Twenty-four adult Sprague–Dawley rats weighing 200–250 grams, available from the animal center of Central South University, were used in the present study. Rats were randomly divided into the control group (n=6) and experimental group (n=18) subjected to induction of acute high intraocular pressure (aHIOP). All animals were housed in acrylic box cages with free access to food and water. Animals were maintained under conditions of constant temperature (25°C), humidity (50±10%) and lighting cycle (12:12 hours). All experimental procedures used in the present study were approved by Ethics Committee of Xiangya School of Medicine, in accordance with the NIH guidelines for use and care of laboratory animals.
Induction of acute high intraocular pressure (aHIOP) and propidium iodide treatment
The animal model was prepared following the procedure described by Tong [13
]. In brief, Animals were anesthetized with 10% chloral hydrate (0.2 ml/kg). A drop of chloramphenicol was administered to the conjunctive sac. A 30-gauge needle connected to the instillation instrument filled with normal saline was inserted into the anterior chamber. The intraocular pressure (IOP) was elevated to 110 mmHg, maintained for 60 min, and then gradually lowered to normal. The rats were allowed to survive for 6, 12 and 24 hrs before terminal use. Thirty minutes prior to animal perfusion, 5 μl PI (1.0 mg/ml in DW, Sigma, MO, USA) was administered by intravitreal injection.
For anatomical examination, animals were deeply anesthetized with 10% Chloral hydrate (Sinopharm Chemical Reagent Co. Ltd, Shanghai, China) in saline (0.4 ml/kg, i.p.), followed by trans-cardiac perfusion with saline and then 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at different survival time points. The eyeballs were enucleated, and the cornea, lens and vitreous body were removed. The remaining eye cups were postfixed in 4% PF overnight, and immersed in ascending sucrose solutions (15% to 30%) in 0.1 M PB at 4°C for cryoprotection. The eye cups were embedded in Optimal-Cutting-Temperature (OCT) medium (Sakura Finetek, Japan), prepared into 20 μm thick cross-sections in a Shanton cryostat (Thermo-Fisher Scientific Inc., CA, USA). Sections were thaw-mounted on positively charged microslides (Thermo-Fisher Scientific Inc., CA, USA), allowed to air-dry and then stored at −20°C before further histological processing. For western blot analysis, eyeballs were removed immediately from deeply anesthetized rats after a vascular rinse with cold saline, and the retinas were dissected out and snap-frozen on dry-ice and stored at −70°C until tissue homogenization.
For immunolabeling with the avidin-biotin complex (ABC) method, sections were treated in 0.3% H2O2 in 0.01M phosphate buffered saline (PBS, pH 7.3) for 15 minute to inactivate endogenous peroxidase. Non-specific antibody binding was blocked by a pre-incubation of sections in 5% normal horse serum (Sigma, MO, USA) in PBS containing 0.3% Triton X-100 (Fluka, CA, USA) for 1 hour at room temperature. Sections were then incubated with a rabbit anti-RIP3 antibody at 4°C overnight (Table ), then reacted with biotinylated horse anti-rabbit IgG (1:400, Vector Laboratories Inc., CA, USA) for 2 hours at room temperature. After 1 hour incubation with the avidin-biotin complex reagents (1:400, Vector Laboratories Inc., CA, USA), immunoreaction product was visualized in PBS containing 0.05% DAB (Sigma, MO, USA) and 0.03% H2O2. Finally, the sections were dehydrated, cleared and coverslippered. The specificity of the RIP3 antibody was evaluated by pre-absorption and omission of the primary antibody in immunohistochemistry. Western blot was also used to confirm the antibody binding product.
Primary antibodies used in the present study
For double immunofluorescence of RIP3 co-localization in normal retina, sections were pre-incubated for 60 minutes in 5% donkey serum (Sigma, MO, USA) in PBS containing 0.3% Triton X-100 at room temperature. Sections were incubated with the rabbit anti-RIP3 antibody and one of the mouse antibodies to neuronal and glial markers overnight (listed in Table ). After several times rinses with PBS, sections were reacted with Cy2 and Cy3-conjugated donkey anti-mouse and anti-rabbit secondary antibodies at 1:200 (Invitrogen, CA, USA). The sections were then briefly incubated in Bisbenzimide (Hoechst 33258, Sigma, MO, USA) at 1:50,000, washed in PBS and covered with an anti-fading mounting medium (Abcam, MA, USA) before microscopic examination.
