Hyperfluorescence of choroidal arteries in the peripheral fundus on late-phase ICGA

To correlate the hyperfluorescent lines in the peripheral fundus on late-phase indocyanine green angiography (ICGA) to infrared and optical coherence tomography (OCT) findings. This is a retrospective, cross-sectional study. Multimodal imaging data, including ICGA, fluorescein angiography, infrared imaging, and OCT were analyzed. The hyperfluorescent lines were categorized into 2 grades according to their extents. In addition, serum levels of apolipoprotein (Apo) A and B were measured by enzyme linked immunosorbent assay. A total of 247 patients who underwent multimodal imaging were reviewed. The hyperfluorescent lines in the peripheral fundus on late-phase ICGA were detected in 96 patients, and were correlated to superficial choroidal arteries by infrared imaging and OCT. The incidence of hyperfluorescent choroidal arteries in the peripheral fundus (HCAP) on late-phase ICGA increased in groups of older ages (0–20 years, 4.3%; 20–40 years, 2.6%; 40–60 years, 48.9%; >60 years, 88.7%; p < 0.001). In addition, the mean age increased with the grades of HCAP (grade 1, 52.3 ± 10.8 years; grade 2, 63.3 ± 10.5 years; p < 0.001). The hyperfluorescence was also detected in posterior choroidal arteries in 11 eyes, all patients in grade 2. There was no significant correlation between grades of HCAP and gender, or serum level of ApoA and ApoB. The occurrence and grades of HCAP increased with age. The superficial location of choroidal arteries in the peripheral fundus exposes their hyperfluorescence on late-phase ICGA. HCAP might reveal the local lipid degeneration of choroidal artery walls, according to ICG binding properties.


INTRODUCTION
Indocyanine green angiography (ICGA) is an important diagnostic tool for choroidal diseases, accessing the choroidal vascular structures through the retinal pigment epithelium (RPE) at near infrared wavelength [1].The pathologic origin of ICGA changes has been verified by histopathology only in few diseases, such as Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, birdshot chorioretinopathy, as well as sarcoidosis [2,3].
Thanks to the development of multi-modal imaging, especially optical coherence tomography (OCT), in vivo interpretation of ICGA performances can be achieved.Under normal conditions, ICG dye could extravasate into the choroidal stroma, pass through Bruch membrane (BrM), and eventually be taken up by the RPE.Therefore, homogeneous background fluorescence was observed on late-phase ICGA.The age-related scattered hypofluorescent spots on late phase indocyanine green angiography (ASHS-LIA) was recently proposed to represent the pre-basal linear deposit (BLinD) and BLinD, a thin layer of soft drusen material.ICG dye may bind preferentially to polar phospholipids over hydrophobic neutral lipids [4], and the deposition of neutral lipids between BrM and the RPE (BLinD and soft drusen) would impede ICG dye through BrM into the RPE [5].Therefore, BLinD should also be hypofluorescent as ASHS-LIA, and neither one was detectable by OCT under current axial resolution (about 5 µm).In the acute stage of multiple evanescent white dot syndrome, the hypofluorescent ICGA spots correlate with the disseminated disruptions in the ellipsoid zone on OCT [6].In this case, a dysfunctional RPE-photoreceptor complex not accumulating the ICG dye in the intermediate and late phase of angiography may account for the hypofluorescence, instead of fluorescence blockage by choroidal inflammatory lesions as previously presumed without OCT correlation.In this study, we correlated the hyperfluorescent lines in the peripheral fundus on late-phase ICGA (>20 min) to infrared imaging and OCT characteristics to reveal their pathology.

