Progressive Age-Related Changes Similar to Age-Related Macular Degeneration in a Transgenic Mouse Model
2002; Elsevier BV; Volume: 161; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64427-6
ISSN1525-2191
AutoresPiroska E. Rakoczy, Dan Zhang, Terry Robertson, Nigel L. Barnett, J. M. Papadimitriou, Ian J. Constable, Chooi‐May Lai,
Tópico(s)Glaucoma and retinal disorders
ResumoAge-related macular degeneration (AMD) is the major cause of blindness in the developed world. Its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atrophy; a transgenic mouse line (mcd/mcd) expressing a mutated form of cathepsin D that is enzymatically inactive thus impairing processing of phagocytosed photoreceptor outer segments in the retinal pigment epithelial (RPE) cells. Pigmentary changes indicating RPE cell atrophy and a decreased response to flash electroretinograms were observed in 11- to 12-month-old mcd/mcd mice. Histological studies showed RPE cell proliferation, photoreceptor degeneration, shortening of photoreceptor outer segments, and accumulation of immunoreactive photoreceptor breakdown products in the RPE cells. An accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice. Transmission electron microscopy demonstrated presence of basal laminar and linear deposits that are considered to be the hallmarks of AMD. Small hard drusen associated with human age-related maculopathy were absent in the mcd/mcd mouse model at the ages analyzed. In summary, this model presents several features of AMD, thus providing a valuable tool for investigating the underlying biological processes and pathomechanism of AMD. Age-related macular degeneration (AMD) is the major cause of blindness in the developed world. Its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atrophy; a transgenic mouse line (mcd/mcd) expressing a mutated form of cathepsin D that is enzymatically inactive thus impairing processing of phagocytosed photoreceptor outer segments in the retinal pigment epithelial (RPE) cells. Pigmentary changes indicating RPE cell atrophy and a decreased response to flash electroretinograms were observed in 11- to 12-month-old mcd/mcd mice. Histological studies showed RPE cell proliferation, photoreceptor degeneration, shortening of photoreceptor outer segments, and accumulation of immunoreactive photoreceptor breakdown products in the RPE cells. An accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice. Transmission electron microscopy demonstrated presence of basal laminar and linear deposits that are considered to be the hallmarks of AMD. Small hard drusen associated with human age-related maculopathy were absent in the mcd/mcd mouse model at the ages analyzed. In summary, this model presents several features of AMD, thus providing a valuable tool for investigating the underlying biological processes and pathomechanism of AMD. Age-related macular degeneration (AMD), a disease of the area centralis or macula, is the leading cause of blindness in the elderly population of the developed world.1Mitchell P Smith W Attebo K Wang JJ Prevalence of age-related maculopathy in Australia. 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5: 27PubMed Google Scholar, 16Bressler NM Bressler SB West SK Fine SL Taylor HR The grading and prevalence of macular degeneration in Chesapeake Bay watermen.Arch Ophthalmol. 1989; 107: 847-852Crossref PubMed Scopus (223) Google Scholar Accumulation of abnormal POS breakdown product in the RPE cells can be the result of a variety of impairments, ie, 1) RPE cell lysosomal abnormalities including loss or decrease of enzymatic activity,17Hayasaka S Lysosomal enzymes in ocular tissues and diseases.Surv Ophthalmol. 1983; 27: 245-258Abstract Full Text PDF PubMed Scopus (55) Google Scholar 2) changes in or related to POS structure,18Cremers FP van de Pol DJ van Driel M den Hollander AI van Haren FJ Knoers NV Tijmes N Bergen AA Rohrschneider K Blankenagel A Pinckers AJ Deutman AF Hoyng CB Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR.Hum Mol Genet. 