June 2001
Volume 42, Issue 7
Free
Retinal Cell Biology  |   June 2001
Age and Topographic Variation of Insulin-like Growth Factor–Binding Protein 2 in the Human RPE
Author Affiliations
  • Noritake Miyamura
    From the Departments of Ophthalmology and
  • Kazuaki Mishima
    From the Departments of Ophthalmology and
  • Shigeru Honda
    From the Departments of Ophthalmology and
  • Amy E. Aotaki-Keen
    From the Departments of Ophthalmology and
    Molecular and Cellular Biology, University of California, Davis.
  • Lawrence S. Morse
    From the Departments of Ophthalmology and
  • James T. Handa
    From the Departments of Ophthalmology and
  • Leonard M. Hjelmeland
    From the Departments of Ophthalmology and
    Molecular and Cellular Biology, University of California, Davis.
Investigative Ophthalmology & Visual Science June 2001, Vol.42, 1626-1630. doi:
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      Noritake Miyamura, Kazuaki Mishima, Shigeru Honda, Amy E. Aotaki-Keen, Lawrence S. Morse, James T. Handa, Leonard M. Hjelmeland; Age and Topographic Variation of Insulin-like Growth Factor–Binding Protein 2 in the Human RPE. Invest. Ophthalmol. Vis. Sci. 2001;42(7):1626-1630.

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

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Abstract

purpose. Previous studies have shown that insulin-like growth factor–binding protein (IGFBP)-2 is markedly upregulated in senescent RPE cells in vitro, and might therefore be a marker of senescent cells in vivo. This study was conducted to determine whether IGFBP-2 expression in human RPE cells from the macula and periphery varies with age in vivo.

methods. Paraformaldehyde (4%)-fixed and optimal cutting temperature (OCT) compound–embedded human eyes from 17 patients were cryosectioned and subjected to high-sensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization to determine the expression of IGFBP-2. Complementary immunohistochemistry experiments using a polyclonal anti-IGFBP-2 antibody were performed to confirm IGFBP-2 protein expression. Specimens were examined by light microscopy, and images were captured with a digital camera. The total numbers of RPE cells and IGFBP-2 mRNA expression–positive RPE cells were counted for each section, and the ratio of labeled RPE cells to total RPE cells counted was calculated for both macular and peripheral regions of each donor.

results. IGFBP-2 mRNA expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner segments of photoreceptor cells in all 17 eyes. In 16 of 17 eyes, IGFBP-2 mRNA expression was detected in the RPE. In 11, the ratio of labeled cells to total RPE cells counted per section in the macula was 1.2 times greater than the ratio in the periphery (P = 0.008). The ratio of labeled RPE cells in the macula decreased with age (P = 0.0064). Immunohistochemistry studies for IGFBP-2 confirmed the expression pattern found by in situ hybridization.

conclusions. There is a topographical and age-related change in IGFBP-2 expression in RPE cells from human donor eyes. This distribution is likely not to represent senescent RPE cells in vivo.

