Adaptive optics scanning light ophthalmoscopy
The AOSLO imaging was performed with a custom instrument, modified to capture light multiply scattered by the retina.27 The multiply scattered light is divided spatially to two separate detectors, and the resulting images are then subtracted to form the nonconfocal split-detector image, which reveals the photoreceptor inner segment mosaic.27 Confocal and split-detector images are recorded simultaneously in perfect spatial register. Photoreceptor image sequences were recorded at the fovea as well as in the periphery to approximately 10° superior and temporal to fixation. In subject KS_0589, an overlying epiretinal membrane in temporal macula obligated imaging to 10° nasal from fixation. Image sequences were corrected for sinusoidal distortion caused by the resonant scanner, then registered and averaged as previously described.27 Using a simplified Gullstrand 2 schematic eye, the predicted 291 μm per degree of visual angle was scaled linearly by the subject's axial length to determine the scale of AOSLO images. Averaged AOSLO images were aligned manually in Adobe Photoshop (Adobe Systems Inc, San Jose, CA) to create a large montage. This montage was manually aligned to the color fundus, line scan ophthalmoscope, en-face OCT, and to previously acquired AOSLO images21 (where available) using blood vessel shadows as landmarks. The location of the fovea was marked on the AOSLO montage, based on the subject's fixation recorded in the Cirrus HD-OCT line scan ophthalmoscope image. All AOSLO images are displayed on the linear intensity scale.
To examine longitudinal changes (approximately 2 years elapsed) in the cone mosaic, previously identified areas of normal cone density were reanalyzed in subjects KS_0600 and KS_0601. At 3 locations in each subject, confocal AOSLO images from both time points were first aligned manually and then registered with rigid translations, using the Stackreg plugin from ImageJ (National Institutes of Health, Bethesda, MA) and finally cropped to the region of overlap. Cones were identified with a previously described semiautomated algorithm.28 Cone density was calculated within 80 × 80 μm regions of interest (ROI).
To determine the effect of retinal lesions on the photoreceptor mosaic, the cone density was measured inside and outside macular lesions in all subjects. Because nonwaveguiding or misaligned photoreceptors are not visualized by confocal AOSLO,27,29 split-detector AOSLO images were chosen for analysis instead. 80 × 80 μm ROIs were identified and analyzed for cone density across the entire span of AOSLO imaging in each subject. An ROI was characterized as intralesional if any of the ROI fell within the limits of the lesion as visualized by en-face OCT segmented at the level of the ellipsoid zone; then each lesion was sampled with 5 to 7 ROI to evaluate for local density variations. Cell locations within the split-detector images were identified manually. The distance between each ROI and the fovea was estimated, and cone densities were compared with published normative in vivo values.30 Normative data were linearly interpolated to cover the range of measurement locations. Patient data were pooled across eccentricity for comparison, because there is no measured difference between temporal and nasal meridians across the eccentricities studied,31–33 and superior and inferior retinal loci are likely to underestimate cone photoreceptor density.31,32 Density data were evaluated using z-scores, calculated as the difference between the subject measurement and the normative mean divided by the standard deviation at that eccentricity. Z-scores of magnitude <2.0 were considered normal, P values < 0.05 were considered significant.
The subjects included in this study had the same disease causing mutation in BEST1 and demonstrated different stages of BVMD, with split-detector AOSLO providing unprecedented views of the photoreceptor pathology (Figures 1–3). Early in the disease, the photoreceptor mosaic remains contiguous but with substantially decreased density (Figures 1 and 3A). Later, after further cell loss has taken place, the photoreceptor packing no longer appears contiguous (Figures 2, 3, B and C). Figure 3 shows the span of photoreceptor mosaic changes across the clinically described pathology1 of subjects in this study from early vitelliform lesion to late-stage atrophy and fibrosis.
In all subjects, the effect of the BVMD lesion on overlying photoreceptors was assessed by comparing photoreceptor density within and outside lesions (Figure 4). Intralesion cone density was significantly reduced in subjects KS_0325, KS_0589, and KS_0601 (z-scores: −5.0 to −2.5). Near the fovea, KS_0599 exhibited reduced density (z-scores: −5.0 to −3.6), but returned to normal at the edges of the lesion (z-scores: −1.5 to −0.6). Intralesion density in subject KS_0600 was preserved (z-scores: 0.1–0.7). Extralesion cone density was near normal in all subjects (z-scores: −1.6 to 1.6) with the exception of one measurement in KS_0589 (z-score: −2.0). Within a lesion, the cone density and cone appearance varied considerably over short distances, with some regions having almost no photoreceptors, as shown in Figure 2.
