The protocol of the present study was approved by the Experimental Animal Ethics Committee of Xiamen University, School of Medicine (Xiamen, China; approval no. 20150306155209) and followed the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research (https://www.arvo.org/About/policies/arvo-statement-for-the-use-of-animals-in-ophthalmic-and-vision-research/). Adult male Sprague-Dawley rats (aged 8-12 weeks old; weight 250±30 g; n=140) were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. The rats were maintained on a 12-h light-dark cycle (~20˚C; humidity ~50%) and were dark-adapted for ≥2 h before any experiments. All animals had access to food (standard lab chow) and water ad libitum. All efforts were made to minimize the number of animals used and their suffering. Prior to AOH induction and drug injection procedures, deep anesthesia was induced by an intraperitoneal injection of pentobarbital sodium (30 mg/kg; Sinopharm Chemical Reagent Co., Ltd.).

The experimental procedure has previously been described (35). Briefly, following the topical administration of 0.5% proparacaine (Alcon), the eye pupil was dilated with 0.5% tropicamide (Alcon). Under a Spot OPMI 11 operation microscope (Carl Zeiss AG), the anterior chamber of the eye was cannulated by 7-scalp infusion acupuncture needle connected to a 500-ml container of sterile saline. Only the right eyes were chosen for the experiment; the left eyes remained untouched and served as control. The IOP was then elevated to ~110 mmHg by raising the height of the saline container to 150 cm above the eye for 60 min. The infusion needle was then removed before further experiments on the rats were performed.

After the needle was removed and AOH was ceased, intravitreal injection of 1 µg/µl LIF was conducted immediately. Topical anesthesia by 0.5% proparacaine eyedrops (Alcon Inc.) was performed as needed. A total volume of 5 µl LIF was injected into the vitreous through the par plana using a 33G microsyringe (Hamilton Co.). For the untreated AOH group, a dose of 5 µl PBS was injected instead of LIF solution.

In another set of experiments, to test the involvement of STAT3 and the PI3K/AKT/mTOR signaling pathway in the effect induced by LIF injection, the pretreatment of intravitreal injection through the par plana of either the STAT3 inhibitor C188-9 (10 mM; Selleck Chemicals) or the PI3K/AKT/mTOR inhibitor LY3023414 (50 nM; Selleck Chemicals), was conducted 3 h prior to LIF injection (~2 h before AOH induction). A dose of 5 µl PBS was used as a control. C188-9, LY3023414 and PBS injections were all performed under topical anesthesia by 0.5% proparacaine and general anesthesia by pentobarbital sodium (30 mg/kg).

One day or 3 days after AOH induction, rats were sacrificed by anesthetic overdose with an intraperitoneal injection of pentobarbital sodium (150 mg/kg), and the eyeball was immediately enucleated and frozen (-20˚C) in the optimal cutting temperature compound (Sakura Finetek Japan Co., Ltd.). The eyeball was sectioned along the meridian to a thickness of 10 mm to assess the histological changes in the anterior part of the eyes. Tissue preparations were stained with hematoxylin (3 min) and eosin (1 min) at room temperature and viewed under a light optical microscope (Nikon Co., cTokyo, Japan). The retinal thickness was measured using Image-Pro Plus 6.0 (Media Cybernetics) and the data were proceeded for statistical analysis.

RGCs were retrogradely labeled with Fluoro-Gold™ (Fluorochrome, LLC) 7 days before the induction of AOH. The procedures were described in our previous reports (9,31). Briefly, the rats were deeply anesthetized with 30 mg/kg pentobarbital sodium (i.p.) and placed in a prone position on the stereotaxic apparatus (RWD Life Science Co. Ltd.). RGC labeling of both eyes was conducted by injecting 4% FG into the superior colliculus at 6.0 mm caudal to the bregma and 1.0 mm lateral to the midline on both sides (3 µl each), to a depth of 5.0 mm from the surface of the skull. A total of 3 days after LIF or PBS injection, the rats were sacrificed by overdose of pentobarbital sodium before the eyes were enucleated and fixed in 4% paraformaldehyde solution for 40 min at room temperature. The retinas were dissected free and flatly mounted onto a glass slide. The FG-labeled RGCs were then identified under a fluorescence microscope (Leica DM2500; Leica Microsystems GmbH) with a wide band ultraviolet filter (0.1% fluorogold solution in distilled water with a pH of 4.5, Ex 414 nm, Em 541 nm). RGCs were manually counted by another investigator (CH) blinded to the experiment protocols. For each quadrant of the retina, three images were captured at 1, 2 and 3 mm radially from the optic disc in the identical retinal preparation (magnification, x10); the total number of RGCs was counted in all four quadrants using the image analysis program Image J (version 1.52; National Institutes of Health).

According to our previous report (35), RGC apoptosis, as well as the protein expression of apoptosis-related cytokines, was detected mostly on day 1 post AOH. One day after AOH induction, rats were deeply anesthetized with 30 mg/kg pentobarbital sodium prior to cardiac perfusion with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd.) for 24 h. The enucleated eyes were embedded in paraffin (65˚C) and sectioned at a thickness of 7 µm using a Leica DM2500 microtome (Leica Microsystems GmbH). TUNEL staining (37˚C; 1 h) of the retina was performed using a TUNEL assay kit (Promega Corporation), whereas the cell nuclei were stained with 50 mM DAPI (37˚C; 15 min, Vector Laboratories, Inc.). As a positive control, sections were incubated (room temperature; 10 min) with 0.5 µg/ml DNase I (Promega Corporation) before adding the equilibration buffer of the TUNEL assay kit. TUNEL-positive cells were observed and counted under fluorescence microscopy. The TUNEL-positive cells in each section were counted and quantified as per mm² of the retina by using the image analysis program Image J. A total of six images on x20 magnification were used from two sections per animal.

