RESEARCH ARTICLE

Comparison between optical coherence
tomography angiography andimmunolabeling for evaluation of laserinduced choroidal neovascularization
Kazuki Nakagawa*, Haruhiko Yamada, Hidetsugu Mori, Keiko Toyama, Kanji Takahashi
Department of Ophthalmology, Kansai Medical University, Hirakata, Osaka, Japan* Nakakazu@hirakata.kmu.ac.jp

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OPEN ACCESS
Citation: Nakagawa K, Yamada H, Mori H, Toyama
K, Takahashi K (2018) Comparison between opticalcoherence tomography angiography andimmunolabeling for evaluation of laser-induced
choroidal neovascularization. PLoS ONE 13(8):e0201958. https://doi.org/10.1371/journal.
pone.0201958
Editor: Alfred S Lewin, University of Florida,
UNITED STATES
Received: March 29, 2018
Accepted: July 25, 2018
Published: August 9, 2018
Copyright: © 2018 Nakagawa et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.

Abstract
This study aimed to investigate the differences between images obtained by optical coherence tomography angiography (OCTA) with those from immunohistochemical labeling oflaser-induced choroidal neovascularization (CNV) in a mouse model. CNV was induced bylaser photocoagulation (GYC-2000, NIDEK; wavelength 532 nm) in the left eyes of 10
female C57BL/6J mice aged 6 weeks. The laser parameters included a 100-μm spot, 100ms pulse duration and 200-mW incident power to rupture Bruch’s membrane. OCT and
OCTA CNV images were obtained using the RS-3000 Advance (NIDEK) 5 days post-laser
photocoagulation. After OCTA imaging, the isolated choroid/retinal pigment epithelium complexes were fluorescently labeled with CD31 (an endothelial cell marker), platelet-derived
growth factor receptor β (PDGFRβ, a pericyte-like scaffold marker), α-smooth muscle actin(α-SMA) and collagen I. Area measurements of the lesions obtained by enface OCTA werecompared with immunolabeled CD31+ CNV lesions in choroid flat-mounts. We also examined structural correlations between the PDGFRβ+ pericyte-like scaffold and OCTA images.
Laser-induced CNV was clearly detected by enface OCTA, appearing as a hyperflow lesion
surrounded by a dark halo. Area measurements of the CNV lesion by immunolabeling weresignificantly larger than those obtained by enface OCTA (p = 0.006). The CNV lesion
beneath the periphery of the pericyte-like scaffold was not clearly visible by enface OCTA
due to the dark halo; however, the lesion was detectable as blood flow by cross-sectional
OCTA and was also highly labeled by CD31. The periphery of the pericyte-like scaffold
appeared to develop into subretinal fibrosis and this region was rich in myofibroblasts.
Enface OCTA was unable to detect the entire area of laser-induced CNV in mice, with anundetectable portion located beneath the fibrotic periphery of the pericyte-like scaffold. Dueto this OCTA fibrosis artifact, OCTA imaging has limited potential for accurately estimating
CNV lesions.

Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declaredthat no competing interests exist.

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OCTA image versus immunolabeling image for laser-induced CNV in mice