MATERIALS AND METHODS
This is a retrospective, cross-sectional study.All patients who were referred to Zhongshan Ophthalmic Center for ICGA, fluorescein angiography (FFA), and OCT examinations as part of the standard care between August 1st and October 31st in 2019 were reviewed.The disease spectrum was recorded according to the eye with higher degree of diagnosis for each patient.Patients who had peripheral lesions, peripheral choroid atrophy, or with vitreous opacity affecting fluorescence judgment were excluded from this study.The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of the Zhongshan Ophthalmic Center at Sun Yat-sen University (2020KYPJ063).Demographic information, medical records and multimodal imaging data were reviewed.ICGA, FFA, infrared imaging and OCT were performed with Spectralis (Heidelberg Engineering, Heidelberg, Germany).Routinely, infrared imaging was performed for the posterior pole.When the hyperfluorescent lines in the peripheral fundus on late-phase ICGA were detected, infrared imaging for the same area was added to help discriminate the layers involved.The extent of hyperfluorescence was categorized into 2 grades, which presenting in all 4 quadrants and more than 3 lines in every quadrant was grade 2, otherwise was grade 1 (Fig. 1).
Blood samples (5 ml) were taken before injecting dye with patients' consent and stored in our blood sample bank.In this study, patients' non-fasting serum samples were used to measure the levels of apolipoprotein A (ApoA, the main apolipoprotein of high density lipoprotein, HDL) and apolipoprotein B (ApoB, the main apolipoprotein of low density lipoprotein, LDL) by enzyme linked immunosorbent assay.
Since the hyperfluorescent lines always occurred binocularly, one eye of each individual was assigned for statistical analysis randomly.Statistical analyses were performed with SPSS Version 21.0 software (SPSS, Inc., Chicago, IL, USA).Significant differences were defined for p < 0.05.
The hyperfluorescent lines corresponding to the terminal segment of the choroidal vessels around vortex veins seen on early-phase ICGA, and therefore thought to be choroidal arteries (HCAP).Infrared imaging revealed that the arteries were superficial.They correlated to the RPE humps on OCT (Fig. 2, yellow arrowheads).No correlated change on FFA was seen.Hyperfluorescent arteries on late-phase ICGA were also detected in posterior pole (Fig. 3, yellow arrowhead) in 11 eyes, all patients in grade 2 HCAP (11 of 67 eyes, 16.4%, Table 1).

