OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY IN GEOGRAPHIC ATROPHY RICCARDO SACCONI, MD,*† ELEONORA CORBELLI, MD,* ADRIANO CARNEVALI, MD,*‡ LEA QUERQUES, MD,* FRANCESCO BANDELLO, MD, FEBO,* GIUSEPPE QUERQUES, MD, PHD* Purpose: To analyze choriocapillaris (CC) vessel density (VD) around geographic atrophy (GA) secondary to non-neovascular dry age-related macular degeneration using optical coherence tomography angiography. Methods: We compared CC VD surrounding GA margin (500 mm radius) with control CC (outside GA margin) in a consecutive series of GA patients presenting between August 2016 and February 2017 at the Medical Retina and Imaging Unit of University Vita-Salute, IRCCS Ospedale San Raffaele in Milan. Images were obtained through thresholding and binarization. We also compared the CC VD in a sample area of 500 mm · 500 mm surrounding GA margin rated as hyperautofluorescent on fundus autofluorescence to a similar area rated as isoautofluorescent. Results: Fifty eyes of 29 patients (19 women and 10 men; mean age 77 ± 6 years) with mean GA area of 9.43 ± 5.08 mm2 and mean subfoveal choroidal thickness of 164 ± 73 mm were included. Choriocapillaris VD surrounding GA margin as detected by optical coherence tomography angiography revealed a significant impairment compared with control CC outside GA margin (0.317 ± 0.083 vs. 0.461 ± 0.054, P , 0.001), which was even greater in patients with foveal involvement (P = 0.013). Furthermore, mean VD in hyperautofluorescent areas was significantly lower compared with isoautofluorescent areas (0.242 ± 0.112 vs. 0.327 ± 0.130, P = 0.001). A positive correlation was disclosed between VD surrounding GA margin and subfoveal choroidal thickness (r = 0.332, P = 0.019). Conclusion: Optical coherence tomography angiography discloses CC impairment surrounding GA margin. Such CC impairment at GA margin seems to precede retinal pigment epithelium alterations at fundus autofluorescence. Optical coherence tomography angiography could be a new valuable tool for detecting CC alterations and to evaluate potential therapeutic responses in clinical studies. RETINA 0:1–6, 2017
G
eographic atrophy (GA) represents the late stage of non-neovascular dry age-related macular degeneration (d-AMD), characterized by a round or oval atrophic area of 175 mm or more in diameter.1 The incidence of GA is almost 4 times that of neo-
vascular AMD in people of 85 years old and may affect up to 22% of population in people older than 90 years.2,3 It is bilateral in most patients and causes a slow irreversible blindness over years.4,5 Geographic atrophy results from the degeneration of photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris (CC). In contrast to the RPE disruption that can be well visualized using fundus autofluorescence (FAF) or en-face optical coherence tomography (OCT), conventional noninvasive technologies have not been able to well identify in vivo the CC disruption.6,7 Even invasive imaging techniques, including fluorescein angiography and indocyanine green angiography, have several limitations to detect the CC vascular network. Although fluorescein angiography and indocyanine green angiography could be
From the *Department of Ophthalmology, University Vita-Salute, IRCCS San Raffaele Hospital, Milan, Italy; †Department of Ophthalmology, University of Verona, Hospital of Verona, Verona, Italy; and ‡Department of Ophthalmology, University of “Magna Graecia,” Catanzaro, Italy. Presented in part at International Retinal Imaging Symposium (IRIS) Annual Meeting 2017, Los Angeles, CA, March 25, 2017. None of the authors has any financial/conflicting interests to disclose. Reprint requests: Giuseppe Querques, MD, PhD, Department of Ophthalmology, University Vita-Salute, IRCCS Ospedale San Raffaele, Via Olgettina 60, 20132 Milan, Italy; e-mail: giuseppe. [email protected]
1
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RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2017 VOLUME 0 NUMBER 0
useful in the study of retinal or choroidal circulation, they play a limited role in the study of CC due to the absence of depth resolution, the limited spatial resolution, and the obfuscation of vascular details because of dye leakage.8–10 Based on histological studies and in vivo findings, there is an open debate in the literature about the first injury in GA that is whether the RPE or CC impairment comes first.10–18 Optical coherence tomography angiography (OCT-A) is a new technique that provides unprecedented clues to the state of retinal vascular flow in a depth resolved manner, thanks to the possibility to visualize the vascular networks in separate layers.19 This revolutionizing technique has allowed to visualize in vivo the CC plexus and to detect GA as loss of CC flow under the atrophic patches.20 To the best of our knowledge, no published studies have quantified in vivo the vessel density (VD) of CC vascular network around the GA area. The aim of this study is to analyze and quantify using OCT-A the VD of CC around the GA margin, where the RPE seems to be still preserved. Moreover, we compared the VD of CC in a sample area surrounding GA margin rated as hyperautofluorescent on FAF to a similar area rated as isoautofluorescent.
