Our study was a prospective interventional case series done to evaluate the effect of three intravitreal bevacizumab injections on the macular perfusion of diabetic patients with DME using OCTA. A total of 40 eyes of 26 patients were successfully imaged before and after 3 monthly intravitreal injections using the 6X6 mm macular scan protocol of OCTA, while 9 eyes of 6 patients were imaged using the 3X3 mm macular scan protocol. All patients were treatment naïve at the beginning of the study and were treated with 3 intravitreal bevacizumab injections for DME during the study.
Using the 6X6 mm scan protocol, we found an 8.1% increase in the mean size of the FAZ area, a 1.3% decrease in the mean FD-Full and mean FD-SCP, a 1.9% decrease in the mean FD-DCP, an 8% decrease in the mean VD-Full, a 9.1% decrease in the mean VD-SCP, a 10.6% decrease in the mean VD-DCP, a 13.3% decrease in the mean skeleton VD-Full, a 12.5% decrease in the mean skeleton VD-SCP, and a 16.3% decrease in the mean skeleton VD-DCP following 3 months of intravitreal bevacizumab injections. These changes were all statistically significant.
Using the 3X3 mm scan protocol, we found a 2.6% decrease in the mean FD-Full, a 3.4% decrease in the mean FD-SCP, an 11.5% decrease in the mean VD-Full, a 14.3% decrease in the mean VD-SCP, and a 25.1% decrease in the mean skeleton VD-SCP following 3 monthly intravitreal bevacizumab injections that were all statistically significant. There was also an 8.3% increase in the mean size of the FAZ area, a 2.6% decrease in the mean FD-DCP, an 8.8% decrease in the mean VD-DCP, an 18.5% decrease in the mean Skeleton VD-Full, and a 19.7% decrease in the mean Skeleton VD-DCP. These changes, however, were not statistically significant, possibly due to the smaller number of eyes imaged using the 3X3 mm protocol.
In the BOLT study, a prospective study that compared the efficacy of 6 weekly intravitreal bevacizumab injections to 4-monthly modified ETDRS macular laser treatment, there was no evidence of worsening of macular ischemia in either group at 4 months after treatment initiation. A total of 40 eyes were evaluated in the bevacizumab group. This study, however, used fluorescein angiography to analyze changes in the macular perfusion, evaluated changes only related to the fovea, and assessed the status of perifoveal capillaries qualitatively using human graders.10
In an analysis of the RIDE and RISE studies, 2 parallel prospective multicenter trials comparing 2 doses of ranibizumab (0.3 and 0.5 mg) to sham injections, there was worsening of posterior retinal nonperfusion in all groups but more in the sham injected group. The authors concluded that monthly injections of ranibizumab can thus slow, but not completely prevent, retinal capillary closure in patients with DME. Again, this study depended on FA images for the pre and post-treatment analyses and used trained human graders.11 In this study, the authors hypothesized that worsening of retinal nonperfusion in patients with DME could be the result of VEGF-induced leukostasis that was partially reversed by the anti-VEGF therapy.11,17 In an electron microscopical investigation of retinal capillaries in VEGF-A-induced retinopathy in monkeys, however, there was no leukocytes adherent to the vascular wall.18 This study, however, identified endothelial cell hypertrophy that is induced by local VEGF-A production as another possible cause of VEGF-induced worsening of capillary nonperfusion by causing progressive luminal narrowing.18
In another study analyzing the effect of aflibercept for DME on the macular perfusion (analysis of VISTA patients), there was both more improvement in retinal non perfusion and slowing of worsening retinal non-perfusion with aflibercept compared to laser treatment.12 This study, however, also used fluorescein angiography and human graders.
OCTA is a new non-invasive modality that is capable of resolving the different retinal vascular layers separately to the capillary level at a higher resolution compared to conventional FA.13,19,20 It has the ability to precisely and reliably delineate areas of capillary drop-out and to image the FAZ without obscuration by dye leakage compared to FA.19,20 It allows quantitative measurements of the vascular density and fractal dimension reliably and reproducibly in the macular area in an objective manner that does not require human graders.16,21,22 Furthermore, unlike FA, OCTA does not require intravenous dye injection which has been associated with side effects including nausea, vomiting, and anaphylaxis.23 This allows OCTA to be repeated frequently with no risks or discomfort to the patient. These advantages may allow OCTA to be more suited for the analysis of changes in macular perfusion following DME treatment.24
In a study investigating the effect of aflibercept injections for treatment naïve wet age-related macular degeneration on the macular vascular density using OCTA, there was a statistically significant decrease in the superficial foveal and parafoveal vascular density after 1, 2, and 3 injections compared to baseline (approximately 31% reduction in foveal vascular density and 6.6% reduction in parafoveal vascular density after 3 injections).25 This is interesting since patients with a history of retinal vascular disease were excluded from the study which suggests that anti-VEGF injections may affect the vascular density of normal retinal vessels. The automated measurements of the AngioVue software were used in this study and the authors hypothesized that aflibercept may result in decreased vascular density as measured by OCTA due to either decrease in nitric oxide, leading to vasoconstriction, or capillary rarefaction, which was described in experimental mouse models treated with VEGF inhibitor.25,26 Limitation of this study included the small number of included eyes (15 eyes) and the lack of a control group.