Age-related macular degeneration (ARMD), a leading cause of blindness in western societies, is a progressive degeneration of the photoreceptors and their underlying retinal pigment epithelium (RPE) in the macular region of the retina. Although influences from genetic to environmental have been implicated in the pathogenesis of ARMD, chronological age is the primary determinant of disease onset. A series of studies have suggested that RPE cells have a phenotype that varies as a function of topography and/or age. For example, Burke and Soref 1 showed that RPE cells from the macula of the human eye or the area centralis of the bovine eye have more limited replicative life-spans in vitro than do cells from more peripheral regions of the posterior pole. Flood et al. 2 observed that the growth rate and numbers of nondividing cells in primary cultures of human RPE were directly correlated with the chronological age of the donor eye. 
Our laboratory has recently investigated the phenomenon of senescence in RPE cells as it might pertain to the pathogenesis of ARMD. 3 4 We first demonstrated that the combined senescence-associated β-galactosidase (SABG)/bromo-deoxyuridine incorporation assay identified senescent RPE cells in vitro. 5 Mishima et al., 6 adapting this same method for use in the posterior pole of the primate eye, showed selective SABG labeling in a group of RPE cells in a 29-year-old, but not a 2-year-old, rhesus macaque eye. Because measurement of SABG activity is dependent on the postmortem and fixation times, this technique is unfortunately not feasible in human donor eyes. Recently, we found that IGFBP-2 mRNA expression is dramatically upregulated in human senescent RPE cells in vitro. 7 Similarly, Shelton et al. 8 reported marked upregulation of IGFBP-2 in senescent RPE 340 cells by microarray analysis. In our attempt to identify a marker of senescence in vivo, we hypothesized that IGFBP-2 is preferentially upregulated in macular RPE cells with age in vivo. Herein, we report topographic differences of IGFBP-2 expression in RPE cells as well as an age-related decline in the macula from human donor eyes. 
Methods
Tissue Processing
Fresh globes from 10 male and 7 female donors, ranging in age from 27 to 83 years at the time of death, were obtained from the Sierra Tissue Eye Bank (Sacramento, CA) within 21 hours of death. Table 1 outlines the age, sex, postmortem time, and systemic and ophthalmic disease histories of each donor. Inspection with a dissecting microscope did not reveal any obvious posterior segment disease. The globes were fixed in phosphate-buffered saline (PBS; pH 7.4) containing 4% paraformaldehyde at 4°C. Using microscopic guidance, the anterior segment was removed, and the posterior segment was divided into five sections (approximately 6 × 6 mm): nasal (nasal side of disc), superior (superior to arcade vessels), inferior (inferior to arcade vessels), macular (centered around the foveola), and temporal region (temporally outside of macula to equator). The tissue was cryoprotected using the technique of Barthel and Raymond 9 and Mishima et al. 6 All tissue blocks were stored at −80°C until used. Cryosections (10 μm) were cut with a cryotome (Bright Instrument Co., Huntingdon, UK), mounted on coated glass slides (Vectabond; Vector Laboratories, Burlingame, CA), and air dried at room temperature for 4 hours. 
In Situ Hybridization
The cDNA containing plasmids for human IGFBP-2 and human opsin were kindly provided by Shunichi Shimasaki and Jeremy Nathans, respectively. The IGFBP-2 cDNA was 446 bp and inserted into a cloning vector (pBluescript II SK(+); Stratagene, La Jolla, CA). Opsin cDNA was inserted into the cloning vector (pBluescript II SK(+); Stratagene) after digestion with EcoRI (Roche, Indianapolis, IN) and ApaI (Roche) to 450 bp. The antisense and sense digoxigenin (DIG)-labeled RNA riboprobes were synthesized according to the labeling kit protocol (Roche). The length and integrity of the synthesized riboprobes were quantified by gel electrophoresis. The concentrations were estimated by the dot-spot test using DIG-labeled control RNA. 