Methods We enrolled consecutive patients with diagnosis of GA secondary to d-AMD that presented at the Medical Retina & Imaging Unit of the Department of Ophthalmology, University Vita-Salute, Ospedale San Raffaele in Milan, between August 2016 and February 2017. This study adhered to the tenets of the Declaration of Helsinki. All patients signed a written consent to participate to observational studies, which was approved by the ethics committee of San Raffaele Hospital. The criteria for inclusion were as follows: 1) age older than 55 years and 2) diagnosis of d-AMD with GA (GA was defined as any sharply demarcated unifocal or multifocal area of absence of the RPE larger than 175 mm in diameter). The exclusion criteria were as follows: 1) signs of choroidal neovascularization (CNV), including intraretinal or subretinal fluid, hemorrhage, and subretinal fibrosis, 2) presence of any other retinal disorders potentially confounding the clinical assessment (e.g., diabetic retinopathy, retinal vein occlusion, retinal artery occlusion), 3) myopia greater than 6 diopters, 4) any previous treatments (e.g., laser photocoagulation, photodynamic therapy, intravitreal injections of anti–vascular endothelial
growth factor or steroids), and 5) presence of significant media opacities (e.g., cataract or corneal opacity) to ensure proper images quality. All patients underwent a complete ophthalmologic examination, including best-corrected visual acuity using Early Treatment Diabetic Retinopathy Study (ETDRS) charts, slit-lamp biomicroscopy, tonometry, indirect fundus ophthalmoscopy, infrared reflectance, FAF, spectral domain-OCT (SD-OCT), fluorescein angiography, indocyanine green angiography, and OCT-A scans of the macula. Infrared reflectance, FAF, SD-OCT, fluorescein angiography, and indocyanine green angiography were performed using Spectralis (Heidelberg Engineering, Heidelberg, Germany). To achieve good visualization of the choroid, enhanced depth imaging–OCT was used in all acquisitions. Central macular thickness in the central 1-mm diameter circle of ETDRS thickness map was recorded with the Spectralis Software (Heidelberg Eye Explorer, version 1.9.14.0; Heidelberg Engineering, Heidelberg, Germany). Choroidal thickness was assessed by manually measuring the subfoveal distance between Bruch membrane interface and the sclerochoroidal interface to identify the inner and outer boundaries of the choroid, respectively. Optical Coherence Tomography Angiography Images Acquisition and Analysis Optical coherence tomography angiography examinations were performed using AngioPlex (CIRRUS HD-OCT model 5000; Carl Zeiss Meditec, Dublin, CA). We used a 6-mm · 6-mm scanning area, which is composed by 350 A-scans in each B-scan, repeated 2 times, along both the horizontal and the vertical direction. All acquisitions were performed using FastTrac retinal-tracking technology to reduce motion artifacts. Minimum strength of OCT-A images was 7 of 10. To ensure a correct visualization and assessment of the CC layer, we used the automatic segmentation provided by the system software with minor manual adjustments. All FAF and 6 · 6 OCT-A images were exported into ImageJ 1.50 (National Institutes of Health, Bethesda, MD) software. Atrophic area was manually outlined using the polygon selection tool in FAF images and transferred in OCT-A images. The dimension of GA was expressed as squared millimeters. To evaluate the CC around the atrophic area, we analyzed the area surrounding GA margin, between the border of GA and a second line drawn circularly to arbitrarily minimum 500 mm distance on the 4 cardinal points (i.e., minimum 500 mm radius). The CC outside this line was used as control. Vessel density was calculated
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OCT-A OF CHORIOCAPILLARIS IN GA SACCONI ET AL
through image thresholding and binarization, according to previous studies.21,22 Specifically, the Mean thresholding was used to binarize each image. To evaluate the VD of the CC surrounding GA margin, atrophic area and control CC were contoured and colored to pure blue (Figure 1). To evaluate the VD of control CC, all area inside the line drawn to 500-mm distance from the border of GA was colored to pure blue (Figure 1). White pixels were considered as vessel, black pixels as background; VD was calculated as the ratio between the white pixel and the total pixels after blue pixel exclusion. We also compared the VD in a CC sample area of 500 mm · 500 mm surrounding GA margin rated as hyperautofluorescent on FAF to a similar area rated as isoautofluorescent in regions not affected by drusen. These two areas have been drawn on FAF images and transferred into binarized OCT-A images (Figure 2). The area outside these samples was colored to pure blue, and the VD was calculated with the same method described above. Statistical Analysis Statistical analyses were performed using SPSS Statistics version 20 (IBM, Armonk, NY). Results of descriptive analyses are expressed as mean ± SD for quantitative variables and as counts and percentages for categorical variables. The Gaussian distribution of continuous variables was verified with the Kolmogorov–Smirnov test. Comparisons of mean VD of different areas were performed using the Student independent samples t-test. Pearson correlation analyses were performed to analyze the correlation between VD and age, best-corrected visual acuity, atrophic area extension, central macular thickness, and choroidal thickness. In all analyses, P values ,0.05 were considered as statistically significant.
