Optical Coherence Tomography Study of Retinal Changes in Normal Aging and After Ischemia

Abstract

Purpose.: Age-related thinning of the retinal ganglion cell axons in the nerve fiber layer has been measured in humans using optical coherence tomography (OCT). In this study, we used OCT to measure inner retinal changes in 3-month-, 1-year-, and 2-year-old mice and after experimental anterior ischemic optic neuropathy (AION).

Methods.: We used OCT to quantify retinal thickness in over 200 eyes at different ages before and after a photochemical thrombosis model of AION. The scans were manually or automatically segmented.

Results.: In normal aging, there was 1.3-μm thinning of the ganglion cell complex (GCC) between 3 months and 1 year (P < 0.0001) and no further thinning at 2 years. In studying age-related inner retinal changes, measurement of the GCC (circular scan) was superior to that of the total retinal thickness (posterior pole scan) despite the need for manual segmentation because it was not contaminated by outer retinal changes. Three weeks after AION, there was 8.9-μm thinning of the GCC (circular scan; P < 0.0001), 50-μm thinning of the optic disc (posterior pole scan; P < 0.0001), and 17-μm thinning of the retina (posterior pole scan; P < 0.0001) in the 3-month-old group. Changes in the older eyes after AION were similar to those of the 3-month-old group.

Conclusions.: Optical coherence tomography imaging of a large number of eyes showed that, like humans, mice exhibited small, age-related inner retinal thinning. Measurement of the GCC was superior to total retinal thickness in quantifying age-related changes, and both circular and posterior pole scans were useful to track short-term changes after AION.

Many changes occur in the eye as part of normal aging, including in the cornea, lens, photoreceptors, and optic nerve. Aging also confers increased risk for some vision-impairing conditions, since many diseases that affect vision occur predominantly in older individuals. We know there is thinning of the optic nerve in normal aging in humans,^1–6 mice,^7,10 rats,^11,12 and monkeys,^13,14 although the causes of age-related changes remain unclear. Some age-related changes^15,16 have been postulated to contribute to optic neuropathies that occur in older individuals, including glaucoma, the most common optic neuropathy in the world, and anterior ischemic optic neuropathy (AION),^17–19 the most common acute optic neuropathy in patients older than 50 years, so changes in normal aging may be relevant to disease pathogenesis. 

Thinning of the optic nerve has been studied in humans using optical coherence tomography (OCT), which provides a noninvasive and reliable way to measure the retinal changes in normal aging^20–27 and in optic neuropathies.^24,28–30 In fact, the circular scan and automatic segmentation of the retinal nerve fiber layer (RNFL) are routinely used clinically, in addition to the recently available posterior pole analysis and segmentation of the macular ganglion cell complex (GCC).^28,31–33 Retinal nerve fiber layer analysis and the macular GCC are routinely used in complementary ways to measure changes in the unmyelinated retinal ganglion cell (RGC) axons and in the cell body layer, especially since the latter is less affected by optic disc swelling from edema or other causes.²⁸ 

Compared with human studies, there have been relatively limited OCT studies in animal models of vision loss,^34–38 although in vivo imaging³⁶ and OCT are powerful research tools and animal studies offer the advantage of precise timing of onset of vision loss and histologic correlation.^7,10,37–42 In our previous study, we use OCT and histology to show that in murine AION, there is significant retinal swelling on day 1 and thinning within weeks.⁴² Several studies of experimental optic neuropathies including optic neuritis, N-Methyl-D-aspartate (NMDA)-related excitotoxicity of the RGCs, and AION have also shown that there is excellent correlation between OCT and retinal histologic changes.^7,39,42,43 

In this study, we used OCT to assess changes in normal aging and in experimental AION in a large number of rodents. Our data allowed us to identify the best OCT parameters to quantify retinal changes in normal aging and following murine AION. 

