Brain tissue oxidative damage as a possible mechanism for the deleterious effect of a chronic high dose of estradiol on learning and memory in ovariectomized rats

Dano oxidativo ao tecido cerebral como possível mecanismo de efeito deletério da alta dose crônica de estradiol no aprendizado e memória de ratas ooforectomizadas

Abstracts

In addition to antioxidative effects, estrogens also exert pro-oxidative actions. The effect of chronic administration of a high dose of estradiol valerate on Morris water maze tasks and brain tissues oxidative damage was investigated. The Sham-Est and OVX-Est groups were treated with estradiol valerate (4 mg/kg) for 12 weeks. Escape latency and traveled path in the Sham-Est and OVX-Est groups were significantly higher than in the Sham and OVX groups (p≪0.01 and p≪0.001). In the probe trial, the animals of the Sham-Est and OVX-Est groups spent lower time in Q₁ compared to Sham and OVX groups (p≪0.05 and p≪0.001). In Sham-Est and OVX-Est groups, the brain tissue total thiol concentration was significantly lower, and malondialdehyde (MDA) concentrations were higher than in the Sham and OVX groups (p≪0.05 and p≪0.001). It is concluded that administration of high exogenous levels of estradiol impairs performance and enhances oxidative stress.

learning; memory; ovariectomy; estradiol; morris water maze; oxidative stress

Além dos efeitos antioxidantes, os estrógenos também têm ação pró-oxidativa. Foi investigado o efeito da administração crônica de alta dose de valereato de estradiol no desempenho do labirinto aquático de Morris e o dano oxidativo ao tecido cerebral. Os grupos Sham-Est e OVX-Est foram tratados com valereato de estradiol (4 mg/kg) por 12 semanas. O tempo de latência para escapada e o caminho percorrido foram significativamente maiores nos grupos Sham-Est e OVX-Est em relação aos grupos Sham e OVX (p≪0,01 e p≪0,001). No estudo probe, os animais dos grupos Sham-Est e OVX-Est levaram menos tempo no Q₁ em comparação aos grupos Sham e OVX (p≪0,05 e p≪0,001). Nos grupos Sham-Est e OVX-Est, a concentração total de tiol foi significativamente menor, enquanto a concentração de malondialdehydo (MDA) for maior do que aquela dos grupos Sham e OVX (p≪0,05 e p≪0,001). Concluiu-se que a administração de altas doses de estradiol exógeno compromete o desempenho e aumenta o estresse oxidativo naqueles animais.

aprendizado; memória; ovariectomia; estradiol; Labirinto Aquático de Morris; estresse oxidativo

Ovarian hormones affect a wide variety of nervous system functions especially the cognitive abilities, learning and memory¹. It has been suggested that estrogen may prevent cognitive impairments due to aging². Increasing in the risk of Alzheimer's disease due to estrogen deficiency has been reported³. It was also found that estrogens have enhancing action on memory tasks⁴. Controversially, some researchers observed no effects or even a negative effect of estrogens on the learning and memory functions⁵,⁶. It seems that these differential effects are related to the used dose and serum estradiol level⁷. Low levels of exogenous estradiol improves learning and memory tasks⁷, whereas administration of high exogenous pharmacological or physiological levels of estradiol impair learning and memory performances⁷,⁸. The results of our previous studies showed that treatment of ovariectomized rats by 2 mg/kg/week of estradiol valerate improved spatial learning and memory while in sham operated rats the effect of estradiol was negative⁴.

The central nervous system (CNS) tissues contain a high level of membranes and fatty acids. It has also been shown that the susceptibility of membrane lipid constituents in the CNS to oxidative injury is very high⁹. It has also been well documented that oxidative damage plays an important role in the pathogenesis of various CNS disorders and neurobehavioral impairments⁹. There is good evidence that oxidative stress and reactive oxygen species contribute in the learning and memory impairments. Stress-induced lipid peroxidation affects learning and memory performances in the rat conversely, and antioxidants have been shown to prevent memory impairments in various experimental models⁹.

