High-resolution adaptive optics retinal imaging of cellular structure in choroideremia
Jessica I W Morgan, Grace Han, Eva Klinman, William M Maguire, Daniel C Chung, Albert M Maguire, Jean Bennett, Jessica I W Morgan, Grace Han, Eva Klinman, William M Maguire, Daniel C Chung, Albert M Maguire, Jean Bennett
Abstract
Purpose: We characterized retinal structure in patients and carriers of choroideremia using adaptive optics and other high resolution modalities.
Methods: A total of 57 patients and 18 carriers of choroideremia were imaged using adaptive optics scanning light ophthalmoscopy (AOSLO), optical coherence tomography (OCT), autofluorescence (AF), and scanning light ophthalmoscopy (SLO). Cone density was measured in 59 eyes of 34 patients where the full cone mosaic was observed.
Results: The SLO imaging revealed scalloped edges of RPE atrophy and large choroidal vessels. The AF imaging showed hypo-AF in areas of degeneration, while central AF remained present. OCT images showed outer retinal tubulations and thinned RPE/interdigitation layers. The AOSLO imaging revealed the cone mosaic in central relatively intact retina, and cone density was either reduced or normal at 0.5 mm eccentricity. The border of RPE atrophy showed abrupt loss of the cone mosaic at the same location. The AF imaging in comparison with AOSLO showed RPE health may be compromised before cone degeneration. Other disease features, including visualization of choroidal vessels, hyper-reflective clumps of cones, and unique retinal findings, were tabulated to show the frequency of occurrence and model disease progression.
Conclusions: The data support the RPE being one primary site of degeneration in patients with choroideremia. Photoreceptors also may degenerate independently. High resolution imaging, particularly AOSLO in combination with OCT, allows single cell analysis of disease in choroideremia. These modalities promise to be useful in monitoring disease progression, and in documenting the efficacy of gene and cell-based therapies for choroideremia and other diseases as these therapies emerge. (ClinicalTrials.gov number, NCT01866371.).
Keywords: adaptive optics; choroideremia; photoreceptors; retinal pigment epithelium.
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
Figures
Retinal images from the right eye of choroideremia patient 13092. (A) The IR fundus SLO imaging shows retinal atrophy in the periphery with scalloped edges along the border. (B) Hypo-AF reveals areas of degenerated RPE at these same locations with centrally intact RPE depicted by remaining AF. The white arrows on the IR fundus image (A) give the horizontal location of the OCT B-scan (C) through the central retina. Centrally, the laminar structure of the retina remains intact, though the RPE and interdigitation layer are thinned and difficult to distinguish. The yellow arrows mark the abrupt transition area between the central retina and area of atrophy. (D) The montage of larger field of view AO images depicts the central retina within approximately 1.5 mm of fixation in all directions. Numerous hyper-reflective clumps can be observed throughout this region. Colored boxes show the locations for each high-resolution AO image given in Figure 2.
The AO images from the right eye of patient 13092 at the locations identified by colored boxes in Figure 1. The cone photoreceptor mosaic is intact and continuous at each location. The cone density for each location is given on each image, each of these cone densities is within the previously reported normal range given by Song et al. Clumps of hyper-reflective cones are readily observed at locations 3, 4, and 5. Locations 1, 2, 3, and 5 are included in Table 1 and the plot of cone density verses eccentricity (Fig. 6). Locations 4, 6, and 7 are not included in Table 1 or Figure 6, because the locations are not along the either horizontal or vertical meridian. Scale bar: 50 μm.
Retinal images from the left eye of choroideremia patient 13045. (A) The IR fundus SLO imaging shows retinal atrophy, which has progressed further centrally as compared with 13092. Large choroidal vessels can be seen in the periphery with the dark central region showing remaining central retina. (B) The AF imaging was difficult in this patient but the contrast enhanced image shown does reveal remaining AF in the central retina corresponding to the dark central region on IR imaging. The white arrows on the IR image (A) give the horizontal location of the OCT b-scan (C) through the fovea. The OCT shows outer retinal tubulation temporal to the central intact retina (yellow arrow). The orange arrowheads show interlaminar bridges similar to those described by Jacobson et al. These bridges mark the abrupt transition from central relatively intact retinal lamination to outer retinal atrophy. Blue arrowhead points to inner retinal microcysts as previously described., The red arrowheads mark the abrupt border to the outer nuclear layer plus Henle's fiber layer, the external limiting membrane, and the ellipsoid band. The interdigitation band is indistinguishable from the RPE throughout the entire central region and the interdigitation band/RPE combination is thin compared to normal. (D) The montage shows the larger field of view AO images within the central 2 mm of fixation, showing the relatively centrally intact retina and surrounding atrophic area. Colored boxes show the locations for each high-resolution AO image given in Figure 4.
