Participants

This prospective study included 21 eyes with resolved BRVO from 21 consecutive patients who were examined at the Department of Ophthalmology of Kyoto University Hospital between November 2012 and July 2013. The BRVO diagnosis was based on fundus examination and fluorescein angiographic findings, as determined by 2 retina specialists (A.T., T.M.). Cone mosaic images were taken from both previously affected and unaffected sides of the retina using an original prototype AO-SLO system. Inclusion criteria included: (1) history of symptomatic BRVO, in which retinal hemorrhage and retinal edema extended to the fovea; (2) complete resolution of retinal hemorrhage and macular edema; and (3) best-corrected VA better than 20/30 following BRVO resolution. Eyes with central retinal vein occlusion or hemicentral retinal vein occlusion were excluded from the current study. Eyes with coexisting ocular disease (eg, epiretinal membrane, glaucoma, diabetic retinopathy) were also excluded.

Ophthalmic Examinations

All subjects underwent comprehensive ophthalmologic examination, including measurement of Landolt best-corrected VA, intraocular pressure, and axial length (IOL Master; Carl Zeiss Meditec, Dublin, California, USA). Indirect ophthalmoscopy, slit-lamp biomicroscopy with a contact lens, color fundus photography, OCT imaging (Spectralis HRA+OCT; Heidelberg Engineering, Heidelberg, Germany), and AO-SLO imaging were also performed. All examinations were performed on the same day for each subject.

Adaptive Optics Scanning Laser Ophthalmoscope System

We developed an original prototype AO-SLO system in collaboration with Canon, Inc. The system is composed of 4 primary optical subsystems, which include the AO subsystem (includes the wavefront sensor and the spatial light modulator), the high-resolution confocal SLO imaging subsystem, the wide-field imaging subsystem, and the pupil observation subsystem. The observation system facilitates initial pupillary alignment with respect to the AO-SLO optical axis by adjusting the chin rest position. The wavefront sensor measures aberrations in the whole eye, and the spatial light modulator compensates for these aberrations. The AO-SLO system is confocal, enabling creation of “en face” images in any plane. These high-resolution images show individual cone photoreceptor cells.

Adaptive Optics Scanning Laser Ophthalmoscope Images

A series of AO-SLO images were taken in each eye at several locations within the macular area. The imaging series at each location was acquired by moving the focus from the retinal nerve fiber layer to the retinal pigment epithelium (RPE), with particular attention paid to the cone mosaic. To obtain cone mosaic images from both the previously affected and unaffected sides of the retina, we used 3 different imaging fields (large field of view [L]: 1700 × 1700 μm; middle field of view [M]: 820 × 820 μm; small field of view [S]: 340 × 340 μm). First, the AO-SLO was centered on the foveal center and the 3 field-of-view images (L, M, and S) were obtained. Next, middle field-of-view images (M) were collected that were centered 0.5 and 1.0 mm away from the foveal center in each direction (superior, nasal, inferior, temporal). Small field-of-view images were then collected with the AO-SLO images centered 0.5 and 1.0 mm superior and inferior to the foveal center. Finally, images centered 0.7 and 1.4 mm superotemporal, superonasal, inferotemporal, and inferonasal to the foveal center were obtained ( Figure 1 ). Images collected from the side previously affected with BRVO are shown in figure parts labeled B, while images from the unaffected side are shown in figure parts labeled A.

(Left) Cone mosaic images, captured with adaptive optics scanning laser ophthalmoscopy (AO-SLO), and the corresponding color fundus photograph are shown. Images were obtained from an eye with a resolved branch retinal vein occlusion (BRVO). (Right) High-resolution AO-SLO image montage from the central fovea out to 1.5 mm. Three different image fields were used (large [L, yellow box]: 1700 × 1700 μm; medium [M, green box]: 820 × 820 μm; small [S, white box]: 340 × 340 μm). The AO-SLO image was first centered on the foveal center and the 3 different image fields (L, M, and S) were obtained. Next, images were centered 0.5 and 1.0 mm away from the foveal center in each direction (superior, nasal, inferior, temporal) and an M image field was obtained. The S image field was also obtained in the superior and inferior directions. Lastly, the AO-SLO image was centered 0.7 and 1.4 mm away from the foveal center in the superotemporal, superonasal, inferotemporal, and inferonasal directions and the S image field was obtained. Images obtained from retina unaffected (A) and affected (B) by the previous BRVO are shown.

Final AO-SLO images were obtained by averaging 22 individual images acquired at each location. Scans were averaged in this way to reduce signal noise. Image location was verified by examining the correspondence between each montage and the area of interest. This was done by comparing high-magnification AO-SLO images with wide-field AO-SLO images. The AO-SLO image montage was also used to verify corresponding locations of other imaging modalities by matching retinal vessel patterns.

