All experimental procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the experimental protocol was approved by the experimental animal ethics committee of Xiamen University (Xiamen, China). A total of 60 Sprague-Dawley rats (200–250 g) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). The rats were maintained on a 12-h light-dark cycle and were dark-adapted for at least 2 h prior to experiments. The animals had ad libitum access to food (standard lab chow) and water. The rats were injected intraperitoneally with chloral hydrate (10 mg/100 g; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) to ensure the animals remained immobile. Pupil dilatation was achieved using 0.5% tropicamide (Alcon, Fort Worth, TX, USA). Following topical administration of 0.5% proparacaine (Alcon), the anterior chamber was cannulated with a 7-scalp acupuncture, which was connected to a container carrying 500 ml sterile normal saline. The IOP was increased to 110 mmHg by elevating the saline reservoir to 150 cm above the eye for 60 min. The body temperature of the rat was maintained at 37°C with a blanket. The opposite eye of each animal served as the normal control. The animals were sacrificed intraperitoneally with chloral hydrate (20 mg/100 g) 12 h, 24 h, 2, 3 or 7 days (n=10/group) following termination of the increase in IOP.

The rats (n=3/group) were anesthetized with an intraperitoneal injection of chloral hydrate (10 mg/100 g) and perfused intracardially with cold 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd.) in 0.1 mol/l phosphate-buffered saline (PBS; pH 7.4). The eyes were immediately enucleated and the globes were postfixed in 4% paraformaldehyde in 0.1 mol/l PBS for 2 h at 4°C. The cornea and the lens of the eye were removed, and the remaining eye cups were placed in the same fixative overnight. Prior to embedding in paraffin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the eye cups were immersed in 70%, 95% and 100% ethyl alcohol in series, and then embedded with paraffin. Paraffin sections of 5-µm thickness were then cut with a semi-motorized rotary microtome (RM2245; Leica, Germany). To assess the end-stage apoptosis of the tissue, in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed on the retinal tissues using an assay kit (DeadEnd Fluorometric TUNEL system G3250; Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. The cellular nuclei were stained with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA), and the apoptotic cells were examined under a laser confocal microscope (Fluoview 1000; Olympus, Tokyo, Japan). The cellular nuclei and apoptotic cells were counted in three sections from each sample. As a positive control, sections were incubated in DNase I (0.5 µg/ml) prior to addition of the equilibration buffer. As a negative control, distilled water was used instead of the TdT reaction mixture.

Retrograde staining of the RGCs of the two eyes was achieved by injecting a fluorescent dye into the superior colliculus bilaterally. The rats (n=3/group) were placed in a stereotactic apparatus (RWD Life Science Co. Ltd., Shenzhen, China), following intraperitoneal injection of 10 mg/100 g chloral hydrate to ensure the animals remained immobile and the skin of the skull was incised. The brain surface was exposed by perforating the parietal bone with a dental drill to facilitate dye injection. Fluorogold (FG; Fluorochrome LLC, Denver, CO, USA) was injected (4%; 3.0 µl each) at a point 5.00 mm caudal to the bregma and 1.00 mm lateral to the midline on the two sides, to a depth of 5.00 mm from the surface of the skull.

Subsequently, 10 days after the injection of FG into the superior colliculus, the eyes were enucleated following sacrifice of the animals with an overdose of intraperitoneal 10% chloral hydrate. The eyes were fixed in 4% paraformaldehyde in PBS for 1 h in the dark at 4°C. The anterior segments were removed and the eye cups were fixed in 4% paraformaldehyde/PBS for 30 min in the same conditions. A total of four radial cuts were made in the periphery of the eye cup, and the retina was carefully separated from the retinal pigment epithelium. A small cut was also made in the peripheral corner of the superior retinal portion in order to correctly identify retinal orientation.

The retina were then flat mounted on a glass slide and preserved in the dark at 4°C. The retinal flat mounts were viewed using a fluorescence microscope (Leica DM2500; Leica Microsystems GmbH, Wetzlar, Germany; magnification, ×100). The FG-labeled RGCs were manually counted in a blinded-manner, by another investigator, in each quadrant at a distance of 1 mm radial from the optic nerve. The total quantity of RGCs per mm² in all four quadrants were calculated. Cells with an irregular shape, intense dye staining, or a smaller or larger size than typical RGCs were considered to be non-RGC cells, including microglia.

