Multiple Effects of Intravitreal Aflibercept

on Microvascular Regression in Eyes with

Diabetic Macular Edema
Masahiko Sugimoto, MD, PhD,1 Atushi Ichio, MD,1 Daiki Mochida,2 Yumiho Tenma, MD,1 Ryohei Miyata, MD,1
Hisashi Matsubara, MD, PhD,1 Mineo Kondo, MD, PhD1

Purpose: To evaluate the effects of intravitreal aflibercept (IVA) on the number of microaneurysms and sizes
of nonperfused areas (NPAs) in eyes with diabetic macular edema (DME).
Design: Interventional, prospective study.
Participants: Twenty-five eyes of 25 DME patients (average age, 64.0
8.8 years) were treated with 3
consecutive monthly IVA injections.
Methods: Fluorescein angiography (FA) and OCT were performed before the IVA injections (baseline) and at
1 week after the IVA treatment. The number of microaneurysms and the ischemic index (ISI), a measure of NPA,
were determined. The correlations between central retinal thickness (CRT) and number of microaneurysms and
the ISI were also determined.
Main Outcome Measures: The mean number of microaneurysms and NPA evaluated as the ISI.
Results: At baseline, the mean CRT was 485.7
90.6 mm. After treatment, the mean CRT was reduced
significantly to 376.9
81.6 mm (P ¼ 0.1  10e5, repeated analysis of variance). The mean number of micro-
aneurysms was decreased significantly from 49.6
33.2 at baseline to 24.8
18.1 at 3 months after the initial
treatment. This was a 50.4
21.2% reduction (P ¼ 0.3  10e5, paired t test). The mean ISI was also decreased
significantly from 55.5
20.4% at baseline to 28.8
16.8% after treatment (P ¼ 0.3  10e5, paired t test). This was
a reduction of 43.3
28.5%. A significant correlation was found between the CRT and number of microaneurysms
at both baseline (r ¼ 0.56; P ¼ 0.004) and after treatment (r ¼ 0.53; P ¼ 0.006). A significant correlation was found
between CRT and ISI at baseline (r ¼ e0.39; P ¼ 0.03) but not after treatment (r ¼ e0.06; P ¼ 0.79).
Conclusions: The reduction in the number of microaneurysms was correlated with reduction in
CRT. Ophthalmology Retina 2019;-:1e9 ª 2019 by the American Academy of Ophthalmology. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Diabetic retinopathy (DR) is a leading cause of blindness,
and it is present in more than one half of patients with
diabetes mellitus of more than 20 years’ duration.1 Various
vascular changes occur during its progression, especially in
the microvasculature, including the formation of
microaneurysms, which are the most characteristic feature
of DR.2,3
Diabetic macular edema (DME) is a significant compli-
cation in eyes with DR.4 The excessive leakage of plasma in
eyes with DR results in a thickening of the retina that can
then damage the photoreceptors.5 Such leakage is
commonly evaluated by fluorescein angiography (FA), and
it is detected as points of hyperfluorescence or focal
leakage. Their importance of FA is not only their
association with the severity of the DR but also as a cause
of DME.6 Histopathologic studies have demonstrated that
microaneurysms are small outpouchings of the capillaries
with focal endothelial cell proliferation and loss of
pericytes resulting from hyperglycemia, which then
weaken the walls of the retinal vessels.7 The
microaneurysms are caused by a loss of the barrier
properties of the vascular walls, leading to abnormal
leakage.5
Many surgical and medical therapies have been reported
to be effective treatments for DME. Several studies have
shown that vascular endothelial growth factor (VEGF) plays
a major role in the vascular proliferation and hyper-
permeability in eyes with DME.8,9 The results of several
multicenter trials have also shown that intravitreal injections
of anti-VEGF agents lead to a rapid resolution of DME and
improvement in vision.10e14
At present, various anti-VEGF agents, for example,
bevacizumab (Avastin; Genentech, South San Francisco,
CA), ranibizumab (Lucentis; Genentech), and aflibercept
(Eylea; Regeneron Pharmaceuticals, Tarrytown, NY), are
used to treat DME, and they have become the first-line
therapy for DME.10e15 However, 0.8% to 3.9% of pa-
tients in the RIDE (NCT00473382) and RISE
(NCT00473330) studies and 0.7% to 3.2% of patients in the
VIVID (NCT01331681) and VISTA (NCT01363440)
studies lost 15 letters or more from baseline on the visual
acuity chart.12,13 In addition, anti-VEGF agents are not