Interestingly, antioxidant actions of estrogens have been long recognized in a variety of in vitro and in vivo models¹⁰. Estrogen has been found to protect against a wide range of toxic insults including free radical generators and excitotoxicity¹¹. The beneficial effects of estrogen in the CNS, especially, on learning and memory have been frequently attributed to its protective properties against oxidative damage¹⁰.

The aim of present study was to evaluate the effect chronic administration of a high dose of estradiol valerate on spatial learning and memory tasks and brain tissues oxidative damage in ovariectomized rats.

METHODS

Animals and drugs

Thirty two female Wistar rats with 16 weeks old and weighing 240±10 g were used. The animals were housed in 4–5 per standard cages, at room temperature (22±2ºC), on a 12 h light/dark cycle. Food and water were available ad libitum properly. Animal handling and all related procedures were approved by the Mashhad Medical University Committee on Animal Research. The animals were divided into four groups: (1) Sham, (2) ovariectomized (OVX), (3) Sham-estradiol (Sham-Est) and (4) ovariectomized-estradiol (OVX-Est). The animals in the Sham-Est and OVX-Est groups were treated by weekly injections of estradiol valerate (4 mg/kg; S.c.) for 12 weeks. The animals of Sham and OVX groups received 1 mL/kg of saline instead of estradiol. All animals were tested in Morris water maze. Ketamin was purchased from Alfasan Company (Holand). β- estradiol was kindly provided by Iran - Hormone Pharm (Tehran, Iran). Other chemicals which were used for malondialdehyde (MDA) and total thiol (SH) concentrations were purchased from Merck Company.

Surgery

Before surgery, the rats were permitted 15 days for acclimatization to the animal house. The animals were ovariectomized under ketamine anesthesia (150 mg/kg, i.p.). Anesthesia was confirmed by reduced respiratory rate and no response to gentle pinching of foot pad. Abdominal incision was made through the skin of the flank of the rats and ovaries and ovarian fats were removed. Ovaries were isolated by ligation of the most proximal portion of the oviduct before removal. The same procedure was performed on the Sham rats, except that the wound was closed without removing the ovaries.

Apparatus

The Morris water maze was a black circular pool with a diameter of 136 cm and a height of 60 cm, filled with 24±1ºC water to a depth of 30 cm. The maze was divided geographically into four equal quadrants and release points were designed at each quadrant as North (N), East (E), South (S) and West (W). A hidden circular platform (10 cm in diameter), made of plexiglass, was located in the center of the southeast quadrant, submerged 1.5 cm beneath the surface of the water. Fixed, outside of the maze, visual cues were present at various locations around the maze (i.e. computer, hardwares and posters). A tracking system was used to measure the escape latency and traveled path.

Behavioral assessment

The Morris water maze task for testing spatial memory was assessed in a water tank as described previously. The animals received a block of four trials during five daily sessions. During five days, the platform, situated in the center of the southeast quadrant, was submerged 1.5 cm below the surface of water and therefore invisible. The platform position remained stable during five days. A trial was started by placing a rat into the pool, facing the wall of the tank. Each of four starting positions (N, E, S, W) was used once in a series of four trials and their order was randomized. Each trial was terminated as soon as the rat had climbed onto the platform or when 60 seconds had elapsed. The animal was allowed to stay on the platform for 15 seconds. Then, it was taken from the platform and the next trial was started after 20 seconds. The rats which did not find the platform within 60 s were put on the platform by the experimenter and were allowed to stay there for 15 seconds. After completion of the 4^th trial, the animals were kept warm for an hour, and then returned to their home cage. The escape latency and traveled path were calculated by a computer. In the retention phase, the platform was removed (in sixth day) and a 60 seconds probe trial was conducted to examine how well the rats had learned the exact location of the platform. The animals were allowed to swim for 60 s. The time spent in the target quadrant (Q1) and non target quadrants (Q2–Q4), as well the traveled path, were compared between groups. All tests were conducted between 16h and 18h o'clock.

