The oral iron chelator deferiprone protects against systemic iron overload-induced retinal degeneration in hepcidin knockout mice

Abstract

Purpose: To investigate the retinal-protective effects of the oral iron chelator deferiprone (DFP) in mice lacking the iron regulatory hormone hepcidin (Hepc). These Hepc knockout (KO) mice have age-dependent systemic and retinal iron accumulation leading to retinal degeneration.

Methods: Hepc KO mice were given DFP in drinking water from age 6 to 18 months. They were then compared to Hepc KO mice not receiving DFP by fundus imaging, electroretinography (ERG), histology, immunofluorescence, and quantitative PCR to investigate the protective effect of DFP against retinal and retinal pigment epithelial (RPE) degeneration.

Results: In Hepc KO mice, DFP diminished RPE depigmentation and autofluorescence on fundus imaging. Autofluorescence in the RPE layer in cryosections was significantly diminished by DFP, consistent with the fundus images. Immunolabeling with L-ferritin and transferrin receptor antibodies showed a decreased signal for L-ferritin in the inner retina and RPE cells and an increased signal for transferrin receptor in the inner retina, indicating diminished retinal iron levels with DFP treatment. Plastic sections showed that photoreceptor and RPE cells were well preserved in Hepc KO mice treated with DFP. Consistent with photoreceptor protection, the mRNA level of rhodopsin was significantly higher in retinas treated with DFP. The mRNA levels of oxidative stress-related genes heme oxygenase-1 and catalase were significantly lower in DFP-treated Hepc KO retinas. Finally, ERG rod a- and b- and cone b-wave amplitudes were significantly higher in DFP-treated mice.

Conclusions: Long-term treatment with the oral iron chelator DFP diminished retinal and RPE iron levels and oxidative stress, providing significant protection against retinal degeneration caused by chronic systemic iron overload in Hepc KO mice. This indicates that iron chelation could be a long-term preventive treatment for retinal disease involving iron overload and oxidative stress.

Keywords: deferiprone; hepcidin; oxidative stress; retinal degeneration.

Figure 1

Fundus imaging showed that DFP-treated Hepc KO mice were protected against retinal degeneration. In untreated mice, there was yellowish retinal depigmentation (A) (black arrows). These depigmented areas were autofluorescent, corresponding to hypopigmented, autofluorescent RPE cells observed previously in cryosections (B). In contrast, DFP-treated Hepc KO mice had a smaller area of depigmentation (C) as well as less autofluorescence (D). The red boxed area in (A) indicates a lens opacity. The opacity is clearly in front of the retina, as it obscures the blood vessels. The white patches in (C) (arrowheads), which are behind the retinal blood vessels, indicate focal retinal degeneration.

Figure 2

Photomicrographs showing autofluorescence and double labeling for L-ferritin and transferrin receptor in Hepc KO retinas with and without DFP. In retina sections of Hepc KO mice with 4′,6-diamidino-2-phenylindole (DAPI) staining only, the RPE layer showed autofluorescence (white asterisks in [A]); however, the autofluorescent RPE cells were significantly fewer in DFP-treated retinas (red asterisks in [B]). Compared to nontreated Hepc KO retinas (C1, C2), the DFP-treated retinas showed increased signal for transferrin receptor in inner retina (D1) and decreased signal for L-ferritin in inner retina and RPE cells (D2). Scale bars: 100 μm. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 3

Bright-field photomicrographs of plastic sections of Hepc KO retinas with or without DFP and WT retinas, and plot showing photoreceptor nuclei preservation of ONL in DFP-treated Hepc KO retinas. Sagittal plane sections pass through optic nerve head. Photomicrographs of plastic sections showing hypertrophic RPE cells (red asterisks in [B]) that are migrating toward the inner retina (black arrowheads in [B]), with degeneration of overlying inner and outer segments and thinning of outer nuclear layer. The DFP-treated Hepc KO retina (A) appears more similar to WT (C). Plot of the thickness of the ONL, measured in numbers of photoreceptor nuclei per column (D). Measurements were made in triplicate every 200 μm from the ONH. n = 3. *Significant difference. Scale bars: 50 μm. RPE, retinal pigment epithelium; OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 4

Graphs showing relative mRNA levels measured by qPCR. The Rho mRNA levels in NSR were higher in DFP-treated Hepc KO mice compared to nontreated mice (A). Rpe65 mRNA levels in the RPE/choroid were not significantly changed. (B) DFP did not alter L-ferritin (Lft) mRNA level but increased transferrin receptor (Tfrc) mRNA levels significantly in NSR (C, D). Hmox1 and catalase mRNA levels in neural retina (E, F) were significantly decreased after DFP treatment (E, F). n = 3. *Significant difference. CHO, choroid; Lft, light chain of ferritin; Tfr, transferrin receptor; Hmox-1, heme oxygenase 1.

Figure 5

DFP protects Hepc KO mice from functional impairment measured by ERG. Saturated rod b-, a-, and cone b-wave responses were significantly higher in DFP-treated Hepc KO mice. n = 3. *Significant difference.