Assessing the Sensitivity of OCT-A Retinal Vasculature Metrics.

Purpose: The purpose of this study was to examine the sensitivity of quantitative metrics of the retinal vasculature derived from optical coherence tomography angiography (OCT-A) images. Methods: Full retinal vascular slab OCT-A images were obtained from 94 healthy participants. Capillary loss, at 1% increments up to 50%, was simulated by randomly removing capillary segments (1000 iterations of randomized loss for each participant at each percent loss). Thirteen quantitative metrics were calculated for each image: foveal avascular zone (FAZ) area, vessel density, vessel complexity index (VCI), vessel perimeter index (VPI), fractal dimension (FD), and parafoveal intercapillary area (PICA) measurements with and without the FAZ (mean PICA, summed PICA, PICA regularity, and PICA standard deviation [PICA SD]). The sensitivity of each metric was calculated as the percent loss at which 80% of the iterations for a participant fell outside of two standard deviations from the sample's normative mean. Results: The most used OCT-A metrics, FAZ area and vessel density, were not significantly different from normative values until 27.69% and 16.00% capillary loss, respectively. Across the remaining metrics, metric sensitivity ranged from 6.37% (PICA SD without FAZ) to 39.78% (Summed PICA without FAZ). Conclusions: The sensitivity of vasculature metrics for detecting random capillary loss varies substantially. Further efforts simulating different patterns of capillary loss are needed for comparison. Additionally, mapping the repeatability of metrics over time in a normal population is needed to further define metric sensitivity. Translational Relevance: Quantitative metrics vary in their ability to detect vascular abnormalities in OCT-A images. Metric choice in screening studies will need to balance expected capillary abnormalities and the quality of the OCT-A images being used.

Long-term retinal imaging of a case of suspected congenital rubella infection.

PURPOSE: Many retinal disorders present with pigmentary retinopathy, most of which are progressive conditions. Here we present over nine years of follow up on a case of stable pigmentary retinopathy that is suspected to stem from a congenital rubella infection. Parafoveal cone photoreceptors were tracked through this period to gain insight into photoreceptor disruption in this pigmentary retinopathy. METHODS: The patient was examined at 8 visits spanning a total of 111 months. Examination at baseline included clinical fundus examination, full-field electroretinography (ERG), kinetic visual field assessment (Goldmann), and best corrected visual acuity; all of these except ERG were repeated at follow up visits. Imaging was performed with fundus photography, spectral-domain optical coherence tomography (SD-OCT) and confocal adaptive optics scanning light ophthalmoscopy (AOSLO). For the latter four time points AOSLO imaging also included split-detector imaging. RESULTS: There were no defects in hearing or cardiac health found in this patient. There were minimal visual deficits found at baseline, with mild rod suppression on ERG; best corrected visual acuity was 20/25 OD and 20/20 OS at baseline, which was stable throughout the follow-up period. Retinal thickness as measured by OCT was within the normal range, though foveal hypoplasia was present and outer nuclear layer thickness was slightly below the normal range at all time points. Cone density was relatively stable throughout the follow-up period. A number of cones were non-reflective when observed with confocal AOSLO imaging and density was markedly lower than expected values (foveal cone density was 43,782 cones/mm2 on average). Genetic analysis revealed no causative variations explaining the phenotype. CONCLUSIONS AND IMPORTANCE: This patient appears to have a stable pigmentary retinopathy. This case is likely due to a congenital insult, rather than progressive retinal disease. This finding of stability agrees with other reports of rubella pigmentary retinopathy. Imaging with AOSLO enabled observation of two notable phenotypic features. First is the observation of dark cones, which are seen in many retinal disorders including color vision defects and degenerative retinal disease. Second, the cone density is well below what is expected - this is especially interesting as this patient has near-normal visual acuity despite this greatly decreased number of normally-waveguiding cones in the fovea.

