|Molecular Vision 2005;
Received 12 August 2004 | Accepted 16 May 2005 | Published 1 June 2005
Changes in the spatial expression of genes with aging in the mouse RPE/choroid
Tsukihiko Ogawa, Sharon A. Boylan, Sharon L. Oltjen,
Leonard M. Hjelmeland
Departments of Biological Chemistry and Ophthalmology, School of Medicine, University of California, Davis, CA
Correspondence to: Leonard M. Hjelmeland, Vitreoretinal Research Laboratory, School of Medicine, University of California, 1 Shields Avenue, Davis, CA, 95616-8794; Phone: (530) 752-2250; FAX: (530) 752-2270; email: email@example.com
Purpose: We recently used microarray and reverse transcriptase PCR (RT-PCR) analysis to show an upregulation of cathepsin S (CatS) and glutathione peroxidase 3 (GPX3) in the aging mouse RPE/choroid. To evaluate the mRNA distribution and levels in the RPE and choroid, in situ hybridizations were performed.
Methods: Eye sections from 2-month-old and 24-month-old C57BL/6 mice were probed for CatS or GPX3 mRNA by in situ hybridization. The ratio of mRNA labeled cells to total cells counted per section was compared between the two age groups for the RPE and choroid separately.
Results: The CatS labeled RPE cell ratio increased significantly with age. The GPX3 labeled RPE cell ratio did not increase with age.
Conclusions: The increases in mRNA levels for CatS and GPX3 found in the aging C57BL/6 RPE/choroid appear to represent an increase in both the numbers of cells expressing these messages and an increase in the level of expression in individual cells.
Aging is associated with various pathologies of the posterior pole, the most prominent and least treatable of which is age related macular degeneration (AMD). Several "normal" changes occur within the posterior pole of the mammalian eye and these changes have been well described [1-3]. More recently, efforts have focused on the changes in gene expression in the posterior pole as the mammalian eye ages . Our own laboratory has recently published a microarray study of the age related changes in gene expression in the C57BL/6J mouse RPE/choroid . In this survey of 2340 genes, we found 150 genes that exhibited altered gene expression after thorough statistical analysis of the results. Almost all of these changes were the result of upregulation in the 24 month old mouse.
Evaluations of total gene expression for given tissues do not provide the next level of insight, however, and it is necessary to follow microarray studies with a cellular based technique such as in situ hybridization. This type of analysis might reveal changes in gene expression in a number of different scenarios at the cellular level. First, the expression of a particular gene might change in selected cell types. For the RPE/choroid this would include the RPE cell, vascular cells, various white blood cells, and melanocytes. Second, the actual number of cells of one given cell type expressing this transcript might also change with age. We have observed this type of change with respect to the ratio of human RPE cells that express HO-1 .
From the 150 genes exhibiting age related expression levels in the mouse RPE/choroid, we chose two specific genes to investigate the age related changes in the pattern of gene expression. Our first choice for study was cathepsin S (CatS). This is a thiol protease which has been proposed to be important in the processing of shed photoreceptor outer segments . Recently, polymorphisms in the inhibitor for CatS (cystatin C) have been associated with the risk of developing both Alzheimer's disease and AMD [8,9]. The second gene we chose to study was glutathione peroxidase 3 (GPX3). This is the secreted or "plasma" form of glutathione peroxidase, and a genome scan performed by Weeks et al.  recently suggested GPX3 as a candidate gene for additional study.
To analyze the age related changes in the pattern of CatS and GPX3 gene expression in the mouse RPE/choroid, we utilized a cell counting method our laboratory previously presented in the literature [6,11]. Multiple animals at 2 months and 24 months of age were analyzed, and the results were tested for statistical significance.
We obtained 6-week-old C57BL/6J male mice from Jackson Labs (Bar Harbor, ME) and 24-month-old C57BL/6 male mice from the National Institute on Aging (Bethesda, MD). After their arrival, the mice were housed in cages with 12 h light-dark cycles for two weeks in order to let them adjust to their new environment. Animal care guidelines comparable to those published by the US Public Health Service (Public Health Service Policy on Humane Care and Use of Laboratory Animals) were followed. The C57BL/6 mice from NIA are derived from founder stocks from the Jackson Labs, and are considered genetically identical. The light history of the 24-month-old mice is identical to the light history of the 2-month-old mice. Because there is a continuum of aging and development in the mouse, we chose sexual maturity, or 2 months for our young mouse age. This is a standard age used in the literature, and one that we used in our previous publication . We performed a brief inspection of the mice for gross abnormalities but did not do a post-mortem examination of the animals for histopathology.