Introduction
Optical coherence tomography angiography (OCTA) is a novel imaging tool which allows thevisualization of the retinal and choroidal vasculature. This method is based on split-spectrum
amplitude-decorrelation angiography (SSADA) and detects blood flow as the motion of erythrocytes [1]. Because of its noninvasive aspects (OCTA does not require the use of dye injection
as in fluorescein angiography [FA] or indocyanine green angiography [ICGA]), this new
method has been successfully applied in daily clinical ophthalmological practice. Moreover,
OCTA has the unique capability of layer analysis and repeatable quantitative analysis. Somestudies have reported that OCTA could be used to detect choroidal neovascularization (CNV)
and evaluate therapeutic responses in exudative age-related macular degeneration (AMD)[2,3]. However, OCTA has undesirable imaging artifacts [4]. Thus, it is important to precisely
explore and understand the benefits and limitations of OCTA for accurate assessment of CNV.
Laser-induced CNV is a reliable animal model for exudative AMD and consists of threestages [5]. The first stage (at 0–3 days post-laser injury) is an inflammatory response which initiates a growth phase. The second stage (at 3–5 days post-laser injury) is the development of
CNV. CNV size reaches a maximum at 5 days post-laser injury. The third stage (after 5 days
post-laser injury) is the regression of CNV. Both the balance of damage to the choroid/Bruch’s
membrane/retinal pigment epithelium (RPE) complex and the ability of RPE cells to encircle
the damaged area determine the evolution of CNV [5,6]. Recently, He et al. [7] suggested thatthe pericyte-like scaffold, also referred to as the myofibroblastic scaffold, forms during theearly wound healing response before the infiltration of CD31+ endothelial cells into the site oflaser injury, and expresses high levels of IL-1β as a proangiogenic stimulus. According to theirstudies, M2 type macrophages infiltrate the site of injury within the first 24 to 40 hours, followed by the appearance of a pericyte-like scaffold strongly expressing smooth muscle actin(SMA) in the CNV lesion 60 hours after injury. Endothelial cells then form the CNV lesion. It
is thought that the pericyte-like scaffold covers and demarcates the site of angiogenesis andsimultaneously leads a colony-like CNV lesion. He et al. also suggested that the pericyte-likescaffold is not derived from transdifferentiated RPE cells, but from cells of the underlying choroid, such as pericyte-like cells [7,8]. These pericyte-like cells show intense expression of SMA
and platelet-derived growth factor receptor β (PDGFRβ). Because the RPE cells adjacent to thesite of laser injury are activated and express SMA in response to laser injury, PDGFRβ is a suitable target for specifically evaluating the pericyte-like cells. Moreover, inhibition of PDGFRβ+cell proliferation prior to neovessel formation suppressed scaffold formation and neovascularization [8].
As described above, the chronological changes in the histology of laser-induced CNV havebeen well characterized. However, the correlation between the histological characteristics of
CNV and OCTA images is poorly understood. In order to accurately assess CNV using
OCTA, it is necessary to understand how the histological characteristics influence OCTA
imaging. In this study, we imaged laser-induced CNV at 5 days post-laser photocoagulation inmice by OCTA and compared the area of the CNV lesion obtained by OCTA imaging withthat obtained by immunolabeling of choroidal flat-mounts. We also evaluated the correlation
between the histological characteristics of the pericyte-like scaffold and OCTA images.

Materials and methods
Animals
All animal experiments followed the guidelines of the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research and were approved by the Animal Care Committee of

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OCTA image versus immunolabeling image for laser-induced CNV in miceKansai Medical University (approval number: 17–013). Wild-type (WT) C57BL/6J mice werepurchased from CLEA Japan (Tokyo, Japan). A total of 10 female mice aged 6 weeks were used
for this study. All mice were kept in pathogen-free plastic cages with 12 hour light-dark cycles
and had continuous free access to water and food. All plastic cages, water and bedding feed
were purified before use. For all procedures, anesthetization was achieved by intraperitoneal
injection of 90 mg/kg ketamine hydrochloride (Daiichi Sankyo Co., Tokyo, Japan) and 40 mg/
kg xylazine (Bayer, Berlin, Germany); pupils were dilated with topical 0.5% tropicamide and0.5% phenylephrine (Santen Pharmaceutical, Osaka, Japan).

Laser-induced CNV model
CNV was induced by laser photocoagulation (GYC-2000; NIDEK Co, Japan; wavelength 532nm) attached to a slit-lamp delivery system (Carl Zeiss SL 130, Jena, Germany) in the left eyeof each mouse, with a spot size of 100-μm, a 100-ms pulse duration and a 200-mW incident
power applied to the posterior pole to rupture Bruch’s membrane. If the laser photocoagulation irradiated only one spot, the CNV lesion was too small to be observed in detail by OCTA
(Fig 1A and 1B). However, when laser photocoagulation irradiated three overlapping spots toenlarge the disruption of the choroid/Bruch’s membrane/RPE complex, the CNV lesion waslarge enough to be observed in detail meby OCTA (Fig 1C and 1D). Thus, we used three spots
per eye for the procedure in this study.

