Retinal Microvasculature and Visual Acuity after Intravitreal Aflibercept in Diabetic Macular Edema: An Optical Coherence Tomography Angiography Study

We investigated changes in retinal vascular area and the foveal avascular zone (FAZ) after intravitreal aflibercept in diabetic macular edema (DME) and the association of these changes with visual outcomes. The retinal vascular area in the superficial capillary plexus (SCP) and the deep capillary plexus (DCP) and the FAZ area were measured using optical coherence tomography angiography (OCTA) in 23 eyes of 23 patients with DME, before and after intravitreal aflibercept. Overall, there was no significant change in retinal vascular area or FAZ. Better BCVA after treatment was significantly associated with larger retinal vascular area in the SCP and the DCP, both at baseline (R2 = 0.512, P < 0.001 and R2 = 0.361, P = 0.002, respectively) and after intravitreal aflibercept (R2 = 0.717, P < 0.001 and R2 = 0.618, P < 0.001, respectively). MAs were observed in the DCP in 20 eyes (87%), but only detected in four eyes (17%) in the SCP before treatment. The number of eyes with MAs in the DCP significantly decreased to 13 (57%) after treatment (P = 0.049). The persistence of DME was associated with persistent MAs (P = 0.019) and less visual gain (P = 0.031) following treatment. Thus, preserving retinal perfusion and the resolution of MAs are associated with better vision and resolution of the DME after intravitreal aflibercept.

retinal vascular density (VD) with visual acuity in DR has been reported [22][23][24] . Changes in the VD and the FAZ area following anti-VEGF therapy in DME have also been reported 25 . However, changes in the microvasculature following anti-VEGF therapy and their association with visual changes have not yet been fully elucidated.
In this study, we assessed the retinal vascular area and the FAZ before and after intravitreal aflibercept injections for DME, and assess the relationship between changes in these measures and visual outcomes.
Association Between Pre-Treatment VA and Retinal Microvasculature. Based on the univariate regression analysis, better BCVA at baseline before intravitreal aflibercept injections was significantly associated with a larger pre-treatment retinal vascular area in the SCP (R 2 = 0.294, P = 0.008) and the DCP (R 2 = 0.264, P = 0.012) although there was no association with the pre-treatment FAZ area in the SCP and the DCP (R 2 = 0.038, P = 0.375 and R 2 = 0.092, P = 0.159, respectively). The better retinal vascular area in both SCP and DCP was significantly associated with NPDR but not PDR (R 2 = 0.243, P = 0.017 and R 2 = 0.179, P = 0.044, respectively) and less macular edema (CRT) before treatment (R 2 = 0.253, P = 0.014 and R 2 = 0.179, P = 0.044, respectively).
Association Between Post-Treatment VA and Retinal Microvasculature. Better BCVA after intravitreal aflibercept treatment was significantly associated with better pre-treatment logMAR BCVA (R 2 = 0.452, P < 0.001), NPDR but not PDR (R 2 = 0.394, P = 0.001), better pre-treatment retinal vascular area in the SCP (R 2 = 0.512, P < 0.001) and the DCP (R 2 = 0.361, P = 0.002), better post-treatment retinal vascular area in the SCP (R 2 = 0.717, P < 0.001) and the DCP ((R 2 = 0.618, P < 0.001), and the smaller post-treatment FAZ in the DCP (R 2 = 0.274, P = 0.010) ( Table 3). Resolution of DME. Resolution of the DME (CRT of 300 µm or less after final intravitreal aflibercept) was observed in 17 eyes (74%), whereas persistence of DME (CRT over 300 µm after final intravitreal aflibercept) was identified in six eyes (26%) ( Table 4). The mean CRT was 249 ± 38 µm in resolved DME and 348 ± 61 µm in persistent DME after treatment (P < 0.001). Although most eyes in both groups had MAs in the DCP before treatment (82% in resolved DME versus 100% in persistent DME), the number of eyes with MAs in the DCP were significantly higher in the persistent DME group (100%) than in the resolved DME group (41%) after treatment (P = 0.019) (Fig. 3). The visual gain was significantly worse in the persistent DME group after treatment (P = 0.031).