Total RNA was extracted from the retina using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Reverse transcription was performed using Oligo 18T primers and RT reagents (Takara Bio Inc., Shiga, Japan), according to the manufacturer's instructions. RT-qPCR was performed with mRNA-specific primers. The following primer sequences were used: LIF, forward 5′-tcaactggctcaactcaacg-3′ and reverse 5′-aaaggtgggaaatccgtcat-3′; and LIFR, forward 5′-gctgacttctcgacctccac-3′ and reverse 5′-ccagttccagtggtgacctt-3′. The qPCR reactions were performed on the StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq (Takara Bio Inc.) at 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 57°C for 30 sec and 75°C for 10 sec. Melt curve analysis was performed immediately between 65°C and 95°C. All reactions were performed in triplicate and average threshold cycle (Ct) values >35 were considered to be negative.

The total protein from the retina at different time-points was extracted using cold radioimmunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was determined with a bicinchoninic acid protein assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal quantities (3 mg/ml; 10 µl) of proteins extracted from the lysates were subjected to electrophoresis on 8% or 9% SDS-PAGE and then electrophoretically transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking for 1 h in 2% bovine serum albumin, the membranes were incubated with primary antibodies against LIF (1:200; rabbit polyclonal anti-rat; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), LIFR (1:200; rabbit polyclonal anti-rat; Santa Cruz Biotechnology, Inc.), STAT3 (1:200, rabbit polyclonal anti-rat; Santa Cruz Biotechnology, Inc.), P-STAT3 (1:200; rabbit polyclonal anti-rat; Santa Cruz Biotechnology, Inc.), AKT (1:2,000; rabbit polyclonal anti-rat; Cell Signaling Technology, Danvers, MA, USA), p-AKT (1:1,000; rabbit polyclonal anti-rat; Cell Signaling Technology), ERK (1:1,000; rabbit polyclonal anti-rat; Cell Signaling Technology), p-ERK (1;1,000; rabbit polyclonal anti-rat; Cell Signaling Technology) and β-actin (1:10,000; Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. Following three washes with Tris-buffered saline containing 0.05% Tween-20 for 10 min each, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 1 h at room temperature. The specific bands were visualized using enhanced chemiluminescence reagents (Lulong Inc., Xiamen, China) and recorded using a transilluminator (ChemiDoc XRS, Bio-Rad Laboratories, Inc.). The data were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). All experiments were performed at least three times with similar results.

All data are expressed as the mean ± standard error of the mean and were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test with SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To assess the changes in retinal histopathology following acute ocular hypertension, hematoxylin and eosin staining was performed on the tissues. At 12 h, 24 h, 2, 3 and 7 days post-reperfusion, the thickness of the inner nuclear layer (INL) and the inner plexiform layer (IPL) gradually decreased, compared with the normal retina. The cell arrangement of the RGCs and the INL was irregular, with a significant reduction in the number of RGCs (Fig. 1). At 7 days post-reperfusion, cytoplasmic vacuolation occurred in the RGCs and cells were absent in the IPL (Fig. 1).

TUNEL staining of normal retinas and retinas at 12 h, 24 h, 2, 3 or 7 days post-reperfusion after 60 min acute ocular hypertension is shown in Fig. 2. TUNEL-positive cells were absent in the normal retina (Fig. 2A), but were observed in the outer nuclear layer (ONL) and the INL in the group subjected to acute ocular hypertension at 12 h post-reperfusion (Fig. 2B–F), and the number of apoptotic cells peaked 24 h after reperfusion.

The results of the FG retrograde staining of RGCs are shown in Fig. 3. Following acute ocular hypertension, significant reductions in the numbers of RGCs were found at 24 h, 2, 3 and 7 days post-retinal reperfusion.

Western blot analysis was performed to assess the protein levels of LIF and LIFR, and RT-qPCR was performed to determine the mRNA expression levels of LIF and LIFR following acute ocular hypertension in the rat retina (Fig. 4). The western blot analysis demonstrated that the protein expression of LIF was significantly increased at 12 h, 24 h, 2, 3 and 7 days, and peaked 12 h after the IOP increase was terminated (Fig. 4A and B). The protein level of LIFR was also significantly higher, compared with the normal control at 12 h post-retinal reperfusion, and peaking 3 days after reperfusion (Fig. 4C and D). The mRNA expression levels of LIF and LIFR revealed the same patterns as the protein expression of LIF and LIFR in the rat retina following acute ocular hypertension (Fig. 4E and F).

Activation of the downstream pathways of LIF were assessed by investigating the expression levels of the phosphorylated forms of STAT3, Akt and ERK (Fig. 5). The results revealed that, at 12 h post-retinal reperfusion, the levels of P-STAT3 were significantly higher than that in the normal retina, and the highest levels of P-STAT3 were observed 24 h after retinal reperfusion (Fig. 5A and B). Upregulation in the phosphorylation of Akt was also observed, which peaked 12 h after retinal reperfusion (Fig. 5A and C). In contrast to P-STAT3 and P-Akt, the level of P-ERK1/2 was reduced from 12 h after retinal reperfusion in the rat retina (Fig. 5A and D).