Optical coherence tomography angiography imaging
OCT and OCTA were performed using the RS-3000 Advance (NIDEK Co, Japan). A mousespecific adapter with a power of 10 diopters was installed for OCT and OCTA and a platform
was built to stably position the mice in front of the RS-3000 Advance, similar to previous
studies [9–11]. This instrument has an A-scan rate of 53,000 scans per second, and uses alight source centered at 880 nm. For OCTA, 8 linear B-scans were acquired from 256 cross sectional locations and retinal areas measuring 900×900 μm were centered on the CNV lesion
and scanned focusing on the retinal vasculature. OCT scans, which have a linear measurementof 900 μm on the retina, were acquired at 50 frames per image. The left eye of each mouse
wore a plain hard contact lens (UNICON, Osaka, Japan) to prevent drying of the cornea andunwanted cataract formation. Laser-induced CNV images were acquired 5 days post-laser
photocoagulation. We obtained 3D enface OCTA images between the deep retina and choroidusing the semi-automatic mode with some modification to the layer settings provided by RS3000 Advance to the first author.

Choroidal flat mount preparation for immunofluorescence imaging
Immediately after in vivo imaging by OCTA, deeply anesthetized mice were sacrificed byintracardial perfusion with PBS after thoracotomy. After PBS perfusion, eyes were enucleated
and fixed in 2% paraformaldehyde/PBS for 10 minutes. The choroid/RPE complex was isolated and permeabilized with 0.5% Triton X-100, 20% DMSO and 0.6% Block Ace blocking
reagent (DS Pharma Biomedical, Osaka, Japan) in PBS for 3 hours at room temperature [9]and subsequently used for immunolabeling. The following antibodies were used for wholemount immunolabeling of the choroid: i) anti-CD31 monoclonal antibody (rat anti-mouse
CD31, MEC 13.3, 1:15; BD Biosciences Pharmingen, San Diego, CA) to detect blood vessels
with Alexa Fluor-488 secondary antibody (1:500); ii) anti-PDGFRβ monoclonal antibodies(rabbit anti-mouse PDGFRβ, ab32570, 1:500; Abcam, Cambridge, MA) to detect the pericytelike scaffold with Alexa Fluor-568 secondary antibody (1:500). In addition, a monoclonal antibody against SMA (mouse anti-mouse α-SMA, ab7817, 1:2000; Abcam, Cambridge, MA) with

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OCTA image versus immunolabeling image for laser-induced CNV in miceFig 1. Alternative method for laser-induced CNV irradiation. (A) Near-infrared reflectance (NIR) image obtained immediately after laser
photocoagulation of one spot. (B) Enface OCTA image at 5 days post-laser photocoagulation (area measuring 900×900 μm). (A) and (B) are thesame eye. (C) NIR image obtained immediately after laser photocoagulation of three spots. (D) Enface OCTA image at 5 days post-laser
photocoagulation (area measuring 900×900 μm). (C) and (D) are the same eye. Note that in the three-spot laser photocoagulation irradiation,
the spots overlapped each other to create a larger disruption of the choroid/Bruch’s membrane/RPE complex, resulting in a larger CNV lesionthat is easier to observe and evaluate in detail by enface OCTA (D) than one-spot laser photocoagulation.
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Alexa Fluor-488 secondary antibody (1:500) and a polyclonal antibody against collagen I (rabbit anti-mouse collagen I, ab34710, 1:200; Abcam, Cambridge, MA) with Alexa Fluor-647 secondary antibody (1:500) were used to compare with an anti-PDGFRβ monoclonal antibody(rat anti-mouse PDGFRβ, CD140b, 1:100; Thermo Fisher Scientific) with Alexa Fluor-568 secondary antibody (1:500). The choroid was incubated in primary antibody solution at 4˚C for 2days. After washing in PBS, the choroid was incubated for 4 hours at room temperature in secondary antibody solution [9]. Four cuts were made from the edge toward the center after thoroughly washing the choroid, which was then flattened and mounted with ProLong Gold antifade reagent (Life Technologies, Eugene, OR, USA) and cover-slipped. Frozen sections cut to
8-μm thickness with a cryostat (Leica CM3050S1; Leica Biosystems, Nuβloch, Germany) werealso processed for staining (for supporting information). The CNV was stained with an anti-

