Choriocapillaris Dropout in Early Age-related Macular Degeneration

Abstract

Loss of choriocapillaris (CC) in advanced age-related macular degeneration (AMD) is well documented but changes in early AMD have not been quantified. Postmortem eyes from donors with clinically documented early AMD were examined in choroidal whole mounts to determine the area, pattern, and severity of CC loss. Choroids from postmortem human eyes without AMD (n=7; mean age=86.1) and from eyes with a Grade 2 clinical classification of early AMD (n=7; mean age=87) were immunolabeled with Ulex europaeus agglutinin (UEA) lectin-FITC to stain blood vessels. Whole mounts were imaged using confocal microscopy and image analysis was performed to determine the area of vascular changes and density of vasculature (percent vascular area, %VA). All areas evaluated had a complete RPE monolayer upon gross examination. In age-matched control eyes, the CC had broad lumens and a homogenous pattern of freely interconnecting capillaries. The mean %VA+/−standard deviation in submacula of control subjects was 78.1 +/− 3.25 %. In eyes with early AMD, there was a significant decrease in mean %VA to 60.1 +/− 10.4% (p<0.0001). The paramacular %VA was not significantly different in eyes with or without AMD. The area of submacular choroid affected by CC dropout was 0.04 +/− 0.09 mm² in control eyes. In eyes with early AMD, the mean area affected by CC dropout was significantly increased (10.4 +/− 6.1 mm²; p<0.001). In some cases, incipient neovascular buds were observed at the border of regions with CC dropout in early AMD choroids. In conclusion, UEA lectin-labeled choroidal whole mounts from donors with clinically documented early AMD has provided a unique opportunity to examine regional changes in vascular pathology associated with choriocapillaris. The study demonstrated attenuation of submacular CC in early AMD subjects but no vascular pathology was observed outside the submacular region. While the affected area in some eyes was quite extensive histologically, these changes may not be detectable clinically using standard in vivo imaging.

1. INTRODUCTION

Age-related macular degeneration (AMD) is the leading cause of severe vision loss in patients over the age of 50 in industrialized countries.(Friedman et al., 2004; Lim et al., 2012) AMD is a complex disease with both behavioral, lifestyle modifiable factors, and a strong genetic component with several common and rare variants.(Seddon, 2017) AMD is defined clinically by fundus examination. Several widely adopted classification schemes require the presence of drusen and/or pigmentary changes (including hyperpigmentation or pigment epithelial atrophy) for clinical diagnosis.(Ferris et al., 2005; Ferris et al., 2013; Seddon et al., 2006) Clinically and histologically, AMD is generally classified into two major subtypes: nonexudative or dry AMD, geographic atrophy (GA) being the advanced form; and exudative or neovascular AMD (nAMD). GA progresses slowly with drusen, geographic or focal atrophy of the retinal pigment epithelium (RPE) and choriocapillaris (CC), and dysfunction and degeneration of photoreceptors. Exudative AMD can develop after various forms of dry AMD (Lim et al., 2012) and is characterized by choroidal neovascularization (CNV), the growth of new blood vessels from the choroid under RPE (Type 1) or into the subretinal space above the RPE (Type 2). The onset of nAMD may be subtle and evident to neither patient nor physician. On clinical examination, signs of NV include hemorrhage, intraretinal or subretinal fluid. OCT and potentially more invasive diagnostic procedures such as fluorescein and indocyanine green angiography are used to document nAMD. Subfoveal leakage on fluorescein angiography is a major inclusion criterion for most therapeutic trials.

Loss of CC occurs in all stages of AMD.(McLeod et al., 2009; McLeod et al., 2002; Mullins et al., 2011; Sohn et al., 2014) A vasculopathy in AMD is not unexpected. Cardiovascular disease risk factors are also associated with AMD, including smoking, adiposity, and systemic inflammatory markers.(Seddon et al., 2003; Seddon et al., 2005; Seddon et al., 1996) Cardiovascular disease, increased cholesterol and hypertension have also been associated with nAMD.(Hogg et al., 2008; Snow and Seddon, 1999) It has been hypothesized that atherosclerosis would be associated with advanced AMD because of defects in the choroidal vasculature as well as deposition of lipids in the Bruch’s membrane, resulting in drusen.(Curcio, 2001; Snow and Seddon, 1999) Hypertension has been positively correlated with risk of early AMD.(Duan et al., 2007) The Beaver Dam Eye Study found that subjects with hypertension at baseline were two to three times more likely to develop nAMD.(Klein et al., 2005)

Using the laser Doppler flowmetry, Grunwald found decreased choroidal blood flow, velocity and volume in the AMD fovea.(Grunwald et al., 2005) Subjects with the lowest choroidal circulatory parameters had increased severity of AMD; reduced flow could result in ischemia, which can subsequently lead to CNV.(Grunwald et al., 2005) Metelitsina et al in a subsequent Doppler study on hypertension found that subjects with hypertension had significantly lower choroid blood flow than subjects without hypertension (Metelitsina et al., 2006; Metelitsina et al., 2008), suggesting that systemic hypertension may contribute to the progression of AMD. A perfusion defect like reduction in CC blood flow could lead to RPE dysfunction because the CC supplies oxygen and nutrients to the RPE cells and the outer retina. Thus, reduction in flow could promote increased debris accumulation in the form of drusen or basal laminar deposits.

