*Contributed equally
Glaucoma is one of the leading causes of irreversible blindness worldwide. As such, neuroprotective therapy is essential for the treatment of this disease. Leukemia inhibitory factor (LIF) is a member of the IL-6 cytokine family and the LIF signaling pathway is considered to be one of the major endogenous factors mediating neuroprotection in the retina. Therefore, the present study aimed to investigate the possible effects of LIF in acute ocular hypertension (AOH). The intraocular pressure in rat eyes was raised to 110 mmHg for 1 h by infusing the anterior chamber with normal saline to establish the AOH model. In the treatment group, LIF was then injected into the vitreous cavity after AOH was ceased. The retinal tissues were obtained after the termination of AOH, and H&E staining was conducted to assess the morphological damage. The number of retinal ganglion cells (RGCs) was counted using the Fluoro-Gold retrograde staining method. TUNEL staining was used to determine the extent of apoptosis among the retinal cells. In addition, the protein expression levels of cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), STAT3 and components of the AKT/mTOR/70-kDa ribosomal protein S6 kinase (p70S6K) signaling pathway were examined by western blotting. The results showed that AOH induced tissue swelling and structural damage in the retina, which were reversed by LIF injection. In the LIF treatment group, RGC loss was significantly inhibited and the quantity of TUNEL-stained cells was also significantly reduced, whereas the expression of cleaved caspase-3 and PARP was decreased. Furthermore, increased phosphorylation of STAT3, AKT, mTOR and p70S6K was observed after LIF treatment. By contrast, pretreatment with the STAT3 inhibitor C188-9 or the PI3K/AKT/mTOR inhibitor LY3023414 reversed the LIF-induced inhibition of RGC loss. These results suggested that exogenous LIF treatment inhibited the retinal damage induced by AOH, which was associated with the activation of STAT3 and mTOR/p70S6K signaling. Therefore, LIF may serve a role in neuroprotection for glaucoma treatment.
Glaucoma is one of the leading causes of irreversible blindness worldwide, the prevalence of which is projected to reach 111.8 million by 2040, with ~10% of patients succumbing to blindness (
A number of causes have been reported to be responsible for RGC damage and death in glaucoma, including IOP elevation, ischemia/reperfusion (I/R) damage of the retina, oxidative stress, glutamate neurotoxicity, neurotrophic growth factor deprivation and immune disturbance (
Several cytokines have been previously found to be involved in the pathophysiology of glaucoma, such as tumor necrosis factor-α, interleukin-1β, interleukin-6 and interleukin-18(
Our previous study on a rat AOH model has revealed that LIF and LIF receptor protein expression is upregulated associated with activation of the STAT3 and AKT signaling pathways (
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 (
The experimental procedure has previously been described (
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 (
According to our previous report (
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
An FG tracer was used to label RGCs to assess the effect of LIF on RGC survival after AOH (
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 (
As reported previously, the expression of STAT3, AKT/mTOR/p70S6K signaling pathways components peaked at around day 3 post AOH (
The expression of the AKT/mTOR/p70S6k signaling pathway components in the retina after AOH was assessed by western blotting (
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;
In the present study, the effects of exogenous LIF on the survival of RGCs was investigated in AOH model rats. When injected into the vitreous, LIF significantly inhibited the retinal atrophy and RGC loss induced by AOH. Furthermore, apoptosis was reduced after LIF injection. Activation of the AKT/mTOR/p70S6K and JAK/STAT signaling pathways may be associated with these neuroprotective effects of LIF.
