To describe the microscopic structure of photoreceptors impacted by subretinal drusenoid deposits, also called pseudodrusen, an extracellular lesion associated with age-related macular degeneration (AMD), using adaptive optics scanning laser ophthalmoscopy (AOSLO).
Observational case series.
We recruited 53 patients with AMD and 10 age-similar subjects who had normal retinal health. All subjects underwent color fundus photography, infrared reflectance, red-free reflectance, autofluorescence, and spectral-domain optical coherence tomography (OCT). Subretinal drusenoid deposits were classified by a 3-stage OCT-based grading system. Lesions and surrounding photoreceptors were examined by AOSLO.
Subretinal drusenoid deposits were found in 26 eyes of 13 patients with AMD and imaged by AOSLO and spectral-domain OCT in 18 eyes (n = 342 lesions). Spectral-domain OCT showed subretinal drusenoid deposits as highly reflective material accumulated internal to the retinal pigment epithelium. AOSLO revealed that photoreceptor reflectivity was qualitatively reduced by stage 1 subretinal drusenoid deposits and was greatly reduced by stage 2. AOSLO presented a distinct structure in stage 3, a hyporeflective annulus consisting of deflected, degenerated or absent photoreceptors. A central core with a reflectivity superficially resembling photoreceptors is formed by the lesion material itself. A hyporeflective gap in the photoreceptor ellipsoid zone on either side of this core shown in spectral-domain OCT corresponded to the hyporeflective annulus seen by AOSLO.
AOSLO and multimodal imaging of subretinal drusenoid deposits indicate solid, space-filling lesions in the subretinal space. Associated retinal reflectivity changes are related to lesion stages and are consistent with perturbations to photoreceptors, as suggested by histology.
Pseudodrusen were first described by Mimoun and associates as a distinctive yellowish pattern that was visible en lumière bleue (visible in blue light) in some eyes with age-related macular degeneration (AMD). Because the lesions appeared to be different from typical drusen and were angiographically silent, the authors thought they were deep to the retinal pigment epithelium (RPE). Arnold and Sarks found that pseudodrusen were more easily visualized with red-free (RF) light or with a helium-neon (He-Ne) laser scanning laser ophthalmoscope (SLO). On the basis of one histologic specimen lacking neurosensory retina, they proposed that pseudodrusen appearance arose from choroidal fibrosis. Lois and associates described a reticular pattern of autofluorescence in eyes with AMD. Smith and associates reported that 87.5% of eyes with reticular autofluorescence patterns also had pseudodrusen corresponding in part with the autofluorescence, later speculating that both phenomena were manifestations of a reticular macular disease involving the RPE, choriocapillaris and inner choroidal. With spectral-domain optical coherence tomography (OCT), Zweifel and coworkers found that pseudodrusen correlated to granular hyper-reflective materials deposited anterior to the RPE in the subretinal space ; other than generalized choroidal thinning, no specific choroidal abnormality was seen. By comparing OCT findings to histologic examination of 1 donor retina with extracellular material between the RPE and photoreceptors called subretinal drusenoid deposits, Zweifel and coworkers attributed the appearance of pseudodrusen to these lesions. Subsequent SLO and OCT imaging studies reached the same conclusion. However, Sohrab and associates stated that subretinal deposits and photoreceptor disturbances shown on OCT did not colocalize with pseudodrusen, and they speculated how pseudodrusen appearance could arise secondarily from choroidal fibrosis. Suzuki and coworkers recently defined 3 subtypes of clinical pseudodrusen, all corresponding to a subretinal reflectivity visible by OCT. Thus the location of pseudodrusen within the chorioretinal layers has been a subject of debate, with evidence accumulating for the subretinal space.
In clinicopathologic studies, Sarks and associates showed that membranous debris, the principal component of soft drusen and basal linear deposit, was also found in the subretinal space. Curcio and associates demonstrated that these subretinal materials shared partial molecular commonality with drusen, including unesterified cholesterol, apolipoprotein E, complement factor H, and vitronectin. Esterified cholesterol, however, was undetectable, as was immunoreactivity for photoreceptor, Müller cell and RPE marker proteins. Subretinal drusenoid deposits were proposed as the correlate to pseudodrusen by these authors because the size, distribution and prevalence of the material seen in a series of 22 donor eyes corresponded so closely to pseudodrusen imaged clinically by several methods. In 1 case reported by Sarks and coworkers, an eye with pseudodrusen was examined histologically and found to correlate with subretinal material. These authors declined to attribute all pseudodrusen to such deposits, however, because the smallest ones were not detectable clinically.