Materials and Methods

Animals

All animal care and experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with approval from the Stanford University Administrative Panel on Laboratory Animal Care. We purchased wild-type adult C57BL/6 mice (Charles River, Hollister, CA, USA; Taconic, Inc., Hudson, NY, USA), which were housed in cages at constant temperature, with a 12-hour light/dark cycle, with food and water available ad libitum. All procedures were performed under sedation, achieved with intramuscular injection of ketamine 50 to 100 mg/kg (Hospira, Inc., Lake Forest, IL, USA), xylazine 2 to 5 mg/kg (Bedford Laboratories, Bedford, OH, USA), and buprenorphine 0.05 mg/kg (Bedford Laboratories). The pupils of anesthetized mice were dilated with 1% tropicamide (Alcon Laboratories, Inc., Fort Worth, TX, USA) and 2.5% phenylephrine hydrochloride (Akorn, Inc., Lake Forest, IL, USA). 

Experimental Anterior Ischemic Optic Neuropathy

We induced a photochemical thrombosis model of AION per published protocol.⁴² The animals were 3 months, 1 year, or 2 years old at the time of AION induction, and they were monitored for 3 weeks after AION. Briefly, following tail vein injection of rose bengal (1.25 mM in phosphate-buffered saline, 2–3 μL/g body weight), the optic nerve head was exposed to low-energy laser light using a frequency-doubled Nd:YAG laser (Pascal; OptiMedica, Santa Clara, CA, USA) at 400 μm, power of 50 mW, duration of 1 second, 15 spots.⁴² Photoactivation of intravascular rose bengal selectively damages vascular endothelium, producing thrombosis while sparing nonvascular tissues.^44–46 

Optical Coherence Tomography

We performed spectral-domain OCT scans on 237 eyes at 3 months (n = 126), 1 year (n = 85), or 2 years (n = 26) of age using Spectralis HRA+OCT (Heidelberg Engineering, GmbH, Heidelberg, Germany), which utilizes a superluminescent diode laser with average wavelength of 870 nm. The OCT scanner has optical axial resolution of 7 μm, digital resolution of 3.5 μm, scan depth of 1.8 mm, and scan rate of 40 kHz. We used the standard objective with a 30° field of view. To correct for the optics of the small mouse eye, we mounted an additional digital high-magnification lens (Volk Optical, Inc., Mentor, OH, USA) in front of the scanning system, similar to the procedure in other OCT studies of rodent eyes.^47–50 The OCT measurements have been shown to be comparable with those of histology.^42,47,48 For imaging of human eyes, OCT measurements assume emmetropia and average axial length, and adjustment for nonhuman primates has been published.⁵¹ In our mouse study, we assumed that there was no significant difference in refraction, axial length, astigmatism, or optical aberrations in the mouse eyes in different age groups measured under ketamine–xylazine anesthesia. 

All animals were measured rapidly following anesthesia and pupillary dilation. We applied lubricating eye drops over the mouse eyes and covered them with custom-made contact lenses to prevent ocular surface issues. For imaging, animals were placed on an adjustable platform, and the camera was aligned perpendicular to the animal directly in front of and very close to the eye using a three-dimensional micromanipulator.^47–50 Once the optic disc was centered and in focus using infrared imaging, we performed the circular scan (scan angle 12°, also referred to as the RNFL scan) (Fig. 1A) using the enhanced depth imaging (EDI) and high-resolution mode, with each B-scan consisting of 1536 A-scans centered around the optic disc. We averaged 16 frames per B-scan (Fig. 1C). We also performed posterior pole scans (scan angle 30° × 25°) (Fig. 1B) using EDI in high-speed mode, with each two-dimensional B-scan consisting of 768 A-scans, average 9 frames per B-scan (Fig. 1D); and 25 line scans (scan angle 25° × 15°) in high-resolution mode, average 16 frames per B-scan. Only images with adequate signal strength index were saved and used for analysis. All OCT scans were performed by one investigator to maximize consistency, and the best image from each eye was selected for segmentation. 

Figure 1

 

Spectral-domain OCT scans of murine retina. (A) Example of circular scan. (B) Example of posterior pole scan. The numbers represent total retinal thickness (in microns) of retina, which is displayed in a grid-like pattern. The center 2 × 2 grid (red dashed box) is used to calculate optic disc thickness. The 6 × 6 box (white dashed box) minus the center 2 × 2 box is used to calculate retinal thickness. (C) Representative B-scan using the circular scan mode. This scan corresponds to the green circle in (A). (D) Representative B-scan using the posterior pole scan mode. The scan corresponds to the green line in (B). GCC, ganglion cell complex; ILM, inner limiting membrane; BM, Bruch's membrane.