 2019 by the American Academy of Ophthalmology https://doi.org/10.1016/j.oret.2019.06.005 1

This is an open access article under the CC BY-NC-ND license ISSN 2468-6530/19
(http://creativecommons.org/licenses/by-nc-nd/4.0/). Published by Elsevier Inc.
 Ophthalmology Retina Volume -, Number -, Month 2019
effective for all patients, and other factors exist that seem to
block the improvements.
Leakage from microaneurysms plays important roles in
DME, and the Early Treatment Diabetic Retinopathy Study
(ETDRS) recommended focal or grid laser photocoagulation
for such leakage.16 However, an important complication of
photocoagulation is its destruction of normal retinal tissue.
Recently, anti-VEGF agents have replaced photocoagula-
tion as a first-line treatment for DME. The results of earlier
studies show that the anti-VEGF agents act by reducing
abnormal leakage, including that from microaneurysms.17,18
In addition, the Diabetic Retinopathy Clinical Research
Network protocol V study reported no significant difference
in vision loss regardless of whether eyes were initally
managed with aflibercept or with laser photocoagulation in
eyes with center-involved DME and good visual acuity. The
authors stated that patients should be given aflibercept only
if visual acuity worsens.19 It will be necessary to examine
whether anti-VEGF therapy affects only some of the
microaneurysms or all of the microaneurysms. The results of
recent studies showed that anti-VEGF agents can improve
the stage of the DR,20e22 which is a new aspect of anti-
VEGF treatment. The aim of this study was to evaluate
the effect of intravitreal aflibercept (IVA) injections on the
number of microaneurysms and size of nonperfused areas
(NPAs) in eyes with DME.
Methods
This prospective study was registered at http://www.umin.ac.jp
(identifier, UMIN 000018315). Its protocol was approved by the
institutional review board of Mie University Hospital (identifier,
2913), and the procedures used conformed to the tenets of the
Declaration of Helsinki. Oral and written consents were obtained
from patients after an explanation of the procedures to be used and
possible complications.
Patient Demographics
The age, gender, creatinine level, estimated glomerular filtration rate,
hemoglobin level, hemoglobin A1c level, systolic and diastolic blood
pressures (BPs), severity of DR, and previous panretinal photocoag-
ulation (PRP) status were determined at baseline and after the third
injection. All patients underwent a complete ophthalmologic exami-
nation before beginning the loading phase of the IVA protocol.
Monthly examinations started from 1 to 2 weeks before the beginning
of the loading phase and continued through the postinjection period.
The examinations included measurements of best-corrected visual
acuity (BCVA) with Snellen charts, slit-lamp biomicroscopy, intra-
ocular pressure measurements, indirect ophthalmoscopy, spectral-
domain OCT, and FA. Fluorescein angiography was performed at
baseline and within 1 week after the third IVA injection.
OCT examinations were performed with the Spectralis OCT
instrument (Heidelberg Engineering, Heidelberg, Germany). The
structural OCT minimum acquisition protocol, which included 19
horizontal raster linear B-scans with each composed of 9 averaged
OCT B-scans (1024 A-scans per line) covering an area of 30 
30 , was used. The CRT in the central 1-mm diameter circle of the
ETDRS thickness map was calculated by Spectralis software
(Heidelberg Eye Explorer, version 1.9.11.0; Heidelberg
Engineering).
2
Inclusion and Exclusion Criteria
The inclusion criteria were: patients 20 years of age or older with
type I or II diabetes, eyes with DR and DME with signs of severe
