Clinical science Choroidal structure in eyes with drusen and reticular pseudodrusen determined by binarisation of optical coherence tomographic images Federico Corvi,1,2 Eric H Souied,2 Vittorio Capuano,2 Eliana Costanzo,2 Lucia Benatti,1 Lea Querques,1 Francesco Bandello,1 Giuseppe Querques1,2

– 1Department of ABSTRACT on integrated imaging6 9 and histopathological , Istituto di Purpose To compare luminal and stromal area of the study,10 it has been proposed that atrophy and Ricovero e Cura a Carattere fi Scientifico (IRCCS) San Raffaele in eyes with drusen and reticular pseudodrusen brosis of the choroid could lead to the derange- Scientific Institute, University (RPD) and to investigate their changes over 24 months. ment of the RPE and secondary accumulation of Vita-Salute San Raffaele, Methods In eyes with drusen and RPD and control photoreceptor outer segments above the RPE as Milan, Italy 2 subjects, total choroidal, luminal and stromal area were RPD deposits (discrete collections of hyper- Department of measured on optical coherence tomography B-scans reflective material). Histological analysis has shown Ophthalmology, University Paris Est Creteil, Centre converted to binary images, at baseline and after that in eyes with AMD, changes of the choroidal Hospitalier Intercommunal de 24 months. interstitial stroma can occur including oedema, Creteil, Creteil, France Results Eighteen eyes of 18 subjects for each group fibrosis and inflammation with cellular infiltra- were included. In drusen and RPD, we found reduction tion.11 However, the histological processing Correspondence to fi Dr Giuseppe Querques, of mean total choroidal (p=0.0005 and p<0.0001, induces artefacts that makes dif cult evaluating the Department of Ophthalmology, respectively), luminal (p=0.003 and p<0.0001, changes in the choroid caused by the disease pro- Istituto di Ricovero e Cura a respectively) and stromal area (p=0.007 and cesses, especially the vascular tone and struc- Carattere Scientifico (IRCCS) p=0.0002, respectively) from baseline to month 24; ture.12 13 Enhanced depth imaging spectral Ospedale San Raffaele, no change of ratio between luminal–stromal and the domain-optical coherence tomography (EDI University Vita Salute San Raffaele, Via Olgettina 60, choroidal area was recorded. Mean luminal, stromal and SD-OCT) is a non-invasive imaging method to Milan 20132, Italy; giuseppe. total choroidal areas were reduced in RPD, as compared visualise with reliable images the full thickness of [email protected] with drusen and controls at both baseline and month 24 the choroid.14 Recently, it has been reported on a (p<0.05 for all). In RPD, the stromal area was more new method with high repeatability to differentiate Received 14 February 2016 Revised 1 April 2016 represented, as we found lower mean ratio of luminal and quantify the choroidal luminal area from the Accepted 30 April 2016 and total choroidal area compared with drusen and stromal area using an open access software named Published Online First control at both baseline and month 24 (p<0.05 for all). ImageJ.15 17 May 2016 Conclusions Mean total choroidal, luminal and The purpose of this study was to compare the stromal area decreased over 24 months similarly in eyes luminal and stromal area of the choroid in eyes with drusen and RPD. Mean total choroidal, luminal and with drusen and RPD and to investigate their stromal area were more reduced in eyes with RPD, as change over 24 months. compared with eyes with drusen and controls; however, stromal area was more represented in eyes with RPD METHODS suggesting a possible role of choroidal vascular depletion Study participants and fibrotic replacement in the pathogenesis and disease This is a retrospective analysis of patients from two progression. institutions (Department of Ophthalmology of the University Paris Est Creteil, and Department of Ophthalmology of the University San Raffaele in INTRODUCTION Milan) who first presented between January 2012 Age-related (AMD) is a pro- and December 2013 with a diagnosis of early gressive retinal disease with genetic, environmental AMD, and for whom a minimum follow-up of 24 and constitutional factors.12Early stages of AMD (±2) months was available. To perform this retro- are usually asymptomatic and have been charac- spective observational study, an informed consent terised, across various classification systems, by the was obtained from all subjects in agreement with presence of drusen and pigmentary alterations the Declaration of Helsinki for research involving within 2 disc diameters of the fovea.3 Drusen are human subjects. This study was institutional review composed of focal deposits of extracellular matrix board (IRB) approved at both sites and was carried and inflammatory components located between the out in compliance with local and national IRB basal lamina of the retinal pigment epithelium guidelines. (RPE) and the inner collagenous layer of Bruch’s Criteria for inclusion were (1) age ≥55 years; (2) membrane. Their formation is thought to be due to presence of five or more medium-large drusen (63– the continued outer segment constituents phagocyt- 124 μm) within the macula due to early AMD in at osis and deposition with an unbalance of produc- least one eye without RPD; (3) RPD (defined by To cite: Corvi F, Souied EH, tion and clearance associated with lipid-rich the peculiar yellowish reticular pattern at the Capuano V, et al. Br J material deposits.45On the other side, reticular macula, whose visibility was enhanced by IR reflect- Ophthalmol 2017;101:348– pseudodrusen (RPD) were identified as an add- ance)10 not accompanied by soft drusen; (4) custo- 352. itional lesion strongly associated with AMD. Based mised high-resolution EDI SD-OCT (Spectralis