3
5.54 and 8.05 ± 3.89, respectively [P = 0.151]). The mean best-corrected visual acuity was less than 20/50 Snellen equivalent (0.45 ± 0.36 logarithm of the minimum angle of resolution; median 0.4; range, 0–1.3), mean central macular thickness was 227 ± 40 mm (median, 232.5; range, 88–284), and the subfoveal choroidal thickness was 164 ± 73 mm (median, 140.5; range, 53–328). Among the 50 eyes analyzed, a hyperautofluorescent area around the GA margin was disclosed in 45 cases on FAF images. Usually, the area around the GA margin was not affected by drusen or reticular pseudodrusen. On OCT-A VD analysis, the CC surrounding the GA margin revealed a qualitative and quantitative impairment compared with control CC (VD: 0.317 ± 0.083 and 0.461 ± 0.054, respectively [P , 0.001]) (Figure 1). This reduction was observed in all cases, but was particularly evident in eyes with foveal involvement GA, which showed a significant lower VD compared with eyes with foveal sparing GA (0.293 ± 0.071 and 0.358 ± 0.089, respectively [P = 0.013]). The reduction of VD in the CC surrounding the GA margin was significant for both hyperautofluorescent and the isoautofluorescent area compared with the control CC (0.242 ± 0.112 and 0.327 ± 0.130 vs. 0.461 ± 0.054, respectively [P , 0.001 in both analysis]). Interestingly, the mean VD in hyperautofluorescent areas was significantly lower compared with isoautofluorescent areas (0.242 ± 0.112 vs. 0.327 ± 0.130, P = 0.001) (Figure 2). A positive correlation was found between VD surrounding GA margin and the subfoveal choroidal thickness (r = 0.332, P = 0.019). No other significant correlation was found between VD around GA lesion and age, best-corrected visual acuity, central macular thickness, and atrophic area extension.
Results
Discussion
Fifty eyes of 29 patients (19 women, 10 men) met the inclusion criteria and were included for the analysis. The fellow eyes of eight patients were not included in the analysis due to the presence of a neovascular AMD (four eyes) or no proper images quality (four eyes). The mean age was 77 ± 6 years (median, 77; range, 65–87) and the ethnicity was white for all patients. Considering FAF images, all patients were affected by GA, with a foveal involvement in 32 eyes and a foveal sparing in 18 eyes. The mean area of GA lesion was 9.43 ± 5.08 mm2 (range, 0.89–20.61), with a larger GA mean size in eyes with foveal involvement than eyes with foveal sparing (10.20 ±
In this study, using OCT-A, we investigated the CC around the GA in patients affected by d-AMD. From the late 60s, many histologic and clinical studies were conducted to assess whether the first injury in GA takes place from the RPE or CC.10–18,23,24 By analyzing 5 postmortem subjects affected by GA, McLeod et al11 found a linear correlation between the RPE loss and CC disruption with a 50% reduction of vascular area in regions of complete RPE atrophy. Based on these results, the authors concluded that the primary insult in GA seems to be at the level of the RPE and secondarily the CC is involved.11 The underlying molecular mechanisms are not completely understood,
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Fig. 1. FAF image and OCT-A of Patient 2 affected by GA showing the experimental design. A and B. FAF image was used to delineate the area affected by GA (A), which was colored with pure blue (B). C–H. CC 6 · 6 en-face OCT-A (C), binarized image (D), and corresponding B-scan with flow (F). The GA area delineated on FAF was overlaid on binarized en-face OCT-A (E). Binarized CC around the atrophic area margin (G) was quantified and compared with CC outside this area (H).
but the authors supported the idea that the production of vascular endothelial growth factor and other trophic molecules are fundamental for the CC survival.11,15,25 However, Biesemeier et al14 reported that, in four postmortem GA eyes, CC is always more damaged compared with RPE at the level of “transition zone”
and concluded that probably the loss of CC precedes the RPE degeneration. The authors sustained that AMD could be considered a vascular disease, in which CC atrophy causes hypoxia resulting probably in RPE and photoreceptor degeneration.14,15 In the past years, the development of new noninvasive tools, in
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OCT-A OF CHORIOCAPILLARIS IN GA SACCONI ET AL
5
Fig. 2. FAF image and binarized OCT-A of Patient 16 affected by GA showing the experimental design. A–D. FAF image was used to delineate the area affected by GA (A), which was colored with pure blue (B). White squares delineating two 500 mm · 500 mm samples of a hyperautofluorescent area (right) and an isoautofluorescent area (left) selected on the GA margin. The area outside these samples was colored with pure blue, and the VD was calculated through image binarization for both isoautofluorescent area (C) and hyperautofluorescent area (D).
particular OCT-A, has provided unprecedented manner to study in vivo the CC, overcoming some limitations of histologic studies and dye angiographies. To the best of our knowledge, no studies have been published about the quantification of CC around GA using OCT-A. By means of OCT-A, we showed a severe impairment of VD in the CC surrounding the GA margin compared with control CC (P , 0.001). This impairment was greater in patients with foveal involvement than in foveal sparing, probably due to the most advanced stage of the disease. It is noteworthy that we disclosed a trend for larger GA mean size in eyes with foveal involvement versus foveal sparing (although no significant changes were recorded). However, no correlation was found between GA area and VD in the CC surrounding the GA margin (data not reported). Therefore, the interpretation of this finding remains to be elucidated. Because FAF did not reveal atrophic RPE in the same area, our data seem to support the theory that the damage of CC vascular layer may precede the RPE atrophy. Together with previous studies, this implicates that other mechanisms unrelated to the RPE disruption may be implicated in the CC impairment, despite an intimate relationship between these two layers. Supporting this theory, Kvanta et al18 reported recently that CC flow was reduced outside the GA extending well beyond the GA margin in a qualitative analysis of CC using OCT-A. Also Kim et al17 described small focal areas of CC dropout at the edge of atrophic area in a GA patient using a noninvasive microvascular imaging technique called phase-variance OCT.