Biochemical assessment

Finally, the animals were sacrificed and the brain tissues (cortical tissues) were removed, weighed and submitted to determination of SH groups and MDA concentrations. Total SH groups were measured using DTNB (2, 2′-dinitro-5, 5′-dithiodibenzoic acid) as the reagent. This reagent reacts with the SH groups to produce a yellow colored complex which has a peak absorbance at 412 nm. Briefly, 1 mL Tris-EDTA buffer (pH=8.6) was added to 50 µL brain homogenate in 1 mL cuvettes, and sample absorbance was read at 412 nm against Tris-EDTA buffer alone (A1). Then, 20 µL DTNB reagents (10 mM in methanol) were added to the mixture and, after 15 min (stored in laboratory temperature), the sample absorbance was read again (A2). The absorbance of DTNB reagent was also read as a blank (B). Total thiol concentration (mM) was calculated from the following equation.

Total thiol concentration (mM) = (A2-A1-B) × 1.07/0.05 × 13.6

MDA levels, as an index of lipid peroxidation, were measured. MDA reacts with thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS) to produce a red colored complex which has peak absorbance at 535 nm. Two mL from reagent of TBA/trichlorooacetic acid (TCA)/hydrochloric acid (HCL) was added to 1 mL of homogenate, and the solution was heated in a water bath for 40 minutes. After cooling, the whole solutions were centrifuged within 1000 g for 10 minutes. The absorbance was measured at 535 nm. The MDA concentration was calculated as follows: C(m) = Absorbance/(1.56×10⁵).

Statistical analysis

All data were expressed as means±standard error median (SEM). The data of different groups during five days were compared using repeated measures ANOVA test with Tukeys' post hoc between groups. The data obtained from probe trial was compared using one-way ANOVA and post hoc test. Data for MDA and total SH groups were evaluated by one-way ANOVA and post hoc test. Differences were considered statistically significant when p≪0.05.

RESULTS

Escape latency and traveled path in the OVX group were significantly higher than in the Sham group (p≪0.05 and p≪0.001, respectively) (Figs 1 and 2). The animals of the Sham-Est group had significantly higher time and path length to reach the platform compared to Sham group (both p≪0.001) (Figs 1 and 2). In the OVX-Est group, escape latency and traveled path were also significantly higher than in the OVX group (p≪0.01 and p≪0.001, respectively) (Figs 1 and 2). There were no differences between the Sham-Est and OVX-Est groups in escape latency and path length (Figs 1 and 2).

Fig 1
Comparison of time latency (sec) to reach the platform between Sham, OVX, Sham-Est and OVX-Est groups.

Fig 2.
Comparison of the length of swimming path (cm) between Sham, OVX, Sham-Est and OVX-Est groups.

In the probe trial, there were no significant differences in the time spent in target quadrant (Q1) between Sham and OVX groups (Fig 3). However, the animals of OVX group spent more time in non-target quadrant (Q4) in comparison with Sham — operated rats (p≪0.01) (Fig 3). The animals of the Sham-Est group spent lower time in Q1 compared to Sham group (p≪0.05) (Fig 3). The time spent in Q1 by the animals of OVX-Est group was significantly lower than of OVX group (p≪0.01) (Fig 3). The animals of Sham-Est group spent more time in non-target quadrants (Q3 and Q4) when compared with Sham group (both p≪0.05) (Fig 3). The animals of OVX-Est group also spent more time in non-target quadrants (Q3) in comparison with OVX group (p≪0.001) (Fig 3). Fig 4 showed the animals of OVX group swam more distance in non target quadrant (Q4) in comparison with Sham group (p≪0.01). The traveled path length by the animals of OVX-Est group in target quadrant was lower than OVX group (p≪0.001) (Fig 4). However, the animals of OVX-Est group traveled longer distance in non target quadrant(Q3) compared to OVX group (p≪0.001). The animals of Sham-Est group traveled longer distances in non-target quadrants (Q3 and Q4) in comparison Sham group (p≪0.01) (Fig 4).

Fig 3.
The results of the time (sec) spent in each quadrant during the probe trial on day 6 (24 h after the last secession of learning).

