En face imaging with scanning laser ophthalmoscope

Near-infrared (NIR) light was used to perform en face imaging with a confocal scanning laser ophthalmoscope (SLO; HRA2, Heidelberg Engineering GmbH, Heidelberg, Germany) without subjecting the diseased retina to undue light exposure [28]. Reflectivity distribution of retinal and subretinal features was imaged with 820 nm NIR light. Health of the RPE was estimated with NIR-autofluorescence (NIR-AF) using 790 nm excitation light and a long-pass blocking filter that allowed detection of fluorescence emissions of >810 nm [16,28-31]. NIR-AF signal is believed to be dominated by the melanolipofuscin in RPE and melanin in the RPE and choroid [16,28-36]. Disease-related changes in RPE melanin content result in spatial variation of the NIR-AF signal intensity, appearance, or both. Imaging was performed in the high-speed mode; 30°×30° of retina was sampled onto a 768×768 pixel image, and video segments of up to 10 s length were obtained at the rate of 8.8 Hz. Detector sensitivity was set to 95% for NIR-AF. Automatic real-time (ART) averaging feature of the manufacturer’s software was used whenever possible. When ART failed, images were exported from the manufacturer’s software and analyzed as previously described [16,28-31].

Optical coherence tomography

Retinal cross-sections were obtained with optical coherence tomography (OCT). Data were acquired in both patients with Fourier-domain (FD) OCT imaging (RTVue-100; Optovue Inc., Fremont, CA). The principles of the method and our recording and analysis techniques have been published [6,10,14,29,37]. For this work, the line protocol of the FD-OCT system, with greater speed of acquisition, was used to obtain 4.5-mm-long scans composed of 1,019 longitudinal reflectivity profiles (LRPs) acquired in approximately 4 ms. Overlapping, nonaveraged, OCT scans were used to produce a digital montage covering up to 9 mm eccentricity from the fovea along the horizontal meridian. A video fundus image was acquired with each OCT scan. Regions of interest in extramacular retina were examined with single horizontal scans; the location and orientation of the scan relative to retinal features (blood vessels, RPE depigmentation, and optic nerve head) were determined by using the digital images of the fundus.

Postacquisition processing of OCT data was performed with custom programs (MATLAB 6.5, MathWorks, Natick, MA). LRPs that composed the OCT scans were aligned by straightening the major RPE reflection [6,10,14,37]. Two nuclear layers, the outer photoreceptor nuclear layer (ONL) and the inner nuclear layer (INL), were defined in regions of scans showing two parallel stereotypical hyporeflective layers sandwiched between the RPE and vitreoretinal interface [6,14,29]. Inner retinal thickness was defined as the distance between the signal transition at the vitreoretinal interface and the sclerad boundary of the INL or the single hyporeflective layer continuous with the INL [6,29,37]. In normal participants, the signal corresponding to the RPE was assumed to be the most sclerad peak within the multi-peaked scattering signal complex [37], deep in the retina. In abnormal retinas, the presumed RPE peak was sometimes the only signal peak deep in the retina; in other cases, it was apposed by other major peaks. In the latter case, the RPE peak was specified manually by considering the properties of the backscattering signal originating from layers vitread and sclerad to it [38]. Also assessed were the presence of photoreceptor inner segment (IS) and outer segment (OS) signal and outer limiting membrane (OLM) signals vitread to the RPE peak [38].

Clinical features and en face infrared fundus images

P1, at age 6 years, had light perception vision, hyperopia (+6.50 sphere), nystagmus, and no corneal or lenticular opacities. An en face montage of the ocular fundus of P1 using NIR reflectance imaging showed a distinctly demarcated dark central island surrounded by alternating elliptical regions of lighter and darker appearance (Figure 1A). A schematic of the darker-appearing regions is shown (Figure 1A, inset left). The reflectance pattern in LCA5 P1 was in contrast to the more homogeneous NIR reflectance view from an age-matched normal subject (Figure 1A, inset right). The lighter regions in LCA5 P1 showed greater visibility of the choroid, suggesting depigmentation of RPE. Darker regions likely correspond to more preserved RPE. NIR-AF provided further information on the RPE with the use of melanosome-specific signals from the fundus [31]. High intensity NIR-AF signal originates from the irregular-shaped central island, suggesting a preserved or hyperpigmented RPE at this location (Figure 1B). There was a region of lower intensity NIR-AF in the parafovea, and this likely corresponds to chorioretinal atrophic change. At greater eccentricities, there was an incremental increase in NIR-AF. In the superotemporal and superonasal near midperiphery, there was a distinct boundary of a further increase in NIR-AF signal with a spatially homogeneous appearance, representing more retained and pigmented RPE.