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OCTA image versus immunolabeling image for laser-induced CNV in mice

CD31 monoclonal antibody (rat anti-mouse CD31, MEC 13.3, 1:50; BD Biosciences Pharmingen, San Diego, CA) and an Alexa Fluor-488 secondary antibody (1:500). The pericyte-likescaffold was stained with an anti-PDGFRβ monoclonal antibody (rabbit anti-mouse PDGFRB,
ab32570, 1:500; Abcam, Cambridge, MA) and an Alexa Fluor-568 secondary antibody (1:500).
The choroid flat-mounts and the frozen sections were visualized with a fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan). Immunolabeled images were obtainedwith a charge-coupled device camera and captured with Basler (Ahrensburg, Germany)
microscopy software.

Quantification of CNV area
To calculate the area of the CNV lesion by either OCTA imaging or CD31+ immunolabeling,
two masked graders (HM and KT) independently determined the outlines of the CNV lesions
using the same exported images. Basler microscopy software was used to quantify these regionsin the OCTA and immunolabeled images. The results of the two graders were compared andthe correlation coefficients were calculated. In addition to assess the CNV lesion, the dark halocomprising the CNV lesion in OCTA images and the PDGFRβ+ pericyte-like scaffold area
determined by immunolabeling were calculated and compared in the same manner. The evaluation method for OCTA images is illustrated in Fig 2.

Statistical analysis
The areas measured by two masked independent graders were analyzed by correlation coefficient and Bland-Altman plot [10]. Results with non-normal distribution are expressed as themedian (interquartile range). A Kruskal-Wallis test was performed using JMP1 (SAS InstituteInc., Caly, NC, USA). A p value of less than 0.05 was considered significant.

Results
Imaging of laser-induced CNV in mice by OCTA
Five days after laser photocoagulation, laser-induced CNV was clearly detected by OCTA (Figs
2 and 3A). CNV was visualized as a hyperflow lesion surrounded by a dark halo by enface
OCTA (Figs 2 and 3A). Blood flow was detected between the deep retina and choroid by crosssectional OCTA (Fig 3B). Laser-induced CNV and the pericyte-like scaffold appeared as a subretinal hyper-reflective lesion by OCT (Fig 3C). A representative OCT image of WT C57BL/6J
mouse is shown in S1 Fig.

Immunolabeled imaging of flat-mounted choroid preparations of laserinduced CNV
Five days after laser photocoagulation, new formation of CD31+ vessels (Fig 3D) and a
PDGFRβ+ pericyte-like scaffold (Fig 3E) could be clearly observed at the site of laser application. Neovessel formation did not extend beyond the pericyte-like scaffold (Fig 3F), which was
positively stained by α-SMA, collagen I and PDGFRβ (Fig 4B–4D). In contrast, the region outside of the pericyte-like scaffold was only stained by α-SMA (white arrowhead in Fig 4E). At
the periphery of the pericyte-like scaffold, the development of subretinal fibrosis was apparent(yellow arrowhead in Fig 4D). In order to assess the positional relationship between CNV andthe pericyte-like scaffold, frozen sections from laser-induced CNV samples were prepared.
The CNV lesions, which were fluorescently labeled with CD31, appeared to be present within
the pericyte-like scaffold (PDGFRβ+ region; S2 Fig).

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OCTA image versus immunolabeling image for laser-induced CNV in miceFig 2. Enface OCTA image of laser-induced CNV at 5 days post-laser photocoagulation. CNV was visualized as a
hyperflow lesion surrounded by a dark halo, indicated by the asterisk ( ). We measured the CNV lesion within the yellowdotted line. The dark halo comprising the CNV lesion was measured within the red-dotted line (OCTA measurement area:
900×900 μm).
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Comparison of OCTA images with fluorescent images of immunolabeledchoroidal flat-mounts in laser-induced CNV
The CNV lesions, which were undetectable by enface OCTA imaging, could be detected byimaging the immunolabelled region (white arrow in Fig 3G). This CNV region was located at
the periphery of the pericyte-like scaffold and within the area that appeared as a dark halo
lesion by enface OCTA (yellow arrowhead in Fig 3I). In the dark halo lesions, blood flow was
detected between the deep retina and choroid by cross-sectional OCTA imaging (yellow
arrowhead in Fig 3B); however, the neovascular network could not be detected by enface
OCTA. The tissue at the periphery of the pericyte-like scaffold was α-SMA+/collagen I+ indicating the development of fibrosis, and this area corresponded with the dark halo lesion visualized by enface OCTA (Fig 4F).