Lindner et al26 reported that GA patients have a thinning of the choroidal thickness extending well beyond the GA margin, along with alterations of CC. In our patients, we also disclosed a significant positive correlation between the VD and the choroidal thickness. Taken together, these findings support the concept that eyes with a thinner choroidal thickness show a lower CC VD at the margin of GA, thus confirming the relationship between CC vascular network and total choroidal circulation. Interestingly, although the reduction of VD in the CC surrounding the GA margin was significant for both the hyperautofluorescent and the isoautofluorescent area compared with the control CC (0.242 ± 0.112 and 0.327 ± 0.130 vs. 0.461 ± 0.054, respectively [P , 0.001 in both analysis]), the mean VD was significantly lower in hyperautofluorescent areas compared with isoautofluorescent areas (0.242 ± 0.112 vs. 0.327 ± 0.130, P = 0.001). Based on these findings and considering the presence of hyperautofluorescence at the GA margin as a sign of significant RPE impairment,6,27 we hypothesize that the first injury in GA could be at the level of CC, and that once the CC is severely impaired, the RPE may develop atrophy. It is noteworthy that because OCT-A may detect vessels only when characterized by a certain range of flow,28 reduced VD in the CC surrounding GA margin does not necessarily mean absence or reduced CC, but rather impaired flow as detected by OCT-A. Although we acknowledge that this study has several limitations, mainly due to the absence of a follow-up and the relative small size of sample, this is the first study to report a quantitative and qualitative CC impairment in vivo beyond the GA margin.
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In conclusion, using OCT-A, we demonstrated significant CC impairment surrounding GA margin, which seems to precede RPE alterations at FAF. In this context, OCT-A could be a new valuable tool for detecting CC alterations and to evaluate potential therapeutic responses in clinical studies. Nevertheless, further studies are warranted to understand better the mutualistic relationship between RPE and CC in GA secondary to non-neovascular d-AMD. Key words: dry age-related macular degeneration, geographic atrophy, optical coherence tomography angiography, multimodal imaging, vessel density. Acknowledgments
9.
10.
11.
12.
13.
F. Bandello is a consultant for Alcon (Fort Worth, TX), Alimera Sciences (Alpharetta, GA), Allergan Inc (Irvine, CA), Farmila-Thea (Clermont-Ferrand, France), Bayer Shering-Pharma (Berlin, Germany), Bausch And Lomb (Rochester, NY), Genentech (San Francisco, CA), Hoffmann-La-Roche (Basel, Switzerland), NovagaliPharma (Évry, France), Novartis (Basel, Switzerland), SanofiAventis (Paris, France), Thrombogenics (Heverlee, Belgium), and Zeiss (Dublin). G. Querques consultant for Alimera Sciences (Alpharetta, GA), Allergan Inc (Irvine, CA), Bayer Shering-Pharma (Berlin, Germany), Heidelberg (Germany), Novartis (Basel, Switzerland), Sandoz (Berlin, Germany), and Zeiss (Dublin).
16.
References
19.
1. Bird AC, Bressler NM, Bressler SB, et al. An International classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiology Study Group. Surv Ophthalmol 1995;39:367–374. 2. Hirvela H, Luukinen H, Laara E, et al. Risk factors of agerelated maculopathy in a population 70 years of age or older. Ophthalmology 1996;103:871–877. 3. Buch H, Vinding T, Nielsen NV, et al. 14-year incidence progression and visual morbidity of age-related maculopaty: The Copenhagen City Eye Study. Ophthalmology 2005;112:787–798. 4. Schatz H, McDonald HR. Atrophic macular degeneration. Rate of spread of geographic atrophy and visual loss. Ophthalmology 1989;96:1541–1551. 5. Sunness JS, Rubin GS, Applegate CA, et al. Visual function abnormalities and prognosis in eyes with age-related geographic atrophy of the macula and good visual acuity. Ophthalmology 1997;104:1677–1691. 6. Holz FG, Bellman C, Staudt S, et al. Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration. Invest Ophthalmol Vis Sci 2001;42:1051–1056. 7. Holz FG, Bindewald-Wittich A, Fleckenstein M, et al; FAMStudy Group. Progression of geographic atrophy and impact of fundus autofluorescence patterns in age-related macular degeneration. Am J Ophthalmol 2007;143:463–472. 8. Pauleikhoff D, Spital G, Radermacher M, et al. A fluorescein and indocyanine green angiographic study of choriocapillaris