1998; 7: 355-362Crossref PubMed Scopus (471) Google Scholar or 3) obstruction of exocytosis because of changes in Bruch's membrane (BM).19Starita C Hussain AA Pagliarini S Marshall J Hydrodynamics of ageing Bruch's membrane: implications for macular disease.Exp Eye Res. 1996; 62: 565-572Crossref PubMed Scopus (139) Google Scholar The exact role of different types of debris in AMD development is unknown but they seem to leadto a common pathway—apoptotic photoreceptor cell death.20Xu GZ Li WW Tso MO Apoptosis in human retinal degenerations.Trans Am Ophthalmol Soc. 1996; 94: 411-430PubMed Google Scholar In developing an animal model that might reproduce at least some features of AMD, the modulation of RPE cell lysosomal enzyme activity is an attractive approach. RPE cells have been shown to express strikingly high levels of cathepsin D (CatD),21Zimmerman WF Godchaux III, W Belkin M The relative proportions of lysosomal enzyme activities in bovine retinal pigment epithelium.Exp Eye Res. 1983; 36: 151-158Crossref PubMed Scopus (44) Google Scholar, 22Rakoczy PE Sarks SH Daw N Constable IJ Distribution of cathepsin D in human eyes with or without age-related maculopathy.Exp Eye Res. 1999; 69: 367-374Crossref PubMed Scopus (38) Google Scholar suggesting an important role for CatD in POS digestion. Inactive proCatD accumulation seems to be an abnormal condition and has been associated with breast and ovarian cancers,23Bazzett LB Watkins CS Gercel-Taylor C Taylor DD Modulation of proliferation and chemosensitivity by procathepsin D and its peptides in ovarian cancer.Gynecol Oncol. 1999; 74: 181-187Abstract Full Text PDF PubMed Scopus (25) Google Scholar24Couissi D Dubois V Remacle C Schonne E Trouet A Western immunoblotting and enzymatic activity analysis of cathepsin D in human breast cancer cell lines of different invasive potential. Regulation by 17beta-estradiol, tamoxifen and ICI 182,780.Clin Exp Metastasis. 1997; 15: 349-360Crossref PubMed Scopus (20) Google Scholar and an age-related increase in the amount of proCatD has been demonstrated in several organs.25Rakoczy PE Baines M Kennedy CJ Constable IJ Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.Exp Eye Res. 1996; 63: 159-167Crossref PubMed Scopus (40) Google Scholar, 26Wiederanders B Oelke B Accumulation of inactive cathepsin D in old rats.Mech Ageing Dev. 1984; 24: 265-271Crossref PubMed Scopus (39) Google Scholar, 27Wilcox DK Vectorial accumulation of cathepsin D in retinal pigmented epithelium: effects of age.Invest Ophthalmol Vis Sci. 1988; 29: 1205-1212PubMed Google Scholar Recently, we demonstrated in vitro and in vivo that presence of proCatD impairs POS proteolysis, resulting in POS-derived breakdown product accumulation in the RPE cells of heterozygous transgenic mice (mcd) that express a form of CatD lacking the Glu44p and Gly1 cleavage site.28Zhang D Lai C-M Constable IJ Rakoczy PE A model for a blinding eye disease of the aged.Biogerontology. 2002; 3: 61-66Crossref PubMed Scopus (14) Google Scholar We hypothesize that any change, genetic or environmental, that might lead to an accelerated POS breakdown product accumulation in the RPE layer will compromise RPE cell function, initiating changes similar to those observed in AMD. To test this hypothesis we produced a homozygous (mcd/mcd) transgenic mouse line and demonstrated that progressive POS-derived breakdown product accumulation could induce hypo- and hyperpigmentary changes in the fundus, the development of basal laminar and linear deposits and photoreceptor and RPE cell proliferation/loss in an age-dependent manner. Animal experimentation was performed in compliance with the Association for Research in Vision and Opthmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The 3.2-kb fragment containing the mutated CatD (CatDm1) gene with a two-amino acid deletion (Glu44p-Gly1) corresponding to the first cleavage site, and driven by the human cytomegalovirus promoter was released from pMCD. After two rounds of gel purification, the DNA fragment was microinjected into fertilized single-cell stage C57BL/6 embryos. The resultant offspring were screened for the presence of CatDm1 by polymerase chain reaction (PCR) and Southern blot analysis of mouse-tail DNA. First generation mcd mice28Zhang D Lai C-M Constable IJ Rakoczy PE A model for a blinding eye disease of the aged.Biogerontology. 