Relationship between RPE and choriocapillaris in age-related macular degeneration

Abstract

Purpose: The purpose of this study was to examine the relationships between choriocapillaris (CC) and retinal pigment epithelial changes in age-related macular degeneration (AMD). Morphologic changes in the retinal pigment epithelium (RPE)/choriocapillaris complex were quantified in dry and wet forms of AMD, and the results were compared with those in aged control eyes without maculopathy.

Methods: Postmortem choroids from three aged control subjects, five subjects with geographic atrophy (GA), and three subjects with wet AMD were analyzed using a semiquantitative computer-assisted morphometric technique developed to measure the percentages of retinal pigment epithelial and CC areas in choroidal wholemounts incubated for alkaline phosphatase activity. The tissues were subsequently embedded in methacrylate and were sectioned so that structural changes could be examined.

Results: There was a linear relationship between the loss of RPE and CC in GA. A 50% reduction in vascular area was found in regions of complete retinal pigment epithelial atrophy. Extreme constriction of remaining viable capillaries was found in areas devoid of RPE. Adjacent to active choroidal neovascularization (CNV) in wet AMD, CC dropout was evident in the absence of retinal pigment epithelial atrophy, resulting in a 50% decrease in vascular area. Lumenal diameters of the remaining capillaries in wet AMD eyes were similar to those in control eyes.

Conclusions: The primary insult in GA appears to be at the level of the RPE, and there is an intimate relationship between retinal pigment epithelial atrophy and secondary CC degeneration. CC degeneration occurs in the presence of viable RPE in wet AMD. The RPE in regions of vascular dropout are presumably hypoxic, which may result in an increase in VEGF production by the RPE and stimulation of CNV.

Figure 1

Flow diagram showing the processing of images for determination of percent RPE or vascular area in a control subject (Case #3). Color selection was used to isolate the RPE (left) or vasculature (right), pixels copied and pasted into new images, converted to grayscale and thresholding was performed. The image was then converted to binary and imported into ImageJ where measurements were made using the “compute percent black and white” command in the measurement macros.

Figure 2

Raw color images of RPE using epi-illumination (A) and APase stained choroidal vessels using transillumination (B) and converted binary images used to make final percent black and percent white determinations from a flat preparation of an 80 year-old Caucasian male aged control subject (Case #2). (scale bar = 100 μm)

Figure 3

Comparison of percent RPE area and percent vascular area in aged control subjects and in the three regions analyzed in geographic atrophy choroids (GA). There was no statistically significant difference between aged control and nonatrophic regions of GA eyes in terms of RPE area (p=0.292) or vascular area (p=0.067). However, in GA choroid, there was a statistically significant decrease in both RPE (<0.0001) and vascular area (p=0.0001) in the border region compared to the nonatrophic region (asterisk). Similarly, there was a significant decrease in the RPE area (<0.0001) and vascular area (p=0.0013) in the atrophic region compared to the border region (asterisk).

Figure 4

APase choroid from an 88 year-old Caucasian male (Case #7) with a well-defined area of geographic atrophy (arrows) measuring 26.3 mm² is shown with epi-illumination (A) and transillumination (B). The optic nerve head is indicated (NH). (scale bar = 2mm)

Figure 5

APase choroid from a 79 year-old Caucasian male with GA (Case #8) showing nonatrophic (A,D,G,J), border (B,E,H,K) and atrophic region (C,F,I,L). Images captured with epi-illumination (A–C) highlight the RPE and were processed to make the binary images used to measure RPE area (D–F). The same three regions are shown with transillumination (G–I) along with the binary images used to measure percent vascular area (J–L). (scale bar = 100 μm)

Figure 6

APase choroid from an 81 year-old Caucasian female with wet AMD (Case #10). A submacular seafan-like CNV formation is shown (arrowheads) using epi-illumination (A) and transillumination (B). Areas of choriocapillaris dropout are located in advance of the CNV (asterisks). Percent RPE and vascular area measurements made in 2 mm intervals from the CNV (C) show that capillary dropout is present well beyond the extent of neovascularization. (NH = nerve head, scale bar = 2 mm)

Figure 7

Area from a 77 year-old Caucasian female with wet AMD (Case #11) 1 mm outside the CNV shown with epi-illumination (A) and transillumination (B) and the processed binary images used for percent RPE (C) and percent vascular area determinations (D). There are a few small areas of drusen and RPE defects present in the epi-illuminated images (A&C) while transmitted light images (B&D) show substantial loss of interconnecting capillary segments. (scale bar = 100 μm)

Figure 8

Compared with aged control eyes, wet AMD eyes showed no significant difference in RPE area outside the area of CNV (P=0.845). However, the percent vascular area was significantly reduced (p=0.0096; asterisk).

