Aqueous humor samples were collected at the Department of Ophthalmology, Konkuk University Medical Center, Seoul, Korea. From July 1, 2013, to January 31, 2015, 44 eyes of 39 patients with DME and 27 eyes of 27 controls were enrolled in this study. All 39 patients had been untreated (i.e., they had not received any treatment for DR except retinal laser photocoagulation at least 3 months before their inclusion in the study) and had experienced recent onset of visual symptoms (<3 months). None of these patients had uncontrolled systemic diseases, such as ischemic heart diseases, chronic kidney diseases, or systemic inflammatory diseases. Eyes with the following characteristics were excluded: DME with epiretinal membrane or foveal traction, severe foveal hard exudate infiltration, vitreous hemorrhage or pre- and sub-retinal hemorrhage, AMD, or a history of uveitis. Aqueous humor samples from patients undergoing cataract surgery were used as control samples. Except for cataracts, the control subjects did not have diabetes or uncontrolled systemic diseases or any ophthalmic diseases. Aqueous humor samples from 44 eyes with DME were obtained immediately before performing the first intravitreal injection of bevacizumab (IVB, 1.25 mg/0.05 mL), and AH samples from 27 control subjects were obtained immediately before cataract surgery. These samples were used for ELISA of the DKK-3 concentration.
All sample collections and intravitreal injections were performed using standard sterile methods. Aqueous humor samples were obtained via anterior chamber paracentesis through a 30-G needle, and no complications occurred. Aqueous humor samples (∼50–100 μL) were extracted into Safe-Lock microcentrifuge tubes (1.5 mL), immediately frozen at −80°C and then stored until ELISA. This study followed the guidelines of the Declaration of Helsinki, and informed written consent was obtained from all patients and control subjects. The procedure for AH collection was approved by the institutional review boards of Konkuk University Medical Center, Seoul, Korea.