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in age-related macular disease. Arch Ophthalmol 1999;117: 1353–1358. Forte R, Querques G, Querques L, et al. Multimodal imaging of dry age-related macular degeneration. Acta Ophthalmol 2012;90:e281–e287. Moult EM, Waheed NK, Novais EA, et al. Swept-SOURCE optical coherence tomography angiography reveals choriocapillaris alterations in eyes with nascent geographic atrophy and drusenassociated geographic atrophy. Retina 2016;36(Suppl 1):S2–S11. McLeod DS, Grebe R, Bhutto I, et al. Relationship between RPE and choriocapillaris in age-related macular degeneration. Invest Ophthalmol Vis Sci 2009;50:4982–4991. Mullins RE, Johnson MN, Faidley EA, et al. Choriocapillaris vascular dropout related to density of drusen in human eyes with early age-related macular degeneration. Invest Ophthalmol Vis Sci 2011;52:1606–1612. Bhutto I, Lutty G. Understanding age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment epithelium/Bruch’s membrane/choriocapillaris complex. Mol Aspects Med 2012;33:295–317. Biesemeier A, Taubitz T, Julien S, et al. Choriocapillaris breakdown precedes retinal degeneration in age-related macular degeneration. Neurobiol Aging 2014;35:2562–2573. Schlingemann RO. Role of growth factors and the wound healing response in age-related macular degeneration. Graefe’s Arch Clin Exp Ophthalmol 2004;242:91–101. Choi W, Moult EM, Waheed NK, et al. Ultrahigh-speed, swept-source optical coherence tomography angiography in nonexudative age-related macular degeneration with geographic atrophy. Ophthalmology 2015;122:2532–2544. Kim DY, Fingler J, Zawadzki RJ, et al. Optical imaging of the chorioretinal vasculature in the living human eye. Proc Natl Acad Sci U S A 2013;110:14354–14359. Kvanta A, Casselholm de Salles M, Amrén U, Bartuma H. Optical coherence tomography angiography of the foveal microvasculature in geographic atrophy. Retina 2017;37:936–942. Gao SS, Jia Y, Zhang M, et al. Optical coherence tomography angiography. Invest Ophthalmol Vis Sci 2016;57:OCT27–OCT36. Waheed NK, Moult EM, Fujimoto JG, Rosenfeld PJ. Optical coherence tomography angiography of dry age-related macular degeneration. Dev Ophthalmol 2016;56:91–100. Chidambara L, Gadde SG, Yadav NK, et al. Characteristics and quantification of vascular changes in macular telangiectasia type 2 on optical coherence tomography angiography. Br J Ophthalmol 2016;100:1482–1488. Battaglia Parodi M, Cicinelli MV, Rabiolo A, et al. Vascular abnormalities in patients with Stargardt disease assessed with optical coherence tomography angiography. Br J Ophthalmol 2016;101:780–785. Hogan MJ. Bruch’s membrane and disease of the macula. Role of elastic tissue and collagen. Trans Ophthalmol Soc U K 1967;87:113–161. Hogan MJ. Role of the retinal pigment epithelium in macular disease. Trans Am Acad Ophthalmol Otolaryngol 1972;76:64–80. Korte GE, Repucci V, Henkind P. RPE destruction causes choriocapillary atrophy. Invest Ophthalmol Vis Sci 1984;25:1135–1145. Lindner M, Bezatis A, Czauderna J, et al. Choroidal thickness in geographic atrophy secondary to age-related macular degeneration. Invest Ophthalmol Vis Sci 2015;56:875–882. Schmitz-Valckenberg S, Bültmann S, Dreyhaupt J, et al. Fundus autofluorescence and fundus perimetry in the junctional zone of geographic atrophy in patients with age-related macular degeneration. Invest Ophthalmol Vis Sci 2004;45:4470–4476. Spaide RF, Fujimoto JG, Waheed NK. Image artifacts in optical coherence tomography angiography. Retina 2015;35:2163–2180.
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G
eographic atrophy (GA) represents the late stage of non-neovascular dry age-related macular degeneration (d-AMD), characterized by a round or oval atrophic area of 175 mm or more in diameter.1 The incidence of GA is almost 4 times that of neo-
vascular AMD in people of 85 years old and may affect up to 22% of population in people older than 90 years.2,3 It is bilateral in most patients and causes a slow irreversible blindness over years.4,5 Geographic atrophy results from the degeneration of photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris (CC). In contrast to the RPE disruption that can be well visualized using fundus autofluorescence (FAF) or en-face optical coherence tomography (OCT), conventional noninvasive technologies have not been able to well identify in vivo the CC disruption.6,7 Even invasive imaging techniques, including fluorescein angiography and indocyanine green angiography, have several limitations to detect the CC vascular network. Although fluorescein angiography and indocyanine green angiography could be
From the *Department of Ophthalmology, University Vita-Salute, IRCCS San Raffaele Hospital, Milan, Italy; †Department of Ophthalmology, University of Verona, Hospital of Verona, Verona, Italy; and ‡Department of Ophthalmology, University of “Magna Graecia,” Catanzaro, Italy. Presented in part at International Retinal Imaging Symposium (IRIS) Annual Meeting 2017, Los Angeles, CA, March 25, 2017. None of the authors has any financial/conflicting interests to disclose. Reprint requests: Giuseppe Querques, MD, PhD, Department of Ophthalmology, University Vita-Salute, IRCCS Ospedale San Raffaele, Via Olgettina 60, 20132 Milan, Italy; e-mail: giuseppe. [email protected]
1
Copyright ª by Ophthalmic Communications Society, Inc. Unauthorized reproduction of this article is prohibited.