After cervical dislocation, three globes were obtained from at least two mice at the ages of 2 and 24 months. Globes were placed in cold phosphate buffered saline (PBS; pH 7.4) containing 4% paraformaldehye. In order to increase the permeability of the globes to paraformaldehye solution, corneas were removed from the globes with a circumferential incision using a stereo zoom microscope (Nikon SMZ800; Tokyo, Japan). The globes were then fixed overnight at 4 °C in phosphate buffered saline (PBS; pH 7.4) containing 4% paraformaldehye. The eye cups (without lens) including RPE and choroidal tissue were cryoprotected using the technique of Barthel and Raymond , and Mishima et al. . All tissue blocks were stored at -80 °C until used. Cryosections (10 μm) were cut with a cryotome (Leica CM3050, Leica Microsystems Inc., Bannockburn, IL), mounted on Vectabond coated glass slides (Vector Laboratories, Burlingame, CA), and air dried at room temperature for 4 h.
In situ hybridization
cDNA clones of CatS (GenBank accession number AI845967) and GPX3 (GenBank accession number AA960521) were purchased from Invitrogen (Carlsbad, CA). cDNA inserts were 1460 bp for CatS, and 1148 bp for GPX3. Antisense and sense digoxigenin (DIG) labeled RNA riboprobes were synthesized according to the manufacturer's protocol (Roche, Indianapolis, IN). The length and integrity of the synthesized ribopobes were analyzed by gel electrophoresis. The concentrations were estimated using a spot blot test and DIG labeled control RNA (Roche).
In situ hybridization was done according to Braissant and Wahli  with slight modifications. Hybridization and wash temperatures were optimized for each individual transcript. After post-fixation in 4% paraformaldehyde-PBS for 10 min at room temperature, sections were immersed in 0.25% acetic anhydride for 10 min and 5X saline sodium citrate (SSC) for 15 min. Prehybridization was carried out at 50 °C (for CatS) or 55 °C (for GPX3) for 2 h in hybridization mixture (50% formamide, 5X SSC, 40 μg/ml salmon sperm DNA). After denaturing the probes for 5 min at 80 °C, hybridization was carried out at 50 °C (for CatS) or 55 °C (for GPX3) for 40 h with a Parafilm cover (Parafilm; American Can Company, Greenwich, CT) in a chamber saturated with the hybridization mixture in a hybridization oven (Fisher Scientific, Los Angeles, CA). Sections were washed in 2X SSC at 55 °C (for CatS) or 60 °C (for GPX3) for 40 min, and then washed in 0.1X SSC at 55 °C (for CatS) or 60 °C (for GPX3) for 30 min. The sections were equilibrated in 100 mM Tris/150 mM NaCl/50 mM MgCl2, pH 7.5 for 5 min, and incubated with 0.5% DIG blocking reagent (Roche)/100 mM Tris/150 mM NaCl/50 mM MgCl2, pH 7.5 for 60 min. The sections were incubated with anti-DIG antibody conjugated to alkaline phosphatase (Roche) diluted 1:500 in 0.5% DIG blocking reagent (Roche)/100 mM Tris/150 mM NaCl/50 mM MgCl2, pH 7.5 at room temperature for 2 h. The sections were equilibrated in 100 mM Tris/100 mM NaCl/50 mM MgCl2, pH 9.5 for 5 min. Color was developed at room temperature with 0.045% nitroblue tetrazolium chloride (NBT; Roche)/0.0175% 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Roche)/100 mM Tris/100 mM NaCl/50 mM MgCl2, pH 9.5 overnight (for CatS) or 3 h (for GPX3). Development was stopped with TE buffer (pH 8.0) for 15 min. Nonspecific background staining was removed in 95% ETOH for 5 min (for CatS) or 60 min (for GPX3).
Because the mouse eyes are pigmented, after post-fixation in 4% paraformaldehyde-PBS at 4 °C overnight, the sections were bleached according to Mishima et al. , with minor adjustments to times and concentrations: Treatments with 0.1% (w/v) KMnO4 for 35 min and 0.5% (w/v) oxalic acid for 5 min were employed. These bleaching conditions retained normal morphology of the mouse eye as shown in the figures. Sections were counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA) for 2 min, dehydrated, and mounted.
Histochemistry and immunohistochemistry
Tissue was fixed in 4% paraformaldehyde-PBS at 4 °C overnight, fix was removed from tissues with PBS washes. Tissues were dehydrated with progressively higher concentrations of ethanol (50%, 75%, 95%, and 100%) followed by xylene. Tissues were then embedded in paraffin. All tissue blocks were stored at 4 °C until used. Sections (6 μm) were cut using a Leica RM2125RT microtome.