In situ hybridization histochemistry was performed according to Braissant and Wahli, 10 with slight modifications. After postfixation in 4% paraformaldehyde-PBS for 10 minutes, sections were immersed in 0.25% acetic anhydride for 10 minutes and 5× SSC for 15 minutes. Prehybridization was performed at 57°C for 2 hours in the hybridization mixture (50% formamide, 5× SSC, and 40 μg/ml salmon sperm DNA). After denaturing the probes for 5 minutes at 80°C, hybridization was performed at 57°C for 40 hours with a 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 and equilibrated in buffer 1 (100 mM Tris, 150 mM NaCl, and 50 mM MgCl2 [pH 7.5]) for 5 minutes and incubated in alkaline phosphatase-coupled anti-DIG antibody (Roche) diluted 1:5000 in buffer 2 (buffer 1 with 0.5% DIG blocking reagent added) at room temperature for 2 hours. The sections were equilibrated in buffer 3 (100 mM Tris, 100 mM NaCl, and 50 mM MgCl2 [pH 9.5]) for 5 minutes and color developed at room temperature in buffer 3 containing 5-bromo-4-chloro-3-indoyl phosphate 4-toluidine salt (BCIP)-nitroblue tetrazolium chloride (NBT; Roche) overnight. Staining was stopped by TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]) for 15 minutes. Nonspecific background staining was removed in 95% ETOH for 1 hour. Selected sections were bleached with potassium permanganate, as previously described. 6 Sections were counterstained with nuclear fast red (Vector Laboratories), dehydrated, and mounted. 
Immunohistochemistry
Eleven eyes (cases 1, 2–5, 9, 10, 12, 14–17) were investigated by immunohistochemistry using a previously published technique. 11 After blocking with 3% goat serum and 3% blocking reagent (Blotto; Santa Cruz Biotechnology, Santa, Cruz, CA), sections were incubated at 4°C overnight with 1:1000 anti-bovine IGFBP-2 rabbit polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY). Detection was performed using an ABC staining kit (Vector Laboratories). Control sections were incubated with 1:1000 normal rabbit immunoglobulin fraction (Dako, Carpinteria, CA) instead of the primary antibody. Some sections were bleached with potassium permanganate, as described earlier. 6 Sections were counterstained with nuclear fast red (Vector Laboratories), dehydrated, and mounted. 
Data Analysis
Specimens were observed under a light microscope (BH-2; Olympus Optical Co., Ltd., Tokyo, Japan) with a charge-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 5.0; Adobe, Mountain View, CA). The number of total RPE cells and in situ hybridization–labeled RPE cells were counted. Each section contained at least 300 RPE cells. The ratio of labeled RPE cells to total RPE cells counted per section was calculated for both macular and peripheral regions in each eye. Statistical significance was determined using the Wilcoxon signed-rank test for the ratio of labeled macular cell versus the ratio of labeled peripheral cells. Spearman’s rank correlation was used to determine the influence of age on each ratio. P < 0.01 was considered significant. 
Results
In Situ Hybridization for IGFBP-2
IGFBP-2 mRNA expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner aspects of photoreceptor cells in all 17 globes (Fig. 1A) . No labeling was detected with the IGFBP-2 sense probe, whereas opsin mRNA expression was detected in the outer nuclear layer and inner segment of photoreceptor cells (Figs. 1B 1C) . Fifteen of 16 eyes (except in case 11) showed IGFBP-2 mRNA labeling in the RPE. The RPE in general showed nonuniform labeling characterized by clumps of labeled cells adjacent to unlabeled cells. In 11 eyes, the macular RPE showed a 1.2-fold greater ratio of labeled cells to total RPE cells counted per section compared with those counted in the periphery (P = 0.008, Wilcoxon signed-rank test). The number of IGFBP-2–labeled RPE cells decreased with age in the macula, whereas the number of IGFBP-2–labeled RPE cells in the periphery did not change (Fig. 2) . The IGFBP-2–labeled RPE cell ratio in the macula decreased with age (Fig. 3A , P = 0.0064, Spearman’s rank correlation). In contrast, the IGFBP-2–labeled RPE cell ratio in the periphery did not change with age (Fig. 3B) . These results suggest that the IGFBP-2 expression in macular RPE cells was preferentially decreased with age compared with those in the periphery. 
Immunohistochemical Staining of IGFBP-2
Immunohistochemical experiments were performed to determine whether there was a corresponding decrease in IGFBP-2 protein in the macula in 11 eyes. IGFBP-2 protein was located in the ganglion cells, inner segment of photoreceptors (data not shown), RPE, and choroid. Sections treated with rabbit IgG showed no staining (data not shown). In the eyes that showed immunohistochemical staining for IGFBP-2 (n = 7), all eyes exhibited more RPE cells labeled with IGFBP-2 in the macula than the periphery (Fig. 4) . This differential immunostaining of IGFBP-2 between the macula and the periphery was more obvious in young eyes. 
Discussion