DISCUSSION
In this study, we detected hyperfluorescent lines in the peripheral fundus on late-phase ICGA, and correlated to superficial choroidal arteries by infrared imaging and OCT.The occurrence (p < 0.001) and grades (p < 0.001) of HCAP increased significantly with age in our study, indicating that age was a very important relevant factor for HCAP.The angiographic procedure of ICGA is composed of 3 main phases.The early-phase, within 2-3 min, demonstrates superimposed retinal and choroidal large vessels and initial dye leakage through the choriocapillaris into the choroidal stroma; the intermediate phase, at about 10 min, reveals maximum choroidal stromal background fluorescence; the late phase, beginning at about 22 ± 4 min, shows wash out of the dye from the general circulation with the large choroidal vessels appearing dark, against the background RPE intake and stromal fluorescence [1,7].Physically, there is no more fluorescence within the choroidal vessels during the late phase.Therefore, we defined HCAP as the hyperfluorescence persisted after 20 min.
Judging from infrared imaging and OCT, the hyperfluorescent segments of choroidal arteries were superficial.RPE humps, which were first reported in pathologic myopia eyes with advanced choroidal atrophy, resulted from the presence of an underlying large choroidal vessel [8].We believed that the underlying choroidal vessels in our study were arteries, for their distinction from the vortex vein in the same location (Fig. 2. teal arrowheads).Choroid thinned substantially in the peripheral fundus, exhibited the minimum and maximum thinning superiorly and nasally, respectively, and was thickest within the perifovea [9].In patients with grade 2 HCAP, hyperfluorescence of choroidal arteries in posterior pole was also seen (16.4%), due to diffuse choroidal atrophy.We believe that the choroidal arteries were hyperfluorescent in entire course in patients with HCAP, while only the superficial segments were detected.
ICGA is of great value on revealing lipid degeneration in the fundus diseases [5,[10][11][12].ICG may bind preferentially to phospholipids, including the ones in vessel walls, circulation (HDL), hard drusen and etc [13,14].We measured the serum levels of ApoA and ApoB between patients with and without HCAP, and there was no difference.HCAP may therefore reveal lipid degeneration of choroidal artery walls.Hyperfluorescence of choroidal arteries on late-phase ICGA was also observed in patients with systemic amyloidosis [15].ICG binding to the circulatory amyloid was supposed to cause the choroidal hyperfluorescence, and therefore it may improve after systemic treatments [16].
Fig. 1 Grading the hyperfluorescence of choroidal arteries in the peripheral fundus (HCAP) according to their extents.A grade 1 HCAP; B grade 2 HCAP.Hyperfluorescence presenting in all 4 quadrants and more than 3 lines in every quadrant was categorized as grade 2 (B), otherwise was grade 1, noticed the line in the inferior quadrant (yellow arrowhead) and peripheral drusen (A).
The relationship between degenerations of choroidal artery coronary artery disease (CAD) is unclear, while there are studies comparing the characteristics of arterial occlusive disease in other lower extremity arteries (i.e., limb arteries) and coronary arteries.Their risk factors, response to pharmacological interventions and plaque characteristics are distinctive.While LDL lowering is beneficial for CAD, the relationship between LDL lowering and peripheral artery disease (PAD) progression remains controversial and there may be other non-LDL targets for risk reduction of PAD.The content of lipid constitution is different between CAD and PAD plaques.CAD plaques have a higher percentage of lipid core named 'necrotic core plaque', whereas calcification and fibrosis dominate the PAD plaques [17].Whether lipid degeneration of choroidal arteries was compatible with a chronic ischemia that could produce retinal degeneration remains further investigation.The prevalence of HCAP was highest in AMD (Table 1), although certain selection bias may exist in our retrospective study.
There are several limitations of our study that need to be considered.Firstly, this is a clinic-based retrospective study, selection bias may occur.Patients needed ICGA for clinical management were mostly AMD, CSC, pathologic myopia and uveitis, and may therefore have differential exposure to environmental factors (e.g., smoking) or tend to have atrophic choriocapillaris.In addition, although we speculated that HCAP might represent phospholipids deposition in the vessel wall, we have no direct pathological evidence.However, considering difficulties in obtaining ICGA images in unremarkable subjects, and our study encompassed a wide age range with a large sample size, our findings are comprehensive, reliable and meaningful.
In conclusion, the occurrence and grades of HCAP increased with age.The superficial location of choroidal arteries in the peripheral fundus exposes their hyperfluorescence on late-phase ICGA.According to the binding property of ICG molecules, HCAP may reveal local lipid degeneration of choroidal artery wall.

SUMMARY
What was known before • Indocyanine green angiography (ICGA) is of great value on revealing lipid degeneration in the fundus diseases.Hyperfluorscence on late-phase ICGA was thought to be the sequel of accumulated phospholipid in the circulation (in the form of HDL), or phospholipid deposits in ocular tissues according to the binding property of ICG.

•
The hyperfluorescent lines in the peripheral fundus on latephase ICGA were correlated to superficial choroidal arteries by

Fig. 2
Fig. 2 Hyperfluorescence of choroidal arteries in the peripheral fundus (HCAP) on late-phase ICGA correlated to RPE humps on OCT.Yellow arrowheads, choroidal arteries; teal arrowheads, vortex veins; green arrowheads, choriocapillaris.A OCT scan crossing HCAP showed the superficial choroidal artery with RPE humps (yellow arrowhead).Noticed the adjacent vortex vein (teal arrowhead).B Choriocapillaris (green arrowhead) presented on the OCT scan crossing area without HCAP.

Fig. 3
Fig. 3 Hyperfluorescent choroidal arteries in the posterior pole on late-phase ICGA (yellow arrowhead) in a 79-year-old man.Diffuse hyperfluorescence of choroidal arteries in the peripheral fundus was also seen.The resolution of ICGA images was partly impacted by the patient's cataract.