We have previously demonstrated, using enzyme histochemical demonstration of endogenous alkaline phosphatase activity (APase), a loss of viable CC in advanced AMD (both GA and exudative).(McLeod et al., 2009; McLeod et al., 2002) In GA eyes, RPE atrophy appeared to occur in advance of CC degeneration while in exudative AMD, CC degeneration was seen in advance of RPE atrophy. Moreover, CNV was always observed in regions with RPE, suggesting that viable but presumed hypoxic RPE were required to initiate or sustain growth of new blood vessels. In this study, we sought to extend our previous observations on vascular changes in AMD by examining the choroidal vasculature in early AMD. Ulex europeaus agglutinin 1 (UEA) lectin staining was used to label viable endothelial cells in wholemounts from the posterior pole region of eyes with early AMD compared to aged donors without clinically documented AMD who were used as controls.

2. MATERIALS AND METHODS

2.1. Donor Eyes

Human donor eyes from 7 aged control subjects and 7 subjects with early AMD from the Eye Donation Program directed by JMS in Boston (n=9) and from the National Disease Research Interchange (NDRI; Philadelphia, PA, n=5) were used in this study. The mean age for control eyes without AMD was 86.14 +/−9.7 years and the mean age for early AMD eyes was 87 +/−6.53 years (p=0.68). All tissues were obtained within 10–35 h of death. Utilization of this human tissue was in accordance with the Declaration of Helsinki with approval of the Joint Committee on Clinical Investigation at Johns Hopkins University School of Medicine and the institutional review boards at Tufts Medical Center and University of Massachusetts Medical School. All donors were Caucasian. Table 1 summarizes the characteristics of each donor subject. The clinical diagnosis and severity of AMD were determined by reviewing and grading ocular examination records and multi-modal imaging of eye donors in the Seddon Longitudinal Cohort by JMS or grading the postmortem gross examination of posterior eyecup, using transmitted and reflected illumination with a dissecting microscope (Stemi 2000; Carl Zeiss Inc., Thornwood, New York). During gross examination, eyes were classified as early AMD according to severity of disease as described previously in the CARMS system.(Seddon et al., 2016; Seddon et al., 2006) The clinical features of early AMD were approximately ≥10 small drusen or <15 intermediate drusen, or pigment abnormalities associated with AMD (Grade 2). Subtypes of Grade 2 include 2a= drusen, 2b= RPE changes (hyperpigmentation and hypopigmentation) and 2c= both drusen and RPE changes. Medical diagnosis were determined by review of medical records of subjects in the JMS cohort or information provided by NDRI and associated eye banks.

Table 1.

Clinical and histopathologic data for ocular tissue from subjects with early stages of age-related macular degeneration (Subjects 8–14) and age-matched controls (Subjects 1–7). (Grade 1 and Normal: no signs of pathology in retina, RPE or choroid, no drusen present; PM: postmortem; pertinent medications: medications affecting hypertension, atherosclerosis, and clotting; Involuting: large radial vessels with very few interconnected capillaries; BLD: basal laminar deposit; dropout: areas with reduced capillary density; COPD: chronic obstructive pulmonary disease; HTN: hypertension; GERD: gastroesophogeal reflux disease)

Donor ID	Age at Death (sex)	Eye		CARMS Grade	PM Grade	Cause of death	Pertinent medications	Medical conditions
1	72 F	OS		1	1	Cerebral vasculitis	Levoxyl	Scleroderma, rheumatoid arthritis, dementia, chronic sinusitis
2	76 F	OS		1	1	Cardiac arrest	Lasix	COPD, sleep apnea, stroke in past, GERD, HTN, anemia, pulmonary HTN, right subclavian stenosis, bilateral cataract surgery
3	87 F	OS		1	1	Bladder cancer, metastatic lung cancer, pneumonia	Simvastatin, Plavix	Bladder cancer, metastatic lung cancer, pneumonia, dementia, high cholesterol, osteoporosis, parathyroid adenoma
4	87 M			1	1	Congestive heart failure	Diabetrix, Glyburide, Lovastatin, Warfarin	HTN borderline, high cholesterol, heart attack or myocardial infarction, arthritis, Diabetes type II, Prostate cancer, Gallbladder removed
5	87 M	OS		1	1	Cardiac arrest secondary to coronary artery disease	Simvastatin, Accupril, Warfarin	High cholesterol, congestive heart failure, angina, stroke, gout, colitis, nephritis
6	94 F	OD		1	1	Cardiogenic shock	Lisinopril, Lebetalol, Cardizem	Hypertension; congestive heart failure; peripheral vascular disease; arthritis; osteoporosis; atrial fibrillation
7	100 F			1	1	Cardiac arrest	Coumadin, Lopressor, Lisinopril, Lasix	HTN, hypothyroidism, dehydration, heart failure, high cholesterol
			Early AMD
8	75 F	OS		2A	2 Small submac ular drusen, retinal hemorr hages	End stage renal failure	Januvia, Levothyroxine, Simvastatin	HTN; borderline high cholesterol; arthritis; atrophic left kidney; abdominal aortic aneurysm;
9	84 M	OD			2	Cardiac arrest	Xaralto, Naproxen	Alzheimer’s, Atrial fibrillation, chronic renal failure, suprapubic catheter
10	86 M	OD		2C	2	Dementia/Alzheimer’s	Hypertensive medicines (exact drugs unknown)	High cholesterol; arthritis; chronic inflammation of the intestine; carpal tunnel; emphysema
11	89 M			1	2	Non-Hodgkin’s lymphoma	Atenolol, Cilostazol, Simvastatin	HTN; stroke; peripheral vascular disease; arthritis; renal failure; abdominal aortic aneurysm; history of hepatitis
12	90 M	OD		2A	2 Subma cular hyperpi gmenta tion, drusen	Heart failure, cardiac arrest, hypoxic respiratory failure, renal failure, pulmonary hypertension	Atenolol, Dilitiazem, Diovan, Furosemide, Warfarin	HTN; congestive heart failure; Stroke/hemorrhage in the brain; Arthritis; osteoporosis; atrial fibrillation
13	91 M	OS		2C	2	Complete heart block	Atenolol, Lisinopril, Glipizide	Diabetes; HTN; high cholesterol; stroke; abdominal aortic aneurysm;
14	94 M	OS		2C	2 Subma cular drusen	Cardiogenic shock	Furosimide, Lisinopril	HTN; congestive heart failure; peripheral vascular disease; arthritis (age 75); osteoporosis (age 70); atrial fibrillation (age 90)