For the circular RNFL scans, we manually segmented the thickness of the GCC,^7,43 which was defined as the combined thickness of the nerve fiber layer, ganglion cell layer, and inner plexiform layer. We used the G or global measurement of GCC thickness, which is the average of all measurements (360°) around the optic disc. The GCC measurements did not include the outer retina and were not affected by outer retinal ischemia or distortion of the outer nuclear layer due to swelling. Because of the subjective nature of the manual segmentation of the GCC, every effort was made to standardize the segmentation process. The segmentation was performed under magnification and was easily and visually distinguished from the adjacent vitreous on one side and the inner nuclear layer on the other side due to obvious change in signal intensities. The segmentation was performed by one well-trained investigator to reduce variability between investigators, and this person was masked to the identity of the eyes. The segmentation was then visually confirmed by a second investigator as needed. We tested the reliability of manual segmentation in a small number of animals, either by reimaging the same eyes and segmenting them under masked condition or by taking the same OCT images and segmenting them several times independently. The R² was 0.92 for reimaging and segmentation of the same eyes on different dates, and the R² was 0.85 to 0.93 for resegmentation of the same OCT images. Based on these values, we were confident of the consistency of the process. 

For the posterior pole scans, the total retinal thickness, which was defined as the inner limiting membrane to the Bruch's membrane, was automatically segmented by the Spectralis software and visually confirmed to correct for poor segmentation as needed. Following posterior pole scan, the coronal view of the retina was displayed to show total retinal thickness in a grid composed of 3° × 3° squares, of which the center 2 × 2 grid was averaged to obtain the optic disc thickness and the 6 × 6 grid minus the center 2 × 2 grid was used for calculation of retinal thickness (Fig. 1B). 

Statistical Analysis

All n refer to the number of eyes rather than the number of animals. All data are presented as mean ± SEM. For statistical significance, we used nonparametric tests (Wilcoxon signed-rank test and Mann-Whitney U test; Prism version 6; GraphPad Software, Inc., La Jolla, CA, USA) and paired or unpaired Student's t-test (Microsoft Office Excel; Microsoft Corporation, Redmond, WA, USA). Statistical significance was defined as P < 0.05. The P values displayed for matched (longitudinal) data were calculated using Wilcoxon signed-rank test and all unmatched comparisons with Mann-Whitney U test. 

Results

Age-Related Thinning Measured With Circular Scan

Using OCT circular scan and manual segmentation of the GCC (Figs. 1A, 1C), we found that there was a significant 1.3-μm thinning of the GCC in comparing 3-month- and 1-year-old eyes (3 months: 76.2 ± 0.3 μm, n = 126 eyes; 1 year: 74.9 ± 0.3 μm, n = 85 eyes; P = 0.0002; Mann-Whitney) (Fig. 2A; Table). There was no change in GCC thickness between 1 and 2 years of age in all eyes (1 year: 74.9 ± 0.3 μm, n = 85; 2 years: 74.2 ± 0.6 μm, n = 26; P = 0.3; Mann-Whitney) (Fig. 2A). In subgroup analysis comparing the same eyes over time, there was also no significant difference in the GCC measurement between 1 and 2 years of age (1 year: 73.9 ± 0.7 μm; 2 years: 74.5 ± 0.8 μm; n = 17 for both groups; P = 0.2; Wilcoxon) (Fig. 2B). 

Figure 2

 

Significant 1.3-μm thinning of the GCC between 3 months and 1 year of age and no change between 1 and 2 years of age. (A) Bar graph showing averaged GCC thickness in different age groups (3 months: 76.2 ± 0.3 μm, n = 126 eyes; 1 year: 74.9 ± 0.3 μm, n = 85 eyes; **P = 0.0002; Mann-Whitney). There was no further thinning between 1 and 2 years (2 years: 74.2 ± 0.6 μm, n = 26 eyes; P = 0.3; Mann-Whitney). (B) Comparison of GCC in the same eyes between 1 and 2 years showed no significant difference between the 1- and 2-year groups (1 year: 73.9 ± 0.7 μm; 2 years: 74.5 ± 0.8 μm; n = 17 eyes for both groups; P = 0.2; Wilcoxon).