Given the paucity of histologic examination of clinically characterized eyes, 2 recent advances in ocular imaging have made it possible to address questions of pseudodrusen localization in vivo. First, the living retina can be imaged with significantly improved resolution and precision in 3 dimensions. Spectral-domain OCT can image cross-sections of retina and choroid with sufficient resolution to reveal cellular and subcellular stratifications. A recent study has demonstrated the feasibility of using adaptive optics (AO) to image cones in the maculae of patients with pseudodrusen. However, the study was conducted with a flood-illumination AO imaging system that did not possess depth-discrimination capability. AO-assisted confocal SLO (AOSLO) has improved depth discrimination ability, so it can form images using light emanating from the selected planes in the fundus. Multimodal imaging featuring AOSLO thus has the potential to answer whether the histologic correlates of pseudodrusen are in the subretinal space. Second, information from multiple imaging technologies can be merged readily to bring the advantages of each individual technique to bear on a single question, resulting in a more comprehensive understanding. Therefore, the purpose of the present study was 2-fold: to correlate AOSLO findings with spectral-domain-OCT so as to determine precisely the laminar localization of pseudodrusen and to investigate how the AOSLO findings inform the imaging characteristics of pseudodrusen obtained by other more established modalities.
The study followed the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board at the University of Alabama at Birmingham. Written informed consent was obtained from participants after the nature and possible consequences of the study were explained. The study complied with the Health Insurance Portabilityand Accountability Act of 1996.
In previous studies, the terms reticular pseudodrusen , pseudo-reticular drusen and pseudodrusen were used for different en face modalities, and the term subretinal drusenoid deposits was used for cross-sectional histology and spectral-domain OCT. In the present article, we use the term pseudodrusen for standard en face ophthalmoscopy (color fundus photography, infrared reflectance [IR], red-free [RF], and autofluorescence [AF]) and the term subretinal drusenoid deposits for the collections of material seen in the subretinal space in histologic specimens, AOSLO and OCT.
Patients and Controls
Study patients with AMD and age-similar subjects with normal retinas were recruited from the clinical research registry of the Department of Ophthalmology of the University of Alabama at Birmingham and through the Retina Service. The patients had been diagnosed with AMD previously. To assess the disease severity, stereoscopic color digital 30 degree fundus photographs were taken with an FF450 Plus fundus camera (Carl Zeiss Meditec, Dublin, California) after pupil dilation, and photographs were graded by a masked, experienced grader using the Age-Related Eye Disease Study 2 (AREDS2) severity scale for AMD. Disease severity ranged from early to advanced (AREDS grade 2–11). Participants in normal macular health met the criteria for AREDS grade 1 in both eyes. Exclusion criteria included diabetes, history of retinal vascular occlusions, and any signs or history of hereditary retinal dystrophy. Subjects were also excluded for reasons that might potentially prevent successful imaging, such as poor fixation, significant media opacity, irregular pupil shape, poor dilation, or refractive errors beyond ±6 diopters (D) spherical and ±3 D cylinder. The inclusion criteria for normal comparison subjects were the same, with the additional criteria of age greater than 50 years, no clinically significant cataract, and best-corrected visual acuity of 20/25 or better.
High-Resolution AOSLO Imaging
Imaging was conducted with a new-generation AOSLO that was developed in our laboratory, as described. This instrument was based on an earlier prototype that used a microelectric-mechanical system–based deformable mirror. The new instrument addressed several major obstacles that impede imaging of older patients with AMD. First, the pupil size decreases and the wavefront aberration increases with aging. Second, many older patients have cataract, which significantly affects wavefront detection and impairs AO wavefront correction. Third, in patients with intraocular lenses, it is very common that the proliferation and transformation of lens epithelial cell remnants lead to posterior capsular opacification or fibrosis over the intraocular lens. Although capsulotomy can make an opening on the opacified posterior capsule, the clear pupil often has an irregular shape for imaging. This may not only reduce the useful pupil size but may also cause complicated light scattering that impedes AO operation.
