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. 2014 Jul 1;55(7):4613-27.
doi: 10.1167/iovs.14-14633.

"V体育官网" Mitochondrial oxidative stress in the retinal pigment epithelium leads to localized retinal degeneration

Haoyu Mao  1 , Soo Jung Seo  1 , Manas R Biswal  1 , Hong Li  1 , Mandy Conners  1 , Arathi Nandyala  1 , Kyle Jones  1 , Yun-Zheng Le  2 , Alfred S Lewin  1
Affiliations

"VSports" Mitochondrial oxidative stress in the retinal pigment epithelium leads to localized retinal degeneration

Haoyu Mao et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Oxidative stress in the RPE is widely accepted as a contributing factor to AMD. We have previously shown that ribozyme-mediated reduction in the antioxidant enzyme manganese superoxide dismutase (MnSOD) leads to some of the features of geographic atrophy in mice. To develop a mouse model independent of viral injection, we used a conditional knockout of the Sod2 gene in the RPE to elevate mitochondrial oxidative stress in that cell layer. VSports手机版.

Methods: Experimental mice in which exon 3 of Sod2 was flanked by loxP sites were also transgenic for PVMD2-rtTA and tetO-PhCMV cre, so that cre recombinase was expressed only in the RPE. Pups of this genotype (Sod2(flox/flox)VMD2cre) were induced to express cre recombinase by feeding doxycycline-laced chow to nursing dams. Controls included mice of this genotype not treated with doxycycline and doxycycline-treated Sod2(flox/flox) mice lacking the cre transgene. Expression of cre in the RPE was verified by immunohistochemistry, and deletion of Sod2 exon 3 in the RPE was confirmed by PCR. Mice were followed up over a period of 9 months by spectral-domain optical coherence tomography (SD-OCT), digital fundus imaging, and full-field ERG V体育安卓版. Following euthanasia, retinas were examined by light and electron microscopy or by immunohistochemistry. Contour length of rod outer segments and thickness of the RPE layer were measured by unbiased stereology. .

Results: Following doxycycline induction of cre, Sod2(flox/flox) cre mice demonstrated increased signs of oxidative stress in the RPE and accumulation of autofluorescent material by age 2 months. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT), which were statistically significant by 6 months. In addition, OCT and electron microscopy revealed increased porosity of the choroid V体育ios版. At the same interval, hypopigmented foci appeared in fundus micrographs, and vascular abnormalities were detected by fluorescein angiography. By 9 months, the RPE layer in Sod2(flox/flox) cre mice was thicker than in nontransgenic littermates, and the rod outer segments were significantly longer over most of the retina, although localized atrophy of photoreceptors was also obvious in some eyes. .

Conclusions: Conditional tissue-specific reduction in MnSOD induced oxidative stress in mouse RPE, leading to RPE dysfunction, damage to the choroid, and death of photoreceptor cells VSports最新版本. The RPE oxidative stress did not cause drusen-like deposits, but the model recapitulated certain key aspects of the pathology of dry AMD and may be useful in testing therapies. .

Keywords: manganese superoxide dismutase; mouse model; reactive oxygen species; retinal degeneration; retinal pigment epithelium. V体育平台登录.

