Effects of long-term administration of 9-cis-retinyl acetate on visual function in mice

Abstract

Purpose: Long-term effects of treatment with 9-cis-retinyl acetate (9-cis-R-Ac), an artificial retinoid prodrug, were tested on changes in rod and cone visual functions in mice.

Methods: The acetyl ester of the functional geometric chromophore 9-cis-retinal was delivered by oral gavage to C57BL/6 female mice. In initial experiments, 10-month-old mice were used for the single treatment with 9-cis-R-Ac or the control vehicle. In long-term experiments, 4-month-old mice were treated with 9-cis-R-Ac monthly for 6 and 10 months. Photoreceptor status was evaluated by various electroretinographic (ERG) techniques, retinoid analyses, and retinal morphology. Opsin, the predicted target of oxidized 9-cis-R-Ac, was purified and its chromophore was characterized.

Results: Age-related changes observed in vehicle-treated mice at 10 months of age, compared with those in 4-month-old mice, included a progressive decline in ERG responses, such as a decreased rate of dark adaptation and a lowered rhodopsin/opsin ratio. Administration of 9-cis-R-Ac increased the rhodopsin regeneration ratio, and improved ERG responses and dark adaptation. Compared with vehicle-treated control animals, 10- and 14-month-old mice treated monthly with 9-cis-R-Ac for 6 or 10 months exhibited improved dark adaptation. In 14-month-old mice treated monthly, changes in the expression of retina-specific genes in the eye were detected by mRNA expression profiling, but no significant effects in gene expression were detected in the liver and kidney.

Conclusions: Deteriorating photoreceptor function documented in mice at 10 and 14 versus 4 months of age was improved significantly by long-term, monthly administration of 9-cis-R-Ac. These findings suggest a potential therapeutic approach to prevent age-related retinal dysfunction.

Fig. 1. Experimental protocol for single dose treatment of 10-month-old mice with 9-cis-R-Ac and timelines for multiple dose long-term treatment regimes

(A) Fully dark-adapted (48 hr) 10-month-old mice were gavaged with 9-cis-R-Ac(~80 mg/kg body weight) or control vehicle vegetable oil solution. One hr after gavage, mice were exposed to continuous strong light at 500 cd·m⁻² for 20 min (~90% rhodopsin bleach) followed by 16 hr of dark adaptation. Mice then were examined by ERG and eyes were analyzed for rhodopsin and retinoid content. ERGs also were recorded prior to the 9-cis-R-Ac treatment. (B)Mice were gavaged monthly with 9 -cis-R-Ac (~80 mg/kg body weight) or control vehicle solution for 6 or 10 months as described in Methods.

Fig. 2. Single flash ERGs of 10-month-old mice treated with a single dose of 9-cis-R-Ac

Mice were treated with 9-cis-R-Ac or control vehicle by gastric gavage as described in Materials and Methods (Fig. 1A). Animals were dark-adaptated for 16 hr after bleaching with intense light and scotopic (A, B) and photopic (C, D) ERGs were recorded. Bars represent ±1 standard errors of the mean, and gray colored background areas represent responses from pretreated 10-month-old mice. The a-wave amplitudes of 9-cis-R-Ac treated mice increased significantly (A, *; p<0.01) whereas b-wave amplitudes showed slight improvement under scotopic conditions (B). No significant differences were observed under photopic conditions (C, D). Statistical analyses were performed by one-way ANOVA.

Fig. 3. Characterization of purified visual pigments from 10-month-old mice treated with a single dose of 9-cis-R-Ac

Mouse eye visual pigments; rhodopsin, isorhodopsin and opsin were copurified as described in Methods from 10-month-old animals treated with a single dose of 9-cis-R-Ac as shown in Fig. 1A. The regeneration level of purified rhodopsin/isorhodopsin/opsin was calculated from the ratio of absorbance at 500 nm (opsin with bound chromophore) to that of total opsin at 280 nm. (A). Above. Normalized representative absorbance spectra of purified rhodopsin from a 10-month-old 9-cis-R-Ac treated mouse (a, solid line) and a control mouse (b, dashed line) are shown. The bar indicates 0.02 AU. Below. The regeneration ratio of the 9-cis-R-Ac treated group was slightly higher than the control group (n = 3, p < 0.02), indicating that the treated group had a lower level of unliganded (free) opsin (a). Means ± S.D. are indicated. (B). Retinoids in the purified mouse visual pigment were extracted as described in Methods. 11-cis-Retinal was detected in the visual pigment from WT mice whereas both 11-cis-retinal and 9-cis-retinal (I) were observed in treated mice. Other minor cis-retinoids that could form during sample preparation (i.e. 13-cis-retinal (II)) also were detected in samples from treated and control mice.

