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Hematologic and biochemical analytes of Sprague-Dawley rats are commonly used to determine effects that were induced by treatment and to evaluate organ dysfunction in toxicological safety assessments, but reference intervals have not been well established for these analytes. Reference intervals as presently defined for these analytes in Sprague-Dawley rats have not used internationally recommended statistical method nor stratified by sex.
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Use of vaginal smear VS technique, allows one to identify the different estrus cycle phases proestrus, estrus, metestrus and diestrus based on the proportion among three types of cells: nucleated epithelial cells, cornified cells and leukocytes, the proportion of which varies according to the plasmatic level of estrogen [ 27 ]. Vertical arrows correspond to the stimulus onset.
The retinal function of 50 normal male and female SD rats was investigated with the ERG at age P30, P60, P, P and P [5—6 rats per sex and age groups] in order to evaluate the effect of sex and age on the retinal function. It may explain the higher incidence of some age-related ocular pathologies such as cataract [ 20 ], glaucoma [ 21 ] and acquired macular degenerations [ 22 ] in postmenopausal women that most probably result from the sudden decline in circulating estrogen.
This is better exemplified at Fig 3 where the ERG responses of male and female rats measured at P were normalized to those obtained at P At P, the growth in amplitude of the rod-cone mediated ERG was ificantly lower in male compared to female rats [scotopic a-wave: females, Data was normalized to P30 values. Afterwards, the retinal function in male and female rats decreased from P to P Fig 4 shows representative vaginal smears of both groups.
Briefly, the rats were kept in the dark for at least 12hr i.
Aging is one of the most important contributors to cumulative oxidative stress that could result in the gradual deterioration in function and structure of different tissues including the retina [ 131415 ]. Rat pups remained with their mom until weaning at P21 when they were separated according to their sex. Group data are shown in Table 2. The maximal rod-mediated b-wave rod V maxrepresenting the maximal function of the rods without the contribution of cones, was defined as the first ERG response of the scotopic luminance response function where a measurable a-wave was obtained.
A typical estrus cycle with the four consecutive phases: proestrus, estrus, metestrus, and diestrus could be identified in premenopausal rats. Adult female SD rats Charles River Laboratories, St-Constant, QC were ordered at 15 days of gestation and kept in the normal environment of animal facility where they were allowed food and water ad libitum. Fig 1 shows representative scotopic rod V max responses Ascotopic mixed rod-cone responses B and photopic cone responses C obtained from male and female rats at age P30, P60, P, P and P from left to right.
Interestingly, the maturation indexes for ERG parameters in female rats gradually increased between P60 and P, while those of males did not show this trend. For each rat, the thickness of the entire retina as well as that of each retinal layer i. The six 18 month-old female rats were housed in standard cages, 2 per cage, in the animal care facility. The effect of age on retinal function and structure has also been demonstrated in human and animals [ 111213 ].
Given that the menopausal age of laboratory rats is reported to be between 15 and 18 month [ 26 ], vaginal smears to determine the stage of the estrous cycle were taken from the female rats before the ERG recordings in order to study the effect of estrus cycle equivalent to menstrual cycle in women on the retinal function.
Although the plasmatic levels of estrogen in men remain relatively constant throughout life, in women it fluctuates over a larger range [ 19 ]. Finally, in order to investigate the possible age-related changes in the retinal function over time, a maturation index of ERG parameters was calculated. Abbreviations: a: a-wave; b: b-wave. were compared to those obtained from 5 male SD rats aged 18 months. It is thought that sex-related functional and structural differences in body tissues, including the retina, might be governed by the remarkable male-female sex in sex hormone profiles.
ERG analysis was performed according to the standard practice [ 34 ] as ly reported by us [ 293035 ]. The want of biological sex on the adult retinal function, as determined with the electroretinogram ERGhas been known for more than 60 years [ 1 ].
Proestrus phase Sprague 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.
The images were taken with a 40X objective.
Competing interests: The authors have declared that no competing interests exist. M: male rat.
Our showed that biological sex and age can influence the retinal function and structure of albino SD rats. Estrus cycle phases were identified in premenopausal female rats, whereas no distinct phase could be observed in menopausal female rats. From the structural point of view, spectral domain optical coherence tomography SD-OCT did reveal sex-related differences in the mean thickness of the retinal layers at the macula.
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. The saline was then flushed into the vagina and sucked back into the pipette. Furthermore, the ERG of premenopausal female rats aged 18 months old P was larger compared to age-matched menopausal female rats as well as that of male rats. Sex and age-related changes in retinal structure and function were observed in our animal model.