leakage indicating abnormal vessels within the vascular arcade area
by FA, BCVA of 20/320 or better, DME involving the fovea, and
CRT of 300 mm or more measured as the mean retinal thickness in
the central 1-mm diameter circle by OCT. The exclusion criteria
were any retinal photocoagulation treatment in the study eye within
3 months preceding the initial IVA injection, eyes with an ischemic
macular region involving the fovea, prior focal or grid laser
photocoagulation, eyes with vitreomacular traction, any history or
presence of other ocular diseases causing vision reduction such as
age-related macular degeneration and severe proliferative DR,
optic nerve atrophy, glaucoma or intraocular pressure of more than
24 mmHg, history of vitreous surgery, aphakia, anti-VEGF
treatment on either eye within 3 months preceding the initial
IVA injection, cloudy optic media including cataract through
which high-quality fundus photographs or OCT images could not
be obtained, history of cataract surgery in the study eye within the
previous 3 months, history of cerebrovascular accident, myocardial
infarction or other systemic disease requiring medications that
could affect the results, severe renal failure with creatinine level of
2.0 mg/dl or more or worse than stage IIb nephropathy defined by
classification of diabetic nephropathy, poorly controlled hyper-
tension with systolic BP higher than 200 mmHg or diastolic BP
higher than 110 mmHg, poorly controlled diabetes mellitus with
hemoglobin A1c level of 12.0% or more, and patients who were
judged to be ineligible for any other reason by the investigators.
Intravitreal Aflibercept Injections
Each eye received 2 mg IVA (40 mg/ml) under topical anesthesia.
The injections were administered with a 30-gauge needle that was
inserted 4 mm posterior to the corneal limbus under sterile con-
ditions. All patients received topical levofloxacin hydrate (1.5%
Cravit ophthalmic solution [Santen, Osaka, Japan]) for 3 days after
the injection. Each eye was treated with 3 consecutive monthly
IVA injections (the loading phase), as described previously.23,24
Fluorescein Angiography
Fluorescein angiography was performed with the Heidelberg
Spectralis HRAþOCT module with the standard intravenous
infusion of 5 ml 10% sodium fluorescein. Images were obtained
using the standard 35 field-of-view lens, and ultra-widefield FA
images were also obtained with the noncontact attachment module,
which enabled the recording of a 150 field centered on the fovea.
Image Analyses
One or more images from the early arteriovenous phase were
selected from each angiographic series. The images were recorded
at between 45 and 60 seconds and also from the late phase at
between 5 and 6 minutes. The images were transferred to Adobe
Photoshop ES software (Adobe Systems, Inc, San Jose, CA) or
ImageJ software (Image processing and analysis in JAVA version
1.46r; Wayne Rasband, National Institutes of Health, Bethesda,
MD). Hyperfluorescent pools resulting from leakage were
considered to be microaneurysms as reported.2 All FA images were
evaluated by 3 masked graders (MS, AI, and DM). One evaluator
counted each image 3 times, and the average value was used for
statistical analyses.
 Sugimoto et al 
Microvasculature regression after aflibercept in DME
Figure 1. Fluorescein angiographic image used to count the number of microaneurysms. Images were selected from the early arteriovenous phase from each
angiographic series at (A) baseline and (B) after treatment. The baseline image was converted to (C) green and the posttreatment image was converted to
(D) red. E, The images were merged, and the high-intensity yellow dot signals were defined and counted as microaneurysms that still had leakages. These