Table

 

OCT Measurements Using Circular and Posterior Pole Scans in Normal Aging and 3 Weeks After AION

 

Age-Related Changes Using the Posterior Pole Scan

We also assessed the retinal changes in 3-month-, 1-year-, and 2-year-old mice using the posterior pole scan and quantification of total retinal thickness of the optic disc and retina (Figs. 1B, 1D). Compared with 3-month-old eyes, the 1-year-old eyes had an 8.6-μm increase in the thickness of the optic disc (3 months: 240.9 ± 0.9 μm, n = 106; 1 year: 249.5 ± 1.1 μm, n = 81; P < 0.0001; Mann-Whitney) and a 6.3-μm increase in the thickness of the retina (3 months: 256.2 ± 0.7 μm, n = 109; 1 year: 262.5 ± 0.7 μm, n = 81; P < 0.0001; Mann-Whitney) (Fig. 3A; Table). These changes were due to thickening of the outer retinal layers (see below) and occurred at the same time as the 1.3-μm thinning of the GCC, providing support that significant retinal changes take place between 3 months and 1 year of age in normal aging. 

Figure 3

 

Changes in retinal thickness measured with posterior pole scan using automatic segmentation of total retinal thickness in 3-month-, 1-year-, and 2-year-old mice. (A) Bar graph showing significant increase in optic disc and retinal thickness at 1 year compared with 3 months and significant thinning of the optic disc and retinal thickness at 2 years (also see Table) (3 months: n = 106; 1 year: n = 81; and 2 years: n = 22; **P < 0.0001 for all measurements). (B) Fundus autofluorescence imaging of a 1.5-year-old mouse with diffuse retinal drusen. (C) Thinning of the peripapillary outer retina (optic disc − GCC) in the same eyes between 1 and 2 years of age (n = 13, **P = 0.008). (D) Thinning of the outer retina (retina-GCC) in the same eyes between 1 and 2 years of age (n = 13, **P = 0.0002).

Outer Retinal Thinning in Older C57BL/6 Mice

Compared with the 1-year-old eyes, the 2-year-old eyes had 8.8-μm thinning of the optic disc (1 year: 249.5 ± 1.1 μm, n = 81; 2 years: 240.7 ± 2.5 μm, n = 22; P = 0.0005; Mann-Whitney) and 18.4-μm thinning of the retina (1 year: 262.5 ± 0.7 μm, n = 81; 2 years: 244.1 ± 2.4 μm, n = 22; P < 0.0001; Mann-Whitney) (Fig. 3A; Table). This thinning was not correlated with findings of retinal drusen on autofluorescence imaging, although retinal drusen were more commonly found in older eyes in general (Fig. 3B). Comparing the same eyes between 1 and 2 years, there was a decrease in the optic disc and retina thickness (Figs. 3C, 3D) and no change in the GCC thickness (Fig. 2B). Subtraction of the GCC thickness from the optic disc or the retinal measurements from the same eyes between 1 and 2 years of age, to estimate changes in the thickness of the outer retina, revealed that significant thinning of the optic disc (n = 13, P = 0.008; Wilcoxon) and retina (n = 13, P = 0.0002; Wilcoxon) could be attributed to outer retinal thinning in 2-year-old eyes (Figs. 3C, 3D), which may be related to the prevalent Rd8 mutation recently reported in commercially available C57BL/6 mice.⁵² 

