analysis of mouse eyes treated with SNC show reduced expression of PVR markers.
A) 4 week fundus images with PVR grades (PG) of each eye used for analysis. Average gene expression of B) EMT markers (Fn1, Col1a1 and Acta2) and C) cytokines and inflammatory markers (Tgfβ, Tnfα, and Mcp1) all show an overall up-regulation in mouse eyes injected with RPE/DMSO (blue bars) after 4 weeks compared to eyes injected with RPE/SNC (orange bars). Uninjected (black bars) and eyes injected with only SNC (grey bars) did not show up-regulation of these genes. Each bar represents the average fold change from all eyes in that treatment groups. Significance between uninjected and RPE/DMSO and RPE/DMSO and RPE/SNC was determined using a one-way ANOVA test followed by Tukey’s post-hoc analysis. *: p<0.05; **: p<0.01; n.s.: not significant.