2002; 3: 61-66Crossref PubMed Scopus (14) Google Scholar from the same founder were mated and the resultant mcd/mcd mice were bred to establish a stable line. Mcd/mcd and age-matched control C57BL/6 mice (6 to 18 months old) were used for subsequent characterization. All animals were maintained in cages at a constant temperature of 22°C and with a 12:12 hour light/dark cycle (light on at 0800 hours). Food and water were available ad libitum. Mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg body weight) and xylazine (8 mg/kg body weight) before pupil dilation with a drop of 1.0% mydriacyl and 2.5% phenylephrine hydrochloride. Fundus photography of mice was performed using a small animal fundus camera (Kowa Genesis, Tokyo, Japan). Ten-month-old mcd/mcd and C57BL/6 mouse eyes were assessed using electroretinography as previously described,29Shen WY Lai MC Beilby J Barnett NL Liu J Constable IJ Rakoczy PE Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina.Arch Ophthalmol. 2001; 119: 1033-1043Crossref PubMed Scopus (20) Google Scholar but using flash stimulus at 0.25 Hz and four consecutive responses. Stimulus-response characteristics were generated by attenuating the flash intensity with neutral density filters. The a-wave amplitude was measured from the baseline to the trough of the a-wave response and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Data were expressed as the mean wave amplitude ±SEM (μV). Two-factor repeated measures analysis of variance was performed to compare the responses from retinas of mcd/mcd mice (n = 4) with those of control mice (n = 4) over the flash stimulus range. The b-wave data were fitted with a Naka-Rushton equation [R/Rmax = I/(I + K)] using SigmaPlot (SPSS, Chicago, IL.) to determine Rmax (maximum amplitude) and K (semisaturation constant) from the response amplitude (R) and the flash intensity (I) over the range of −6 to −1.2 log neutral density units. CatDm1 expression in the transgenic mice was detected by RT-PCR. Total retinal RNA from C57BL/6, mcd, and mcd/mcd were isolated using Trizol Reagent (Life Technologies, Inc., Gaithersburg, MD). A primer-specific one-step RT-PCR was performed with Qiagen One-Step RT-PCR Kit (Qiagen, Hilden, Germany) using 1 μg of total RNA plus a pair of CatDm1-specific primers, 5′-ATGCAGCCCTCCAGCCTTCT-3′ and 5′-TACTTGTGGTGGATCCAGCA-3′. A mouse hypoxanthine phosphoribosyl-transferase (HPRT) primer pair, 5′-CACAGGACTAGAACACCTGC-3′ and 5′-GCTGGTGAAAAGGACCTCT-3′, was used as positive control for quality of the RT and the amount of cDNA added to each PCR reaction. The cycles used were: 1 cycle at 50°C for 30 minutes and 95°C for 15 minutes; followed by 35 cycles of 94°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute. Western blot analysis was performed as previously described.22Rakoczy PE Sarks SH Daw N Constable IJ Distribution of cathepsin D in human eyes with or without age-related maculopathy.Exp Eye Res. 1999; 69: 367-374Crossref PubMed Scopus (38) Google Scholar Briefly, the mouse retina was homogenized in phosphate-buffered saline and loading buffer before being used for polyacrylamide gel electrophoresis. A mouse anti-human CatD mAb (Calbiochem, San Diego, CA), diluted 1/200, was used as the primary antibody and a goat anti-mouse IgG-horseradish peroxidase (Amersham, Uppsala, Sweden), diluted 1/2000, as the secondary antibody. The blots were developed with the ECL Western blotting analysis system (Amersham). Enucleated mouse eyes were fixed in 4% paraformaldehyde (pH 7.4) for 4 hours and embedded in paraffin. Five-μm-thick sections of eyes from 11- to 14-month-old mcd/mcd and C57BL/6 mice were stained with hematoxylin and eosin (H&E) for light microscopy. Some eyes were embedded in OCT compound for frozen section preparation for fluorescence microscopy. For immunohistochemistry, sections were deparaffinized, rehydrated, and bleached to remove melanin by incubation of sections in 0.25% potassium permanganate for 20 minutes and in 1% oxalic acid for 5 minutes before incubation with rabbit anti-bovine rod outer segment (ROS) polyclonal