title: MB and AAV-SIRT1 8 weeks
design: Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (9 mg/kg) and treated by topical application of proparacaine (0.5%). Elevation of intraocular pressure (IOP) was induced unilaterally by injection of polystyrene microbeads (MBs) (15-m diameter) into the anterior chamber of the right eye of each animal using a 33 gauge  needle. MBs were prepared at a concentration of 5.0  106 beads/ml in sterile PBS. IOPs were measured weekly using Icare TONOLAB tonometry (Icare TONOVET, Vantaa, Finland) to ascertain IOP spike. An average of 3 measurements per eye were taken at each time point. For viral vector injection, 8-week-old mice were anesthetized by isoflurane inhalation, and their pupils were dilated by topical application of tropicamide 1% for 1 min, followed by application of phenylephrine 2.5%. A 10-L Hamilton syringe attached to a 33-gauge blunt-end needle was inserted ~1.5 mm into the vitreous cavity. Two microliters of SIRT1-AAV vector preparation (1  1010 vector genomes) were intravitreally injected into the right eye of each mouse. Vehicle-treated left eyes were injected with an equivalent volume of vector dilution buffer (0.001% Pluronic F68 in balanced salt solution aka BSS).  Mice injected treated with AAV-SIRT1 and/or injected with MBs and their respective controls were euthanized. Retinas were then rapidly isolated and snap frozen in liquid nitrogen. Total RNA was extracted from retinas using RNeasy purification kit according to the manufacturers instructions (Qiagen).