The AO images from the left eye of patient 13045 at the locations identified by colored boxes in Figure 3. The central AO images show the cone mosaic is intact and continuous throughout this region. The AO images outlined in red and yellow (locations 4 and 6) show retinal locations at the border of atrophy. The cone mosaic is intact and visible up to the edge of the atrophic region. The AO image outlined in orange (location 5) shows the choroidal blood vessel network, visible because of the atrophic retina at this location. The cone mosaic is not observed at this location. Locations 1, 2, 3, 4. and 6 are included in Table 1 and the plot of cone density verses eccentricity (Fig. 6). Scale bar: for AOSLO images outlined in color, 50 μm.
High-resolution AO images of the cone mosaic at approximately 1 mm nasal and 1 mm temporal in normal control subjects (NCS), carriers of choroideremia (CAR), and choroideremia patients (CHM). Cone densities for each of these images fell within the previously reported normal range (Supplementary Fig. S1). Though cone density is within the range of normal cone densities for each of the choroideremia patient images shown here, the cones appeared less reflective and with lower contrast in patient images compared to normal and choroideremia carriers. Scale bar: 20 μm.
Plot of cone density versus eccentricity along each meridian of the retina. Open triangles are choroideremia patient data, open circles are choroideremia carrier data, pluses are normal control data. Solid black line with closed circles is replotted histology data from Curcio et al.Solid gray lines are the previously reported range of cone densities measured with AOSLO replotted from Song et al.Solid squares with error bars are the previously reported normal densities from Park et al. The data from our eight normal controls match previously reported control data, thus showing the AOSLO used in this study is capable of resolving the cone mosaics at these retinal eccentricities. S, superior; I, inferior; N, nasal; T, temporal.
The AOSLO, IR, AF, and OCT images showing border regions of retinal atrophy, where the cone mosaic abruptly ends at the same location as the border of RPE atrophy (marked with an asterisk in the AOSLO images). The AF images show preserved AF in locations corresponding to intact RPE and cone mosaics in AOSLO. The OCT shows an abrupt end to the outer nuclear layer plus Henle's fiber layer, the ellipsoid band, and the RPE with increased choroidal backscatter at the border of atrophy, consistent with hyper-reflection at the same location on IR. Again the interdigitation band cannot be distinguished from the thinned RPE. The white squares on IR and AF images show the location of the AOSLO image. Arrow on IR image shows the location and direction of the OCT line.
The IR (A), AF (B), and OCT (C) images from patient 13071, right eye. The IR image (A) shows central area of remaining retina, with scalloped edges at the border of the atrophic region. The AF image (B) shows the central retinal area still retaining AF and, thus, still containing functional RPE. (C) The OCT image shows the central laminar structure of the retina. The interdigitation layer is difficult to distinguish from the RPE/Bruch's membrane layer; however, the combination of these two layers become thinner at the location marked by the red arrow and slight increase in choroidal backscatter. A second increase in choroidal backscatter is observed at the orange arrow. The ellipsoid band is present beyond (further nasal than) the orange arrow and the ellipsoid band is disrupted though visible at the yellow arrow. The blue arrow marks the edge of the outer nuclear layer plus Henle's fiber layer. The wide extent of horizontal locations marking the loss of the different outer retinal layers on OCT in this eye, is different from the sharp borders of atrophy shown in the OCTs of Figures 1, 3, and 7. (D) The OCT image magnified by a factor of 3 (area outlined by the black rectangle in [C]). (E) The AOSLO image of the cone photoreceptors at the retinal location outlined by the white box. Evidence that the reflections seen in the AOSLO image at this location are due to the presence of cones is as follows: The cone mosaic is visible at the same focus level throughout the entire image. The reflections are similar in size, shape, pattern, and density as what would be expected for the cone mosaic at this location. The OCT images show the ellipsoid band is present though disrupted. Evidence that the RPE may be a primary site of disease at this location is as follows: The increase in choroidal backscatter at this location shows that the RPE is at minimum less pigmented than locations further central. The less pigmented RPE also is observed in the IR image at this location. The AF is not present at this location, suggesting at minimum that the RPE is not functioning normally.