microaneurysms were counted as RG microaneurysms.
Number of Microaneurysms
We used the color merge technique to determine the number of
microaneurysms. To do this, an early-phase baseline FA image
(Fig 1A) was converted to a green image (Fig 1C), and a
posttreatment image (Fig 1B) was converted to a red image (Fig
1D) using Photoshop. These 2 images were overlapped, and the
number of high-intensity yellow dots was counted as the leakage
points in the merged image (Fig 1E). Thus, the posttreatment
microaneurysms were designated as red and green (RG)
microaneurysms.
We also counted the number of microaneurysms in the central
area that was associated with the origin of the DME. The 6.0-mm
diameter (outer ring of the ETDRS circle centered on the fovea)
of the OCT thickness map was delineated by Photoshop (Fig
2A). These OCT images and the FA images (Fig 2B) obtained
at the same time were merged (Fig 2C). The hyperfluorescent
dot signals in the area with retinal thickness of more than 400
mm (color coded as red or warmer) were identified and counted
as leakage points related to the DME. We compared the
baseline images (Fig 2C) and posttreatment images (Fig 2D).
These microaneurysms were counted as map microaneurysms.
Ischemic Index
The degree of peripheral retinal ischemia of all eyes was quantified
by the ischemic index (ISI), which was calculated as described in
detail previously.25 The ISI was used in other studies as an
indicator of retinal nonperfusion. The degree of capillary
nonperfusion was determined by masked graders in the macula-
centered ultra-widefield FA frames as described in detail.26
Briefly, the images were exported in a JPEG format without
changes in the contrast, g value, or brightness and were imported
into ImageJ software. The total retinal surface and capillary
nonperfused areas seen in the late phase were outlined manually
using the area measurement function and divided by the area of
the total image area in pixels. The capillary NPA was defined as
the area where a dropout of the retinal capillary bed was
detected in the images (Fig 3, asterisk). The ISI was calculated
as the ratio between ischemic retina and total retinal area. All
results were evaluated by 3 masked graders (MS, AI, and DM).
One evaluator counted each image 3 times, and the average
value was used for the statistical analyses.
Statistical Analyses
To evaluate the relative changes in CRT, the number of
microaneurysms and degree of ISI were determined. We used
d values that were expressed as a rate of change from the
baseline values. The d values or reduction rates were defined as
follows:
d values (%) ¼ 100 e (value after IVA / value before
IVA  100).
The results are presented as the means
standard deviations.
The significance of the differences of the data was determined by
2-way nonrepeated analysis of variance followed by Bonferroni
post hoc tests for the comparison of the means. Paired t tests were
used to determine the significance of differences between 2 groups.
Spearman’s rank-order correlation coefficient was used to deter-
mine the significance of the correlations among the variables. The
strength of the correlations (r value) was classified as: 0.0 to 0.2,
not correlated or very weak; 0.2 to 0.4, weak or low; 0.4 to 0.7,
moderate; 0.7 to 0.9, strong or high; and 0.9 to 1.0, very strong.
The results were considered statistically significant when the P
value was less than 0.05.
Results
Thirty eyes of 30 patients met the inclusion and exclusion criteria
and were studied between September 2015 and March 2017 in the
3
 Ophthalmology Retina Volume -, Number -, Month 2019

