Müller cells are the principal glial cells of the retina playing important roles in normal retinal physiological activities. Under pathological conditions such as retinal detachment, PVR (proliferative vitreous retinopathy) and PDR (peripheral diabetic retinopathy), Müller cells can be activated to perform a series of changes in morphology, as well as protein expression and production (Bringmann and Reichenbach 2001; Ortrud et al. 2003; Steven and Geoffrey 2003; Clyde 2005). However, it is still unclear whether Müller cells were also activated during retinitis pigmentosa. Royal College of Surgeons (RCS) rats, an animal model of retinitis pigmentosa, possess a naturally occurring deletion mutation in the receptor tyrosine kinase gene, which impairs the ability of the retinal pigmented epithelium (RPE) to phagocytose the photoreceptor outer segments, resulting in a buildup of debris in the subretinal space ultimately leading to photoreceptors death (Steven and Geoffrey 2003). Our aim was to determine if RCS rat Müller cells are activated during retinal degeneration and any changes in Müller cell morphology and protein expression.

66.2 Materials and Methods

66.2.1 Animal

All experiments were carried out in accordance with applicable Chinese laws and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. RCS rats at different developmental stages (postnatal days, PND15, PND30, PND60, PND90 and PND120, 3 rats at each age) were deeply anesthetized with urethane (2.0 g/kg) before decapitation and enucleation. Take the RCS-rdy rats of same age and quantity as control.

66.2.2 Immunohistochemical Staining

After fixation in 4% paraformaldehyde for 24 h and cryosectioning (thickness of 10um), immunostaining on sections or cover slips was performed as following: several washes in phosphate-buffered saline (PBS), followed by incubation in 10% normal goat serum plus 0.3% Triton X-100 in saline for 1 h, then incubation with a primary antibody overnight at 4◦C. Sections were washed several times and incubated with a secondary antibody for 1 h at 37◦C, washed in PBS, counterstained with DAPI, and then mounted with fluorescent mounting medium (Dako corporation) and examined under a confocal microscope (Zeiss, Germany). The following antibodies were used: mouse anti-vimentin (1:100; Sigma-Aldrich), mouse antiextracellular signal-regulated kinase (ERK) (1:100; Santa Cruz), rabbit anti-GFAP (1:100; Sigma-Aldrich). Appropriate fluorescence conjugated secondary antibodies were used. Müller cells count: count those vimentin positive processes with the length over 1/2 of the retina thickness as number of Müller cells.

66.2.3 Western Blot Test

The retinal tissues (4 eyes for each age) were treated with test substances for 10 minutes before harvesting. The tissues were washed twice with cold PBS (pH 7.4; Biochrom), and moved into 200 μl of lysis buffer (Mammalian Cell Lysis-1 Kit; Sigma). The total lysates were centrifuged at 10,000 rpm for 10 min, and the supernatant then analyzed by immunoblotting. Equal amounts of protein (30 μg) were separated by 12% SDS-polyacrylamide gel electrophoresis. Immunoblots were probed with primary and secondary antibodies, and immunoreactive bands visualized with diaminobenzidine (peroxidase substrate kit; Vector Laboratories) or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma-Aldrich). Chemiluminescence development and software Labworks 4.6 were used to analyze the gray scale of protein electrophoresis.

66.2.4 Müller Cell Cultures

Müller cells were isolated according to an established protocol (Hicks and Courtois 1990). Ten PND15 control rats were killed by decapitation and their eyes were removed and stored overnight at room temperature in the dark in Dubelcco’s Modified Eagles medium (DMEM) containing 2 mM glutamine and 1/1000 penicillin/streptomycine. The intact globes were incubated in DMEM containing 0.1% trypsin at 37◦C for 45 min. The retinas were isolated, chopped into 1 mm2 pieces and cultured in DMEM with 10% fetal bovine serum (FBS) and 1% penicilline/streptomycine. The use of pigmented eyes in all experiments permitted easy monitoring of any possible contaminating tissue such as the RPE or ora serrata. The cultures were maintained at 37◦C in a 5% CO2/95% air in a humidified incubator. To obtain a purified cell population, retinal aggregates and non-adherent cells were removed by vigorous rinsing and fresh medium added when cell outgrowth had acquired semiconfluence. Medium was changed every 3–4 days. When the cells became fully confluent the cultures were washed twice with DMEM, twice with Ca2+-free PBS and incubated in D-Hank’s with 1% trypsin for 1 min at 37◦C. The action of trypsin was stopped by adding 10% FBS in DMEM. The suspension was centrifuged at 1,000g for 10 min at room temperature, the pellet re-suspended and the Müller cells cultured on poly-L-lysine-coated coverslips until they became adherent. Coverslips containing normal Müller cells were co-cultured in a transwell system with mixed cell cultures from PND30 RCS rat retinas.

66.2.5 Data Analysis

Quantitative analysis of the data was preformed using SPSS13.0 software. Mean values with standard deviations are given in the text. Unless stated otherwise, statistical analysis was performed using a Student’s t-test. Differences with a P-value ≤0.05 were considered significant.