Fasudil hydrochloride, a potent ROCK inhibitor, inhibits corneal neovascularization after alkali burns in mice

Animals

Female BALB/c mice aged 4 to 6 weeks and weighting between 16 and 20 g were purchased from the Guangdong Provincial Center for Animal Research in Guangzhou, China. The right eye of each mouse was selected for experimentation. All experiments on animals were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The research protocol was approved by the Animal Care Committee of the Zhongshan Ophthalmic Center at Sun Yat-sen University in China.

Alkali burn- induced CNV

CNV was induced by alkali burns using a method outlined in previous reports [17]. In brief, after the mice were sedated with an intraperitoneal injection of general anesthesia consisting of 4.3% chloral hydrate [10 ml/kg] and a topical anesthesia consisting of a drop of 0.5% proparacaine hydrochloride (Alcaine eye drops, Alcon Inc., Fort Worth, TX), a 2 mm diameter filter paper soaked with 2 μl of 0.1 M NaOH solution was placed on the central cornea for 40 s, followed by immediate rinsing with 30 ml of 0.9% saline solution for 10 s. The entire corneal limbus and epithelium were then scraped off with a surgical blade under a microscope. Tobramycin ophthalmic ointment (Tobrex, Alcon Inc.) was administered after the operation.

Treatment with fasudil hydrochloride eye drops

The fasudil hydrochloride (Asahi Kasei Inc., Tokyo, Japan) was diluted in phosphate-buffered saline (PBS) to make different concentrations of fasudil eye drops. To observe the antiangiogenic effects of fasudil, 75 mice with alkali burns were randomly assigned to either the experimental groups to be treated topically with 30, 100, 300, and 1000 μM fasudil eye drops or to the control groups to be treated with PBS eye drops four times daily for 14 consecutive days (n=15 in each group). All the mice were killed on day 14 for immunohistopathological examination and reverse quantitative real-time polymerase chain reaction (PCR) analysis.

To detect the production of HO-1 in the murine corneas, another 50 mice with alkali burns were randomly treated with 100 μM fasudil eye drops or PBS eye drops four times daily for 4 consecutive days (n=25 in each group). To detect the production of ROS, 15 mice with alkali burns were randomly assigned to be treated with 100 μM fasudil eye drops, PBS eye drops, or nothing every 2 h for six consecutive hours (n=5 in each group), while another five normal mice without burns were assigned to a control group. The murine corneas were then procured for the analysis of ROS production.

Quantification of CNV and measurement of corneal epithelial defects

On days 4, 7, 10, and 14 after receiving alkali burns, the murine corneas were examined and photographed with a digital camera (Cannon, Tokyo, Japan) attached to a slit-lamp microscope (SL-120, Zeiss Inc., Jena, Germany). After the cycloplegia with a tropicamide eye drop (Wujing Pharmaceutical Company, Wuhan, China), CNV was observed under a white light. Corneal epithelial defects were then revealed using 0.5% fluorescein staining and were observed under a cobalt blue light. Three consecutive photos with satisfactory full-face imaging were used for image analysis. Image J software (ver. 1.62) was downloaded from the US National Institutes of Health (NIH) website and used for quantitative analysis, as documented in a previous report [18]. The consecutive photos of each mouse were used to measure the areas of CNV, epithelial defects, and the entire cornea, and then their percentages were calculated.

Measurement of ROS

Six hours after the alkali burn, the murine eyeballs were enucleated and immediately frozen in optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA). The cryopreserved blocks were cut until the first slide with corneal tissue was observed. The next five 30th slides of 10 μm thickness were then collected for immunostaining. when the first slide was observed, then the last section of every 30 consecutive cuts was procured to get five sections. Each section was 10 μm thickness.Unfixed cryosections were incubated with 5 μM dihydroethidium (DHE, ROS Fluorescent Probe, Eugene, OR) for 15 min at 37 °C, as previously reported [19]. The negative control was created by incubating sections with PBS instead of DHE. Sections were examined using a fluorescence microscope at 400× magnification (Olympus IX51, Tokyo, Japan) and photographed with an exposure time of 695 ms. The intensity of fluorescein staining was measured in the corneal epithelium and stroma using Image J 1.62 software.