2.2. Tissue preparation

Globes were opened at the limbus, the anterior segments removed, and the posterior eyecups were grossly examined. Digital images of the eye cup were captured (Micropublisher, QImaging, Surrey BC, Canada) using reflected and transmitted illumination prior to dissection. Vitreous was removed and retinas were then excised from the RPE/choroid. Typically, the neural retina was loosely attached to the RPE because of postmortem time. The eyecup was soaked in Tris buffer saline solution and then the edge of the sclera was gently grasped with a suture forceps to stabilize the eyecup. The neural retina was gently teased from the RPE/choroid with a cyclodialysis spatula. This procedure was continued until the entire neural retina was separated from RPE. At the optic nerve head, the retina was cut from nerve using a blunt edge curved Tenotomy scissor and then the neural retina was teased from the rest of the eyecup.

After removing the retina, each eye cup containing the choroid with the RPE intact was reimaged. All specimens had an intact RPE monolayer in submacula. Eye cups were then soaked in 1% EDTA (disodium salt; dihydrate crystal; Baker Chemical Co.) in distilled water for 2 h at room temperature to remove RPE. Any adherent RPE cells were removed by squirting the choroid with EDTA solution from a syringe with a blunted 25-gauge needle. Gross digital images of choroids were captured again without RPE. RPE-denuded choroids were then dissected from the sclera, washed briefly in 0.1 M cacodylate and fixed overnight in 2% paraformaldehyde in 0.1 M cacodylate buffer at 4°C.

2.3. Wholemount Immunohistochemistry

Choroids were washed twice in 0.1 M cacodylate buffer at 4°C and then in Tris buffered saline (TBS) with 0.1% Triton X-100 (TBS-T) for 10 minutes. After washes, choroids were incubated with 5% normal goat serum in TBS-T with 1% bovine serum albumin (BSA) overnight at 4°C. Tissues were then w ashed with TBS-T and incubated for 48 hrs at 4°C with Ulex Europaeus agglutinin (UEA lectin) conjugated to FITC (1:100; Sigma-Aldrich Corp. St. Louis, MO, Cat#L9006; or GeneTex, Inc., Irvine, CA, Cat#GTX01512). UEA lectin binds the fucose-residues. Tissues were washed in TBS and then imaged with a Zeiss LSM 710 confocal microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY) at 488 nm excitation.

2.4. Image Acquisition

An approximately 8X10 mm² area from the posterior pole region was excised from each intact choroid. The trimmed tissue included the region just nasal to the optic nerve opening, beyond the inferior and superior vascular arcades of retina, and several millimeters beyond the macula temporally as previously described.(McLeod et al., 2016) The tissue was flat mounted in TBS on a glass microscope slide, coverslipped, and imaged at 5, 10 and 20X magnification with Bruch’s membrane (BrMb) nearest the objective. The submacular region was centered in the oculars and 5X7 mm tiled overlapping fields (10% overlap) at 2048X2048 pixel resolution were collected as Z stacks using Zen Software (2010, Carl Zeiss Inc., Thornwood, New York). Laser power, pinhole, gain, and other capture parameters were saved and used for imaging each choroid under identical conditions. The number of Z-slices collected varied somewhat depending on the tissue thickness. The inner boundary of the Z stack was set at the focus level just above the CC and the outer limit of the Z stack was set where large choroidal vessels disappeared into the tissues.

2.5. Image Analysis for Percent Vascular Area (%VA)

Maximum intensity projections, 5X stitched images were exported from Zen Software as full resolution Tiff images and opened in Adobe Photoshop (CS6, Adobe Systems Incorporated, San Jose, CA). Three 1204X1204 pixel dimension selections (equivalent to1 mm²) were randomly made of regions in submacular and paramacular choroid, as described previously, and pasted into new image documents.(McLeod et al.,2016) Each image was adjusted using levels and thresholding and saved for analysis in ImageJ.(McLeod et al., 2016) The images were converted to binary, noise reduction applied, and the calculate black and white pixel macro in ImageJ was used to perform %VA (percent vascular area) as described previously.(McLeod et al., 2009)

2.6. Embedment of tissue in glycol methacrylate (JB4)

After image analysis, areas were excised from the submacular area and the tissue was dehydrated and embedded in glycol methacrylate as previously published.(McLeod et al., 2009) Two micron sections were cut and stained with periodic acid-Schiff’s reagent and hematoxylin (PAS and H) as previously reported.(McLeod et al., 2009)

2.7. Statistical analysis

Data are reported as means +/− standard deviation. Statistical evaluation of the data involved calculating probability values using the Student’s t-test for two samples with unequal variances assumed. A p-value of 0.05 or less was considered statistically significant.