The rat retinas were dissected free after the global enucleation and homogenized with lysis buffer (Solarbio Science & Technology, China), and the protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). SDS-PAGE (12%) of the protein (20 mg per lane) was performed for 1-2 h and then transferred onto a PVDF membrane (MilliporeSigma). After blocking with 2% bovine serum albumin (Ameresco, Inc.) for 2 h at room temperature, the PVDF membranes were incubated with primary polyclonal antibodies against cleaved-caspase-3 (1:1,000), poly (ADP-ribose) polymerase (PARP; 1:1,000), STAT3 (1:500), phosphorylated (p-)-STAT3 (STAT3; 1:500), AKT (1:1,000), p-AKT (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), p70S6K (1:1,000), p-p70S6K (1:1,000) or β-actin (1:10,000; cat. no. A5441, Sigma-Aldrich; Merck KGaA) overnight at 4˚C. The antibodies of cleaved-caspase-3 (cat. no. 9579), PARP (cat. no. 9542), AKT (cat. no. 9272), p-AKT (cat. no. 31957), mTOR (cat. no. 2972), p-mTOR (cat. no. 5536), p70S6K (cat. no. 9202) and p-p70S6K (cat. no. 9208) were purchased from Cell Signaling Technology, Inc., whilst the antibodies of STAT3 (cat. no. APR13562G) and p-STAT3 (cat. no. APR11162G) were bought from Santa Cruz Biotechnology, Inc. After washing with TBS + 1% Tween 20 three times, the membranes were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:10,000; cat. no. 172-1050; Bio-Rad Laboratories, Inc.) for 2 h at room temperature. The protein bands were visualized with Enhanced Chemiluminescence reagents (SuperSignal; cat. no. 46641; Thermo Fisher Scientific, Inc.) and images captured using a transilluminator (ChemiDoc XRS; Bio-Rad Laboratories). Image Lab software (version 6.1; Bio-Rad Laboratories, Inc.) was used for the densitometry of the bands.

All data are presented as the mean ± standard deviation. One-way ANOVA followed by Tukey's multiple comparisons were performed using SPSS (version 17.0; SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

H&E staining was used to assess the effect of LIF on retinal histopathology induced by AOH. As shown in Fig. 1A (n=3), on day 1 after AOH, the thickness of the inner nuclear layer (INL) was decreased, whereas that of the inner plexiform layer (IPL) was markedly increased. The nuclei in the RGC layer also appeared larger compared with those in the normal control retina. On day 3 after AOH, a marked decrease in the IPL was observed After LIF treatment, the change in the thickness of the IPL was reversed compared with AOH group without LIF treatment. The change in the total retinal thickness (TRT) is presented in Fig. 1B and C (n=3/group). At 1 day after AOH, the TRT increase in both AOH and LIF treatment groups was statistically significant compared with the control (both P<0.05). At 3 days after AOH, significant decrease in the TRT was noticed in AOH rats compared with the control (P<0.001), while the TRT in AOH rats treated with LIF was significantly reversed compared with the AOH group (P<0.001).

An FG tracer was used to label RGCs to assess the effect of LIF on RGC survival after AOH (Fig. 2A). Quantitative analyses indicated that the FG-labeled RGC density in the AOH model retina (1,572.6±21.3/mm²) was significantly less compared with that in the normal control retina (2,390.4±68.8/mm²; P<0.001) (Fig. 2B). In retinae that received intravitreal LIF treatment, the RGC density was significantly higher compared with those in the AOH group without treatment (2,131.2±85.6/mm²; P<0.001; Fig. 2).

TUNEL staining was used to measure the degree of apoptosis in the retina 1 day after AOH, particularly in the RGC and inner nuclear layers (Fig. 3A, n=3). As shown in Fig. 3B, the number of TUNEL-positive cells was increased significantly in the AOH group (127.6±30.0/mm², n=3) compared with that in the normal control group (1.8±3.3/mm², n=3) (P<0.001). Accordingly, 1 day after AOH, the protein expression levels of cleaved-caspase-3 and PARP, indicators of apoptotic activity in the tissue, were significantly increased (Fig. 3C-E). After treatment with intravitreal LIF injection, the number of apoptotic cells (26.9±13.3/mm², n=3) was reduced significantly compared with that in the AOH group (P<0.001; Fig. 3B). In addition, the expression of cleaved-caspase-3 (P<0.001) and PARP (P<0.01) were significantly reduced compared with that in the AOH group (Fig. 3C-E).

As reported previously, the expression of STAT3, AKT/mTOR/p70S6K signaling pathways components peaked at around day 3 post AOH (35). The level of STAT3 phosphorylation in the rat retina was tested 3 days after AOH. A significant increase in the phosphorylation of STAT3 was observed only in the LIF treatment group compared with the AOH group (P<0.001, n=3/group; Fig. 4).

The expression of the AKT/mTOR/p70S6k signaling pathway components in the retina after AOH was assessed by western blotting (Fig. 5A). AOH didn't induce any significant change in the expression of any signaling pathway components compared with the control. After LIF treatment, a slight but insignificant increase in the phosphorylation of AKT was observed (Fig. 5B, n=3). By contrast, the phosphorylation levels of mTOR (P<0.05; Fig. 5C, n=3) and p70S6K (P<0.001; Fig. 5D, n=3) were significantly upregulated in the LIF treatment group compared with the untreated AOH group.

Compared with that in the AOH retina treated with LIF and PBS intravitreal injection, the RGC density was significantly lower in AOH rats receiving intravitreal injection of the JAK/STAT3 inhibitor C188-9 or with the PI3K/AKT/mTOR inhibitor LY3023414 (both P<0.01; Fig. 6, n=4/group).