Oestrogen receptor α agonist improved long-term ovariectomy-induced spatial cognition deficit in young rats Free

Abstract

Ovariectomy is known as ‘surgical menopause’ with decreased levels of oestrogen in female rodents and its reported risks and adverse effects include cognitive impairment. In the brain, oestrogen exerts effects through its receptors, oestrogen receptor α (ERα) and β (ERβ). However, the role of ERα or ERβ in ovariectomy-induced cognitive impairment needs further investigation. Here, we observed that bilaterally ovariectomized 3-month-old rats showed obvious spatial learning and memory deficits in the Morris water maze with significant loss of neurons and synapses in the hippocampus. In addition to the rapid decline in serum oestradiol levels, the expression of ERα, but not ERβ, was decreased in the hippocampus starting 1 wk after ovariectomy. Prompt 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) treatment (1 mg/kg.d), an agonist of ERα, improved the spatial learning and memory ability of ovariectomized rats and rescued ovariectomy-induced neuron loss by up-regulating the level of BCLxl, an important anti-apoptosis protein. Furthermore, PPT treatment also improved ovariectomy-induced hippocampal synapse loss and up-regulated the levels of synaptic proteins (synapsin I, NR2A and GluR1) and the activates of CaMK Πα, ERK and Akt. Thus, these results demonstrated that ERα plays an important role in neuroprotection and that prompt ERα rescue is effective to improve hippocampal-dependent cognition deficit after long-term ovariectomy.

Introduction

Oestrogen, an important gonadal steroid hormone, exhibits important roles in brain structure and function by exerting actions on synaptic plasticity, neurogenesis and cognition (Correia et al.2010; Li et al.2004; Walf et al.2011). Its neuroprotective effects have been widely demonstrated in various models against different kinds of neuronal toxicity, such as β-amyloid toxicity, oxidative stress, bio-energetic deficiency, mitochondrial failure and excitotoxicity (Acosta et al.2009; Li et al.2004; Rocca et al.2010). Oestrogen deficiency may be linked with cognitive impairment. For instance, menopausal women are reported to be more vulnerable to neurodegeneration in age-related cognitive decline, such as Alzheimer's disease (AD) and vascular dementia (Li et al.2011; Pike et al.2009; Simpkins et al.2009). Most premenopausal women who accepted bilateral ovariectomy to treat or prevent ovarian diseases enter a menopause-like period, known as ‘surgical menopause’ (Stout Steele & Bennett, 2011), with cognitive impairment being one of the reported adverse effect of the procedure (Rocca et al.2007). Oestrogen replacement therapy has proven effective against ovariectomy-induced cognitive impairment in previous basic and clinical studies (Bonomo et al.2009; Shuster et al.2008; Turgeon et al.2006). However, the Women's Health Initiative (WHI) studies published in 2003 not only failed to support such positive effects, but reported an increased risk of cognitive decline and dementia with hormone therapy (Bonomo et al.2009; Daniel & Bohacek, 2010; Sherwin, 2009; Shuster et al.2008; Turgeon et al.2006). To address the inconsistencies between these findings, recent analyses supported a hypothesis of a ‘critical period’, such that oestrogen replacement therapy needs to be initiated shortly after oestrogen depletion.

In the brain, oestrogen acts through oestrogen receptor α and β (ERα and ERβ), both receptors are generally expressed in a similar distribution throughout the brain, including the pre-optic area, the bed nucleus of the stria terminalis, the medial and cortical amygdaloid nuclei. Some regions show selective expression, such as ERα is the predominant subtype in the pre-optic area and most of the hypothalamus, meanwhile ERβ is primarily localized in the olfactory bulb, cerebral cortex and septum (McEwen & Alves, 1999; Mitra et al.2003; Shughrue et al.1997; Thakur & Sharma, 2006). ERα and ERβ are both indicated in learning and memory (Dubal et al.2001; Moriarty et al.2006; Srivastava et al.2010; Tiwari-Woodruff et al.2007). It was reported that ERα is necessary for the formation of normal short-term social recognition memory in females (Choleris et al.2003, 2004, 2006; Cordey & Pike, 2005; Frye et al.2007) and oestrogen exerted neuroprotection by suppressing inflammation through ERα-mediated mechanisms (Vegeto et al.2003, 2006). However, ERβ was reported to regulate hippocampal synaptic plasticity and improved hippocampus-dependent cognition, while its agonist had the capability to modulate synaptic structure and synapse-related proteins (Liu et al.2008; Wang et al.2003; Zhao et al.2011) and ERβ knockout mice have shown signs of impaired learning on hippocampal-dependent spatial tasks (Liu et al.2008; Rissman et al.2002). Although these two receptors are expressed in the CA3, CA1 and dentate gyrus (Mitra et al.2003), ERα mRNA was very weak compared to ERβ mRNA (Shughrue et al.1997), while the nuclear ERα immunoreactivity was found to be the predominant subtype in hippocampus (Mitra et al.2003). Indeed, the specific contributions of these two ER subtypes for oestrogen-induced neuroprotection in vivo need more understanding.

