Quantitative Retinal Layer Analysis in Rats

OCT was performed using a Micron^® IV (Phoenix Research Labs, Pleasanton, CA) with a contact lens specifically designed for rat OCT. In the RCS^-/- rats, OCT was conducted at 12 time points between postnatal (PN) day 17 and PN day 111, while in the RCS^+/+ rats, OCT was carried out at eight time points between PN day 18 and PN day 67. At each time point, four to eight rats (eight to 16 eyes) were evaluated. The rats were anesthetized by intraperitoneal injection of a mixture of medetomidine hydrochloride (0.315mg/kg), midazolam (2.0mg/kg), and butorphanol tartrate (2.5mg/kg). To alleviate the pain associated with injection, the rats were pre-anesthetized by inhalation of 80% carbon dioxide and 20% oxygen prior to the intraperitoneal injection. The researchers monitored the physical conditions of the rats including heart beat and respiratory pattern, by inspection and gentle palpation every minute during the experiment. The pupils were dilated using eyedrops that contained a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride. The corneal surface was protected using a 1.5% hydroxyethylcellulose solution. The rat ocular fundus was monitored using the fundus camera of the Micron^® IV, and the position of the retinal OCT image was set horizontally at one disc diameter superior to the optic disc. Fifty images were averaged to eliminate projection artifacts. The acquired OCT images were quantitatively analyzed using the InSight^® software (Phoenix Research Labs). Five images from five rats in each genotype group were selected at each time point on the basis of image sharpness; importantly to avoid selection bias, the pictures were not selected by thickness or reflectivity. We measured the thicknesses of the inner (A, Fig 1), middle (B, Fig 1), and outer (C, Fig 1) layers of the neural retina, as well as that of the combined RPE and choroid (D, Fig 1). The middle layer consists of the combined outer plexiform and outer nuclear layers, and the outer layer consists of the photoreceptor inner segment (IS) and OS layers (Fig 1).