Diabetic macular edema (DME) is one of the most significant causes of visual disturbance in patients with diabetic retinopathy (DR). DME may be caused by damage to the blood–retina barrier induced by metabolic changes and inflammation.[1,2] DME is affected by not only cells in the retina but also the expressions of various molecules, including interleukins (ILs), vascular endothelial growth factors (VEGFs), tumor necrosis factor-α, and matrix metalloproteinase.[2–4]
Classification of DME into several types using various imaging tools, including fluorescein angiography, optical coherence tomography (OCT), and OCT angiography, has been investigated to identify causes, predict prognosis, and select appropriate treatments.[5–8] Of these techniques, OCT is a fast and noninvasive tool to quantify macular profiles. Recent studies using OCT have morphologically classified DME as cystoid macular edema (CME), diffuse retinal thickening (DRT), or serous retinal detachment (SRD). Of these morphological classifications, the prognosis and mechanism of SRD is controversial. Some studies have reported that DME with SRD has a better responsiveness or better prognosis after anti-VEGF treatments, suggesting that there could be an association between VEGF and SRD.[10,11] However, there are other studies that reported contradicting results showing no association between inflammation and SRD.[12–14]
Therefore, in this study, we grouped naïve DME patients according to the presence of SRD, and compared their systemic and ocular factors including the levels of VEGFs and inflammatory cytokines in the aqueous humor. Then, we identified factors associated with SRD.
The study protocol adhered to the tenets of the Declaration of Helsinki. The protocol was approved by the institutional review/ethics board of the Catholic University of Korea. All participants gave written informed consent for the use of their clinical records.
We enrolled treatment-naïve center-involving DME (CIDME) eyes of central subfield thickness (CST) ≥300 μm from June, 2016 to November, 2016. The criteria of exclusion included macular edema due to other causes including an epiretinal membrane or vitreo-macular traction. We also excluded eyes with histories of uveitis or intraocular surgery.
We measured glycated hemoglobin levels, and all patients took ophthalmic examinations, including measurements of the best corrected visual acuity (BCVA) and fundus examination. The CST was measured using OCT (Cirrus High-Definition OCT; Carl Zeiss Meditec, Dublin, CA) and axial length was measured using an IOL Master (Carl Zeiss Meditec). The hyperreflective foci (HF) were manually measured within 1500 μm, and ellipsoid zone (EZ) disruptions were manually measured within 1000 μm using a horizontal scan centered on the fovea.[6,16] EZ disruption was graded as 0 in case of intact, 1 in case of focal disruption ≤200 μm in length, and 2 in case of disruption >200 μm in length.
2.1 Assay of cytokines and growth factors
We collected aqueous fluid specimens before first intravitreal bevacizumab (IVB) injection, and measured the concentrations of IL-1β, -2, -8, -10, -17, placental growth factor (PlGF), and VEGF in 75 μL aqueous humor. The antibodies were immobilized on beads, and samples with 75 μL Calibrator Diluent RD6–52 (R&D Systems, Minneapolis, MN) were added to the preparations. And the samples were incubated for 2 hours after adding beads, for 1 hour after adding detection antibodies, and for half-hour after adding the streptavidin-phycoerythrin reagent. Samples were read using the Luminex xMAP system (Luminex, Austin, TX).
2.2 Statistical evaluation
Statistical analyses were performed using SPSS statistical software for Windows, version 20.0 (SPSS, Chicago, IL). The t test, Mann–Whitney U test, and chi-square test were used to compare the values or the ratios of the patient subgroups. Logistic regression was employed to identify factors associated with SRD occurrence.
We enrolled 64 treatment-naïve CIDME eyes of 64 patients. The mean age was 55.85 ± 9.35 years, and there were 30 men and 34 women. In the DR staging, 47 patients had proliferative DR (73.44%) and 17 patients had non-proliferative DR (26.56%). All the patients who showed proliferative DR had received panretinal photocoagulation. The mean BCVA (LogMAR) was 0.45 ± 0.27, and the mean CST was 416.03 ± 81.14 μm at baseline. Based on DME morphology, 33 patients had CME and 31 had DRT. The systemic and ocular characteristics of the patients are summarized in Table 1.
There were 50 eyes with DME without SRD and 14 eyes with SRD. There was a significant difference in sex distribution, but no significant differences were found in age, glycated hemoglobin level, duration of diabetes, DR stage, BCVA, or CST between the 2 groups. The OCT findings such as number of HF and EZ grade also showed no significant differences. The BCVA and CST after 3 consecutive monthly IVB injections did not significantly differ (P = .238 and P = .314, respectively). In the comparison of cytokine levels in the aqueous humor, VEGF and PlGF levels were significantly higher in the SRD group (P = .022 and P = .041, respectively) (Table 2).
The factors identified as associated with SRD are summarized in Table 3. In univariate and multivariate logistic analyses, VEGF level was the only factor associated with the existence of SRD (odds ratio [OR]: 1.01, P = .009 and OR: 1.03, P = .038, respectively).