In order to visualize mast cells, sections were deparaffinized, bleached as described in the previous section, stained with a 1:1000 dilution of toluidine blue O (Sigma, St. Louis, MO) for 5 min, dehydrated, and mounted. Macrophages were visualized using a monoclonal antibody (Serotec, Oxford, UK) to the mouse F4/80 antigen, a 160 kDa glycoprotein expressed by murine macrophages. Sections were first deparaffinized, and digested with 1 mg/ml pepsin (Sigma) in 2.8% glacial acetic acid for 10 min at room temperature. Sections were washed three times for 3 min each in H2O, followed by PBS. After blocking for 1 h in 5% normal rabbit serum diluted in PBS, sections were incubated overnight at 4 °C with 10 μg/ml rat anti-F4/80 monoclonal antibody (Serotec). Negative controls were incubated in 10 μg/ml rat IgG2b (Serotec). Antibody was removed by three washes for 3 min each in PBS containing 0.1% Tween 20. Sections were incubated for 30 min at room temperature with 7.5 μg/ml biotinylated rabbit anti-rat IgG (mouse adsorbed; Vector Laboratories). Detection was performed using an ABC-AP staining kit (Vector Laboratories) and BCIP/NBT substrate (Vector Laboratories). Sections were bleached, and counterstained with Nuclear Fast Red (Vector Laboratories) for 10 min, dehydrated, and mounted.
Analysis and statistical treatment of data
Two to three sections from each eye were examined, and the best sample in terms of morphology was used for statistical analysis. The sections were observed under a light microscope (Olympus BH-2, Olympus Optical Co., LTD, Tokyo, Japan) with a charged coupled device camera (ProgRes 3012, Kontron Elektronik GmbH, Eching, Germany). Digitalized images were captured through the digital camera plug-in directly to graphic software (Photoshop 6.0, Adobe Systems Inc., Mountain View, CA). Photoshop was used to increase the resolution of the images and to assign colors in order to distinguish stained cells from the background. All cells on each section in the RPE or choroid that contain a nucleus stained by Nuclear Fast Red were counted for the total number of cells according to our previously published method . RPE or choroidal cells with both red stained nuclei and blue-purple stained cytoplasm were counted as labeled cells. For CatS, from 184 to 233 total RPE cells and 612 to 1131 total choroidal cells were counted in each section. For GPX3, between 134 and 195 RPE cells and 403 to 891 choroidal cells were counted in each section. The ratio of labeled cells to total cells counted per section was calculated. The data were analyzed using the StatView program version 5 (SAS Institute, Cary, NC). The ratios were averaged to obtain the mean and standard error of the mean (SEM) values. Statistical significance was determined using Student's t-test for the comparison of the ratios between sections from 2-month-old mice and 24-month-old mice. A p value <0.05 was considered significant.
In situ hybridization for CatS
The expression of mRNA for CatS appeared in a mosaic pattern in the RPE from both 2-month-old and 24-month-old C57BL/6 mice (Figure 1A,B). The cells could be classified as negative or positive and enumerated. The difference in the fraction of CatS positive RPE cells between 2-month-old and 24-month-old mice was statistically significant (p<0.01; Table 1). Compared to the RPE, fewer cells in the choroid showed expression of CatS mRNA either in 2 month old or in 24-month-old mice (Figure 1A,B). There was no clear difference in CatS positive cells between 2-month-old and 24-month-old mice in the choroid (p=0.33; Table 1). No labeling of cells was detected in RPE or choroid with the CatS sense probe (Figure 1C).
In situ hybridization for GPX3
The expression of GPX3 mRNA also appeared in a mosaic pattern for both 2-month-old and 24-month-old C57BL/6 mice (Figure 2A,B). In RPE, the increase of the ratio of GPX3 mRNA labeled cells to total cells with age showed borderline significance (p=0.05; Table 2). Few cells in the choroid showed expression of GPX3 mRNA either in 2-month-old or in 24-month-old mice (Figure 2A,B). In choroid, there was no apparent statistical difference between 2 month old mice and 24-month-old mice with respect to the ratio of labeled cells (p=0.14; Table 2). No labeled cells were detected in RPE or choroid with the GPX3 sense probe (Figure 2C).
Identification of macrophages and mast cells in the choroid
It was not possible to clearly identify the cells that were positively stained for either CatS or GPX3 in the choroid without additional study. First, we examined the distribution of macrophages using the F4/80 antigen as described in the literature. Clumps of large cells near the sclera were positively stained (Figure 3A). To identify mast cells, we stained tissue with toluidine blue. Results of this stain are shown in Figure 3B, in which Photoshop was used to enhance the image to help in the discrimination of cell types. The pattern of staining (red cells) appears to be different from the distribution of macrophages as illustrated in Figure 3A.