Discussion
Our results reveal that both retinal vascular area and the FAZ within the 9 (3 × 3) mm 2 area did not significantly differ before and after intravitreal aflibercept treatment in patients with DME. That is, repeated administration of intravitreal aflibercept with a mean of 2.6 injections over the course of 8.5 months maintained the retinal perfusion at the macula in eyes with DME. This result is consistent with previous studies showing no significant change in vessel density and the FAZ area 1 month after anti-VEGF treatment 25 . Since we could not evaluate potential changes in retinal vascular area and the FAZ in eyes without treatment, it remains unclear whether intravitreal aflibercept has a preventative effect on the progression of macular ischemia. However, the current study suggests that intravitreal aflibercept injections following a PRN regimen may at least not lead to a deterioration of retinal perfusion in most eyes with DME. Before treatment with intravitreal aflibercept, worse BCVA was significantly associated with less retinal vascular area in the SCP and the DCP. In addition, less retinal vascular area in the SCP and the DCP was significantly correlated with the severity of the DR and the macular edema. High intraocular concentration of VEGF is associated not only with the macular edema but also with the progression of DR, including the progression of ischemia 26,27 . VEGF overexpression contributes to the increase in permeability as well as to the increase in the intercellular adhesion molecule-1 (ICAM-1) expression in retinal vascular endothelium, and promotes ICAM-1-mediated leukocytes binding to the retinal vasculature, resulting in downstream non-perfusion [28][29][30] . Therefore, we speculate that macular edema and macular ischemia may thus progress concomitantly in eyes with DME, resulting in visual impairments as shown in the current study. Therefore, early initiation of the anti-VEGF therapy may help the prevention of the concomitant progression of macular edema and capillary ischemia 31 .
In the current study, BCVA significantly improved after intravitreal aflibercept administration, as expected. Better BCVA after intravitreal aflibercept administration was significantly associated with a larger retinal vascular area in the SCP and the DCP before treatment, indicating that baseline retinal vascular area not only correlates with baseline BCVA but may also be useful to predict visual outcomes after intravitreal aflibercept treatment. In addition, because better BCVA after intravitreal aflibercept treatment was also significantly associated with a larger retinal vascular area in the SCP and the DCP and a smaller FAZ area in the DCP after treatment, the preservation of retinal perfusion after treatment seems to be crucial for better visual outcomes. Therefore, strategies that maintain or increase retinal perfusion are expected to further improve visual outcomes following intravitreal aflibercept administration in eyes with DME.
It has been reported that MAs are seen more frequently in the DCP than in the SCP and are associated with the development of DME 17,[32][33][34][35] . Here, consistent with previous studies, MAs were observed in the DCP in 20 eyes (87%), although they were only detected in four eyes (17%) in the SCP before treatment. The number of eyes with MAs in the DCP significantly decreased to 13 eyes (P = 0.049), indicating that the anti-VEGF therapy resolved the MA formation in some eyes. Because increased VEGF expression has been shown to play a significant role in MA formation by inducing focal endothelial proliferation 36 , it is reasonable to assume that the inhibition of VEGF may suppress MAs 27 . However, more than half of the patients had persistent MAs, even after anti-VEGF treatment, that were associated with persistent DME. In our case series, MAs in the DCP were identified in all six eyes with persistent DME, even after repeated aflibercept administration. In contrast, a significantly lower number of eyes (seven of 17 eyes; 41%) had MAs in the DCP in resolved DME (P = 0.019). The visual gain was significantly worse in persistent DME than in resolved DME (P = 0.031). Therefore, we speculate that a smaller effect of VEGF inhibition on MA regression may be responsible for persistent DME and poor visual improvement. Further studies are required to elucidate the differences in MA responses and strategies for the treatment of persistent DME associated with non-resolved MA. This study has several limitations, including its retrospective design, the variable follow-up period, and a relatively small sample size. We could not evaluate vessel diameter. Furthermore, we only analyzed a limited area (3 × 3 mm 2 ) of retinal microvasculature, which is important for central vision but may not reflect the entire disease process in DME. The inability to fully eliminate autosegmentation errors may also have influenced the measurements of microaneurysms and retinal vascular area especially in the DCP. Therefore, further studies with large number of patients are necessary to validate current results and to improve understanding of microvascular changes before and after intravitreal aflibercept in DME. Nevertheless, the current study reveals that vascular perfusion in both the SCP and the DCP is maintained after intravitreal aflibercept administration, and that preservation of retinal vascular area seems crucial for better visual outcomes. In addition, we identified a significant association between persistent MA formation and persistent DME, responsible for lower visual gain after intravitreal aflibercept treatment. These findings are valuable for our understanding of the changes in microvasculature associated with diabetes and for the improvement of the management of DME.
In conclusion, our study shoewed that the retinal vascular area and the FAZ were maintained after intravitreal aflibercept administration in patients with DME. A larger retinal vascular area and a smaller FAZ area were associated with better visual acuity after treatment. Persistent MAs were associated with persistent DME and poor visual improvement. Further studies are needed to develop strategies to maintain or increase retinal blood flow and resolve MAs, for better long-term visual prognosis in eyes with DME.

