Multimodal Imaging in Choroideremia.

Choroideremia (CHM) is associated with progressive degeneration of the retinal pigment epithelium (RPE), choriocapillaris (CC), and photoreceptors. As animal models of CHM are lacking, most information about cell survival has come from imaging affected patients. This chapter discusses a combination of imaging techniques, including fundus-guided microperimetry, confocal and non-confocal adaptive optics scanning laser ophthalmoscopy (AOSLO), fundus autofluorescence (FAF), and swept-source optical coherence tomography angiography (SS-OCTA) to analyze macular sensitivity, cone photoreceptor outer and inner segment structure, RPE structure, and CC perfusion, respectively. Combined imaging modalities such as those described here can provide sensitive measures of monitoring retinal structure and function in patients with CHM.

The spectral identity of foveal cones is preserved in hue perception.

Organisms are faced with the challenge of making inferences about the physical world from incomplete incoming sensory information. One strategy to combat ambiguity in this process is to combine new information with prior experiences. We investigated the strategy of combining these information sources in color vision. Single cones in human subjects were stimulated and the associated percepts were recorded. Subjects rated each flash for brightness, hue, and saturation. Brightness ratings were proportional to stimulus intensity. Saturation was independent of intensity, but varied between cones. Hue, in contrast, was assigned in a stereotyped manner that was predicted by cone type. These experiments revealed that, near the fovea, long and middle wavelength sensitive cones produce sensations that can be reliably distinguished on the basis of hue, but not saturation or brightness. Taken together, these observations implicate the high-resolution, color-opponent parvocellular pathway in this low-level visual task.

Rod hue biases for foveal stimuli on CRT displays.

Signals from rod photoreceptors bias (shift) the hues determined by cone photoreceptors for extrafoveal mesopic stimuli, creating green, blue, and red rod hue biases at long, middle, and short wavelengths, respectively. The fovea contains far fewer rods and S cones but may not be immune to rod hue biases. Here, we determine the biases found for mesopic foveal stimuli presented on a CRT display. The rod green bias was observed at unique yellow for all but one observer with 2° tests and persisted for most observers with 0.5° tests. The rod red bias typically seen at unique blue in extrafoveal studies was not apparent for either size of foveal test stimulus, and it was sometimes replaced by a rod green bias. The rod blue bias typically seen at unique green and unique red in extrafoveal studies was weak on average and inconsistent for both sizes of foveal test stimuli. Thus, small mesopic foveal stimuli permit rod influence on M- and L-cone color pathways but disadvantage rod influence on S-cone pathways, perhaps because of the sparseness of foveal S-cones. However, some observers did show idiosyncratic foveal rod hue biases that do not follow the general trends.

Comparison of the Richmond HRR 4th edition and Farnsworth-Munsell 100 Hue Test for quantitative assessment of tritan color deficiencies.

Drugs and environmental factors can induce tritan deficiencies. The Farnsworth-Munsell (FM) 100 Hue Test has become the gold standard in measuring these acquired defects. However, the test is time consuming, and color discrimination is confounded by concentration and patience. Here, we describe a test that compares six tritan plates from the HRR Pseudoisochromatic Plates 4th edition to 16 FM 100 Hue tritan caps. CIE Standard Illuminant C was reduced over five light intensities to simulate the effects of acquired losses in the S-cone pathway. Both tests showed quantitative differences in error rates with all light levels; thus they could serve equally well for assessing acquired deficiencies. However, compared to the FM 100, the HRR took subjects about 20-40 s per trial, making it more practical.

Comparing Cone Structure and Function in RHO- and RPGR-Associated Retinitis Pigmentosa.

Purpose: To study cone structure and function in patients with retinitis pigmentosa (RP) owing to mutations in rhodopsin (RHO), expressed in rod outer segments, and mutations in the RP-GTPase regulator (RPGR) gene, expressed in the connecting cilium of rods and cones. Methods: Four eyes of 4 patients with RHO mutations, 5 eyes of 5 patients with RPGR mutations, and 4 eyes of 4 normal subjects were studied. Cone structure was studied with confocal and split-detector adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral-domain optical coherence tomography. Retinal function was measured using a 543-nm AOSLO-mediated adaptive optics microperimetry (AOMP) stimulus. The ratio of sensitivity to cone density was compared between groups using the Wilcoxon rank-sum test. Results: AOMP sensitivity/cone density in patients with RPGR mutations was significantly lower than normal (P < 0.001) and lower than patients with RHO mutations (P < 0.015), whereas patients with RHO mutations were similar to normal (P > 0.9). Conclusions: Retinal sensitivity/cone density was lower in patients with RPGR mutations than normal and lower than patients with RHO mutations, perhaps because cones express RPGR and degenerate primarily, whereas cones in eyes with RHO mutations die secondary to rod degeneration. High-resolution microperimetry can reveal differences in cone degeneration in patients with different forms of RP.