Recent work indicates a decreased expression of ERα, but not ERβ, in the CA1 region at 10 wk after ovariectomy in rats (Zhang et al.2009, 2011), indicating that ERα and ERβ might have different actions in different oestrogen-related pathological states, including ovariectomy-induced oestrogen deficit. To confirm this hypothesis, rats, aged 3 months, were bilaterally ovariectomized in this study and the serum oestradiol levels and the expression of oestrogen receptors were measured at five different time-points, namely, 24 h, weeks 1, 2, 4 and 8 after ovariectomy. We found that the expression of ERα in the hippocampus, but not ERβ, decreased together with declining serum oestradiol levels starting the first week after ovariectomy. The rats showed spatial learning and memory deficits in the Morris water maze within 7–8 wk after the operation, with obvious hippocampal neuron loss and synapse loss. Then, we used 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT; 1 mg/kg.d), an agonist of ERα, to treat the rats shortly after ovariectomy for 8 wk. It was found that PPT treatment significantly improved the spatial learning and memory ability of ovariectomized rats and rescued ovariectomy-induced neuron loss by up-regulating the level of BCLxl, an important anti-apoptosis protein. Furthermore, it also rescued the synapse loss and up-regulated the expression of synaptic proteins including synapsin I, N-methyl d-aspartate receptor 2A (NR2A) and glutamate receptor 1 (GluR₁) and activated calcium/calmodulin-dependent protein kinase Π (CaMK Π α), ERK and Akt in hippocampus. These results indicated an important role of ERα in ovariectomy-induced cognitive impairment and the validity of prompt ERα rescue in neuroprotection after ovariectomy.

Materials and method

Antibodies and chemicals

The primary antibodies used in this study are listed in Table 1. PPT was from Tocris Bioscience (USA). Bicinchoninic acid (BCA) protein detection kit and phosphocellulose units were from Pierce Chemical Company (USA). Anti-rabbit or anti-mouse IgG conjugated to IRDye^™ (800CW) (1: 10 000) was from Lincoln (USA). Toluidine Blue and dimethyl sulfoxide (DMSO) were from Sigma (USA).

Animals and treatments

Female Sprague–Dawley (SD) rats (aged 3 months) weighing 250–280 g were supplied by Experimental Animal Central of Tongji Medical College, Huazhong University of Science and Technology. Rats were allowed free access to food and water and maintained at a constant temperature of (25 ± 2 °C ). All efforts were made to minimize animal suffering and to reduce the number of rats used. All experimental procedures in this research have been approved by the Animal Care and Use Committee of Huazhong University of Science and Technology.

Initially, 30 rats were randomly divided into two groups, namely, sham-operated group (Sham, n = 15) and ovariectomized group (Ovx, n = 15). Bilateral ovaries were removed from the Ovx rats, as described previously (Stout Steele & Bennett, 2011) and the Sham rats received the same incisions and sutures, but the ovaries were palpated instead of removed. Brain tissue and peripheral blood were taken at 24 h, weeks 1, 2, 4 and 8 after the operation (n = 3/time-point) in both Sham and Ovx groups. Peripheral blood was collected via caudal vein and the serum was prepared by centrifugation (10 000 g, 10 min). By Western blotting analysis, we detected the expression of hippocampal oestrogen receptors at those five different time-points and found that the expression of ERα had clearly decreased from week 1 to week 8 after ovariectomy together with the decreased serum oestradiol levels. Therefore, we chose 7–8 wk as the observation time-frame to further investigate the role of ERα in ovariectomy-induced cognition deficit in the following experiment.