Comparing Retinal Structure in Patients with Achromatopsia and Blue Cone Monochromacy Using OCT.

Purpose: To compare foveal hypoplasia and the appearance of the ellipsoid zone (EZ) at the fovea in patients with genetically confirmed achromatopsia (ACHM) and blue cone monochromacy (BCM). Design: Retrospective, multi-center observational study. Subjects: Molecularly confirmed patients with ACHM (n = 89) and BCM (n = 33). Methods: We analyzed high-resolution spectral domain optical coherence tomography (SD-OCT) images of the macula from aforementioned patients with BCM. Three observers independently graded SD-OCT images for foveal hypoplasia (i.e. retention of one or more inner retinal layers at the fovea) and four observers judged the integrity of the EZ at the fovea, based on an established grading scheme. These measures were compared with previously published data from the ACHM patients. Main Outcome Measures: Presence of foveal hypoplasia and EZ grade. Results: Foveal hypoplasia was significantly more prevalent in ACHM than in BCM (p<0.001). In addition, we observed a significant difference in the distribution of EZ grades between ACHM and BCM, with grade II EZ being by far the most common phenotype in BCM (61% of patients). In contrast, ACHM patients had a relatively equal prevalence of EZ grades I, II, and IV. Interestingly, grade IV EZ was 2.6 times more prevalent in ACHM compared to BCM, while grade V EZ (macular atrophy) was present in 3% of both the ACHM and BCM cohorts. Conclusions: The higher incidence of foveal hypoplasia in ACHM than BCM supports a role for cone activity in foveal development. Although there are differences in EZ grades between these conditions, the degree of overlap suggests EZ grade is not sufficient for definitive diagnosis, in contrast to previous reports. Analysis of additional OCT features in similar cohorts may reveal differences with greater diagnostic value. Finally, the extent to which foveal hypoplasia or EZ grade is prognostic for therapeutic potential in either group remains to be seen, but motivates further study.

Interocular Symmetry of Foveal Cone Topography in Congenital Achromatopsia.

Purpose: To determine the interocular symmetry of foveal cone topography in achromatopsia (ACHM) using non-confocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Methods: Split-detector AOSLO images of the foveal cone mosaic were acquired from both eyes of 26 subjects (mean age 24.3 years; range 8-44 years, 14 females) with genetically confirmed CNGA3- or CNGB3-associated ACHM. Cones were identified within a manually delineated rod-free zone. Peak cone density (PCD) was determined using an 80 × 80 µm sampling window within the rod-free zone. The mean and standard deviation (SD) of inter-cell distance (ICD) were calculated to derive the coefficient of variation (CV). Cone density difference maps were generated to compare cone topography between eyes. Results: PCD (mean ± SD) was 17,530 ± 9,614 cones/mm2 and 17,638 ± 9,753 cones/mm2 for right and left eyes, respectively (p = .677, Wilcoxon test). The mean (± SD) for ICD was 9.05 ± 2.55 µm and 9.24 ± 2.55 µm for right and left eyes, respectively (p = .410, paired t-test). The mean (± SD) for CV of ICD was 0.16 ± 0.03 µm and 0.16 ± 0.04 µm for right and left eyes, respectively (p = .562, paired t-test). Cone density maps demonstrated that cone topography of the ACHM fovea is non-uniform with local variations in cone density between eyes. Conclusions: These results demonstrate the interocular symmetry of the foveal cone mosaic (both density and packing) in ACHM. As cone topography can differ between eyes of a subject, PCD does not completely describe the foveal cone mosaic in ACHM. Nonetheless, these findings are of value in longitudinal monitoring of patients during treatment trials and further suggest that both eyes of a given subject may have similar therapeutic potential and non-study eye can be used as a control.

Assessing the Interocular Symmetry of Foveal Outer Nuclear Layer Thickness in Achromatopsia.