Figure 9

There was no significant difference in choriocapillaris diameters between the aged control, the nonatrophic region of GA eyes and the wet AMD eyes. However, the choriocapillaris diameters were significantly less in the border regions compared to nonatrophic and in the atrophic regions compared to border regions in GA eyes.

Figure 10

APase choroid from a 88 year-old Caucasian male with GA (Case #9) showing the nonatrophic region (A,D&G), border region (B,E&H) and atrophic region (C,F&I) in flat view prior to embedment (A–C) and in cross sections stained with PAS and hematoxylin (D–I). APase stained capillaries in the nonatrophic region (A) have broad diameter lumens filled with serum APase (arrow in D&G), with endothelial cells and pericytes (paired arrows) underlying viable RPE (arrowhead in D&G). At the border region, RPE appear hypertrophic (arrowhead in E&H), capillaries appear constricted (arrow in E&H), and some have completely degenerated (paired arrows H). A thin basal laminar deposit is associated with Bruch’s membrane (open arrow). In the atrophic region, many capillaries have degenerated leaving only remnants of basement membrane material (arrows in F&I). Some highly constricted viable capillaries had serum APase activity suggesting flow at some level.(scale bar = 100 μm in A–C, 30 μm in D–F, 10 μm in G–I)

Figure 11

APase choroid from an 81 year-old Caucasian female with wet AMD (Case #10) showing submacular CNV using epi-illumination (A&C) and transillumination (B&D). The front of growing vessels is closely associated with viable RPE (arrows A–D). Areas of CC dropout are evident in advance of the CNV (asterisks A&B). In PAS and hematoxylin stained sections, the equatorial region (E&H), has broad capillaries (arrows) containing serum APase with both endothelial cells and pericytes. The RPE has a normal morphology (arrowhead) and Bruch’s membrane is free of deposits. In sections taken 1 mm beyond the CNV (F&I), only a few capillaries are viable (arrows) and many degenerative capillaries are seen (asterisks in I). The RPE is hypertrophic (arrowheads) and a basal laminar deposit is present. Sections taken through the edge of the CNV (G&J) show degenerative capillaries (asterisk in J), sub-RPE neovascularization (open arrow) and hypertrophic RPE overlying the leading edge of the CNV. (scale bar = 0.5mm A&B, 100 μm C&D, 30 μm E–G, and 10 μm H–J)

Figure 12

Arteriosclerotic changes shown in PAS and hematoxylin-stained sections from GA (A&B) and wet AMD subjects (C&D). (A) An artery in a GA subject (Case #5) showing hyperplastic changes consisting of concentric laminations of smooth muscle cells and basement membranes. (B) An artery in a GA subject (Case #4) showing formation of nodules that replace the media during hyalinosis. (C) Hyperplastic arteriosclerotic changes in a wet AMD subject (Case #10) and (D) hyaline arteriolosclerosis in a wet AMD subject (Case #9). (arrows indicate the outer vessel wall and asterisks indicate the lumen in all, scale bar = 20 μm in A, B & D and 30 μm in C)

Figure 13

Fenestrations (arrowheads) along the inner aspect of the choriocapillaris endothelium in an aged control eye (A), and in a GA eye where RPE were present (B), near the border region (C) and in the region of atrophy (D). (scale bar = 500nm)

Figure 14

Ultrastructure of choriocapillaris in a GA eye in TEM sections from a region with RPE (A), border region (B&C) and area of atrophy (D). Capillaries from the region with RPE had fine endothelial cell cytoplasmic processes surrounded by a thin basement membrane. Endothelial cells in capillaries from the border region (B) were often vacuolated (arrow) with thickened processes. Others were highly vacuolated with extensive cytoplasmic infoldings (C) that had fenestrations (arrowhead). In atrophic regions (D), degenerative capillaries were collapsed and consisted almost entirely of basement membrane material (arrowheads). (scale bar = 1μm)