Analysis of the Cone Mosaic

Cone integrity was evaluated by applying an automated cone labeling process, previously established by Li and Roorda. When the algorithm failed to properly identify cones, cone labeling results were manually corrected. An experienced observer, who was masked to clinical findings, examined each image after automated cone labeling was completed. The observer then manually labeled areas where cones were visible but had not been identified by the software.

Cone density was estimated in selected areas where vascular component shadows were not present. The computer software was then instructed to estimate cone density by dividing the number of cones in each imaging area by the size of the imaging area. The area for quantification (80 × 80 μm) was identified manually by selecting the area without vascular shadows and by placing the computer cursor on the area to be quantified. This method has been used in previous studies, and interobserver reproducibility was high in normal eyes, as well as in eyes with retinitis pigmentosa. The location of the foveal center, defined here as the center of the foveal avascular zone, was determined using AO-SLO image montages. Cone density was measured in each location and overall cone density was taken as the mean of all measurements in all sections. Accurate scan lengths were determined in each eye using the adjusted axial length method to correct for the eye’s inherent magnification effect.

Spatial organization of the cone mosaic was determined by nearest-neighbor distances (NNDs) and Voronoi domains. Voronoi domains were constructed for each cell by defining points in the region that were closer to that cell than to any other cell in the mosaic. The NNDs were determined by calculating the minimum distance from the cell center to every other cell center in the mosaic. The expected NND was calculated as that of a perfect hexagonally packed mosaic, with a density equal to that in each location. The ratio of the observed average NND and the expected NND was calculated in each field of view.

Parafoveal Outer Photoreceptor Layer Integrity and Retinal Thickness

Vertical B-scan images (30 degrees) through the foveal center were obtained in each eye with the Spectralis HRA+OCT. To reduce speckle noise, 100 OCT images at the same location were acquired and averaged. Total retinal thickness and outer retinal thickness were measured 0.5 and 1.0 mm away from the foveal center ( Figure 2 ). Total retinal thickness was defined as the distance between the vitreoretinal interface and the inner border of the RPE. Outer retinal thickness was defined as the distance between the outer border of the inner nuclear layer and the inner border of the RPE. Thickness was measured with digital calipers built into the OCT software. Inner retinal thickness was calculated by subtracting outer retinal thickness from total retinal thickness.

Retinal thickness measurements and outer photoreceptor layer integrity in eyes with resolved branch retinal vein occlusion. A vertical section through the foveal center, obtained with optical coherence tomography, illustrates thickness parameter definitions. Total retinal thickness and outer retinal thickness were measured 0.5 and 1.0 mm away from the foveal center in both unaffected and previously affected side in the same eye. Total retinal thickness was defined as the distance between the vitreoretinal interface and the inner border of the retinal pigment epithelium (RPE). Outer retinal thickness was defined as the distance between the outer border of the inner nuclear layer and the inner border of the RPE. Inner retinal thickness was calculated by subtracting outer retinal thickness from total retinal thickness. The integrity of the external limiting membrane (ELM) and the photoreceptor inner and outer segment junction (IS/OS) line was also determined 0.5 and 1.0 mm away from the foveal center.

In averaged OCT cross-sections through the fovea, the integrity of the external limiting membrane (ELM) and the IS/OS line was determined 0.5 and 1.0 mm away from the foveal center by masked evaluators. Eyes in which the ELM and IS/OS lines were deemed intact were classified as ELM(+) and IS/OS(+), respectively. Eyes in which the ELM and IS/OS lines were deemed discontinuous were classified as ELM(±) and IS/OS(±), respectively. When the ELM and IS/OS lines could not be detected, eyes were classified as ELM(–) and IS/OS(–), respectively.

Statistical Analyses

Best-corrected VA was measured using the Landolt chart and converted to the logarithm of the minimal angle of resolution (logMAR) for analyses. We compared means of cone density, NNDs, and Voronoi parameters using the Mann-Whitney U test. Bivariate relationships were analyzed using the Pearson correlation coefficient. Differences between 2 retinal regions were tested for statistical significance using the paired t test. Statistical analyses were performed using SPSS software (version 19.0; SPSS, Inc, Chicago, Illinois, USA). Statistical significance was defined as P < .05.

Retinal Vascular Abnormalities Visible With Adaptive Optics Scanning Laser Ophthalmoscopy

Retinal capillaries were seen as black shadows on the cone mosaic on AO-SLO images. In the BRVO-unaffected side, retinal capillaries appeared identical to those seen in a normal retina. However, in the BRVO-affected side, most capillaries were dilated, varied in caliber, and seemed to have a longer, more tortuous path than capillaries in the normal retina ( Figure 3 ).