The AOSLO images depicting choroidal vessels in locations corresponding to retinal atrophy in choroideremia patients. The cone mosaic is not observed at these locations. Corresponding IR and OCT images are shown at the same locations. The white squares on the IR images show the locations of the AOSLO images. The arrows on IR images show the locations and directions of the OCT lines.
Examples of the bubble-like features observed in the AOSLO images of a subset of choroideremia patients. These features are observed only over regions corresponding to retinal atrophy; the cone mosaic is not observed at these locations. The bubble-like features can be observed in the IR images. The white box in the IR image outlines the area marked by the AOSLO image above. The arrows on IR images show the locations and directions of the OCT lines. The OCTs at the same locations show the bubble-like features co-locate with hyporeflective space in the choroid (white arrowheads). The OCT of patient 13086 shows there are no inner retinal microcysts and that the bubble-like features do not co-locate with inner retinal cysts. The OCTs from patients 13012, 13057, and 13086 show the bubble-like features do not co-locate with interlaminar bridges. Supplementary Video S1 also shows the features are located posterior to the photoreceptors.
The IR and OCT images from patient 13062. The IR image shows numerous clearly visible bubble-like features spread throughout the atrophic macula. Four OCT lines are shown in precise alignment with the IR image. The bubble-like features outlined in red, yellow, white, and blue boxes on the IR image correspond to the red, yellow, white, and blue boxes on each OCT line. The bubble-like features within each of these boxes co-locate with hypo-reflective space in the choroid (white arrows on OCT). A different retinal feature (teal arrow on IR and OCT) was used to verify the alignment of each OCT line on the IR image separate from the bubble-like features. The bubble-like features do not align with inner retinal microcysts, interlaminar bridges, or outer retinal tubulations. A different retinal feature (teal arrow on IR and OCT: OCT line 1, pigment clump; OCT line 2, outer retinal tubulation; OCT line 3, retinal blood vessel; OCT line 4, retinal perforation) was used to verify the alignment of each OCT line on the IR image separate from the bubble-like features. The bubble-like features do not align with inner retinal microcysts, interlaminar bridges, or outer retinal tubulations.
Examples of inner retinal microcysts observed in the AOSLO images of a subset of choroideremia patients. These features are only observed over regions corresponding to retinal atrophy; the cone mosaic is not observed at these locations. The inner retinal microcysts cannot be readily identified in the IR images. The white box in the IR image outlines the area marked by the AOSLO image. The arrows on IR images show the locations and directions of the OCT lines. OCTs at the same locations show the features co-locate with hyporeflective space in the inner retina (white arrowheads).
The IR, OCT, AF, and AOSLO montage images of the right eye of choroideremia carrier 13036. The fundus shows patchy RPE atrophy in the IR (A) and AF (B) images. The white arrows on the IR fundus image (A) give the horizontal location of the OCT B-scan (C) through the central retina. The layer corresponding to the interdigitation zone is lost and the ellipsoid band is disrupted (yellow arrows) at locations corresponding to patchy atrophy on IR and AF. (D) The montage of the larger field of view AO images depicts the central retina within approximately 1.5 mm of fixation in all directions. Colored boxes show the locations for each high-resolution AO image given in Figure 14.
Most of the AOSLO images show a complete and contiguous cone mosaic (locations 1, 3, 4, 6, and 7). The cone density for each location counted is given on each image, each of these cone densities is within the previously reported normal range given by Song et al. Some local disruption of the cone mosaic can be observed in locations 2 and 5 (asterisks on the AOSLO images). These locations also show local patches of hypo-AF corresponding to RPE degeneration (Fig. 13). Location 4 is not included in Table 1 or Figure 6, because the location is not along the either horizontal or vertical meridian. Scale bar: 50 μm.
Source: PubMed