OCTA was also used to evaluate the effect of a single anti-VEGF injection on the macular perfusion in patients with macular edema secondary to DR or central retinal vein occlusion (CRVO) in a prospective non-comparative case series.27 This study found that the FAZ area and the foveal and parafoveal vessel density of the SCP and DCP did not significantly change after the single injection. Further analysis of data from the study, however, revealed an increase in the size of the FAZ and a decreased in the vascular density of the foveal area following the single injection which, however, was not statistically significant. This may be due to the relatively small number of eyes included in the study (18 eyes), the variability in the anti-VEGF agent used (bevacizumab in 14 eyes, aflibercept in 3 eyes, and ranibizumab in 1 eye), the inclusion of 2 different causes of macular edema (DME in 13 eyes and CRVO in 5 eyes), the short duration of treatment (1 month), and the use of vascular density measurements from the machine software. The study also did not exclude patients with a history of previous anti-VEGF injections which may have influenced the results.
In another retrospective study evaluating the effect of repeated intravitreal anti-VEGF injections for treatment of DME or PDR on the macular perfusion using OCTA 3X3 mm and 6X6 mm scan protocols, there was no statistically significant difference in the macular perfusion after 1, 2, and 3 injections using both scanning protocols.28 Although the study included 46 eyes which performed OCTA after the first injection, only 28 and 26 eyes performed OCTA after the 2nd and 3rd injections respectively. Unlike our study, the type of anti-VEGF agent used was variable (45.7% bevacizumab, 42.4% aflibercept, and 11.9% ranibizumab) and the mean interval between injections was 47 days. Half of the eyes (23 eyes) that performed OCTA after the 1st injection were not treatment naïve. Quantification of the vascular density was performed automatically using the proprietary built-in machine software (AngioVue software) using the ETDRS grid.
Commercially available OCTA is currently limited to evaluating the perfusion of the macular area but the technology is rapidly evolving. Using ultrawide field imaging, anti-VEGF injections were found to be associated with improved DR severity score (DRSS) on ultrawide field color fundus photographs but with no evidence of reperfusion of either arterioles or venules in or around non-perfusion areas on ultrawide field FA following 3 monthly injections.29 On the contrary, following the last injection, 83% of studied eyes showed occlusion of few vessel segments (mean: 6 ± 11 per eye) passing through the non-perfusion areas. Interestingly, similar to our study, this study also used i2k Align retina software to align pre and post-treatment images allowing only areas common to both images to be compared. Future technology enabling ultrawide field OCTA imaging can help confirm these results using the advantages of OCTA imaging.30
An important factor that could influence the measurements of vascular density using OCTA is the scan signal strength index (SSI). In a study of the effect of signal strength on OCTA metrics, differences in signal strength were associated with statistically significant differences in measurements of vascular density, perfusion density, and FAZ metrics with increased signal strength being associated with a significantly increased value of all measurements.31 Consideration of this data is essential for correct analysis of OCTA data. In our study, there was no significant difference between pre and post-treatment imaging signal strength, which further supports the validity of our results.
Another factor that could also influence OCTA measurements is IOP. In a study evaluating macular and peripapillary vascular density changes immediately following an intravitreal anti-VEGF injection for various pathologies, there was a statistically significant decrease in angiographic perfusion density in most areas of the superficial and deep layer macular vascular density, with more affection of the superficial layer, and the overall optic nerve head and the radial peripapillary capillary layer, preferentially temporal.32 In that study, the mean pre-treatment IOP was 17.15 mmHg while the mean immediate post-treatment IOP, taken approximately 15 seconds after the injection, was 46.35 mmHg as measured by Tonopen (Reichert, Depew, NY). The superficial macular density decreased by 7.8% while the deep macular density decreased by 3.5% immediately (within 3 minutes) following the injection. The authors hypothesized that the acute rise in IOP was the cause of reduced vascular density observed in the study. In our study, there was no statistically significant difference between pre and post-treatment IOP suggesting that IOP did not have a significant effect on our measurements.
