Quantitative analysis of subretinal injections in the rat

Background: Experimental therapeutic approaches to retinal degenerations often require the subretinal injection of a therapeutic agent. The injected volume and the age of the animal can influence the proportion of the retinal surface affected by the subretinal injection. We have investigated the quantitative effect of a single injection in the subretinal space. Methods: Normal and Royal College of Surgeons rats aged 1 week, 3 weeks or 2 months received subretinal transscleral injections of 1, 3, 5 or 10 µl China ink. After 24 h, animals were killed, injected eyes were enucleated and fixated, and the retinas flattened. An image analyzing program was used to measure the total retinal surface and the retinal surface affected by the dye. Results: The mean retinal surface affected by the injection ranged from 5.24±2.76 mm2 to 14.8±2.3 mm2, depending on ani- mal age and injected volume. The injection affected 8.79±0.89 to 36.9±8.13% of total retinal surface. There was no statistically significant difference between normal and Royal College of Surgeons rats. Intravitreal leakage of the dye was more frequent with increasing injection volumes. Conclusion: The retinal surface affected by a single subretinal injection increases with the injected volume, but this increase is not proportional. Higher volumes lead to a loss of injected solution, either in the vitreous body or through the sclero-tomy. In 2-month-old rats, a 3-µl subretinal injection appears to have the best reproducibility, with 20–30% of retinal surface covered by the injected dye.