To address these challenges, we developed an advanced wavefront detection and correction strategy. We designed a high-speed Shack-Hartmann wavefront sensor based on a complementary metal-oxide-semiconductor (CMOS) camera (MicroVista-NIR; Intevac, Santa Clara, California). The camera’s spectral response is optimized for the imaging light used by AOSLO, enabling the AO system to be operated at a loop frequency up to 100 Hz. Most previously reported AO systems for retinal imaging run under 30 Hz. High-speed wavefront sensing significantly facilitates advanced control strategy based upon Zernike mode correction, thereby allowing for AO correction of wavefronts in eyes with the optical problems described above. Furthermore, we adopted a high-speed deformable mirror (Hi-Speed DM97-15; ALPAO SAS, Montbonnot St. Martin, France) with 97 actuators with stroke up to 30 μm, which provides improved ability to compensate for increased high orders and amplitudes of wavefront aberration due to aging. The AOSLO pupil size was set at 5.6 mm in diameter. After AO correction, the root-mean-square wave aberration was reduced to less than 0.05 μm in most eyes, reaching the criterion for diffraction-limited resolution for the light used in imaging. A low-coherence light source, a superluminescent diode (Broadlighter S840-HP; Superlum, Carrigtwohill Co, Cork, Ireland) was employed for producing high-fidelity retinal images. The imaging light power measured at the cornea was 500 μW, which is about 1/26 of the maximum permitted exposure limits set by the American National Standards Institute. The AOSLO records continuous videos from the eye with a frame rate of 15 Hz.
All participants underwent best-corrected visual acuity measurement by the Electronic Visual Acuity protocol. Pupils were dilated with 1.0% tropicamide and 2.5% phenylephrine hydrochloride. The subject’s head was aligned and stabilized using a head mount with a chin rest. A fixation target consisting of a moving bright green dot formed by the light from a laser diode (DJ532-10- 532; Thorlabs, Newton, New Jersey) on a calibrated grid. The grid, on white paper, was placed in front of the eye via a pellicle beam splitter (BP208, Thorlabs) to help the subject’s fixation. The wavelength of the diode laser was 532 nm, and the light was coupled into a single-mode fiber and collimated to form a light dot of 1.5 mm diameter on the grid paper. The light power was adjusted to 2 mW at the output of the fiber. The subject saw the light dot on the back of the grid paper through a pellicle beam splitter. During imaging, the dot was moved on the grid paper to direct the subject’s view angle. At each grid point, the light dot stopped for 3–5 seconds so that 45–75 frames were acquired. Videos were recorded continuously across an area of 15 × 15 degrees. An AOSLO imaging session lasted approximately 1 hour. Data presented were collected from 1 imaging session for each subject. Before images were recorded, the gain of the AOSLO photodetector was titrated to obtain proper image brightness and contrast according to a real-time histogram of a retinal video, and it remained constant through the whole imaging session.
AOSLO Image Processing and Analysis
Image distortions caused by nonlinearities in the resonant scanner and by eye movements were eliminated by customized software. Registered images were averaged to enhance signal-to-noise ratio. Images of various retinal locations were manually aligned on a cell-to-cell basis to create a continuous montage (Photoshop; Adobe Systems, Mountain View, California).
AOSLO image pixel size was computed from an image of a precisely calibrated dot grid placed at the retinal plane of a model eye. The extent of retina affected by the underlying subretinal drusenoid deposits at different progression stages was measured at multiple locations, using the Ruler Tool of Photoshop, and then averaged.
In addition to stereoscopic color digital 30 degree fundus photographs, enface IR (λ = 830 nm); RF (λ = 560 nm); and AF (excitation, 488 nm; emission >600 nm) images were acquired with the confocal SLO of the Spectralis (Heidelberg Engineering, Carlsbad, California). Fields of view of 30 × 30 degrees were digitized at 768 × 768 pixels. Retinal cross-sections were imaged using the Spectralis spectral-domain OCT (λ = 870 nm; acquisition speed, 40,000 A-scans per second; scan depth, 1.9 mm; digital depth resolution, 3.5 μm per pixel in tissue; lateral resolution in tissue 14 μm). In each study eye, 97 B-scans were acquired across a 15 × 10 degree area of the central macula to create a volume.
Multiple imaging modalities can disclose pseudodrusen with differing specificity and sensitivity. It is recommended that detection should be confirmed using more than 1 modality so as to improve accuracy. The identification of pseudodrusen in our study was based on their presence in at least 2 en face imaging modalities and in spectral-domain OCT. In en face imaging, pseudodrusen appear as an interlacing collection of ribbons or a dotlike pattern of yellow-white lesions (in color fundus photography); as a pattern of hyporeflective or hyper-reflective spots (in IR reflectance); or as a pattern of small hypoautofluorescent areas against a background of mild hyperautofluorescence (in AF). Although a reticular pattern was often seen in color photography, it was not a criterion for diagnosis. In spectral-domain OCT, subretinal drusenoid deposits were hyper-reflective mounds internal to the RPE. Axial microstructure and location of subretinal drusenoid deposits were evaluated by spectral-domain OCT, using the nomenclature of Spaide and Curcio for the 4 outer retinal hyper-reflective bands.