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RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2017 VOLUME 0 NUMBER 0
useful in the study of retinal or choroidal circulation, they play a limited role in the study of CC due to the absence of depth resolution, the limited spatial resolution, and the obfuscation of vascular details because of dye leakage.8–10 Based on histological studies and in vivo findings, there is an open debate in the literature about the first injury in GA that is whether the RPE or CC impairment comes first.10–18 Optical coherence tomography angiography (OCT-A) is a new technique that provides unprecedented clues to the state of retinal vascular flow in a depth resolved manner, thanks to the possibility to visualize the vascular networks in separate layers.19 This revolutionizing technique has allowed to visualize in vivo the CC plexus and to detect GA as loss of CC flow under the atrophic patches.20 To the best of our knowledge, no published studies have quantified in vivo the vessel density (VD) of CC vascular network around the GA area. The aim of this study is to analyze and quantify using OCT-A the VD of CC around the GA margin, where the RPE seems to be still preserved. Moreover, we compared the VD of CC in a sample area surrounding GA margin rated as hyperautofluorescent on FAF to a similar area rated as isoautofluorescent.
Methods We enrolled consecutive patients with diagnosis of GA secondary to d-AMD that presented at the Medical Retina & Imaging Unit of the Department of Ophthalmology, University Vita-Salute, Ospedale San Raffaele in Milan, between August 2016 and February 2017. This study adhered to the tenets of the Declaration of Helsinki. All patients signed a written consent to participate to observational studies, which was approved by the ethics committee of San Raffaele Hospital. The criteria for inclusion were as follows: 1) age older than 55 years and 2) diagnosis of d-AMD with GA (GA was defined as any sharply demarcated unifocal or multifocal area of absence of the RPE larger than 175 mm in diameter). The exclusion criteria were as follows: 1) signs of choroidal neovascularization (CNV), including intraretinal or subretinal fluid, hemorrhage, and subretinal fibrosis, 2) presence of any other retinal disorders potentially confounding the clinical assessment (e.g., diabetic retinopathy, retinal vein occlusion, retinal artery occlusion), 3) myopia greater than 6 diopters, 4) any previous treatments (e.g., laser photocoagulation, photodynamic therapy, intravitreal injections of anti–vascular endothelial
growth factor or steroids), and 5) presence of significant media opacities (e.g., cataract or corneal opacity) to ensure proper images quality. All patients underwent a complete ophthalmologic examination, including best-corrected visual acuity using Early Treatment Diabetic Retinopathy Study (ETDRS) charts, slit-lamp biomicroscopy, tonometry, indirect fundus ophthalmoscopy, infrared reflectance, FAF, spectral domain-OCT (SD-OCT), fluorescein angiography, indocyanine green angiography, and OCT-A scans of the macula. Infrared reflectance, FAF, SD-OCT, fluorescein angiography, and indocyanine green angiography were performed using Spectralis (Heidelberg Engineering, Heidelberg, Germany). To achieve good visualization of the choroid, enhanced depth imaging–OCT was used in all acquisitions. Central macular thickness in the central 1-mm diameter circle of ETDRS thickness map was recorded with the Spectralis Software (Heidelberg Eye Explorer, version 1.9.14.0; Heidelberg Engineering, Heidelberg, Germany). Choroidal thickness was assessed by manually measuring the subfoveal distance between Bruch membrane interface and the sclerochoroidal interface to identify the inner and outer boundaries of the choroid, respectively. Optical Coherence Tomography Angiography Images Acquisition and Analysis Optical coherence tomography angiography examinations were performed using AngioPlex (CIRRUS HD-OCT model 5000; Carl Zeiss Meditec, Dublin, CA). We used a 6-mm · 6-mm scanning area, which is composed by 350 A-scans in each B-scan, repeated 2 times, along both the horizontal and the vertical direction. All acquisitions were performed using FastTrac retinal-tracking technology to reduce motion artifacts. Minimum strength of OCT-A images was 7 of 10. To ensure a correct visualization and assessment of the CC layer, we used the automatic segmentation provided by the system software with minor manual adjustments. All FAF and 6 · 6 OCT-A images were exported into ImageJ 1.50 (National Institutes of Health, Bethesda, MD) software. Atrophic area was manually outlined using the polygon selection tool in FAF images and transferred in OCT-A images. The dimension of GA was expressed as squared millimeters. To evaluate the CC around the atrophic area, we analyzed the area surrounding GA margin, between the border of GA and a second line drawn circularly to arbitrarily minimum 500 mm distance on the 4 cardinal points (i.e., minimum 500 mm radius). The CC outside this line was used as control. Vessel density was calculated
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OCT-A OF CHORIOCAPILLARIS IN GA SACCONI ET AL
through image thresholding and binarization, according to previous studies.21,22 Specifically, the Mean thresholding was used to binarize each image. To evaluate the VD of the CC surrounding GA margin, atrophic area and control CC were contoured and colored to pure blue (Figure 1). To evaluate the VD of control CC, all area inside the line drawn to 500-mm distance from the border of GA was colored to pure blue (Figure 1). White pixels were considered as vessel, black pixels as background; VD was calculated as the ratio between the white pixel and the total pixels after blue pixel exclusion. We also compared the VD in a CC sample area of 500 mm · 500 mm surrounding GA margin rated as hyperautofluorescent on FAF to a similar area rated as isoautofluorescent in regions not affected by drusen. These two areas have been drawn on FAF images and transferred into binarized OCT-A images (Figure 2). The area outside these samples was colored to pure blue, and the VD was calculated with the same method described above. Statistical Analysis Statistical analyses were performed using SPSS Statistics version 20 (IBM, Armonk, NY). Results of descriptive analyses are expressed as mean ± SD for quantitative variables and as counts and percentages for categorical variables. The Gaussian distribution of continuous variables was verified with the Kolmogorov–Smirnov test. Comparisons of mean VD of different areas were performed using the Student independent samples t-test. Pearson correlation analyses were performed to analyze the correlation between VD and age, best-corrected visual acuity, atrophic area extension, central macular thickness, and choroidal thickness. In all analyses, P values ,0.05 were considered as statistically significant.