Fig 4.
The results of the length of the swimming path (cm) in each quadrant during the probe trial on day 6 (24 h after the last secession of learning).

The total thiol concentration in cortical tissues of ovariectomized rats were significantly lower than Sham animals (p≪0.01). In both OVX-Est and Sham-Est groups, the total thiol concentration were significantly lower than OVX and Sham groups respectively (p≪0.001 and p≪0.05 respectively). There was no significant difference between OVX-Est and Sham-Est groups in thiol group (Fig 5). MDA concentration in cortical tissues of ovariectomized animals was higher than sham operated ones (p≪0.05). Chronic treatment of both the ovariectomized and sham-operated rats by 4 mg/kg estradiol increased MDA concentration in cortical tissues in comparison with saline treated Sham and OVX rats (p≪0.05 and p≪0.01 respectively) (Fig 6). The results also showed that the serum 17 β-estradiol concentration in OVX group was significantly higher than that of sham operated rats (p≪0.001) (Fig 6). Fig 7 shows treatment by 4 mg/kg estradiol increased 17 β-estradiol serum concentration in ovariectomized and sham operated animals (p≪0.001).

Fig 5.
The total thiol concentrations in cortical tissues of four groups.

Fig 6.
Comparison of MDA concentrations in cortical tissues between four groups.

Fig 7.
Comparison of serum estradiol level between four groups.

DISCUSSION

The results of the present study indicated that OVX rats had more time and path length to reach the platform in comparison with Sham group. The results also showed that deprivation of rats from ovarian hormones affects retention phase of Morris water maze in rats. However, there were no significant differences between OVX and Sham-operated rats in the time spent and traveled distance in target quadrant in probe trial when the platform was removed from the tank; the animals of OVX rats spent more time and traveled longer distance in non target quadrant (Q4) in comparison with sham animals. The results of the present study confirmed the results of previous studies showing that ovariectomy impairs spatial memory⁴. However, it was found no effect of surgical menopause on cognitive functioning³.

Estrogen replacement therapy has been reported to prevent or delay cognitive decline in postmenopausal women and in estrogen-depleted animals³,⁴. It has been suggested that the effect of exogenous administration of estradiol on learning and memory of ovariectomized female rats depends on the dose and the estradiol plasma concentration. Low, but physiological, levels of exogenous estradiol enhances performance whereas administration of high exogenous pharmacological or physiological levels of estradiol impairs performance⁷. The results of our previous study also showed that treatment by 2 mg/kg estradiol valerate for eight weeks improves Morris water maze tasks of ovariectomized rats, however deleterious effects of estradiol was seen in the same dose and time when administered to Sham-operated rats⁴. It has also been suggested that, during the breeding season (high estradiol), female voles show poor performance in comparison with males in the spatial version of the water escape task¹². Besides of the dose of estradiol, the discrepancies of the data may be due to the differences in type of memory which is studied or the age of OVX animals.

Estrogen exerts its memory-related effect via multiple pathways including antioxidant properties, which may also have a role in its neuroprotective effects¹⁰. The antioxidant property of the estradiol has been attributed to its free phenolic hydroxyl group on the A-ring of the steroid. Removal or blocking of the phenolic hydroxyl group eliminates the antioxidant effect, as well the neuroprotection properties¹⁰. The results of the present experiment demonstrated that SH contents in cortical tissues were decreased and MDA concentration increased after ovariectomy. Ovariectomy model has been widely used as an in vivo model to mimic post-menopausal pathophysiological changes in women related to learning and memory and brain oxidative stress¹³. Therefore, it is suggested that learning and memory impairments which were seen in ovariectomized rats in comparison to the sham animals at least in part may be due to brain tissues oxidative damage. The cognitive impairments due to human menopausal conditions may also be related to brain oxidative damage. The results of previous studies have also confirmed that ovariectomy of animals or postmenopausal conditions in women increase lipid peroxidation in brain, erythrocytes and plasma¹³.