Multimodal Image Registration
Color fundus photographs and IR, RF and AF images taken with the Spectralis SLO were registered manually by use of retinal vessels and capillaries as invariant landmarks. Color fundus photographs and the SLO IR image were then magnified and registered with the AOSLO montage by using retinal vessels and capillaries as landmarks. Then pseudodrusen apparent on standard funduscopy were localized in AOSLO images and examined with high resolution.
Subretinal Drusenoid Deposits Classification
Lesions were scored by the 3-stage grading system introduced by Zweifel and coworkers. Specific lesions at each stage and surrounding photoreceptors were examined by AOSLO and by en face OCT.
A total of 63 subjects (33 males and 30 females) were enrolled between October 2010 and January 2013, including 53 patients with AMD (73.50 ± 8.08 years of age, mean ± standard deviation) and 10 control subjects (64.0 ± 9.93 years of age). All subjects were white and non-Hispanic. Subretinal drusenoid deposits were found in 26 eyes of 13 patients with AMD (13/53; 24.5%). Of the eyes, 1 eye (1/26; 4%) was at AREDS grade 4 (early stage); 16 eyes (16/26; 62%) were at AREDS grade 5–8 (intermediate stages); 4 eyes (4/26; 15%) were at AREDS grades 9–10 (advanced stage, geographic atrophy [GA]); 5 eyes (5/26; 19%) were at AREDS grade 11 (advanced stage, choroidal neovascularization [CNV]). There were 11 patients who had both subretinal drusenoid deposits and conventional sub-RPE drusen, and 2 patients who had only subretinal drusenoid deposits. AOSLO imaged 18 eyes of 11 patients. Two patients (4 eyes) were not imaged by AOSLO due to small pupil (subject 2, AREDS OD:5, OS:5) and poor fixation (subject 7, AREDS, OD 11, CNV; OS 9, GA). No subretinal drusenoid deposits were found in eyes at AREDS grade 1.
Figure 1 shows images of a normal subject. AOSLO revealed clearly the mosaic of cone and rod inner segments across the macula. Although individual photoreceptors manifest varying brightness, brightness is similar across clusters of adjacent photoreceptors; accordingly, spectral-domain OCT reveals even and well-aligned outer retinal hyper-reflective bands without discernible hyper-reflective material between the RPE and ellipsoid zone (EZ) bands.
The effects of subretinal drusenoid deposits on surrounding cells are readily imaged with AOSLO at differing lesion stages. At stage 1 ( Figure 2 ), the EZ band undulates due to a granular hyper-reflective material accumulated between it and the RPE band. The overlying photoreceptors exhibit reduced reflectivity, and the mosaic is undetectable by AOSLO. At stage 2 ( Figure 3 ), the EZ band is appreciably deflected inwardly by mounds of accumulated material. The retina superjacent to each lesion shows further reduced overall reflectance, and individual photoreceptors are no longer visible by AOSLO. At stage 3 ( Figure 4 ), subretinal drusenoid deposits have interrupted the EZ band and extended to the inwardly deviated external limiting membrane (ELM). Surrounding the lesion apex is a region of absent OCT signal from the EZ and a lack of visualized cones. By AOSLO, the retina immediately adjacent to subretinal drusenoid deposits shows a hyporeflective annular zone with indistinct photoreceptors, which corresponds to the hyporeflective EZ gaps. As discussed in the next section, this annular zone may contain missing, degenerated or deflected photoreceptors. AOSLO also reveals within the annulus a reflective center area with a granular structure that is similar to the surrounding retina ( Figure 4 , bottom left; Figure 5 , top panel). Outside the annulus, the cone mosaic resumes, albeit at variable reflectivity levels relative to normal photoreceptors ( Figure 1 ). En face OCT imaging ( Figure 5 , bottom row) of a large solitary subretinal drusenoid deposit also shows a hyporeflective annulus of the stage 3 lesion like that revealed by AOSLO. The hyper-reflective center in AOSLO imaging is the subretinal drusenoid deposit material itself, as confirmed by en face reconstruction of OCT scans ( Figure 5 , bottom row), as well as AO-assisted spectral-domain OCT.