Functional and Anatomic Consequences of Subretinal Dosing in the Cynomolgus Macaque

Abstract

Purpose: : There is growing interest in subretinal delivery of therapeutically targeted viral vectors to treat ocular disease. In this study, we characterized the functional and anatomic consequences of the subretinal injection per se using multifocal electroretinography (mfERG), optical coherence tomography (OCT), immunohistochemistry (IHC) and transmission electron microscopy (TEM).

Methods: : The right eyes of 3 cynomolgus macaques were given subretinal injections (100 µl) of Balanced Saline Solution (BSS^TM). The control (left) eyes received intravitreous injections (100 µl) of BSS. Fundus photography, OCT and mfERG were obtained before and immediately after injection and again 9 and approximately 35, 60 and 90 days later. IHC for GFAP, rhodopsin, S-cone opsin and M/L-cone opsin, and TEM of the retina were also done.

Results: : Subretinal blebs included the superotemporal macula. Expected mild retinal hemorrhage occurred at the injection sites. On the day of injection, mfERG was similar to baseline in the eyes given intravitreous injections. However, there was transient suppression of mfERG responses in the bleb and also in other non-bleb regions. The retinas were re-attached by 2 days after injection. Partial recovery of mfERG amplitudes occurred by 9 days post-injection and was largely, but not completely recovered after 90 days. OCT showed a marked decrease in the inner segment/outer segment (IS/OS) line in the region of the former blebs. There was no change in GFAP and S-cone staining but the rhodopsin and M/L-cone opsins were partially displaced into the inner segments. TEM revealed disorganization of the outer segment rod (but not cone) disks.

Conclusions: : Subretinal injections were well tolerated although some residual effects remained after 90 days. Transient suppression of mfERG responses spatially remote from the bleb was unexpected. While, the mfERG responses nearly recovered, disorganization of the outer segment rod disks remained and may explain the decrement in the IS/OS line on OCT seen in the region of the former blebs.

Keywords: retinal detachment • drug toxicity/drug effects • pathology: experimental