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5.54 and 8.05 ± 3.89, respectively [P = 0.151]). The mean best-corrected visual acuity was less than 20/50 Snellen equivalent (0.45 ± 0.36 logarithm of the minimum angle of resolution; median 0.4; range, 0–1.3), mean central macular thickness was 227 ± 40 mm (median, 232.5; range, 88–284), and the subfoveal choroidal thickness was 164 ± 73 mm (median, 140.5; range, 53–328). Among the 50 eyes analyzed, a hyperautofluorescent area around the GA margin was disclosed in 45 cases on FAF images. Usually, the area around the GA margin was not affected by drusen or reticular pseudodrusen. On OCT-A VD analysis, the CC surrounding the GA margin revealed a qualitative and quantitative impairment compared with control CC (VD: 0.317 ± 0.083 and 0.461 ± 0.054, respectively [P , 0.001]) (Figure 1). This reduction was observed in all cases, but was particularly evident in eyes with foveal involvement GA, which showed a significant lower VD compared with eyes with foveal sparing GA (0.293 ± 0.071 and 0.358 ± 0.089, respectively [P = 0.013]). The reduction of VD in the CC surrounding the GA margin was significant for both hyperautofluorescent and the isoautofluorescent area compared with the control CC (0.242 ± 0.112 and 0.327 ± 0.130 vs. 0.461 ± 0.054, respectively [P , 0.001 in both analysis]). Interestingly, the mean VD in hyperautofluorescent areas was significantly lower compared with isoautofluorescent areas (0.242 ± 0.112 vs. 0.327 ± 0.130, P = 0.001) (Figure 2). A positive correlation was found between VD surrounding GA margin and the subfoveal choroidal thickness (r = 0.332, P = 0.019). No other significant correlation was found between VD around GA lesion and age, best-corrected visual acuity, central macular thickness, and atrophic area extension.
Results
Discussion
Fifty eyes of 29 patients (19 women, 10 men) met the inclusion criteria and were included for the analysis. The fellow eyes of eight patients were not included in the analysis due to the presence of a neovascular AMD (four eyes) or no proper images quality (four eyes). The mean age was 77 ± 6 years (median, 77; range, 65–87) and the ethnicity was white for all patients. Considering FAF images, all patients were affected by GA, with a foveal involvement in 32 eyes and a foveal sparing in 18 eyes. The mean area of GA lesion was 9.43 ± 5.08 mm2 (range, 0.89–20.61), with a larger GA mean size in eyes with foveal involvement than eyes with foveal sparing (10.20 ±
In this study, using OCT-A, we investigated the CC around the GA in patients affected by d-AMD. From the late 60s, many histologic and clinical studies were conducted to assess whether the first injury in GA takes place from the RPE or CC.10–18,23,24 By analyzing 5 postmortem subjects affected by GA, McLeod et al11 found a linear correlation between the RPE loss and CC disruption with a 50% reduction of vascular area in regions of complete RPE atrophy. Based on these results, the authors concluded that the primary insult in GA seems to be at the level of the RPE and secondarily the CC is involved.11 The underlying molecular mechanisms are not completely understood,
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RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2017 VOLUME 0 NUMBER 0
Fig. 1. FAF image and OCT-A of Patient 2 affected by GA showing the experimental design. A and B. FAF image was used to delineate the area affected by GA (A), which was colored with pure blue (B). C–H. CC 6 · 6 en-face OCT-A (C), binarized image (D), and corresponding B-scan with flow (F). The GA area delineated on FAF was overlaid on binarized en-face OCT-A (E). Binarized CC around the atrophic area margin (G) was quantified and compared with CC outside this area (H).
