Analysis of Cytokines in the Aqueous Humor During Intravitreal Treatment With Ranibizumab in Diabetic Macular Edema

Purpose: This study aimed to analyze the concentrations, in aqueous humor, of VEGF, b-FGF, TNF, and interleukins 1, 6, 8, 10, and 12 in patients with diabetic macular edema, with and without peripheral retinal ischemia, and to analyze the variation of the levels of these molecules, during the treatment with ranibizumab. Methods: A therapeutic, prospective, randomized interventional study was carried out. Twenty-four eyes from 24 patients were studied, divided into 3 groups. Group 1, (9 eyes), patients with EMD without peripheral ischemia. Group 2 (10 eyes), patients with EMD with peripheral ischemia. Group 3, (5 eyes), control group, formed by patients without systemic and/or eye diseases. Patients in groups 1 and 2 were treated with 3 intravitreal injections of 2 mg/0.05 ml of ranibizumab, with intervals of approximately 30 days. Before performing the injections, the aqueous humor was collected. In the control group, the material was collected before the facectomy procedure. Results: During treatment, the medians of IL-6 concentrations showed a statistically signicant increase, in group 1 and group 2, there was a slight decrease, not statistically signicant. Interleukin 8 showed statistically signicant variations in groups 1 and 2, at the end of treatment. TNF, IL-1, IL-10, and IL-12 had their concentrations practically unchanged, in both groups. VEGF showed a statistically signicant reduction in groups 1 and 2 at the end of the study. B-FGF was not detected in most of the studied patients, and in those that were found, they did not present statistically signicant numbers. Conclusion: There were statistically signicant variations in the increase of their median levels in interleukin 6, in the group without ischemia and a decrease in VEGF in both groups. The cytokines TNF, IL-1, IL-10, and IL-12 did not show statistically signicant variations.


Introduction
Diabetic retinopathy (DR) is one of the main causes of vision loss in the world 1 .
Macular edema (ME) is considered the main responsible for visual impairment in patients with diabetic retinopathy, which can appear in all stages of the disease, affecting approximately 30% of diabetic patients, with more than 20 years of the disease 2,3,4, 5 .
Tests such as optical coherence tomography (OCT) and uorescein angiography (FA) are performed for diagnosis and monitoring of patients with DR 6 .
Fluorescein angiography is essential for the ability to acquire information about the stages of the disease 7,8 . New devices called wide-angle uorescein angiography can capture images with angles between 55º and 200º 9 . This new technology has aroused the interest in analyzing the retinal periphery and correlating the changes found with the emergence and progression of diabetic retinopathy 10,11,12,13,14 .
We believe that regions of poor perfusion stimulate the production of vascular endothelial growth factor (VEGF) and that their high levels, in the vitreous, induce the formation of ME 14,15,16,17 .
It is not only VEGF that is increased in patients with diabetic retinopathy. Cytokines such as interleukins, TNF, and b-FGF are also altered in this disease 18,19,20,21,22,23,24 .
We performed an unprecedented study in which we recruited patients with diabetic macular edema, with and without peripheral retinal ischemia, for analysis and comparison of the levels of concentrations, of the molecules Interleukin 1, 6, 8, 10, and 12, TNF, VFGF, and b-FGF, during treatment with intravitreal injections of ranibizumab.

Methods
All methods were performed according to relevant guidelines.
The consent form was signed by all research participants as well as by their legal guardian.

Study population and design
Therapeutic, prospective, randomized interventional study. The sample consisted of 24 eyes, 24 patients, divided into 3 groups. Group 1, (9 eyes), with DME without peripheral retinal ischemia. Group 2, (10 eyes), with DME, with peripheral retinal ischemia. Group 3 was (5 eyes) a control group, formed by a patient without systemic and eye diseases.
Patients in groups 1 and 2 underwent 3 intravitreal injections of ranibizumab, with intervals of approximately 30 days. Before each procedure, we collected samples of the aqueous humor ( Figure 1). In group 3, samples were collected on the day they underwent cataract surgery.
We performed optical coherence tomography in the Heidelberg Spectralis® HRA + OCT for the diagnosis of edema and angiography with a non-contact lens, in the Ultra-Wide eld Spectralis® module, with 102° angle, to detect the presence of peripheral retinal ischemia.
The study was approved by the Ethics Committee in the Medical Research Ethics Council of Hospital Agamenon Magalhães, Recife, Pernambuco.

Analysis of cytokines
For Interleukins 1, 6, 8, 10, 12, and TNF, the Human In ammatory Cytokine (BD) kit was used and for human VEGF and bFGF molecules, the dosage was through CBA FLEX SET, both kits from the manufacturer BD Medical.