Immunohistochemistry

Fourteen days after the alkali burns, seven mice in each of the 100 μM and PBS groups were randomly killed and their eyeballs were enucleated for immunohistochemistry. The eyeballs were fixed in a 10% neutral buffered formaldehyde solution and embedded in paraffin. The paraffin blocks containing the samples were cut until the first slide with corneal tissue was observed. Then the next three 20th slides of 5 μm thickness were collected for immunostaining, and another three 25th slides were collected for hematoxylin-eosin (H-E) staining. When the first slide was observed, then the 20th and 25th sections of every 30 consecutive cuts was procured to get three sections each for immunostaining and HE staining. For the immunostaining of polymorphonuclear neutrophils (PMNs), sections were deparaffinized and boiled in antigen retrieval solution (Dako, Glostrup, Denmark) for 15 min. Nonspecific staining was blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) for 1 h at room temperature. Sections were then incubated with the primary antibody (monoclonal rat antibody against mouse neutrophil marker NIMP-R14; sc59338, Santa Cruz Biotechnology, Santa Cruz, CA) diluted with 1% BSA (1:300) overnight at 4 °C. After three washes with PBS for 15 min, sections were incubated with HRP-conjugated secondary anti-rat IgG antibody (ZSGB-BIO Institute of Biotechnology, Beijing, China) for 1 h at room temperature and then washed again with PBS. The negative control was created by incubating sections with PBS instead of the primary antibody. The sections were then counterstained with Hoechst 33342 for 2 min. The sections were then washed three times in PBS for 3 min before being mounted on glass slides. The corneal sections were procured for histopathological study, just as in the procedures for immunostaining, and the slides were deparaffinized and regularly stained with H-E. Corneal morphology and the infiltrated PMNs in the corneal stroma were visualized and photographed with a fluorescence microscope (Olympus IX51). The number of infiltrated inflammatory cells in the cornea, including PMNs, was counted in five randomly selected fields (at 400× magnification) of a slide.

Analysis of mRNA expression

Real-time quantitative reverse transcription PCR (real-time qRT-PCR) was performed to detect the mRNA expression levels of the VEGF, VEGFR1, VEGFR2, TNF-α, IL-1β, MMP-2, MMP-8, and MMP-9 genes in the murine corneas. On day 14 after the burns, 8 mice from each of the 100μM group and the PBS group were euthanized by cervical dislocation, and their right corneas were procured. The total RNAs of each cornea were immediately isolated using an RNeasy Micro Kit (Qiagen, Valencia, CA). After quantification of the RNA concentration, the total RNAs were treated with DNase I (Sigma-Aldrich) to remove any contaminated genomic DNA. A total of 0.5 μg RNA was reverse transcribed into cDNA in a 20 μl volume reaction system using a Maxima First Strand cDNA Synthesis Kit (Fermentas International Inc., Burlington, ON, Canada). Samples of synthesized cDNA were divided into aliquots and stored at -80 °C.

Real-time qRT-PCR was performed and the results analyzed using an ABI PRISM 7000 sequence detection system (Applied Biosystems Inc., Foster City, CA). PCR was performed in a 20 μl volume reaction system containing 10 μl 2 × SYBR Green Reaction Mix (Invitrogen, Carlsbad, CA), 0.4 mmol/l paired primers, and 1 μl cDNA. Each sample was simultaneously run in triplicate. The sequences of the PCR primer pairs are listed in Table 1. Thermal cycling consisted of denaturation for 3 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The PCR amplification efficiency of the primer sets was determined to be 100% before qPCR. A comparative Ct (ΔΔCt) method was used to compare the mRNA expression levels of the genes of interest. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was chosen to be an internal control gene.

Western blot

The protein levels of HO-1 in the murine corneas were measured by western blots. Briefly, the corneas procured from the mice in the 100 μM group and the PBS group were homogenized in a 1.5 ml microtube with a lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) on day 4 after the alkali injury. Twenty-five corneas in each group were randomly divided and pooled into five samples, each of which had five corneas. Supernatants from each sample homogenate were collected after centrifugation at 24949 ×g for 30 min at 4 °C. Protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Next, 20 μg of protein from each sample was mixed with a 2× sample buffer and heated to 100 °C for 5 min. After electrophoresis, the proteins were electrotransferred onto 0.2 mm PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% skimmed milk and incubated overnight at 4 °C with primary antibodies (1:500, sc-10789, Santa Cruz Biotechnology). The staining specificity was assessed by the omission of the primary antibody. After three washes with tris-buffered saline (TBS), the membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody solution (1:1000, sc-2004, Santa Cruz Biotechnology) and then diluted in TBS with 5% skim milk for 2 h at room temperature. The membranes were then washed in TBS and placed in the chemiluminescent working solution (ECL detection reagents, Thermo Fisher Scientific, Waltham, MA) for 5 min at room temperature. The membrane was imaged with a G:BOX BioImaging system (Syngene, Cambridge, England) at the same time, and the pictures were analyzed with Image J 1.62 software to measure the densitometric intensities of the respective bands. The experiment was repeated once.