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OCTA image versus immunolabeling image for laser-induced CNV in miceFig 3. Representative OCT, OCTA and immunolabeling images of laser-induced CNV at day 5 post-laser photocoagulation. (A) By enface OCTA,
laser-induced CNV was observed as a hyperflow lesion surrounded by a dark halo (indicated by an asterisk  ; measurement area: 900×900 μm). (B) By
cross-sectional OCTA imaging (corresponding to the horizontal blue line in Fig 3A) blood flow was detected between the deep retina and choroid(measurement width: 900 μm). (C) By OCT, laser-induced CNV and the pericyte-like scaffold appeared as a subretinal hyper-reflective lesion(measurement width: 900 μm). (D) Laser-induced CNV lesions were stained with CD31. (E) The pericyte-like scaffolds were stained with PDGFRβ. The
periphery of the pericyte-like scaffold appeared to develop into subretinal fibrosis (yellow arrowhead). (F) Immunohistochemistry for CD31 mergedwith the PDGFRβ image. Neovascularization did not extend beyond the pericyte-like scaffold. (G) Enface OCTA image merged with the CD31
immunohistochemistry. The CNV lesion, which was undetectable by enface OCTA imaging, was apparent by immunolabeling (white arrow). (H)Enface OCTA image merged with PDGFRβ immunohistochemistry. The periphery of the pericyte-like scaffold, which developed into subretinal
fibrosis, corresponded with the dark halo lesion visible by enface OCTA (yellow arrowhead). (I) Triple-merged image of enface OCTA, CD31, andPDGFRβ immunohistochemistry. Note that the CNV lesion, which was undetectable by enface OCTA imaging, was detectable by immunolabeling(white arrow in Fig 3G). This portion of the CNV lesion, at the periphery of the pericyte-like scaffold, is within the lesion area comprising the dark haloby enface OCTA (yellow arrowhead in Fig 3I). Within this dark halo, blood flow could be detected between the deep retina and the choroid by crosssectional OCTA (yellow arrowhead in Fig 3B). Scale bar, 500 μm (D-F).
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Comparison of the CNV area between OCTA and immunolabelingTo determine the comparability of the areas visualized by histological assessment versus
OCTA, measurements of the CNV lesions determined by OCTA imaging were compared withmeasurements obtained by imaging the CD31+ immunolabeled lesions. In addition to evaluating the differences in CNV size, measurements of the dark halo comprising the CNV lesion in
OCTA images and area measurements of the immunolabeled PDGFRβ+ pericyte-like scaffold
were investigated. The areas measured by two masked independent graders were plottedagainst each other (Fig 5A–5D). The measured r2 values for these data were 0.97 (CNV lesion
by OCTA), 0.94 (dark halo comprising CNV lesion by OCTA), 0.95 (CD31+ CNV lesion byimmunolabeling) and 0.98 (PDGFRβ+ pericyte-like scaffold by immunolabeling). Thus, there
was good correlation between the area measurements of the two graders. Furthermore, aBland-Altman plot of the area measurements was created, with the difference in grader measurements on the y-axis and the average grader measurements on the x-axis (Fig 5E–5H). No
obvious trend was found in this plot, suggesting that there was no systematic bias. The dashedlines in Fig 5E–5H indicate the 95% confidence interval for the difference in measurements,