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Figures

Figure 1
Figure 1
Expression of cre in the RPE leads to deletion of Sod2. (A, B) Immunostaining of cre recombinase in flat mounts of the RPE of Sod2flox/floxVMD2cre mice. In the absence of doxycycline treatment (A), cre recombinase was not detectable at age 1 month. However, after 2-week feeding of doxycycline via the mother's milk (B), cre recombinase accumulated in nuclei of the RPE from Sod2flox/floxVMD2cre mice. Cre has a eukaryotic nuclear localization signal. (C) The PCR analysis of the Sod2 gene using primers flanking exon 3. In the absence of the cre transgene (Sod2flfl), a 1.1-kb amplicon expected of intact Sod2 was observed in Sod2flox/flox mice; less than 5% of the 0.4-kb–deleted allele was detected by densitometry of the image following this end point analysis. These are biological replicates from three individual mice. In three Sod2flox/floxVMD2cre mice, most of the product represented the intact gene in the absence of doxycycline treatment (−dox), and detection of the 0.4-kb band suggests leaky expression of cre in noninduced mice. Following doxycycline treatment of three mice (+dox), the 0.4-kb band representing the deleted allele was predominant. (D–E) Distribution of active cre in the RPE. Detection of β-galactosidase expression in doxycycline-treated Rosa26-lacZ VMD2-cre mice. Flat mounts of the RPE were briefly bleached, and the enzyme activity was detected by blue staining using X-gal. The original magnification in (D) was ×4, and the original magnification in (E) and (F) was ×10. (G–I) Rosa26-lacZ control mice without the cre transgene resulted in no enzymatic conversion of X-gal.
Figure 2
Figure 2
Induction of cre leads to a reduction in MnSOD and an increase in oxidative stress in the RPE. (A) The MnSOD was detected by immunohistochemistry in RPE flat mounts prepared from 2-month-old Sod2flox/floxVMD2cre mice not treated with doxycycline as pups. Despite the specificity of the antibody (Supplementary Fig. S1), immune reactivity was detected in the cytoplasm but also surrounding the nuclei and along the surfaces of cells. (B) In mice of the same age and genotype, but induced with doxycycline as neonates, immune reactivity was dramatically reduced in greater than 90% of the RPE cells. (C) By ELISA, there was no significant difference in nitrotyrosine between doxycycline-induced mice and control mice at age 1 month, but by 2 months there was a significant elevation of nitrotyrosine in doxycycline-induced mice. n = 6; P < 0.05. (D–F) To examine oxidative damage to DNA, RPE flat mounts were decorated with primary antibodies to 8-OHdG and with antibodies to ZO-1 to visualize cell boundaries. There was a significant increase in 8-OHdG in Sod2flox/floxVMD2cre mice induced with doxycycline (E). The higher-magnification image (F) reveals that most of the immune reactivity was in the cytoplasm (red), although some oxidative damage could also be detected in nuclei (yellow).
Figure 3
Figure 3
Deletion of Sod2 leads to increased autofluorescence in the RPE. (A, B) The RPE/choroid was dissected from 4-month-old doxycycline-treated Sod2flox/flox mice (no cre) (A) and from doxycycline-treated Sod2flox/floxVMD2cre mice (B), cryosectioned, and examined with a laser scanning confocal microscope (Leica TCS SP2 AOBS). Wavelength scans were performed at the 488-nm laser line (incident light), and fluorescence spectra were made from the regions of the image corresponding to the RPE and the choroid, as well as from a background region of the slide.
Figure 4
Figure 4
Gradual decline in ERG response following deletion of Sod2. (A) Dark-adapted ERG responses were measured in doxycycline-induced Sod2flox/floxVMD2cre mice at age 1, 2, 3, 6, and 9 months. The average a-wave amplitudes and the average b-wave amplitudes recorded at 0-dB (2.68 scot cd/m2) flash intensity are shown. The same cohort of mice was used for all data points. n = 20 for months 1 through 3; n = 16 for month 6; n = 12 for month 9. (B) Light-adapted ERG responses were recorded in doxycycline-induced Sod2flox/floxVMD2cre mice at age 2, 6, and 9 months. These were from a different group of mice than those used for scotopic ERG. n = 38 for 2 months; n = 30 for 6 months; n = 26 for 9 months. (C–G) Representative scotopic ERG waveforms of doxycycline-induced Sod2flox/floxVMD2cre mice at intensities of 0 dB (2.68 scot cd/m2), −10 dB (0.18 scot cd/m2), and −20 dB (0.02 scot cd/m2) at age 1, 2, 3, 6, and 9 months. *P = 0.05; **P < 0.01.
