Patients with naive ICNV were recruited from the Eye Center, the Second Affiliated Hospital，Zhejiang University School of Medicine between May 2017 and March 2018. Inclusion criteria were: newly diagnosed active ICNV undergoing first-time ranibizumab treatment, and age less than 50 years at the time of diagnosis. Diagnosis of ICNV was based on funduscopy, spectral domain optic coherence tomography (SD-OCT, Cirrus OCT; Carl Zeiss Meditec, Dublin, CA, USA) and fundus fluorescein angiography (FFA, Heidelberg Engineering HRA Spectralis, Heidelberg, Germany).
All eyes underwent a full ophthalmological examination at baseline, including best corrected visual acuity (BCVA) testing using a Snellen chart, intraocular pressure measurement, ophthalmoscopic fundus examination, OCT and FFA. For statistical analysis, BCVA were converted to logarithm of the minimum angle of resolution (logMAR) units. Central retinal thickness (CRT, thickness of the 1-mm central retina) was measured by OCT; lesion area，including dye leakage area, pigment epithelial detachment, subretinal hemorrhage, and hyperfluorescent staining of fibrous tissue, was measured on fluorescein angiograms, as described previously. Our exclusion criteria are as follows: (1) clinical features suggesting that CNV was secondary to other causes such as AMD, PM, trauma, choroiditis, or hereditary diseases in the studied or contralateral eye; (2) axial length >26.0 mm or myopia> -6 diopter; (3) prior local or systemic treatment for ICNV, including intravitreal drug injection, photodynamic therapy, or steroids; (4) previous intraocular surgery; (5) systemic inflammatory diseases such as autoimmune disease, diabetes mellitus or malignoma.
For comparison, 30 healthy volunteers with no evidence of active ocular or systemic disease were recruited as a control group. Blood samples were collected aseptically, and serum was prepared and stored at -80°C until cytokine analysis. For patients with ICNV, blood samples were collected before FFA or ranibizumab treatment.
The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital，Zhejiang University School of Medicine, and the research followed the tenets of the Declaration of Helsinki. Written consent was obtained from all patients and controls.
Cytokine determination in serum samples
The Bio-Plex multiplex assay (Bio-Plex Human Cytokine 7-plex panel; Bio-Rad, Hercules, CA, USA) and the multiplex bead analysis system (Bio-Plex Suspension Array System; Bio-Rad) were used simultaneously to measure serum levels of IL-2, IL-10, IL-15, IL-17, basic FGF, GM-CSF, and VEGF as in a previous study. The levels of serum cytokines were set to 0 if the levels were below detection.
Statistical analyses were performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). The χ2 test or Fisher exact test was used to compare categorical variables. The unpaired t test or Mann-Whitney U test was used to determine the differences between two groups and the Spearman’s correlation analysis was used to evaluate the relationship between numerical data. P < 0.05 was considered to be statistically significant.