Figure 2. OCT image used to count abnormal leakages related to the origin of diabetic macular edema. The (A) OCT thickness color-coded map images
were merged with (B) the fluorescein angiography images obtained simultaneously. Merged images were created at (C) baseline or (D) after treatment. The
microaneurysms were counted as map microaneurysms. E, After treatment, the mean number of map microaneurysms decreased to 24.8
18.1 (P < 0.01,
paired t test). F, A significant and moderate correlation was found between the number of RG microaneurysms and d value of map microaneurysms (r ¼ 0.51;
P < 0.01, Spearman test).

Department of Ophthalmology, Mie University Hospital, Tsu,
Japan. There were no IVA-related ocular complications, including
intraocular pressure elevations, infections, or any adverse systemic
events. Five patients among the 30 patients could not complete the
entire injection protocol or entire examination schedules for
financial reason (3 patients) or because of difficulty with trans-
portation to the hospital.
filtration rate was 74.0
23.6 ml/minute per 1.73 m2 (range,
20.3e106.7 ml/minute per 1.73 m2), the average hemoglobin
level was 14.0
2.2 g/dl (range, 9.3e17.2 g/dl), and the average
hemoglobin A1c level was 7.8
1.3% (range, 6.4%e10.5%). The
average systolic BP was 145.8
19.3 mmHg (range, 101e180
mmHg), and the average diastolic BP was 72.4
13.0 mmHg
(range, 51e95 mmHg).

Baseline Characteristics Ocular Examinations

In the end, 25 eyes of 25 DME patients comprising 14 men and
11 women were studied. Their mean age was 64.0
8.8 years.
Fifteen of the eyes had undergone PRP. Twelve patients had
moderate nonproliferative DR and 13 patients had severe non-
proliferative DR. The average creatinine level was 0.8
0.5 mg/dl
(range, 0.4e2.7 mg/dl), the average estimated glomerular
The mean baseline BCVA was 0.45
0.35 logarithm of the mini-
mum angle of resolution, and the mean baseline CRT was
485.7
90.6 mm. After the loading phase, the mean BCVA improved
significantly to 0.40
0.38 logarithm of the minimum angle of res-
olution (P ¼ 0.02, repeated analysis of variance), and the mean CRT
was reduced significantly to 376.9
81.6 mm (P ¼ 0.1  10e6).

4
 Sugimoto et al 
Microvasculature regression after aflibercept in DME

Figure 3. Ischemic index (ISI) changes during diabetic macular edema treatment. The degree of peripheral retinal ischemia was quantified by the ISI. The
retinal surface and capillary nonperfused areas (asterisks) seen in the late phase of ultra-widefield fluorescence angiography were outlined manually (A) at
baseline and (B) after treatment. C, The mean baseline ISI was 55.5
20.4%, and the mean ISI after treatment decreased significantly to 28.8
16.8% (P <
0.01, paired t test).

Number of Microaneurysms Correlation between Microaneurysms and

The mean number of map microaneurysms at baseline was
49.6
33.2, and the mean number after the loading phase was
reduced to 24.8
18.1 (P ¼ 0.3  10e6, paired t test; Fig 2E). The
reduction rate of the d value for map microaneurysms was
50.4
21.2%. A moderate and significant correlation was found
between the number of RG microaneurysms and d value for map
microaneurysms (r ¼ 0.51; P ¼ 0.009, Spearman test; Fig 2F).
Ischemic Index Values
The mean ISI at baseline was 55.5
20.4%, and the mean was
reduced significantly to 28.8
16.8% (P ¼ 0.3  10e6, paired t
test; Fig 3C). The reduction percentage was 43.3
28.5%.
Ischemic Index before and after AntieVascular
Endothelial Growth Factor Treatment
A moderate significant correlation was found between the CRT
and number of microaneurysms at both baseline (r ¼ 0.56;
P ¼ 0.004, Spearman test; Fig 4A) and after IVA treatment
(r ¼ 0.53; P ¼ 0.006, Spearman test; Fig 4B). A significant
correlation was found between CRT and the ISI at baseline
(r ¼ e0.39; P ¼ 0.03, Spearman test; Fig 4C), but no
significant correlation was found between them after IVA
treatment (r ¼ e0.06; P ¼ 0.79, Spearman test; Fig 4D). No
significant correlation was found between the reduction rate of
the CRT and the d value for map microaneurysms (r ¼ 0.08;
P ¼ 0.53, Spearman test).