Open in a separate window

Figure 1

En face near-infrared reflectance and autofluorescence images of the LCA5 patients. A: Near-infrared (NIR) reflectance (REF) image of the left fundus of P1 is shown. Inset to the left is a schematic drawing of the retinal regions corresponding to low reflection (black), intermediate reflection (hatched), and high reflection and choroidal visibility (white). Inset to the right is a NIR reflectance view of the fundus of a 6 year-old child with normal vision. B: Near-infrared-autofluorescence (NIR-AF) image of the left fundus of P1 is shown. Black arrows indicate the boundaries of the midperipheral transitions to healthier retinal pigment epithelium; and gray arrow points to the parafoveal annular region of low intensity. Inset is a normal image. C: NIR reflectance image of the left fundus of P2 is shown. Inset is an enlarged view of the optic nerve head (ONH) region with ONH drusen. D: NIR-AF image of the left fundus of P2 is shown. Inset is an enlarged view of the ONH region. All images are shown contrast stretched for visibility of features.

LCA5 patient 2 (P2) was followed from age 6 months through age 25 years. Visual acuity loss increased from 7/200 (right eye) and 20/400 (left eye) at age 6 years to light perception in both eyes at age 25. A high hyperopic refractive error was present at all visits (+10.00 sphere); there was nystagmus but no corneal or lenticular opacities. Funduscopic examinations throughout the years noted retina-wide granular-appearing pigmentary disturbances and optic disc drusen in both eyes, a finding previously noted in LCA5 [1]. An en face montage of the ocular fundus of P2 at age 25, using NIR reflectance imaging, showed a light appearance with visibility of the choroid, suggesting depigmentation of RPE (Figure 1C). NIR-AF displayed a choroidal-appearing pattern; specifically there was no evidence of a central region of hyperautofluorescence or peripheral boundary to a relatively increased signal (Figure 1D). The optic disc drusen were evident in the NIR reflectance image (Figure 1C, inset) and they revealed hyperautofluorescence under NIR excitation (Figure 1D, inset). Optic disc drusen are known to show AF under short-wavelength excitation [40], but their NIR-AF signals have not been described.

Between ages 8 and 25 years, P2 was able to perform kinetic perimetry and showed only a central island of perception in each eye of roughly 2–3 degrees in diameter (with a large bright target, V-4e). Static threshold perimetry, using an achromatic target (size V, but 1 log unit brighter than the kinetic perimeter 4e) in the dark-adapted state, was performed in the left eye of P2. This was initially done when the patient was 17 years old and then repeated at 25 years of age. The target was only detected in the central field, and thresholds were elevated by >5.5 log units at both visits [27].

Cross-sectional retinal imaging: preserved photoreceptors adjacent to retinal disorganization

Retinal laminar architecture of LCA5 P1 at age 6 years and P2 at 25 years was examined by high resolution cross-sectional OCT imaging (Figure 2). The central 14 mm scan of retina along the horizontal meridian in a normal subject (Figure 2A, upper) illustrates the foveal depression and the hyporeflective and hyperreflective layers that have been shown to have a predictable relationship to histologically defined layers [14,29,37]. Lamination was also present in the patient scans, but there were abnormalities (Figure 2A, middle and lower). Both patients had a foveal depression; this was identified as the deepest pit on raster scanning. P2 had far less depth to the foveal pit than P1; there was notable epiretinal membrane in this scan and in others (data not shown). Unlike the representative normal scan, both patients also showed some tissue vitread to the foveal ONL. The epifoveal tissue in P1 appeared continuous with parafoveal INL and inner plexiform and retinal ganglion cell layers. Foveal ONL thickness in P1 measured 43 µm, which is reduced to about 48% of normal (normal mean±SD=90±8.6 µm; n=5; ages 5–15 years), suggesting loss of foveal cone cells. Foveal ONL thickness in P2 measured 101 µm, which is within normal limits. Whether this hyporeflectivity measured at the fovea in P2 represents only cone nuclei or is a more complex structure with, for example, Müller cell hypertrophy is uncertain [38]. Eccentric to roughly 1.5 mm nasal and temporal to the fovea, the ONL in both patients is barely discernible. At approximately 7 mm eccentricity into the temporal retina, normal lamination is no longer present, and the patient scans have almost a “bilaminar appearance” with a thick vitread hyperreflective layer and a deep thickened hyporeflective layer. The thick superficial layer likely includes inner plexiform layer and retinal ganglion cell layer, and the deeper layer may be an amalgam of thickened INL with remnant photoreceptor nuclei [16,29]. Retinal thickness and especially inner retinal thickness is remarkably greater in P2 than in P1.

