T75 culture flasks were used to construct the pressure mechanism (Fig. 1). An air mixture of 95% air and 5% CO₂ was pumped to obtain the relevant pressure. The pressure level of the model was 0, 20, 40, 60 and 80 mmHg, respectively, in accordance with human IOP in glaucoma, and it was adjusted every 4 h. The time period of the pressure was 24 h. All of the flasks were placed in an incubator at 37˚C.

The possibility that the elevated hydrostatic pressure could alter the gas exchange was assessed by analyzing the blood gas in the culture medium in the pressure (20, 40, 60 and 80 mmHg) and control (0 mmHg) culture groups before and after 12 h of pressurization. The tightness was assessed by a pressure gauge.

Experiments were performed on newborn (0–3 days) Sprague-Dawley (SD) rats (Slaccas Laboratory Animal Co., Ltd.). SD rats were decapitated, and their retinas were quickly dissected in cold D-Hank’s solution (Anresco). The mixture was transferred to sterile centrifuge tubes and centrifuged at 1,000 rpm for 10 min. The supernatant was discarded. Trypsin (0.125%) (Anresco) was added for digestion. The mixture was incubated at 37˚C for 15 min. At the end of the designated time, DMEM/F12 medium (Gibco) supplemented with 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and 10% fetal bovine serum (FBS; Sijiqing) was added to terminate the digestion. The digests were filtered using a 200-mesh nylon sieve and centrifuged at 1,000 rpm for 10 min. The supernatant was discarded. The cell suspension was cultured in T75 culture flasks at 37˚C in humidified air containing 5% CO₂. When confluent, the cells were washed once with D-Hank’s solution, detached from the flask by treatment with 0.125% trypsin, washed with complete cell culture medium and split 1:2 into fresh flasks.

The cells on the coverslips were incubated at 37˚C for 1 day and washed three times (10 min/wash) with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 10 min and incubated with 0.3% Triton X-100 at 37˚C for 10 min. The cells were washed three times (10 min/wash) with PBS, blocked with 10% goat serum in PBS and subsequently incubated with a rabbit anti-rat polyclonal antibody against GS (1:5,000; Abcam) as an identity marker for Müller cells. The cells were then incubated overnight at 4˚C. The following day, the cells were incubated with the secondary anti-rabbit IgG-Cy3 antibodies (1:200; BioLegend) at 37˚C in darkness for 1 h. After three washes with PBS, the cells on the coverslips were mounted on glass slides with Histomount. The cells were viewed under an Axio microscope, and images were acquired with a digital camera.

Fifteen samples, three at each pressure (0, 20, 40, 60 and 80 mmHg/24 h), were used for Western blotting. Cells were washed twice in PBS. The protein concentration was determined by a radioimmunoprecipitation assay and lysed in 2X Laemmli buffer. Protein extracts were boiled for 10 min and centrifuged at 16,000 x g. Proteins were separated on 12% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were soaked in Tris-buffered saline (20 mmol/l Tris-Cl, 140 mmol/l NaCl, pH 7.5) containing 5% skimmed milk and 0.1% Tween-20 for 1 h at room temperature. The primary antibody used was GS (1:2,500; Abcam). Blots were incubated with the primary antibody overnight at 4˚C. Anti-β-actin antibody (1:3,000; Abcam) was used as a reference to normalize the intensities of the immunoreactions with the different antibodies. Then, after several washes, the membranes were incubated with a secondary antibody against rabbit-IgG (Invitrogen) for 1 h at room temperature in darkness. The protein bands were scanned, and images were quantified with Odyssey (Li-CDR).

Total RNA was isolated from the individual samples using the TRIzol^® reagent (Invitrogen). The concentration and purity of the RNA preparations were determined by measuring the absorbance at 260/280 nm in a spectrophotometer (Beckman). Total RNA was reversetranscribed into cDNA in a 20-μl reaction containing 2 μg RNA, 4 μl 5X M-MLV buffer, 2 μl of dNTP, 1 μl random hexamer primer, 0.5 μl of RNase inhibitor and 1 μl of M-MLV RTase. Reactions were performed at 25˚C for 10 min, at 42˚C for 60 min and at 70˚C for 10 min.

The following targets were analyzed by RT-PCR. The nucleotide sequences of the primers were based on published cDNA sequences (Gene Bank). The primer sequences used for RT-PCR were as follows: GS, 5′-ccgctcttcgtctcgttc-3′ and 5′-ctg cttgatgcctttgtt-3′; β-actin, 5′-cccatctatgagggttacgc-3′ and 5′-ttta atgtcacgcacgatttc-3′. RT-PCRs were performed on a LightCycler instrument (Rotor Gene) with SYBR Green I, according to the manufacturer’s recommendations. Cycling conditions were as follows: initial denaturation at 94˚C for 5 min, followed by 40 cycles at 94˚C for 30 sec, 55˚C for 30 sec and 72˚C for 30 sec. The melting curve analysis of the final PCR products was carried out from 50 to 99˚C at 1˚C intervals.

Data are presented as the mean ± SD (n=3–6 in each group). Data were analyzed using one-way ANOVA followed by the Turkey’s test. A p-value of <0.05 was accepted as indicative of statistical significance.

We analyzed pH, PCO₂ or PO₂ from samples of the culture medium 12 h after exposure to the different pressure levels (0, 20, 40, 60 and 80 mmHg). There was no significant difference in these values between the control (0 mmHg) and each pressure (20, 40, 60 and 80 mmHg) group (Table I).

We characterized the cultured rat Müller cells by their expression of GS as assessed by immunocytochemical staining. Cells in the culture system showed positive labeling for GS (Fig. 2). Using this immunocytochemical labeling, the cultured cells were determined to be Müller cells.

Western blot analyses of the samples at different pressure showed that the expression of GS protein was variable. The expression of the GS protein in the 20 mmHg/24 h group was decreased compared to the control (0 mmHg/24 h) group. The expression of GS protein was increased in the 40 mmHg/24 h and the 60 mmHg/24 h groups. The expression of GS protein was slightly decreased in the 80 mmHg/24 h group (Fig. 3).

RT-PCR was performed to examine whether the mRNA expression levels of GS were different among the different pressure groups. Data showed that the expression of GS mRNA was decreased in the 20 mmHg/24 h group compared to the control (0 mmHg/24 h) group. Hereafter, the GS mRNA expression was increased in the 40 mmHg/24 h group which further increased in the 60 mmHg/24 h group, and then decreased in the 80 mmHg/24 h group. However, the difference was only statistically significant between the control (0 mmHg/24 h) and the 40 mmHg/24 h and 60 mmHg/24 h groups, and there was no statistical significance between the control (0 mmHg/24 h) group and the 20 mmHg/24 h and 80 mmHg/24 h groups (Fig. 4). The results of the expression of GS mRNA were in accord with the results of the expression of the GS protein.