Evaluation of results and statistical analysis
In this study, data were previously evaluated for parametric or non-parametric approaches. Thus, the variables involved in the study were tested for their distribution of normality and homogeneity using the tests by Shapiro Wilk and Bartlett respectively.
The signi cance level of 5% was used to con rm the conclusions. We used the software R (R DEVELOPMENT CORE TEAM, 2016) to evaluate the results of the study.

Inclusion criteria
People with diabetic macular edema diagnosed by wide-angle uorescein angiography exams and optical coherence tomography; age equal to or above 18 years old; without previous treatment with laser photocoagulation antiangiogenic drugs, less than 3 months ago.

Exclusion criteria
Cataract patients, corneal or vitreous opacities that prevented adequate visualization of the retina; patients with macular edema that was not due to diabetic retinopathy; patients undergoing previous vitreoretinal surgery in the eye to be studied and the patient's refusal to participate in the study.

Performing clinical and complementary examinations
Patients underwent ophthalmic clinical evaluation. Visual acuity exams were performed with better correction by ETDRS, applanation tonometry, biomicroscopy, and indirect binocular ophthalmoscopy.
The optical coherence tomography and uorescein angiography examinations were performed using the Heidelberg Spectralis® HRA + OCT. A non-contact lens was used in the Ultra-Wide eld Spectralis® module to capture the 102º wide-eld angiographic image.

Collection and analysis of aqueous humor
Limbar paracentesis was performed with a 30-gauge needle attached to a 1 ml syringe. Sample volumes ranged between 0.10 ml and 0.3 ml.
We quanti ed the cytokine levels using the Cytometric Bead Array (CBA) system, following the manufacturer's instructions (BD Biosciences, USA). The cytokines IL-1, IL-6, IL-8, IL-10, IL-12, and TNF were analyzed using the Human In ammatory Cytokine (BD) kit, and the human VEGF and b-FGF molecules dosed through CBA FLEX SET also from BD.

Visual acuity and macular thickness
At the end of the research, 8 showed improvement in visual acuity. One patient showed no visual improvement, remaining with the same initial vision ( Figure 2).
Upon OCT examination, all patients presented a reduction in central macular thickness upon analysis of the macular thickness map, as illustrated in two patients ( Figure 3).

Clinical examinations and uorescein angiography
Upon biomicroscopy examination, one patient was pseudophakic and 08 were phakic. No lens had progressed to cataracts during the study.
At gonioscopy, the presence of neovessels at an angle or iris was not seen before and at the end of the research.
Upon examination of angiography, no patient presented vascular changes of the staining type or retinal neovascularization during the treatment period.

Group 2:
The group had 10 patients (7 males and 3 females), aged between 47 and 68 years (± 7.06), all with DME with peripheral retinal ischemia, undergoing monthly treatment with ranibizumab.

Visual acuity and macular thickness
At the end of the research, 8 patients showed improvement in visual acuity. Those who did not improve maintained initial visual acuity ( Figure 2). Upon OCT examination, all patients presented a reduction in central macular thickness upon analysis of the macular thickness map, as illustrated in two patients ( Figure 4).

Clinical examinations and uorescein angiography
Upon biomicroscopy examination, 9 patients were phakic. No lens had progressed to cataracts during the survey. At gonioscopy, the presence of neovessels at an angle or iris was not seen before and at the end of the research.
At the beginning of the study, on angiography, two patients had retinal neovessels and one of them also had disc neovessels at the beginning of treatment. At the end of the study, both patients presented regression of the new vessels ( Figure 5).

Analysis of cytokines in aqueous humor
Comparison of the medians between the beginning and the end of the treatment, in groups 1 and 2. In the data of the control group, we used the values at the beginning of the treatment such as the baseline levels ( Table 1).
In analysis, of the variation of medians of IL-6, during treatment, we observed that, in group 1, it presented a statistically signi cant increase, at the end of the research in group 1 (10.78 -26.35 pg/ml, p = 0.0148).
In group 2, there was a slight decrease in the median of its concentrations, but it was not statistically signi cant (28.02 -27.41 pg/ml, p = 0.0194) (Figure 6).
The presence of b-FGF was only detected in only 3 patients in group 1 and none in group 2. Thus, we could analyze its variations during the research (Table 2).