However, irrespective of age, no ificant difference was observed in ERG and retinal histology obtained from male and female rats. 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.
Our showed that in female rats there was an increase in ERG response amplitudes noticed between 2 and 6.
The retinal samples were collected right after each recording session at P30, P60, P and P [2—4 rats per sex at each age] to assess the role of sex and age on the retinal structure. The resulting fluid was placed on glass slides, stained with 0. The experimental procedure was done under a dim-red light. Given that estrogen has anti-oxidant effects on different tissues of the body and exerts neuroprotection without having to involve a receptor-mediated process [ 1718 ], it could be proposed that sex might also influence the normal aging process.
Furthermore, a possible modulatory effect of the menstrual cycle and the accompanying hormonal fluctuations, especially estrogen, was observed on several ocular structures, including the retina [ 910 ]. Representative scotopic and photopic ERG and rod V max obtained from premenopausal and menopausal groups as well as from age-matched males are presented at Fig 5A. Retinal pictures were taken with a Zeiss microscope Zeiss Axiophot, Zeiss microscope, Germany: 40X equipped with a digital camera.
The latter would suggest a beneficial effect of the estrus cycle on the retinal function.
Age and biological sex are two of the most important regulator of our day-to-day body functions, including retinal function. In this study the VS technique was used in order to determine the menstrual status in female rats through observation of different cell types in the vaginal secretions. Following each recording session P30, P60, P and P two to four male and female rats per age groups were euthanized with CO 2 asphyxiation and their retinas were collected for histology. 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 animals were laid on their right side in a want box [ 32 ] in which a rod desensitizing background light and a photo-stimulator had been installed model PS 22, Grass Instrument, Quincy, MA. Following the scotopic ERG recordings, a background light of 30 cd. The mixed rod-cone a-wave and mixed rod-cone b-wave refer to scotopic ERGs evoked to the highest flash intensity available 0. Fig 2 shows the maturation index of the retinal function in adult and female rats. After the removal of the cornea and lens, the eyecups were placed in 3.
At menopause, the ovaries stop producing sex hormones, while in men the testes never stop producing testosterone, which is partly converted into estradiol in the neural tissues [ 19 ]. In view of the above, the Sprague of the present study was to investigate the effect of biological sex and age on the retinal function and structure of aging male and female albino Sprague-Dawley SD rats.
No equivalent distinct estrus phase could be observed in menopausal group. It has been reported that the antioxidant properties of some tissues such as the heart, kidneys, liver and brain was ificantly higher in female rats explaining the longer lifespan in females [ 16 ]. Following the ERG recording sessions, retinal histology was performed in both sexes. This is an open access article distributed under the terms of the Creative Commons Attributionwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
In sex, the retinal function of premenopausal and menopausal female rats aged P were also compared. Briefly, an orientation suture was placed on the nasal conjunctiva of both eyes then the eyes were enucleated and immersed for 3 hours in 3.
Furthermore, our also showed that premenopausal female rats had larger ERG amplitudes compared to menopausal rats, further confirming the role of the estrus cycle on the retinal function. Horizontal calibration: 40 msec. All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology ARVO statement for the use of animals in ophthalmic and vision research and were approved by the McGill University-Montreal Children's Hospital animal care committee.
Supporting the latter claim, studies reported a reduced occurrence of cataract and age-related macular degeneration in women on estrogen replacement therapy compared to those not receiving hormonal supplementation [ 22232425 ]. Premenopausal females showed higher ERG responses compared to the menopausal females. The funders had no role in study de, data collection and analysis, decision to publish, or preparation of the manuscript.
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Amplitude measurements of ERG parameters obtained from male and female rats at five different ages are reported in Table 1. On each section thickness measurement was done at two to three randomly selected locations. The amplitude of the a-wave was measured from baseline to the most negative trough, while the amplitude of the b-wave was measured from the trough of the a-wave to the most positive peak of the retinal response.
Electroretinograms are usually reported to be of larger amplitudes in women compared to men [ 2 ]. Of interest, the presence of estrogen receptors in different retinal layers [ 56 ] suggests that this sexual hormone might play an important role in the normal functioning of this tissue [ 78 ]. F: female rat. Notwithstanding the above we did however notice that between P60 and P there was a gradual increase in ERG amplitudes of female rats compared to males.
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In the present investigation, we examined the influence of both genetic background and sex factors in the rat hypothalamo-pituitary-adrenal HPA axis function under both basal and post adrenalectomy ADX conditions.
research in our laboratory indicates that the psychotomimetic drug phencyclidine PCP reduces voluntary sucrose consumption in male rats, potentially modeling the schizophrenic symptom of anhedonia.