March 2011
Volume 52, Issue 3
Free
Retina  |   March 2011
Light-Induced Retinal Degeneration Correlates with Changes in Iron Metabolism Gene Expression, Ferritin Level, and Aging
Author Affiliations & Notes
  • Emilie Picard
    From INSERM (Institut National de la Santé et de la Recherche Médicale), U872, Paris, France;
    Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, France;
    Université Paris Descartes, UMR S 872, Paris, France;
  • Isabelle Ranchon-Cole
    Laboratoire de Biophysique Sensorielle, Université Clermont 1, Clermont-Ferrand, France; and
  • Laurent Jonet
    From INSERM (Institut National de la Santé et de la Recherche Médicale), U872, Paris, France;
    Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, France;
    Université Paris Descartes, UMR S 872, Paris, France;
  • Carole Beaumont
    INSERM U773, Centre de Recherche Biologique Bichat Beaujon CRB3, Equipe 4: Fer et Synthèse d'Hème/Génétique, Physiologie et Pathologie, Université Paris 7, Paris, France.
  • Francine Behar-Cohen
    From INSERM (Institut National de la Santé et de la Recherche Médicale), U872, Paris, France;
    Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, France;
    Université Paris Descartes, UMR S 872, Paris, France;
  • Yves Courtois
    From INSERM (Institut National de la Santé et de la Recherche Médicale), U872, Paris, France;
    Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, France;
    Université Paris Descartes, UMR S 872, Paris, France;
  • Jean-Claude Jeanny
    From INSERM (Institut National de la Santé et de la Recherche Médicale), U872, Paris, France;
    Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, France;
    Université Paris Descartes, UMR S 872, Paris, France;
  • Corresponding author: Emilie Picard CRC, UMR S872, team 17, 15 Rue de l'Ecole de Médecine, 75006 Paris, France; picardemilie@gmail.com
Investigative Ophthalmology & Visual Science March 2011, Vol.52, 1261-1274. doi:10.1167/iovs.10-5705
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      Emilie Picard, Isabelle Ranchon-Cole, Laurent Jonet, Carole Beaumont, Francine Behar-Cohen, Yves Courtois, Jean-Claude Jeanny; Light-Induced Retinal Degeneration Correlates with Changes in Iron Metabolism Gene Expression, Ferritin Level, and Aging. Invest. Ophthalmol. Vis. Sci. 2011;52(3):1261-1274. doi: 10.1167/iovs.10-5705.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron.

Methods.: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt+/+) and heterozygous H ferritin (HFt+/−) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt+/+ and HFt+/− mouse retinas.

Results.: Retinal degeneration in the 4-month-old HFt+/− mice was more extensive than in the HFt+/+ mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt+/− and HFt+/+ mice.

Conclusions.: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Iron is an essential element for cell survival and is a cofactor of various enzymes. 1 However, it can generate free radicals and is harmful when present in excess. Homeostatic mechanisms maintain iron levels by regulating the proteins involved in iron import (transferrin and transferrin receptor), storage (ferritins), and export (ferroportin, ceruloplasmin, and hephaestin), through various central regulators, including iron regulatory proteins 1 and 2, hepcidin, human hemochromatosis protein (HFE), hemopexin, and hemojuvelin. Iron and several proteins involved in its homeostasis have been reported in various retinal layers. 2 7 A specific iron cycle involving phagocytosis of outer segment (OS) tips by retinal pigmented epithelium (RPE) cells and transferrin-dependent redistribution of iron to photoreceptors has been described. 8 Retinal cell dysfunction has been recently associated with deregulation of gene expression of hemopexin, 9 human hemochromatosis protein, 5 hemojuvelin, 7 and ferroportin 1. 10 However, the role of ferritin has not been directly investigated, despite reports that its level increases with age in various animal models, as in Hfe−/− mice, 11 in cytomegalovirus-infected retina, 7 and in RPE–choroid complex and neural retina of aged rodent. 12,13 It was also reported that inadequate ferritin expression was found in hemopexin null mice brain. 14 Most of the retinal degeneration occurring in these animal models appears with aging, which reflects additional factors such as age-dependent oxidative stress. In addition, age-related macular degeneration (AMD) is associated with iron accumulation 15 and high levels of ferritin and ferroportin in the retina. 16  
The role of the ferritin complex is to bind iron in a nontoxic and readily available form. 17 It consists of two functionally and genetically different chains, termed heavy (H) and light (L). H and L ferritins are present in all the outer retinal layers. 2 In addition to its role in normal iron homeostasis, H ferritin appears to act as a protector against oxygen-derived free-radical–mediated damage. 18 20 Since homozygous H ferritin deletion is lethal, 21 to evaluate whether H ferritin is involved in retinal degeneration associated with aging, we used a mouse model heterozygous for the H ferritin gene (HFt+/−). These mice produce 50% less H ferritin protein and mRNA than do littermates homozygous for H ferritin (HFt+/+), 22 but seem unaffected. 
To evaluate the sensitivity of HFt+/− mice compared with HFt+/+ mice to oxidative stress, we exposed the mice to light. Exposure to acute and continuous visible light results in an increased production of H2O2 in the outer retina 23 and leads to dose-dependent retinal degeneration. 24 27 This stress-related oxidative condition causes retinal cell dysfunction 28 31 and leads to photoreceptor death. 
First, 4- and 16-month-old HFt+/+ and HFt+/− mice were exposed to excessive light for 24 hours, and their retinas were analyzed 1 and 12 days later. We studied nuclear layer thickness as a measurement of photoreceptors degeneration, H and L ferritin protein and mRNA profiles, and mRNAs of the other proteins involved in iron homeostasis. Second, to analyze the early effects of excessive light on neural retinal iron status in the 4-month-old HFt+/− and HFt+/+ mice, we assayed the mRNAs of iron-regulating proteins—H ferritin, L ferritin, transferrin, transferrin receptor, ceruloplasmin, ferroportin, hephaestin, hepcidin, and iron-regulatory proteins—as well as that of the antioxidative stress protein heme oxygenase-1 at various times after illumination. We found substantial time-dependent variations in the expression of genes of several proteins involved in iron homeostasis. 
Methods
Animals
HFt+/+ and HFt+/− mice of both genders (an equal number of males and females) and in an albino BALB/c genetic background were provided by Carole Beaumont (Faculté Bichat). They were screened for the presence of one or two allele(s) by PCR. All mice were fed a standard laboratory diet and tap water ad libitum and were maintained in a temperature-controlled room at 21°C to 23°C with a 12-hour/12-hour light–dark photoperiod. Experimental procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in accordance with policies on animal use in neuroscience research. They were approved by each author's institutional animal care review board. 
Mouse Light-Induced Injury
Four- and/or 16-month-old HFt+/+ and HFt+/− mice were dark adapted, exposed to light (13,000 lux) in nonreflective cages for 24 hours, and killed 12 days after exposure to light. Their eyes were then enucleated and fixed (see the following section) for histologic analyses. Also, for histologic and molecular analyses, some 4-month-old HFt+/+ and HFt+/− mice were killed immediately after 1 day of exposure to light. To study the early effects of exposure to light, we dark adapted 4-month-old HFt+/+ and HFt+/− mice; exposed them to light (13,000 lux) for 0, 2, 12, and 24 hours; and immediately killed them for analysis. All mice were killed in the morning during the light photoperiod. 
For each experiment, control group mice were dark adapted and exposed to a light–dark photoperiod (200 lux), and their eyes were prepared for analysis as just described. The chamber temperature during the experiments was 25 ± 1.5°C. The mice were allowed free access to food and water during exposure to light. 
Immunohistofluorescence Analysis
Freshly enucleated eyes were fixed for 2 hours with 4% paraformaldehyde (PFA; Inland Europe, Conflans sur Lanterne, France) in 1× phosphate-buffered saline (PBS; Invitrogen-Gibco, Cergy Pontoise, France). Then, the eyes were washed with PBS and mounted in optimal cutting temperature (OCT) compound (Tissue Tek; Siemens Medical, Puteaux, France). The samples were then frozen with dry ice. The frozen sections (10 μm) were cut on a cryostat (model CM3050S; Leica, Rueil Malmaison, France). 
For the immunodetection of H ferritin, L ferritin, and mitochondrial ferritin, the sections were first blocked with 1% bovine serum albumin (BSA; Sigma, Saint Quentin Fallavier, France) and 10% normal goat serum (Invitrogen-Gibco). All primary antibodies were diluted in PBS and incubated for 1 hour with the eye sections. Rabbit polyclonal antibodies for the H and L subunits of ferritin and rabbit polyclonal mitochondrial ferritin (the generous gift of Paolo Santambrogio, Department Biological and Technological Research, Instituto de Ricovero e Cure a Carattere Scientifico, San Rafaelle, Milan, Italy) were diluted 1:250; rabbit monoclonal anti-Iba1 was diluted 1:400 (Wako, Neuss, Germany); rabbit polyclonal anti-4-HNE was diluted 1:200 (Chemicon, distributed by Millipore, Saint Quentin, France), and rabbit anti-glial fibrillary acidic protein (GFAP) was diluted 1:250 (DakoCytomation, Trappes, France). Control sections were incubated with rabbit nonimmune serum (Invitrogen-Gibco) or with no primary antibodies. 
The sections were washed with PBS and incubated for 1 hour with secondary antibodies (goat anti-rabbit), labeled with Texas Red (Jackson Laboratories, TebuBio, Le Perray en Yvelines, France) or Alexa 596 (Molecular Probes-Invitrogen, Cergy Pontoise, France) diluted 1:200 in PBS. The nuclei were counterstained with 4′,6-diamidino-2-phenyl-indole (DAPI; Sigma). The sections were analyzed by fluorescence microscopy (Olympus, Rungis, France) and photographed using identical exposure parameters for all samples to be compared. 
Preparation of Retinal Extracts
The eyes were enucleated, and the neural retinas were extracted and immediately stored in RNA stabilization reagent (Qiagen, Courtaboeuf, France) at −80°C for qRT-PCR analysis. 
For enzyme-linked immunosorbent assays (ELISAs), the mice were perfused through the left cardiac ventricle with 1× PBS. Then, the eyes were enucleated and dissected to isolate the neural retinas. They were immediately frozen in liquid nitrogen and stored at −80°C. 
Analysis by qRT-PCR
Neural retinas were homogenized with a motorized pestle (Dutscher, Issy les Moulineaux, France) in lysis buffer and RNA was isolated on a mini column (RNeasy; Qiagen). 
First-strand cDNA was obtained by a standard reverse transcription (RT) reaction (Invitrogen). Next, 150 ng RNA was reverse transcribed for 50 minutes at 42°C with 200 U of reverse transcriptase (SuperScript II; Invitrogen) and oligo-dT. One microliter of RNase H was added to each sample and incubated for 20 minutes at 37°C before target cDNA amplification. A qPCR master mix was prepared to a final volume of 18 μL: 7.5 μL water, 0.1 μL mixed forward and reverse primers (0.25 μM each; MWG Biotech France, Champlan, France), 0.4 μL Rox reference dye, and 10 μL SYBR green qPCR master mix (Platinum Super-Mix-UDG; Invitrogen). Master mixes (18 μL) were added to 2 μL of cDNA (1:10 dilution), and the samples were placed on a reaction support (Applied Biosystems, Inc. [ABI], Applera France SA, Courtaboeuf, France). See Table 1 for detailed information about the primers. 
Table 1.
 