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OCTA image versus immunolabeling image for laser-induced CNV in miceFig 4. Images from OCTA and immunolabeling experiments in laser-induced CNV at 5 days post-laser photocoagulation. (A) By enface OCTA, laserinduced CNV was observed as a hyperflow lesion surrounded by a dark halo ( ; areas measured 900×900 μm). (B-D) The pericyte-like scaffold was stained with
α-SMA (B), PDGFRβ (C), and collagen type I (D; shown in gray scale). The periphery of the pericyte-like scaffold appeared to develop into subretinal fibrosis(yellow arrowhead). (E) Merged image of immunohistochemistry for α-SMA, PDGFRβ and collagen type I. The region outside of the pericyte-like scaffold was
only stained by α-SMA (white arrowhead). (F) Merged enface OCTA image and immunohistochemistry of the α-SMA positive pericyte-like scaffold. Bothoutside of and at the periphery of the pericyte-like scaffold, α-SMA+ staining correlated with the dark halo lesion visualized by enface OCTA imaging. Scale bar,
500 μm (B-E).
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which ranged from -5310 to 2618 (CNV lesion by OCTA), -5705 to 7635 (dark halo comprising CNV lesion by OCTA), -6009 to 5819 (CD31+ CNV lesion by immunolabeling) and -5347
to 2870 (PDGFRβ+ pericyte-like scaffold by immunolabeling). Given the lack of apparent bias
and good agreement between the graders, we determined that averaging the graders’ measurements would be suitable for our analysis. A comparison of the measured areas between OCTA
images and those obtained by immunolabeling is shown in Fig 6. Area measurements of theCD31+ CNV lesions determined by immunolabeling were significantly larger than those fromenface OCTA imaging (p = 0.006). Area measurements of the immunolabeled PDGFRβ+ pericyte-like scaffold were significantly larger than those of the CD31+ CNV lesion (p = 0.03).
Area measurements of the immunolabeled PDGFRβ+ pericyte-like scaffold tended to be larger
than those of the dark halo comprising CNV lesion by enface OCTA; however, this difference
was not statistically significant (p = 0.38).

Discussion
OCTA, a novel imaging tool, has been used in daily ophthalmological practice and has become
indispensable for assessing exudative AMD. However, OCTA has the potential to generate
undesirable imaging artifacts [4] which may lead to overestimating or underestimating CNV

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OCTA image versus immunolabeling image for laser-induced CNV in miceFig 5. Inter-grader reliability (A-D) and a Brand-Altman plot (E-H) of the area measurements from OCTA and immunolabeling.
(A and E) CNV lesion by OCTA. (B and F) dark halo comprising CNV lesion by OCTA. (C and G) CD31+ CNV lesion byimmunolabeling. (D and H) PDGFRβ+ pericyte-like scaffold by immunolabeling. The r2value was 0.97 (A: CNV lesion by OCTA), 0.94
(B: dark halo comprising CNV lesion by OCTA), 0.95 (C: CD31+ CNV lesion by immunolabeling) and 0.98 (D: PDGFRβ+ pericyte-likescaffold by immunolabeling). A Bland-Altman plot showed agreement between the measurements of the two graders (E-H). No
particular trend was found in this plot suggesting that there was no systematic bias.
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lesions in exudative AMD. Projection artifacts are one of the most critical artifacts which canaffect evaluation of OCTA images. This artifact results in vessels that may appear deeper within
the eye than they actually are [4]. These projection artifacts may result in overestimation of the
CNV lesion. Alternatively, shadowing is an attenuation of signal behind an absorbing or scattering opacity or obstruction [4]. Shadowing artifacts may lead to false-negative flow and resultin the underestimation of CNV lesions. Media opacities, pigment in the RPE and fibrosis also
have the potential to cause shadowing. Although there have been some reports of OCTA imaging in laser-induced CNV, these studies did not compare the OCTA images with histologicalmarkers other than CD31 or evaluate the presence of OCTA artifacts [9–11]. In this study, we
not only compared CNV lesions, but also evaluated the dark halo lesions between OCTA

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OCTA image versus immunolabeling image for laser-induced CNV in miceFig 6. Comparison of area measurements from OCTA and immunolabeled images. Median (interquartile range). Kruskal-Wallis test with asignificance level of 0.05 ( ).
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images and those obtained from immunolabeled samples. Our results will be helpful in eliminating OCTA artifacts and more accurately evaluating OCTA images.
Laser-induced CNV has been shown to be a reliable animal model for exudative AMD. As
previously reported [9–11], laser-induced CNV in mice could be clearly detected by OCTA at
5 days post-laser photocoagulation in the second stage of CNV development, characterized by
new-vessel formation (Fig 3A). The dark halo around the CNV lesion could also be observedby enface OCTA (Fig 3A) and corresponded with the periphery of the pericyte-like scaffold
that had developed fibrosis (Fig 3H). In the dark halo lesion, the CD31-labeled region comprising the CNV was undetectable by enface OCTA. The size of the CD31+ CNV lesions by immunolabeling was significantly larger than those of the CNV lesion by enface OCTA (Fig 6).
Although it is possible that the CNV lesions are artificially enlarged during the preparation of