3. RESULTS

3.1. Aged Control Eyes

Examination of aged control eyes revealed a dense homogeneous pattern of freely inter-anastomosing capillaries throughout the submacular and paramacular region (Figures 1& Supplemental Fig. 1). The vasculature was very dense in posterior pole region and there was no easily distinguishable lobular CC pattern (Fig. 1D–E and Supplemental Fig. 1E–F). The percent vascular are (%VA) was 78.1 +/− 3.2% in the submacular choroid and 79.69 +/− 5.1% in paramacula choroid (Fig. 2A). Very small areas of CC degeneration (0.04 +/− 0.09 mm²) were observed in submacular choroid in some aged control eyes (Fig. 2B). The mean CC luminal diameters in the submacular region were 14.7 +/− 1.6 microns in control subjects (data not shown). Numerous connections between the CC and feeding arterioles and draining venules were observed in Z Stacks of flat mounts (Fig. 1E, Supplemental Figs. 1E and 2). Precapillary arterioles made connections to the CC at right angles while postcapillary venules were oriented more obliquely. These connections were unevenly distributed with some being widespread and others being in extremely close proximity to each other (Supplemental Fig. 2). In sections, Bruch’s membrane (BrMb) was generally free of drusen and deposits. The CC lumen were broad and endothelialized (Fig. 1F). Feeding arterioles and draining venules were located in Sattler’s layer and larger arteries and veins were found in Haller’s layer (Fig. 1F).

Fig. 1. Case #7.

Gross photo of the left eye of a 100-year-old Caucasian female without hypertension with the retina intact (A) and after removal of retina (B). The missing RPE in the peripapillary region is a dissection artifact. The submacular choroid shows an intact RPE layer without drusen or pigmentary clumping. Stitched panorama of confocal images of the posterior pole region of choroid labeled with UEA lectin showing uniform appearance of choriocapillaris (C) at low magnification and the submacular CC at higher magnification (D). Note the seemingly disorganized intermediate vessels in this collapsed z-stack (D-E). The percent vascular area was 78.6% (+/−1.9%) in the submacular region (E) and 79.5% (+/−3.2) in the paramacular region. (F) A cross section of submacular choroid stained with PAS and H shows normal angioarchitecture and no deposits on Bruch’s membrane. (small arrows = CC, arrowheads = Sattler’s Layer, large arrows = Haller’s Layer) Scale bars = 1mm in A-C, 250 μm in D, 100μm in E and 20μm in F.

Fig. 2:

A) Percent Vascular Area (%VA); (B) Area of Involvement in all subjects.

3.2. Early AMD eyes

Eyes with early AMD showed CC dropout specific to the submacular region (Figs. 3–9). All of the areas analyzed had a complete RPE monolayer cover when examined and photographed after removing the retina. The area of involvement in early AMD subjects ranged from 1.04 mm² (Fig. 3) to as much as 19.5 mm² (Fig. 7). The dropout was diffuse in that viable capillary segments remained in the involved area, however, the density (%VA) was significantly reduced compared to aged controls (Fig.2A). The defects in the CC pattern could be detected in the 5X images (Fig. 3C), but the details and extent of capillary loss were not fully appreciated until 10X (Fig. 3D) and 20X images were acquired (Fig. 3E–F). In images of a 94 year old (YO) hypertensive subject’s choroidal vasculature, there were a few defects in paramacula (Fig. 4F) but in submacula it is obvious that many capillary segments were not stained with UEA lectin (Fig. 4D–E) in a small but distinct area (4.6 mm²) and were presumably not viable.(Mullins et al., 2011; Seddon et al., 2016; Sohn et al., 2014) The area of affected submacular choroidal vasculature was much larger (13.03 mm²) in an 84 YO hypertensive subject shown in Figure 5. In this subject there is a significant attenuation in CC which resulted in a reduction from 83.2 %VA in paramacula (Fig. 5F) to 57.8 %VA in submacula (Fig. 5D–E). The CC in a 89 YO hypertensive subject had small round areas that lacked viable CC (Fig. 6). When paramacular and submacular areas were sectioned, the CC loss was apparent with PAS and H staining of the sections (Fig. 6G). In submacula there were constricted intermediate blood vessels in Sattler’s layer and a basal laminar deposit (Fig. 6G). The subject with the largest area of CC attenuation in submacula (19.5 mm²) was a 90 year old hypertensive (Fig. 7). This subject had areas of hyperpigmentation and drusen (Fig. 7A–B). The density of submacular CC in this subject was 61.8% (Fig. 7E) compared to 79.9% in paramacula (Fig. 7F).

Fig. 3. Case #13.

