Choroidal Vascular Density on C-Scans: Macular (6×6 mm) versus Regional (500×500 microns).

<p>AMD = age-related macular degeneration; Control = epiretinal membranes; early AMD patients = drusen, advanced reticular patients = reticular pseudodrusen with advanced atrophic or neovascular AMD.</p>Ac<p> = Analysis of covariance (ANCOVA) p-value. Model 1. Adjusting for choroidal thickness. Model 2. Adjusting for age and gender. When the ANCOVA was statistically significant (p<0.05), Bonferroni adjusted p-values were calculated for the pairwise comparisons. NS = non-significant (p>0.05).</p>*<p>When comparing Drusen vs early reticulars, p = 0.004, while all other comparisons were non-significant.</p>∧<p>When comparing Drusen vs early reticulars, p = 0.04, while all other comparisons were non-significant.</p

A Pilot Study of Morphometric Analysis of Choroidal Vasculature <em>In Vivo</em>, Using En Face Optical Coherence Tomography

<div><h3>Purpose</h3><p>To study the ability of volumetric spectral domain optical coherence tomography (SD-OCT) to perform quantitative measurement of the choroidal vasculature <em>in vivo</em>.</p> <h3>Methods</h3><p>Choroidal vascular density and vessel size were quantified using en face choroidal scans from various depths below the retinal pigment epithelium (RPE) in 58 eyes of 58 patients with either epiretinal membranes (ERM), early age-related macular degeneration (AMD), or reticular pseudo-drusen (RPD). For each patient, we used the macular volume scan (6×6 mm cube) for vessel quantification, while high-definition (HD) cross-section raster scans were used to qualitatively assess vascularity of the choroidal sub-layers, and measure choroidal thickness.</p> <h3>Results</h3><p>Of the 58 patients, more were female (66% versus 34% male), of whom 14 (24%) had ERM, 11 (19%) early AMD, and 33 (57%) RPD. Compared to intact choriocapillaris in all ERM (100%), none of the RPD and only 5/11 (45%) early AMD eyes had visible choriocapillaris on either cross section or C-scans (p-value<0.001). When comparing select regions from the most superficial C-scans, early AMD group had lowest vascular density and RPD had highest (p-value 0.04). Qualitative evaluation of C-scans from all three groups revealed a more granular appearance of the choriocapillaris in ERM versus increased stroma and larger vessels in the RPD eyes.</p> <h3>Conclusions</h3><p>SD-OCT can be used to qualitatively and quantitatively assess choroidal vascularity <em>in vivo</em>. Our findings correlate to previously reported histopathologic studies. Lack of choriocapillaris on HD cross-sections or C-scans in all RPD and about half of early AMD eyes suggests earlier choroidal involvement in AMD and specifically, RPD.</p> </div

Selecting Pixel Intensity Threshold for Choroidal Vessel Density Analysis.

<p>Choroidal C-scans (top image) were obtained using the advanced visualization feature of macular cubes consisting of 512×128 optical coherence tomography volume scans over a 6 mm square grid. Each C-scan was a 2 micron-thick scan from each of the three choroidal layers. A customized image analysis program was used for both full C-scans (top image) and a selected region of each scan (inset) for all patients. A strict threshold of R = 65, G = 65, and B = 65 pixel intensity combination was selected for vessel versus stroma (center).</p

Qualitative Choriocapillaris Assessment Comparing C-Scans and B-Scans (Example 2).

<p>Comparing another example of control (top panel), early age-related macular degeneration (AMD, middle panel) and reticular (bottom panel) patients, reveals qualitatively different appearance of representative C-scans obtained just below the retinal pigment epithelium in reticular groups (bottom right) than in the control and AMD groups (top right and center right, respectively). Red boxes on the B-Scans (left) show magnified and colorized selected areas of choroidal vasculature from the regions contained by the smaller red boxes on the C-Scans (right) for each group. Blue boxes on the B-Scans (left) show magnified areas of selected stroma from the regions contained by the smaller blue boxes on the C-Scans (right). Stromal sections demonstrate more patchy whitish regions in the RPD group (bottom panel) as compared to the control and AMD groups (top and middle panels) but similar vessel density due to the presence of larger vessels.</p

Glial fibrillary acid protein (GFAP) immunofluorescence representative cross-sectional proliferative diabetic retinopathy and idiopathic epiretinal membranes.