IgG, rabbit anti-bovine CatD polyclonal IgG, mouse anti-human CatD mAb (Calbiochem) or control nonimmune serum (rabbit or mouse) diluted 1/200. Sections were incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase (Life Technologies, Inc.) or goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma, St. Louis, MO) diluted 1/100, followed by alkaline phosphatase substrate (Fast Red, Sigma). Sections were counterstained with Mayer's hematoxylin for light microscopy. Eyes from C57BL/6 (n = 4) and mcd/mcd (n = 4) mice were fixed in 10% neutral buffered formalin for 4 hours for paraffin embedding, followed by section preparation. The TUNEL technique was performed using ApopTag Plus Peroxidase in Situ Apoptosis Detection Kit (Intergen Discovery Products, Purchase, NY) according to the manufacturer's protocol. From each control C57BL/6 and mcd/mcd retina, five 60-μm-long sections of the outer nuclear layer (ONL) were randomly selected under a light microscope (Leica DM RBE, Solms, Germany) using a ×100 objective lens and the images captured using an attached video camera (Olympus DP-10, Tokyo, Japan) and digitized. The total number of nuclei and total number of TUNEL-positive nuclei was counted using the Scion Image package (Scion Corp., Frederick, MA). Enucleated eyes from 12- to 18-month-old mcd/mcd and C57BL/6 mice were fixed in 2.5% glutaraldehyde in 0.05 mol/L of cacodylate buffer (pH 7.4). The eyes were then trimmed into 1-mm3 blocks and re-immersed into a fresh fixative for a further 24 hours. After postfixing in 1% osmium tetroxide, the tissues were processed for transmission electron microscopy by conventional methods and embedded in Araldite. Semithin sections (1 μm thick) were stained with 0.5% toluidine blue in 5% borax and examined with a light microscope. After selecting the areas of interest, the blocks were trimmed under a dissecting microscope and ultra-thin sections (70 nm thick) prepared on an ultramicrotome (LKB Nova, Sweden), stained with Reynolds lead citrate and examined in a Philips 410LS Transmission Electron Microscope at an accelerating voltage of 80 kV. Five transgenic founders, designated D2, D8, D28, D29, and D30, were generated and identified by PCR and Southern blot analysis from a total of 47 pups produced from microinjected eggs. D8, however, did not contain the cytomegalovirus promoter region, and D28, D29, and D30 failed to produce a positive transgenic offspring. Hence, only D2 was used to established stable mcd and mcd/mcd transgenic lines and only mcd/mcd mice were used in the subsequent characterization.28Zhang D Lai C-M Constable IJ Rakoczy PE A model for a blinding eye disease of the aged.Biogerontology. 2002; 3: 61-66Crossref PubMed Scopus (14) Google Scholar The level of CatDm1 expression in the retina was assessed by RT-PCR, immunohistochemistry, and Western blot analysis. From RT-PCR analysis using human CatDm1 transgene-specific primers, a 379-bp cDNA fragment of the human CatD was amplified in mcd/mcd mice, but not in age-matched C57BL/6 mice (Figure 1A). The 260-bp HPRT bands had similar intensities, demonstrating equal loading of sample. As expected, the mRNA level of CatDm1 in mcd/mcd mice was higher than in mcd mice and no band was detected in the C57BL/6 mice. Western blot analysis demonstrated the presence of human proCatD, appearing at 52 kd, in mcd and mcd/mcd mice (Figure 1B, lanes 2 and 3). In addition, bands probably representing the human proCatD-opsin-like complex appeared at 75 kd in mcd and mcd/mcd mice. There were no human proCatD-immunoreactive bands present in the control C57BL/6 mice (Figure 1B, lane 1) demonstrating the lack of human CatD expression in these animals. Transgene expression was also confirmed by immunohistochemistry. Using the rabbit anti-bovine CatD polyclonal antibody, CatD-positive immunostaining was detected in the RPE cell layer of mcd/mcd and C57BL/6 mice (data not shown). However, using a mouse anti-human CatD mAb that was specific to human CatD, CatD-positive immunostaining was not observed in the C57BL/6 (Figure 1C) but was detected in mcd/mcd mice where it was localized to the cytoplasm of the RPE