but the authors supported the idea that the production of vascular endothelial growth factor and other trophic molecules are fundamental for the CC survival.11,15,25 However, Biesemeier et al14 reported that, in four postmortem GA eyes, CC is always more damaged compared with RPE at the level of “transition zone”
and concluded that probably the loss of CC precedes the RPE degeneration. The authors sustained that AMD could be considered a vascular disease, in which CC atrophy causes hypoxia resulting probably in RPE and photoreceptor degeneration.14,15 In the past years, the development of new noninvasive tools, in
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OCT-A OF CHORIOCAPILLARIS IN GA SACCONI ET AL
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Fig. 2. FAF image and binarized OCT-A of Patient 16 affected by GA showing the experimental design. A–D. FAF image was used to delineate the area affected by GA (A), which was colored with pure blue (B). White squares delineating two 500 mm · 500 mm samples of a hyperautofluorescent area (right) and an isoautofluorescent area (left) selected on the GA margin. The area outside these samples was colored with pure blue, and the VD was calculated through image binarization for both isoautofluorescent area (C) and hyperautofluorescent area (D).
particular OCT-A, has provided unprecedented manner to study in vivo the CC, overcoming some limitations of histologic studies and dye angiographies. To the best of our knowledge, no studies have been published about the quantification of CC around GA using OCT-A. By means of OCT-A, we showed a severe impairment of VD in the CC surrounding the GA margin compared with control CC (P , 0.001). This impairment was greater in patients with foveal involvement than in foveal sparing, probably due to the most advanced stage of the disease. It is noteworthy that we disclosed a trend for larger GA mean size in eyes with foveal involvement versus foveal sparing (although no significant changes were recorded). However, no correlation was found between GA area and VD in the CC surrounding the GA margin (data not reported). Therefore, the interpretation of this finding remains to be elucidated. Because FAF did not reveal atrophic RPE in the same area, our data seem to support the theory that the damage of CC vascular layer may precede the RPE atrophy. Together with previous studies, this implicates that other mechanisms unrelated to the RPE disruption may be implicated in the CC impairment, despite an intimate relationship between these two layers. Supporting this theory, Kvanta et al18 reported recently that CC flow was reduced outside the GA extending well beyond the GA margin in a qualitative analysis of CC using OCT-A. Also Kim et al17 described small focal areas of CC dropout at the edge of atrophic area in a GA patient using a noninvasive microvascular imaging technique called phase-variance OCT.
Lindner et al26 reported that GA patients have a thinning of the choroidal thickness extending well beyond the GA margin, along with alterations of CC. In our patients, we also disclosed a significant positive correlation between the VD and the choroidal thickness. Taken together, these findings support the concept that eyes with a thinner choroidal thickness show a lower CC VD at the margin of GA, thus confirming the relationship between CC vascular network and total choroidal circulation. Interestingly, although the reduction of VD in the CC surrounding the GA margin was significant for both the hyperautofluorescent and the isoautofluorescent area compared with the control CC (0.242 ± 0.112 and 0.327 ± 0.130 vs. 0.461 ± 0.054, respectively [P , 0.001 in both analysis]), the mean VD was significantly lower in hyperautofluorescent areas compared with isoautofluorescent areas (0.242 ± 0.112 vs. 0.327 ± 0.130, P = 0.001). Based on these findings and considering the presence of hyperautofluorescence at the GA margin as a sign of significant RPE impairment,6,27 we hypothesize that the first injury in GA could be at the level of CC, and that once the CC is severely impaired, the RPE may develop atrophy. It is noteworthy that because OCT-A may detect vessels only when characterized by a certain range of flow,28 reduced VD in the CC surrounding GA margin does not necessarily mean absence or reduced CC, but rather impaired flow as detected by OCT-A. Although we acknowledge that this study has several limitations, mainly due to the absence of a follow-up and the relative small size of sample, this is the first study to report a quantitative and qualitative CC impairment in vivo beyond the GA margin.
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In conclusion, using OCT-A, we demonstrated significant CC impairment surrounding GA margin, which seems to precede RPE alterations at FAF. In this context, OCT-A could be a new valuable tool for detecting CC alterations and to evaluate potential therapeutic responses in clinical studies. Nevertheless, further studies are warranted to understand better the mutualistic relationship between RPE and CC in GA secondary to non-neovascular d-AMD. Key words: dry age-related macular degeneration, geographic atrophy, optical coherence tomography angiography, multimodal imaging, vessel density. Acknowledgments
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F. Bandello is a consultant for Alcon (Fort Worth, TX), Alimera Sciences (Alpharetta, GA), Allergan Inc (Irvine, CA), Farmila-Thea (Clermont-Ferrand, France), Bayer Shering-Pharma (Berlin, Germany), Bausch And Lomb (Rochester, NY), Genentech (San Francisco, CA), Hoffmann-La-Roche (Basel, Switzerland), NovagaliPharma (Évry, France), Novartis (Basel, Switzerland), SanofiAventis (Paris, France), Thrombogenics (Heverlee, Belgium), and Zeiss (Dublin). G. Querques consultant for Alimera Sciences (Alpharetta, GA), Allergan Inc (Irvine, CA), Bayer Shering-Pharma (Berlin, Germany), Heidelberg (Germany), Novartis (Basel, Switzerland), Sandoz (Berlin, Germany), and Zeiss (Dublin).
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