Abstract
purpose. In this study, the hypothesis that increased intraocular levels of iron cause oxidative damage to the retina was tested.
methods. Adult C57BL/6 mice were given an intravitreous injection of saline or 0.10, 0.25, or 0.50 mM FeSO4. Scotopic electroretinograms (ERGs) were performed 3, 7, and 14 days after injection, and photopic ERGs were performed on day 14. Hydroethidine was used to identify superoxide radicals and lipid peroxidation was visualized by staining for hydroxynonenal (HNE). Retinal cell death was evaluated by TUNEL and measurement of inner nuclear layer (INL) and outer nuclear layer (ONL) thickness. Levels of rhodopsin and cone-opsin mRNA were measured by quantitative real time RT-PCR. Cone density was assessed by peanut agglutinin staining and confocal microscopy.
results. Compared with retinas in saline-injected eyes, retinas from eyes injected with FeSO4 showed greater fluorescence after intravenous injection of hydroethidine due to superoxide radicals in photoreceptors, greater photoreceptor staining for HNE, a marker of lipid peroxidation, and increased expression of Heme oxygenase 1, an indicator of oxidative stress. ERG b-wave amplitudes were reduced (photopic > scotopic) in FeSO4-injected eyes compared with those in saline-injected eyes. Numerous TUNEL-stained nuclei were seen along the outer border of the ONL, the location of cone cell nuclei, at 1 and 2 days after injection of FeSO4. In FeSO4-injected eyes, the thickness of the ONL, but not the INL, was significantly reduced, and 17 days after injection, there were 3.8- and 2.6-fold reductions in the mRNAs for M-cone and S-cone opsin, respectively, whereas there was no significant difference in rhodopsin mRNA. Confocal microscopy of peanut agglutinin–stained sections showed dose-dependent FeSO4-induced cone drop out.
conclusions. Increased intraocular levels of FeSO4 cause oxidative damage to photoreceptors with greater damage to cones than rods. This finding suggests that the oxidative defense system of cones differs from that of rods and other retinal cells, and that cones are more susceptible to damage from the type of oxidative stress imposed by iron.
Intraocular foreign bodies containing iron are toxic to the retina and, if left in the eye for prolonged periods, result in an extinguished electroretinogram (ERG) and blindness. This condition is referred to as siderosis bulbi, because there is progressive deposition of iron in ocular tissues.
1 Dense collections of ferritin particles are seen in the cytoplasm and organelles of ocular cells, and it has been hypothesized that the large accumulations cause physical damage that kills retinal cells.
2 It has also been hypothesized that iron liberated from intraocular hemorrhage causes damage by inducing inflammation.
3 4 A third hypothesis relating to iron-induced retinal damage is that iron liberated from hemoglobin after intraocular hemorrhage or from other sources causes oxidative damage in the retina.
5
The effects of iron in the eye are complicated, because there are multiple forms of iron with different reactivity and several proteins that modulate reactivity and/or levels. Ceruloplasmin is a copper-binding serum protein that oxidizes highly reactive Fe
2+ ions to less reactive Fe
3+ ions.
6 This effect not only alters the nature of iron, but also promotes its export from tissues by allowing it to bind to transferrin, the major iron transporter that only binds the Fe
3+ form. Patients with aceruloplasminemia have defective transport of iron out of some tissues including the retina and some brain regions, resulting in retinal degeneration and dementia usually occurring in an age range of 40 to 60 years.
7 8 9 At 5 months of age and older, mice deficient in ceruloplasmin and hephaestin, another protein that oxidizes Fe
2+ to Fe
3+, develop high levels of iron in the retina and retinal pigmented epithelium (RPE), and beyond 6 months of age they show areas of RPE hyperplasia and atrophy, with some areas of photoreceptor atrophy and occasional subretinal neovascularization.
10 Thus, increased levels of iron in the eye, whether from a metallic foreign body, high levels of hemoglobin, or defective export of iron, cause retinal degeneration. As noted earlier, there are several hypotheses as to the mechanism of iron-induced damage, but since iron is capable of reacting with oxygen to generate reactive oxygen species,
11 recent studies seem to assume oxidative damage plays a role, although this has not been conclusively proven. In this study we sought to test this hypothesis and determine whether the damage is homogeneous or localized to a particular cell type.