cells (Figure 1C). Fundus photography was performed on mcd/mcd (n = 17) and C57BL/6 (n = 17) mice every 1 to 2 months from the age of 6 months. In C57BL/6 mice, the fundus appeared normal and no changes were detected with progress in age (data not shown). In mcd/mcd mice, the fundus appeared normal up to 9 months (Figure 2A) after which light, pinkish-yellowish patches (Figure 2B), suggestive of retinal atrophy (Figure 4, C and D), were detected in 16 (94%) of the mcd/mcd mice. A typical atrophy emerged at the peripheral part of the retina. The size of these patches increased with age, reaching a maximum size covering between 3 to 10% of the fundus at 10 to 12 months and remaining stable thereafter (Figure 2C, Table 1). In addition to these patches, multiple small yellowish spots (probably pigmentary disturbance) in the periphery and at the posterior pole were also observed and their numbers increased with age around 18 months. In three 18-month-old mice, these small yellowish spots were scattered throughout the retina (Figure 2C, Table 1). At the same time, signs of hyperpigmentary changes were also noted (Figure 2, B and C).Figure 4Light microscopy of 12-month-old mcd/mcd (A–C and E–H) and C57BL/6 (A–C and E–H) mouse and an 18-month-old mcd/mcd (D) retinae. The RPE layer in each figure is indicated by a black or white arrow. A: Morphology of the normal region of an mcd/mcd mouse retina. B: Morphology of mcd/mcd mouse retina showing abnormal layers of pigmented cells (arrowheads). C: Abnormal hyper- and hypopigmented RPE cells (arrowheads) in mcd/mcd mouse retina and thinning of photoreceptor layer above the abnormal RPE cells. D: Toluidine blue-stained resin section showing abnormal RPE cells (arrowheads) and thinned photoreceptor cell layers (note: the area centralis is on the left side of the micrograph) (original magnification, ×200). E: ROS-immunohistochemistry of C57BL/6 mouse retina. F: ROS-immunohistochemistry of mcd/mcd mouse retina (note the shortening of the ROS and ROS-immunoreactivity localized in the RPE cells). Width of ROS is indicated by double-headed white arrows. G: Fluorescent microscopy of retinas of C57BL/6. H: mcd/mcd mouse. Note the higher fluorescent signal in the RPE cells and in the clumped cells (white arrow) (original magnification, ×400).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Summary of Ophthalmic, Histological, and Immunohistochemical Analysis of mcd/mcd MiceExaminationAge (months)Observation (no. of eyes)Fundus photographymcd/mcd 6HypopigmentationHyperpigmentationYellow Dots0/340/340/3410/1232/3432/340/34186/66/64/6Histologymcd/mcd 6RPE proliferationRPE clumpingONL thinning0/80/80/810/1210/107/104/10184/44/44/4IHC (anti-BROS Ab)mcd/mcd 12Strong staining in RPEPOS shortening3/33/3RPE; retinal pigment epithelium; ONL, outer nuclear layer; IHC, immunihistochemistry; anti-BROS Ab, bovine rod outer segment antibody; POS, photoreceptor outer segment. Open table in a new tab RPE; retinal pigment epithelium; ONL, outer nuclear layer; IHC, immunihistochemistry; anti-BROS Ab, bovine rod outer segment antibody; POS, photoreceptor outer segment. As a consequence of the geographic distribution of photoreceptor degeneration and the constant rod/cone ratio throughout the mcd/mcd mouse retinas, retinal function/dysfunction could be assessed easily by full-field electroretinography in this transgenic mouse model. Typical flash ERGs recorded from four dark-adapted 12-month-old mcd/mcd and four C57BL/6 eyes are shown in Figure 3A. The negative a-wave was generated by hyperpolarization of the photoreceptors and the positive b-wave resulted from depolarization of outer nuclear bipolar cells. The waveform of the experimental response was similar to that of C57BL/6 mice but the amplitude of the response was much smaller. A two-way repeated measures analysis of variance of the stimulus-response characteristics demonstrated a significant (P < 0.01) difference in the a-wave amplitude between the C57BL/6 and mcd/mcd mice. Post hoc Bonferroni tests revealed a significant (P < 0.001, n = 4) suppression of the a-wave response at all flash intensities greater than −1.2 log neutral