PURPOSE: We examine the interocular symmetry of foveal outer nuclear layer (ONL) thickness measurements in subjects with achromatopsia (ACHM). METHODS: Images from 76 subjects with CNGA3- or CNGB3-associated ACHM and 42 control subjects were included in the study. Line or volume scans through the fovea of each eye were acquired using optical coherence tomography (OCT). Image quality was assessed for each image included in the analysis using a previously-described maximum tissue contrast index (mTCI) metric. Three foveal ONL thickness measurements were made by a single observer and interocular symmetry was assessed using the average of the three measurements for each eye. RESULTS: Mean (± standard deviation) foveal ONL thickness for subjects with ACHM was 79.7 ± 18.3 µm (right eye) and 79.2 ± 18.7 µm (left eye) compared to 112.9 ± 15.2 (right eye) and 112.1 ± 13.9 µm (left eye) for controls. Foveal ONL thickness did not differ between eyes for ACHM (P = 0.636) or control subjects (P = 0.434). No significant relationship between mTCI and observer repeatability was observed for either control (P = 0.140) or ACHM (P = 0.351) images. CONCLUSIONS: While foveal ONL thickness is reduced in ACHM compared to controls, the high interocular symmetry indicates that contralateral ONL measurements could be used as a negative control in early-phase monocular treatment trials. TRANSLATIONAL RELEVANCE: Foveal ONL thickness can be measured using OCT images over a wide range of image quality. The interocular symmetry of foveal ONL thickness in ACHM and control populations supports the use of the non-study eye as a control for clinical trial purposes.

Characterization of Retinal Structure in ATF6-Associated Achromatopsia.

Purpose: Mutations in six genes have been associated with achromatopsia (ACHM): CNGA3, CNGB3, PDE6H, PDE6C, GNAT2, and ATF6. ATF6 is the most recent gene to be identified, though thorough phenotyping of this genetic subtype is lacking. Here, we sought to test the hypothesis that ATF6-associated ACHM is a structurally distinct form of congenital ACHM. Methods: Seven genetically confirmed subjects from five nonconsanguineous families were recruited. Foveal hypoplasia and the integrity of the ellipsoid zone (EZ) band (a.k.a., IS/OS) were graded from optical coherence tomography (OCT) images. Images of the photoreceptor mosaic were acquired using confocal and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Parafoveal cone and rod density values were calculated and compared to published normative data as well as data from two subjects harboring CNGA3 or CNGB3 mutations who were recruited for comparative purposes. Additionally, nonconfocal dark-field AOSLO images of the retinal pigment epithelium were obtained, with quantitative analysis performed in one subject with ATF6-ACHM. Results: Foveal hypoplasia was observed in all subjects with ATF6 mutations. Absence of the EZ band within the foveal region (grade 3) or appearance of a hyporeflective zone (grade 4) was seen in all subjects with ATF6 using OCT. There was no evidence of remnant foveal cone structure using confocal AOSLO, although sporadic cone-like structures were seen in nonconfocal split-detection AOSLO. There was a lack of cone structure in the parafovea, in direct contrast to previous reports. Conclusions: Our data demonstrate a near absence of cone structure in subjects harboring ATF6 mutations. This implicates ATF6 as having a major role in cone development and suggests that at least a subset of subjects with ATF6-ACHM have markedly fewer cellular targets for cone-directed gene therapies than do subjects with CNGA3- or CNGB3-ACHM.

Adaptive Optics Retinal Imaging in CNGA3-Associated Achromatopsia: Retinal Characterization, Interocular Symmetry, and Intrafamilial Variability.

