Study Subjects

Inclusion criteria were 1) central foveal thickness (CFT) that was 250 μm or greater by optical coherence tomography (OCT), 2) type 2 diabetes, 3) clinically significant macular edema, and 4) DR. Exclusion criteria were 1) previous vitreous surgery, 2) previous retinal photocoagulation, 3) previous intravitreal injection of triamcinolone acetonide or bevacizumab, 4) vitreoretinal pathology other than diabetic retinopathy, and 5) signal strength (max 10) in OCT image less than 5.

Eleven consecutive patients with bilateral DME were recruited as a DME group at the eye center, Gachon University Gil Hospital, from June 3, 2008 through March 25, 2009. The control group was composed of 6 patients who had undergone cataract surgery without other ocular or systemic diseases.

Before and after the treatment, all patients underwent a comprehensive ophthalmic examination that included best-corrected visual acuity (BCVA) by Snellen chart recording, intraocular pressure (IOP) measurement, slit-lamp examination, fundus examination, and CFT measurement. The CFT measurements were taken with the STRATUS OCT3 version 4.0 software (Carl Zeiss Meditec, Dublin, California, USA) using the fast macular scan protocol. This protocol consists of 6 line scans that are 6.0 mm long, centered on fixation, and spaced 30 degrees apart around the circumference of a circle. The foveal or central 1-mm thickness is an average generated value based on central 21 scans of each of the 6 lines that pass through the patient’s fixation.

Intravitreal Injections

Intravitreal injections of 4 mg/0.1 mL triamcinolone acetonide (Kenalog; Dongkwang Pharm, Seoul, Korea) or 1.25 mg/0.05 mL bevacizumab (Avastin; Genentech, Inc, San Francisco, California, USA) were done with the generally used concentration within 7 days of baseline.

In each DME patient, the eye with thicker foveal thickness was defined as the primary eye and the other eye as the secondary eye. To make possible equal conditions of macular edema between bevacizumab- and triamcinolone-injected groups, bevacizumab and triamcinolone were injected into the primary eyes of consecutive patients by turn. For example, in the first patient, bevacizumab was injected into the primary eye and triamcinolone in the secondary eye in 1 patient, and subsequently in the next patient, triamcinolone was injected in the primary eye and bevacizumab in the secondary eye. Intravitreal injections for the second eye were performed within a week of the primary eye. Eyes assigned to intravitreal triamcinolone acetonide constituted the IVTA group and those assigned to bevacizumab constituted the IVBe group.

All injections were performed by a retinal specialist (H.J.S.) using topical anesthesia. Under sterile condition in the operation room, triamcinolone or bevacizumab was injected via 30-gauge needle through the pars plana at 3.5 mm posterior to the limbus. For the 2 weeks after injection, levofloxacin (Cravit; Santen, Osaka, Japan) ophthalmic solution was administered 4 times a day.

Sample Collection

Undiluted aqueous humor samples (50-100 μL) were obtained just before an intravitreal injection of triamcinolone acetonide or bevacizumab and then 4 weeks after the injection in the DME group, and at the beginning of cataract surgery in the control group. All sample collections were performed by 3 physicians (H.J.S., D.Y.L., K.H.K.) under sterile conditions in the operating room. Aqueous humor was withdrawn through a limbal paracentesis site using a 30-gauge needle with a tuberculin syringe. Special care was taken to avoid touching intraocular tissues and to prevent mixing of aqueous samples with other fluids. The specimens were immediately transferred to a sterile plastic tube and stored at -70 C until assayed.

Measurement of Cytokines Using Multiplex Analysis

IL-6, IL-8, IP-10, MCP-1, PDGF-AA, and VEGF were measured in aqueous samples by the Luminex 100 multiplex array assay (Luminex Corporation, Austin, Texas, USA). This technique uses microspheres that are 5.5 μm in diameter as a vehicle for molecular reactions. The microspheres, which are available in 100 distinct sets, have a unique spectral domain by virtue of the unique orange/red emission profile of each set. Antibody specifically directed against the mediator of interest was covalently coupled to a set of color-coded microspheres. Antibody-conjugated microspheres are allowed to react with the sample (eg, the aqueous humor) and then with a biotinylated detection antibody (in principle, similar to a sandwich ELISA). The reaction mixture is detected by the addition of streptavidin-phycoerythrin, which binds to the biotinylated detection antibodies. The assay solution is drawn into the Luminex reader, which illuminates and reads the sample.

In the measurement of 7 factors in aqueous humor, instructions from the manufacturer were followed strictly (Lincoplex cytokine/chemokine multiplex kit, #HCYTO-60K; Millipore Corporation, Billerica, Massachusetts, USA). In brief, the desired number of wells on a 96-filter plate was blocked by pipetting 200 μL of the Lincoplex assay buffer and sealed and mixed on a plate shaker for 10 minutes at room temperature (20-25 C). The plate shaker was set at a speed of approximately 500 to 800 rpm. The buffer was removed by vacuum filtration. A serial dilution of standards (3.2-10 000 pg/mL) was used for processing a standard curve for each cytokine. The assay buffer (25 μL) was added to the sample plate and the 0 standard (background). Each standard or control (25 μL) was pipetted into the appropriate wells and the appropriate matrix diluent (25 μL) was added to the background, standards, and control wells. Aqueous humor (25 μL) was added to the appropriate wells. Mixed beads containing antibody-immobilized beads were added to each well and the plate was sealed and covered with aluminum foil, and incubated with agitation on a plate shaker for 1 hour at room temperature (20-25 C). Fluid was gently removed by vacuum and the plate was twice washed and vacuum-filtrated with 200 μL of Lincoplex wash buffer. Next, 25 μL of Lincoplex detection antibody cocktail was added to each well and the plate was sealed and covered with aluminum foil, and incubated with agitation on a plate shaker for 30 minutes at room temperature (20-25 C). Then, 25 μL of streptavidin-phycoerythrin solution was added to each well containing the 25 μL of Lincoplex detection antibody cocktail and the plate was sealed and covered with aluminum foil and incubated with agitation on a plate shaker for 30 minutes at room temperature (20-25 C). The samples/standards were gently removed by vacuum filtration, and were twice washed and vacuum-filtered with 200 μL/well of wash buffer. Then, 100 μL of sheath fluid was added to all wells and the plate was covered with aluminum foil and the beads in each well were resuspended on a plate shaker for 5 minutes. Next, the plate was run on a Luminex instrument. The median data using a 5-parameter logistic or spline curve-fitting method were saved and evaluated for calculation of the cytokine/chemokine concentrations in the samples.

Statistical Analysis

All statistical analyses were carried out using SPSS 12.0 for Windows (SPSS, Inc, Chicago, Illinois, USA) by physician authors (H.J.S., D.H.H., D.H.N.). The aqueous levels of IL-6, IL-8, IP-10, MCP-1, PDGF-AA, and VEGF were expressed as a mean with standard deviation (SD). To analyze the statistical differences, the Wilcoxon signed rank test was used between preinjection and postinjection clinical data, and the Mann-Whitney U test was used both between control and DME groups and between IVTA and IVBe groups. A P value < .05 was judged as statistically significant.