Rabbit anti-HNE antibody was used for indirect immunofluorescent staining (Alpha Diagnostic International, Inc., San Antonio, TX). The frozen sections were dried at room temperature and postfixed in 4% paraformaldehyde for 15 minutes. The sections were washed with phosphate-buffered saline (PBS), blocked with 10% normal goat serum in PBS for 30 minutes at room temperature, and incubated overnight at 4°C with 1:100 rabbit anti-HNE diluted with PBS and containing 2% goat serum. After they were washed three times with PBS/0.1% Triton X-100, the sections were incubated for 1 hour at room temperature in 1:800 donkey anti-rabbit IgG conjugated with Cy3 (Jackson ImmunoResearch Laboratory, West Grove, PA). They were washed three times with PBS/0.1% Triton X-100 and then incubated at room temperature for 4 minutes with the nuclear dye Hoechst 33258 (1:1200; Sigma-Aldrich). The slides were viewed with a fluorescence microscope (Nikon Instruments Inc., New York, NY). Images were captured at the same exposure time for each section with a digital camera (Nikon) and software (SPOT RT 3.4; Diagnostic Instruments, Sterling Heights, MI).
The mice were euthanatized and eyes were fixed in 4% paraformaldehyde for 1 to 2 hours. Twenty-micrometer ocular frozen sections were dried, placed in 10% normal goat serum in PBS for 30 minutes at room temperature, and incubated for 1 hour at room temperature in 1:30 rhodamine-conjugated peanut agglutinin (PNA; Vector Laboratories, Burlingame, CA) and Hoechst nuclear dye (Sigma-Aldrich) in PBS containing 1% normal goat serum. The sections were viewed with a confocal microscope (LSM 510 META; Carl Zeiss Meditec, Oberkochen, Germany, with a 20×/0.75 numeric aperture objective; Plan-Apochromat; Carl Zeiss Meditec) using excitation wavelengths of 543 nm (PNA) or 405 nm (Hoechst). Images were acquired in the frame-scan mode, and stack size was 230.3 × 230.3 μm (512 × 512 pixels).
In this study, intravitreous injection of FeSO4 caused increased generation of superoxide radicals and oxidative damage in photoreceptors. This effect was associated with reduced retinal function determined by ERGs, with the greatest reduction in the photopic signaling pathway, which is initiated by cones. There was loss of photoreceptors by apoptosis, resulting in thinning of the ONL and no significant change in the INL. Apoptotic nuclei were concentrated along the outer border of the ONL, the location of cone cell nuclei, and whereas the mRNAs for M- and S-cone opsins were reduced 17 days after injection, there was not a significant reduction in rhodopsin mRNA. Staining of cone matrix sheaths with PNA showed reduced cone density, somewhat greater in the superior midperiphery of the retina than in the inferior midperiphery. These data suggest that FeSO4 causes oxidative damage to photoreceptors and that cones are more susceptible than rods.
This is an intriguing finding, because it indicates that with respect to the endogenous antioxidant defense system, photoreceptors differ from other retinal cells and cones differ from rods. The oxidative stress posed by FeSO
4 is different from that posed by paraquat, an herbicide that generates reactive oxygen species, because the inner retina is more susceptible to paraquat than it is to FeSO
4.
18 We hypothesize that different components of the endogenous antioxidant defense system may provide different levels of protection against various types of oxidative stress and that the cell types in the retina differ with respect to their antioxidant defense system. The results of this study are consistent with this hypothesis, but to prove the hypothesis, it is necessary to define how well components of the antioxidant defense system protect against specific types of oxidative stress and determine their roles in various cell types. We are currently defining which components of the endogenous antioxidant defense system protect retinal cells against paraquat-induced damage and have found that SOD1 provides some protection,
19 whereas SOD3 does not (Dong A, Campochiaro PA, unpublished data). It will be useful to determine whether protection against FeSO
4 is provided by different components of the defense system than those that protect against paraquat.
Intraocular injection of FeSO
4 provides a model of oxidative damage-induced retinal degeneration that may have relevance to particular ocular diseases in which Fe overload has been demonstrated. Intraocular implantation of Fe particles in rats
20 or rabbits
21 provides similar models, but requires a surgical procedure, and the Fe dose is unknown. Intraocular Fe wires caused selective damage to photoreceptors and after implantation of Fe particles, rats showed apoptosis only in the ONL and not in any other layers of the retina. Our findings confirm those previous observations and extend them to show that cones are more susceptible than rods.
The FeSO
4-injection model is relevant to aceruloplasminemia in which increased levels iron in the retina may be associated with midperipheral atrophic changes in the retina and RPE
22 or drusenlike yellow spots and atrophic changes in the macula.
23 Agents that protect against FeSO
4-induced retinal damage could provide benefit in patients with aceruloplasminemia. Mice deficient in ceruloplasmin and hephaestin provide a highly relevant model for aceruloplasminemia, but the retinal degeneration occurs slowly over several months, making it impractical for screening a large number of agents, but reasonable to use for confirmatory testing of agents found to protect against intraocular FeSO
4-induced retinal damage.
Iron overload has also been shown in a few patients with age-related macular degeneration.