Several mechanisms are involved in the pathological changes in the retina after AOH. In the early stages, direct stress on the inner retina leads to the death of RGCs and axonal damage (
Apoptosis inhibition is reported to be associated with the neuroprotective effect of LIF in the retina. In a previous model of light-mediated retinal injury, apoptosis of photoreceptor cells triggers the expression of LIF from Müller cells (
Our previous study showed that AOH activated the expression of intrinsic LIF and LIF receptors in the retina, which is accompanied by the upregulation of STAT3 activity and AKT protein expression (
The present study has several limitations. First of all, the effect of LIF on RGC survival after AOH needs to be monitored for a longer time, and a combination of any functional assessment would be valuable. Additionally, the mechanism underlying the neuroprotection of LIF injection needs further investigation and the involvement of the signaling pathways need to be clarified. Nevertheless, the present study demonstrated the neuroprotective effects of exogenous LIF treatment against retinal damage observed in AOH, which suggests that LIF may serve a role in the neuroprotective treatment for glaucoma. Further studies are needed to confirm this, as well as the neuroprotective effects of LIF treatment in patients with glaucoma.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JL, CH and RW designed the study. JL and RW were responsible for the data collection. JL, RG, CH and YW conducted the experiments. JL, RG and RW were responsible for data analysis. JL and CH confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The present study was approved by the Ethics Review Committee of Xiamen University (Xiamen, China; approval no. 20150306155209).
Not applicable.
The authors declare that they have no competing interests.
Protective effect of LIF on the rat retina 1 and 3 days after AOH. (A) Morphological changes in the rat retina. On day 1 after AOH, the thickness of the INL was decreased, whereas that of the IPL was increased. On day 3 after AOH, the IPL decreased markedly. After LIF treatment, the change in the thickness of the IPL was reversed compared with AOH group without LIF treatment. Scale bar, 100 µm. Changes in retinal thickness (B) 1 day and (C) 3 days after AOH. n=3/group. *P<0.05 and #P<0.001. AOH, acute ocular hypertension; LIF, leukemia inhibitory factor; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR (IS/OS), photoreceptor (inner segment/outer segment).
FG retrograde labeling to determine the effects of LIF on RGC survival after AOH. (A) FG retrograde labeling of RGCs; 1, 2 and 3 mm indicate the radial distances to the optic disc in the identical retinal preparation for each treatment condition. Scale bar, 200 µm. (B) The results of RGC counting under the fluorescence microscope (Leica DM2500). 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). RGCs were manually counted by an investigator blinded to the experiment protocols. *P<0.001, n=4/group. (C) Partial enlargement (magnification, x10) of an FG-labeled image. The red arrows refer to examples of RGCs. Scale bar, 40 µm. AOH, acute ocular hypertension; FG, Fluoro-Gold; LIF, leukemia inhibitory factor; RGC, retinal ganglion cell.
Effects of LIF on the apoptosis of retinal cells in AOH model rats. (A) TUNEL staining of the retinae. Green, TUNEL staining; blue, DAPI nuclear staining. Scale bar, 100 µm. (B) Quantitative analysis of the number of TUNEL-stained cells in the retinae. (C) Representative western blotting images and semi-quantitative analysis of (D) c-caspase-3 and (E) PARP protein expression in the retina 1 day after AOH. *P<0.01 and #P<0.001, n=3/group. AOH, acute ocular hypertension; c-caspase-3, cleaved-caspase-3; LIF, leukemia inhibitory factor; PARP, poly (ADP-ribose) polymerase; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR (IS/OS), photoreceptor (inner segment/outer segment).
Effects of LIF on the activation of STAT3 after AOH modeling in rats. (A) Representative images of western blot analysis. (B) Semi-quantitative analysis of p-STAT3 and STAT3 protein levels in the retinal samples. *P<0.001, n=3/group. AOH, acute ocular hypertension; LIF, leukemia inhibitory factor; p-, phosphorylated.
Effects of LIF on the expression and activation of AKT/mTOR/p70S6k signaling pathway components after AOH. (A) Representative western blotting images, and semi-quantitative analysis of the (B) p-AKT/AKT, (C) p-mTOR/mTOR and (D) p-p70S6K/p70S6K ratios. *P<0.05, #P<0.001, n=3/group. AOH, acute ocular hypertension; LIF, leukemia inhibitory factor; p-, phosphorylated; p70S6K, ribosomal protein S6 kinase.
Effect of Janus kinase/STAT3 and PI3K/AKT/mTOR pathway inhibitor pretreatment on LIF-induced neuroprotection in the retina of rats after AOH. (A) Representative images and (B) quantitative analysis of Fluoro-Gold retrograde labeling of RGCs in the retina 3 mm distant to the optic disc. Scale bar, 100 µm. *P<0.01, n=3/group. AOH, acute ocular hypertension; LIF, leukemia inhibitory factor; RGC, retinal ganglion cell.