Purpose: To investigate retinal structure in subjects with CNGA3-associated achromatopsia and evaluate disease symmetry and intrafamilial variability. Methods: Thirty-eight molecularly confirmed subjects underwent ocular examination, optical coherence tomography (OCT), and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). OCT scans were used for evaluating foveal hypoplasia, grading foveal ellipsoid zone (EZ) disruption, and measuring outer nuclear layer (ONL) thickness. AOSLO images were used to quantify peak foveal cone density, intercell distance (ICD), and the coefficient of variation (CV) of ICD. Results: Mean (±SD) age was 25.9 (±13.1) years. Mean (± SD) best corrected visual acuity (BCVA) was 0.87 (±0.14) logarithm of the minimum angle of resolution. Examination with OCT showed variable disruption or loss of the EZ. Seven subjects were evaluated for disease symmetry, with peak foveal cone density, ICD, CV, ONL thickness, and BCVA not differing significantly between eyes. A cross-sectional evaluation of AOSLO imaging showed a mean (±SD) peak foveal cone density of 19,844 (±13,046) cones/mm2. There was a weak negative association between age and peak foveal cone density (r = -0.397, P = 0.102), as well as between EZ grade and age (P = 0.086). Conclusions: The remnant cone mosaics were irregular and variably disrupted, with significantly lower peak foveal cone density than unaffected individuals. Variability was also seen among subjects with identical mutations. Therefore, subjects should be considered on an individual basis for stratification in clinical trials. Interocular symmetry suggests that both eyes have comparable therapeutic potential and the fellow eye can serve as a valid control. Longitudinal studies are needed, to further examine the weak negative association between age and foveal cone structure observed here.

Deep learning based detection of cone photoreceptors with multimodal adaptive optics scanning light ophthalmoscope images of achromatopsia.

Fast and reliable quantification of cone photoreceptors is a bottleneck in the clinical utilization of adaptive optics scanning light ophthalmoscope (AOSLO) systems for the study, diagnosis, and prognosis of retinal diseases. To-date, manual grading has been the sole reliable source of AOSLO quantification, as no automatic method has been reliably utilized for cone detection in real-world low-quality images of diseased retina. We present a novel deep learning based approach that combines information from both the confocal and non-confocal split detector AOSLO modalities to detect cones in subjects with achromatopsia. Our dual-mode deep learning based approach outperforms the state-of-the-art automated techniques and is on a par with human grading.

An Automated Reference Frame Selection (ARFS) Algorithm for Cone Imaging with Adaptive Optics Scanning Light Ophthalmoscopy.

PURPOSE: To develop an automated reference frame selection (ARFS) algorithm to replace the subjective approach of manually selecting reference frames for processing adaptive optics scanning light ophthalmoscope (AOSLO) videos of cone photoreceptors. METHODS: Relative distortion was measured within individual frames before conducting image-based motion tracking and sorting of frames into distinct spatial clusters. AOSLO images from nine healthy subjects were processed using ARFS and human-derived reference frames, then aligned to undistorted AO-flood images by nonlinear registration and the registration transformations were compared. The frequency at which humans selected reference frames that were rejected by ARFS was calculated in 35 datasets from healthy subjects, and subjects with achromatopsia, albinism, or retinitis pigmentosa. The level of distortion in this set of human-derived reference frames was assessed. RESULTS: The average transformation vector magnitude required for registration of AOSLO images to AO-flood images was significantly reduced from 3.33 ± 1.61 pixels when using manual reference frame selection to 2.75 ± 1.60 pixels (mean ± SD) when using ARFS (P = 0.0016). Between 5.16% and 39.22% of human-derived frames were rejected by ARFS. Only 2.71% to 7.73% of human-derived frames were ranked in the top 5% of least distorted frames. CONCLUSION: ARFS outperforms expert observers in selecting minimally distorted reference frames in AOSLO image sequences. The low success rate in human frame choice illustrates the difficulty in subjectively assessing image distortion. TRANSLATIONAL RELEVANCE: Manual reference frame selection represented a significant barrier to a fully automated image-processing pipeline (including montaging, cone identification, and metric extraction). The approach presented here will aid in the clinical translation of AOSLO imaging.

REPEATABILITY AND LONGITUDINAL ASSESSMENT OF FOVEAL CONE STRUCTURE IN CNGB3-ASSOCIATED ACHROMATOPSIA.