24 25 Although it is unlikely that Fe overload is a primary problem in most patients with AMD, it may occur secondary to photoreceptor damage from another cause and contribute to ongoing oxidative damage. Iron overload has been seen in association with several neurodegenerative disorders, including the RCS rat model of retinitis pigmentosa, in which the primary defect is known to be a mutation that has nothing to do with Fe metabolism or transport.
26 It appears that damaged neurons may accumulate Fe. One possible mechanism is loss of transferrin on damaged cells, compromising export of Fe. Whatever the source of Fe overload, the present study supports the hypothesis that its presence leads to exacerbation of oxidative stress and the nature of that oxidative stress differs from that due to other sources of oxidative stress, such as paraquat. Defining the components of the oxidative defense system that best protect against Fe-induced damage to photoreceptors and RPE cells may help in the development of treatments for retinal degenerations associated with iron overload.
Contributed equally to the work and therefore should be considered equivalent authors.
Supported by the Macula Vision Research Foundations and Dr. and Mrs. William Lake. PAC is the George S. and Dolores Dore Eccles Professor of Ophthalmology.
Submitted for publication May 13, 2006; revised September 1 and 21, 2006; accepted November 14, 2006.
Disclosure:
B.S. Rogers, None;
R.C.A. Symons, None;
K. Komeima, None;
J.-K. Shen, None;
W. Xiao, None;
M.E. Swaim, None;
Y.Y. Gong, None;
S. Kachi, None;
P.A. Campochiaro, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Peter A. Campochiaro, Maumenee 719, The Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287-9277;
[email protected].
Table 1. Gene Expression Analysis for Hmox1 in Mouse Retina after Intravitreous Injection of Normal Saline or 0.25 mM FeSO4
Table 1. Gene Expression Analysis for Hmox1 in Mouse Retina after Intravitreous Injection of Normal Saline or 0.25 mM FeSO4
Gene | Time (days) | Eyes (n) | | Average Δ(CtS16 − CtHmox1) | | SD (ΔCt) | | ΔΔCt | Increase Due to FeSO4 (x-fold) | P (t-test) |
| | Control | FeSO4 | Control | FeSO4 | Control | FeSO4 | | | |
Hmox1 | 1 | 4 | 4 | −8.6 | −4.2 | 0.2 | 0.3 | 4.4 | 20.4 | <0.001 |
Hmox1 | 3 | 8 | 8 | −9.2 | −6.0 | 1.0 | 0.8 | 3.2 | 9.5 | <0.001 |
Table 2. Gene Expression Analysis for Cone Opsins and Rhodopsin in Mouse Retina after Intravitreous Injection of Normal Saline or 0.25 mM FeSO4
Table 2. Gene Expression Analysis for Cone Opsins and Rhodopsin in Mouse Retina after Intravitreous Injection of Normal Saline or 0.25 mM FeSO4
Gene | Time (d) | Eyes (n) | | Average Δ(CtS16 − Ctgene) | | SD (ΔCt) | | ΔΔCt | Decrease Due to FeSO4 (x-fold) | P (t-test) |
| | Control | FeSO4 | Control | FeSO4 | Control | FeSO4 | | | |
Opn1mw | 1 | 4 | 4 | −2.5 | −5.6 | 0.7 | 1.1 | −3.0 | 8.0 | <0.004 |
Opn1mw | 17 | 12 | 12 | −3.8 | −5.8 | 0.5 | 1.3 | −1.9 | 3.8 | <0.001 |
Opn1sw | 1 | 4 | 4 | −0.8 | −3.3 | 0.6 | 1.9 | −2.5 | 5.8 | <0.05 |
Opn1sw | 17 | 12 | 12 | −2.0 | −3.3 | 0.6 | 1.1 | −1.4 | 2.6 | <0.001 |
Rho | 1 | 4 | 4 | 4.7 | 3.4 | 0.7 | 0.6 | −1.3 | 2.5 | <0.05 |
Rho | 17 | 12 | 12 | 4.4 | 4.2 | 0.3 | 0.5 | −0.2 | 1.2 | 0.19 |
Table 3. Reduction in Photopic and Scotopic ERG b-Waves 14 Days after Injection of Various Concentrations of FeSO4
Table 3. Reduction in Photopic and Scotopic ERG b-Waves 14 Days after Injection of Various Concentrations of FeSO4
Dose | Wave | Percent of Control | n | P |
0.10 mM | Photopic b-wave | 53.6 ± 6.7 | 8 | |
0.10 mM | Scotopic b-wave | 119.6 ± 28.7 | 8 | 0.042 |
0.25 mM | Photopic b-wave | 30.2 ± 7.1 | 8 | |
0.25 mM | Scotopic b-wave | 52.8 ± 3.8 | 8 | 0.014 |
0.50 mM | Photopic b-wave | 29.8 ± 5.0 | 13 | |
0.50 mM | Scotopic b-wave | 64.6 ± 11.5 | 13 | 0.010 |
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