Figure 3. Increase in area and level of GFP expression after a second suprachoroidal injection of AAV8.GFP in rats.

Brown Norway rats (n = 21) were given a 3 μL suprachoroidal injection containing 2.85 × 10¹⁰ GCs of AAV8.GFP vector superiorly in both eyes. Three days later, one eye was given an inferior 3 μL suprachoroidal injection containing 2.85 × 10¹⁰ GCs of AAV8.GFP and the other eye received no injection. After 11 days, RPE flat mounts showed high GFP fluorescence in 1 localized region of eyes that had received 1 injection (A) and in 2 regions of eyes that received 2 injections (B); scale bars: 1000 μm. The mean percentage of total RPE area that was fluorescent was significantly greater in eyes that received 2 injections (30.52%) compared with those that received 1 injection (18.85 %, n = 6, P = 0.026 by Mann-Whitney U test). (C) For the remaining 15 rats, 2 eyes in the single injection group had been traumatized and could not be used, but for the remainder, homogenates of RPE/choroid and retinas were used to measure total protein and then GFP protein was measured by ELISA. The mean (± SEM) level of GFP per mg protein was significantly higher in retinas and RPE/choroid from eyes that received 2 injections compared with those that received 1 injection. *P = 0.0008; **P = 0.005 by Mann-Whitney U test and unpaired t test.