In this study, we showed a topographic and age-related variation in IGFBP-2 mRNA expression in human RPE cells by determining the number of labeled cells and calculating the ratio of IGFBP-2–labeled RPE cells to the total number of cells counted per section. We identified two important associations between IGFBP-2 expression and human RPE cells in vivo: First, more cells expressed IGFBP-2 in the macula than the periphery at all ages in our collection, and second, the number of labeled cells decreased with age in the macula but remained unchanged in the periphery. Our immunohistochemical experiments supported these findings. 
To quantify topographic and age-related changes in IGFBP-2 expression, we developed a method to count RPE cells. This method, by using the ratio of labeled cells to total cells counted per section, quantifies the expression differences that were qualitatively seen when examining the sections. An advantage of using the ratio of labeled cells to total cells is that the confounding influences, such as an age-related decline in cell density, are excluded. We also considered the effect of variable postmortem and tissue processing times on the overall patterns of IGFBP-2 expression. One effect of prolonged postmortem and processing times may be the loss of both IGFBP-2 mRNA and protein. This effect would make processing time a significant variable in our results. To exclude the effect of postmortem and processing times, we examined specific correlations between the ratio of labeled cells in the macula versus the ratio of labeled cells in the periphery for individual eyes (i.e., the ratio of the two labeling ratios). This analysis yielded the same results concerning topographic and age-related variations in IGFBP-2 expression (data not shown). To further exclude the effect of postmortem and processing times, we incubated freshly enucleated rhesus macaque eyes at room temperature from 0 to 24 hours before fixation. We found no difference in RPE cell IGFBP-2 mRNA labeling in pattern and extent. 
Our studies add to the topographical and age-dependent morphologic changes to the RPE that have been reported in the literature, 1 12 13 14 15 16 and suggest that the phenotype of the RPE differs depending on its location and age. For example, Liles et al. 16 found that catalase activity was higher and show a greater decrease with age in the macula than in the periphery in normal eyes, whereas superoxide dismutase (SOD) activity does not change. Furthermore, Burke and Soref 1 demonstrated that RPE cells from the macula of the human eye or the area centralis of the bovine eye have more limited replicative life-spans in vitro than do cells from more peripheral regions of the posterior pole. In a separate study, the same laboratory showed that cytochrome oxidase activity in bovine and human RPE cells was lower in area centralis-macular cells at all donor ages and that the highest activities were from the oldest donors. 15 Finally, this group observed phenotypic variations in the staining of tight-junction complexes of RPE cells in situ, noting a chimeric pattern reminiscent of our current observations. 17  
The rationale for studying IGFBP-2 came from recent in vitro work from our laboratory and others who showed marked upregulation of IGFBP-2 with replicative senescence using quantitative Northern and microarray analyses, respectively. 7 8 Our data contradict our hypothesis that IGFBP-2 may be a useful marker of senescence in RPE cells in vivo, because we found decreased IGFBP-2 expression with age. The reason for this in vivo and in vitro difference in expression of IGFBP-2 is unresolved, but our results highlight the potential deficiencies of in vitro culture systems. 18 19  
Previously published studies indicate that the expression of IGFBP-2 appears to be affected by oxidative stress and chronological age. 20 21 22 23 Arnold et al. 20 showed that IGFBP-2 progressively decreases with age in human cerebrospinal fluid, and speculate that IGFBP-2 is an important regulator of neuronal function, especially during aging. Tham et al. 24 showed that IGF-II and IGFBP-2 were elevated in the cerebrospinal fluid of patients with Alzheimer disease compared with normal subjects. Although the exact role of IGFBP-2 in the RPE is unclear at this time, these findings offer the intriguing possibility that IGFBP-2 helps to maintain the RPE in a differentiated state in health, and during aging or exposure to critical levels of oxidative stress, IGFBP-2 expression could decline and promote the loss of RPE differentiation. Further investigations are needed to determine IGFBP-2–specific functions in the RPE cell during health and aging. 
 
