Association between Aqueous Humor Cytokines and Structural Characteristics Based on Optical Coherence Tomography in Patients with Diabetic Macular Edema

Purpose To investigate the relationship between aqueous humor cytokines and structural characteristics based on optical coherence tomography (OCT) in patients with diabetic macular edema (DME). Methods Forty eyes of 28 patients with DME diagnosed in the Affiliated Eye Hospital of Wenzhou Medical University at Hangzhou were included. All patients collected aqueous humor during anti-VEGF treatment, and the IL-6, IL-8, IL-10, VEGF, VCAM-1, ICAM-1, TGF-β1, FGF, and MCP-1 concentrations were detected. OCT examination was performed before anti-VEGF treatment and 1 month after anti-VEGF operation. Central macular thickness (CMT), macular volume (MV), choroidal thickness (CT), and the number of hyperreflective foci (HRF) were obtained for analysis. Each eye was determined whether there is subretinal effusion (SRD), cystoid macular edema (CME), and diffuse retinal thickening (DRT). Results The levels of IL-6 and FGF in DME patients with SRD were significantly higher than those without SRD (all P < 0.05). The level of VEGF in DME patients with CME was significantly higher than that in DME patients without CME (P = 0.005); IL-6, TGF-β1, and MCP-1 were significantly higher in DME patients with DRT than that without DRT (all P < 0.05). There was no significant correlation between aqueous humor cytokines and retinal thickness and retinal volume. However, the thinner the CT, the higher the level of aqueous humor cytokines IL-6 (r = −0.313, P = 0.049) and FGF (r = −0.361, P = 0.022). A multivariate linear regression analysis showed that IL-6 was significantly correlated with CT (P = 0.002) and SRD (P = 0.017), FGF was also significantly correlated with CT (P = 0.002) and SRD (P = 0.005), and TGF-β1 was correlated with triglycerides (P = 0.030) and HRF (P = 0.021). Conclusion DME patients with significant macular cystoid edema changes may be related to high VEGF concentrations and thin CT; meanwhile, the presence of SRD or a high number of HRF on OCT macular scans in DME patients may indicate high levels of intraocular inflammatory factors. Thus, OCT morphology characteristics to some extent reflect intraocular inflammatory factors and VEGF levels and may guide treatment alternatives.


Introduction
Diabetic macular edema (DME) is one of the major causes of vision loss in diabetic patients. Vascular endothelial growth factor (VEGF) plays an important role in the development of DME; anti-VEGF drugs have become the frst-line treatment for DME. However, 30%-40% of patients are still insensitive to anti-VEGF therapy [1,2]. Both clinical and experimental studies have confrmed that in addition to VEGF, infammatory factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), VEGF, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-β1 (TGF-β1), fbroblast growth factor (FGF), and monocyte chemoattractant protein-1 (MCP-1) also play important roles in the pathogenesis of DME [3,4]. Terefore, patients with DME who are insensitive to anti-VEGF therapy may be treated with dexamethasone implant alone or in combination. Hence, it has been suggested that intraocular cytokine levels could be used to predict the outcomes of anti-VEGF therapy and to enhance treatment accuracy [5,6].
However, intraocular cytokine testing is an invasive test that may raise the risk of infection and is currently mainly gathered at the time of surgical treatment; thus, it ofers limited guidance on treatment options. Optical coherence tomography (OCT), a noninvasive test that provides highresolution images of retinal microstructures in a noncontact and rapid manner, plays an important role in the diagnosis and follow-up of DME. Depending on the OCT morphology and pathogenesis, DME can manifest in one of the three forms: subfoveal neuroretinal detachment (SRD), cystoid macular edema (CME), and difuse retinal thickening (DRT) [7,8]. Tis study aims to analyze the relationship between aqueous humor cytokines and OCT structures in patients with DME, and to estimate the intraocular cytokine levels in patients with DME before treatment, to give clinicians more information to make better treatment plans.

Methods
Tis study adhered with the Declaration of Helsinki and was approved by the Ethics Committee of the Optometry Hospital of Wenzhou Medical University (No: 2021-003-K-03-01). All patients were informed and signed written informed consents. Consecutive patients with outpatient diagnosed DME and received anti-VEGF treatments were collected from March 2021 to December 2021 at the Afliated Eye Hospital of Wenzhou Medical University. Inclusion criteria were as follows: (1) age 18-80 years; (2) OCT recording of central macular thickness (CMT) > 250 μm. Exclusion criteria were as follows: (1) previous history of ocular trauma and uveitis; (2) combined macular degeneration, glaucoma, iris neovascularization, and other eye diseases; (3) previous history of vitreous surgery; (4) patients who had undergone cataract surgery, anti-VEGF injection, laser therapy, and other treatments.