An increase in the FAZ size following intravitreal bevacizumab injections for DME has also been previously reported using FA in several noncomparative studies.33,34 In one of these studies, there was a 19.7% increase in the FAZ area 6–8 weeks following a single bevacizumab injection for DME,33 while another study showed a 13% increase in the FAZ area following 3 monthly bevacizumab injections which was greater in patients with milder DR.34 This was in agreement with our results and could be due to progression of ischemia due to the underlying DR or due to VEGF inhibition or both.34 Controlled studies are needed to further explain these findings.
Several explanations could account for our findings. First, although long term treatment with anti-VEGF agents have been shown to improve the DRSS, studies have shown that they are not able to completely halt the progressive retinal capillary closure associated with DR but to only slow it down compared to sham or laser.11,12,29 This was consistent with our findings where we found a significant decrease in retinal vascular density associated with anti-VEGF injections, however, whether this decrease could have been more in the absence of injections or using other treatment modalities could not be assessed in our study due to the absence of a control group. Second, in a study using doppler ultrasonography, there was a statistically significant decrease of 10%, 20%, and 20% in the mean blood flow velocity of the central retinal artery, temporal posterior ciliary arteries, and ophthalmic arteries, respectively, 4 weeks following a single intravitreal bevacizumab injection in patients with wet age-related macular degeneration.35 The authors concluded that bevacizumab may result in hypoperfusion of the whole globe through vasoconstriction and decrease in capillary density. This could explain the decreased vascular density and flow detected by OCTA following bevacizumab injections in our study. Third, in a recent study of eyes with DME, both ranibizumab and bevacizumab injections were found to result in a significant constriction in the retinal blood vessels diameter measured using a semiautomated system.36 In that study, following a single injection of treatment naïve eyes, the central retinal artery diameter decreased by 4.5% and the central retinal vein diameter decreased by 7.8% in the ranibizumab group, while the central retinal artery diameter decreased by 2.9% and the central retinal vein diameter decreased by 3.2% in the bevacizumab group one month after the injection. There was no change in the vessel diameter in the untreated control group. This effect maybe due to inhibition of nitric oxide following VEGF inhibition which results in vasoconstriction and possible decrease in retinal blood flow which may explain the decreased vascular density and flow observed following bevacizumab injections in our study. Fourth, VEGF-A plays an important role in both physiological vascular development and pathological neovascularization in the retina and choroid, and the retinal vessels are initially dependent on VEGF as a survival factor,37 but such dependence is lost as soon as capillaries are covered by pericytes.38,39 In DR, there is early loss of pericytes,40 and this may perhaps render capillary endothelial cells in patients with DR susceptible to anti-VEGF inhibition,41 leading to endothelial apoptosis and decreased vascular density.
Using univariate and multivariate analysis, we have found that pre-treatment FD, VD, and skeleton VD at each capillary layer significantly negatively correlate with the change in FD, VD, and skeleton VD at the corresponding capillary layer respectively. This may be due to a floor effect, where eyes with more ischemia show less change over time due to a lower baseline vascularity as previously suggested.34
Strengths of our study include the prospective nature of the study, the strict exclusion criteria including previous treatment for macular edema, inclusion of patients with a single diagnosis (DME), use of a single type of anti-VEGF agent, use of 2 scanning protocols, use of automated image alignment for pre and post-treatment images, and the previously unreported analysis of post-treatment changes in fractal dimension, a measure of complexity and branching of retinal vessels, and skeleton vascular density, which is a more sensitive approach for estimation of retinal non-perfusion.16 Limitations of our study include the relatively small number of patients, inclusion of both eyes of some patients, the relatively short period of treatment, and the absence of a control group. Having a proper control group, however, could be difficult, as most patients with DME benefit from, and are currently being treated at least initially with, anti-VEGF agents. In the light of our findings, however, having a sham control group in a future study may seem proper for at least a short-term period. Several imaging artifacts are currently associated with OCTA imaging and may have influenced our results,42,43 however, we have taken several measures to limit the effects of these artifacts on the results of our study by: excluding patients with media opacities, repeating scans until no major obvious artifacts were present in the image, excluding patients with persisting major artifacts, including only images with an SSI higher than 5, manually correcting minor segmentation errors, using full thickness retinal slabs which are less affected by edema and segmentation errors, and using complex thresholding and filtering processes to create the final binarized images.