The suture-induced mouse model of corneal senescence. Balb/c mice (male, 6–8 weeks; Beijing Pharmacology Institute, Beijing, China) were used for the study. All animal experiments were performed according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and approved by the ethics committee of Shandong Eye Institute, and all animal experiments were performed accordance with ARRIVE guidelines. After systemic and topical anesthesia, five interrupted stitches of the 11 − 0 polypropylene suture (Mani, Togichi, Japan) were placed in the central, superior, inferior, nasal, and temporal cornea, respectively. Ofloxacin eye ointments were administered immediately after the injury and once daily for 1 week to avoid infection. Only one eye was operated in each mouse for all experiments. The sutures were removed on day 14. At 12 h, and 3, 5, 7, 14, and 21 days after the operation, corneal edema, neovascularization, and scarring were observed under a slit lamp microscope with a digital camera.
Cell culture and senescence induction. New Zealand white rabbits (male, 2–4 months; Kangda, Qingdao, Shandong, China) were used for corneal fibroblasts culture. The peripheral and central corneal tissues were isolated using a trephine of 8 mm and 5 mm in diameter, respectively. The corneal epithelium and endothelium were digested with 2.4 U/ml dispase II (Roche, Basel, Switzerland) overnight at 4°C. The corneal stroma was cut into pieces and incubated with 2 mg/ml collagenase I (Invitrogen, Carlsbad, CA) for 2–4 hours at 37°C. Then the cells were collected and cultured in DMEM/F-12 medium supplemented with 10% FBS. A hydrogen peroxide (H2O2)-induced in vitro model of senescent corneal fibroblasts was established as previously described.6 Briefly, the cells were exposed three times to 200 µM H2O2 for 1 h daily. After each treatment, the cells were washed with PBS and incubated in complete medium for 24 h.
Senescent cell assay. The mouse eyeballs were embedded in the Tissue-Tek optimum cutting temperature (OCT) compound, and 8-µm sections were made (n = 5). The cryosections were fixed with ice methanol at -20°C for 10 min. The rabbit keratocytes were fixed with 4% paraformaldehyde at room temperature for 10 min. Cellular senescence was identified by SA-β-gal staining (Beyotime, Haimen, China). In brief, the samples were washed with PBS, fixed at room temperature for 15 min, and then washed 2–3 times again, before they were incubated in the SA-β-gal staining solution (pH 6.0) overnight at 37°C and observed under a Nikon microscope.
Real-time RT-PCR. Total RNA was extracted from the H2O2-treated peripheral and central rabbit corneal fibroblasts using Nucleospin RNA kits (BD Biosciences, Palo Alto, CA). cDNA was acquired from total RNA using the first strand cDNA synthesis kit (TaKaRa, Dalian, China). The real-time PCR analysis was then performed with the SYBR Green PCR reagent (Invitrogen, Carlsbad, CA) in an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The cycling system was an initial denaturation cycle at 95°C for 10 sec, followed by 45 cycles at 95°C for 15 sec and 60°C for 1 min. The nucleotide sequences of primers used in this assay are listed in Table 1, and GAPDH was used as an endogenous control gene.
Statistical analysis. Data in this study were representative of more than three different experiments and were presented as mean ± SEM. The differences between control and treated groups were compared with the Student’s t test. P < 0.05 was considered significant.