Postnatal day 14 Sprague Dawley rats or P21-28 mice were anaesthetized with 2.5% inhaled isoflurane in 2.0 L O2/min. Without incision to the orbital rim, the supero-external orbital contents were blunt-dissected, the superior and lateral rectis muscles teased apart, and the left optic nerve exposed. The nerve was crushed for 3–5 seconds at approximately 2 mm distal to the lamina cribrosa. After surgery, the eye fundi were checked to ensure retinal blood flow was intact. Some rats also received a 2 μl intravitreal injection of neutralizing antibodies to Il-1α (150 μg/μl, abcam, ab9614), TNFα (150 μg/μl, Cell Signaling Technology, #7321), and C1q (Quidel, A301), rabbit IgG control (150 μg/μl, abcam, ab27472), or PBS at day 0 (the time of optic nerve crush, for 7 and 14 day experiments) or day 7 (for some 14 day experiments). Retinas were collected for qPCR analysis and immunofluorescent analysis at 7 and 14 days.