<p>Confocal images of both diabetic (top) and idiopathic ERMs (bottom) show GFAP (green) immunostaining within the tissue stroma. Nuclei are labeled with DAPI (blue). Patient numbers are indicated in the upper-right corners. Scale bars: 25 μm.</p

Co-localization of Endothelin-1 (ET-1) and S100A4 in representative cross-sectional proliferative diabetic retinopathy and idiopathic epiretinal membranes.

<p>Merged confocal images of diabetic ERMs (A) show co-localization of ET-1 (green) and S100A4 (red). Nuclei are labeled with DAPI (blue). Patient numbers are indicated in the upper-right corners. Scale bars: 50 μm.</p

Choroidal Thickness.

<p>AMD = age-related macular degeneration; crude versus adjusted for age and gender; Control = epiretinal membranes; early AMD patients = drusen, advanced reticular patients = reticular pseudodrusen with advanced atrophic or neovascular lesions.</p>*<p>Analysis of variance (ANOVA) p-values. Pairwise comparisons were statistically significant between Drusen and early reticulars (p = 0.04), but not significant when comparing other subtypes of AMD (p>0.05).</p>Ac<p>: Analysis of covariance (ANCOVA) p-value, adjusting for age and gender. When the ANOVA or ANCOVA were statistically significant (p<0.05), Bonferroni adjusted p-values were calculated for the pairwise comparisons.</p

Qualitative Choriocapillaris Assessment Comparing C-Scans and B-Scans (Example 3).

<p>Comparisons of control (top panel), early age-related macular degeneration (AMD, middle panel) and reticular (bottom panel) patients reveals qualitatively different appearance of representative C-scans obtained just below the retinal pigment epithelium in reticular groups (bottom right) than in the control and AMD groups (top right and center right, respectively). Red boxes on the B-Scans (left) show magnified and colorized selected areas of choroidal vasculature from the regions contained by the smaller red boxes on the C-Scans (right) for each group. Blue boxes on the B-Scans (left) show magnified areas of selected stroma from the regions contained by the smaller blue boxes on the C-Scans (right). Stromal sections demonstrate more patchy whitish regions in the RPD group (bottom panel) as compared to the control and AMD groups (top and middle panels) but similar vessel density due to the presence of larger vessels.</p

Qualitative High Density (HD) Raster Scan Assessment of Choriocapillaris.

<p>HD-raster scans of the control group (top) had a distinct granular-appearing choriocapillaris layer, in contrast to only 45% of the early age-related macular degeneration group (center) and none of the reticular group (bottom), in whom this layer was lacking.</p

Reduced Photoreceptor Heterogeneity Packing Index (HPi) in an Eye with Capillary Non-Perfusion Contiguous with the Foveal Avascular Zone (FAZ).

<p>Case 7, right eye. (A) Infrared (IR) image with location of adaptive optics scanning laser ophthalmoscopy (AOSLO) montage (green box). (B) Fluorescein angiography (FA) with location of AOSLO montage (green box). (C) Enlarged 1° x 1° AOSLO image from montage (HPi = 0.328). (D) Enlarged FA from B reveals an enlarged and irregular contour of the FAZ with surrounding contiguous areas of capillary non-perfusion. Green box shows location of AOSLO montage in F. Red box shows the location of enlarged AOSLO image (C) in the area of the enlarged FAZ. (E) Spectral-domain optical coherence tomography (SD-OCT) registered to the IR image showing the retinal area covered by the AOSLO montage. B-scan shows focal points where inner segment / outer segment junction is interrupted with decreased intensity of the outer segment / retinal pigment epithelium junction. Red line shows location of enlarged AOSLO image in C. (F) AOSLO montage stitched from 2° x 2° images with location of OCT B-scan (yellow line) and enlarged AOSLO inset (red box).</p