OCT was performed via the previously reported method with slight modifications [25 (link)]. In brief, using a Micron® IV (Phoenix Research Labs, Pleasanton, CA, USA) with a contact lens specifically designed for mouse OCT, OCT was carried out at 17 time points starting from the postnatal day (P) 15 until P294 for P23H rats and at 6 points from P26 until P247 for SD rats. Although we employed a contact lens designed for rats in previous experiments, we found that the contact lens designed for mice provided a better resolution and clearer images, even for rats, in the OCT [25 (link)]. We therefore employed the contact lens designed for mice in the present study. Two to four rats (four to eight eyes) were evaluated at each time point. In addition, to keep the rats’ cornea sufficiently clear during the OCT examination, we prepared four different age groups of the P23H rats and two age groups of SD rats (three to six rats in each group) and performed OCT measurements alternately using these groups of rats (two to three times per group). Rats were anesthetized with an intraperitoneal injection of a mixture of medetomidine hydrochloride (0.315mg/kg), midazolam (2.0mg/kg), and butorphanol tartrate (2.5mg/kg). To prevent any pain associated with injections, rats were first anesthetized by inhalation of 80% carbon dioxide and 20% oxygen prior to the intraperitoneal injection. The physical conditions, including heartbeat and respiratory pattern, of rats were frequently monitored during experiments by inspection and gentle palpation by the researchers. Pupils were dilated by instillation of a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride eyedrops. Corneal surface was protected using a 1.5% hydroxyethylcellulose solution. The rat ocular fundus was simultaneously monitored using a fundus camera, and the position of the retinal OCT image was set horizontally at 1 disc diameter superior to the optic disc. Fifty images were averaged to eliminate the projection artifacts. The acquired OCT images were quantitatively analyzed using the InSight® software program (Phoenix Research Labs). Three to eight images from two to five rats each from the both P23H rat and SD rat groups were selected based on the quality of the pictures in terms of the image sharpness at each time point in order to perform as precise a segmentation analysis using InSight® as possible. The pictures deemed unsuitable for the segmentation analysis because of blurring from rats’ respiratory body movement during OCT experiments were eliminated.
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