Overview of All Primers Used in the qRT-PCR
Table 1.
 
Overview of All Primers Used in the qRT-PCR
Gene Name Forward Primer Reverse Primer
Beta-actin 5′ GAC GGC CAA GTC ATC ACT ATT G 3′ 5′ CCA CAG GAT TCC ATA CCC AAG A 3′
H ferritin 5′ TCA GTC ACT ACT GGA ACT GC 3′ 5′ CGT GGT CAC CCA GTT CTT TA 3′
L ferritin 5′ TGG CCA TGG AGA AGA ACC TGA ATC 3′ 5′ GGC TTT CCA GGA AGT CAC AGA GAT 3′
Transferrin 5′ GGA CGC CAT GAC TTT GGA TG 3′ 5′ GCC ATG ACA GGC ACT AGA CC 3′
Transferrin receptor 5′ GAG GAA CCA GAC CGT TTA GTT GT 3′ 5′ CTT CGC CGC AAC ACC AGC A 3′
Iron-regulatory protein 1 5′ TGC TTC CCC AGG TGA TTG GCT ACA 3′ 5′ TAG CTC GGT CAG CAA TGG ACA GCT 3′
Iron-regulatory protein 2 5′ ACC AGA GGT GGT TGG ATG TGA GCT 3′ 5′ ACG CCT ACT TGC CTG AGG TGC TTT 3′
Ferroportin 5′ GCT GCT AGA ATC GGT CTT TGG 3′ 5′ CAG CAA CTG TGT CAC CGT CAA 3′
Ceruloplasmin 5′ GGG AGC CGT CTA CCC TGA CAA 3′ 5′ GCA CTT TGT CAT CAG CCC GTT 3′
Hepcidin 5′ AGA GCT GCA GCC TTT GCA C 3′ 5′ GAG GTC AGG ATG TGG CTC TA 3′
Hephaestin 5′ GCC TGG GCA CAG AGA CTG AT 3′ 5′ GGT GTG AGG AAA GAG CAT GAC TG 3′
Heme oxygenase 1 5′ GCC ACC AAG GAG GTA CAC AT 3′ 5′ GCT TGT TGC GCT CTA TCT CC 3′
Amplification was performed on a real-time PCR system (model 7300l; ABI). The following conditions were used: 95°C for 10 minutes (denaturation); 40 cycles of 95°C for 15 seconds and 60°C for 1 minute (amplification and quantification); and 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds (melting curve). Crossing points for each transcript (the point at which the fluorescence is significantly higher than background fluorescence) were determined for subsequent analysis. 
ELISA for Tissue H and L Ferritin Subunit Determination
Neural retinas were incubated in lysis buffer (15 mM Tris [pH 7.9], 60 mM KCl, 15 mM NaCl, 2 mM EDTA, and 0.4 mM phenylmethylsulfonyl fluoride [PMSF]; Perbio Science, Brebiers, France). After four successive freeze–thaw cycles (liquid nitrogen–room temperature), the lysates were centrifuged at 5000g for 10 minutes. The supernatants were then stored at −20°C. 
Ferritin concentration was determined by ELISA specific for the H and L chains, as previously described. 32 Briefly, microtiter plates were coated with 1 μg polyclonal antibody specific for mouse H or L ferritin. Soluble mouse tissue homogenate or standard ferritin was diluted in PBS-Tween-BSA (50 mM sodium phosphate [pH 7.4], 150 mM NaCl, 0.05% vol/vol Tween-20, and 1% BSA) and added to the plates. The presence of ferritin was revealed by incubation with the same antibody labeled with horseradish peroxidase. Peroxidase activity was developed with o-phenylenediamine dihydrochloride (Sigma). Recombinant mouse H and L subunits were used as ferritin standards. Supernatants were used to determine the ferritin content. Protein concentrations were determined with the BCA protein assay kit (Pierce Biotechnology, Brebieres, France), with BSA used as the standard. Results are reported in nanograms per milliliter of supernatant. 
Histology
Enucleated eyes from mice were fixed with 4% paraformaldehyde (LADD; Inland Europe, Conflans-sur-Lanterne, France) and 0.5% glutaraldehyde (LADD) in PBS for 2 hours. After fixation, the samples were washed, dehydrated, transferred to the infiltration solution in a resin embedding kit (Historesin; Leica), and incubated overnight at 4°C. Samples were embedded (Leica) and attached to a block holder after polymerization. Plastic sections (5 μm thick) were prepared on a microtome (RM 2055; Leica Jung). The sections were mounted on slides coated with gelatin, stained for 2 minutes with 1% toluidine blue solution, analyzed by microscope (Aristoplan; Leica), and photographed with a digital camera (Leica) with identical exposure settings, so that the images could be compared. 
Measurement of Nuclear Layer Thickness
We studied retinal morphology in the 4- and 16-month-old HFt+/+ and HFt+/− mouse retinas. The outer nuclear layer (ONL) thickness was measured at 200-μm intervals in both superior and inferior poles and averaged to give a single value. 
We used two histologic protocols for retinal analysis after exposure to light. First, the ONL thickness was measured in similar central regions: from 100 to 600 μm of the optic nerve head, in both superior and inferior poles in retinas of 4-month-old HFt+/+ and HFt+/− mice exposed to light for 24 hours and killed 1 day later. Second, the thicknesses of the ONL and inner nuclear layer (INL) were measured at 200-μm intervals on each side of the optic nerve and across the whole retina in the 4- and 16-month-old HFt+/+ and HFt+/− mice killed 12 days after being exposed to light for 24 hours. 
For each protocol, three separate sections collected from two to six eyes from separate animals were measured. Results were averaged to give a single value. 
Statistical Analyses
Results are presented as the mean ± SEM. We used the Mann-Whitney U test to evaluate the significance of the differences between control and illuminated HFt+/+ and HFt+/− mice. For qRT-PCR data analysis, HFt+/+ mice were compared to HFt+/− mice, unless the bracket on the graph indicates another comparison. Two-way ANOVA was used to analyze retinal nuclear layer thickness after illumination (Prism; GraphPad, San Diego, CA). P < 0.05 was considered statistically significant. We performed three or four replicates for each experimental condition. 
Results
Comparative Analysis of 4- and 16-Month-Old HFt+/+ and HFt+/− Mouse Retinas
Histologic Analysis.
We performed histologic analysis of the retinas of HFt+/− mice and control HFt+/+ littermates. There were no gross regional differences in the retinal structures (Figs. 1A–D). Nevertheless, the ONLs in the 4- and 16-month-old HFt+/− mice was slightly but significantly less thick than those in the HFt+/+ mice (7.7% in the 4-month-old mice and 9.9% in the 16-month-old mice). The ONL was also thinner (by 25%) in both the 16-month-old HFt+/+ and HFt+/− mice than in the 4-month-old mice (Fig. 1E) reflecting a similar age-related cell loss. 
Figure 1.
 
Histology of retinas in 4- and 16-month-old HFt+/+ and HFt+/− mice. (AD) Retinal sections from HFt+/+ (A, C) and HFt+/− mice (B, D) at 4 (A, B) and 16 (C, D) months. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (E) Mean thickness of the ONL in the 4- and 16-month-old HFt+/+ and HFt+/− mouse retinas. ONL thickness decreased with age in the HFt+/+ and HFt+/− mice and was slightly lower in the HFt+/− than in the HFt+/+ mice. The measurements of ONL thicknesses every 200 μm, on each pole of the retina, were averaged. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Figure 1.
 