PURPOSE: Congenital achromatopsia is an autosomal recessive disease causing substantial reduction or complete absence of cone function. Although believed to be a relatively stationary disorder, questions remain regarding the stability of cone structure over time. In this study, the authors sought to assess the repeatability of and examine longitudinal changes in measurements of central cone structure in patients with achromatopsia. METHODS: Forty-one subjects with CNGB3-associated achromatopsia were imaged over a period of between 6 and 26 months using optical coherence tomography and adaptive optics scanning light ophthalmoscopy. Outer nuclear layer (ONL) thickness, ellipsoid zone (EZ) disruption, and peak foveal cone density were assessed. RESULTS: ONL thickness increased slightly compared with baseline (0.184 µm/month, P = 0.02). The EZ grade remained unchanged for 34/41 subjects. Peak foveal cone density did not significantly change over time (mean change 1% per 6 months, P = 0.126). CONCLUSION: Foveal cone structure showed little or no change in this group of subjects with CNGB3-associated achromatopsia. Over the time scales investigated (6-26 months), achromatopsia seems to be a structurally stable condition, although longer-term follow-up is needed. These data will be useful in assessing foveal cone structure after therapeutic intervention.

High-Resolution Imaging of Intraretinal Structures in Active and Resolved Central Serous Chorioretinopathy.

Purpose: We improved our understanding of central serous chorioretinopathy (CSC), we performed an analysis of noninvasive, high-resolution retinal imaging in patients with active and resolved CSC. Methods: Adaptive optics scanning light ophthalmoscopy (AOSLO) and spectral-domain optical coherence tomography (SD-OCT) were performed on five subjects with CSC. A custom AOSLO system was used to simultaneously collect confocal and split-detector images. Spectral domain-OCT volume scans were used to create en face views of various retinal layers, which then were compared to montaged AOSLO images after coregistration. Results: Three distinct types of intraretinal hyperreflective clusters were seen with AOSLO. These clusters had a well-demarcated, round, and granular appearance. Clusters in active CSC over areas of serous retinal detachment were termed type-1. They were found primarily in the outer nuclear layer (ONL) and were associated with large defects in the photoreceptor mosaic and ellipsoid zone. Clusters in areas where the retina had reattached were termed type-2. They also were located primarily in the ONL but showed stability in location over a period of at least 8 months. Smaller clusters in the inner retina along retinal capillaries were termed type-3. Conclusions: Retinal imaging in CSC using en face OCT and AOSLO allows precise localization of intraretinal structures and detection of features that cannot be seen with SD-OCT alone. These findings may provide greater insight into the pathophysiology of the active and resolved phases of the disease, and support the hypothesis that intraretinal hyperreflective foci on OCT in CSC are cellular in nature.

Evaluating outer segment length as a surrogate measure of peak foveal cone density.

Adaptive optics (AO) imaging tools enable direct visualization of the cone photoreceptor mosaic, which facilitates quantitative measurements such as cone density. However, in many individuals, low image quality or excessive eye movements precludes making such measures. As foveal cone specialization is associated with both increased density and outer segment (OS) elongation, we sought to examine whether OS length could be used as a surrogate measure of foveal cone density. The retinas of 43 subjects (23 normal and 20 albinism; aged 6-67years) were examined. Peak foveal cone density was measured using confocal adaptive optics scanning light ophthalmoscopy (AOSLO), and OS length was measured using optical coherence tomography (OCT) and longitudinal reflectivity profile-based approach. Peak cone density ranged from 29,200 to 214,000cones/mm2 (111,700±46,300cones/mm2); OS length ranged from 26.3 to 54.5µm (40.5±7.7µm). Density was significantly correlated with OS length in albinism (p<0.0001), but not normals (p=0.99). A cubic model of density as a function of OS length was created based on histology and optimized to fit the albinism data. The model includes triangular cone packing, a cylindrical OS with a fixed volume of 136.6µm3, and a ratio of OS to inner segment width that increased linearly with increasing OS length (R2=0.72). Normal subjects showed no apparent relationship between cone density and OS length. In the absence of adequate AOSLO imagery, OS length may be used to estimate cone density in patients with albinism. Whether this relationship exists in other patient populations with foveal hypoplasia (e.g., premature birth, aniridia, isolated foveal hypoplasia) remains to be seen.