OCT Angiography to Predict Geographic Atrophy Progression using Choriocapillaris Flow Void as a Biomarker.

Purpose: To investigate the relationship between choriocapillaris (CC) flow void (FV) percentage and geographic atrophy (GA) growth rate, and study how variations in FV percentage surrounding GA predict regional GA growth. Methods: This prospective, longitudinal study enrolled subjects with GA secondary to nonexudative age-related macular degeneration. Optical coherence tomography angiography imaged the CC and FV percentage was evaluated using a validated algorithm. GA growth rate was measured as the difference in the square root of GA area divided by the months between baseline and follow-up imaging. Results: Twelve eyes from 7 subjects with a mean age of 80 ± 5 years (range 74-86) were studied once at baseline and 7 to 16 months later. GA expansion rate was positively correlated with increased CC FV percentage (Spearman rank correlation coefficient r = 0.69 [P = 0.038] and 0.76 [P = 0.013]) within the 6 x 6 mm scanned macular region and the 2° margin surrounding each GA lesion, respectively. Regions with CC FV at baseline located within 480 µm from the GA margin showed 33% greater chance of becoming atrophic compared with regions within 480 µm from the GA margin that did not show CC FV at baseline. Conclusions: GA expansion rate and CC FV density throughout the macular region and surrounding the GA margin were significantly correlated. The regional magnitude of FV immediately surrounding GA was associated with GA growth into that region. Translational Relevance: CC FV analysis may facilitate prediction of GA growth over time for patients with advanced nonneovascular age-related macular degeneration.

Cone Structure Persists Beyond Margins of Short-Wavelength Autofluorescence in Choroideremia.

Purpose: We studied the relationship between structure and function of the choriocapillaris (CC), retinal pigment epithelium (RPE), and photoreceptors in patients with choroideremia (CHM). Methods: Six CHM patients (12 eyes) and four normal subjects (six eyes) were studied with fundus-guided microperimetry, confocal and nonconfocal adaptive optics scanning laser ophthalmoscopy (AOSLO), near-infrared and color fundus photos, short wavelength fundus autofluorescence (SW-AF), and swept-source optical coherence tomography (SS-OCT) and angiography (SS-OCTA) images. Cone spacing was represented using Z-scores (standard deviations from the mean at that eccentricity). CC flow voids were defined using a threshold of 1 SD below the normal mean. Results: Cone spacing Z-scores were not significantly correlated with distance from the borders of preserved RPE, determined using either the SS-OCT or SW-AF scans. Cone spacing Z-scores were significantly correlated with CC flow voids and retinal sensitivity. Flow voids were abnormal in regions of preserved RPE and increased progressively from within -2° of the preserved area to +2° beyond the border. Visual sensitivity decreased as CC flow voids increased approaching and beyond the border of preserved structure. Conclusions: In CHM, cone spacing Z-scores correlated with CC flow voids, and were negatively correlated with retinal sensitivity, suggesting cone degeneration accompanied reduced CC perfusion. Functional cones were found outside the presumed borders of preserved outer-retina/RPE as defined by SW-AF, but not outside the borders determined by SS-OCT. The use of SW-AF to identify the border of preserved structures may underestimate regions with cells that may be amenable to treatment.

Cone Spacing Correlates With Retinal Thickness and Microperimetry in Patients With Inherited Retinal Degenerations.

Purpose: To determine whether high-resolution retinal imaging measures of macular structure correlate with visual function over 36 months in retinal degeneration (RD) patients and normal subjects. Methods: Twenty-six eyes of 16 RD patients and 16 eyes of 8 normal subjects were studied at baseline; 15 eyes (14 RD) and 11 eyes (6 normal) were studied 36 months later. Adaptive Optics Scanning Laser Ophthalmoscopy (AOSLO) was used to identify regions of interest (ROIs) with unambiguous cones at baseline to measure cone spacing. AOSLO images were aligned with spectral-domain optical coherence tomography (SD-OCT) and fundus-guided microperimetry results to correlate structure and function at the ROIs. SD-OCT images were segmented to measure inner segment (IS) and outer segment (OS) thickness. Correlations between cone spacing, IS and OS thickness and sensitivity were assessed using Spearman correlation coefficient ρ with bootstrap analyses clustered by person. Results: Cone spacing (ρ = 0.57, P < 0.001) and macular sensitivity (ρ = 0.19, P = 0.14) were significantly correlated with eccentricity in patients. Controlling for eccentricity, cone spacing Z-scores were inversely correlated with IS (ρ = -0.29, P = 0.002) and OS thickness (ρ = -0.39, P < 0.001) in RD patients only, and with sensitivity in normal subjects (ρ = -0.22, P < 0.001) and RD patients (ρ = -0.38, P < 0.001). After 36 months, cone spacing increased (P < 0.001) and macular sensitivity decreased (P = 0.007) compared to baseline in RD patients. Conclusions: Cone spacing increased and macular sensitivity declined significantly in RD patients over 36 months. High resolution images of cone structure correlated with retinal sensitivity, and may be appropriate outcome measures for clinical trials in RD.