Statistics

All data was expressed as mean ± standard deviation. The percentages of CNV and corneal epithelium defects were compared using one-way ANOVA between the four experimental groups and the control. A least-significant difference (LSD) analysis was performed to compare each experimental group and the control group. The expression levels of mRNAs, ROS production, and HO-1 proteins, as well as the number of infiltrated inflammatory cells, including PMNs, were compared using a Student t test for both the 100 μM fasudil group and the PBS group. A p value of less than 0.05 was considered to be statistically significant.

Effects of fasudil on alkali burn-induced CNV

The area of CNV was found to increase with time in both the experiment groups and the control group after alkali burns (Figure 1A). The percentages of CNV areas in the 100 μM fasudil treated group were significantly lower than those in the PBS treated group at every check point time (all p<0.05, Figure 1B). The percentages of CNV areas in the 300 μM fasudil treated group were lower than in the PBS treated group on days 10 and 14 (all p<0.05, Figure 1B). The areas of CNV in the 100 μM fasudil treated group were smaller than in the 300 μM fasudil treated group. There were no statistical differences in the percentages of CNV areas between either the 30 μM or the 1000 μM fasudil treated groups and the control group at any checkpoint after injury.

Fasudil promotes corneal epithelial healing

The percentages of corneal epithelial defect area to the entire cornea in the fasudil treated groups were lower than those in the PBS treated group on days 4 and 7 after the alkali burns (all p<0.01), with the exception of the 30 μM fasudil treated group on day 4. By day 10, the murine corneas had re-epithelialized in all groups (Figure 2).

The production of ROS in the cornea was enhanced after alkali burns and was then inhibited after treatment with 100 μM fasudil

To evaluate the effect of fasudil on ROS formation after the alkali burns, ROS production was examined using a DHE assay, in which DHE reacts with o 2 -   to show fluorescent imaging. The results show that the production of ROS in the corneal epithelium and stroma after an alkali injury without treatment was 2.29±0.21 and 2.0±0.20 times that of ROS production in the normal cornea (all p<0.01, n=5/group, Figure 3). After treatment with 100 μM fasudil eye drops, the intensity of ROS-associated DHE fluorescence was significantly reduced in the corneal epithelium and stroma (all p<0.01, n=5/group, Figure 3).There was no statistical difference in the production of ROS between the mice with and without PBS treatment after injury, either in the corneal epithelium or in the stroma.

100 μM fasudil reduced the infiltration of inflammatory cells in the alkali-burned murine corneas

The histopathological analysis reveals a significant decrease in the number of infiltrated macrophages, lymphocytes, and PMNs in murine corneas after treatment with 100 μM fasudil eye drops compared to corneas treated with PBS (18.4±5.31 versus 47.8±9.01 per field, p<0.01; Figure 4A-C).The number of monoclonal antibody stained PMNs in the corneal stroma was significantly lower in the 100 μM fasudil treated group than in the PBS treated group (5.0±4.6 versus 16.0±5.0 per field, p<0.01; Figure 4D-F).

Effects of 100 μM fasudil on the mRNA expression of angiogenic genes in alkali-burned murine corneas

The mRNA expression levels of VEGF, VEGFR1/2, IL-1β, TNF-a, and MMP-2, MMP-8, and MMP-9 were detected and compared by quantitative RT-PCR. Gene expression was significantly lower in the 100 μM fasudil treated group; the expression levels were VEGF, 34.14%; TNF-α, 18.82%; MMP-8, 27.63%; and MMP-9, 12.57% compared to those in the PBS treated group. However, there were no statistical differences in the expression of VEGFR1/2, IL-1β, and MMP-2 between these two groups (Figure 5).

Effect of 100 μM fasudil on the expression of heme oxygenase-1 (HO-1) protein in the murine corneas after the alkali burn

To assess the effect of fasudil on HO-1 expression in the murine corneas, we examined the HO-1 protein on day 4 after the injury using western blotting. The results indicated that the optical intensity of HO-1 protein bands in the 100 μM fasudil treated group was 1.52±0.34 times that of HO-1 protein bands in the PBS treated group (p<0.05, Figure 6).