Subretinal Administration of Fluorescent Microspheres in the Minipig

Abstract

Purpose: : Anatomical characteristics of the minipig eye make it a useful model for development of ocular therapeutics administered by intraocular injection. Subretinal delivery of therapeutic agents allows direct access to photoreceptor or RPE cell targets avoiding barriers associated with intravitreous administration. In this study, we evaluated technical aspects of subretinal dosing in the minipig. Fluorescent microspheres approximating the size of cells were administered to confirm anatomical location of subretinal doses.

Methods: : Both eyes of three Göttingen minipigs were given subretinal injections of fluorescent polystyrene microspheres (15 µm; 3 x10⁴/150 µl dose) in the peripapillary region. Two 23 ga cannulae were inserted through the sclera 3.5 mm posterior to the limbus. A fiber optic light was passed through one of the cannulae. An initial subretinal bleb was made with a balanced salt solution injected through a DORC^® extendible 41 ga subretinal injection needle. The microspheres were then injected via a 30 ga bent needle into the existing bleb. Fundus photography and spectral domain optical coherence tomography (sdOCT) were performed on the day of dosing, 1 and 6 days later. Microscopic evaluation of the dose sites was performed.

Results: : Subretinal blebs containing microspheres could be reliably produced in both eyes of 2 of the 3 animals. Deeply set eyes of 1 animal prevented access to the subretinal space. Some microspheres (<5% of the total) were seen in the vitreous. sdOCT showed resolution of the fluid in the subretinal blebs 1 day following injection with subretinal microspheres present and a small number remaining in the vitreous. Light microscopic evaluation confirmed the presence of subretinal microspheres. Other histomorphologic changes were consistent with previous descriptions of short-term retinal detachments.

Conclusions: : Injection of cell-sized microspheres into the subretinal space of minipigs using minimally invasive techniques and instruments approved for human use is achievable on a fairly consistent basis. However, animals with deep-set globes may be poor candidates for the procedure.

Keywords: ocular irritancy/toxicity testing • retina