Residual Foveal Cone Structure in CNGB3-Associated Achromatopsia.

PURPOSE: Congenital achromatopsia (ACHM) is an autosomal recessive disorder in which cone function is absent or severely reduced. Gene therapy in animal models of ACHM have shown restoration of cone function, though translation of these results to humans relies, in part, on the presence of viable cone photoreceptors at the time of treatment. Here, we characterized residual cone structure in subjects with CNGB3-associated ACHM. METHODS: High-resolution imaging (optical coherence tomography [OCT] and adaptive optics scanning light ophthalmoscopy [AOSLO]) was performed in 51 subjects with CNGB3-associated ACHM. Peak cone density and inter-cone spacing at the fovea was measured using split-detection AOSLO. Foveal outer nuclear layer thickness was measured in OCT images, and the integrity of the photoreceptor layer was assessed using a previously published OCT grading scheme. RESULTS: Analyzable images of the foveal cones were obtained in 26 of 51 subjects, with nystagmus representing the major obstacle to obtaining high-quality images. Peak foveal cone density ranged from 7,273 to 53,554 cones/mm2, significantly lower than normal (range, 84,733-234,391 cones/mm2), with the remnant cones being either contiguously or sparsely arranged. Peak cone density was correlated with OCT integrity grade; however, there was overlap of the density ranges between OCT grades. CONCLUSIONS: The degree of residual foveal cone structure varies greatly among subjects with CNGB3-associated ACHM. Such measurements may be useful in estimating the therapeutic potential of a given retina, providing affected individuals and physicians with valuable information to more accurately assess the risk-benefit ratio as they consider enrolling in experimental gene therapy trials. (www.clinicaltrials.gov, NCT01846052.).

Cone Photoreceptor Structure in Patients With X-Linked Cone Dysfunction and Red-Green Color Vision Deficiency.

PURPOSE: Mutations in the coding sequence of the L and M opsin genes are often associated with X-linked cone dysfunction (such as Bornholm Eye Disease, BED), though the exact color vision phenotype associated with these disorders is variable. We examined individuals with L/M opsin gene mutations to clarify the link between color vision deficiency and cone dysfunction. METHODS: We recruited 17 males for imaging. The thickness and integrity of the photoreceptor layers were evaluated using spectral-domain optical coherence tomography. Cone density was measured using high-resolution images of the cone mosaic obtained with adaptive optics scanning light ophthalmoscopy. The L/M opsin gene array was characterized in 16 subjects, including at least one subject from each family. RESULTS: There were six subjects with the LVAVA haplotype encoded by exon 3, seven with LIAVA, two with the Cys203Arg mutation encoded by exon 4, and two with a novel insertion in exon 2. Foveal cone structure and retinal thickness was disrupted to a variable degree, even among related individuals with the same L/M array. CONCLUSIONS: Our findings provide a direct link between disruption of the cone mosaic and L/M opsin variants. We hypothesize that, in addition to large phenotypic differences between different L/M opsin variants, the ratio of expression of first versus downstream genes in the L/M array contributes to phenotypic diversity. While the L/M opsin mutations underlie the cone dysfunction in all of the subjects tested, the color vision defect can be caused either by the same mutation or a gene rearrangement at the same locus.

Assessing Photoreceptor Structure in Retinitis Pigmentosa and Usher Syndrome.

