Adult, female 200–250 g Wistar rats (n=4 eyes per treatment) were anaesthetized intraperitoneally with Hypnorm/Hypnovel anaesthetic (Janssen Pharmaceuticals, Oxford, UK) and the ON of both eyes were crushed (ONC) intraorbitally to completely transect all RGC axons leaving sheath and artery intact. All reagents were intravitreally injected using glass micropipettes in a final volume of 10 μl. Animals were treated with either PBS or with 20 μg control siCNL or with 5, 10 or 20 μg siCASP2 or with 5 μg BDNF (Peprotech Ltd, London, UK) immediately after ONC and within 5 min post-surgery.
At 5 days post-ONC, 2 μl of 4% FG (Cambridge Bioscience, Cambridge, UK) retrograde tracer was injected into the nerve mid-way between the lamina cribrosa and the site of ONC. After 48 h, animals were killed by exposure to CO2 and intracardially perfused with 4% formaldehyde (TAAB Laboratories, Aldermaston, UK). Retinas were dissected out and immersion fixed in 4% formaldehyde (TAAB Laboratories) for 30 min and whole-mounted onto glass slides (VWR International, Lutterworth, UK). Four equidistal radial cuts were made to give four equally sized quadrants, attached together around the optic disc. Retinal whole mounts were air dried and mounted in Vectashield mounting medium (Vector Laboratories, Peterborough, UK). An observer was blinded to the treatments and groups and samples were randomly assigned numbers before image capture and analysis. Images were captured at × 20 magnification using a Zeiss epifluorescent microscope (Zeiss, Hertfordshire, UK) equipped with a Axiocam HRc camera (Zeiss) running the Axiovision software (Zeiss). Images were captured from three different areas of each quadrant (total=12 counts per quadrant, n=4 retinas per treatment, to account for variation of RGC numbers in the different areas and quantified using the built-in particle counting facilities in ImagePro; Media Cybernetics, Bethesda, MD, USA) and expressed as the number of RGC per mm2±S.E.M.
Ahmed Z., Kalinski H., Berry M., Almasieh M., Ashush H., Slager N., Brafman A., Spivak I., Prasad N., Mett I., Shalom E., Alpert E., Di Polo A., Feinstein E, & Logan A. (2011). Ocular neuroprotection by siRNA targeting caspase-2. Cell Death & Disease, 2(6), e173-.