Retinal microvascular abnormalities in eyes with resolved branch retinal vein occlusion (BRVO), as shown on adaptive optics scanning laser ophthalmoscopy (AO-SLO) images. (Top row left) Initial fundus photograph from a 49-year-old woman with an acute BRVO in the left eye. (Second row left) An optical coherence tomography (OCT) vertical section from the initial visit shows a thick retinal hemorrhage and macular edema in the macular area (visual acuity [VA] = 20/22). (Top row right) Three years later, the retinal hemorrhage had been completely absorbed. (Second row right) A vertical OCT section confirmed complete resolution of macular edema (VA = 20/13). (Third row) The AO-SLO images from physiologically normal retinal capillaries in the BRVO-unaffected side. (Bottom) The AO-SLO images from the side of the retina previously affected by BRVO showing abnormal retinal capillaries. Retinal vessel pathways are longer and more tortuous and vessel dilations, similar to small aneurysms, were identified (arrows). Images were obtained from both the BRVO-affected (A) and BRVO-unaffected (B) sides.

The Cone Mosaic on Adaptive Optics Scanning Laser Ophthalmoscope Images

Figure 4 shows AO-SLO images from an eye with a resolved BRVO. In the BRVO-unaffected side, cone mosaic patterns were regular, with only a minimal number of dark regions. However, in the affected side, cone mosaic patterns were disorganized and had numerous dark areas that were missing wave-guiding cones. Table 2 shows the cone density of each measured area. In both unaffected and affected sides, cone density decreased as the distance from the foveal center increased. At each eccentricity examined, cone density was significantly lower in the affected side than in the corresponding unaffected side ( P < .001). Additionally, hexagonal Voronoi domain ratio and average NND/expected NND ratio were significantly lower on the affected side than on the unaffected side (both P < .05).

Cone density in eyes with resolved branch retinal vein occlusion (BRVO). (Top left) Initial fundus photograph of a 59-year-old man with an acute BRVO in the left eye (visual acuity [VA] = 20/30). An optical coherence tomography (OCT) vertical section shows macular edema and a discolored retinal hemorrhage at the fovea. (Top right) Sixteen months later, the retinal hemorrhage had completely absorbed. A vertical OCT section confirmed complete resolution of macular edema. Both the photoreceptor inner segment/outer segment junction and the external limiting membrane lines are clearly visible 0.5 and 1.0 mm away from the foveal center in the previously affected retina. (Second row) Adaptive optics scanning laser ophthalmoscopy (AO-SLO) images in the BRVO-unaffected side. The image labeled A(0, 0.5) shows a high-magnification image 0.5 mm away from the foveal center. Cone density was 25 763 cells/mm ² , hexagonal Voronoi domain ratio was 36.9%, and average nearest-neighbor distance (NND)/expected NND ratio was 0.53. The image labeled A(0, 1.0) shows a high-magnification image 1.0 mm away from the foveal center. Cone density was 16 575 cells/mm ² , hexagonal Voronoi domain ratio was 38.0%, and average NND/expected NND ratio was 0.60. (Bottom) The AO-SLO images in the side of the retina previously affected by BRVO show many dark patches, suggestive of cone loss. The image labeled B(0, 0.5) revealed a cone density of 6215 cells/mm ² , a hexagonal Voronoi domain ratio of 21.0%, and an average NND/expected NND ratio of 0.30. The image labeled B(0, 1.0) shows a high-magnification AO-SLO image 1.0 mm away from the foveal center. Cone density was 10 398 cells/mm ² , hexagonal Voronoi domain ratio was 21.4%, and average NND/expected NND ratio was 0.40.

Relationship Between Outer Photoreceptor Layer Integrity and Cone Density

In vertical OCT cross-sections through the fovea, the integrity of the ELM and the IS/OS lines was examined 0.5 mm away from the fovea. The IS/OS line integrity 0.5 and 1.0 mm away from the foveal center was significantly correlated with regional cone density ( Table 3 , Supplementary Figure ). In addition, both the hexagonal Voronoi domain ratio and the average NND/expected NND ratio were positively correlated with IS/OS line integrity 0.5 and 1.0 mm away from the foveal center. The ELM line integrity at the same locations also showed similar correlations with regional cone density, hexagonal Voronoi domain ratio, and average NND/expected NND ratio.

Table 3

Relationship Between Photoreceptor Integrity and Adaptive Optics Scanning Laser Ophthalmoscope Measurements in the Parafovea in Eyes With Resolved Branch Retinal Vein Occlusion

	Cone Density (Cones/mm 2 )		Hexagonal Voronoi Domain (%)		Average NND/Expected NND
	r	P Value	r	P Value	r	P Value
0.5 mm away from the foveal center
Integrity of ELM line	0.39	.011	0.21	.071	0.36	.018
Integrity of IS/OS line	0.52	<.001	0.45	.002	0.54	<.001
1.0 mm away from the foveal center
Integrity of ELM line	0.41	.007	0.50	<.001	0.46	.002
Integrity of IS/OS line	0.46	.002	0.63	<.001	0.53	<.001

ELM = external limiting membrane; IS/OS = photoreceptor inner and outer segment junction; NND = nearest-neighbor distance.

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