PURPOSE: The purpose of this study was to examine cone photoreceptor structure in retinitis pigmentosa (RP) and Usher syndrome using confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO). METHODS: Nineteen subjects (11 RP, 8 Usher syndrome) underwent ophthalmic and genetic testing, spectral-domain optical coherence tomography (SD-OCT), and AOSLO imaging. Split-detector images obtained in 11 subjects (7 RP, 4 Usher syndrome) were used to assess remnant cone structure in areas of altered cone reflectivity on confocal AOSLO. RESULTS: Despite normal interdigitation zone and ellipsoid zone appearance on OCT, foveal and parafoveal cone densities derived from confocal AOSLO images were significantly lower in Usher syndrome compared with RP. This was due in large part to an increased prevalence of non-waveguiding cones in the Usher syndrome retina. Although significantly correlated to best-corrected visual acuity and foveal sensitivity, cone density can decrease by nearly 38% before visual acuity becomes abnormal. Aberrantly waveguiding cones were noted within the transition zone of all eyes and corresponded to intact inner segment structures. These remnant cones decreased in density and increased in diameter across the transition zone and disappeared with external limiting membrane collapse. CONCLUSIONS: Foveal cone density can be decreased in RP and Usher syndrome before visible changes on OCT or a decline in visual function. Thus, AOSLO imaging may allow more sensitive monitoring of disease than current methods. However, confocal AOSLO is limited by dependence on cone waveguiding, whereas split-detector AOSLO offers unambiguous and quantifiable visualization of remnant cone inner segment structure. Confocal and split-detector thus offer complementary insights into retinal pathology.

Noninvasive imaging of the thirteen-lined ground squirrel photoreceptor mosaic.

Ground squirrels are an increasingly important model for studying visual processing, retinal circuitry, and cone photoreceptor function. Here, we demonstrate that the photoreceptor mosaic can be longitudinally imaged noninvasively in the 13-lined ground squirrel (Ictidomys tridecemlineatus) using confocal and nonconfocal split-detection adaptive optics scanning ophthalmoscopy using 790 nm light. Photoreceptor density, spacing, and Voronoi analysis are consistent with that of the human cone mosaic. The high imaging success rate and consistent image quality in this study reinforce the ground squirrel as a practical model to aid drug discovery and testing through longitudinal imaging on the cellular scale.

Correlating Photoreceptor Mosaic Structure to Clinical Findings in Stargardt Disease.

PURPOSE: To demonstrate a method for correlating photoreceptor mosaic structure with optical coherence tomography (OCT) and microperimetry findings in patients with Stargardt disease. METHODS: A total of 14 patients with clinically diagnosed Stargardt disease were imaged using confocal and split-detection adaptive optics scanning light ophthalmoscopy. Cone photoreceptors were identified manually in a band along the temporal meridian. Resulting values were compared to a normative database (n = 9) to generate cone density deviation (CDD) maps. Manual measurement of outer nuclear layer plus Henle fiber layer (ONL+HFL) thickness was performed, in addition to determination of the presence of ellipsoid zone (EZ) and interdigitation zone (IZ) bands on OCT. These results, along with microperimetry data, were overlaid with the CDD maps. RESULTS: Wide variation in foveal structure and CDD maps was seen within this small group. Disruption of ONL+HFL and/or IZ band was seen in all patients, with EZ band preservation in regions with low cone density in 38% of locations analyzed. Normality of retinal lamellar structure on OCT corresponded with cone density and visual function at 50/78 locations analyzed. Outer retinal tubulations containing photoreceptor-like structures were observed in 3 patients. CONCLUSIONS: The use of CDD color-coded maps enables direct comparison of cone mosaic local density with other measures of retinal structure and function. Larger normative datasets and improved tools for automation of image alignment are needed. TRANSLATIONAL RELEVANCE: The approach described facilitates comparison of complex multimodal data sets from patients with inherited retinal degeneration, and can be expanded to incorporate other structural imaging or functional testing.

Reliability and Repeatability of Cone Density Measurements in Patients with Congenital Achromatopsia.