Changes of GCC After Experimental AION

We compared the effects of normal aging to those after experimental AION. The largest group we studied for AION effects was the 3-month-old animals, and some of these data were taken from a prior, smaller study.⁴² In the 3-month group, both circular and posterior pole scans were useful to track changes after AION. On day 1 after AION, the 3-month-old eyes exhibited thickening of the GCC by 29 μm (baseline: 76.3 ± 0.4 μm, n = 48; AION: 105.6 ± 2.8 μm, n = 24; Mann-Whitney). This was significantly different from the control group value at day 1 (control: 80.3 ± 1.1 μm, n = 44; P < 0.0001; Mann-Whitney). In the same eyes 3 weeks after AION, there was a significant 8.9-μm thinning in the GCC measurement in the AION group compared with control eyes (control 3 weeks: 76.0 ± 0.5 μm, n = 34; AION 3 weeks: 67.1 ± 0.7 μm, n = 31; P < 0.0001; Mann-Whitney) and compared with same eyes at baseline (76.3 ± 0.4 μm, n = 48; P < 0.0001; Mann-Whitney) (Fig. 4A; Table). 

Figure 4

 

GCC changes following experimental AION in 3-month-, 1-year-, and 2-year-old animals. (A) Line graph showing GCC thickness over time after AION in 3-month-old mice. There was significant swelling in the AION group at day 1 (control: 80.3 ± 1.1 μm, n = 44; AION: 105.6 ± 2.8 μm, n = 24; P < 0.0001) and progressive thinning that stabilized by week 3 (control: 76.0 ± 0.5 μm, n = 34; AION: 67.1 ± 0.7 μm, n = 31; P < 0.0001). (B) Bar graph of normalized GCC in 3-month-, 1-year-, and 2-year-old mice showing similar pattern of significant GCC thickening at day 1 and significant thinning at week 3.

Similar to the 3-month-old animals, older animals after AION also exhibited optic disc swelling and thickening of the GCC on day 1 (1 year: n = 5; P < 0.0001; 2 years: n = 8; P < 0.0001; Mann-Whitney), improved swelling at week 1 (1 year: 78.0 ± 0.0 μm, n = 3; P = 0.5; 2 years: 77.0 ± 1.0 μm; n = 5; P = 0.2; Mann-Whitney), and significant thinning at week 3 (1 year: n = 3; P = 0.0009; 2 years: n = 4; P = 0.002; Mann-Whitney). We did not find differences in these GCC measurements between the different age groups, but we performed GCC analysis in a small number of year 1 and year 2 animals after AION due to the limited number of available animals with high-quality images at different time points. The similar pattern of retinal changes within 3 weeks after AION in different age groups supported the continued use of the more available 3-month-old animals to study AION despite the fact that human AION typically occurs in those older than 50 years of age. 

Changes in Total Retinal Thickness Measurements at the Optic Disc and Retina After AION

We performed posterior pole scans of mice of all ages after AION. Three weeks after AION, the posterior pole scans of the optic disc in 3-month-old mice revealed significant 50-μm thinning of the optic disc (control: 240.9 ± 0.9 μm, n = 106; AION: 190.9 ± 5.3 μm, n = 10; P < 0.0001; Mann-Whitney) and 17-μm thinning of the retina compared with control group (control: 256.2 ± 0.7 μm, n = 109; AION: 239.1 ± 5.0 μm, n = 10; P < 0.0001; Mann-Whitney) (Table). This thinning in chronic AION was similar to that of the GCC (Fig. 5). 

Figure 5

 

Normalized OCT data comparing circular scan GCC and posterior pole scan optic disc and retinal thickness measurements in different age groups and in 3-month-old mice 3 weeks after AION. This showed that retinal thinning occurring in the GCC (8.9 μm; 12%), optic disc (50 μm; 21%), and retina (17 μm; 7%) after AION was substantially more severe than what occurred in normal aging. **P < 0.0001, Mann-Whitney.