Methods
This study represents a retrospective consecutive case series. All procedures were in adherence with the tenets of the declaration of Helsinki, and were approved by the institutional review board of Osaka University Graduate School of Medicine (10039). Patients with macular edema secondary to diabetic retinopathy who had been treated with intravitreal aflibercept between December 2014 and February 2016 at the Department of Ophthalmology, Osaka University Hospital, were enrolled in this study. All patients provided written informed consent before intravitreal aflibercept. Written informed consent was not considered to be necessary by the Ethics Committee to participate in this retrospective study.
OCTA Examination. OCTA was performed using an AngioVue instrument (Optovue RTVue XR Avanti; Optovue, Inc., Fremont, CA, USA). Automated segmentation using the AngioVue module was performed on the same day as the clinical examination. In each eye, a 3 × 3-mm 2 area centered on the fovea was scanned. The retinal vascular area was automatically segmented into four layers: the superficial capillary plexus (SCP), which includes vasculature from the internal limiting membrane to 15 μm beneath the inner plexiform layer; the deep capillary plexus (DCP), which includes vasculature from 15 to 70 μm beneath the inner plexiform layer; the outer retina; and the choroid. We quantitatively assessed the retinal vascular area of the SCP and the DCP in the fovea using the AngioVue instrument. The retinal vascular area was defined as the percentage of the entire area occupied by large vessels and microvasculature in a particular region, and was calculated within the 3 × 3-mm 2 scan area in  Fig. 1  each eye. The FAZ area was also measured within the 3 × 3-mm 2 area in the SCP and the DCP. Microaneurysms was defined as a saccular enlargement of the retinal capillaries. The number of microaneurysms in the SCP and the DCP was examined by two masked investigators (CB, AW). In the event of disagreement, a third investigator (TW) was consulted to reach a consensus. OCTA images with poor quality (signal strength index ≤ 50), motion artifacts, or incorrect autosegmentation were excluded from all analyses. The segmentation error with irregularly wavy border was excluded by two masked investigators (CB, AW) and a third investigator (TW) was consulted to derive a final determination in the event of disagreement.

SD-OCT Examination.
Patients were examined with a Cirrus SD-OCT (Carl Zeiss Meditec, Inc., Dublin, CA, USA) before and after intravitreal aflibercept injections. Central retinal thickness measurements were acquired by measurements within a 1-mm diameter of the fovea.
Intravitreal Aflibercept. All patients were examined monthly at the Osaka University hospital and received treatment with intravitreal aflibercept. The patients received one to three initial intravitreal anti-VEGF injections decided by retina specialists, followed by a pro re nata (PRN) regimen with monthly monitoring. Retreatment with intravitreal aflibercept was considered if a patient showed macular edema on SD-OCT imaging with a central retinal thickness (CRT) of more than 300 µm with intraretinal or subretinal exudation. Data Collection and Statistical Analyses. The following data were collected: ophthalmic history, baseline best corrected visual acuity (BCVA), baseline CRT, baseline OCTA images, post-treatment BCVA, post-treatment CRT, post-treatment OCTA images. Post-treatment examination was conducted at final follow-up visit in each patient. The main outcome measures were post-treatment BCVA and OCTA parameters in the SCP and the DCP. All pre-treatment and post-treatment data were collected on the same day before and after treatment, respectively. For statistical analysis, the BCVA was measured using the Landolt C acuity chart and analyzed on a logarithm of the minimum angle of resolution (logMAR) scale. Univariate regression analysis was performed to investigate the associations between logMAR BCVA and OCTA parameters for the vascular area and the FAZ area. All analyses were conducted using SigmaStat software version 3.1 (SPSS Inc., Chicago, IL, USA) and JMP Pro Software (SAS Inc., Cary, NC, USA). P < 0.05 indicated statistical significance.