The experimental protocol was approved from the Institutional Animal Care and Use Committee of Seoul National University Hospital Biomedical Research Institute (Seoul, Korea) and followed the guidelines stated by ARVO for ophthalmic and vision research.

Eight-week-old male BALB/c mice (KOATECH, Pyeongtaek, Korea) were used for the study. Under anesthesia with intraperitoneal injection of zolazepam-tiletamine (Zoletil®, Virbac, Carros, France) and topical administration of 0.5% proparacaine hydrochloride ophthalmic solution (Hanmi Pharm Co., Ltd., Seoul, Korea), the cornea was marked using a 2-mm-diameter trephine, and three 10 − 0 nylon sutures were evenly placed 120° apart from each other, through the epithelial and stromal layers, and with both points of each stitch going in and out of the cornea along the mark. The knots were left unburied.

Immediately after the placement of corneal sutures, either 200 µg aflibercept (5 µL) (Eylea®, Regeneron Pharmaceuticals, Inc.) or the same volume of phosphate-buffered saline (PBS) was injected subconjunctivally using a 33-gauge needle (Hamilton, Reno, NV). Seven days later, the mice were subjected to assays.

Corneal new vessels were examined under slit-lamp biomicroscopy and photographed with a camera mounted on a microscope. The extent of corneal neovascularization was graded independently by two individuals (C.H.Y. and J.Y.O.) in a blinded manner using the standardized scale system: the growth of corneal new vessels was graded from 0 to 3 in each quadrant of the cornea, and scores for each quadrant were summed to obtain the clinical score (range 0 to 12) for each eye [19,20,21].

The corneas were extracted and fixed in 4% paraformaldehyde overnight at 4˚C. After washing with PBS, the tissues were treated with proteinase K for 5 min at room temperature, followed by treatment with methanol for 30 min. After PBS washing, the tissues were blocked with 2% BSA overnight at 4˚C, and then were incubated with primary antibodies against CD31 (BD Pharmingen, San Diego, CA), CD11b (BD Pharmingen), VEGFR-2 (R&D Systems, Minneapolis, MN), or VEGFR-3 (R&D Systems) for 16 h at 4˚C. The next day, the corneas were incubated with secondary antibodies overnight at 4˚C in the dark. The stained corneas were flat-mounted onto slides and examined under a fluorescence microscope (Nikon, Tokyo, Japan).

For quantification of the CD31-stained area, the digital fluorescence images of corneal flat mounts were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) [20, 22]. The total area of the cornea was manually delineated by outlining the innermost vessels of the limbal arcade and removing areas outside the delineated margin. After background subtraction and thresholding, the percentage of CD31+ area out of the total corneal area was calculated.

For quantification of VEGFR-2+CD11b+ or VEGFR-3+CD11b+ myeloid cells in the cornea, the numbers of VEGFR-2+CD11b+ and VEGFR-3+CD11b+ cells were counted in the regions adjacent to the limbus along the sutures of the cornea under a fluorescence microscope, at a magnification of X400. For each quantification, the corneal stroma with approximate size of 280 μm X 450 μm was covered.

The corneal tissue was dissected into small pieces using microscissors, lysed in RNA isolation reagent (RNA Bee, Tel-Test, Friendswood, TX), and homogenized using an ultrasound sonicator (Ultrasonic Processor, Cole Parmer Instruments, Vernon Hills, IL). Total RNA was extracted from the lysates using the RNeasy Mini kit (Qiagen, Valencia, CA) and converted to first-strand cDNA by reverse transcription using the High Capacity RNA-to-cDNA™ Kit (Applied Biosystems, Carlsbad, CA). RT-qPCR amplification was performed using TaqMan® Universal PCR Master Mix (Applied Biosystems) and specific TaqMan® probe sets for Cd31, Vegfc, Angpt1 (angiopoietin-1), Vegfa, Tek/Tie2, Mrc1 (mannose receptor C-type 1), Mrc2, and Il6 (all from Applied Biosystems) in an automated instrument (ABI 7500 Real Time PCR System, Applied Biosystems). Assay ID for each TaqMan®probe was as follows: Mm01242576_m1 for Cd31; Mm00456503_m1 for Angpt1; Mm00437306_m1 for Vegfa; Mm00437310_m1 for Vegfc; Mm00443243_m1 for Tek/Tie2; Mm00485148_m1 for Mrc1; Mm00485184_m1 for Mrc2; Mm00446190_m1 for Il6. The data obtained were normalized to Gapdh (Mm99999915_g1) and expressed as fold changes relative to the controls.

Whole blood was collected using a 26-gauge needle with a heparin-precoated syringe via cardiac puncture and preserved in EDTA tube. The collected blood was treated with red blood cell lysis buffer (BD Biosciences, San Diego, CA) for 5 min and centrifuged at 2,000 rpm for 10 min. Ocular draining cervical lymph nodes (DLNs) were minced into small pieces between the frosted ends of two glass slides in RPMI 1640 medium (WelGENE, Daegu, Korea) containing 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin-streptomycin (Gibco) on ice. The resultant single-cell suspension from the blood and DLNs were stained with fluorescence-conjugated antibodies against CD11b-FITC, VEGFR-2-APC, and VEGFR-3-PE (all from eBioscience, San Diego, CA) for 30 min at 4˚C. The stained cells were analyzed using the S1000EXi Flow Cytometer (Stratedigm, San Jose, CA), and the obtained data were analyzed using the FlowJo program (Tree Star, Ashland, OR).

Prism software (GraphPad, San Diego, CA) was used for statistical tests and generation of graphs. The Shapiro-Wilk test or Kolmogorov-Smirnov test was used to determine a normal distribution of data in each group. The unpaired t-test or Mann-Whitney test was used for comparisons of mean values between two groups. One-way ANOVA followed by Tukey’s Honestly Significant Difference test or Kruskal–Wallis test followed by Dunn’s multiple-comparisons test was employed for comparisons of the means between more than two groups. The data were presented as mean ± SD. Differences were considered significant at p < 0.05.