A total of 130 newborn C57BL/6 mice (Purchased from Shanghai SLAC Laboratory Animal Co.,Ltd) were used in the current study. All animal protocols were reviewed and approved by the institutional animal care committee of Zhejiang Chinese Medicine University and in accordance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research.
The mouse ROP model was established by Smith et al . Briefly, mice were placed into an oxygen-enrichment device (Hangzhou APU Instrument and Equipment Co., Ltd.) at postnatal day of life 7 (P7) with their nursing mothers. In the device, mice were exposed to 75 ± 0.5% O2 for 5 days (through P12), then were returned to room air and raised to 17 days (P17). Control animals were raised in normal room air for 17 days. Mice were further divided into a control group (n = 75) and an oxygen-exposed group (n = 75). Mice in both groups were supplied with standard mouse water and chow. Temperature and humidity were maintained at 25 °C and 75–80%, respectively. Day/light cycles of 12 hours were used. After the experiment, all mice were killed with carbon dioxide. The mice were placed in the container and carbon dioxide was injected into the container until the mice died.
Fluorescein Dextran Perfusion of the Retinal Blood Vessels
To observe the retinal vascular pattern, the perfusion in six mice (P17) was performed using fluorescein-conjugated dextran (MW = 2,000,000, Sigma, St. Louis, MO, USA) in phosphate buffered saline (PBS) [11, 12]. After injecting chloral hydrate in abdomen, the left ventricle of the heart was perfused with 0.3 mL fluorescein-conjugated dextran (50 mg / ml in 4% PBS). When the color of the mouse's lips turned yellow, the eyeballs were enucleated immediately and then placed in 4% paraformaldehyde for 3 h. The retina was removed and four radial cuts were performed to flatten the retina under a surgical microscope. The flat-mounted retinals were viewed and photographed by a Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
Hematoxylin and eosin (H&E)
On P17, 12 eyes of oxygen-exposed and control group mice were fixed immediately in 1% formaldehyde and 1.25% glutaraldehyde after enucleation for 48 h. Then, they were embedded in Optimum Cutting Temperature compound. Serial sections (5 µm thick) were cut through the cornea, parallel to the optic disc in a sagittal plane. The tissue sections were stained with hematoxylin and eosin (H&E). Under light microscopy, we observed the nuclei of the vascular endothelial cells of the neovascular vessels extending beyond the inner limiting membrane of the retina into the vitreal space, and we observed the thickness of retinal layers, as described by Smith and colleagues.10
Eyes of oxygen-exposed (four eyes) and control group (four eyes) mice were enucleated and fixed immediately in 2% glutaraldehyde and 4% paraformaldehyde on P17. Then the retinas were dissected and post-fixed in 2% aqueous osmium tetroxide for 1.5 h. After embedding in Epon, ultra-thin sections were cut at 1 µm and stained in uranyl acetate. The ultrastructures of retinal cells were observed under an electron microscope (Type 1400plus, Tokyo, Japan).
Quantitative real-time polymerase chain reaction (RT-qPCR)
Sixty eyes in each group, there are 10 time points, 6 eyes at each time point. Retinas from oxygen-exposed eyes and control eyes were dissected from the retinal pigment epithelial (RPE)-choroid at different time points (P8–P17). Total RNA of retinas was extracted using Trizol Reagent (Invitrogen, USA). Complementary DNAs (cDNAs) were synthesized with 4 µg of total RNA, according to the manufacturer’s protocol for the Reverse Transcription Kit (TaKaRa, Japan). Based on the sequences reported in the GenBank database (Beclin-1: 5’-ggaccaggaggaagctcagtacc-3’,5’-cgctgtgccagatgtggaagg-3’;ULK1:5’-cggaccaggcagacattgagaac-3’,5’-aggttggcagcaggtagtcagg-3’;Atg-5:5’-gcaagccaaggaggagaagattcc-3’,5’-gtgtctcagagaagcagtggtg-3’; β-actin:5’- gtgctatgttgctctagacttcg-3’,5’-atgccacaggattccatacc-3’.), Beclin1 (mammalian Atg6), Uncoordinated-51 like kinase 1 (ULK1), and Autophagy 5 (Atg5), and β-actin primers were designed, selected, and ordered from Sangon (China). Quantitative real-time polymerase chain reaction (RT-qPCR) was performed with an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). A typical reaction was performed in 20 µL, consisting of 1 µL cDNA, 10 µL 2 × SYBR Green PCR buffer, and primer pairs (final volume 20 µmol each). The PCR temperature cycle was performed for 3 min at 95.0 °C, followed by 48 cycles with primer annealing for 30 seconds at 60.0 °C, and extension for 30 seconds at 55.0 °C. ∆CT was calculated by subtracting the average threshold cycle (CT) of β-actin mRNA from the average CT of the target genes. All experiments were performed in duplicate. The comparative quantification values were obtained from the CT number at which the increase in signal was associated with an exponential growth of PCR products.
Western blot analysis
Total protein extract of retinas was obtained from mice eyes at P8–P17 (54 eyes in oxygen-exposed group and 54 eyes in control group), and protein concentrations were measured using a BCA Protein Assay kit (Beyotime, China). Samples containing 50 µg of protein were subjected to SDS-polyacrylamide gel electrophoresis gels (PAGE) using 10% and 15% polyacrylamide gel, then transferred onto a polyvinylidene difluoride membrane (Bio-Rad, USA). The membrane was blocked in 5% TBSA (10 mM Tris; pH 8.0, 150 mM NaCl, 0.5% Tween 20 and 5% fat-free dry milk) for 1 h at room temperature, then probed overnight at 4 °C with specific primary antibodies against Beclin-1, ULK1, Atg5, Bax, Bcl-2, and β-actin (1:1000; Cell Signaling Technology, USA), and incubated in blocking buffer with β-actin as an internal control. Immunoblots were then washed and incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, USA). Membranes were developed with the ECL substrate (Pierce, Thermo Scientific, USA). Images of labeled specimens were obtained with a ChemiDoc XRS imageing system (Bio-Rad Laboratories). Quantitation of bands were performed by densitometry using the Image Lab software (Bio-Rad Laboratories) and normalized to the expression of β-actin.
Statistical analyses were performed using SPSS 18 (SPSS Inc., Chicago, IL, USA). Each group of animals was studied as an independent unit of analysis. Student’s independent t-test was used to determine the significance of differences between the groups. Statistical significance was defined as a p value < 0.05.