Subjects
This prospective consecutive randomized clinical study was conducted in the Eye Hospital of Wenzhou Medical University from July 2018 to December 2018, approved by the Institutional Review Board of the Affiliated Eye Hospital of Wenzhou Medical University (No. KYK[2018]24), and has been retrospectively registered in the Chinese Clinical Trial Registry (No.
ChiCTR1900028374). The whole procedure adheres to CONSORT guidelines with a complete CONSORT checklist as an additional file. The written informed consent was obtained from all subjects before joining in the project.
Patients that were diagnosed with age-related cataract with or without type 2 diabetes and had the intention to fulfill the bilateral cataract surgeries in a short period were included, while the ones with a history of previous eye surgery, trauma, high intraocular pressure, glaucoma or shallow anterior chamber, high myopia, proliferative diabetic retinopathy or other retinal diseases were excluded. Similarly, eyes that failed to obtain > 100 μl aqueous humor (AH) or encountered intraopertative complications such as posterior capsule rupture and endothelial injury were excluded. Patients with or without type 2 diabetes were randomly assigned to 2 groups respectively: 1-day or 1-week surgical interval for the two eyes, so 4 groups were included in this study. The patient enrollment was conducted by two senior doctors, and the random sequence was determined by an assistant using the random number table.
All the cataract surgeries were fulfilled by the same surgeon (XH.G.). Aqueous humor samples (100–200 μl) were collected by inserting a 26-gauge needle into the anterior chamber before starting the surgery, and were immediately stored within a − 80 °C refrigerator. A standard surgical protocol included: a 2.0 mm clear corneal incision, standard phacoemulsification procedure, and a foldable IOL implantation. Postoperative medication is standardized using topical antibiotic-corticosteroid combination eye drops (Tobramycin 0.3%/ dexamethasone 0.1%; Alcon Laboratories, Fort Worth, Texas, USA) 4 times daily for 1-week. The eye drops were tapered and discontinued over 3 weeks. Follow-up visits were scheduled on 1-day, 1-week, and 1 and 3 months intervals postoperatively.
Magnetic Luminex assays
We used magnetic Luminex assays (Human Premixed Multi-Analyte Kit, R&D Systems, Inc., Minneapolis, MN, USA) to analyze 10 selected proteins simultaneously, including IL-1β, IL-1ra,IL-6, IL-8, IL-10, MCP-1, VEGF-A, MIP-1a, interferon-inducible protein 10 (IP-10) and RANTES. Analyte-specific antibodies were pre-coated onto color-coded magnetic microparticles. A total of 102 AH samples were drawn from both eyes of the 29 ARC patients and 22 diabetic patients, 50 μl AH was pipetted into the wells and the immobilized antibodies binded the specific proteins. After washing away the unbound substances, a biotinylated antibody cocktail was added to each well. Following a wash, streptavidin-phycoerythrin conjugate was added, then after a final wash, the microparticles were detected using the FLEXMAP 3D® Analyzer. Densitometric analysis of each spot was then performed using a xPONENT® software (Luminex® Corporation).
Elisa
The SP concentrations of the AH samples were tested by the Human SP ELISA kits (ab133029, Abcam, MA, USA). Specifically, 50 μl AH sample or standards were added to the wells, along with an alkaline phosphatase (AP) conjugated-SP antigen and a polyclonal rabbit antibody specific to SP. The plate was incubated at room temperature for 2 h, then after a wash, the pNpp substrate was added. After a incubation of 1 h at room temperature, stop solution was added and the plate was read immediately at 405 nm. The optical density was inversely proportional to the amount of SP captured in the plate.
Statistical analysis
SPSS software version 20.0 (SPSS Inc., Chicago, IL) was used. Comparisons of both eyes of the same patients utilized the paired t test to determine if the data is of normal distribution, or otherwise the Wilcoxon signed rank test was used with the Bonferroni correction. Comparisons of the differences (the second surgical eye minus the first one) between the ARC patients and diabetic patients were conducted using the independent t test to determine if the data is of normal distribution, or otherwise the Mann-Whitney U rank test was utilized. Two-tailed P < 0.05 were considered to indicate a statistically significant difference.