Neuroprotective effect of estrogen has been attributed to its antioxidant properties of estrogen. It has been shown that the low nanomolar doses of estrogen has neuroprotection effect in rodent models and reduces brain lipid peroxidation¹⁴. It was also shown that estrogens prevent intracellular peroxide accumulation in an endoplasmic reticulum (ER)-independent manner¹⁵, decrease reactive oxygen species (ROS) production¹⁶, limit lipid peroxidation and decrease hydrogen peroxide concentrations¹⁷. Indirect anti-oxidative effects of estrogens have been reported, including attenuation of microglial superoxide release¹⁸, increase of glutathione reductase, gamma-glutamylcystein synthetase, glutaredoxin and glutathione¹⁰, increased manganese superoxide dismutase (MnSOD) activity¹⁹ and reduction of free radical production via an increase mitochondrial efficiency²⁰. The ability of estrogens for regeneration of vitamin E has also been suggested²¹. It has also been shown that estrogens potentiated lipid peroxidation inhibitors²². Estrogens are highly lipid soluble and largely reside in the membrane component of cells²³. In fact, there is evidence that estrogen prevents lipid peroxidation by sacrificing itself to oxidation²⁴. This estrogen redox cycle is operative in the brain and serves, together with the classic antioxidant mechanism, as a defense mechanism against ROS.

The results of the present study indicated that administration of a high dose (4 mg/kg) of exogenous estradiol valerate impaired learning and memory in sham and ovariectomized which was accompanied by low level of total SH groups in cortical tissues.

This result was in contrast with the findings mentioned and showed beneficial effects of estradiol on learning and memory and its antioxidant properties. In consistent with the results of present study, it has been shown that high doses of estradiol have deleterious effects on memory and learning. Varga et al. showed that Sprague-Dawley ovariectomized rats treated by the high dose of estradiol displayed a week performance in the water maze task in comparison with non treated ones²⁵. It has also been reported that chronic administration of high doses of estradiol impairs performance on a working/reference memory in the eight-arm radial arm maze⁷. Fugger et al. also showed that treatment of the female, but not male, mice with estradiol impairs acquisition of water maze tasks²⁶.

We hypothesized that the negative effects of a high dose of estradiol seen in the present study may be related to its pro-oxidative effects. The results showed that in both Sham-Est and OVX-Est rats the MDA concentrations was higher and total thiol concentration was lower than that of sham and ovariectomized rats. It should be noted that in at least one study, estradiol administration was resulted in a time-dependent increase in lipid peroxidation²⁷. It seems that the differential actions of estrogens as a pro-oxidant or an antioxidant depends in part on their metabolism and the subsequent actions of the metabolites²⁸. It has been shown that estrogen administration to rodents results in the initiation of tumors in several organs²⁹. The induction of renal tumors in Syrian hamsters due to chronic exposure to estrogens is an extensively studied model of carcinogenesis. It has been shown that estrogen-induced free radicals play a tumor-initiating role in this process²⁷,³⁰. It was shown that renal lipid peroxide concentrations were raised by chronic treatment with estradiol²⁷. It was also shown that certain metabolites of estradiol or estrone can directly produce DNA damage in target tissues, independent of their interaction with the estrogen receptor²⁹. The genotoxic effect of hydroxyestrogens has also been related to the generation of free radicals²⁹. Indeed, hydroxyestrogen-semiquinone intermediates can react in vitro with molecular oxygen yielding the superoxide anion (O2•−). Finally it seems that estrogens have dual actions in reference to their oxidative effects³¹.

In conclusion, the results of present study show that a high dose of estradiol impairs spatial learning and memory. Our results also indicate that the oxidative stress in brain cortical tissues might take part in spatial learning and memory impairments due to high levels of estradiol, but further investigations using more methods need to be done.

The results described in this paper were from a M.Sc. student thesis proposal. The authors would like to thank the Vice Chancellor of Research Affairs of Mashhad University of Medical Science for financial assistance.

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Publication Dates

• Publication in this collection
  May 2013

History

• Received
  18 Sept 2012
• Received
  5 Dec 2012
• Accepted
  12 Dec 2012