density units (Figure 3B). Figure 3C shows the significant difference (P < 0.01) between the stimulus intensity b-wave response characteristics of the C57BL/6 and mcd/mcd mice. The b-wave amplitude was significantly attenuated (P < 0.002, n = 4) in mcd/mcd mice at all flash intensities greater than −3 log neutral density units. A Naka-Rushton equation was fitted to the b-wave stimulus response curves (Figure 3C, inset) for flash intensities up to −1.2 log neutral density units.30Ren JC LaVail MM Peachey NS Retinal degeneration in the nervous mutant mouse. III. Electrophysiological studies of the visual pathway.Exp Eye Res. 2000; 70: 467-473Crossref PubMed Scopus (26) Google Scholar The Rmax value (±SEM) of 370 ± 29 μV for mcd/mcd mice was significantly lower than the C57BL/6 mice value of 740 ± 73 μV (P < 0.005). Similarly, a significant reduction in retinal sensitivity of 0.85 log neutral density units was seen in mcd/mcd mice. The calculated values of log K were −3.30 ± 0.08 (C57BL/6, n = 4) and −2.45 ± 0.23 neutral density units (mcd/mcd, P < 0.05, n = 4). The kinetic properties of the maximum intensity, dark-adapted a-waves recorded from four control and four mcd/mcd eyes were analyzed to assess rod phototransduction. The leading edges of the a-waves were normalized to the deepest a-wave trough31Pardue MT McCall MA LaVail MM Gregg RG Peachey NS A naturally occurring mouse model of X-linked congenital stationary night blindness.Invest Ophthalmol Vis Sci. 1998; 39: 2443-2449PubMed Google Scholar and the averaged responses plotted in Figure 3D. The averaged response recorded from mcd/mcd mice overlaps the averaged response recorded in control mice. Light microscopy of H&E-stained paraffin sections and toluidine blue-stained resin sections from 10- to 18-month-old mcd/mcd mice demonstrated that while the RPE cell layer appeared normal and continuous throughout the retina of C57BL/6 mice (data not shown) and most parts of the retina in mcd/mcd mice (Figure 4A), areas of atrophy and hypertrophy were evident in mcd/mcd mice (Table 1). These include clumps of pigmented cells in the subretinal space (Figure 4B, arrowheads) in the central or peripheral retina. The putative RPE cells beneath these pigmented cells were attenuated, enlarged, and sometimes less pigmented (Figure 4, B and C). Obvious changes in the peripheral and central parts of the retina (Figure 4, C and D, respectively) were present. In some eyes, there were also regions where RPE cells were disorganized, swollen (Figure 4, C and D), clumped (Figure 4C), atrophic, or absent. The ONLs in these regions were also significantly thinner or sometimes missing (Figure 4D) with large stunted inner segments. In some parts, the outer segments were shortened or missing (Figure 4; C, D, and F). Progressive degeneration of photoreceptors, thus sometimes aggressive thinning of the ONL became visible by 12 months with subsequent disappearance of the ONL by 18 months. In these animals some disorganization in the inner nuclear layers accompanied the disappearance of the ONL (Figure 4D). However at 18 months of age the ganglion cell layer remained normal (data not shown). ROS immunohistochemistry was performed to determine whether or not there was any abnormal ROS-immunoreactive breakdown product accumulation in the RPE cell layer of mcd/mcd mice. In C57BL/6 mice, ROS-positive immunostaining was localized predominantly to the ROS with weak immunostaining in the RPE (Figure 4E). In mcd/mcd mice, the ROS were shortened (Figure 4, E and F, double-headed arrow) but they continued to demonstrate ROS-positive immunostaining (Figure 4F). In addition, strong ROS-positive immunostaining was also detected in the cytoplasm of the RPE cells (Figure 4F, arrow). Fluorescence microscopy of frozen sections showed autofluorescent deposits resembling lipofuscin in the C57BL/6 and mcd/mcd mouse eyes (Figure 4, G and H). Some increase in autofluorescent signal was detected in RPE cell layers of mcd/mcd mice (Figure 4H, white arrow) and the abnormal regions containing clumped, enlarged RPE cells were also autofluorescen
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