Then, 48 rats were divided into four groups randomly, namely, Sham group (n = 12), Ovx group (n = 12), ovariectomized treated with vehicle group (Ovx + Veh, n = 12) and ovariectomized treated with PPT group (Ovx + PPT, n = 12). In the PPT-treated group, rats received daily subcutaneous injections of PPT (1 mg/kg.d) for 8 wk and the first injection was performed 2 h after ovariectomy. The Veh-treated group received an equal volume of solvent (50% DMSO/50% Dulbecco's PBS) by daily subcutaneous injection. In the seventh week after the operation, all rats were given 7-d training in a Morris water maze and the memory ability of all rats was tested on the last day of week 8 in the maze. After memory testing, peripheral blood, brain tissue and uterus were taken. The body weight of all rats was measured every morning.

Spatial learning and memory in the Morris water maze

During week 7 after ovariectomy, all rats from the Sham group, Ovx group, Veh-treated group and PPT-treated group began 7-d training of spatial learning in the Morris water maze. Rats tested in the water maze were extensively handled (2 min every day). Before each experiment (2 h), the rats were brought to the site to allow them to become acclimatized. The test subjects were kept in cages on outer-room shelves to eliminate directional olfactory and auditory cues. The temperature of the room and water was kept at 26 ± 2 °C . The water in the pool was made opaque with ink to hide the escape platform. The Plexiglas platform was 40 cm high and 10 cm in diameter and its surface was scarred to help the rats climb. The water surface was 18 cm from the rim of the pool and the inner wall was consistently carefully wiped to eliminate any local cues. The rim of the pool was 1.0 m from the nearest visual cue of red and blue marks. A camera was fixed to the ceiling of the room, 1.5 m from the water surface. A computer with the water-maze software then processed the tracking information.

The submerged platform (2 cm under water) was located at a fixed position throughout training. A training session consisted of three trials altogether (one trial per quadrant) with a 30 s interval, lasting for 7 d. The first quadrant was included as the first trial every day. On each trial, the rat started from the middle of the quadrant facing the wall of the pool and the trial ended when it climbed the platform. The rats were not allowed to search for the platform for >60 s, after which they were guided to the platform. The rats were allowed 6 d rest before the memory test on the last day of week 8. For the learning and memory testing, we chose the first quadrant as the testing area and the swimming pathways and latencies of testing were recorded.

Western blotting analysis

The rats were killed by 6% chloral hydrate (0.06 ml/kg) and the hippocampus was immediately removed from the brain and homogenized at 4 °C using a Teflon glass homogenizer in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 10 mm NaF, 1 mm Na₃VO₄, 10 mm β-mercaptoethanol, 5 mm EDTA, 2 mm benzamidine, 1.0 mm phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin and 2 µg/ml pepstatin. The homogenate was mixed in 2:1 (v/v) ratio with lysis buffer containing 200 mm Tris-HCl (pH 7.6), 8% SDS, 40% glycerol and boiled for 10 min in a water bath, stored at −80 °C for Western blotting analysis. The concentration of protein in the extracts was measured using a BCA kit according to manufacturer's instruction (Pierce Chemical Company). The protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 2% non-fat milk dissolved in TBS (50 mm Tris-HCl, pH 7.6, 150 mm NaCl) for 1 h and probed with primary antibodies (Table 1) for 18 h. The blots were incubated with anti-rabbit or anti-mouse IgG conjugated to IRDye^™ (800 CW; 1:10 000) for 1 h at room temperature. The intensity of the protein bands was quantified by Odyssey system (Li-Cor Bioscience, USA).

Nissl staining

For Nissl staining studies, 12 rats from four groups (n = 3/group) were killed by 6% chloral hydrate (0.06 ml/kg) and perfused through the aorta with 100 ml 0.9% NaCl, followed by 400 ml phosphate buffer containing 4% paraformaldehyde after undergoing the spatial memory test. Brains were removed and post-fixed in perfusate overnight and then cut into 30 µm sections with a vibratome (Leica, Germany; S100, TPI). The sections of rat brain were collected consecutively in PBS for Nissl staining. Selected slices were stained in 0.1% Toluidine Blue solution for 20 min and then quickly rinsed in distilled water and differentiated in 95% ethyl alcohol for 15 min. The slices were observed by a microscope (Nikon Eclipse 90i; Nikon, Japan) and analysed with Image-Pro Plus 4.5 system (Media Cybernetics Inc., USA).