For immunofluorescence of RIP3 and double labeling of RIP3 with Bax or cleaved caspase-3 following aHIOP, sections were pre-incubated for 60 minutes in 5% donkey serum (Sigma, MO, USA) in PBS containing 0.3% Triton X-100 at room temperature. Sections were incubated with the rabbit anti-RIP3 antibody (for RIP3 expression test) or goat anti-RIP3 and Bax or cleaved caspase-3 antibodies overnight. After several times rinses with PBS, sections were reacted with Cy2 and Cy3-conjugated donkey anti-rabbit/donkey anti-goat/donkey anti-mouse secondary antibodies at 1:200 (Invitrogen, CA, USA). The sections were then briefly incubated in Bisbenzimide, washed in PBS and covered with an anti-fading mounting medium (Abcam, MA, USA) before microscopic examination.
For double labeling of PI-labeled cells and RIP3 following aHIOP. 12 hr of PI-pretreated retinal sections were selected to carry out RIP3 immunofluorescence staining. The protocol of RIP3 immunofluorescence staining have illustrated before. Photograph were using a confocal microscope (Nikon, DIGITAL ECLIPSE C1 plus, Japan) fitted with excitation/emission filters 568/585 for PI.
Retinas were homogenized by sonication on ice in a digestion buffer [150 mM NaCl, 25 mM Tris- HCl (pH 7.4), 2 mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS] containing a cocktail of protease inhibitors (Sigma, MO, USA). Homogenates were centrifuged at 10,000×g for 20 min at 4°C. The supernatants were collected, and protein concentration determined by Bicinnchoninic acid (BCA) assay (Pierce, IL, USA). A total of 100 μg of protein in 62.5 mM Tris loading buffer (pH 6.8, containing 25% glycerol, 2% SDS, 0.01% bromophenol blue and 5% β-mercaptoethanol, Bio-Rad, CA, USA), was boiled for 10 min, and loaded in each lane of 4-20% linear gradient Tris–HCl ready gel (Bio-Rad, CA, USA). The polypeptides were electrotransferred to Trans-Blot® pure nitrocellulose membrane (Bio-Rad, CA, USA). Non-specific binding was blocked with PBS containing 5% nonfat milk (Bio-Rad, CA, USA) and 3% bovine serum albumin (Sigma, MO, USA). Membranes were incubated with RIP3 or β-tubulin antibodies overnight, and then in HRP-conjugated secondary antibodies (1:20000, Bio-Rad, CA, USA) for 1 hour. Immunoblotting products were visualized with an ECL Plus™ Western Blotting Detection kit according to manufacturer’s instruction (GE Healthcare Life Sci., NJ, USA), and images captured in a Molecular Dynamics Phosphor imager (Nucleo Tech Inc., CA, USA). For RIP3 antibody specific test, nitrocellulose membranes were blotted for RIP3 with the presence of the immunogenic peptide corresponding to amino acids 473 to 486 of murine RIP3 (US Biological, R2031-74P, at 1:10 dilution).
Imaging and data analysis
Sections were examined on an Olympus (BX53, Tokyo, Japan) microscope equipped with a digital camera and imaging system (CellSens Standard, Olympus, Tokyo, Japan) for single (DAB) immunolabeling and on a confocal microscope (Nikon, DIGITAL ECLIPSE C1 plus, Japan) for RIP3 distribution double immunolabeling and RIP3 immunofluorescence following aHIOP. For double labeling of Bax or cleaved caspase-3 with RIP3, the slices were examined with a confocal microscope (Carl Zeiss, Axiovert 200M, Germany). In the latter case, immunofluorescence was scanned from 1 μm depth of tissue (i.e., 5–6 μm below the section surface) around the middle segment of retina between the optic nerve disk and periphery. Semi-quantitative analyses were conducted using approximately 20 merged images (40×) from 4 animals (5 sections per animal) to estimate the frequency of colocalization. Figure panels were assembled using Photoshop CS5 (Adobe Systems Incorporated, CA, USA). Image J (National Institutes of Health, MD, USA) were used to analyze the optical density value of the RIP3 bands by western blot. The average values of RIP3 and β-Tubulin were compared, the average relative value was obtained. One-way analysis of variance was performed to test differences between group averages. All results were presented as mean ± SD. A value of p<0.05 was considered statistically significant.