The Cognitive Effects Of Dydrogesterone On Rat Menopausal Models Of Different Ages

Cognitive function deteriorates across aging in humans, especially for women. As we all know, sex hormones can modulate reproductive functions by affecting the reproductive related brain regions (such as the hypothalamus, pituitary). Brain is a target organ of sex hormones. Cognitive domains can also be regulated by the sex hormones. Estrogen and progesterone can modulate the metabolism of neurons, the release of neurotransmitters, the generation of amyloid (Aβ) and lipid peroxidation, etc. Along with the menopause, the ovarian production of estrogen (E2) and progesterone (P4) decreases rapidly. The changes have the potential to influence the function of the central nervous system. The incidence of mild cognition impairment (MCI) and Alzheimer’s disease (AD) is dramatically increased in postmenopausal women. Results from multiple large-scale survey studies also show that 62—70% of the postmenopausal women report noticeable cognition and memory complaints. Furthermore, premature menopause, especially the iatrogenic menopause, can increase the risk of cognitive decline and dementia. The younger when the menopause occurs, the higher the morbidity of cognitive decline. The global consensus of’The International Menopause Society (IMS)’ recommends that hormone replacement therapy should be taken by the premature menopausal women until the natural menopause. The duration of hormone loss, rather than the senility, appears to be the determinant factor of the cognitive status. Thus the cognitive decline is not only an issue of aging, but also associated with gonadal steroid deficiency.Hormone replacement therapy (HRT) is expected to show efficacy in preventing degenerative cognitive decline. HRT has been used in clinical for decades. It has experienced a flexuous process and has made some achievements. At present, HRT is the most effective treatment to alleviate climacteric symptoms (such as vasomotor symptoms, urogenital atrophy, neuropsychiatric symptoms, osteoporosis, etc.). However, its use has brought some potential threats, such as the possibility to increase the incidence of Alzheimer’s disease (AD), thrombosis, breast cancer and endometrial cancer. With the aging of the population, there have been more than one hundred million menopausal women in China. On clinical and preclinical, the cognitive effects of HRT have been extensively studied, but the results are divergent. It has important significance for further research on HRT.The type of the progestin and the age of the experimental participants may affect HRT’s cognitive effect. Estrogen (E2) has been generally considered as effective to improve cognition and to delay the onset of AD. In addition, its efficacy on alleviating hot flashes, osteoporosis and other symptoms has been affirmed. Besides, it has been gradually realized that, the use of progestin is necessary to antagonize estrogen’s proliferative effect on the uteri. However, we know little about the cognitive effect of progestin. There have been numerous researches in this field. Nevertheless, results are equivocal. Results from the WHI indicate that medroxyprogesterone acetate (MPA), the most commonly used synthetic progestin in HRT, combined with CEE fails to prevent the age-related cognitive decline and even increases the risk of dementia in menopausal women. Some studies show that different progestin exert different neural effects. Maybe, when other progestins (such as natural progesterone) combined with estrogen, the cognitive results will be different. In addition, people enrolled in WHI (mean 63.3 years old) are too old to be benefit from the treatment. HRT is suggested to be used within 10 years after menopause. The risks and benefits of MHT differ for women of different ages. It’s meaningful to further study the choice of progestin and the best timing of treatment.DG, dydrogesterone (6-dehydro-retroprogesterone) is a synthetic retro-progesterone. It is very close to the natural P4 in structure but with greater oral availability and receptor selectivity. It is 10-20 times more orally potent than the natural P4. The unique retro-structure makes it different from other progestins in that it binds almost exclusively to the progesterone receptor (PR). Thus, it shows limited side effects.DG has been widely used in HRT. Clinical data show that DG provides good endometrial protection and shows advantages over the other kindof progestins in preventing breast cancer. Moreover, E2/DG therapy shows to be effective in relief of cardiovascular risk, osteoporosis-related