Histology of retinas in 4- and 16-month-old HFt+/+ and HFt+/− mice. (AD) Retinal sections from HFt+/+ (A, C) and HFt+/− mice (B, D) at 4 (A, B) and 16 (C, D) months. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (E) Mean thickness of the ONL in the 4- and 16-month-old HFt+/+ and HFt+/− mouse retinas. ONL thickness decreased with age in the HFt+/+ and HFt+/− mice and was slightly lower in the HFt+/− than in the HFt+/+ mice. The measurements of ONL thicknesses every 200 μm, on each pole of the retina, were averaged. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Ferritin Proteins and mRNAs.
We used qRT-PCR to assay the corresponding mRNAs in the neural retina (Figs. 2A, 2B). H ferritin mRNA was less abundant in the HFt+/− mice than in the HFt+/+ mice at all ages studied (4 months, 52.7%; 16 months, 45.9%; Fig. 2A). However, the amounts of L ferritin mRNA were similar in the 4- and 16-month-old HFt+/− and HFt+/+ mice (Fig. 2B). The concentration of H ferritin protein in the 4-month-old HFt+/− mice was 37.0% of that in the HFt+/+ mice. Similarly, the concentration of H ferritin protein in the 16-month-old HFt+/− mice was 44.5% lower than in the HFt+/+ mice (Fig. 2C), indicating that there was no age effect. Conversely, L ferritin protein was significantly more abundant (168.0%) in the 4-month-old HFt+/− mice than in the 4-month-old HFt+/+ mice. However, the amount of L ferritin in the 16-month-old HFt+/− mice was similar in the WT mice (Fig. 2D). The amount of L ferritin mRNA and protein decreased in the HFt+/− mice between 4 and 16 months (Figs. 2B, 2D). That a significant increase in L ferritin protein level occurred only at 4 months seems to indicate a mechanism of compensation that was not present at 16 months. 
Figure 2.
 
Ferritin proteins and mRNAs in 4- and 16-month-old HFt+/+ and HFt+/− mice. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. In the 4- and 16-month-old HFt+/− mice, the amounts of H ferritin protein and mRNA were nearly half those in the HFt+/+ mice. L ferritin protein was more abundant in the 4-month-old HFt+/− than HFt+/+ mice, but was similar in the 16-month-old HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; ** P < 0.01. (EM) Immunolocalization of H (FI) and L (JM) ferritin on frozen sections of retina from 4- and 16-month-old HFt+/+ and HFt+/− mice. (E) Omission of the primary antibody was used as the control. (FI) H ferritin in the HFt+/+ (F, H) and HFt+/− (G, I) mouse retina from 4-month-old (F, G) and 16-month-old (H, I) mice. (JM) L ferritin in the HFt+/+ (J, L) and HFt+/− (K, M) mouse retina from 4-month-old (J, K) and 16-month-old (L, M) mice. In the HFt+/+ and the HFt+/− mice, the signals for H and L ferritin were in the photoreceptor inner segments (IS), ONL, OPL, and INL and in the inner top of the IPL. Sections were counterstained with DAPI (blue). Scale bar, 100 μm.
Figure 2.
 
Ferritin proteins and mRNAs in 4- and 16-month-old HFt+/+ and HFt+/− mice. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. In the 4- and 16-month-old HFt+/− mice, the amounts of H ferritin protein and mRNA were nearly half those in the HFt+/+ mice. L ferritin protein was more abundant in the 4-month-old HFt+/− than HFt+/+ mice, but was similar in the 16-month-old HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; ** P < 0.01. (EM) Immunolocalization of H (FI) and L (JM) ferritin on frozen sections of retina from 4- and 16-month-old HFt+/+ and HFt+/− mice. (E) Omission of the primary antibody was used as the control. (FI) H ferritin in the HFt+/+ (F, H) and HFt+/− (G, I) mouse retina from 4-month-old (F, G) and 16-month-old (H, I) mice. (JM) L ferritin in the HFt+/+ (J, L) and HFt+/− (K, M) mouse retina from 4-month-old (J, K) and 16-month-old (L, M) mice. In the HFt+/+ and the HFt+/− mice, the signals for H and L ferritin were in the photoreceptor inner segments (IS), ONL, OPL, and INL and in the inner top of the IPL. Sections were counterstained with DAPI (blue). Scale bar, 100 μm.
We used immunostaining with cryosections to test whether the location of H and L ferritin in the retina was modified in the 4- and 16-month-old HFt+/+ and HFt+/− mice (Figs. 2E–M). H ferritin was found throughout the retina, with the strongest signals in the RPE, photoreceptor inner segments, ONL, outer plexiform layer (OPL), inner nuclear layer (INL), and in the inner top of the inner plexiform layer (IPL; Figs. 2F–I). With the same exposure time, H ferritin labeling in the HFt+/− mice was very faint compared with that in the HFt+/+ mice, but was nevertheless present (Figs. 2G, 2I). L ferritin had the same distribution as H ferritin (Figs. 2J–M). Immunodetection of mitochondrial ferritin gave the same pattern in the HFt+/+ and HFt+/− mice at both developmental stages studied. Mitochondrial ferritin was present in all the layers of the retina, with the strongest immunolabeling in the RPE and photoreceptor inner segment ellipsoids (data not shown). 
Iron-Regulating Protein Expression.
We assayed the mRNAs for several proteins important in regulating iron homeostasis in the 4- and 16-month-old HFt+/+ and HFt+/− mouse retinas: ferroportin, transferrin, transferrin receptor, ceruloplasmin, hephaestin, hepcidin, and iron regulatory proteins 1 and 2. Ferroportin mRNA was significantly more abundant in the 16-month-old mice than in the 4-month-old mice (by 35% in the HFt+/+ mice and 37% in the HFt+/− mice), but was unaffected by genotype (data not shown). The amounts of other mRNAs for iron-regulating proteins were not modified by either the age or the genotype of the mice (data not shown). 
Although the concentration of H ferritin in the HFt+/− mice was half that in the HFt+/+ mice, the thickness of the ONL in the retina was only slightly affected. There were no other differences in the amounts of mRNAs for the iron-regulating proteins between the HFt+/+ and the HFt+/− mice. 
Thus, the allelic diminution of H ferritin does not appear to affect retinal histology and iron-regulating protein expression during normal aging. To test whether the response to oxidative stress of the retina was affected by H ferritin level, we used light-induced retinal degeneration experiments. 
Comparison of Light-Induced Retinal Degeneration in 4- and 16-Month-Old HFt+/+ and HFt+/− Mouse Retinas
We used two different experimental protocols to compare the light sensitivity of the HFt+/− and the HFt+/+ mice. The first was to study retinal structure after 24 hours of illumination. For retinal histology analyses, the mice were killed 12 days after the end of illumination. For evaluating modifications to several iron homeostasis proteins and mRNA, the mice were killed 1 day after illumination. For the second protocol, the mice were exposed for 2, 12, and 24 hours and killed immediately. Iron-regulating protein mRNAs were assayed after these various times of exposure. 
Histologic Analysis of the Effects of Light on 4- and 16-Month-Old HFt+/+ and HFt+/− Mice 12 Days after Illumination.
After 24 hours of dark adaptation, the 4- and 16-month-old HFt+/+ and HFt+/− mice were subjected to 13,000 lux white light for 24 hours and then returned to a 12-hour/12-hour light–dark photoperiod for 12 days. The illumination caused photoreceptor death characterized by a decrease in ONL thickness in the retinas of the 4- and 16-month-old HFt+/+ and HFt+/− mice (Figs. 3A, 3B). However, INL thickness was unaffected (Figs. 3C, 3D). In the 4-month-old mice, exposure to light caused a decrease in ONL thickness over most of the retina, with the largest effect in the central part. The thinning of the ONL was greater in retinas from the HFt+/− mice than in those from HFt+/+ illuminated mice. In the central part of the retinas (1000 μm from each pole), the mean decrease in ONL thickness compared with that in the nonilluminated controls was 93% in the HFt+/− mice and 60% in the HFt+/+ mice. The effects of light decreased (ONL thickness increased) with the distance from the optic nerve. Light had significantly different effects on HFt+/+ and HFt+/− mouse retinas in each pole, between 1000 and 2200 μm from optic nerve. ONL thickness in the HFt+/− mouse retinas was 45.0% of that in the HFt+/+ retinas in the superior pole and 49.6% in the inferior pole (Fig. 3A). Illumination led to a significant reduction of ONL thickness in the entire retina in the 16-month-old HFt+/+ and HFt+/− mice (84.7% in the HFt+/+ mice and 78.0% in the HFt+/− mice; Fig. 3B). 
Figure 3.
 
Retinal modification in 4- and 16-month-old HFt+/+ and HFt+/− mice 12 days after illumination. (A, B) Measurement of the ONL thickness, every 200 μm, throughout the retina of 4-month-old (A) and 16-month-old (B) HFt+/+ and HFt+/− mice exposed or not exposed to light for 24 hours and killed 12 days later. The decrease in ONL thickness after exposure to light was much higher in the 4-month-old HFt+/− than HFt+/+ mice at both poles, especially toward the periphery. The decrease in ONL thickness was more apparent in the 16-month-old HFt+/+ and HFt+/− illuminated mice. (C, D) Measurement of the INL thickness, every 200 μm, throughout the retinas of 4-month-old (C) and 16-month-old (D) HFt+/+ and HFt+/− mice illuminated or not illuminated for 24 hours and killed 12 days later. Solid gray line: HFt+/+ control mice; solid black line: HFt+/− control mice; dashed gray line: HFt+/+ illuminated mice; dashed black line: HFt+/− illuminated mice. Data are the mean ± SEM of results from 6 to 18 independent sections. ***P < 0.0001; ns: not significant.
Figure 3.
 
