34 gauge needle

Intravitreal injection of NMDA (Sigma) was performed as described previously²³ (link) with minor modifications. Briefly, 7-week-old rats were anesthetized with 5% isoflurane, then maintained with 2.5% isoflurane. The pupil was dilated with phenylephrine hydrochloride and tropicamide eye-drops (Wakamoto Pharmaceuticals Co., Ltd., Tokyo, Japan). Subsequently, PBS (vehicle control) or 20 nmol NMDA/eye with or without 5 ng/eye E-LP in a total volume of 4 µl was injected into the vitreous cavity. Injections were performed under a microscope using a 34-gauge needle (Nanopass, Terumo, Tokyo, Japan) connected to a micro syringe (80008; Hamilton, Reno, NV, USA) with an infusion pump (Fusion 200; Chemyx Inc., Stafford, TX, USA). The needle was inserted approximately 1.0 mm behind the corneal limbus. The eyes were enucleated 3 days after NMDA injection, and retinae were collected for immunoblotting and quantitative RT-PCR.

Timolol maleate 0.5%, latanoprost 0.005%, and vehicle solutions were kind gifts from Nitto Medic Co. Ltd. (Toyama, Japan), all of which were administered by eye drop (5 µl) immediately and at 3, 6, 12, and 18 h after laser irradiation. In another independent experiment, an integrated stress response inhibitor (ISRIB; 9 ng/2 µl; Cayman Chemical, Ann Arbor, MI) was injected (2 μl) into the vitreous body of the right eye immediately after laser irradiation using a sterile 34-gauge needle (Terumo, Tokyo, Japan) attached to a Hamilton glass syringe (701 N; Hamilton Co., Reno, NV). For controls, mice were IV injected with 2 μl of 0.01 M PBS into the right eye, after which 0.5% levofloxacin ophthalmic solution (Santen Pharmaceuticals Co., Ltd.) was applied topically to the treated eyes.