Discussion
The role of cytokines in the progression of diabetic retinopathy and the formation and maintenance of diabetic macular edema has long been studied.
Molecules such as interleukins, TNF, VEGF, b-FGF are important components that are involved in the pathophysiology of diabetic retinopathy and have become the object of study to determine the function of each one in this disease.
In this study, we produced an unprecedented analysis of variations in the levels of these cytokines during the treatment of diabetic macular edema, in patients with and without peripheral retinal ischemia, using an antiangiogenic drug called ranibizumab.
The purpose of selecting patients, with or without peripheral retinal ischemia, was to investigate how much the hypoxia condition can in uence the expression of these cytokines and interfere with the result of the treatment of diabetic macular edema as well as to analyze their variations on the action of an anti-VEGF drug.
Knowing the importance of the role of these cytokines, we chose to study their concentrations during the treatment of diabetic macular edema with a drug that has the function of stabilizing vascular permeability and acting by inhibiting one of these molecules.
According to studies, the intraocular levels of TNF and IL-1 must be elevated, when there is a state of edema because they have the power to induce an increase in vascular permeability with the breakdown of the internal hematoretinal barrier 19,21,22 .
In this study, when comparing the levels of IL-1 and TNF, before treatment, they were slightly higher than the control group. When doing a longitudinal analysis, we observed that the levels of IL-1 and TNF varied little, during the research, in both groups.
Interleukin 6 is considered a potent proin ammatory cytokine that plays an important role in the mechanisms of chroni cation of in ammatory processes 25 .
Chernykh and collaborators showed that patients with proliferative diabetic retinopathy have high levels of IL-6 in the vitreous, being signi cantly higher than in patients without diabetes 23 . An important aspect of the research carried out by the Chernykh group was in the selected patients with RDP, that is, patients who have pictures of retinal ischemia.
In this study, the median values of IL-6, before treatment, were approximately 2.5 times higher in ischemic patients than the non-ischemic patients and 3.4 times higher than the control group at the beginning of treatment. Thus, we can think that this cytokine may have a great relationship with the state of retinal hypoxia.
VEGF is considered to be one of the main agents in the progression of retinal vascular diseases. They stimulate the in ammatory process, increase vascular permeability, and the appearance of new vessels 26,27,28,29 .
Osamu and collaborators studied the variation in VEGF concentrations in aqueous humor, before and after intravitreal injection of bevacizumab, in patients with proliferative diabetic retinopathy. The medications were performed one week before the patients were submitted to vitrectomy surgery through pars plana. The samples were collected before the injection and during the surgery. They concluded that there was a signi cant decrease in the concentration of vitreous VEGF, after one week of its administration 30 .
Another study also presented data on VEGF concentrations in patients with DR. The results suggested a signi cant relationship between VEGF levels and disease progression, showing its importance in the pathogenesis of diabetic retinopathy 31 .
VEGF was the molecule that showed the greatest variation in concentration levels, a decrease of approximately 5 times, in both groups (Figure 8).
Although we observed higher levels in patients with peripheral ischemia, the numbers were not statistically signi cant than those without peripheral retinal ischemia ( Table 1).
The expression of b-FGF is related to the time of ischemia that tissue is exposed to. Normally, its production is increased when there is a prolonged period of ischemia. One of its functions is to stimulate cell proliferation and induce angiogenesis 32 .
A study published in 2015 analyzed the levels of VEGF and b-FGF in patients with proliferative diabetic retinopathy treated with bevacizumab. We studied 68 eyes, divided into 2 groups. Group 1: received bevacizumab before vitrectomy, 5 days before, and 14 days before surgery. Group 2: patients with a macular hole without retinopathy and previous treatment with bevacizumab. They concluded that patients with proliferative diabetic retinopathy had higher concentrations of VEGF than those without the disease. When analyzing the concentrations of b-FGF, the results showed that those who underwent surgery later had high levels of b-FGF than other groups 33 .
In our study, b-FGF was only detected in only 3 patients in group 1 and none in group 2. We did not nd justi cations for having been detected only in group 1. In group 2, the duration of ischemia or neovascular condition presented may not have been su cient to induce the production of this cytokine (Table 1). Conclusion 1. In patients with diabetic macular edema, without peripheral retinal ischemia, detected by wide-angle uorescein angiography, treated with ranibizumab, there were statistically signi cant variations with an increase in their median levels in interleukin 6 and a decrease in VEGF levels. Interleukins 1, 8, 10, and 12 Informed consente: Informed consent was obtained from all individual participants included in the study. Table 1: Values of medians and standard deviation of cytokines in aqueous humor in the initial time of treatment (D1) and after treatment (D3), for the groups: non-ischemic and ischemic and values of the medians of the control group at baseline treatment. D1: First collection; D3: Third Collection. Study design.

Figure 2
Comparison of the variation in visual acuity in the non-ischemic and ischemic groups.

Figure 3
Page 16/19 OCT showing reduction of macular edema after treatment in Group 1.

Figure 4
Upon OCT examination, all patients presented a reduction in central macular thickness upon analysis of the macular thickness map, as illustrated in two patients ( Figure 4).

Figure 8
Variation of medians of VEGF before and after treatment in groups 1 and 2. D1: First collection; D3: Third Collection.