Retinal Pigment Epithelium Remodeling in Mouse Models of Retinitis Pigmentosa

Abstract

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.

Keywords: Tvrm4 mouse; blood retinal barrier; dextranes; immunocytochemistry; rd10 mouse; rd9 mouse; retinal pigment epithelium; retinitis pigmentosa; zonula occludens.

Figure 1

Retinal and RPE morphologies in wild type (wt) mice aged 1 year. (A) Retinal vertical section, DAPI nuclear staining. (B) Whole-mount RPE, ZO-1 immunostaining. Arrows show short and rare interruptions of the RPE (hexagonal) array. In this and in other Figures: onl—outer nuclear; inl—inner nuclear; and gcl—ganglion cell layer.

Figure 2

Retinal and RPE morphologies in rd9 mice aged 12 (A,B) and 20 months (C,D). (A,C) Retinal vertical sections. DAPI nuclear staining. Note the progressive thinning of the ONL from (A–C) and the poor retinal layering compared with the wt of Figure 1A. (B,D) ZO-1 staining of RPEs. Arrows point to large discontinuities, evident at 20 months (D). All images are from the central retina/RPE.

Figure 3

Retinal and RPE morphologies in rd10 mice aged 45 days. (A) DAPI nuclear staining of retinal vertical sections. Note the persistence of only one row of nuclei in the ONL. (B) ZO-1 staining of RPE from a similar 45 day old mouse in which several wide discontinuities can be appreciated (arrows). Both images are obtained from the central retina/RPE.

Figure 4

Retinal and RPE morphologies in light-induced Tvrm4 mice. (A) DAPI nuclear staining of retinal vertical sections from Tvrm4 mouse one week post light-induction. Note the presence of gigantic apoptotic bodies in the ONL, which is composed of few disorganized rows of nuclei compared with the wt. (B) ZO-1 staining of RPE from Tvrm4 mouse one week post light-induction. Arrows show wide discontinuities in ZO-1.

Figure 5

Density of ZO-1-positive profiles in the RPE of different mouse models of RP. (A) Comparison between 20 m old rd9 (n = 5) and age-matched wt mice (n = 6). Unpaired t test, p = 0.0158. (B) Comparison between rd10 (n = 6) and age-matched wt mice (n = 3) (all 45–50 days old). Unpaired t test, p = 0.0168. (C) Comparison between central (cen Tvrm4, n = 3) and peripheral (per Tvrm4, n = 3) zones of the RPE of Tvrm4 mice. Paired t test, p = 0.0138. Error bars represent ±SEM. * p < 0.05, ** p < 0.01.

Figure 6

ZO-1 density distribution in the three mouse strains. Comparison among ZO-1 density profiles in the RPE of rd9 (n = 5), rd10 (n = 6), and Tvrm4 (n = 3, central RPE). One-way ANOVA p = 0.0327. Post hoc Tukey’s test. rd9 vs. rd10, p = 0.9999; rd9 vs. Tvrm4, p = 0.0457; rd10 vs. Tvrm4, p = 0.0400. Error bars represent ±SEM. * p < 0.05; ns: not significant.

Figure 7

Aging contribution to changes of ZO-1 density. Comparison between RPEs of wt animals, aged 12 (n = 3) and 20 (n = 6) months, respectively, distinguishing central (cen RPE) and peripheral (per RPE). Two-way ANOVA p = 0.0162. Post hoc Sidak’s test. wt 12 m cen RPE vs. wt 20 m cen RPE, p = 0.0095; wt 12 m per RPE vs. wt 20 m per RPE, p > 0.999. Error bars represent ±SEM. ** p < 0.01; ns: not significant.

Figure 8

ZO-1 discontinuities are a stereotyped form of RPE remodeling. Comparison among the same groups of data used to generate Figure 5, each normalized to the respective control group (i.e., wt aged 20 months for rd9 mutants; wt aged 45–50 days for rd10 mice and for central Tvrm4 RPE). One-way ANOVA p = 0.0566. Post hoc Tukey’s test. rd9 vs. rd10, p = 0.479; rd9 vs. Tvrm4, p = 0.2614; rd10 vs. Tvrm4, p = 0.0465. Error bars represent ±SEM. * p < 0.05; ns: not significant.

Figure 9

Functional and structural breakdown of the outer BRB. Whole-mount RPEs from rd10 (A,E), wt (B) and light-induced (C,D) Tvrm4 mice after intravenous injection of FITC-Dextran. Arrowheads show leakage of the fluorescent probe from choroidal vessels between the RPE cells (asterisks). Leakage of the bright, green-fluorescent probe occurs only in mutant mice (A,C), showing that the wt RPE (B) and the peripheral RPE of Tvrm4 mice (D) are intact. E—ZO-1 immunostaining (red signal) and FITC-Dextran (green) in the central RPE portion (rd10 mutant). A large ZO-1 discontinuity overlaps the area of main leakage.

Figure 10

Vacuolization of RPE cells. Ocular vertical section from Tvrm4 mice, 4 weeks following light-induction (A,B) and in a non-induced littermate (C,D). Transmission electron microscopy shows membranous vacuoles which accumulate at the basal side (abutting Bruch’s membrane, BM) in the proximity of the basal infoldings of RPE cells of the light-induced samples (asterisks in (A,B)); the photoreceptor layer is absent or completely disorganized. In the non-induced control, outer segments (OS) are clearly visible. N—nucleus of RPE cell.

Figure 11

Whole-mount RPE stained for ZO-1 obtained from P45, rd10 mice administered with saline, control solution (A) and dexamethasone (B). ZO-1 interruptions (arrowheads) are visibly fewer in (B) and the density of ZO-1 stained profiles correspondingly higher (C). *** p < 0.001.