Relationship Between Foveal Cone Structure and Visual Acuity Measured With Adaptive Optics Scanning Laser Ophthalmoscopy in Retinal Degeneration.

Purpose: To evaluate foveal function in patients with inherited retinal degenerations (IRD) by measuring visual acuity (VA) after correction of higher-order aberrations. Methods: Adaptive optics scanning laser ophthalmoscopy (AOSLO) was used to image cones in 4 healthy subjects and 15 patients with IRD. The 840-nm scanning laser delivered an "E" optotype to measure AOSLO-mediated VA (AOSLO-VA). Cone spacing was measured at the preferred retinal locus by two independent graders and the percentage of cones below the average density of 47 age-similar healthy subjects was computed. Cone spacing was correlated with best-corrected VA measured with the Early Treatment of Diabetic Retinopathy Study protocol (ETDRS-VA), AOSLO-VA, and foveal sensitivity. Results: ETDRS-VA significantly correlated with AOSLO-VA (ρ = 0.79, 95% confidence interval [CI] 0.5-0.9). Cone spacing correlated with AOSLO-VA (ρ = 0.54, 95% CI 0.02-0.7), and negatively correlated with ETDRS letters read (ρ = -0.64, 95% CI -0.8 to -0.2). AOSLO-VA remained ≥20/20 until cones decreased to 40.2% (CI 31.1-45.5) below normal. Similarly, ETDRS-VA remained ≥20/20 until cones were 42.0% (95% CI 36.5-46.1) below normal. Cone spacing z scores negatively correlated with foveal sensitivity (ρ = -0.79, 95% CI -0.9 to -0.4) and foveal sensitivity was ≥35 dB until cones were 43.1% (95% CI 39.3-46.6) below average. Conclusions: VA and foveal cone spacing were weakly correlated until cones were reduced by 40% to 43% below normal. The relationship suggests that VA is an insensitive measure of foveal cone survival; cone spacing may be a more sensitive measure of cone loss.

Dysflective cones: Visual function and cone reflectivity in long-term follow-up of acute bilateral foveolitis.

PURPOSE: Confocal adaptive optics scanning laser ophthalmoscope (AOSLO) images provide a sensitive measure of cone structure. However, the relationship between structural findings of diminished cone reflectivity and visual function is unclear. We used fundus-referenced testing to evaluate visual function in regions of apparent cone loss identified using confocal AOSLO images. METHODS: A patient diagnosed with acute bilateral foveolitis had spectral-domain optical coherence tomography (SD-OCT) (Spectralis HRA + OCT system [Heidelberg Engineering, Vista, CA, USA]) images indicating focal loss of the inner segment-outer segment junction band with an intact, but hyper-reflective, external limiting membrane. Five years after symptom onset, visual acuity had improved from 20/80 to 20/25, but the retinal appearance remained unchanged compared to 3 months after symptoms began. We performed structural assessments using SD-OCT, directional OCT (non-standard use of a prototype on loan from Carl Zeiss Meditec) and AOSLO (custom-built system). We also administered fundus-referenced functional tests in the region of apparent cone loss, including analysis of preferred retinal locus (PRL), AOSLO acuity, and microperimetry with tracking SLO (TSLO) (prototype system). To determine AOSLO-corrected visual acuity, the scanning laser was modulated with a tumbling E consistent with 20/30 visual acuity. Visual sensitivity was assessed in and around the lesion using TSLO microperimetry. Complete eye examination, including standard measures of best-corrected visual acuity, visual field tests, color fundus photos, and fundus auto-fluorescence were also performed. RESULTS: Despite a lack of visible cone profiles in the foveal lesion, fundus-referenced vision testing demonstrated visual function within the lesion consistent with cone function. The PRL was within the lesion of apparent cone loss at the fovea. AOSLO visual acuity tests were abnormal, but measurable: for trials in which the stimulus remained completely within the lesion, the subject got 48% correct, compared to 78% correct when the stimulus was outside the lesion. TSLO microperimetry revealed reduced, but detectible, sensitivity thresholds within the lesion. CONCLUSIONS AND IMPORTANCE: Fundus-referenced visual testing proved useful to identify functional cones despite apparent photoreceptor loss identified using AOSLO and SD-OCT. While AOSLO and SD-OCT appear to be sensitive for the detection of abnormal or absent photoreceptors, changes in photoreceptors that are identified with these imaging tools do not correlate completely with visual function in every patient. Fundus-referenced vision testing is a useful tool to indicate the presence of cones that may be amenable to recovery or response to experimental therapies despite not being visible on confocal AOSLO or SD-OCT images.