PLOS ONE | https://doi.org/10.1371/journal.pone.0201958 August 9, 201810 / 14OCTA image versus immunolabeling image for laser-induced CNV in micethe choroid as a flat-mount, we assume that the enface OCTA images of laser-induced CNV
are actually underestimated due to shadowing artifacts caused by the dark halo which resultsfrom subretinal fibrosis of the pericyte-like scaffold. In previous study, Shah RS et al. reported
that laser-induced CNV area measurements taken from the isolectin B4 stained flatmounts
tended to be larger than those of OCTA images at 5 and 7 days post-laser photocoagulation
without statistically significant, because isolectin B4 stained not only endothelial cells of theperfused blood vessels but also those of the nascent vessels [10]. It is also thought that slow
blood flow is a major cause of underestimation of laser-induced CNV by OCTA due to theinability of OCTA to detect slow blood flow [4]. However, we assume that slow blood flow
does not lead to underestimation of laser-induced CNV by OCTA because in the dark halo
lesions, blood flow was detected between the deep retina and choroid by cross-sectional
OCTA imaging (yellow arrowhead in Fig 3B). Moreover, El Ameen et al. reported that thedark ringed area observed in OCTA was present around the CNV lesion in exudative AMD
with type 2 CNV and this area corresponded to the dark ring in the early phase of FA [12].
Because type 2 CNV often involves subretinal fibrosis, OCTA may underestimate type 2 CNV
due to artifactual shadowing in a clinical setting.
The pericyte-like scaffold is also referred to as the myofibroblastic scaffold [7]. Myofibroblasts are mesenchymal cells that express SMA, PDGFRβ and collagen type I and III [7,8,13–15]. These cells contribute to the formation of fibrosis and play important roles in corneal stromal wound healing, anterior subcapsular cataract, proliferative vitreoretinopathy, proliferative
diabetic retinopathy and exudative AMD [16–19]. Myofibroblasts are derived from an epithelial or endothelial-mesenchymal transition, bone marrow-derived fibroblasts and tissue local
fibroblasts [15,20,21]. In the laser-induced CNV model, the pericyte-like scaffold is not derived
from transdifferentiating RPE cells but from cells of the underlying choroid, such as pericytelike cells [7]. In this study, the pericyte-like scaffold was stained not only with PDGFRβ but
also with α-SMA and collagen I (Fig 4). This suggests that the scaffold is composed of myofibroblasts that will later develop into subretinal fibrosis. Measurements of the pericyte-like scaffold area based on immunolabeling tended to be larger than the measurements of the dark
halo comprising CNV lesion by OCTA, although this difference was not significant (p = 0.38).
It is possible that during tissue processing the pericyte-like scaffold could become extended
during the choroid flat-mounting. However, it is also possible that the dark halo visualized by
OCTA imaging does not precisely correlate with the periphery of the pericyte-like scaffold.
The peripheral border of the dark halo lesion by OCTA imaging was often blurry (Fig 4A)
while the border of the pericyte-like scaffold was sharp (Figs 3E, 4C and 4D). As a previous
study reported [8], the RPE cells adjacent to the site of laser injury are activated and express αSMA (Fig 4B and white arrowhead in Fig 4E). While there is some accumulation of activatedRPE cells around the periphery of the pericyte-like scaffold corresponding to the dark halo
lesion in OCTA images (Fig 4F), it is possible that the OCTA artifact resulting from activatedRPE cells did not affect the estimation of CNV size, as the CD31+ CNV region never extended
to the PDGFRβ+ pericyte-like scaffold (Fig 3F).
We acknowledge that this study has several limitations. Because of the small number ofsamples, it was not possible to fully determine the reliability and accuracy of this study’s
results. However, in all samples, the immunolabeled CD31+ CNV lesions were larger thanthose measured by OCTA, and the size of the immunolabeled PDGFRβ+ pericyte-like scaffolds was larger than the CD31+ CNV lesions (S1 Table). Furthermore, we did not use electron
microscopy to visualize the pericyte-like scaffold and without such ultrastructural assessment
it is difficult to confirm the presence of myofibroblasts within the scaffold. While the sole useof immunohistochemistry may be insufficient to confirm the localization and identification ofthese cells, the result that the pericyte-like scaffold was stained by PDGFRβ, α-SMA andPLOS ONE | https://doi.org/10.1371/journal.pone.0201958 August 9, 201811 / 14OCTA image versus immunolabeling image for laser-induced CNV in micecollagen type I would support our hypothesis that myofibroblasts in the pericyte-like scaffold
play a role in the development of subretinal fibrosis. Furthermore, we obtained enface OCTA
images between the deep retina and choroid using the semi-automatic mode, which assessed
CNV without automatic removal of projection artifacts. Although we calculated the area of the
CNV lesions by enface OCTA imaging confirming obvious projection artifacts, it is possible
there is a tendency to overestimate the size of the CNV lesions by enface OCTA. Moreover, it
is also possible there is a bias of field angle for OCTA images obtained by the only first author.
This might affect area measurements of OCTA images.