Fig. 3. Characterization of purified visual pigments from 10-month-old mice treated with a single dose of 9-cis-R-Ac

Mouse eye visual pigments; rhodopsin, isorhodopsin and opsin were copurified as described in Methods from 10-month-old animals treated with a single dose of 9-cis-R-Ac as shown in Fig. 1A. The regeneration level of purified rhodopsin/isorhodopsin/opsin was calculated from the ratio of absorbance at 500 nm (opsin with bound chromophore) to that of total opsin at 280 nm. (A). Above. Normalized representative absorbance spectra of purified rhodopsin from a 10-month-old 9-cis-R-Ac treated mouse (a, solid line) and a control mouse (b, dashed line) are shown. The bar indicates 0.02 AU. Below. The regeneration ratio of the 9-cis-R-Ac treated group was slightly higher than the control group (n = 3, p < 0.02), indicating that the treated group had a lower level of unliganded (free) opsin (a). Means ± S.D. are indicated. (B). Retinoids in the purified mouse visual pigment were extracted as described in Methods. 11-cis-Retinal was detected in the visual pigment from WT mice whereas both 11-cis-retinal and 9-cis-retinal (I) were observed in treated mice. Other minor cis-retinoids that could form during sample preparation (i.e. 13-cis-retinal (II)) also were detected in samples from treated and control mice.

Fig. 4. Retinoid levels in eyes from 10-month-old mice gavaged with a single dose of 9-cis-R-Ac and exposed to intense light followed by full dark-adaptation

(A) HPLC separation of retinoids from 9-cis-R-Ac treated and vehicle treated control mice. Fatty acid all-trans-retinyl esters eluted first (peak 1) followed by syn-11-cis-retinal oxime (2), syn-all-trans-retinal oxime (3), syn-9-cis-retinal oxime (4) and all-trans-retinol (5). Minor peaks on the chromatograms represent syn-oximes and the asterisk (*) indicates a spike related to a solvent change. Inset(a), an expanded scale of the chromatogram shows peaks 3 and 4 corresponding to levels of syn-all-trans-retinal and syn-9-cis-retinal oximes. Online spectra of these oximes are shown below (3 and 4). Retinoid levels in the eyes from treated and control mice (Fig. 1A) were analyzed by HPLC. Extraction procedures and derivatization with hydroxylamine to improve the recovery of retinals are described in Methods. (B and C) Quantification of retinals and esters. Amounts of 11-cis-retinal and all-trans-retinyl esters were similar in eyes of 9-cis-R-Ac treated and control mice but 9-cis-retinal and 9-cis-retinyl esters were detected only in the eyes of treated mice (n = 3, p < 0.0001). Levels of other non-polar retinoids were similar in 9-cis-R-Ac treated and untreated animals. A significant amount of 9-cis-retinal (peak 4) was detected in the samples from treated mice. Means ± S.D. are indicated.

Fig. 5. ERG responses of 10- and 14-month-old mice treated monthly with 9-cis-R-Ac or control vehicle for various periods

Scotopic and photopic ERGs were recorded from dark-adapted mice as described in Methods. The a- and b-wave amplitudes are plotted as a function of light intensity and error bars are shown (n = 10) and gray colored background areas represent responses from C0 mice. Compared to control 10-month-old mice (C1), responses of 10-month-old mice treated monthly for 6 months with 9-cis-R-Ac (N1) were increased significantly under scotopic (A, B) and photopic (D) conditions (p<0.01) except for a-wave amplitudes under photopic conditions (C). No significant differences between 14-month-old mice treated monthly with 9-cis-R-Ac for either 10 or 4 months (groups N2 and N3) relative to vehicle treated controls (C3) were observed under either scotopic (E, F) or photopic (G, H) conditions.