Open in a separate window

Figure 2

Dysmorphology in the retina of LCA5. A: Cross-sectional OCT images across the horizontal meridian are shown for a normal 6-year-old subject (upper) and LCA5, P1 (middle) and P2 (lower). Layers or structures are labeled as individual or combined laminae. IPL+RGC, inner plexiform and retinal ganglion cell layers; INL, inner nuclear layer; ONL, outer nuclear layer; OLM, outer limiting membrane; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium. N: Nasal, T: Temporal retina. B: Central scans are shown for a normal subject, P1, P2, and an RP patient with a residual and abnormally reduced central island of retinal structure. ONL layer is highlighted in blue. C: Photoreceptor nuclear layer thickness horizontally across the central 4 mm of retina is graphically displayed for a group of normal subjects (gray represents mean±2SD; n=26; ages 5–58 years), 19 patients with retinal degeneration but not LCA5, and the LCA5 data. For comparison with LCA5 P1 and P2 with light perception (LP) vision, the ONL data from the patients with retinal degeneration are color-coded by their visual acuity levels. D: Magnified (1.2 mm across) horizontal cross-sections through the fovea of LCA5 P1 and P2 are compared to those of the 6-year-old normal subject (left panel) and an RP patient (right panel) with similarly reduced foveal ONL. E: Cross-sectional, 0.9 mm-long, extramacular images from LCA5 P1 and P2 are compared to a normal subject. Longitudinal reflectivity profiles (LRP, white traces) overlaid on the scans show signal features corresponding to the different retinal laminae. The ONL is highlighted (blue) next to the corresponding LRP signal feature. LCA5 P1 (left) at 7 to 7.8 mm in nasal retina shows remnants of ONL, retained retinal lamination and a thickened inner retina (bracketed to the left of the scans) compared to the normal subject at the same eccentricity. Scans from 7 mm in temporal retina from both LCA5 patients show complete loss of ONL signal and retinal disorganization with a bilaminar appearance of the LRPs.

We determined whether the residual foveal ONL in the LCA5 patients was expected to be associated with light perception vision. In vivo histopathology of the LCA5 patients was compared to a group of patients with retinal degeneration and similar foveal ONL thickness, and who had been found, by dark-adapted chromatic perimetry, to be at a stage of disease that had reduced vision to a central island with only abnormal cone function [41] (Figure 2B-D).

The 19 patients with retinal degeneration compared with LCA5 patients were in the general clinical category of retinitis pigmentosa (RP) and showed a range of visual acuities from 20/20 to 20/200. These patients were 21 to 59 years of age with diagnoses that included X-linked RP due to RPGR mutations, Usher syndrome 1B and 2A, and ungenotyped RP. Their ONL profiles were similar to those of the LCA5 patients (Figure 2C). This suggested that the foveal cone cells in the LCA5 patients were not functioning optimally.

Magnified views of the foveal center of P1 and P2 (Figure 2D) demonstrate that the IS/OS signal appears ill-defined in P1 or nearly not discernible in P2. For comparison are similar views of the foveal center for a normal subject and for an RP patient. The RP patient has thinned ONL but more definable IS/OS signal and better foveal function (visual acuity, 20/40). This abnormal signal deep to the foveal cone ONL may indicate structural abnormalities in the cone IS/OS, possibly a consequence of a ciliopathy, with resultant visual dysfunction. At greater eccentricities there was severe ONL reduction. Remnants of ONL observed in the nasal retina of P1 were accompanied by a thickened inner retina (Figure 2E, left P1 panel) suggestive of retinal remodeling. Total photoreceptor layer loss in both patients resulted in a disorganized retinal structure with a bilaminar appearance (Figure 2E, right two panels) with a hyper-thick (P1=148 µm; P2=212 µm) inner retina (normal mean±SD=108±20 µm).

This study was supported by the Foundation Fighting Blindness, Hope for Vision, Macula Vision Research Foundation, and the Grousbeck Family Foundation.