Retinal modification in 4- and 16-month-old HFt+/+ and HFt+/− mice 12 days after illumination. (A, B) Measurement of the ONL thickness, every 200 μm, throughout the retina of 4-month-old (A) and 16-month-old (B) HFt+/+ and HFt+/− mice exposed or not exposed to light for 24 hours and killed 12 days later. The decrease in ONL thickness after exposure to light was much higher in the 4-month-old HFt+/− than HFt+/+ mice at both poles, especially toward the periphery. The decrease in ONL thickness was more apparent in the 16-month-old HFt+/+ and HFt+/− illuminated mice. (C, D) Measurement of the INL thickness, every 200 μm, throughout the retinas of 4-month-old (C) and 16-month-old (D) HFt+/+ and HFt+/− mice illuminated or not illuminated for 24 hours and killed 12 days later. Solid gray line: HFt+/+ control mice; solid black line: HFt+/− control mice; dashed gray line: HFt+/+ illuminated mice; dashed black line: HFt+/− illuminated mice. Data are the mean ± SEM of results from 6 to 18 independent sections. ***P < 0.0001; ns: not significant.
The same protocol of illumination as that used for the 4-month-old mice led to retinal degeneration so massive in the 16-month-old mice that we were unable to detect any difference between mice of the two genotypes. 
To describe further the effect of H ferritin haploid deficiency in the HFt+/− light-exposed mice, we concentrated our study on the 4-month-old HFt+/− mice and the HFt+/+ controls 1 day after the same light treatment. 
Effects of Light on the 4-Month-Old HFt+/+ and HFt+/− Mouse Retinas 1 Day after Illumination.
Histologic Analysis.
We tested the difference in photoreceptor loss 1 day after illumination compared with that after 12 days, by measuring the ONL thickness. The animals were subjected to the same exposure to light, but were allowed only 1 day of 12-hour/12-hour light–dark photoperiod recovery before analysis. After treatment, the photoreceptor nuclei layer was not regular, showing several folds. RPE monolayer was disorganized, with hypertrophic cells compared with that in the nonilluminated controls. The disturbances of the ONL and RPE were more pronounced in the HFt+/− than in the HFt+/+ mice (Figs. 4A, 4B). Mean ONL thickness in the exposed HFt+/+ and HFt+/− mice was significantly lower (41.0%) than in the unexposed controls. However, there was no difference between the two genotypes (Fig. 4C). Thus, the H ferritin deficiency in the HFt+/− mice was not associated with greater immediate susceptibility of photoreceptors during the initial light insult. 
Figure 4.
 
Early (1 day) retinal damage in 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to 13,000-lux light. (A, B) Representative histology of the central sections of retina from 4-month-old HFt+/+ (A) and HFt+/− (B) mice exposed for 24 hours to light and observed 1 day later. After illumination, the ONL was irregular and the RPE disorganized. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (C) Mean thickness of the ONL of retinas from the 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to light and the mean ONL thickness in the control mice. The thickness of the first 500 μm at each pole of the retina was measured. The ONL thickness decreased similarly in the HFt+/+ and HFt+/− mice 1 day after exposure to light. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Figure 4.
 
Early (1 day) retinal damage in 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to 13,000-lux light. (A, B) Representative histology of the central sections of retina from 4-month-old HFt+/+ (A) and HFt+/− (B) mice exposed for 24 hours to light and observed 1 day later. After illumination, the ONL was irregular and the RPE disorganized. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (C) Mean thickness of the ONL of retinas from the 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to light and the mean ONL thickness in the control mice. The thickness of the first 500 μm at each pole of the retina was measured. The ONL thickness decreased similarly in the HFt+/+ and HFt+/− mice 1 day after exposure to light. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Light-Induced Injury Marker Analysis.
Exposure to light induces oxidative stress. 23 We examined several markers of processes associated with exposure to light: apoptosis, lipid peroxidation, glial activation, and microglia migration. 27,33 36 We used TUNEL staining to study apoptosis 1 day after exposure to light. Most ONL nuclei in the retinal center were TUNEL positive in both the HFt+/+ and the HFt+/− mice, in agreement with our findings for ONL thickness (Figs. 5B, 5D). 4-HNE is a product of lipid peroxidation, a specific marker of oxidative stress in the retina 37 (Figs. 5E–J). In nonexposed mice, 4-HNE staining was weak and essentially localized in segments of photoreceptors in both mouse groups. No significant difference was detected for 4-HNE between the HFt+/+ and HFt+/− nonilluminated mice (Figs. 5E, 5H). 4-HNE immunostaining was observed throughout the retinal layers of the HFt+/+ and HFt+/− illuminated mice, but was clearly more intense in the ONL and IPL of the illuminated HFt+/− than in the HFt+/+ mouse retinas (compare Figs. 5F, 5G to Figs. 5I, 5J). We used an antibody against GFAP to investigate glial activation in the retina of the HFt+/+ and HFt+/− mice after illumination. Labeling after illumination was strong in Müller glial cells and astrocytes, inside the ganglion cell layer (GCL) throughout the retina (Figs. 5K, 5L). Since GFAP staining was identical for the two genotypes in control or exposure conditions, only images of HFt+/− retina staining are shown. We used an antibody against Iba1 in the HFt+/+ and HFt+/− mouse retinas to reveal light-induced mobilization of microglial cells from the inner retina (OPL, IPL, and GCL) to the ONL and RPE. Activation of microglial cells is characterized by a round morphology without extensions (Figs. 5M, 5N). Since no gross differences in microglial cell distribution or number were seen between the HFt+/+ and the HFt+/− mice, only images of stained retinas of HFt+/− mice are shown. 
Figure 5.
 
Apoptotic cells, membrane lipid peroxidation, and glial activation in the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light. (AD) TUNEL staining of retinal sections from the HFt+/+ (A, B) and the HFt+/− (C, D) mice not exposed (A, C) or exposed (B, D) for 24 hours and killed 1 day later. (E, H) 4-HNE immunostaining of retinal sections from the HFt+/+ (E) and the HFt+/− (H) nonilluminated mice. (F, G, I, J) 4-HNE staining of retinal sections from the HFt+/+ (F, G) and the HFt+/− (I, J) illuminated mice. (F, I) Enlargement of the inner part of the retina from the HFt+/+ and the HFt+/− illuminated mice. (G, J) Enlargement of the outer part of the retina from HFt+/+ and HFt+/− illuminated mice. 4-HNE immunostaining was very weak in the HFt+/+ and HFt+/− nonexposed mice. In illuminated mice, the 4-HNE staining was stronger in IPL of HFt+/− than HFt+/+ mice. (K, L) GFAP staining of the retinas of HFt+/− mice in control conditions (K) and exposed to light (L). (M, N) Iba1 staining of the retinas of the HFt+/− mice in control conditions (M) and exposed to light (N). Müller glial cell were activated (GFAP) and microglia cells (Iba1) migrated to the outer retina after exposure to light in the HFt+/+ and HFt+/− two mice. Sections were counterstained with DAPI (blue). Scale bar, 50 μm.
Figure 5.
 
Apoptotic cells, membrane lipid peroxidation, and glial activation in the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light. (AD) TUNEL staining of retinal sections from the HFt+/+ (A, B) and the HFt+/− (C, D) mice not exposed (A, C) or exposed (B, D) for 24 hours and killed 1 day later. (E, H) 4-HNE immunostaining of retinal sections from the HFt+/+ (E) and the HFt+/− (H) nonilluminated mice. (F, G, I, J) 4-HNE staining of retinal sections from the HFt+/+ (F, G) and the HFt+/− (I, J) illuminated mice. (F, I) Enlargement of the inner part of the retina from the HFt+/+ and the HFt+/− illuminated mice. (G, J) Enlargement of the outer part of the retina from HFt+/+ and HFt+/− illuminated mice. 4-HNE immunostaining was very weak in the HFt+/+ and HFt+/− nonexposed mice. In illuminated mice, the 4-HNE staining was stronger in IPL of HFt+/− than HFt+/+ mice. (K, L) GFAP staining of the retinas of HFt+/− mice in control conditions (K) and exposed to light (L). (M, N) Iba1 staining of the retinas of the HFt+/− mice in control conditions (M) and exposed to light (N). Müller glial cell were activated (GFAP) and microglia cells (Iba1) migrated to the outer retina after exposure to light in the HFt+/+ and HFt+/− two mice. Sections were counterstained with DAPI (blue). Scale bar, 50 μm.
H and L Ferritin Proteins and mRNAs.
We assessed the direct effects of exposure to light (24 hours) on ferritin expression 1 day after illumination (Figs. 6A–D). H ferritin mRNA was significantly less abundant (50%) in the exposed than in the unexposed HFt+/+ and HFt+/− mice (Fig. 6A), but L ferritin mRNA was unchanged after illumination in both mouse retinas (Fig. 6B). Illumination had no effect on the amount of H ferritin protein, but L ferritin protein was significantly (twofold) more abundant after illumination than in the controls (Figs. 6C, 6D). Immunolocalization experiments indicated that the distributions of H and L ferritin proteins in the retinas of mice were unaffected by exposure to light (data not shown). Thus, illumination decreased the availability of H ferritin, which may be compensated by an increase in L ferritin protein content. This strongly suggests that the illumination protocol used in these experiments modified the dynamics of iron storage in ferritin protein. We therefore studied various iron-regulating protein gene expression. 
Figure 6.
 