fractures and neuropsychic symptoms. There are few published clinical or pre clinical studies that have investigated the effects of DG in a combined HRT regimen on cognition.It’s important to use a qualified animal model which could maximally reflect the physiological characteristics of the menopause-induced cognitive changes. HRT has not been widely used in clinical, and the results of the’Cognitive Abilities Test’can be affected by many factors. Thus, clinical research is difficult to carry out. Rats have stable physiological characteristics, and are cheap and easy to obtain. In addition, animal experiment has good controllability.The objective of the present study is to investigate the chronic effects of DG on the cognitive functions in the menopausal rat models and also the impact of aging on DG’s effect. The establishment of an appropriate animal model is important for the further understanding of the prevention and treatment of degenerative cognitive decline. First, the ovarian function was reflected by vaginal smear. Then we evaluated the cognitive function of each age group. These help to establish climacteric rat model. In the second part, the chronic cognitive effect of DG and its mechanism was investigated. To make clear these issues would help to provide new ideas for the treatment of postmenopausal cognitive decline.Part I The Establishment of Menopausal Rat Model1. Assessment of the ovarian function and cognitive ability of rats in different age groupsObjective:Select appropriate rats to establish animal model.Methods:1. Evaluation of the estrous cycle’s effect on cognitive ability. Adult female rats were divided into four groups according to the vaginal cytology: proestrus group, estrus group, metestrus group and diestrus group.1-day Morris water maze task and the open field test were used to detect the cognitive abilities of rats in different phases.2. Assessment of the ovarian function statusHealthy female rats distributed over 5 different age groups (3,7,12,18,24-months old) were used. Changes of estrous cycles with aging were monitored by vaginal smear cytology.3. Detection of the cognitive function Classical Morris water maze was used to detect the spatial learning and memory abilities of rats; the open field test was carried out to evaluate the locomotor activity, exploratory behavior and anxiety.Results:1. The cognitive ability of healthy adult rats showed no significant difference during the estrous cycle.2. The estrous cycle changed with age. The adult rats showed regular estrous cycles, with 4-6 days cycle length; estrous cycle of the 12-month old rats still exist, but with prolonged or irregular cycle length; the cycle disappeared in 18-month old rats and characterized as persistent diestrus or pseudopregnancy; 24-month old rats were in persistent diestrus.3. The cognitive ability of rats deteriorated across aging.Conclusion:1. The estrous cycles of SD rats gradually become irregular by aging, which suggested that the ovarian function declined gradually. The process of ovarian dysfunction is relatively slow.2. With aging, the cognitive function declined, which may be due to the dysfunction of the ovary.3.7,18-month old healthy female SD rats were chosen for animal model establishment. 2 Establishment of rat menopausal modelObjectiveTo establish rat menopausal model by ovariectomyMethods:1. OvariectomyFemale Sprague-Dawley rats (7-month and 18-month old) were randomly divided into three groups respectively, the control group, the Sham group and the OVX group. Rats in the OVX group were ovariectomized.2. vaginal smearTo ensure the estrous cycle had stopped, vaginal smears were taken daily. With Eosin and Methylene Blue staining, cellular typology was observed under the light microscope. Constant leukocyte predominance in smears indicated that OVX rats were in persistent-diestrus. The surgery was successful.3. Behavioral tests1 month after surgery, Morris water maze and the open field test were used to test the cognitive function of each group.After behavioral tests, rats were sacrificed. The morphological changes and the weight of the uteri were observed.Results:1. OVX led to the loss of ovarian function. After bilateral ovariectomy (OVX), estrous cycles gradually disappeared. Rats got into persistent diestrus. The estrous cycle disappeared aboutl5 days after the surgery.2. OVX impaired the cognitive function.The escape latencies in OVX rats were much longer than that of the Sham group (P<0.01). In the probe trial, the OVX group spent less time in the target quadrant than the Sham group (P<0.01). The rearings in the open