All patients underwent comprehensive ophthalmic examinations before anti-VEGF treatment, including best corrected visual acuity (BCVA), intraocular pressure, slit lamp microscopy, and dilated fundus examination. OCT three-dimensional macular scans were performed using Heidelberg OCT (Heidelberg Spectralis, Heidelberg, Germany) with 30°× 25°horizontal line scan mode, and the build in software was used to automatically calculate the CMT and macular volume (MV) at the end of the scan. Specifcally, the macular area was divided into 1 circle and 2 annular areas according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) Group division method, i.e., a 3 circular area with a diameter of 1 mm, 2 mm, and 3 mm centered on the fovea, and the mean CMT within a diameter of 1 mm circle and the mean MV within a diameter of 3 mm circle were calculated. OCT horizontal line scan centered at the macular with 100 frames per scan was also acquired to count hyperrefective foci (HRF) and measure choroidal thickness (CT). Te intraretinal HRF was defned as isolated, well-defned foci with refectance intensity not weaker than the retinal pigment epithelium (RPE) layer refectance intensity, and with a diameter >20 μm and <50 μm. Two scorers manually counted HRF in the retina, disagreements between scorers were solved by open adjudication, and the mean value was taken for statistical analysis [9]. CT was defned as the distance between the outer border of the RPE and the inner border of the choroidal-scleral interface under the central macular. Te presence of SRD, DRT, and CME was determined for each eye based on previous studies. SRD demonstrated as a plasmacytic separation between retinal neuroepithelium and pigment epithelium; CME was detected as intraretinal cystic hyporefective lesion; DRT was identifed as retinal spongy thickening and reduced intraretinal refectivity (Figure 1).
Te aqueous humor specimens were collected during anti-VEGF treatment, and 0.05-0.1 ml aqueous humor was extracted using a 30-gauge insulin syringe via limbal paracentesis before intravitreal injection. Te sample was immediately transferred to a plastic tube and kept at −84 until assayed. Aqueous humor concentrations of IL-6, IL-8, IL-10, VEGF, VCAM-1, ICAM-1, TGF-β1, FGF, and MCP-1 were tested with CBA fex set (BD Cytometric Flex Set, San Diego, USA) with FACSCanto plus fow cytometer (BD Biosciences). SPSS 26.0 statistical software was used to analyze the data. Te Shapiro-Wilk test was used to evaluate the normality of data distribution. Non-normally distributed data were expressed as median ± quartiles, and the Mann-Whitney U test was used for comparison between groups. Normally, distributed data were expressed as the mean-± standard deviation, and the independent samples t test was used for comparison between the groups. Te relationship between aqueous humor cytokines and retinal choroidal structures was analyzed by Spearman rank correlation. Aqueous humor cytokine data were logtransformed to reduce the efect of outliers; univariate and multiple linear regression analyses were performed to determine the correlation between variables. Variables with a signifcance of P < 0.15 in the univariate analysis were entered into the multivariate analysis. P < 0.05 was considered statistically signifcant.

Results
Forty eyes of 28 patients with a mean age of 62.5 ± 9.3 years were included in this study. Among them, nine (22.5%) eyes exhibited SRD, thirty two (80%) eyes exhibited CME, and thirty (75%) eyes exhibited DRT. All DME eyes were grouped according to whether they presented with DRT, CME, and SRD, the baseline characteristics were compared between the groups in Table 1, the aqueous humor cytokine levels and OCT parameters were compared and shown in Table 2. Tere were no signifcant diferences in baseline characteristics in eyes with and without SRD, however, IL-6 and FGF levels were signifcantly higher in eyes with SRD than those without SRD (all P < 0.05). Similarly, there were no signifcant differences in baseline characteristics in eyes with and without CME, however, VEGF levels were signifcantly higher in eyes with CME than those without CME (P � 0.005). Meanwhile, the CT in DME patients with CME was signifcantly smaller than those without CME (P � 0.042). Tere were no signifcant diferences in baseline characteristics in eyes with and without DRT; however, TGF-β1 levels were signifcantly higher in eyes with DRT than those without DRT (P � 0.039). Tere were no signifcant diferences in CMT, MV among diferent types of DME; however, HRF was signifcantly higher in DME patients with SRD than those without SRD (P < 0.001), and HRF was higher in DME patients with DRT than those without DRT (P > 0.05).