348 Corvi F, et al. Br J Ophthalmol 2017;101:348–352. doi:10.1136/bjophthalmol-2016-308548 Clinical science

Heidelberg Engineering, Heidelberg, Germany) for a minimum and each parameter was estimated as the mean of the measure- follow-up of 24 (±2) months; (5) axial length between 23.5 ments performed on the three lines of the EDI OCT protocol. and 26.5 mm. The exclusion criteria were (1) presence of neo- vascular AMD in the study eye; (2) presence of atrophy in the Statistical analyses study eye; (3) any previous treatment in the study eye including Statistical calculations were performed with GraphPad Prism intravitreal injection of antivascular endothelial growth factor V.5.0(GraphPad Software, San Diego, California, USA). All vari- agents, photodynamic therapy or laser photocoagulation; (4) ables were tested for normal distributions, according to the history of ocular inflammation in the study eye; (5) significant Kolmogorov–Smirnov test. The data were summarised with the media opacities; (6) any other retinal disease (such as retinal mean±SD. The significance of differences between patients with vein occlusion, diabetic retinopathy or macular dystrophy) in drusen and RPD and control subjects was determined by the study eye. Age-matched and sex-matched control subjects Mann–Whitney rank sum test for continuous data and the χ2 with no ocular diseases and axial length between 23.5 and test for categorical data. The differences in patients and controls 26.5 mm were also included in the current analysis. In all sub- during follow-up were analysed using Wilcoxon signed-rank jects, a review of paper and electronic medical records was per- test. Univariate linear regression models and Pearson correlation formed. Demographic data and findings from the clinical were used to study the relationship among variables (each chor- examination including EDI SD-OCT were collected at baseline oidal structure and the age, sex). All tests were two-sided and a and at 24 months. p value <0.05 was considered significant.

