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. 2016 Nov 1;57(14):5843-5855.
doi: 10.1167/iovs.16-20049.

Distribution and Quantification of Choroidal Macrophages in Human Eyes With Age-Related Macular Degeneration

D Scott McLeod  1 , Imran Bhutto  1 , Malia M Edwards  1 , Rachel E Silver  2 , Johanna M Seddon  3 , Gerard A Lutty  1
Affiliations

Distribution and Quantification of Choroidal Macrophages in Human Eyes With Age-Related Macular Degeneration

D Scott McLeod et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Increasing evidence suggests a role for macrophages in the pathogenesis of age-related macular degeneration (AMD). This study examined choroidal macrophages and their activation in postmortem eyes from subjects with and without AMD.

Methods: Choroids were incubated with anti-ionized calcium-binding adapter molecule 1 (anti-IBA1) to label macrophages, anti-human leukocyte antigen-antigen D-related (anti-HLA-DR) as a macrophage activation marker, and Ulex europaeus agglutinin lectin to label blood vessels. Whole mounts were imaged using confocal microscopy. IBA1- and HLA-DR-positive (activated) cells were counted in submacula, paramacula, and nonmacula, and cell volume and sphericity were determined using computer-assisted image analysis.

Results: In aged control eyes, the mean number of submacular IBA1+ and HLA-DR+ macrophages was 433/mm2 and 152/mm2, respectively. In early AMD eyes, there was a significant increase in IBA1+ and HLA-DR+ cells in submacula compared to those in controls (P = 0.0015 and P = 0.008, respectively). In eyes with neovascular AMD, there were significantly more HLA-DR+ cells associated with submacular choroidal neovascularization (P = 0.001). Mean cell volume was significantly lower (P ≤ 0.02), and sphericity was significantly higher (P ≤ 0.005) in all AMD groups compared to controls.

Conclusions: The average number of IBA1+ macrophages in submacular and paramacular choroid was significantly higher in early/intermediate AMD compared to that in aged controls. HLA-DR+ submacular macrophages were significantly increased in all stages of AMD, and they were significantly more round and smaller in size in the submacular AMD choroid, suggesting their activation. These findings support the concept that AMD is an inflammatory disease.