Analysis of H and L ferritin proteins and mRNAs in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after 24 hours of exposure to 13,000-lux light. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from the controls. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from nonexposed controls. In the HFt+/+ and HFt+/− mice, the concentration of H ferritin protein was unaffected by light treatment, whereas the amount of H ferritin mRNA decreased. The amount of L ferritin protein increased after light treatment, whereas L ferritin mRNA was unaffected in both the HFt+/+ and the HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 6.
 
Analysis of H and L ferritin proteins and mRNAs in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after 24 hours of exposure to 13,000-lux light. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from the controls. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from nonexposed controls. In the HFt+/+ and HFt+/− mice, the concentration of H ferritin protein was unaffected by light treatment, whereas the amount of H ferritin mRNA decreased. The amount of L ferritin protein increased after light treatment, whereas L ferritin mRNA was unaffected in both the HFt+/+ and the HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Profile of mRNAs for Other Iron-Regulating Proteins.
We studied mRNAs for several proteins (transferrin, transferrin receptor, ceruloplasmin, hephaestin, ferroportin, hepcidin, and iron regulatory proteins 1 and 2) involved in iron homeostasis 1 day after 24 hours illumination in the HFt+/+ and HFt+/− mice. Transferrin mRNA was significantly more expressed in illuminated retinas than control (2.74- and 1.71-fold for HFt+/+ and HFt+/−, respectively; Fig. 7A). Light significantly decreased transferrin receptor mRNA in the HFt+/+ and HFt+/− mice by 80% (Fig. 7B). The mRNAs of the two ferroxidases ceruloplasmin and hephaestin, which export iron from cells, were compared after exposure to light. The amount of mRNA for ceruloplasmin increased significantly, by nearly fourfold in the HFt+/+ mice and threefold in the HFt+/− mice, whereas hephaestin mRNA significantly decreased by 29% in the HFt+/+ mice and by 27% in the HFt+/− mice (Figs. 7C, 7D). Ferroportin mRNA was unaffected by light treatment in the HFt+/+ and HFt+/− mice (Fig. 7E). Hepcidin mRNA was significantly decreased by exposure to light (by 4.0- and 3.6-fold) in the HFt+/+ and HFt+/− mouse retinas, respectively (Fig. 7F). The mRNAs for iron regulatory proteins 1 and 2 were both 50% downregulated after illumination in the HFt+/− and HFt+/+ mouse retinas (Figs. 7G, 7H). Thus, for all these iron-regulating proteins, the light-exposure protocol had substantial effects on the patterns of expression of their mRNAs, but the effects were independent of the H ferritin genotype. We tested different initial illumination periods to study the timing of these effects. 
Figure 7.
 
mRNAs for iron-regulating proteins in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after exposure for 24 hours to 13,000-lux light. (A–H) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), ceruloplasmin (C), hephaestin (D), ferroportin (E), hepcidin (F), iron-regulatory protein 1 (G), and iron-regulatory protein 2 (H) in lysates of retinas from HFt+/+ and HFt+/− mice killed 24 hours after 24 hours of light treatment and from the nonexposed controls. The light treatment increased the amounts of transferrin and ceruloplasmin mRNAs, whereas it decreased those of transferrin receptor, hephaestin, hepcidin, iron-regulatory protein 1, and iron-regulatory protein 2 in the 4-month-old HFt+/+ and HFt+/− mouse retinas. mRNA for ferroportin was unaffected. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 7.
 
mRNAs for iron-regulating proteins in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after exposure for 24 hours to 13,000-lux light. (A–H) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), ceruloplasmin (C), hephaestin (D), ferroportin (E), hepcidin (F), iron-regulatory protein 1 (G), and iron-regulatory protein 2 (H) in lysates of retinas from HFt+/+ and HFt+/− mice killed 24 hours after 24 hours of light treatment and from the nonexposed controls. The light treatment increased the amounts of transferrin and ceruloplasmin mRNAs, whereas it decreased those of transferrin receptor, hephaestin, hepcidin, iron-regulatory protein 1, and iron-regulatory protein 2 in the 4-month-old HFt+/+ and HFt+/− mouse retinas. mRNA for ferroportin was unaffected. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Progressive Illumination Time Protocol: Effects of Light on the 4-Month-Old Mouse Retina Immediately after Various Times of Illumination
HFt+/+ and HFt+/− mice were exposed to 2, 12, and 24 hours of light, and the retinas were analyzed immediately thereafter. We assayed various mRNAs and proteins involved in iron metabolism: H and L ferritin, transferrin, transferrin receptor, ferroportin, ceruloplasmin, hephaestin, hepcidin, and iron regulatory proteins 1 and 2. 
H and L Ferritin mRNA and Protein Profiles.
In the HFt+/+ and HFt+/− mouse retinas, H ferritin mRNA abundance was not affected after 2 hours of illumination, but had significantly decreased (60%) after 12 hours and 24 hours. (Fig. 8A). The duration of exposure to light had no effect on H ferritin protein amounts in the HFt+/+ and HFt+/− mice (Fig. 8C). In this short period of illumination, L ferritin mRNA and protein levels were not modified in the two mouse strains (Figs. 8B, 8D). 
Figure 8.
 
The effects of various lengths of exposure to light. H and L ferritin mRNAs and proteins in retinas of HFt+/+ and HFt+/− mice exposed to light treatment for 2, 12, and 24 hours. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. (C, D) H (C) and L (D) ferritin protein assays in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, or 24 hours of light treatment and from the nonexposed controls. Ferritins were assayed by ELISA. The amounts of H ferritin mRNAs in the HFt+/+ and HFt+/− mice decreased after 12 hours of light treatment, whereas H ferritin protein was unaffected by the duration of exposure. The amounts of L ferritin mRNAs and proteins in two mice were unaffected during light treatment. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.0001.
Figure 8.
 
The effects of various lengths of exposure to light. H and L ferritin mRNAs and proteins in retinas of HFt+/+ and HFt+/− mice exposed to light treatment for 2, 12, and 24 hours. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. (C, D) H (C) and L (D) ferritin protein assays in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, or 24 hours of light treatment and from the nonexposed controls. Ferritins were assayed by ELISA. The amounts of H ferritin mRNAs in the HFt+/+ and HFt+/− mice decreased after 12 hours of light treatment, whereas H ferritin protein was unaffected by the duration of exposure. The amounts of L ferritin mRNAs and proteins in two mice were unaffected during light treatment. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.0001.
Profiles of mRNAs for Other Iron-Regulating Proteins.
The mRNAs for transferrin, transferrin receptor, hephaestin, and ferroportin were rapidly activated after 2 hours of illumination and then returned to their initial level or lower after 12 hours of exposure (Figs. 9A–D). Ceruloplasmin mRNA increased steadily by 12 hours and further by 24 hours of exposure (Fig. 9E). Hepcidin mRNA was less abundant at 12 hours after exposure in both the HFt+/+ and the HFt+/− mice (Fig. 9F). After 2 hours of illumination, there was no significant difference in iron regulatory protein 1 mRNA between exposed and unexposed mouse retinas. However, after longer periods of illumination (from 12 hours), iron regulatory protein 1 mRNA levels decreased in the HFt+/+ and HFt+/− mice (Fig. 9G). Iron regulatory protein 2 mRNA was significantly upregulated after 2 hours of illumination in the HFt+/+ and HFt+/− mice, but in both type of mouse after 12 hours of illumination, iron regulatory protein 2 mRNA was less abundant than in the control condition and after 2 hours of illumination (P ≤ 0.05; Fig. 9H). 
Figure 9.
 
mRNAs for iron-regulating proteins in retinas from 4-month-old HFt+/+ and HFt+/− mice after 2, 12, and 24 hours of light treatment. (AI) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), hephaestin (C), ferroportin (D), ceruloplasmin (E), hepcidin (F), iron-regulatory protein 1 (G), iron-regulatory protein 2 (H), and heme-oxygenase 1 (I) in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. The amounts of mRNAs for transferrin, transferrin receptor, hephaestin, ferroportin, and iron-regulatory proteins 1 and 2 increased after 2 hours of exposure to light and decreased to the initial level after 12 and 24 hours. mRNA for ceruloplasmin increased, whereas that of hepcidin decreased with duration of exposure. The amount of heme-oxygenase 1 mRNA increased after 12 hours of illumination. No difference was detected between the mRNA levels in the HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ns: not significant.
Figure 9.
 