Te aqueous humor cytokine data were log-transformed to remove the impact of outliers and multiple linear regression analysis was carried out to better understand the relationship between aqueous humor cytokine levels and retinal choroidal structures. Univariate and multivariate Figure 1: OCT images of diferent DME types. (a) DME primarily exhibits DRT, with macular spongy thickening indicated by arrowheads; (b) DME primarily exhibits CME, with intraretinal hyporefective cystic cavity indicated by arrows; (c) DME primarily exhibits SRD, with an optically clear gap between retinal neuroepithelium and pigment epithelium indicated by asterisks; (d) DME exhibits CME and DRT; (e) DME exhibits DRT and SRD; (f ) DME consists of a mixture of CME, DRT, and SRD types.   linear regression analyses were performed for IL-6, FGF, and TGF-β1, separately (Table 3). Te univariate regression analysis showed that CT (P � 0.015), SRD (P � 0.030), CME (P � 0.032), and DRT (P � 0.098) correlated with the increase of IL-6. Te multivariate regression analysis showed that CT (P � 0.002) and SRD (P � 0.017) was signifcantly correlated with IL-6. Te univariate regression analysis showed that CT (P � 0.001), DM duration (P � 0.091), total cholesterol (P � 0.091), SRD (P � 0.036), and CME (P � 0.065) correlated with the increase of FGF. Te multivariate regression analysis showed that CT(P � 0.002) and SRD (P � 0.005) was signifcantly correlated with FGF. Te univariate regression analysis showed that CT (P � 0.001), triglycerides (P � 0.097), and HRF (P � 0.008) correlated with the increase of TGF-β1. Te multivariate regression analysis showed that triglycerides (P � 0.030) and HRF (P � 0.021) was signifcantly correlated with TGF-β1.

Discussion
Depending on the OCT morphology and pathogenesis, DME can manifest as SRD, DRT, CME, or mixed type. SRD mainly forms as the result of extraretinal barrier dysfunction; DRT presents as difuse retinal edema mainly caused by retinal cell hypoxia and intracellular edema; CME primarily related to intraretinal barrier dysfunction and manifest as intraretinal fuid, muller cell, intracytoplasmic swelling, and necrosis [10,11].
In this study, we found that IL-6 and FGF levels were signifcantly higher in DME patients with SRD than DME  Journal of Ophthalmology patients without SRD. A multivariate linear regression analysis showed that both aqueous humor IL-6 and FGF levels were associated with the presence of SRD. Tis result is consistent with previous studies, suggesting that infammation plays an important role in the development of SRD. Abcouwer [12] reported that IL-6 was closely associated with SRD-type DME. Sonoda [13] et al. divided DME into groups with and without SRD, and demonstrated that IL-6 and IL-8 levels were signifcantly greater in eyes with SRD than in eyes without SRD, IL-6 levels were signifcantly associated with the presence of SRD after adjusting for age and sex. In one study, DME was divided into DRT, CME, and SRD types. Te IL-6 levels between the three groups were also signifcantly diferent, with the SRD group having signifcantly higher IL-6 levels than the CME group [14]. IL-6 is a proinfammatory cytokine that regulates the immune response and initiates the acute phase response as a factor necessary for tissue injury healing. Injury-activated cytokines draw infammatory cells to the site of injury, which stimulates the secretion of IL-6 and in turn induces the production of VEGF [15]. FGF is another growth factor that promotes cell proliferation, maintains tissue integrity, anticipated in wound healing, and is associated with the fbrotic process in DR [16]. Terefore, elevated levels of IL-6 and FGF in the SRD group may be associated with impairment of the outer retinal barrier, RPE cells and the choroid; however, the exact mechanism remains to be further investigated. Hwang et al. [17] reported that VEGF  levels in aqueous humor were greater in the SRD group than in the no-SRD group. Our study was consistent with Hwang et al., as we demonstrated higher VEGF levels in DME patients with SRD than DME patients without SRD, although the diference was not statistically signifcant. Previous studies found that IL-6 and IL-8 levels were signifcantly lower in CME eyes than in non-CME eyes [13]. However, the present study found that IL-6 levels in DME patients with CME were lower than those without CME, and IL-8 levels were higher in DME patients with CME were lower than those without CME, although the diferences were not statistically signifcant. Meanwhile, this study showed that VEGF levels were signifcantly higher in DME patients with CME than in DME patients without CME. It has been reported that VEGF can induce conformational changes in the tight junctions of vascular endothelial cells and increase retinal vascular permeability, thus possibly causing intraretinal barrier dysfunction and extracellular edema between retinal layers [18]. Kim et al. [14] also reported signifcant diferences in VEGF levels between the DRT, CME, and SRD groups; however, a post hoc comparison did not show statistically signifcant diferences between any two groups.