(A) Fundus photo of the posterior pole region of the left eye of a 91-year-old Caucasian male with hypertension taken 8 years prior to death. (B) Gross photo after removal of retina shows an area of RPE hypopigmentation in the submacular region. (C, D) Stitched panorama of confocal images of the posterior pole region of choroid labeled with UEA lectin showing a region of choriocapillaris degeneration (arrows)(1.04 mm²) corresponding to the area of RPE hypopigmentation. Boxes in D show areas in images in E and F. The percent vascular area was 68.2% (+/−3.3%) in the submacular region (E) and 77.7% (+/−5.86) in the paramacular region (F). (G, H) Cross sections show degeneration of choriocapillaris lumen in submacular choroid (arrows in G) and normal structure of choriocapillaris in paramacular choroid (arrows in H). Scale bars = 1mm in B&C, 250 μm in D, 100μm in E&F.

Fig. 9. Case #8.

Fundus photo at low (A) and higher magnification (B) of the posterior pole region of the right eye of a 75-year-old Caucasian female with hypertension taken 2 years prior to death showing drusen in the macula. This eye was last graded a 2A clinically 2 years prior to death. (C) Gross photo of the eyecup after removal of retina showing drusen in the submacular region. (D) Stitched panorama of confocal images from the posterior pole region of choroid labeled with UEA lectin showing a 13.04 mm² region of choriocapillaris degeneration in the submacular choroid (arrows) at low magnification. The boxed regions in “D” are shown a higher magnification from the paramacular region (F) and submacular region (E). The percent vascular area was 59.3% (+/−5.8%) in the submacular region and 81.1% (+/−3.6%) in the paramacular region. Bulbous vascular formation in the submacular choroid (arrows in E&G) shown in flat perspective (G) and in cross section (H) demonstrating that the structure represents early CNV with numerous endothelial cells in clustered lumens extending through a break in Bruch’s membrane (arrows). (Scale bars = 1 mm in C&D, 200 μm in E&F and 20 μm in G&H).

Fig. 7. Case #12.

Fundus photo (A) of the posterior pole region of the right eye of a 90-year-old Caucasian female with hypertension taken 13 years prior to death showing drusen in the macula (arrows) and hyperpigmentation in the inferior choroid. This eye was last graded a 2C clinically 17 months prior to death. Gross photo after removal of retina (B) showing drusen in the submacular region (arrows in B) and hyperpigmentation inferior to submacula. Stitched panorama of confocal images from the posterior pole region of choroid labeled with UEA lectin (C) showing a 19.5 mm² region of choriocapillaris degeneration in the submacular choroid (arrows in C) at low magnification. The border of normal paramacular and degenerative submacular CC is shown at higher magnification (D). The boxed regions in “D” are shown a higher magnification from the submacular region (E) and paramacular region (F). The percent vascular area was 75% (+/−2.5%) in the submacular region and 86% (+/−3.1%) in the paramacular region. Scale bars = 1mm in B&C, 500 μm in D, 100μm in E&F.

Fig. 4. Case #14.

(A) Fundus photo of the posterior pole region of the left eye of a 94-year-old Caucasian female with hypertension taken 14 months prior to death showing several drusen in the macula (arrows). This eye was last graded a 2C. (B) Gross photo after removal of retina showing the same drusen in the submacular region (arrows). The missing RPE in the peripapillary region is a dissection artifact. (C, D) Stitched panorama of confocal images from the posterior pole region of choroid labeled with UEA lectin showing a 4.6 mm² region of choriocapillaris degeneration in the submacular choroid (arrows in C) at low and higher magnification (D). The percent vascular area was 86% (+/−3.1%) in the paramacular region (F) and 75% (+/−2.5%) in the submacular region (E). Scale bars = 1mm in B&C, 500 μm in D, 100μm in E&F.

Fig. 5. Case #9.

Gross photos of the posterior pole region of the left eye of an 84-year-old Caucasian male with hypertension with the retina intact (A) and after removal of retina (B). A few indistinct drusen were noted in the submacular region (arrows in B). The missing RPE in the peripapillary region is a dissection artifact. (C, D) Stitched panorama of confocal images from the posterior pole region of choroid labeled with UEA lectin showing a 13.03 mm² region of choriocapillaris degeneration in the submacular choroid (arrows in C) at low and higher magnification. Boxes in C show areas imaged at higher magnification in E and F. The percent vascular area was 83.2% (+/−3%) in the paramacular region (F) and 57.8% (+/−2.2%) in the submacular region (E). Scale bars = 1 mm in A-C, 500 μm in D, 100 μm in E&F.

Fig. 6. Case #11.

Fundus photo (A) of the posterior pole region of the left eye of an 89-year-old Caucasian male with hypertension taken 14 years prior to death. This eye was last graded clinically a “1” two years prior to death. Gross photo after removal of retina (B) shows no drusen or pigmentary changes in the submacular region. This subject was graded 2 histologically after sectioning (see G below). The missing RPE in the peripapillary region is a dissection artifact. Stitched panorama of confocal images from the posterior pole region of choroid labeled with UEA lectin (C) showing a 9.2 mm² region of choriocapillaris degeneration in the submacular choroid (arrows in C) at low and higher magnification (D). The percent vascular area was 84.8 % (+/−0/8%) in the paramacular region (E) and 55.3% (+/−2.9%) in the submacular region (F). Cross sections show degeneration of choriocapillaris lumen in submacular choroid (arrows in G), a constricted vein (arrowhead) and a BLD on Bruch’s membrane. Choriocapillaris (arrows) and larger choroidal vessels (arrowheads) in paramacular choroid have normal structure (H). Scale bars = 1mm in B&C, 250 μm in D, 50μm in E&F, and 20μm in G&H.

