The animal housing and light-exposure conditions used in this study have been described in detail previously,
19 and are briefly summarized. Dogs were maintained in kennel runs in a cyclic light environment (7 AM lights on/7 PM lights off) with light intensities that varied between 175 and 350 lux at the level of the “standard” dog eye. For the clinical light exposures, the dark-adapted dogs had the pupils dilated (1% tropicamide, 1% atropine, 10% phenylephrine) in both eyes, and one eye was completely shielded from light (shielded). Under dim red illumination, the other eye was exposed to a series of overlapping retinal photographs (exposed) obtained with a hand-held, manual advance fundus camera (model RC-2; Kowa Company Ltd., Nagoya, Japan) within 4 to 5 hours of light onset (7 AM). With one exception, the right eye was shielded, and the left eye was exposed
(Table 1) . Because the dog retina has regions where the pigment epithelium is (inferior, nontapetal region) and is not (superior, tapetal region) pigmented, different light intensities were used for viewing and photographing these two regions. These adjustments were made with neutral density or gray polarizing film filters and/or adjusting the settings of the photographic flash. For viewing, the approximate retinal illuminances produced by the tungsten bulb were 0.95 and 3.8 mW · cm
−2, respectively, for the tapetal and nontapetal regions. For photography, microsecond duration flashes of a xenon lamp produced approximate retinal doses/flash of 0.6 and 11 mJ · cm
−2, respectively, for the tapetal and nontapetal regions. Fifteen to 17 pictures were taken of the eye during an ∼5-minute period, and these were equally distributed between the tapetal and nontapetal zones.
Based on the light intensities used and activation of AP-1 by the exposure to light,
19 we propose that the light exposure paradigm used causes photoreceptor degeneration via the bright-light pathway, although the genetic tools for testing independence from transducin signaling presently are not available for such studies in the dog model.
3 5 The use of multiple microsecond-duration, bright-light flashes suggests damage by intermittent exposure to light, which has been shown to be more damaging to the retina.
1 However, the short interflash interval used in our studies (∼17–20 seconds) differed from the 1-hour dark-adapted intervals used by Noell et al.
1 in their study of intermittent exposure to light
1 ; thus a direct translation of those studies is not possible.
After exposure to light, the dogs were returned to the dark and killed 1 to 3 hours later for biochemical studies. For immunohistochemistry and conventional morphology, samples were collected at 1 hour and 2 weeks, or at 2 weeks, respectively, after exposure. For the longer postexposure time point, the dogs were returned to the regular kennel environment and maintained under cyclic light conditions until they were killed 2 weeks after exposure.
For collections of retinas from exposed and shielded eyes, the dogs were anesthetized with intravenous pentobarbital sodium in a dark room with dim red illumination, the eyes were enucleated, and the dogs were euthanatized with a barbiturate overdose. The globes were opened with a razor-blade cut anterior to the ora serrata, the posterior segment isolated, and the vitreous removed. The retina was then manually separated from the pigment epithelium and frozen at −80°C until use. For immunohistochemistry, the eyes were processed by using standard techniques previously described.
22