Fig. 5. ERG responses of 10- and 14-month-old mice treated monthly with 9-cis-R-Ac or control vehicle for various periods

Scotopic and photopic ERGs were recorded from dark-adapted mice as described in Methods. The a- and b-wave amplitudes are plotted as a function of light intensity and error bars are shown (n = 10) and gray colored background areas represent responses from C0 mice. Compared to control 10-month-old mice (C1), responses of 10-month-old mice treated monthly for 6 months with 9-cis-R-Ac (N1) were increased significantly under scotopic (A, B) and photopic (D) conditions (p<0.01) except for a-wave amplitudes under photopic conditions (C). No significant differences between 14-month-old mice treated monthly with 9-cis-R-Ac for either 10 or 4 months (groups N2 and N3) relative to vehicle treated controls (C3) were observed under either scotopic (E, F) or photopic (G, H) conditions.

Fig. 6. Recovery of dark adaptation after intense light bleaching by 10- and 14-month-old mice treated monthly with 9-cis-R-Ac or control vehicle for various periods

(A) Recovery of 10-month-old mice from intense retinal bleaching. Different groups of 48 hr dark-adapted mice were bleached with intense constant illumination (500 cd·m⁻²) for 3 min and a-wave amplitude recovery was monitored by recording single-flash ERGs (−0.2 log cd·s·m⁻²) over a 60 minute dark adaptation period. The rate of recovery was significantly higher in 10-month-old mice treated monthly with 9-cis-R-Ac (N1) than in similarly aged mice gavaged with control vehicle (C1). Moreover, treatment with 9-cis-R-Ac even restored the rate of recovery to that seen in 4-month-old mice. Gray colored background areas represent recovery responses from C0 mice. (B) Recovery of 14-month-old mice from intense retinal bleaching. A significantly higher recovery rate occurred in 14-month-old 9-cis-R-Ac treated mice (N2 and N3) than in similarly aged vehicle gavaged (C2) animals (**, n=5; p<0.001; ***, p<0.0001, respectively). Again, 9-cis-R-Ac treated mice showed the same response as did young 4-month-old mice. Error bars are indicated.

Fig. 7. A2E accumulation in the eyes of control and long-term 9-cis-R-Ac treated mice

(A) Chromatographic separation and spectra of A2E and iso-A2E. A representative HPLC chromatogram of eluted A2E and iso-A2E is shown from a group of 10-month-old N1 mice treated monthly with 9-cis-R-Ac for 6 months (left panel in A). Inset. Magnified elution profiles of A2E and iso-A2E are highlighted. Spectra of these peaks (I and II) represent A2E and iso-A2E, respectively (top right). (B) Amounts of A2E (black bars) and iso-A2E (gray bars) from different experimental groups are shown. Amounts of A2E did not differ significantly among all groups of mice with the exception of N3, where they were slightly decreased (p<0.05). Iso-A2E levels were similar among all groups. Neither compound was detected at significant levels in young 4-month-old untreated mice (C0). Means ± S.D. are indicated (n = 5), ND, not determined.

Fig. 8. Retinal morphology of 9-cis-R-Ac gavaged mice

(A) A representative cross- section of retina from a 4-month-old untreated mouse (C0) is shown. RPE, retinal pigment epithelium; PR, photoreceptors, ROS, rod outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; and GCL, ganglion cell layer. (B) Thicknesses of the ROS and IS and numbers of ONL nuclei in retinas of control and 9-cis-R-Ac treated mice of various ages are shown. Data points are plotted as a function of the distance from the optic nerve head (ONH). Lengths of the ROS and IS of young 4-month-old control mice (C0) were significantly greater than those of all other groups of 10- and 14-month-old mice (*, n=5; p<0.01). No other significant differences were detected. Means ± S.D. are indicated.

Fig. 9. RNA array analyses of control and long-term 9-cis-R-Ac treated mice

Expression levels of 37,364 genes from the eye, liver and kidney of two groups of 10-month-old mice, C2 and N2, were examined with cDNA arrays (Nimblegen). Two independent RNA samples were prepared for microarray hybridization. Representative normalized values of mRNA expression were plotted (control vs. the 9-cis-R-Ac treated group) as scatter plots with Sigma Plot v9.0. Genes expressed more than 2.0 fold and less than 0.5 fold are indicated in red and blue respectively. Further information is available from supplemental Tables S1 and S2. Immunoblots of eye extracts from different groups of mice were probed with various antibodies as described in Methods.