Loss of RGCs was assessed in labeled flat mounts by two procedures: retrograde labeling and immunocytochemistry. For the retrograde labeling, rats were deeply anesthetized by intramuscular injections of a ketamine/xylazine cocktail to a dose of 70 mg/kg ketamine and 30 mg/kg xylazine. Rats were then immobilized on a stereotaxic apparatus (KOPF, Tujunga, CA), their head fur shaved, and exposed skin infiltrated with 1% bupivacaine (Hospira, Inc., Lake Forest, IL). A 1- to 2-cm incision was made to expose the dorsal surface of the skull and sutures. With the help of a caliper, microinjection points were drawn on the skull 6 mm posterior to bregma and 2.0 mm lateral to the sagittal suture. Openings were made through the skull at the marked sites to expose the superior colliculus using a Micro Drill system (World Precision Instruments, Sarasota, FL). A 5-μL Hamilton 65RN syringe, equipped with a 2-inch 30G RN NDL point 4 needle (Hamilton, Reno, NV) was loaded with a 3% Dil dye solution (Molecular Probes/Life Technologies, Carlsbad, CA) (dissolved in dimethyl sulfoxide–ethanol 1:1) and placed in the stereotaxic frame. Using the stereotaxic's micromanipulator (KOPF), the needle was lowered 4.5 mm to deliver 1.5 μL of the tracer. The needle was kept in place for 10 minutes, slowly withdrawn, antibiotic ointment (erythromycin, 5 mg/g; E. Fougera & Co., Melville, NY) applied and the skin incision sutured with 2-0 silk, nonabsorbable suture (Oasis, Mettawa, IL). Rats were placed on top of deltaphase isothermal pads at 37°C (Braintree Scientific, Braintree, MA) for recovery. Seven days after tracer labeling, rats' eyes were enucleated immediately after euthanization and immersed in fresh 4% PFA for 1 hour with a corneal puncture. Eyes were then opened at the limbus, lens and ciliary body removed, and retinal cups rinsed with PBS and treated with 20 μg/mL hyaluronidase (Sigma-Aldrich) for 30 minutes at room temperature. Consecutively, retinal cups were postfixed for 30 minutes and washed in PBS overnight. The next morning, retinas were dissected from the underlying sclera/choroid, flattened by four radial cuts, mounted vitreal side up on Superfrost Plus slides (Thermo Fisher Scientific), and coverslipped with a drop of antifade mounting medium containing 0.1 mg/mL p-phenylenediamine (Sigma-Aldrich) in 90% glycerol/PBS, pH 8.0 (PPD). Images were taken in an Olympus XI71 fluorescence microscope equipped with an Olympus DP70 digital camera and software. For each retina, images were taken from 20, ×20 fields (0.394 mm2) located at 0.5 mm from the periphery (two per quadrant). Automated cells counts in each field were obtained on the gray scale mode using MetaMorph software for Olympus (Olympus) and using the same threshold value. For each individual retina, the total count of surviving RGC neurons was obtained by averaging the eight counts for each retina. Results are expressed in percentage of RGC loss in the Ad5BMP2-treated eyes versus the Ad5.CMV-Null-control. The two groups in each experiment were analyzed for differences using the Student's paired t-test.
For the immunohistochemistry procedure, rats were either immersed in 4% PFA for 1 hour as indicated above, or perfused through the heart using a gravity system (In Vivo Perfusion System, 70 cm high; AutoMate Scientific, San Francisco, CA) following the manufacturer's recommendations. Briefly, rats were deeply anesthetized with intraperitoneal injection of 100 to 400 mg/kg of euthasol (Butler Schein) and their chest cavities opened. A 20G × 3/4-inch scalp vein set (EXEL International, Los Angeles, CA) was inserted into the left ventricle and approximately 150 mL 0.9% NaCl was perfused through the circulatory system after making a slit on the right atrium to allow perfusion. Perfusion continued with 400 to 600 mL 4% PFA made in 0.1 M phosphate buffer pH 7.2 until the rat's body was stiff. Eyes were then immediately enucleated and treated like above for flat mounting on Superfrost Plus slides (Thermo Fisher Scientific). Subsequently, a hydrophobic ring was drawn around the retina using a Liquid Blocker super pap-pen (Newcomer Supply, Middleton, WI), retinas were rinsed with PBS/0.5% Triton X-100 and permeabilized at −80°C for 15 minutes. Alternatively, the nonperfused, hyaluronidased/postfixed retinas were separated from the sclera/choroid, kept in solution in 24 wells for all immunohistochemistry steps, and flat mounted after the last wash. For this, after thawing them at room temperature, in-well retinas were washed and blocked with 2% donkey serum/PBS/0.5% Triton X-100 for 1 hour. Retinas were then incubated with goat antihuman Brn-3a antibody (1:550, sc-31984; Santa Cruz Biotechnologies, Santa Cruz, CA) overnight followed by an additional 2 hours with donkey antigoat Alexa Fluor 568 (1:550; Molecular Probes/Life Technologies). All antibody solutions were made in 2% donkey serum/PBS/0.5% Triton X-100, and three washes (PBS/0.5% Triton X-100) were performed between incubation steps. Slides with retinal flat mounts were briefly dried on the slide warmer and coverslipped with a drop of Fluoromount G (SouthernBiotech). Images were taken with either a Zeiss LSM 510 Meta confocal (Carl Zeiss Group, Oberkochen, Germany) or an Olympus XI71 microscope equipped with its own digital camera software (LSM 510 for the confocal and DP70 for the Olympus). For each retina, images were taken from eight, ×20 fields (0.212 and 0.394 mm2, respectively) at half field from the periphery. Quantification was subsequently performed as indicated above.