RESULTS High-resolution EDI SD-OCT assessment A total of 18 eyes from 18 patients with drusen (12 female The method to obtain EDI OCT images has been previously (66.6%), mean age 76.3±7.2 years) and 18 eyes from 18 described in detail.14 A 19-horizontal-line protocol (6×6 mm patients with RPD (13 female (72.2%), mean age 76.6 area), each consisting of 1.024 A-scans per line, was performed. ±10.9 years) met inclusion criteria and were included in the Line scans were saved for analysis after up to 100 frames were study. A total of 18 eyes from 18 control subjects (12 female averaged, using the automatic averaging and eye tracking fea- (66.6%), mean age 75.4±6.1 years) were also included in the tures of the proprietary device. Given that medium-large drusen study. The mean follow-up was 24.7±0.7 months in eyes with are most prevalent in the central macula, while RPD superiorly drusen, 24.8±0.8 months in eyes with RPD and 25 and inferiorly to the macula,16 in all eyes we analysed the ±0.8 months in controls. central (the 10th line of this protocol, in fovea), the upper (the In eyes with drusen we observed, after 24 months, a signifi- first line of this protocol) and the lower line (the last line of this cant reduction of mean total choroidal (from 2065908 protocol) scans in order to have a more exhaustive overview on ±652018 to 1943579±616875; p=0.0005), luminal (from macular choroidal changes. Follow-up mode was used to 1438420±547387 to 1351658±474675; p=0.003) and compare the baseline examination at month 24. stromal area (from 627488±141255 to 591920±172466; p=0.007); no change of ratio between luminal–stromal and Evaluation of total choroidal area, luminal area and stromal total choroidal area was recorded (p=0.1 and p=0.1, respect- area by binarisation technique ively) (table 1)(figure 1). Similarly, in eyes with RPD we found The examined area was determined for a large 3000 μm-wide a significant reduction in mean total choroidal (from 1512251 area because preliminary study showed that the sampling of a ±399661 to 1385979±430932; p<0.0001), luminal (from small area tended to have a large diversity of the ratio of 1007640±290166 to 924802±335829; p<0.0001) and luminal/stromal area.17 EDI SD-OCT images were displayed on stromal area (from 504611±121907 to 461176±113626; a computer screen and evaluated by three masked graders inde- p=0.0002) at month 24 compared with baseline; no change of pendently (GQ, EHS and FB). When two or more graders ratio between luminal–stromal and total choroidal area was determined that the choroidal images at baseline and at month recorded (p=0.1 and p=0.1, respectively) (table 1)(figure 2). 24 of follow-up were clearly distinguishable, images were In eyes of control subjects, from baseline to month 24, we did deemed acceptable and used for the following analysis by two not record any significant change of mean total choroidal (from readers (FC and VC). The upper margin of the region of inter- 2113163±620464 to 2033480±529013; p=0.08), luminal est (ROI) was the RPE line and the lower margin was the chor- (from 1475130±507254 to 1410308±439604; p=0.1) and ioscleral border of the EDI SD-OCT images. The binarisation of stromal area (from 637959±155297 to 623171±125901; the choroidal area of the OCT image was done by a modified p=0.4); in a similar fashion we did not find any change of ratio Niblack method as previously reported.15 Briefly, the OCT between luminal–stromal and total choroidal area (p=0.9 and image was analysed by ImageJ V.1.47 (National Institutes of p=0.9, respectively) (table 1)(figure 3). Health, Bethesda, Maryland, USA; available at http://imagej.nih. Interestingly, mean luminal, stromal and total choroidal areas gov/ij/). An ROI was selected and set by the ROI manager of the were significantly reduced in eyes with RPD, as compared with OCT image. The average reflectivity of three choroidal vessels eyes with drusen and controls at both baseline and month 24 with lumens larger than 100 μm was settled as average bright- (table 1). Moreover, in eyes with RPD the stromal area was ness to the minimum value to minimise the noise of the OCT more represented, as we found lower mean ratio of luminal and image. Then, the image was converted to 8 bits and adjusted by total choroidal area compared with eyes with drusen and con- the auto local threshold of Niblack. The binarised image was trols at both baseline and month 24 (table 1). No significant dif- reconverted to an RGB image, and the luminal area was deter- ferences were found between eyes with drusen and control mined using the threshold tool. After adding the data for the subjects at both baseline and month 24 (table 1). Correlation distance of each pixel, the total choroidal, luminal and stromal analysis did not reveal any significant relationship at baseline areas were automatically calculated. The inter-rater agreement and at month 24 between the three groups (data not shown). was examined for each case by two readers. For each partici- Intrarater agreement was high with an intraclass correlation pant, examinations were performed at baseline and at month 24 coefficient (ICC) of 0.990 (CI 0.983 to 0.994) for the total