The areas with submacular attenuation of CC often had basal laminar deposits (Fig. 6 and 8). The severe attenuation (43 %VA) of CC in an 84 year old subject with hypertension (Fig. 8) was accompanied by a thick basal laminar deposit in an area that had no viable CC (Fig. 8G). Another feature which was striking in the areas of CC attenuation was perhaps the earliest forms on CNV (Fig. 9). These hypercellular structures were apparent because they had intense UEA labeling and appeared almost as buds in the flat perspective (Fig. 9E and G). The CNV breaking through BrMb in Fig. 9H has a thick basal laminar deposit over it. These early CNV formations were observed in areas of submacular CC attenuation in 28% of the early AMD eyes.

Fig. 8. Case #10.

(A) Fundus photo of the posterior pole region of the right eye of a 91-year-old Caucasian female without hypertension taken 7 years prior to death showing drusen in the macula. This eye was last graded a 2C clinically 3 years prior to death. (B) Gross photo after removal of retina showing drusen, hypopigmented RPE and pigment clumping in the submacular region. (C) Stitched panorama of confocal images from the posterior pole region of choroid labeled with UEA lectin showing a 12.3 mm² region of choriocapillaris degeneration in the submacular choroid (arrows) at low magnification. (D) The border of normal paramacular and degenerative submacular CC is shown at higher magnification (D). The boxed regions in “C” are shown a higher magnification from the paramacular region (F) and submacular region (E). The percent vascular area was 43.08% (+/−8.7%) in the submacular region and 76.5% (+/−2.8%) in the paramacular region. (G, H) Cross sections show degeneration of choriocapillaris lumens in submacular choroid (small arrows in (G) and a thick BLD is present in regions with CC atrophy. Normal structure of choriocapillaris was present in paramacular choroid (small arrows in H) [Sattler’s layer (arrowhead in H) and Haller’s layer (large arrow in H).] Scale bars = 1 mm in B&C, 500 μm in D, 100μm in E&F and 20μm in G&H.

3.3. JB4 cross sections of choroid

Another striking feature of these areas of submacular CC attenuation was seen when the tissues were embedded in JB4, sectioned, and stained with PAS and H (Fig. 10). In submacula of control subjects, the three layers of the choroidal vasculature were easily apparent: CC, Sattler’s layer intermediate blood vessels, and large blood vessels in Haller’s layer (Fig. 10A). Fig.10 has 5 submacular sections of early AMD subjects as well (Fig. 10B–F). Arteries in Haller’s layer often had sclerosed walls and constriction of arterioles in Sattler’s layer (Fig. 10B–D). The outcome of these arteriosclerotic changes was certainly constriction of the lumens (Fig. 10D) and in the worst-case scenarios, stenosis of the arteries (Fig. 10E–F). These effected arterioles often demonstrated the effects of hypertension with reduplication of intima (Fig. 10E–F). All subjects with hypertension and early AMD had significant attenuation of CC in submacula.

Fig. 10. Changes in Sattler’s and Haller’s Vessels.

Periodic acid–Schiff (PAS) stained sections from submacular choroids showing choriocapillaris (CC), medium size blood vessels of Sattler’s Layer (SL) and large vessels of Haller’s Layer (HL). No pathological changes were evident in the choroidal vasculature of a 100-year-old Caucasian female without hypertension (Case #7) (A). Thickened PAS-positive material is present within the walls of blood vessels in SL and HL as well as CC dropout (asterisks) in early AMD eyes (B-F). Those subjects are shown in cross sections: 91-year-old Caucasian male (Case #13) with hypertension (B), 94-year-old Caucasian female (Case #14) with hypertension (C), 89-year-old Caucasian male (Case #11) with hypertension (D), 90-year-old Caucasian female (Case #12) with hypertension (E) and an 84-year-old Caucasian male (Case #9) with hypertension (F). Scale bar = 20 μm for all

DISCUSSION

The loss of CC in AMD has been documented by many investigators.(McLeod et al., 2009; McLeod and Lutty, 1994; Mullins et al., 2011; Sarks, 1976; Seddon et al., 2016) Most studies have focused on the advanced forms of AMD, nAMD and GA. The current study focused only on early AMD specimens and compared paramacular CC to submacular CC with the striking conclusion that there was significant loss in viability of CC in submacula in all 7 early AMD subjects studied. UEA lectin was used to label blood vessels so only viable blood vessels were visualized. The loss of vasculature occurred in areas that had a complete monolayer of RPE cells. Furthermore, the loss in CC was often associated with apparent dysfunctional intermediate blood vessels in Sattler’s layer. The area of loss was as large as 19.5 mm² and vascular density was reduced from around 80% in control subject submaculas to 60% in submacula of early AMD subjects.

