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Estrogen or 17β-estradiol (17β-E2) is known to act as an antioxidant and has neuroprotective effects. The purpose of this study is to evaluate the effects of estrogen and estrogen receptor beta (ERβ) on photoreceptor cell structure and function in an experimental model for light-induced photoreceptor degeneration.
Estrogen has a protective function. The protective efficacy of estrogen has been found in a variety of tissues. Different investigators have shown that 17 beta-estradiol protects RPE from oxidative stress. Additionally, reports also suggest that 17β-E2 pretreatment reduces RPE degeneration by up-regulating the apoptosis-related proteins.
Animals
Balb/c mice were generated from breeding pairs obtained from Harlan Laboratories (Indianapolis, IN). Mice were housed in the Medical University of South Carolina animal care facility under a 12-hour light/12-hour dark cycle with access to food and water ad libitum. The ambient light intensity at the eye level of the mice was 85 ± 18 lux. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee.
Light exposure experiments were performed on 3-month-old Balb/c mice. Such experiments consisted of constant fluorescent illumination of approximately 1500 lux for 10 days, as described previously, ensuring that all cages were equidistant to the light source. Furthermore, the control group of mice was kept in the same cages as their experimental littermates.
Baseline and endpoint ERGs were performed on the experimental and control BALB/c mice. ERG recordings were performed as described previously. In short, mice were dark-adapted overnight. After dark adaptation, mice were anesthetized using a ketamine-xylazine cocktail.
Eyes were fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose solution. The tissues were then cut into 12-μm cryostat sections. Slides of sections were stained with 0.1% toluidine blue and rows of photoreceptors in the outer nuclear layer were counted in ten different regions of the entire retina.
Tissues were fixed and cryoprotected as described in histology. After the slides were washed in phosphate buffered saline (PBS); they were blocked with 10% normal goat serum and 3% bovine serum albumin. The tissues were incubated overnight in blocking solution containing the UV-opsin antibody (generously provided by Jeannie Chen, University of Southern California, Los Angeles, CA).
We investigated the effect of estrogen in light-damaged Balb/c mice. After 10 days of constant light, control Balb/c mice had ~4.5 rows of photoreceptors. Rows of photoreceptors were counted in 10 different locations to obtain an average row count per retina. Ten days treatment of 17- beta-estradiol (0.2mg/kg body weight) caused a reduction in rod photoreceptor cell degeneration in light-damaged Balb/c mice.
Discussion
In the present study, we have demonstrated that estrogen has a protective role against light-induced photoreceptor degeneration. It has been shown previously that Balb/c mice are susceptible to constant light exposure which leads to loss of nuclei in the outer nuclear layer (ONL) and loss of retinal function as assessed by electroretinography. Mice with estrogen supplementation (17β estradiol) lose fewer nuclei in the ONL and exhibited higher rod and cone photoreceptor function.
Balb/c mice treated with 17β estradiol also exhibited more UV cone opsin immunoreactivity in the cone outer segments. Functionally, 17β estradiol-treated retinas exhibited better rod photoreceptor function after the 10-day light-damage period. The involvement of estrogen receptor beta (ERβ)-signaling in the protective effects of 17β estradiol treatment was confirmed, documenting that mice co-treated with 17β estradiol and the ERβ-specific antagonist PHTPP no longer exhibited structural and functional improvements.