Corvi F, et al. Br J Ophthalmol 2017;101:348–352. doi:10.1136/bjophthalmol-2016-308548 349 Clinical science

Table 1 Results of choroidal binarisation method analyses in patients with drusen and RPD and in control subjects at baseline and at 24 months Drusen Pseudodrusen Control p D-RPD p D-C p RPD-C

Baseline Luminal area (μm2) 1438420±547387 1007640±290166 1475130±507254 <0.0001 0.7 <0.0001 Stromal area (μm2) 627488±141255 504611±121907 637959±155297 0.0001 0.7 <0.0001 Choroid area (μm2) 2065908±652018 1512251±399661 2113163±620464 <0.0001 0.7 <0.0001 Luminal ratio 0.68±0.05 0.66±0.03 0.69±0.06 0.02 0.8 0.03 Stromal ratio 0.32±0.05 0.34±0.03 0.31±0.05 0.02 0.8 0.03 24 months Luminal area (μm2) 1351658±474675 924802±335829 1410308±439604 <0.0001 0.3 <0.0001 p Value 0.003 <0.0001 0.1 Stromal area (μm2) 591920±172466 461176±113626 623171±125901 0.0001 0.2 <0.0001 p Value 0.007 0.0002 0.4 Choroid area (μm2) 1943579±616875 1385979±430932 2033480±529013 <0.0001 0.3 <0.0001 p Value 0.0005 <0.0001 0.08 Luminal ratio 0.69±0.05 0.66±0.04 0.68±0.06 0.005 0.9 0.007 p Value 0.1 0.1 0.9 Stromal ratio 0.31±0.05 0.34±0.04 0.32±0.06 0.005 0.9 0.007 p Value 0.1 0.1 0.9 Values are expressed as mean±SD. C, control subjects; D, drusen; RPD, reticular pseudodrusen.

choroidal, 0.991 (CI 0.984 to 0.995) for the luminal and 0.990 The metabolic support for the RPE and outer retina is pro- (CI 0.982 to 0.994) for the stromal area. The intersession agree- vided by the choroid.18 In particular the choriocapillaris has ment was also high with an ICC of 0.962 (CI 0.954 to 0.968) unique features including high flow rate and low oxygen extrac- for the total choroidal, 0.958 (CI 0.95 to 0.961) for the luminal tion that are mandatory to sustain normal photoreceptor metab- and 0.953 (CI 0.941 to 0.966) for the stromal area. olism.19 Any reduction in blood flow of choroidal circulation would have meaningful clinical effects as it is the sole supply of DISCUSSION nutrients to the outer retina. One of the negative consequences In this study, using the image binarisation method we compared may be the development of drusen and RPD. A recent study the luminal and stromal area of the choroid in eyes with drusen showed that in patients with dry AMD, there is an association and RPD and investigated their change over 24 months. In eyes between increased drusen extent and decreased choroidal blood 18 with drusen and RPD we found significant reduction of the velocity and flow. Another recent study found that eyes with 20 mean total choroidal, luminal and stromal area from baseline to more drusen had the lowest choriocapillaris density. In month 24; however, no change of ratio between luminal– patients with RPD an overall thinned choroid along with chor- stromal and the total choroidal area was recorded. oidal atrophy and fibrosis underlying RPD was demonstrated,

Figure 1 Enhanced depth imaging-optical coherence tomography (EDI-OCT) of the choroid converted to binary images in a patient with drusen at baseline (left panels) and month 24 (right panels). EDI-OCT of the fovea (the 10th line of the protocol) shows drusen, as collections of hyper-reflective material located under the retinal pigment epithelium. The area surrounded by yellow lines defines the region of interest of the choroid (first row). The EDI-OCT was converted to a binary image using ImageJ software, which can allow the visualisation of the luminal area (black) and the stromal area (white) (second row). In the region of interest the luminal area is excluded by the yellow colourisation (third row). Overlay of binary images to EDI-OCT showing the colourised (yellow) stromal area and the non-colourised luminal area (fourth row).