Adaptive optics scanning light ophthalmoscopy (AOSLO) allows non-invasive assessment of the cone photoreceptor mosaic. Confocal AOSLO imaging of patients with achromatopsia (ACHM) reveals an altered reflectivity of the remaining cone structure, making identification of the cells more challenging than in normal retinas. Recently, a "split-detector" AOSLO imaging method was shown to enable direct visualization of cone inner segments in patients with ACHM. Several studies have demonstrated gene replacement therapy effective in restoring cone function in animal models of ACHM and human trials have on the horizon, making the ability to reliably assess cone structure increasingly important. Here we sought to examine whether absolute estimates of cone density obtained from split-detector and confocal AOSLO images differed from one another and whether the inter- and intra-observer reliability is significantly different between these modes. These findings provide an important foundation for evaluating the role of these images as tools to assess the efficacy of future gene therapy trials.

ASSESSING PHOTORECEPTOR STRUCTURE ASSOCIATED WITH ELLIPSOID ZONE DISRUPTIONS VISUALIZED WITH OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: To compare images of photoreceptor layer disruptions obtained with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO) in a variety of pathologic states. METHODS: Five subjects with photoreceptor ellipsoid zone disruption as per OCT and clinical diagnoses of closed-globe blunt ocular trauma (n = 2), macular telangiectasia type 2 (n = 1), blue-cone monochromacy (n = 1), or cone-rod dystrophy (n = 1) were included. Images were acquired within and around photoreceptor lesions using spectral domain OCT, confocal AOSLO, and split-detector AOSLO. RESULTS: There were substantial differences in the extent and appearance of the photoreceptor mosaic as revealed by confocal AOSLO, split-detector AOSLO, and spectral domain OCT en face view of the ellipsoid zone. CONCLUSION: Clinically available spectral domain OCT, viewed en face or as B-scan, may lead to misinterpretation of photoreceptor anatomy in a variety of diseases and injuries. This was demonstrated using split-detector AOSLO to reveal substantial populations of photoreceptors in areas of no, low, or ambiguous ellipsoid zone reflectivity with en face OCT and confocal AOSLO. Although it is unclear if these photoreceptors are functional, their presence offers hope for therapeutic strategies aimed at preserving or restoring photoreceptor function.

Genotype-dependent variability in residual cone structure in achromatopsia: toward developing metrics for assessing cone health.

PURPOSE: Gene therapy trials for inherited photoreceptor disorders are planned. Anatomical metrics to select the best candidates and outcomes are needed. Adaptive optics (AO) imaging enables visualization of photoreceptor structure, although analytical tools are lacking. Here we present criteria to assess residual photoreceptor integrity in achromatopsia (ACHM). METHODS: Two AOSLOs, at the Medical College of Wisconsin and Moorfields Eye Hospital, were used to image the photoreceptor mosaic of 11 subjects with ACHM and 7 age-matched controls. Images were obtained, processed, and montaged using previously described methods. Cone density and reflectivity were quantified to assess residual cone photoreceptor structure. RESULTS: All subjects with ACHM had reduced numbers of cone photoreceptors, albeit to a variable degree. In addition, the relative cone reflectivity varied greatly. Interestingly, subjects with GNAT2-associated ACHM had the greatest number of residual cones and the reflectivity of those cones was significantly greater than that of the cones in the subjects with CNGA3/CNGB3-associated ACHM. CONCLUSIONS: We present cone reflectivity as a metric that can be used to characterize cone structure in ACHM. This method may be applicable to subjects with other cone disorders. In ACHM, we hypothesize that cone numerosity (and/or density) combined with cone reflectivity could be used to gauge the therapeutic potential. As gene replacement would not be expected to add cones, reflectivity could be a more powerful AO-metric for monitoring the cellular response to treatment and could provide a more immediate indicator of efficacy than behavioral measures, which may take longer to change.