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Hui Li, Stephen Poor, Chad Bigelow, Vivian Choi, Shawn Hanks, Joanna Vrouvlianis, Michael Maker, Steve Louie, Sha-Mei Liao, Bruce Jaffee; A comparison of the effects of subretinal injection of scAAV2-CMV-GFP on retinal structure, visual function, and GFP expression by two injectors. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2740.
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© ARVO (1962-2015); The Authors (2016-present)
To compare the effects of subretinal (SR) injection of adeno-associated virus (AAV) expressing GFP and saline by two scientists, using optical coherence tomography (OCT) and electroretinography (ERG) and RPE/Choroid tissue flat mount for GFP expression.
1μl of scAAV2-CMV-GFP (5.67x108 DNase resistant particle/μl) or saline, both mixed with sodium fluorescein was injected subretinally into the eyes of C57Bl/6 mice (16 eyes /treatment group/operator). Injection quality was evaluated at the time of the procedure using a 3-level scoring system (1= no hemorrhage minimal intravitreal backflow, score 2 = minor hemorrhage and mild backflow, score 3 = moderate hemorrhage, significant backflow). Fundus images were acquired immediately after the SR injection. OCT was performed 1 week post-injection and ERG recordings were performed 2 weeks post-injection. RPE-choroidal flatmounts were made from ocular tissues collected 3 weeks post-injection. Levels of GFP expression were quantified using Axiovision software to delineate fluorescent area on the flat mounts.
59 of 64 injections were scored a “1”, 3 injections were scored “2”, and 2 injections were scored “3”. Eyes with an injection score of 1 or 2 were used for OCT and ERG quantification. Retinal structure 1 week post subretinal injection was overall similar by OCT examination with a slight increase in incidence of injection site trauma and reduced media clarity for one operator. Photoreceptor function measured with a single flash intensity ERG showed an average drop of approximately 18% vs. uninjected controls (no statistically significant) for both operators and injection materials. No statistically significant difference in function was detected between groups injected by each operator or between materials injected (AAV vector vs. saline). GFP expression per percentage of flatmount area for score 1 was 74%, score 2 was 60%, and score 3 was 25%. No significant difference in GFP coverage was observed between operators.
Subretinal injections disrupted retinal structure to a similar degree for both injection materials (vector and saline). ERGs from injected eyes were reduced in amplitude vs. uninjected controls, but the reduction was independent of individual operator or injection material. Poor quality injections predicted a smaller area of GFP expression.
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