Sonoda et al. [13] reported no signifcant diferences in IL-6, IL-8, and VEGF levels between eyes with and without DRT in DME patients. Te present study also found no signifcant diference in the VEGF levels between DME eyes with and without DRT. However, the IL-6, TGF-β1, and MCP-1 levels were signifcant greater in DME eyes with DRT than DME eyes without DRT, suggesting that infammation plays an important role in macular difuse edema.
HRF are thought to be activated microglia in the retina and are an imaging marker of retinal infammation [19,20]. Previous studies reported that HRF level was signifcantly greater in DME patients with SRD than in DME patients without SRD [20,21]. TGF-β1 plays an important role in the process of angiogenesis, endothelial cell proliferation, extracellular matrix deposition, and tissue fbrosis [22]. We found that higher TGF-β1 levels were associated with greater HRF numbers. TGF-β1 has been reported to play a role in regulating microglia-mediated synaptic pruning in retina [23]. Terefore, TGF-β1 may promote migration and transformation of microglia, which were noted to manifest as HRF in DME.
Only a limited number of studies have analyzed the relationship between aqueous humor cytokines and CMT and MV. Hillier et al. [24] analyzed aqueous humor levels of VEGF, TGF-β, ICAM-1, IL-2, IL-3, IL-6, IL-8, IL-10, IL-17, VCAM-1, MCP-1, and EGF in 49 DME patients and analyzed their relationship with CMT and MV. A multiple regression analysis showed that only the increased level of ICAM-1 was found to be associated with an increase of MV, other cytokines were not signifcantly related to either the CMT or MV. Jonas et al. [25] measured macular thickness (defned as the distance between the inner retinal boundary membrane and the RPE) in 15 patients with DRT and found that central macular thickness was signifcantly associated with ICAM-1 levels. Kwon and Jee [26] analyzed the relationship between IL-1β, IL-2, IL-8, IL-10, IL-17, PlGF, and VEGF levels and CMT in 64 DME patients, and found that only IL-10 levels were correlated with CMT. In this study, we found that aqueous humor cytokine IL-6, IL-8, IL-10, VEGF, VCAM-1, ICAM-1, TGF-β1, FGF, and MCP1 levels lacked signifcant correlation with CMT or MV.
As far as we are aware, there are currently no research studies available that discuss the association between the CT and aqueous humor cytokine levels. Te relationship between CT and the severity of diabetic retinopathy also remains unclear. Some studies reported that patients with diabetic retinopathy have higher CT than controls, while other studies reported that CT decreases as the severity of diabetic retinopathy increases or a lack of signifcant correlation between CT and diabetic retinopathy [27,28]. Recently, it has been suggested that diabetic retinopathy causes an early increase in CT, which then decreases as diabetic retinopathy progresses. However, there was no statistically signifcant relationship between CT and DME. [29]. Gerendas et al. [30] studied 142 fuorescent angiographic images of patients with DME and showed that the thinner the CT, the greater the area of retinal leakage. In DME recurrence, Mathis et al. [31] discovered an increase in CT. In DME eyes with higher CT, Rayess et al. [32] discovered a better short-term morphological and functional response following anti-VEGF. All these studies suggest that the choroid may be involved in the pathogenesis of diabetic retinopathy and DME; however, their defnite roles and mechanisms are not clear. In this study, we found that the thinner the CT, the higher the aqueous humor IL-6 and FGF levels. However, more studies are needed to confrm the exact association and to explore the mechanism.
Tere are several limitations in our current study. First, the number of patients included is relatively small, which may have attenuated the statistical power for detecting diferences between the groups. Second, the vitreous fuid may be a better sample to refect the posterior segment pathology than aqueous fuid. However, aqueous sampling is less invasive than vitreous fuid sampling and currently the most used intraocular specimen for the study of retinal diseases. Another limitation is a short followup, and the response to anti-VEGF treatment has varied in diferent studies and may be subjective in the present study.
In conclusion, this study suggested that signifcant macular cystoid edema changes may be related to high VEGF concentrations, and thin CT, the presence of SRD, or a high number of HRF on OCT macular scans in DME patients may indicate high levels of intraocular infammatory factors. We believe that the reorganization of these characteristic OCT structures will facilitate medical professionals to estimate the levels of VEGF and intraocular infammatory factors noninvasively in DME patients and to make better treatment strategies.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.