Attenuation of CC in advanced AMD has been documented using many techniques.(McLeod et al., 2009; McLeod and Lutty, 1994; Mullins et al., 2011; Sarks, 1976; Seddon et al., 2016) Ramrattan and associates in a series of 95 eyes quantified elements of the choroid using H&E stained paraffin sections of human maculas (Ramrattan et al., 1994) and found that the length of intact CC per length of BrMb significantly decreased in AMD, consistent with CC dropout. Spraul and colleagues used similar methods to study 80 donor eyes and found the opposite, an increased vascular density in the eyes with AMD.(Spraul et al., 1996) The difference between these studies could be that Ramrattan et al had subjects with more advanced AMD (geographic atrophy, disciform scar) than Spraul et al whose subjects had active nAMD and “nonneovascular” AMD. The discrepancies between these reports could also be reliance on H&E stained sections of a complex lobular vasculature where ghost vessels may not be readily distinguished from healthy vessels. Thus, even a well powered study cannot provide information on the state of the choroid without distinguishing ghost from healthy vessels using H&E staining. Sarks observed hyalinization of the blood vessel walls in AMD choroid that was associated with loss of CC and expansion of the extracellular matrix in intercapillary pillars.(Sarks, 1976) It appeared that spaces previously occupied by CC were filled with various cells and collagen.(Sarks, 1976) We have observed the presence of both macrophages and mast cells in the spaces left by degenerated CC.(Bhutto et al., 2016; Grebe et al., 2019; McLeod et al., 2016) The Lutty lab has used endogenous alkaline phosphatase (APase) enzymatic activity in human choroid to characterize the healthy and diseased choroidal vasculature.(McLeod et al., 2009; McLeod and Lutty, 1994; McLeod et al., 2002) This technique permitted partial bleaching of melanin to simultaneously quantify RPE pigment and alkaline phosphatase reaction product.(McLeod et al., 2009; McLeod and Lutty, 1994; McLeod et al., 2002) This provided evidence for loss of CC endothelial cells beneath an intact RPE monolayer adjacent to CNV, whereas, in GA CC loss and constriction were associated with RPE loss. These results suggest that different forms of AMD exhibit different rates and timing of RPE and CC loss.

Ulex europaeus agglutinin-I (UEA) was used in the current study to assess the human choroidal vasculature using laser confocal scanning microscopy. UEA was first used by Mullins et al to label viable choroidal blood vessels.(Mullins et al., 2011) They found that a subset of CC endothelial cells lost CD34 expression but retained both UEA-binding glycoconjugates, suggesting that morphometric studies relying on CD34 expression for viable endothelium may underrepresent the actual number of viable cells (Sohn et al., 2014). In the current study, UEA staining demonstrated complexity of the inner choroidal vasculature and reconfirmed our previous report on the density of the CC (80% in submacula).(McLeod et al., 2009; McLeod et al., 2002) The results using UEA were similar to the study by Biesemeier et al in which quantitative histology of serial sections noted that there was substantial loss (27%) of the choriocapillaris in areas with intact RPE in AMD compared to controls.(Biesemeier et al., 2014)

The cause of the CC loss is unknown but Mullins found in UEA-stained cross sections a 45% attenuation of CC associated with drusen.(Mullins et al., 2011) They found an inverse relationship between the cross-sectional area of deposits and vascular density of the CC. Because ghost vessels were formerly viable capillaries, they suggested that this relationship was most likely due to capillary loss rather than a congenitally lower vascular density in individuals prone to develop drusen. Curcio and associates also found CC ghost vessels associated with basal linear deposits and subretinal drusenoid deposits.(Curcio and Johnson, 2013) In the current study, CC loss was associated with basal laminar deposits but this was not quantified. Additionally, we have never observed CC loss associated with hard drusen in UEA flat mount choroids or with APase labeled choroids. Mullins also suggested another cause of CC loss in AMD, membrane attack complex (MAC) deposition.(Mullins et al., 2014) They found that MAC was deposited at an early age around CC but the level increased with age. Furthermore, Cabrera et al demonstrated that aged choroidal endothelial cells are more susceptible to lysis by MAC than younger cells due to their rigidity.(Cabrera et al., 2016)

Another possible reason for CC loss could be a poor blood supply. The current study demonstrated atherosclerotic and hypertensive changes in the arterioles of Sattler’s layer, the blood supply for CC. All early AMD subjects in this study had hypertension, while only 3 of 7 control subjects had hypertension. Unfortunately, we have no actual blood pressure record for any subjects. We observed these changes previously in blood vessels of Sattler’s layer in subjects with geographic atrophy and nAMD.(McLeod et al., 2009) Spraul observed that large vessel density was lower in AMD.(Spraul et al., 1996) Woods has recently observed thinning of Sattler’s layer arterioles and Haller’s layer arteries with OCT.(Wood et al., 2011) The Grunwald group has demonstrated a reduction in choroidal blood flow in AMD subjects using laser doppler flowmetry.(Grunwald et al., 1998; Grunwald et al., 2005) They also found reduction in choroidal blood flow was further reduced with drusen extent and with CNV.(Berenberg et al., 2012; Metelitsina et al., 2008) This measurement is an assessment mostly of blood flow in foveolar CC where the current study found loss in CC density. A physiological postulate is that capillary density decreases with decreased blood flow. This applies to muscles with varying levels of exercise(Hotta et al., 2018) and even occurs after 14 days of bed rest.(Arentson-Lantz et al., 2016) Therefore, reduction in choroidal blood flow could contribute to reduced capillary density.