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Figures

Figure 1
Figure 1
Higher magnification image of a submacular field in the aged control choroid in Supplementary Figure S3 showing a homogeneous pattern of choriocapillaris with broad lumen and freely interconnecting channels (blue [A]; gray [B]). IBA1-labeled macrophages (green) were generally ramified, with several thick limbs, bearing short fine processes. HLA-DR+ macrophages (red) were much fewer in number than IBA1+ cells. (A) Merged channels; (B) desaturated UEA channel; (C) IBA1 channel; (D) HLA-DR channel. Scale bar: 25 μm.
Figure 2
Figure 2
Graphs showing cell counts/mm2 in three regions of choroid. (A) Submacula; (B) paramacula; (C) nonmacular, analyzed in aged control eyes, early/intermediate AMD eyes (CARM grades 2/3), GA eyes (CARMS grade 4), and neovascular AMD eyes (CARMS grade 5). There was a significant increase in HLA-DR+ macrophages (red) in the submacula of all AMD groups compared to those in controls. Additionally, there was a significant increase in the number of IBA1+ macrophages (blue) in early AMD in submacula and paramacular choroid and in nonmacular region in GA and neovascular AMD (*P < 0.05; **P < 0.001).
Figure 3
Figure 3
Representative volume renderings of IBA1+ macrophages in the submacular choroid of an aged control eye (A), early AMD eye (B), an eye with neovascular AMD (C), and in a subject with GA eye (D). Macrophages in the aged control have a large cell volume and a ramified cellular morphology. In eyes with early AMD, macrophages have fewer processes and reduced cell volumes. In advanced AMD (neovascular AMD and GA), macrophages have very few processes, are more rounded, and are much smaller in size. Scale bar: 10 μm.
Figure 4
Figure 4
(A) Cell volume of submacular choroidal macrophages in aged control eyes, eyes with early/intermediate AMD, GA, and neovascular. There was a statistically significant decrease in cell volume in a comparison between control subjects and each AMD group (*P = 0.03; **P < 0.0009). (B) Sphericity of submacular choroidal macrophages in aged control eyes, eyes with early/intermediate AMD, eyes with neovascular AMD, and GA eyes. There was a statistically significant decrease in cell sphericity in a comparison between control subjects and AMD group (**P < 0.005).
Figure 5
Figure 5
Low-magnification view of the posterior pole region of the choroid from an early AMD subject (subject 6) showing a localized 11-mm2 area of CC dropout in the submacular region (arrows). At this magnification, there was no obvious increase in either IBA1+ (C) or HLA-DR+ (D) macrophages in the submacular choroid. (A) Merged channels; (B) desaturated UEA channel; (C) IBA1 channel; (D) HLA-DR channel. *Optic nerve. Scale bar: 1 mm.
Figure 6
Figure 6
Higher magnification images of the paramacular (A, C, E, G) and submacular field (B, D, F, H) of choroid in the early AMD subject (subject 6) shown in Figure 6 demonstrates a homogeneous pattern of choriocapillaris with broad lumen and freely interconnecting channels in the paramacular region (A, C) and an abbreviated pattern with loss of interconnecting capillaries in the submacular region (B, D). IBA1-labeled macrophages are somewhat more ramified in paramacular choroid (E) than those in the submacula (F). HLA-DR+ macrophages were more numerous in both of these regions (G, H) compared to aged controls. (B) Merged channels; (C, D) desaturated UEA channel; (E, F) IBA1 channel; (G, H) HLA-DR channel. Scale bar: 25 μm.
Figure 7
Figure 7
Low-magnification image of the posterior pole region of choroid from a subject with GA (subject 14, OD) showing a well-demarcated area of choriocapillaris attenuation in the region corresponding to RPE atrophy (arrows). There appear to be no significant differences in the number of IBA1+ macrophages (F) in the atrophic, border, or nonatrophic regions of choroid. (A) Merged channels; (B) desaturated UEA channel; (C) IBA1 channel; (D) HLA-DR channel. Scale bar: 1 mm.
Figure 8
Figure 8
Higher magnification images of the nonmacular, nonatrophic region (A, D, G, J), a paramacular/border region to atrophy (B, E, H, K), and submacular atrophic region (C, F, I, L) in the GA subject shown in Figure 7 (subject 14, OD) demonstrates normal choriocapillaris pattern in the nonmacular region (D), an attenuated choriocapillaris at the border of RPE atrophy (E), and a severely attenuated choriocapillaris in the region of RPE atrophy (F). IBA1+ macrophages were more ramified in nonmacular choroid (G) then they were at the border of atrophy (H) or in the area of atrophy (I). There were fewer HLA-DR+ macrophages in the nonmacula (J) than at the border (K) and in the atrophic region (L). (A–C) Merged channels; (D–F) desaturated UEA channel; (G–I) IBA1 channel; (J–L) HLA-DR channel. Scale bar: 50 μm.
Figure 9
Figure 9
Low-magnification image of the posterior pole region of choroid from a subject (subject 14, OS) with neovascular AMD showing a CNV membrane in the submacular region (arrows). There was an increase in both IBA1+ (C) and HLA-DR+ macrophages (D) associated with the CNV in neovascular AMD eyes. (A) Merged channels; (B) desaturated UEA channel; (C) IBA1 channel; (D) HLA-DR channel. Scale bar: 1 mm.
Figure 10
Figure 10
Higher magnification images of the paramacular choroid adjacent to CNV (A, C, E, G) and submacular field of choroid (B, D, F, H) in the neovascular AMD demonstrate attenuated choriocapillaris in advance of CNV (C) and a nonlobular pattern of CNV in submacular region (D). Very few IBA1- or HLA-DR+-labeled macrophages were ramified in paramacular choroid (E, G) or within the CNV membrane (F, H). There were significantly more HLA-DR+ macrophages in the CNV than in the non-CNV region. (B) Merged channels; (C, D) desaturated UEA channel; (E, F) IBA1 channel; (G, H) HLA-DR channel. Scale bar: 25 μm.
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