Table 1.
 
Case Data
Table 1.
 
Case Data
Case Age (y) Sex Past Systemic Diseases Past Ophthalmic Disease Postmortem Time (hr)
1 27 F 11
2 47 M HT 11
3 51 F 6
4 53 M HT 10
5 54 M HT, HD 8
6 63 M HT, HD 8
7 63 M DM 8
8 64 F HT, HD 10
9 65 M DM 11
10 66 F 11
11 68 M DM, HD IOL 6
12 69 F Sepsis 10
13 69 F HT 10
14 71 M DM, HT 21
15 72 M IOL, glaucoma 7
16 72 F DM, HD 13
17 83 M 4
Figure 1.
 
In situ hybridization using anti-sense IGFBP-2 (A), sense IGFBP-2 (B), and anti-sense opsin (C) mRNA probes in the macula of a 65-year-old eye (case 9). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. (A) IGFBP-2 expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner photoreceptor segments. Arrowheads: RPE cells with IGBP-2 labeling. (B) No labeling was seen with the sense probe. (C) Opsin mRNA expression was detected only in the outer nuclear layer and inner segment of photoreceptor cells ( Image not available ). Scale bar, 50 μm.
Figure 1.
 
In situ hybridization using anti-sense IGFBP-2 (A), sense IGFBP-2 (B), and anti-sense opsin (C) mRNA probes in the macula of a 65-year-old eye (case 9). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. (A) IGFBP-2 expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner photoreceptor segments. Arrowheads: RPE cells with IGBP-2 labeling. (B) No labeling was seen with the sense probe. (C) Opsin mRNA expression was detected only in the outer nuclear layer and inner segment of photoreceptor cells ( Image not available ). Scale bar, 50 μm.
Figure 2.
 
Labeling of IGFBP-2 mRNA by in situ hybridization in RPE cells by region and age. RPE sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. More IGFBP-2–labeled cells (arrowheads) were seen in the macula than the periphery at all ages. The number of IGFBP-2–labeled RPE cells decreased with age in the macula, whereas the number of IGFBP-2–labeled cells in periphery remained unchanged. Scale bar, 50 μm.
Figure 2.
 
Labeling of IGFBP-2 mRNA by in situ hybridization in RPE cells by region and age. RPE sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. More IGFBP-2–labeled cells (arrowheads) were seen in the macula than the periphery at all ages. The number of IGFBP-2–labeled RPE cells decreased with age in the macula, whereas the number of IGFBP-2–labeled cells in periphery remained unchanged. Scale bar, 50 μm.
Figure 3.
 
Graph of the ratio of labeled to total cells counted as a function of age in the macula (A) and periphery (B). The ratio of labeled cells in the macula decreases with age (A; P = 0.0064 Spearman’s rank correlation). The ratio of labeled cells in the periphery showed no age-related change (B).
Figure 3.
 
Graph of the ratio of labeled to total cells counted as a function of age in the macula (A) and periphery (B). The ratio of labeled cells in the macula decreases with age (A; P = 0.0064 Spearman’s rank correlation). The ratio of labeled cells in the periphery showed no age-related change (B).
Figure 4.
 
Immunohistochemical staining of IGFBP-2 in RPE cells by region and age. The sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The immunohistochemical staining with BCIP-NBT appears blue-purple. More RPE cells were labeled for IGFBP-2 (arrowheads) in the macula than in the periphery in the young and middle-aged specimens. More IGFBP-2–labeled macular RPE cells were visualized in the 27-year-old than in the 63-year-old eye, whereas the immunoreactivity in the peripheral RPE was the same for both ages. The RPE in the oldest eye did not show immunoreactivity in either the macula or periphery. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical staining of IGFBP-2 in RPE cells by region and age. The sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The immunohistochemical staining with BCIP-NBT appears blue-purple. More RPE cells were labeled for IGFBP-2 (arrowheads) in the macula than in the periphery in the young and middle-aged specimens. More IGFBP-2–labeled macular RPE cells were visualized in the 27-year-old than in the 63-year-old eye, whereas the immunoreactivity in the peripheral RPE was the same for both ages. The RPE in the oldest eye did not show immunoreactivity in either the macula or periphery. Scale bar, 50 μm.
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Figure 1.
 
In situ hybridization using anti-sense IGFBP-2 (A), sense IGFBP-2 (B), and anti-sense opsin (C) mRNA probes in the macula of a 65-year-old eye (case 9). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. (A) IGFBP-2 expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner photoreceptor segments. Arrowheads: RPE cells with IGBP-2 labeling. (B) No labeling was seen with the sense probe. (C) Opsin mRNA expression was detected only in the outer nuclear layer and inner segment of photoreceptor cells ( Image not available ). Scale bar, 50 μm.
Figure 1.
 