field test decreased (P<0.05).3. Changes of the uteriThe uterine volume reduced obviously after OVX. The uterine weight was significantly decreased (P<0.01), compared with the control group. There was no significant difference between the control group and the sham group.Conclusion:The estrous cycle disappeared and the uterus was atrophy after OVX. Moreover, the Cognitive abilities were declined. These suggested that the model establishment was successful.Part II The Cognitive Effects of Dydrogesterone on Rat Menopausal ModelsObjectives:Investigate the chronic cognitive effect of dydrogesterone on OVX rats of different ages as well as the related mechanism.Methods:Each age cohort was randomly divided into five groups:Sham (Sham operation +placebo, as positive control), OVX (OVX+ placebo, as negative control),OVX+E2 (OVX+ estrogen),OVX+E2/DG (OVX+ estrogen +DG) and OVX+DG. The corresponding therapy was administered.After 20 weeks of treatments, the behavioral tests began to evaluate the cognitive function. Morris water maze task was used to detect the spatial learning and memory abilities; the open field test was carried out to evaluate the locomotor activity, exploratory behavior and anxiety. After behavioral tests, rats were sacrificed for the following researches.After sacrifice, the uteri and the fourth mammary gland were removed. The "Y" shaped uteri were weighed and the fourth left mammary gland were fixed in 4% paraformaldehyde overnight before paraffin embedding. Hematoxylin and Eosin staining (H&E staining) was used to observe the morphology of the breast. The uterine weight was used to evaluate the effect of hormones on the uteri.After sacrifice, the brain was removed. Detect the expression of hippocampal pro-inflammatory factors (IL-1 and TNF-a) by real-time quantitative PCR and ELISA; Immunohistochemical stain for NeuN, Iba-1 and Aβ1-42; Detect cell apoptosis in the hippocampus by Tunel.Results:1. The escape latencies in Adult-OVX rats were much longer than that of the Adult-Sham group (P<0.01). In the probe trial, the Adult-OVX group spent less time in the target quadrant than the Adult-Sham group (P<0.01).2. The escape latency showed no significant changes between the OVX rats and the OVX+DG rats. The progestin-alone treatment has no significant benefit for the OVX rats of both age cohorts on the behavioral tests.3. Both the OVX+E2 group and the OVX+E2/DG groups spent less time than the OVX group to find the platform. The probe trial showed the similar trend.4. For both age groups, the uterine weight decreased after ovariectomy obviously. The progestin treated groups showed the similar data as the OVX rats. The weight increased under the influence of the E2. The addition of progestin alleviated the uterine weight gain.5. The breast atrophied after OVX. The use of E2 can ease the mammary gland atrophy; mammary lobular and ductal is well-developed. When DG was used alone, the mammary gland showed no difference with the OVX rats. The mammary glands of the aged Sham rats were highly hyperplasia. Ovariectomy could protect the breast.6. After OVX, The inflammatory cytokines increased in the hippocampus. The results showed that, the expression of IL-1β and TNFa was elevated after OVX, especially for the aged rats. Level of cytokines in rats of OVX+E2/DG group was lower than that of the OVX group. However, the DG-alone treatment showed little effect.7. The number of the microglia in hippocampus increased significantly in aged rats. OVX induced activation of microglia in middle-aged rats; the number of microglia cells of rats in the OVX+E2/DG group was less than that in the OVX group. There was no significant difference for the aged rats.8. After OVX, the Aβ1-42 level increased significantly in middle-aged rats. The A(3 1-42 level in OVX+E2 group and OVX+E2/DG group was significantly lower than that of the OVX group.The number of neurons in middle-aged rats significantly reduced after OVX. E2/DG was effective in protecting neurons from apoptosis.Conclusion:The DG-alone treatment has no significant benefit for the OVX rats of both age cohorts on the behavioral tests. When combined with E2, the performance is comparable to, if not better than, the E2-alone treatment. The E2/D treatment could ameliorate cognitive performance in adult rats with uterus protection and without breast harm. The cognitive improve effects are more remarkable for the adult rats than the aged. The effect of DG on cognition may be related to its regulation on neuro-inflammation. These results suggest that E2/DG is relatively effective and safe in preventing cognitive decline.