Compared with 3-month-old mice, the older mice also exhibited a similar pattern of thinning after AION. Three weeks after ischemia, there was thinning of the optic disc in the 1-year-old eyes (control: 244.6 ± 1.4 μm, n = 3; AION: 214.6 ± 5.7 μm, n = 3; P = 0.007; Mann-Whitney) and in the 2-year-old eyes (control: 242.3 ± 7.4 μm, n = 4; AION: 218.5 ± 8.3 μm, n = 3; P = 0.09; Mann-Whitney) as well as thinning of the retina in the 1-year-old group (control: 258.7 ± 3.4 μm, n = 3; AION: 247.3 ± 2.1 μm, n = 4; P = 0.03; Mann-Whitney). At 3 weeks after AION in the 2-year-old eyes, there was no significant thinning of the retina (control: 243.9 ± 6.0 μm, n = 4; AION: 236.6 ± 3.5 μm, n = 3; P = 0.4; Mann-Whitney) because there was thinning in the control group due to outer retinal thinning and the small number of animals studied. The posterior pole data demonstrated the utility of different parameters in tracking changes after AION. 

Comparison of Data From Normal Aging and After AION

Comparing all OCT data in all age groups, the most significant age-related finding was a relatively small change in normal aging—a GCC thinning of 1.3 μm from 3 months to 1 year—compared with a relatively large change of 8.9-μm thinning 3 weeks after AION. Looking at the normalized OCT data, the circular scan and manual segmentation of the GCC was the most useful measurement of age-related changes in optic neuropathy because this measurement was relatively stable over time in C57BL/6 mice (Fig. 5). All OCT parameters assessed were useful to monitor changes after AION, including the circular scan and GCC manual segmentation and posterior pole scan and automatic segmentation of the total retinal thickness measurements of the optic disc and retina (Fig. 5). In all age groups, there was significant thinning of the GCC, optic disc, and the retina 3 weeks after AION (Fig. 5, also see Fig. 4). There was a relative 12% thinning of the GCC, 21% thinning of the optic disc, and 7% thinning of the retina in 3-month-old mice 3 weeks after AION. 

Discussion

We report here the first comprehensive mouse OCT study aimed at measuring age-related retinal changes relevant to the optic nerve and after experimental AION. In normal aging, there was a significant 1.3-μm thinning of the GCC between 3 months and 1 year, which confirmed that a small amount of inner retinal thinning occurred in normal aging. In chronic AION, there was an 8.9-μm thinning of the GCC, which was 6.8 times that for normal aging. 

Our study is the first to measure inner retinal thinning in normal aging in the mouse using OCT in a large number of eyes, and our finding is consistent with the modest thinning of the RNFL in human aging measured with OCT^{20,22,23,25–27} and other modalities, which show RNFL thinning of 0.16 to 0.33 μm annually^{20,22,23,25–27,53–59} or 2 to 3 μm/decade.²³ Age-related thinning of the optic nerve has also been seen in histologic studies in humans,^1–6,11 mice,^7–10 rats,^12,43,60 and monkeys.^13,14 Humans have on average 1.2 million axons per optic nerve,⁵ and age-related thinning in humans has been estimated at an average of 4000 to 5000 axons lost per year.^3,5,61 Balazsi et al.³ estimate that normal aging may account for a loss of 400,000 RGC axons during a 70-year life span, with loss of 2500 axons per year in those younger than 50 years old and 7500 axons per year in those older than 50 years. Many animals also exhibit age-related thinning of the optic nerve and loss of RGCs. In C57BL/6J adult mice (6–8 months), which have 45,000 ± 4000 axons per optic nerve,⁶² aging is associated with an increase in number of necrotic and swollen fibers at 22 months.⁸ Cenni et al.⁶² show that the number of ganglion cells in murine eyes is 112,000 ± 17,400 at birth and declines to 45,000 ± 4000 by 6 to 8 months of age. Samuel et al.⁶³ show that the overall thickness of the entire retina decreases by 15% between young (3 to 5 months) and old (24 to 48 months) animals, with thinning of the ganglion cell layer and the inner plexiform layer. In rats, Cepurna et al.¹² show that rodent eyes (5 months old) have 112,000 to 132,000 axons per optic nerve, which decline to 107,000 by 31 months. Primates have on average 122,000 axons per optic nerve,¹² and one study of 28 monkeys 1.5 to 29 years of age, which is equivalent to 4.5 to 87 human years, reveals an annual loss of 4319 axons per optic nerve.¹³ 

We found that after ischemia, there was significantly more thinning of the optic nerve than in normal aging, and that murine AION exhibited a pattern of acute swelling and chronic thinning similar to that in humans. In human AION studies, there is a 93% to 160% increase in RNFL thickness acutely^29,64,65 and 32% to 53% thinning chronically.^64,66–69 This pattern of OCT changes after murine AION was found using the circular and the posterior pole scan modes. Optical coherence tomography measurements of the RNFL or the macular GCC have been correlated with loss of vision in human AION^29,65,70,71 and loss of RGCs and optic nerve degeneration in animal models of AION^42,46,72,73; therefore, OCT is a useful technique to track anatomic changes in preclinical and clinical testing of potential therapies. 