5
 Ophthalmology Retina Volume -, Number -, Month 2019

Figure 4. Scatterplots showing the correlation between the baseline and posttreatment number of microaneurysms and the ischemic index (ISI). Significant
and moderate correlations were found between the central retinal thickness (CRT) and microaneurysms both (A) at baseline (r ¼ 0.56, P <0.01; Spearman
test) and (B) after treatment (r ¼ 0.53; P < 0.01). A weak but significant correlation is present between CRT and the ISI (C) at baseline (r ¼ e0.39; P <
0.05), but no significant correlation is present (D) after treatment (r ¼ e0.06; P > 0.05).

Discussion addition, the Study of the Efficacy and Safety of Intravitreal

The results of this study show that the IVA injections
reduced the number of microaneurysms and size of the
NPA. These results suggest that the reduction of the ISI did
not play a role in the reduction of CRT.
The ETDRS showed that focal macular photocoagulation
is effective in treating microaneurysms.8 However, focal
photocoagulation does not necessarily work immediately,
and complications resulting from the damage caused by
the damaged tissues, for example, creeping atrophy, can
occur. In addition, only 3% of the patients show an
improvement of 15 letters or more.27 At present, anti-
VEGF therapy has largely replaced photocoagulation and
become the first line of therapy for eyes with DME.
There have been at least 2 reports of a reduction in the
number of microaneurysms after anti-VEGF therapy, as we
found.17,18 However, these studies did not examine the re-
sults of more than 3 consecutive injections. Many other
studies have shown that multiple injections of anti-VEGF
agents can improve the severity of the DR.20e22 In
(IVT) Aflibercept for the Improvement of Moderately Se-
vere to Severe Nonproliferative Diabetic Retinopathy
(NPDR) (PANORAMA), a phase 3, double-masked, ran-
domized study, was designed to test the efficacy and safety
of intravitreal aflibercept injections in patients with moder-
ately severe to severe nonproliferative DR compared with
sham injections (ClinicalTrials.gov identifier,
NCT02718326). The study reported that the proportion of
patients with 2-step or more improvement in the severity of
DR was significantly higher in eyes after aflibercept injec-
tion than in the sham-injected eyes (paper presented at:
Bascom Palmer Eye Institute’s Angiogenesis, Exudation,
and Degeneration Symposium, February 9, 2019; Miami).
However, all of these studies did not specifically quantify
the changes in the number of microaneurysms during
treatment. Our results showed that 3 consecutive anti-VEGF
injections reduced the number of microaneurysms and the
ISI. These changes occurred because of the biological as-
pects of the anti-VEGF agents. Thus, the alterations reduced
the number of microaneurysms by changes in the structural