Figure 5
Figure 5
Deletion of Sod2 in the RPE leads to thinning of the ONL and to choroidal abnormalities. (A–E) Representative SD-OCT images were captured to monitor the posterior retina in doxycycline-induced Sod2flox/floxVMD2cre mice at age 1, 2, 3, 6, and 9 months. White arrows indicate increased porosity of the choroid at 6 and 9 months. (F) Averaged ONL thickness measured between the outer plexiform layer and the external limiting membrane was measured at four standard locations relative to the optic nerve head at time points of 1, 2, 3, 6, and 9 months to demonstrate progressive degeneration of photoreceptor layers. These were the same mice and numbers of mice at each interval used for scotopic ERG (Fig. 4). **P < 0.01. (G) Electron micrographs reveal widened blood vessels and distended layers of the choroid in 9-month-old Sod2flox/floxVMD2cre mice treated with doxycycline (H) compared with untreated controls (scale bars: 10 μm).
Figure 6
Figure 6
Deletion of Sod2 in the RPE leads to localized atrophy of the retina. (A–F) Fundus images were captured at age 1, 2, 3, 6, 9, and 10 months in doxycycline-induced Sod2flox/floxVMD2cre mice using a Micron III digital fundus microscope. By 6 months, hypopigmented areas (blotches) and white spots reflected thinning of the retina.
Figure 7
Figure 7
Vascular leakage following Sod2 deletion. (A) A small atrophic region was apparent in a 6-month-old Sod2flox/floxVMD2cre mouse in fundus imaging. (B–F) Fluorescein angiography (FA) was performed to detect fluorescein leakage. The FA images were captured at 1, 3, 5, 10, and 15 minutes after fluorescein injection. Leaking fluorescein came from the same area of atrophic region from the fundus image; gradual enlargement of green fluorescein started at 3 minutes and reached maximum size at 15 minutes. In this and other retinas from doxycycline-induced Sod2flox/floxVMD2cre mice, we noted twisted major blood vessels, vascular beading, and an increase in branching and dilation of deeper blood vessels. Such changes were not observed at earlier time points (Supplementary Fig. S5).
Figure 8
Figure 8
Increased thickness of the RPE in doxycycline-induced Sod2flox/floxVMD2cre mice. Left: Light micrographs of the retina and RPE were prepared from glutaraldehyde/paraformaldehyde–fixed tissue, and the RPE thickness was measured at 10 evenly spaced locations measuring from the center of the optic nerve head. The RPE thickness was averaged for the inferior hemisphere, the superior hemisphere, and the overall RPE. For each sample, four mice were used. *P < 0.05 (9-month time point compared with 1-month and 4-months); #P < 0.05 (9-month time point with doxycycline compared with 9 months without doxycycline). Right: The average RPE thickness at each measured point and age is displayed as a “spidergram.” *P < 0.05 (9-month time point compared with 1 month and 4 months); **P < 0.01 (9-month time point relative to 1-month time point); #P < 0.05 (9 months with doxycycline compared with 9 months without doxycycline).
Figure 9
Figure 9
Ultrastructural evidence of the RPE and photoreceptor atrophy. Electron micrographs were prepared at low (top) and high (bottom) magnification (scale bars: 2 μm) from eyes of 4-month-old and 9-month-old Sod2flox/floxVMD2cre mice with or without doxycycline induction. (A, B) Even at 4 months, the photoreceptor outer segments appeared disordered in doxycycline-induced mice, and basal infoldings of the RPE were disordered. (C, D) By 9 months, disc membranes were more widely spaced, and fragmented mitochondria had accumulated along the basal surface of the RPE. (E, F), In some areas of the retina, membrane-filled vacuoles were apparent in the RPE, and inner and outer segments of the photoreceptors were truncated and disordered. (G, H) In Sod2flox/floxVMD2cre mice of the same age but not treated with doxycycline, Bruch's membrane retained its normal laminar appearance, basal infoldings were compact, elongated mitochondria and apical microvilli were visible, and photoreceptor disc membranes maintained their tight, parallel packing.
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