Group investigates effects of hormone replacement on brain

Estradiol treatment improved the performance of rats with induced menopause in memory tests as well as mitigating symptoms of depression.

By Karina Toledo, in Rio de Janeiro

Agência FAPESP – Estrogen replacement in women during peri- and post-menopause has been associated in observational studies of humans with an improvement in concentration, memory, mood and sleep, as well as a delayed cognitive decline.

To investigate the molecular mechanisms by which sex hormones affect the brain, researchers at the Federal University of Minas Gerais (UFMG) in Brazil have performed experiments with female mice, in which a condition similar to menopause is induced by surgical removal of the ovaries, the most important organs for the production of female sex hormones.

Findings from the study were presented by Grace Schennato Pereira Moraes, a researcher affiliated with UFMG, during the Ninth World Congress of the International Brain Research Organization (IBRO 2015) held in Rio de Janeiro on July 7-11.

“We evaluated the animals by means of behavioral tests and observed the onset of a decline in memory only 12 weeks after surgery, alongside evidence of depression and anxiety,” Moraes told Agência FAPESP. “Treatment with estradiol, the most active form of estrogen, was capable of reversing the symptoms, even when begun after 12 weeks.”

According to Moraes, the findings contradict the theory that there is a window of opportunity for hormone replacement therapy to prevent cognitive impairment. The benefits can apparently be obtained even by late replacement.

“In practice, that’s what happens to many women who only start treatment when they’re disturbed by the symptoms,” Moraes said.

One of the tests used to assess memory consisted of placing a female mouse in a box for about ten minutes, with two identical objects in the center of the box. The researchers measured how long the mouse spent exploring the objects. A few days later it was put in the same environment with one object identical to the object used for the training session and a second object that was different.

“We assumed the animal recalled the object seen before whenever it spent more time exploring the new one,” Moraes explained. “It’s a simple standardized test and widely cited in the literature.”

To evaluate conditioned fear memory, researchers put mice in a box and applied a mild shock to their paws every time a certain sound was emitted. In this test, known as footshock, the animals typically associate the sound with discomfort and freeze whenever they hear it.

“We then put the mice in a different box where they could move about normally,” Moraes said. “The control animals immediately froze when they heard the tone. We quantified the ovariectomized animals’ manifestation of this freezing behavior. The more closely it resembled the behavior of the control animals, the better we assumed their memory to be.”

Signs of depression were evaluated by the forced swimming test, widely used to screen antidepressants. Mice are placed in a water tank, and the researchers measure how long they swim in an effort to escape. Depressed animals typically give up sooner and start to float. Animals given drugs with antidepressant effects tend to keep swimming longer.

The elevated plus maze test was used to gauge anxiety. In this model, mice are placed in a plus-shaped maze with two open and two enclosed arms. The more anxious the animals, the longer they tend to remain in the enclosed arms and the less they explore the open sections.

The next step was to measure the females’ performance in the same behavioral tests after two different treatment protocols with estradiol. In the first, a single injection of the hormone was administered directly to the hippocampus in the twelfth week after surgery. In the second, the hormone was given orally for five weeks, also starting in the twelfth week after ovariectomy.

The acute protocol improved the animals’ performance in the object recognition test and the forced swimming test, which measures depression, so that the results were equivalent to those of the control group. However, there was no improvement either in the conditioned fear test or in the elevated maze test, which assesses anxiety. “These two symptoms appear to be unrelated to the presence of estradiol, at least in the hippocampus,” Moraes said. “Now we’re investigating whether application of this hormone to the amygdala has any effect.”

After chronic treatment lasting five weeks, the researchers observed an improvement in object recognition memory. The other behavioral tests have not yet been applied in this treatment protocol.

Analysis of brain tissue from the mice in the twelfth week after ovariectomy showed a decrease in the expression of estrogen receptors in the hippocampus. For Moraes, however, it would be premature to conclude that this caused the cognitive decline observed.

“What we plan to do next is assess the expression of these receptors in the sixth week after surgery, before the symptoms are manifested,” she said.

According to Moraes, there are two types of estrogen receptor (ER) in this brain region, ER alpha and ER beta. The observed improvement in memory tests appears to be associated with stronger activation of ER alpha, whereas the reduction in symptoms of depression appears to be associated with activation of ER beta.

“We assume activation of estrogen receptors in the hippocampus allowed more adequate recruitment of the neurons involved in memory consolidation, but this is speculative; ur next experiments will be designed to try to disprove this theory,” she said.

The latest findings were published in July in the journal Psychoneuroendocrinology. Data from the experiments were also published in two articles in Neurobiology of Learning and Memory, in October 2014 and January 2013.

The findings suggest that hormone replacement treatment may be beneficial for the brains of menopausal women. It is not suitable for all such women, however.

“A thorough medical assessment, including family history, is necessary first,” Moraes said. “The main contraindication is when there are cases of breast cancer among close relatives.”

Perimenopause

Ovariectomy was a requisite part of the experiments because in mice, unlike women, menopause does not occur naturally. Thus, one of the limitations of the study conducted at UFMG is that it could not accurately mimic the slow and gradual decline in sex hormone production during the period known as perimenopause.

The perimenopause begins when a woman is approximately 40 and persists for roughly a decade, until her last menstruation. During this phase, most women experience unpleasant symptoms such as hot flashes, depression, insomnia, anxiety and aggressiveness.

Moreover, the fact that the brains of ovariectomized animals are still young could bias the results of experiments designed to identify the action of hormone replacement in the central nervous system.

To address these issues, Moraes is beginning a collaboration with a group of researchers at the University of São Paulo’s Ribeirão Preto Dentistry School (FORP-USP), led by Professor Janete Franci, whose research includes experiments designed to validate an animal model that mimics the perimenopausal process in rats.

FAPESP has awarded scholarships to several members of her research group, such as Paulo de Tarso Silva Barros, Karin Viana Weissheimer, Arikawe Adesina Paul and Cristiane Mota Leite.

According to Franci, some years ago it was discovered that 4-vinylcyclohexene diepoxide (VCD), a chemical compound released during the manufacturing of synthetic rubber, insecticides and some plastics, destroys ovarian follicles, the structures in which eggs are stored, and can cause infertility.

“We start treating female rats with injections of VCD when they’re 30 days old; he treatment lasts a fortnight,” Franci said. “We wait 80 days to see if symptoms of ovarian failure appear. We’re interested in studying mood disorders in these animals during this transition period, to see whether they’re similar to those observed in women.”

The behavioral tests performed to date have shown that VCD-induced perimenopausal rats display more symptoms of depression (forced swimming) and anxiety (elevated maze) than a control group.

“We’re now doing new tests that suggest they also become more aggressive,” Franci said. “We plan to investigate why this happens, in partnership with the group at UFMG. We can use the animal model to study changes in the brain, which we can’t do with humans, and also to find out what type of drug might prevent these undesirable symptoms from appearing.”