mRNAs for iron-regulating proteins in retinas from 4-month-old HFt+/+ and HFt+/− mice after 2, 12, and 24 hours of light treatment. (AI) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), hephaestin (C), ferroportin (D), ceruloplasmin (E), hepcidin (F), iron-regulatory protein 1 (G), iron-regulatory protein 2 (H), and heme-oxygenase 1 (I) in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. The amounts of mRNAs for transferrin, transferrin receptor, hephaestin, ferroportin, and iron-regulatory proteins 1 and 2 increased after 2 hours of exposure to light and decreased to the initial level after 12 and 24 hours. mRNA for ceruloplasmin increased, whereas that of hepcidin decreased with duration of exposure. The amount of heme-oxygenase 1 mRNA increased after 12 hours of illumination. No difference was detected between the mRNA levels in the HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ns: not significant.
The mRNA for the antioxidative stress protein heme-oxygenase 1 started to increase at 12 hours, and it remained high thereafter; the mRNA for ceruloplasmin showed a similar pattern (Fig. 9I). Finally, there was no significant difference between the HFt+/+ and HFt+/− mice in levels of all the iron-regulating proteins mRNAs. 
The effect of light on the expression of iron-regulating proteins is summarized in Supplementary Figure S1. The hypotheses that iron increases after light-induced stress is based on publications that describe iron accumulation during retinal degeneration. 23,27,30,38 Iron homeostasis modifications, even after the end of exposure to light, caused us to suppose that the iron level was increased. However, it is difficult at this time to determine in what tissue or cell compartment this increase took place. 
Several iron-regulating protein mRNAs were increased rapidly after the beginning of the exposure. An extensive light-exposure period correlated with the increase of reactive oxygen species formation and modified expression of other iron-regulating protein mRNAs. At the end of illumination, the iron level should continue to increase due to cell death. 1,4 Thus, the iron homeostasis cycle was mobilized at each step of oxidative stress, which was enhanced by illumination. 
Discussion
Iron accumulates in the outer retina during normal aging, 39 and in some retinal degenerative animal models. 3,8,40 Recently, age-dependent and gender-specific changes in mouse retina and RPE/choroid iron levels by strain have been reported. 12,41 A proton-induced x-ray emission study showed accumulation of iron in the RPE and photoreceptors during aging in C57Bl/6J mice. 42 To investigate the role of ferritin, the main iron-regulating protein, we used a mouse strain with a haploid deletion in the H ferritin gene (HFt+/−). These HFt+/− mice had 63% less H ferritin than did the WT control mice. This deficit resulted in a significant decrease in the number of photoreceptor cells in the 4- and 16-month-old HFt+/− mice compared with WT mice. Ferroportin mRNA was more abundant in the 16-month-old mice than in the younger mice in both strains. This finding is consistent with what is observed in aging mouse 12 or AMD patients' retinas. 16  
We also examined whether HFt+/+ and HFt+/− mouse retinas are equally resistant to stress by using excessive light to provoke retinal degeneration. Light-induced oxidative stress has been extensively used in various experimental systems 25,43 45 to study early and late events affecting the retina. Little information is available about the regulation of iron homeostasis protein expression in the retina after exposure to light, except for ceruloplasmin in mice. 38 We used an illumination protocol causing relatively fast degeneration, starting with 1 day of dark adaptation, followed by 24 hours of illumination with 13,000 lux of white light. Twelve days of recovery after illumination, the ONL was thicker in the 4-month-old HFt+/+ mice than in the HFt+/− mice. However, the massive retinal degeneration in the central part of the retina in the 16-month-old mice (which have normally lost 20% of the ONL thickness by this age) after treatment with light was similar in the HFt+/+ and HFt+/− mice. 
In normal retinal physiological conditions or under the moderate stress associated with normal retinal aging, the iron-sequestering capacity of the half-normal amounts of H ferritin is sufficient. Indeed, a minimum of one or two H subunits in ferritin polymers is thought to be sufficient to allow formation of ferritin molecules, which have the capacity to sequester iron. 46 Overexpression of L ferritin can compensate for the deficiency of H ferritin. L ferritin contributes to the nucleation of iron cores in the ferritin molecule. 47 Recently, it has been shown that the conditional deletion of H ferritin in mouse liver had no immediate or long-term effect unless the animals were previously overloaded with iron. Thus, it is the combined effect of iron excess and aging that is toxic in these animals with totally inactivated H ferritin expression. 48 This effect is in accordance with our results on the combined role of aging and iron metabolism with exposure to light in the retina. The capacity of the aged retina to respond to additional stress may depend on the cells' ability to accumulate more L ferritin, a property that is higher in young than in old retinas (Figs. 2B, 2D). 
The retinal degeneration in the 16-month-old HFt+/+ and HFt+/− mice 12 days after illumination was too great for them to be useful in these studies, and so we used only 4-month-old mice for subsequent analyses. We studied management of iron by iron-regulating protein during exposure to light (2, 12, or 24 hours) and 1 day after the end of 24 hours of exposure. The illumination protocol that we used revealed different time courses of modifications of the expression of mRNA for the various iron-regulating proteins (see Supplementary Fig. S1). 
At the beginning of illumination (2 hours), the mRNAs for transferrin mainly, but also for transferrin receptor, hephaestin, ferroportin, and iron regulatory proteins 1 and 2 were increased. Thus, the retinas reacted quickly to oxidative stress-caused, light-induced injury by regulating the mRNA level of these iron metabolism proteins. Moreover, photoreceptor phagocytosis is under the control of the circadian rhythm; such disturbance by aberrant illumination triggers modification of OS phagocytosis by the RPE, 49,50 decreasing iron recycling throughout. Transferrin, synthesized by RPE and Müller glial cells, 2,51 and its receptor must be increased for the egress of free iron to occur across the retina from the photoreceptor layers into the circulation. 52 Ferroportin is a protein that exports iron from cells and is coupled to a ferroxidase hephaestin. Hephaestin converts iron in a form available for transferrin binding. 
Effects on ceruloplasmin and hepcidin were dependent on the duration of illumination. As the duration of exposure to light increases, oxidative stress and therefore free reactive iron increase, and the need for ceruloplasmin to oxidize the iron increases. Reduced hepcidin expression should promote iron export by increasing ferroportin on the cell surface. 53 The modification of ceruloplasmin and hepcidin gene expression over the same time period as heme-oxygenase 1 suggests that there also may be a later stress response of these proteins to light. 26,54  
After exposure to light, the central part of HFt+/− retinas was more susceptible than that of the HFt+/+ retinas to oxidative stress, as revealed by the presence of 4-HNE and TUNEL. After light treatment, iron flux in the retina was modified as early as 1 day after 24 hours of illumination. Iron was transformed into a bioavailable form by ceruloplasmin, which was increased and was chelated by transferrin, which also increased. The decrease in hephaestin mRNA and the absence of modification of ferroportin mRNA showed a reduction in iron exportation. However, iron export was always essential, since mRNA of hepcidin was decreased. These observations thus confirm a drastic change in iron metabolism, as a response of retinal cells against damage from light-induced stress. 
The excess iron flux in the retina after exposure to excessive light mobilizes retinal cells, including microglia and Müller glial cells. Indeed, Müller glial cells are activated after light-induced degeneration, 35 and microglia are recruited from the inner retina 55 through the release of chemokines produced by photoreceptors and Müller glial cells. 35,56 Activation of Müller glial cells may be part of the early response to light-induced injury. Indeed, they produce and secrete transferrin 51,57 and ceruloplasmin, 31,38 which are increased after exposure. 
This study demonstrated the difference in transcription and posttranscription regulation of H and L ferritin. During and even after exposure, H ferritin mRNA was decreased, whereas its protein level was unmodified. The decrease started at 12 hours of illumination, at the same time that mRNA of heme-oxygenase 1 increased. H ferritin, like heme-oxygenase 1, has antioxidant-responsive element (ARE) sequences in its gene, sensitive to oxygen species, which represses H ferritin and enhances heme-oxygenase 1 transcription. 58 L ferritin mRNA was unmodified during and after exposure, but its protein was significantly elevated after exposure to light. The iron regulatory proteins are sensitive to high iron levels, and binding iron regulatory element (IRE) sequences on the mRNA of L ferritin enhances its transcription. We noted that iron regulatory protein 2 mRNA increased after 2 hours of illumination. 
We can suppose that the increase in iron reacts with iron regulatory protein and enhances L ferritin protein for rapid nucleation of iron in the ferritin complex. Thereafter, high oxidative stress, starting at 12 hours of illumination, decreases mRNA of H ferritin to avoid overexpression (by iron regulatory protein) and to maintain H ferritin level at a steady threshold. Indeed, H ferritin overexpression is known to result in reduced cell growth and iron deficiency. 59,60 This may also explain why the H ferritin level does not change dramatically in brain samples from iron-fed mice, despite significant changes in the H/L ratio. 61  
In conclusion, we demonstrated that iron-regulating proteins were able to manage photo-oxidation resulting from light-induced injury as early as the beginning of illumination. The response is regulated after exposure to light, so as to allow retinal remodeling and management of excess iron. 
The early oxidative stress (heme-oxygenase 1 expression) during exposure was similar in both mice. Nevertheless, 24 hours after illumination, the oxidative stress (4-HNE staining) was increased in the HFt+/− mice, resulting in more photoreceptor cell death (reduction in photoreceptor nuclear layer thickness) in this mouse 12 days after illumination. The sensitivity of the HFt+/− mouse retina to excessive light seems to be due to a decreased capacity to manage iron, rather than to more oxidative stress. Even the modification of gene expression of iron-regulating proteins (which are similar in both mouse groups) did not induce redistribution of iron across the retina cells. 
Therefore, therapeutic strategies involving the reduction of photo-oxidative stress and thus iron toxicity in various retinal diseases including AMD may be beneficial to retinal health. This outcome may be obtained by the use of iron chelators 62 or by modulating iron homeostasis mechanisms. 
Supplementary Materials
Figure sf01, PDF - Figure sf01, PDF 
Footnotes
 Supported by Association pour les aveugles et handicapés visuels de France (EP) and Centre de Recherche d'Ophtalmologie.
Footnotes
 Disclosure: E. Picard, None; I. Ranchon-Cole, None; L. Jonet, None; C. Beaumont, None; F. Behar-Cohen, None; Y. Courtois, None; J.-C. Jeanny, None
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Figure 1.
 