It was assumed historically that CC was so vast that there was sufficient redundancy in the vasculature that hypoxia was not possible in early AMD. However, we know from the studies of Linsenmeier and associates that all of the oxygen provided by CC is consumed by photoreceptors in the dark.(Wangsa-Wirawan and Linsenmeier, 2003) Therefore, just a small attenuation in vasculature might yield a hypoxic state in the dark, making RPE hypoxic. The current study demonstrated a 20% reduction in vascular density in submacula in early AMD eyes. Only 2 of 7 AMD eyes had early CNV associated with areas of CC attenuation. However, there were many structures that were intensely stained with UEA in most of the early AMD eyes but only limited areas were sectioned; so, the current study did not completely assess all areas in the entire submacula. Therefore, the incidence of early CNV was likely underestimated. Although these hypercellular capillaries that appeared to be “buds” of neovascularization were present in areas of submacular capillary dropout in 28% of the early AMD eyes, they were also in 40% of the eyes with intermediate AMD in a prior study using UEA.(Seddon et al., 2016) Because all of these areas had a complete RPE monolayer, we hypothesize that RPE associated with areas of CC loss might be hypoxic causing RPE to increase expression of VEGF, which could stimulate CNV formation. We have observed VEGF immunoreactivity in basal RPE in such areas.(Bhutto et al., 2006) In addition, there are less endogenous inhibitors of angiogenesis in AMD BrMb.(Bhutto et al., 2008) Our finding that the CC endothelium are attenuated adjacent to CNV (McLeod et al., 2009; Seddon et al., 2016) may seem paradoxical but, as in the current study, there was a complete monolayer over this attenuated CC.

Choriocapillaris has become a clinical focus for swept source optical coherence tomography angiography (SS-OCTA). With this technology RBC movement in blood vessels can be detected. Recent studies demonstrate that a loss in this signal in succinct areas in choroid, an observation originally called flow voids and now termed flow deficits.(Borrelli et al., 2018) Two groups have found that signal attenuation is greater in submacula and that the percentage of CC with flow deficits increases with age.(Nassisi et al., 2018; Zheng et al., 2019) So the question arises concerning the relationship of flow deficits to the CC loss observed herein. We observed very small areas of CC loss in the images of control subjects that were not recognized numerically by our image analysis technique. Also, do the dysfunctional arterioles in Sattler’s layer observed herein create flow deficits in CC that were fed by those arterioles? The answer to these questions is probably not possible until the eyes of a subject with clinically documented flow deficits are evaluated with the UEA technique postmortem. A final question is whether the early forms of CNV revealed by UEA staining can be detected with SS-OCTA. Subclinical CNV has been detected with SS-OCTA by some authors (de Oliveira Dias et al., 2018; Lane et al., 2016), so perhaps the structures documented herein could be detected and used as a biomarker for prophylactic treatment of the CNV.

5. Conclusions

In conclusion, a significant loss in viable submacular CC segments was observed using UEA stained human choroids where the RPE monolayer anterior to these areas was intact. Considering the size of these lesions, they should be recognizable by SS-OCTA. This suggests that longitudinal studies on flow deficits might determine if the loss in CC that we observed in submacular CC becomes future sites of RPE loss or CNV formation. Early CNV formations associated with some of these sites suggests that they may be future sites of clinically detectable CNV. The observation of dysfunctional arterioles associated with these sites suggests that actual flow deficits to CC may occur and this provides a challenge for developers of future SS-OCTA machines to image these intermediate blood vessel flow deficits. The histopathological changes observed in arterioles associated with CC loss support Ephraim Friedman’s hemodynamic theory of AMD etiology(Friedman, 1997, 2004) and also lend credence to the recent modification of the hypothesis: there is heterogeneity in hemodynamic changes, i.e. small areas may have the vascular dysfunction and flow.(Gelfand and Ambati, 2016) The current study certainly suggests that CC dysfunction occurs early in AMD and could become a biomarker for future advanced AMD and, perhaps, a target for future therapies.

Supplementary Material

1

Supplemental Fig1. Case #5 OS

Fundus photo of the posterior pole region of the left eye of an 87-year-old Caucasian male without hypertension taken 28 months prior to death (A). Gross photo with the retina intact (B) and after removal of retina (C) shows an intact submacular RPE without drusen or pigmentary changes. The missing RPE in the peripapillary region is a dissection artifact. Stitched panorama of confocal images of the posterior pole region of choroid labeled with UEA lectin showing uniform appearance of choriocapillaris (D). The percent vascular area was 76.14% (+/−4.5%) in the submacular region (E) and 74.5% (+/−2.2%) in the paramacular region (F). In (E), the intermediate vessels are the non-uniform positioned linear segments in this collapsed z stack. Scale bars = 1 mm in B-D and 100 μm in E and F.

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Supplemental Fig. 2

Confocal images from the submacular choroid of an 87 yr old Caucasian male, aged control subject (Case #5). The images are parts of a Z stack that were segregated at the level of the choriocapillaris (A) and at the level of Sattler’s layer feeding precapillary arterioles and draining postcapillary venules (arrowheads in B). Even though the choriocapillaris (CC) shows remarkable homogeneity, the precapillary arterioles and postcapillary venules have a highly irregular arrangement and often occur in close proximity to each other. Precapillary arterioles generally made connections at a 90-degree angle to the CC while postcapillary venules drained the CC at 45 degree or less angles. (UEA lectin staining, Scale bar = 30 microns)

Highlights:

There is significant loss of submacular choriocapillaris in clinically documented subjects with early AMD compared to control subjects, but no vascular pathology in paramacular choriocapillaris of these AMD subjects.

The reduction in choriocapillaris was associated with arterosclerotic and hypertensive changes in arterioles of Sattler’s layer.

Some of the areas of choriocapillaris loss had early choroidal neovascularization formations.