Only KS_0600 and KS_0601 showed clear disease progression in OCT B-scan over 32 months and 30 months, respectively (See Figure, Supplemental Digital Content 1 http://links.lww.com/IAE/A496, which shows longitudinal OCTs for all subjects). To determine the effect of lesion enlargement on photoreceptor number, previously analyzed areas were recounted. Three extralesional locations were analyzed in 2 subjects, at approximately 1° from the fovea and just nasal to the BVMD lesion, where cone density was previously determined to be normal.21 In KS_0601, the cone density was found to change −2.4%, −1.7%, and 1.2% over a period of 30 months. In KS_0600, the cone density was found to change 0.0%, 0.5%, and −2.6% over a period of 32 months. These small variations in cone density are within the 95% confidence interval for the repeatability of the method of parafoveal density measurements (2.6%–2.8%)28 and are, therefore, consistent with no significant changes.
Split-detector AOSLO imaging within BVMD lesions is repeatable, even in subjects with advanced retinal degeneration as illustrated by the ability to track individual cells, shown in Figure 5. Here, the same clusters of photoreceptors were visualized over 4 months follow-up. There were, however, structures that appeared and disappeared from the images over this time scale (arrows, Figure 5). These round features had a lumpy appearance, were on average 20 μm in diameter, and appeared in areas that previously contained isolated photoreceptors or apparently empty space. Structures of similar size and appearance were also found to change in appearance on much shorter time scales, as short as an hour. These features were only noted in KS_0325, the subject with the most advanced disease.
Accurate assessment of cellular structure in inherited retinal degenerations in vivo can provide invaluable information about the pathology of these degenerations. In this study, we used newly developed split-detector AOSLO to further assess photoreceptor structure associated with BVMD in 5 individuals with the same previously reported BEST1 mutation (p.Arg218Cys). Compared with confocal imaging, nonconfocal split-detector AOSLO allows for a more accurate assessment of photoreceptor structure in BVMD, especially in areas of the photoreceptor mosaic overlying subretinal pathology (Figures 1–3 and see Figure, Supplemental Digital Content 2, http://links.lww.com/IAE/A497, which shows split-detector and OCT imaging within and outside vitelliform lesions).
Cone photoreceptor packing within vitelliform lesions can range from normal appearing mosaic (Figure 3, D and G) to significant disruption (Figure 2). As highlighted in patients KS_0589 and KS_0599 (Figure 3), significant intralesional variability also exists with focal areas of near-normal density present next to areas with severe disruption. In the fibrotic stages of BVMD as seen in KS_0325, cone photoreceptors remain, although sparsely packed and with focal areas entirely devoid of photoreceptors (Figures 2 and 5). We hypothesize that this loose packing allows some photoreceptors to freely pivot so that they are oriented horizontally, allowing visualization of both inner and outer segments of the photoreceptors (Figure 2—teardrop shaped structures in split-detector image). This irregular packing underscores the need for caution when reporting cone photoreceptor densities within areas of pathology as visualized by AOSLO, as these can vary dramatically, even if measurements are taken within 100 μm of each other.
It has been long debated whether BVMD has only focal clinically apparent fundus effects or is a true panretinal photoreceptor disorder. The results presented here show that within clinically apparent lesions, cone photoreceptor inner segments are enlarged and cone density is reduced. In agreement with previous AOSLO studies,21 immediately adjacent to the lesions, both density and appearance of cone inner segments return to normal (Figure 4 and see Figure, Supplemental Digital Content 2, http://links.lww.com/IAE/A497, which shows split-detector and OCT imaging within and outside vitelliform lesions), lending support to BVMD causing focal photoreceptor lesions. Interestingly, patient KS_0325 has been followed clinically for 5 years with the imaged lesion exhibiting detachment of the retina from the RPE over this span. Despite this change, split-detector AOSLO confirms photoreceptors overlying these lesions still exist, and combined with stable fixation within the lesions, suggests an alternate pathway for maintenance of the photoreceptors viability than from the RPE alone.
Split-detector imaging also revealed mobile disk-like structures consistent in size with cells (Figure 5, C and D). Previous histological studies have hypothesized that these cells represent subretinal macrophages,14,34 but their lineages were not rigorously confirmed. Alternative explanations for these cells include migratory microglia35 and RPE.36 The significance of this finding is unknown, but these may represent the first in vivo images of reactive subretinal cells in a human eye.
Recent work by Milenkovic et al37 suggests that the shared mutation identified in all participants in this study may affect volume-regulated anion channels in the RPE differently than other BEST1 mutations. Although the individuals imaged represent the spectrum of stages of BVMD, the clinical and subclinical phenotypes described here cannot necessarily be extended to other mutations in BEST1. Conversely, the diverse findings displayed above are more likely related to the stage of the disease rather than differential pathophysiology.
In summary, the improved resolution possible with split-detector AOSLO allows for increased understanding of cellular disease processes and could potentially be useful in monitoring therapeutic response on a cellular level in diseases such as BVMD. Future studies should be expanded to include high-resolution imaging in individuals with other mutations in BEST1 to further explore the genotype–phenotype correlations in photoreceptor morphology in BVMD.
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adaptive optics; genetics; imaging; photoreceptor; retina
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