Histology of retinas in 4- and 16-month-old HFt+/+ and HFt+/− mice. (AD) Retinal sections from HFt+/+ (A, C) and HFt+/− mice (B, D) at 4 (A, B) and 16 (C, D) months. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (E) Mean thickness of the ONL in the 4- and 16-month-old HFt+/+ and HFt+/− mouse retinas. ONL thickness decreased with age in the HFt+/+ and HFt+/− mice and was slightly lower in the HFt+/− than in the HFt+/+ mice. The measurements of ONL thicknesses every 200 μm, on each pole of the retina, were averaged. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Figure 1.
 
Histology of retinas in 4- and 16-month-old HFt+/+ and HFt+/− mice. (AD) Retinal sections from HFt+/+ (A, C) and HFt+/− mice (B, D) at 4 (A, B) and 16 (C, D) months. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (E) Mean thickness of the ONL in the 4- and 16-month-old HFt+/+ and HFt+/− mouse retinas. ONL thickness decreased with age in the HFt+/+ and HFt+/− mice and was slightly lower in the HFt+/− than in the HFt+/+ mice. The measurements of ONL thicknesses every 200 μm, on each pole of the retina, were averaged. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Figure 2.
 
Ferritin proteins and mRNAs in 4- and 16-month-old HFt+/+ and HFt+/− mice. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. In the 4- and 16-month-old HFt+/− mice, the amounts of H ferritin protein and mRNA were nearly half those in the HFt+/+ mice. L ferritin protein was more abundant in the 4-month-old HFt+/− than HFt+/+ mice, but was similar in the 16-month-old HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; ** P < 0.01. (EM) Immunolocalization of H (FI) and L (JM) ferritin on frozen sections of retina from 4- and 16-month-old HFt+/+ and HFt+/− mice. (E) Omission of the primary antibody was used as the control. (FI) H ferritin in the HFt+/+ (F, H) and HFt+/− (G, I) mouse retina from 4-month-old (F, G) and 16-month-old (H, I) mice. (JM) L ferritin in the HFt+/+ (J, L) and HFt+/− (K, M) mouse retina from 4-month-old (J, K) and 16-month-old (L, M) mice. In the HFt+/+ and the HFt+/− mice, the signals for H and L ferritin were in the photoreceptor inner segments (IS), ONL, OPL, and INL and in the inner top of the IPL. Sections were counterstained with DAPI (blue). Scale bar, 100 μm.
Figure 2.
 
Ferritin proteins and mRNAs in 4- and 16-month-old HFt+/+ and HFt+/− mice. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retinas from 4- and 16-month-old HFt+/+ and HFt+/− mice. In the 4- and 16-month-old HFt+/− mice, the amounts of H ferritin protein and mRNA were nearly half those in the HFt+/+ mice. L ferritin protein was more abundant in the 4-month-old HFt+/− than HFt+/+ mice, but was similar in the 16-month-old HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; ** P < 0.01. (EM) Immunolocalization of H (FI) and L (JM) ferritin on frozen sections of retina from 4- and 16-month-old HFt+/+ and HFt+/− mice. (E) Omission of the primary antibody was used as the control. (FI) H ferritin in the HFt+/+ (F, H) and HFt+/− (G, I) mouse retina from 4-month-old (F, G) and 16-month-old (H, I) mice. (JM) L ferritin in the HFt+/+ (J, L) and HFt+/− (K, M) mouse retina from 4-month-old (J, K) and 16-month-old (L, M) mice. In the HFt+/+ and the HFt+/− mice, the signals for H and L ferritin were in the photoreceptor inner segments (IS), ONL, OPL, and INL and in the inner top of the IPL. Sections were counterstained with DAPI (blue). Scale bar, 100 μm.
Figure 3.
 
Retinal modification in 4- and 16-month-old HFt+/+ and HFt+/− mice 12 days after illumination. (A, B) Measurement of the ONL thickness, every 200 μm, throughout the retina of 4-month-old (A) and 16-month-old (B) HFt+/+ and HFt+/− mice exposed or not exposed to light for 24 hours and killed 12 days later. The decrease in ONL thickness after exposure to light was much higher in the 4-month-old HFt+/− than HFt+/+ mice at both poles, especially toward the periphery. The decrease in ONL thickness was more apparent in the 16-month-old HFt+/+ and HFt+/− illuminated mice. (C, D) Measurement of the INL thickness, every 200 μm, throughout the retinas of 4-month-old (C) and 16-month-old (D) HFt+/+ and HFt+/− mice illuminated or not illuminated for 24 hours and killed 12 days later. Solid gray line: HFt+/+ control mice; solid black line: HFt+/− control mice; dashed gray line: HFt+/+ illuminated mice; dashed black line: HFt+/− illuminated mice. Data are the mean ± SEM of results from 6 to 18 independent sections. ***P < 0.0001; ns: not significant.
Figure 3.
 
Retinal modification in 4- and 16-month-old HFt+/+ and HFt+/− mice 12 days after illumination. (A, B) Measurement of the ONL thickness, every 200 μm, throughout the retina of 4-month-old (A) and 16-month-old (B) HFt+/+ and HFt+/− mice exposed or not exposed to light for 24 hours and killed 12 days later. The decrease in ONL thickness after exposure to light was much higher in the 4-month-old HFt+/− than HFt+/+ mice at both poles, especially toward the periphery. The decrease in ONL thickness was more apparent in the 16-month-old HFt+/+ and HFt+/− illuminated mice. (C, D) Measurement of the INL thickness, every 200 μm, throughout the retinas of 4-month-old (C) and 16-month-old (D) HFt+/+ and HFt+/− mice illuminated or not illuminated for 24 hours and killed 12 days later. Solid gray line: HFt+/+ control mice; solid black line: HFt+/− control mice; dashed gray line: HFt+/+ illuminated mice; dashed black line: HFt+/− illuminated mice. Data are the mean ± SEM of results from 6 to 18 independent sections. ***P < 0.0001; ns: not significant.
Figure 4.
 
Early (1 day) retinal damage in 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to 13,000-lux light. (A, B) Representative histology of the central sections of retina from 4-month-old HFt+/+ (A) and HFt+/− (B) mice exposed for 24 hours to light and observed 1 day later. After illumination, the ONL was irregular and the RPE disorganized. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (C) Mean thickness of the ONL of retinas from the 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to light and the mean ONL thickness in the control mice. The thickness of the first 500 μm at each pole of the retina was measured. The ONL thickness decreased similarly in the HFt+/+ and HFt+/− mice 1 day after exposure to light. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Figure 4.
 
Early (1 day) retinal damage in 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to 13,000-lux light. (A, B) Representative histology of the central sections of retina from 4-month-old HFt+/+ (A) and HFt+/− (B) mice exposed for 24 hours to light and observed 1 day later. After illumination, the ONL was irregular and the RPE disorganized. Nuclei were stained with toluidine blue. Scale bar, 100 μm. (C) Mean thickness of the ONL of retinas from the 4-month-old HFt+/+ and HFt+/− mice after 24 hours of exposure to light and the mean ONL thickness in the control mice. The thickness of the first 500 μm at each pole of the retina was measured. The ONL thickness decreased similarly in the HFt+/+ and HFt+/− mice 1 day after exposure to light. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. ***P < 0.0001.
Figure 5.
 
Apoptotic cells, membrane lipid peroxidation, and glial activation in the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light. (AD) TUNEL staining of retinal sections from the HFt+/+ (A, B) and the HFt+/− (C, D) mice not exposed (A, C) or exposed (B, D) for 24 hours and killed 1 day later. (E, H) 4-HNE immunostaining of retinal sections from the HFt+/+ (E) and the HFt+/− (H) nonilluminated mice. (F, G, I, J) 4-HNE staining of retinal sections from the HFt+/+ (F, G) and the HFt+/− (I, J) illuminated mice. (F, I) Enlargement of the inner part of the retina from the HFt+/+ and the HFt+/− illuminated mice. (G, J) Enlargement of the outer part of the retina from HFt+/+ and HFt+/− illuminated mice. 4-HNE immunostaining was very weak in the HFt+/+ and HFt+/− nonexposed mice. In illuminated mice, the 4-HNE staining was stronger in IPL of HFt+/− than HFt+/+ mice. (K, L) GFAP staining of the retinas of HFt+/− mice in control conditions (K) and exposed to light (L). (M, N) Iba1 staining of the retinas of the HFt+/− mice in control conditions (M) and exposed to light (N). Müller glial cell were activated (GFAP) and microglia cells (Iba1) migrated to the outer retina after exposure to light in the HFt+/+ and HFt+/− two mice. Sections were counterstained with DAPI (blue). Scale bar, 50 μm.
Figure 5.
 