In this study, we investigated mosaic expression patterns for CatS and GPX3 mRNAs in the C57BL/6 RPE/choroid. Cellular mosaicism is defined as a variable pattern of genotype, epigenotype, or phenotype among a population of cells either in vivo or in vitro. These types of mosaicism can arise during development or with ageing . For our studies, mosaicism refers to the variable gene expression as revealed by in situ hybridization or the variable protein expression as revealed by immunohistochemistry within the RPE monolayer. The mechanisms giving rise to the mosaicism we have observed are unknown.
We demonstrated a significant increase with age in the numbers of cells in the RPE labeled for CatS mRNA. The labeling index for the choroid with respect to CatS expression did not show a statistically significant age related change. We also found no statistically significant age related change in the labeling index for GPX3 either in the RPE or in the choroid. We have previously shown however, by semi-quantitative competitive PCR, that CatS and GPX3 mRNA levels increase with age in the RPE/choroid of the C57BL/6 mouse  (Table 3). The data from this study show that total changes of mRNA expression in the RPE/choroid can be accounted for by either an increase in the number of cells expressing the message or an increase in the level of mRNA expression within a set of cells that does not change in number. The data for CatS expression in the RPE, for example, clearly shows a significant change in the ratio of labeled cells in the RPE as a function of age. This can be contrasted with the data for GPX3 that do not show a statistically different change in the number of cells labeled as a function of age for either the RPE or the choroid. Changes in overall expression of GPX3 in this tissue are likely to represent changes in the level of expression among individual cells.
Using a labeling index method to quantify age related changes in mRNA or protein expression should be interpreted with caution. We have used this method in previous studies [6,11], and it is clear that a number of alternatives may account for the data. First, it is essential to differentiate mRNA from levels of protein or activity as the relationships among these parameters may vary with respect to each gene studied. Second, the binary approach to counting labeled cells does not account for much of the change in labeling of individual cells, and thus undoubtedly is a simplification of the true values for mRNA expression within all individual cells.
CatS is expressed in the RPE [7,16,17]. CatS has been proposed to be the major cysteine protease participating in the lysosomal processing of rod outer segments in RPE . Cystatin C is the major inhibitor of CatS  and functional polymorphisms in this gene are associated with the risk of developing AMD and Alzheimer's disease [8,9].
Oxidative stress has been hypothesized to be important in the development of AMD. Levels of GPX3 in the blood show a significant increase in late AMD patients, presumably due to oxidative stress . One of the AMD susceptibility loci, mapped to a location near GPX3 (chromosome 5q32) .
As in our previous study of insulin-like growth factor binding protein 2 , heme oxygenase-1, and catalase , the expressions of CatS and GPX3 mRNAs were shown to exhibit a mosaic pattern in the RPE. Our finding from RPE65 in situ hybridization, however, did not show a mosaic pattern (data not shown). Judging from the pattern of staining of RPE65, we believe that the mosaic expression patterns of CatS and GPX3 are not artifacts, but may represent true functional heterogeneity with respect to gene expression. Similar to our study, Burke et al.  reported a mosaic of functionally heterogeneous RPE cells in vivo and in cell culture. They found several morphological and biochemical properties that were heterogenous.
Our studies do not clearly identify the cells staining either for CatS or GPX3 in the choroid. We utilized approaches suggested by McMenamin . Clumps of large cells near the choroid stained for both CatS and F4/80 suggesting but not proving that these are the large resident macrophages of the choroid referred to as "clump cells" in human tissue specimens. Cells stained for GPX3, however, were not convincingly found in the same distribution as the resident macrophage cells stained by the toluidine blue procedure. McMenamin has commented extensively on the nature of these types of cells in the rodent uveal track .
In conclusion, the ratio of cells labeled for CatS mRNA showed a significant increase with age in the mouse RPE. However, this was not observed for GPX3. How the changes of expression of these two genes are related to the aging phenotype of the RPE/choroid, or whether these changes are relevant to the development of AMD are not known. These studies would be interesting to pursue in relation to the development of our understanding of the cellular events leading to this disease.
This research was supported by NIH grant R01 EY06473 (LMH), Foundation for Fighting Blindness Grant (LMH), an unrestricted grant from Research to Prevent Blindness (Department of Ophthalmology, University of California, Davis, CA), and NEI Core Grant P30EY12576.
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