In situ hybridization using anti-sense IGFBP-2 (A), sense IGFBP-2 (B), and anti-sense opsin (C) mRNA probes in the macula of a 65-year-old eye (case 9). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. (A) IGFBP-2 expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner photoreceptor segments. Arrowheads: RPE cells with IGBP-2 labeling. (B) No labeling was seen with the sense probe. (C) Opsin mRNA expression was detected only in the outer nuclear layer and inner segment of photoreceptor cells ( Image not available ). Scale bar, 50 μm.
Figure 2.
 
Labeling of IGFBP-2 mRNA by in situ hybridization in RPE cells by region and age. RPE sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. More IGFBP-2–labeled cells (arrowheads) were seen in the macula than the periphery at all ages. The number of IGFBP-2–labeled RPE cells decreased with age in the macula, whereas the number of IGFBP-2–labeled cells in periphery remained unchanged. Scale bar, 50 μm.
Figure 2.
 
Labeling of IGFBP-2 mRNA by in situ hybridization in RPE cells by region and age. RPE sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The DIG-labeled in situ hybridization reaction with BCIP/NBT appears blue-purple. More IGFBP-2–labeled cells (arrowheads) were seen in the macula than the periphery at all ages. The number of IGFBP-2–labeled RPE cells decreased with age in the macula, whereas the number of IGFBP-2–labeled cells in periphery remained unchanged. Scale bar, 50 μm.
Figure 3.
 
Graph of the ratio of labeled to total cells counted as a function of age in the macula (A) and periphery (B). The ratio of labeled cells in the macula decreases with age (A; P = 0.0064 Spearman’s rank correlation). The ratio of labeled cells in the periphery showed no age-related change (B).
Figure 3.
 
Graph of the ratio of labeled to total cells counted as a function of age in the macula (A) and periphery (B). The ratio of labeled cells in the macula decreases with age (A; P = 0.0064 Spearman’s rank correlation). The ratio of labeled cells in the periphery showed no age-related change (B).
Figure 4.
 
Immunohistochemical staining of IGFBP-2 in RPE cells by region and age. The sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The immunohistochemical staining with BCIP-NBT appears blue-purple. More RPE cells were labeled for IGFBP-2 (arrowheads) in the macula than in the periphery in the young and middle-aged specimens. More IGFBP-2–labeled macular RPE cells were visualized in the 27-year-old than in the 63-year-old eye, whereas the immunoreactivity in the peripheral RPE was the same for both ages. The RPE in the oldest eye did not show immunoreactivity in either the macula or periphery. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical staining of IGFBP-2 in RPE cells by region and age. The sections from a 27-year-old (case 1) macula (A) and periphery (B), a 63-year-old (case 6) macula (C) and periphery (D), and an 83-year-old (case 17) macula (E) and periphery (F). The immunohistochemical staining with BCIP-NBT appears blue-purple. More RPE cells were labeled for IGFBP-2 (arrowheads) in the macula than in the periphery in the young and middle-aged specimens. More IGFBP-2–labeled macular RPE cells were visualized in the 27-year-old than in the 63-year-old eye, whereas the immunoreactivity in the peripheral RPE was the same for both ages. The RPE in the oldest eye did not show immunoreactivity in either the macula or periphery. Scale bar, 50 μm.
Table 1.
 
Case Data
Table 1.
 
Case Data
Case Age (y) Sex Past Systemic Diseases Past Ophthalmic Disease Postmortem Time (hr)
1 27 F 11
2 47 M HT 11
3 51 F 6
4 53 M HT 10
5 54 M HT, HD 8
6 63 M HT, HD 8
7 63 M DM 8
8 64 F HT, HD 10
9 65 M DM 11
10 66 F 11
11 68 M DM, HD IOL 6
12 69 F Sepsis 10
13 69 F HT 10
14 71 M DM, HT 21
15 72 M IOL, glaucoma 7
16 72 F DM, HD 13
17 83 M 4
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