Apoptotic cells, membrane lipid peroxidation, and glial activation in the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light. (AD) TUNEL staining of retinal sections from the HFt+/+ (A, B) and the HFt+/− (C, D) mice not exposed (A, C) or exposed (B, D) for 24 hours and killed 1 day later. (E, H) 4-HNE immunostaining of retinal sections from the HFt+/+ (E) and the HFt+/− (H) nonilluminated mice. (F, G, I, J) 4-HNE staining of retinal sections from the HFt+/+ (F, G) and the HFt+/− (I, J) illuminated mice. (F, I) Enlargement of the inner part of the retina from the HFt+/+ and the HFt+/− illuminated mice. (G, J) Enlargement of the outer part of the retina from HFt+/+ and HFt+/− illuminated mice. 4-HNE immunostaining was very weak in the HFt+/+ and HFt+/− nonexposed mice. In illuminated mice, the 4-HNE staining was stronger in IPL of HFt+/− than HFt+/+ mice. (K, L) GFAP staining of the retinas of HFt+/− mice in control conditions (K) and exposed to light (L). (M, N) Iba1 staining of the retinas of the HFt+/− mice in control conditions (M) and exposed to light (N). Müller glial cell were activated (GFAP) and microglia cells (Iba1) migrated to the outer retina after exposure to light in the HFt+/+ and HFt+/− two mice. Sections were counterstained with DAPI (blue). Scale bar, 50 μm.
Figure 6.
 
Analysis of H and L ferritin proteins and mRNAs in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after 24 hours of exposure to 13,000-lux light. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from the controls. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from nonexposed controls. In the HFt+/+ and HFt+/− mice, the concentration of H ferritin protein was unaffected by light treatment, whereas the amount of H ferritin mRNA decreased. The amount of L ferritin protein increased after light treatment, whereas L ferritin mRNA was unaffected in both the HFt+/+ and the HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 6.
 
Analysis of H and L ferritin proteins and mRNAs in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after 24 hours of exposure to 13,000-lux light. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from the controls. (C, D) Quantification by ELISA of H (C) and L (D) ferritin proteins in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice 1 day after 24 hours of exposure to 13,000-lux light and from nonexposed controls. In the HFt+/+ and HFt+/− mice, the concentration of H ferritin protein was unaffected by light treatment, whereas the amount of H ferritin mRNA decreased. The amount of L ferritin protein increased after light treatment, whereas L ferritin mRNA was unaffected in both the HFt+/+ and the HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 7.
 
mRNAs for iron-regulating proteins in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after exposure for 24 hours to 13,000-lux light. (A–H) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), ceruloplasmin (C), hephaestin (D), ferroportin (E), hepcidin (F), iron-regulatory protein 1 (G), and iron-regulatory protein 2 (H) in lysates of retinas from HFt+/+ and HFt+/− mice killed 24 hours after 24 hours of light treatment and from the nonexposed controls. The light treatment increased the amounts of transferrin and ceruloplasmin mRNAs, whereas it decreased those of transferrin receptor, hephaestin, hepcidin, iron-regulatory protein 1, and iron-regulatory protein 2 in the 4-month-old HFt+/+ and HFt+/− mouse retinas. mRNA for ferroportin was unaffected. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 7.
 
mRNAs for iron-regulating proteins in 4-month-old HFt+/+ and HFt+/− mouse retinas 1 day after exposure for 24 hours to 13,000-lux light. (A–H) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), ceruloplasmin (C), hephaestin (D), ferroportin (E), hepcidin (F), iron-regulatory protein 1 (G), and iron-regulatory protein 2 (H) in lysates of retinas from HFt+/+ and HFt+/− mice killed 24 hours after 24 hours of light treatment and from the nonexposed controls. The light treatment increased the amounts of transferrin and ceruloplasmin mRNAs, whereas it decreased those of transferrin receptor, hephaestin, hepcidin, iron-regulatory protein 1, and iron-regulatory protein 2 in the 4-month-old HFt+/+ and HFt+/− mouse retinas. mRNA for ferroportin was unaffected. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 8.
 
The effects of various lengths of exposure to light. H and L ferritin mRNAs and proteins in retinas of HFt+/+ and HFt+/− mice exposed to light treatment for 2, 12, and 24 hours. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. (C, D) H (C) and L (D) ferritin protein assays in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, or 24 hours of light treatment and from the nonexposed controls. Ferritins were assayed by ELISA. The amounts of H ferritin mRNAs in the HFt+/+ and HFt+/− mice decreased after 12 hours of light treatment, whereas H ferritin protein was unaffected by the duration of exposure. The amounts of L ferritin mRNAs and proteins in two mice were unaffected during light treatment. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.0001.
Figure 8.
 
The effects of various lengths of exposure to light. H and L ferritin mRNAs and proteins in retinas of HFt+/+ and HFt+/− mice exposed to light treatment for 2, 12, and 24 hours. (A, B) Quantification by qRT-PCR of H (A) and L (B) ferritin mRNAs in lysates of retinas from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. (C, D) H (C) and L (D) ferritin protein assays in lysates of retina from 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, or 24 hours of light treatment and from the nonexposed controls. Ferritins were assayed by ELISA. The amounts of H ferritin mRNAs in the HFt+/+ and HFt+/− mice decreased after 12 hours of light treatment, whereas H ferritin protein was unaffected by the duration of exposure. The amounts of L ferritin mRNAs and proteins in two mice were unaffected during light treatment. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.0001.
Figure 9.
 
mRNAs for iron-regulating proteins in retinas from 4-month-old HFt+/+ and HFt+/− mice after 2, 12, and 24 hours of light treatment. (AI) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), hephaestin (C), ferroportin (D), ceruloplasmin (E), hepcidin (F), iron-regulatory protein 1 (G), iron-regulatory protein 2 (H), and heme-oxygenase 1 (I) in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. The amounts of mRNAs for transferrin, transferrin receptor, hephaestin, ferroportin, and iron-regulatory proteins 1 and 2 increased after 2 hours of exposure to light and decreased to the initial level after 12 and 24 hours. mRNA for ceruloplasmin increased, whereas that of hepcidin decreased with duration of exposure. The amount of heme-oxygenase 1 mRNA increased after 12 hours of illumination. No difference was detected between the mRNA levels in the HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ns: not significant.
Figure 9.
 
mRNAs for iron-regulating proteins in retinas from 4-month-old HFt+/+ and HFt+/− mice after 2, 12, and 24 hours of light treatment. (AI) Quantification by qRT-PCR of mRNAs for transferrin (A), transferrin receptor (B), hephaestin (C), ferroportin (D), ceruloplasmin (E), hepcidin (F), iron-regulatory protein 1 (G), iron-regulatory protein 2 (H), and heme-oxygenase 1 (I) in lysates of retina from the 4-month-old HFt+/+ and HFt+/− mice, immediately after 2, 12, and 24 hours of light treatment and from the nonexposed controls. The amounts of mRNAs for transferrin, transferrin receptor, hephaestin, ferroportin, and iron-regulatory proteins 1 and 2 increased after 2 hours of exposure to light and decreased to the initial level after 12 and 24 hours. mRNA for ceruloplasmin increased, whereas that of hepcidin decreased with duration of exposure. The amount of heme-oxygenase 1 mRNA increased after 12 hours of illumination. No difference was detected between the mRNA levels in the HFt+/+ and HFt+/− mice. (□) HFt+/+ mice; (■) HFt+/− mice. Data are the mean ± SEM. *P < 0.05; **P < 0.01; ns: not significant.
Table 1.
 
Overview of All Primers Used in the qRT-PCR
Table 1.
 
Overview of All Primers Used in the qRT-PCR
Gene Name Forward Primer Reverse Primer
Beta-actin 5′ GAC GGC CAA GTC ATC ACT ATT G 3′ 5′ CCA CAG GAT TCC ATA CCC AAG A 3′
H ferritin 5′ TCA GTC ACT ACT GGA ACT GC 3′ 5′ CGT GGT CAC CCA GTT CTT TA 3′
L ferritin 5′ TGG CCA TGG AGA AGA ACC TGA ATC 3′ 5′ GGC TTT CCA GGA AGT CAC AGA GAT 3′
Transferrin 5′ GGA CGC CAT GAC TTT GGA TG 3′ 5′ GCC ATG ACA GGC ACT AGA CC 3′
Transferrin receptor 5′ GAG GAA CCA GAC CGT TTA GTT GT 3′ 5′ CTT CGC CGC AAC ACC AGC A 3′
Iron-regulatory protein 1 5′ TGC TTC CCC AGG TGA TTG GCT ACA 3′ 5′ TAG CTC GGT CAG CAA TGG ACA GCT 3′
Iron-regulatory protein 2 5′ ACC AGA GGT GGT TGG ATG TGA GCT 3′ 5′ ACG CCT ACT TGC CTG AGG TGC TTT 3′
Ferroportin 5′ GCT GCT AGA ATC GGT CTT TGG 3′ 5′ CAG CAA CTG TGT CAC CGT CAA 3′
Ceruloplasmin 5′ GGG AGC CGT CTA CCC TGA CAA 3′ 5′ GCA CTT TGT CAT CAG CCC GTT 3′
Hepcidin 5′ AGA GCT GCA GCC TTT GCA C 3′ 5′ GAG GTC AGG ATG TGG CTC TA 3′
Hephaestin 5′ GCC TGG GCA CAG AGA CTG AT 3′ 5′ GGT GTG AGG AAA GAG CAT GAC TG 3′
Heme oxygenase 1 5′ GCC ACC AAG GAG GTA CAC AT 3′ 5′ GCT TGT TGC GCT CTA TCT CC 3′
Figure sf01, PDF
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