Conclusions
OCTA is a useful and noninvasive tool that allows for the visualization of CNV lesions without
dye injection. However, OCTA images can have shadowing artifacts resulting from the presence of fibrosis and these artifacts may lead to an underestimation of CNV lesions in exudative
AMD. When assessing CNV lesions by OCTA, it is necessary to confirm whether histologicalcharacteristics such as fibrosis result in imaging artifacts.

Supporting information
S1 Fig. OCT Image of wild-type C57BL/6J mouse. RNFL: retinal nerve fiber layer, GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform
layer, ONL: outer nuclear layer, RPE: retinal pigment epithelium.
(TIF)
S2 Fig. Images of OCT, OCTA and immunolabeling experiments with frozen sections fromlaser-induced CNV at 5 days post-laser photocoagulation. (A) By enface OCTA, laserinduced CNV was observed as a hyperflow lesion surrounded by a dark halo ( ; measured area:
900×900 μm). (B) By cross-sectional OCTA, corresponding to the horizontal blue line in S2 Fig
A, blood flow was detected between the deep retina and the choroid (measured width: 900 μm).
(C) By OCT, laser-induced CNV and the pericyte-like scaffold appeared as a subretinal hyperreflective lesion (measured width: 900 μm). (D) Vessels were stained with CD31. The CNV
lesion is inside a yellow circle. (E) The pericyte-like scaffold and pericytes were stained withPDGFRβ. The pericyte-like scaffold is inside a yellow circle. (F) Merged image of CD31 andPDGFRβ immunohistochemistry. The CNV lesion and the pericyte-like scaffold are inside ayellow circle. CNV was detected within the pericyte-like scaffold. Scale bar, 500 μm (D-F).
(TIF)
S1 Table. Area measurements for all samples. In all samples, the areas measured by immunolabeling for the CD31+ CNV lesion were larger than those obtained by OCTA imaging. The
size of the immunolabeled PDGFRβ+ pericyte-like scaffolds were larger than the CD31+ CNV
lesions.
(TIF)

Acknowledgments
The authors thank Kenya Ozaki for excellent technical assistance in taking OCTA images.

Author Contributions
Conceptualization: Kazuki Nakagawa.
Data curation: Kazuki Nakagawa, Hidetsugu Mori, Keiko Toyama.

PLOS ONE | https://doi.org/10.1371/journal.pone.0201958 August 9, 201812 / 14OCTA image versus immunolabeling image for laser-induced CNV in miceFormal analysis: Kazuki Nakagawa.
Investigation: Kazuki Nakagawa.
Methodology: Kazuki Nakagawa.
Project administration: Kazuki Nakagawa.
Writing – original draft: Kazuki Nakagawa.
Writing – review & editing: Haruhiko Yamada, Kanji Takahashi.

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