Because three low daily doses of 9-
cis-R-Ac improved ERG responses after 6 days of light exposure
(Figs. 6A 6B 6C 6D 6E 6F) , we tested whether a prolonged intermittent dosing regimen might improve retinal function as well.
Rpe65 −/− mice were split into two groups, each treated for a total of 8 weeks with 1 or 4 mg/kg of 9-
cis-R-Ac. One group was dosed daily for 3 days followed by a 4-day drug holiday during each week of the 8-week regimen (intermittent group), and the other was dosed daily for the full 8-week period (daily group;
Fig. 7 ). Mice were exposed to a daily cycle of 8 hours of fluorescent light with a luminance range of 500 to 1500 lux followed by 16 hours of darkness. Electroretinograms were recorded at day 28 and again at day 56, after which tissues were collected for retinoid analyses of the eye and liver and for histologic examination of the eye. Intermittent dosing and daily dosing regimens evoked dose-dependent increases in the amplitudes of a- and b-waves on days 28 and 56
(Figs. 8A 8B 8C 8D 8E 8F 8G 8H) . Responses were more pronounced in the daily dosed than in the intermittently dosed group. The lower dose (1 mg/kg) was sufficient to cause significant improvement in ERG responses over the control group at high-intensity stimuli, irrespective of the treatment schedule. In addition, a- and b-wave amplitudes were similar at days 28 and 56, suggesting that equilibrium might have been achieved between the intake and storage of 9-
cis-retinol on one hand and its mobilization in the retina to support the retinoid cycle on the other. In agreement with these ERG results, 9-
cis-retinal was detected in a dose-dependent manner in the eyes in which levels were higher in mice dosed daily (Supplementary Fig. S4A). Fatty acid 9-
cis-retinyl esters at low variable levels also were found in the eyes of both sets of treated animals (Supplementary Fig. S4B). A dose-dependent slight increase in fatty acid all-
trans-retinyl esters also was noted in the eyes of treated mice regardless of the regimen. In the liver, 9-
cis-retinol was essentially stored in the form of fatty acid 9-
cis-retinyl esters in a dose- and regimen-dependent manner (Supplementary Figs. S5A, S5B). Levels of fatty acid all-
trans-retinyl esters were not significantly affected by these regimens, though there might have been a slight increase in mice receiving 4 mg/kg 9-
cis-R-Ac. Long-term administration of 9-
cis-R-Ac had a dose-dependent protective effect on the retina, as assessed by the lengths of the photoreceptor outer segments
(Figs. 9A 9C)and the number of nuclei in the outer nuclear layer
(Figs. 9B 9D) ; however, >50% less ROS length and >30% less nuclei numbers were observed in mice receiving 4 mg/kg 9-
cis-R-Ac daily compared with wild-type mice of similar ages in our previous study.
34 These effects were more pronounced in the superior than the inferior retinas of mice treated at the 4-mg/kg dose. Highly magnified images of retinal cross-sections showed improvement in rod outer segment (ROS) morphology and fewer oil droplet-like structures in parts of the superior and inferior portions of retinas from mice treated with the 4-mg/kg daily or the 4-mg/kg intermittent regimens
(Figs. 9E 9F) . However, no significant change was observed in retinas of mice receiving the 1-mg/kg 9-
cis-R-Ac dose by either schedule
(Figs. 9G 9H)compared with retinas of control mice
(Fig. 9I) . These results are consistent with those of our previous study.
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