Many studies have reported that DME is mediated by inflammation and VEGFs, and their actions are closely interconnected.[2,18] DME treatment by suppressing VEGF levels or the controlling inflammation has been spotlighted. Studies using imaging tools or biomarkers to find early and effective treatments among the various options have been reported.[16,19] This study also focused on finding more effective treatment regimen among various treatment options by using an imaging tool and biomarker; we are the first study to find that SRD in DME is associated with VEGF levels.
Several treatment options for DME are now available. Photocoagulation of the leaking point with a focal laser is used to treat non-CIDME patients. Removal of traction, clearing the inflammatory cytokines and growth factors, and increasing the oxygen levels of the vitreous and retina via vitrectomy have also been used to treat refractory DME.[8,21–23] However, the main treatment of DME is intravitreal injection of anti-VEGF agents or steroids.[24–26] Many studies have shown that visual disturbances are associated with the degree of macular thickness, and long-lasting and chronic DME can compromise visual functions.[27–29] Thus, early and optimal treatments are required to recover and acquire a normal macular contour. If proper treatments are delayed, permanent visual disturbance can occur.[29,30] Thus, it is necessary to identify more relevant mechanisms in DME subtypes, so that customized treatments can be selected. Although SRD is one of the most common manifestations in DME, the mechanism of action is still unclear. We found SRD group had significantly higher aqueous levels of VEGF and PlGF (P = .022 and P = .041, respectively). Additionally, in the logistic analyses, the occurrence of SRD was significantly associated with VEGF levels (OR: 1.03; P = .038). The PlGF, a member of the VEGF family, is induced by ischemic reinal condition, and has a key role in angiogenesis and vasculogenesis in retina. Based on these results, SRD seems to be more associated with VEGFs than with inflammation.
Although vitreous samples adequately reflect retinal status, obtaining vitreous samples is invasive and difficult because few patients are treated with vitrectomy due to DME. In addition, posterior vitreous detachment or blood contamination could compromise data quality of vitreous samples. The aqueous humor, which can easily be obtained during intravitreal injection, can also reflect retinal status. The levels of many cytokines are increased under conditions such as retinal hypoxia or inflammation,[33,34] and most studies that have used aqueous humor samples have shown that the concentrations of various molecules from DME patients differ from those of controls.[17,35] However, few studies have investigated the associations between responsiveness of treatment and the levels of these biomarkers. We suggest that aqueous humor samples can be used to identify the major mechanisms of DME. Our results could be useful in selecting appropriate treatments or predicting the prognoses of patients.
There is a consensus that the subretinal fluid in DME is resolved with anti-VEGF treatment, but the prognosis regarding BCVA and CST is controversial. Some studies have reported that SRD patients showed better responsiveness or prognoses in CST reduction or BCVA improvement with anti-VEGF treatments,[10,36,37] but others have reported no better responsiveness with the same treatments.[11,14] The mechanisms of SRD are also controversial. In contrast to our results, some studies have reported that there is an association between SRD and inflammation[12,38] and that steroid implants can result in a positive response.[39,40] On the other hand, other studies have shown that there is an association between systemic status or glycemic control and SRD.[41,42] Although there was no remaining SRD after 3 consecutive IVBs in all patients in the SRD group (Fig. 1), the degree of CST reduction in these patients did not differ from that of the DME patients without SRD in our study, and we did not detect a difference in glycated hemoglobin levels between the 2 groups. As the mechanism of DME is complicated and there is a lack of studies about the responsiveness of DME, more investigations and studies are needed to clarify the origin and responsiveness of SRD in DME.
Our study had some limitations. First, our sample size was relatively small and the follow-up duration was short. The aqueous levels showed no significant difference in SRD group may be attributable to the small number of control patients. We also judge that there is significantly higher ratio of women in SRD group by chance due to small sample size. Long-term changes in CST and BCVA must be evaluated with the treatments. In addition, although we made an effort to statistically factor in the systemic and ocular status of patients, other factors we did not consider might have affected our results. Second, the relationships between cytokine levels and other imaging techniques like fluorescein angiography or OCT angiography should be studied in terms of DME pathogenesis.
In summary, the occurrence of SRD was associated with VEGF levels. Additional studies with more patients are required to confirm our results and to elucidate the pathogenesis of DME, which may provide the basis for novel therapeutic approaches.
The authors acknowledge financial support from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1C1B5017408). This research was also supported by a Grant of Translational R&D Project through the Institute for Bio-Medical Convergence, Incheon St. Mary's Hospital, The Catholic University of Korea.
Conceptualization: Jin-woo Kwon.
Data curation: Jin-woo Kwon.
Formal analysis: Jin-woo Kwon.
Funding acquisition: Hyung Bin Hwang.
Investigation: Hyung Bin Hwang.
Supervision: Hyung Bin Hwang, Donghyun Jee.
Validation: Jin-woo Kwon.
Visualization: Jin-woo Kwon.
Writing – original draft: Jin-woo Kwon.
Jin-woo Kwon orcid: 0000-0003-2093-4284.
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