Differences in Retinal Structure and Function between Aging Male and Female Sprague-Dawley Rats are Strongly Influenced by the Estrus Cycle

Abstract

Purpose: Biological sex and age are considered as two important factors that may influence the function and structure of the retina, an effect that might be governed by sexual hormones such as estrogen. The purpose of this study was to delineate the influence that biological sex and age exert on the retinal function and structure of rodents and also clarify the effect that the estrus cycle might exert on the retinal function of female rats.

Method: The retinal function of 50 normal male and female albino Sprague-Dawley (SD) rats was investigated with the electroretinogram (ERG) at postnatal day (P) 30, 60, 100, 200, and 300 (n = 5-6 male and female rats/age). Following the ERG recording sessions, retinal histology was performed in both sexes. In parallel, the retinal function of premenopausal and menopausal female rats aged P540 were also compared.

Results: Sex and age-related changes in retinal structure and function were observed in our animal model. However, irrespective of age, no significant difference was observed in ERG and retinal histology obtained from male and female rats. Notwithstanding the above we did however notice that between P60 and P200 there was a gradual increase in ERG amplitudes of female rats compared to males. Furthermore, the ERG of premenopausal female rats aged 18 months old (P540) was larger compared to age-matched menopausal female rats as well as that of male rats.

Conclusion: Our results showed that biological sex and age can influence the retinal function and structure of albino SD rats. Furthermore, we showed that cycled female rats have better retinal function compared to the menopausal female rats suggesting a beneficial effect of the estrus cycle on the retinal function.

Fig 1. Representative scotopic rod V_max (A) and mixed rod-cone (B) ERGs and photopic ERG (C) recorded from a male and a female rat aged 30, 60, 100, 200 and 300 days old (age identified as P at the top up each column).

Vertical arrows correspond to the stimulus onset. Horizontal calibration: 40 msec. Vertical calibration: 200 μV. Abbreviations: a: a-wave; b: b-wave. F: female rat. M: male rat.

Fig 2. Maturation of retinal function in male and female rats (expressed of P30 value in %) at 30, 60, 100, 200 and 300 days of age.

Data was normalized to P30 values. Values are given as mean ± SD.

Fig 3. Percent changes in retinal function of male and female rats between P60 and P200 as determined with the retinal maturation index.

The retinal maturation index represents the average (percentage) of the ratios (P200/P60 X 100) for ERG parameters (scotopic a-wave, scotopic b-wave, photopic b-wave, rod V_max). ** P < 0.01 indicates significant difference between male and female rats at P200. Values are given as mean ± SD.

Fig 4. Representative examples of vaginal smears obtained from premenopausal (the upper images) and menopausal (the bottom images) female rats aged 18 months collected over five consecutive days.

A typical estrus cycle (with the four consecutive phases: proestrus, estrus, metestrus, and diestrus) could be identified in premenopausal rats. Proestrus phase was identified with the presence of nucleated epithelial cells (solid line in image taken at day 4); Estrous phase was recognized with the presence of cornified epithelial cells (dashed line in image taken at day 1); the other two phases (Metestrus and Diestrus) were mainly distinguished with the leukocyte infiltration (images taken at day 2 and 3). No equivalent distinct estrus phase could be observed in menopausal group. The images were taken with a 40X objective.

Fig 5. (A) Representative scotopic (mixed rod-cone and rod V_max) and photopic ERGs recorded from a male rat (first column), premenopausal and menopausal female rats (second and third column, respectively) at age 18 months. (B) Comparison of ERG amplitudes (scotopic a-wave, scotopic b-wave, photopic b-wave, and rod V_max) measured from male rats, premenopausal and menopausal female rats.

**P < 0.01; *** P < 0.001 indicate significant differences between premenopausal and menopausal female rats. Values are given as mean ± SD. Vertical arrows correspond to the stimulus onset. Horizontal calibration: 40 msec. Vertical calibration: 200 μV. Abbreviations: a: a-wave; b: b-wave.

Fig 6. Representative retinal sections obtained at 30, 60, 100, and 300 days of age from female (A) and male adult SD rats (B).

Images were taken between 1020 μm and 1700 μm from the optic nerve head in the superior retina. Abbreviations: RPE: retinal pigmented epithelium; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; RGCL/FL: retinal ganglion cell layer/fiber layer. Calibration bar: 75μm.

Fig 7. Quantitative assessment of retinal structure in male and female rats.

Retinal layer thicknesses in female and male rats (N = 2-4/age) were measured at 30, 60, 100, and 300 days of age. * P < 0.05; *** P < 0.001 indicate significant differences between the thicknesses measured at P300 and those at P30. Values are given as mean ± SD.