6
 Sugimoto et al 
Microvasculature regression after aflibercept in DME
features, namely, a loss of pericytes and astrocytes, and they
also altered the hemodynamic properties, namely, increased
capillary intramural pressure and production of vaso-
proliferative factors such as VEGF.7
Our results showed that anti-VEGF therapy can reduce
the number of map microaneurysms. Central macular
thickening, quantified by OCT maps as red or gray areas,
may be considered a variable DME outcome.28 Our results
also showed that there were still nonresponding
microaneurysms even after 3 consecutive loading
injections. Although it is not clear whether all of the
microaneurysms disappeared after the multiple injections,
many earlier studies have reported an improvement in DR
after multiple injections. The subgroup analysis of the
RIDE and RISE and the VIVID and VISTA studies
showed that DR improved in the eyes with DME. The
mean injection number was high compared with that in
our study.
Our results show that the residual number of micro-
aneurysms was correlated significantly with CRT after the
loading injections. Earlier studies showed that consecutive
injections will decrease the edema and improve the vision in
eyes with DR progression; however, 3 consecutive in-
jections were not sufficient, and continued treatment during
a maintenance phase may be needed to reduce the number of
residual microaneurysms.
When we consider the problems of anti-VEGF treatment,
its costs, its inconveniences for doctors and healthcare
providers, and the poor results with inadvertent complica-
tions, the need for a proper treatment regimen leading to
optimal visual outcomes with fewer injections becomes
clear.29,30 Stabilization with combined laser photocoagula-
tion31,32 or other agents33,34 could also lead to earlier visual
gains and reduced anti-VEGF re-treatment numbers. The
Navilas Laser System (NAVILAS) demonstrated that there
is a significantly higher accuracy in laser spot applications
and that this system has a potential of reducing retreatment
rates, indicating that it could be used for combination
therapy.31 Therefore, the use of combination therapy for the
nonresponding microaneurysms means that fewer anti-
VEGF injections may be effective with minimum stress.
Large NPAs are generally associated with the most
resistant cases of DME.26,35 The hemodynamic changes
resulting from DR can lead to reduced production of VEGF,
which may lead to DME. A blockade of excess VEGF by
photocoagulation of the retina and RPE or by anti-VEGF
agents leads to substantial improvements in edema,36 and
targeted photocoagulation of the NPA can also reduce the
edema.37,38 However, the Efficacy & Safety Trial of Intra-
vitreal Injections Combined With PRP for CSME Secondary
to Diabetes Mellitus (DAVE) study, a 3-year randomized
trial of combination therapy with ranibizumab and targeted
photocoagulation, reported a lack of improvement in visual
outcomes.39 The disadvantages of PRP are decreased vision,
macular edema, and development of visual field defects
resulting from damage to the retina and RPE.40,41 Our re-
sults showed that the correlation between the IVA
treatments and ISI was not significant. Although we found a
reduction in NPA, it was not correlated significantly with
the reduction in CRT. We suggest that the reduction in NPA
is a separate effect of anti-VEGF agents.
There are some limitations in our study, including the
small sample size. Our study also lacked controls. Because
anti-VEGF therapy has become the standard of care, it is
difficult to recruit a control DME group without any treat-
ment for ethical reasons. Furthermore, screening by human
graders is not necessarily accurate.42 To resolve these
difficulties, an automated system for screening would be
better.43,44 Microaneurysms are dynamic lesions with a
significant turnover of less than 6 months.45e47 These
changes occur over time, disappearing by closing down
because of thrombosis, with new ones appearing at different
locations of the vascular tree. This turnover indicates dy-
namic processes and changes in the disease activity. Leicht
et al18 reported the specific effects of anti-VEGF therapy on
microaneurysm turnover or resolution and its value for
DME patients. In addition, the disappearance rate of
microaneurysms (50.4%) was higher than that reported by
Allingham et al48 (17.9%). They reported that most of the
hyperfluorescence that disappeared in patients was the
result of an improvement in the diffuse edema and not of
the focal loss of microaneurysms. A control group
consisting of untreated patients is needed to compare the
turnover of the microaneurysms to show the effects of
aflibercept on the number of microaneurysms. We need to