We found that OCT study of experimental optic neuropathy was challenging because of the difficulty of segmenting the RNFL consistently and automatically using commercially available software in small animals. In the mouse, which has a relatively thin RNFL layer that is not well distinguished from the inner plexiform layer, the easiest way to approximate changes relevant to the optic nerve was a circular scan followed by manual segmentation of the GCC, which was defined as the RNFL, ganglion cell layer, and the inner plexiform layers. However, manual segmentation of the GCC was time-consuming and required consistency in data analysis. Despite the high resolution of spectral-domain OCT, less than 5 μm of retinal thinning over a long period of time means that a large number of animals would need to be used to assess significant changes in aging studies. 

In contrast to the manual segmentation needed after performance of the circular scan, the posterior pole scan provides an automatic way to track changes in the total retinal thickness. Current software also allows for analysis of the optic disc separately from the rest of the retina, which is useful for different studies. Our data supported the use of the posterior pole scan and automatic segmentation of the optic disc total thickness to study AION, since the optic disc measurements changed similarly and were proportionally greater than those of the GCC. The posterior pole scan and automatic segmentation of the retina minus the GCC may be useful to assess age-related outer retinal degeneration, since this measurement is predominantly affected by changes in the photoreceptors, retinal pigment epithelium, choroid, or connective tissues. 

A limitation of this study is the use of C57BL/6 mice in an aging study, since this strain of mice has been shown to harbor a Rd8 mutation.⁵² Consistent with this finding, our OCT measurements showed significant outer retinal thinning between 1 and 2 years old. This mutation did not have an obvious effect on GCC measurement. Similarly, the AION study tracking serial GCC and posterior pole measurements was still valuable despite this caveat since there was only approximately 1 month between the first and the last time points. We could not rule out a potential contribution of the Rd8 mutation in conferring selective vulnerability of the outer retinal neurons, but there was no significant difference between the pattern of post-AION changes in all age groups. 

Another limitation of our study is the small number of animals used to study OCT changes after AION in older mice. This is an important issue because age is a significant risk factor in the development of AION.^74,75 We did not find differences in post-AION changes in 3-month-, 1-year-, and 2-year-old animals, but we studied only a small number of animals in the older groups. This was because the development of ocular surface tissues and lens opacities were more prevalent in older animals, and we had to be vigilant about monitoring ocular clarity, which can affect image quality. These changes are known to be more common in aging mice.⁷⁶ Bell et al.⁷⁶ report in a study of 450 mice of multiple genetic backgrounds that fundus abnormalities increase in number in normal aging. The frequency of fundus abnormality in 2- to 4-month-old mice is 1.5% in C57BL/6J and as high as 100% in BALB/cJ mice. The frequency of fundus abnormality in C57BL/6J mice increases to 5.9% (range, 0%–13.6%) in 6- to 14-month-old mice.⁷⁶ The significance of these age-related changes in rodents is of unclear relevance to human studies because there are major differences in the genetic background. However, age-related vision changes and loss are part of normal aging, and further human and animal studies using technologies such as OCT to quantify changes associated with important risk factors will make us better able to identify ways to maintain good vision in aging and in disease. 

Acknowledgments

Supported by a Career Award in Biomedical Sciences from the Burroughs Wellcome Foundation, a Weston Havens Foundation grant, a Center for Biomedical Imaging at Stanford grant, and a Vice Provost Undergraduate Education grant from Stanford University (YJL). 

Disclosure: M.A. Shariati, None; J.H. Park, None; Y.J. Liao, None