350 Corvi F, et al. Br J Ophthalmol 2017;101:348–352. doi:10.1136/bjophthalmol-2016-308548 Clinical science

Figure 2 Enhanced depth imaging-optical coherence tomography (EDI-OCT) of the choroid converted to binary images in a patient with reticular pseudodrusen (RPD) at baseline (left panels) and month 24 (right panels). EDI-OCT of the fovea (the 10th line of the protocol) shows RPD, as collections of hyper-reflective material located above the retinal pigment epithelium. The area surrounded by yellow lines defines the region of interest of the choroid (first row). The EDI-OCT was converted to a binary image using ImageJ software, which can allow the visualisation of the luminal area (black) and the stromal area (white) (second row). In the region of interest the luminal area is excluded by the yellow colourisation (third row). Overlay of binary images to EDI-OCT showing the colourised (yellow) stromal area and the non-colourised luminal area (fourth row). favouring the hypothesis that the choroid may be involved in luminal–stromal and the total choroidal area. On the other RPD pathogenesis.7 In the current study, we found progressive hand, in control subjects, we did not find any significant change thinning of the total choroidal area in patients with drusen and from baseline to month 24. In agreement with previous studies7 RPD, involving in a similar manner both luminal and stromal the choroid was significantly thinner in eyes with RPD claiming area whether we did not find change of the mean ratio between the possible role of choroidal vascular depletion in their

Figure 3 Enhanced depth imaging optical coherence tomography (EDI-OCT) of the choroid converted to binary images in a control subject at baseline (left panels) and month 24 (right panels). EDI-OCT of the fovea (the 10th line of the protocol) shows any retinal alteration. The area surrounded by yellow lines defines the region of interest of the choroid (first row). The EDI-OCT was converted to a binary image using ImageJ software, which can allow the visualisation of the luminal area (black) and the stromal area (white) (second row). In the region of interest the luminal area is excluded by the yellow colourisation (third row). Overlay of binary images to EDI-OCT showing the colourised (yellow) stromal area and the non-colourised luminal area (fourth row).

Corvi F, et al. Br J Ophthalmol 2017;101:348–352. doi:10.1136/bjophthalmol-2016-308548 351 Clinical science development. At both baseline and 24 months, mean luminal, agreement to be accountable for all aspects of the work in ensuring that questions stromal and total choroidal area were reduced in eyes with related to the accuracy or integrity of any part of the work are appropriately RPD, as compared with eyes with drusen and controls. investigated and resolved). Interestingly, in eyes with RPD, we found lower mean ratio of Competing interests None declared. luminal and total choroidal area, and higher mean ratio of Patient consent Obtained. stromal and total choroidal area, compared with patients with Ethics approval This study was institutional review board (IRB) approved at both drusen and control subjects. These findings suggest that the sites and was carried out in compliance with local and national IRB guidelines. luminal area (vascular component) was less represented in Provenance and peer review Not commissioned; externally peer reviewed. patients with RPD (choroidal vascular depletion). Arnold et al10 in their histopathological report showed a loss of the small chor- oidal vessels and increased spacing between the large choroidal REFERENCES fi 1 Chew EY, Clemons TE, Agrón E, et al. Ten-year follow-up of age-related macular veins, and proposed that a loss of vascularity and brotic degeneration in the age-related eye disease study: AREDS report no. 36. JAMA replacement of the choroidal stroma may be responsible for Ophthalmol 2014;132:272–7. RPD. Querques et al7 proposed that in RPD development and 2 Congdon N, O’Colmain B, Klaver CC, et al. Causes and prevalence of visual progression there may be first a diffuse loss of small choroidal impairment among adults in the United States. 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352 Corvi F, et al. Br J Ophthalmol 2017;101:348–352. doi:10.1136/bjophthalmol-2016-308548