consider the effects of anti-VEGF on this turnover.
Although we excluded patients who had received any focal
or grid photocoagulation, PRP-treated eyes were included.
From a post hoc analysis of the RIDE and RISE studies,22
patients who had undergone prior PRP achieved limited
DR improvement compared with patients who had not
undergone PRP. The patients underwent prior PRP at each
doctor’s clinical discretion before entry. Because there was
no specific guidance for prior PRP, this may have affected
the results. Finally, although FA may be more sensitive
than ophthalmoscopy in identifying areas of retinal NPA,
small areas of neovascularization, and vascular leakage, it
carries a risk of anaphylaxis. Technical improvements in
the evaluations of the microaneurysms, such as OCT
angiography (OCTA), are needed. OCT angiography is a
newly developed method that enables the clinician to
evaluate the retinal vasculature without invasion. From en
face images of different retinal or choroidal layers, OCTA
has enabled clinicians to detect conformational changes in
more detail.49 However, we did not evaluate our patients
by OCTA. In addition, we cannot fully state that the
reduction in the number of microaneurysms was the
reason for the reduction in CRT based on only a
significant correlation between them. On this point, OCTA
can give us additional information to support our
hypothesis.
In conclusion, our results showed that IVA can improve
BCVA and CRT in eyes with DME. It can also improve DR,
as shown by a reduction in the number of microaneurysms
7
 Ophthalmology Retina Volume -, Number -, Month 2019
and size of the NPAs. Intravitreal aflibercept exerts multiple
effects on the improvements of the microvasculature in eyes
with DME. This reduction in the number of microaneurysms
is correlated with CRT reduction, but some nonresponding
microaneurysms remained.
Acknowledgments. The authors thank Professor Emeritus
Duco Hamasaki, Bascom Palmer Eye Institute, University of
Miami, for critical discussion and final manuscript revisions, and
Dr. Yoshihiro Takamura, Fukui University, for kind technical
advice.
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Footnotes and Financial Disclosures
Originally received: December 17, 2018.
Final revision: May 29, 2019.
Accepted: June 10, 2019.
Available online: ---. Manuscript no. ORET_2018_483.
1
Department of Ophthalmology, Mie University Graduate School of
Medicine, Tsu, Japan.
2
Faculty of Medicine, Mie University Graduate School of Medicine, Tsu,
Japan.
Financial Disclosure(s):
The author(s) have made the following disclosure(s): M.S.: Financial sup-
port e Alcon Pharma, Bayer; Alcon Pharma, Kowa Pharma, Senjyu
Pharma, Daiichi Yakuhin Sangyo, Bayer, Wakamoto Pharma
A.I.: Lecturer e Alcon Pharma, Bayer, Santen
R.M.: Lecturer e Alcon Pharma, Bayer, Nihon Tengan, Santen
H.M.: Lecturer e Alcon Pharma, Bayer, Nihon Tengan, Santen
M.K.: Lecturer e Alcon Pharma, Bayer, Senjyu Pharma
Presented at: 121st Congress of Japanese Society for Ophthalmology, April
2017, Tokyo, Japan.
Supported by Bayer Yakuhin, Ltd., Osaka, Japan.
HUMAN SUBJECTS: Human subjects were included in this study. The
human ethics committees at Mie University Hospital approved the study.
All research adhered to the tenets of the Declaration of Helsinki. All par-
ticipants provided informed consent.
No animal subjects were included in this study.
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Author Contributions:
Conception and design: Sugimoto, Kondo
Analysis and interpretation: Ichio, Mochida, Tenma, Miyata, Matsubara
Data collection: Sugimoto
Obtained funding: N/A
Overall responsibility: Sugimoto
Abbreviations and Acronyms:
BCVA ¼ best-corrected visual acuity; BP ¼ blood pressure;
CRT ¼ central retinal thickness; DME ¼ diabetic macular edema;
DR ¼ diabetic retinopathy; ETDRS ¼ Early Treatment Diabetic Reti-
nopathy Study; FA ¼ fluorescein angiography; ISI ¼ ischemic index;
IVA ¼ intravitreal aflibercept; NPA ¼ nonperfused area; OCTA ¼ OCT
angiography; PRP ¼ panretinal photocoagulation; RIDE ¼ A Study of
Ranibizumab Injection in Subjects With Clinically Significant Macular
Edema (ME) With Center Involvement Secondary to Diabetes Mellitus;
RISE ¼ A Study of Ranibizumab Injection in Subjects With Clinically
Significant Macular Edema (ME) With Center Involvement Secondary to
Diabetes Mellitus; VISTA-DME ¼ Study of Intravitreal Aflibercept In-
jection in Patients with Diabetic Macular Edema; VIVID-
DME ¼ Intravitreal Aflibercept Injection in Vision Impairment due to
DME.
Correspondence:
Masahiko Sugimoto, MD, PhD, Department of Ophthalmology, Mie Uni-
versity Graduate School of Medicine, 2-174, Edobashi, Tsu, 514-8507,
Japan. E-mail: sugimochi@clin.medic.mie-u.ac.jp.