Golgi impregnation

Golgi staining was performed as described (Ramon-Molinier, 1970).The rats from four groups (n = 3/group) were killed by 6% chloral hydrate (0.06 ml/kg) and perfused through the aorta with 150 ml 0.9% NaCl containing 0.5% sodium nitrite followed by 400 ml 4% formol-fixed. The brain was then removed from the skull and immersed in a block of formol-fixed brain tissue into a 2% aqueous solution of potassium dichromate for 3 d in 37 °C . Blocks were dried with filter paper and immersed in a 1% aqueous solution of silver nitrate for another 3 d in 37 °C . Golgi-Cox stained brains were cut into 35 µm thick sections with a vibratome (Leica; S100, TPI) and observed by a microscope (Nikon Eclipse 90i; Nikon) with identical settings (×100 objective lens). The number of apical and basal spines on hippocampal CA1 pyramidal neurons was counted. For each group, a minimum of 40 cells per slice was analysed. The images were analysed with Image-Pro Plus 4.5 system (Media Cybernetics Inc.).

Serum oestradiol assay

Serum was prepared from peripheral blood by centrifuging blood at 10 000 g for 10 min. Serum oestradiol levels were analysed as the introduction of a commercial oestradiol assay kit (Cayman Chemical Company, USA). Results were expressed in pg/ml and each sample was assayed in duplicate.

Statistical analysis

Statistical analysis was performed using SPSS 12.0 statistical software (SPSS Inc., USA). Data are expressed as means±s.e.m. Differences between the Sham group and Ovx group in the initial experiment were tested with Student's t tests. The differences among the Sham group, Ovx group, Ovx + Veh group and Ovx + PPT group were tested with the one-way analysis of variance procedure followed by Student–Newman–Keuls test. The level of significance was set at p < 0.05.

Results

Ovariectomy-induced hippocampal ERα decrease in 3-month-old rats

To investigate the effect of ovariectomy on the expression of oestrogen receptors, 3-month-old rats received either bilateral ovariectomy or sham operation. The expression of hippocampal ERα and ERβ was tested respectively at 24 h, weeks 1, 2, 4 and 8 after the operation (n = 3/time-point) by Western blotting and the serum oestradiol levels were assayed simultaneously by a commercial oestradiol assay kit. The serum oestradiol levels of Sham rats remained around 63.9 ± 1.4 pg/ml for 8 wk after ovariectomy without any difference from that of normal same-aged female rats. The oestradiol levels of rats in the Ovx group significantly decreased to 16.8 ± 3.5 pg/ml at 24 h and the low oestradiol levels remained until the eighth week after ovariectomy (p < 0.01, Fig. 1a). By Western blotting, the expression of hippocampal ERα was found to be decreased, starting the first week after ovariectomy and decreased to nearly 40% at the end of week 8 (p < 0.01, Fig. 1b, c). The expression of hippocampal ERβ did not show any alteration after ovariectomy (Fig. 1b, c) by Western blotting, whereas, in Sham rats, the expressions of hippocampal ERα and ERβ were maintained after the operation (data not shown). These results demonstrated that ovariectomy reduced serum oestradiol levels and the expression of hippocampal ERα, but not ERβ expression.

PPT rescued ovariectomy-induced spatial cognitive deficit in rats

To confirm the role of hippocampal ERα, PPT, a ligand specific for ERα, was used to treat the rats (1 mg/kg.d) according to previous research (Le Saux et al.2006; Morissette et al.2008b) and the learning and memory tests in the Morris water maze were performed as shown in Fig. 2a. Ovariectomy was associated with spatial learning and memory deficits in rats. During the learning test, the latency for the Sham rats to find the hidden platform from the first quadrant on day 6 of learning was 9 s and it was 6 s on day 7 (Fig. 2b). For the memory test, delivered after a 6-d rest period, the latency for the Sham rats to find the platform was 10 s, indicating that they retained a spatial reference memory. In contrast, the Ovx rats took more time to find the hidden platform on days 6 and 7 of learning compared to Sham rats (p < 0.05, Fig. 2b). Furthermore, for the memory test, the latency for Ovx rats was increased compared to their latency on day 7 of learning (p < 0.05, Fig. 2b), indicating possible memory impairments. The increased latency in Ovx rats was due to impaired spatial learning and memory rather than simply floating, as evidenced by their swimming path (Fig. 2c). The Sham rats in the memory test found the hidden platform as quickly and directly as on day 7 of learning (Fig. 2c).

After daily PPT treatment (Fig. 2a), the latency for the rats in PPT-treated group to find the hidden platform from the first quadrant was 8 s on day 6 and 6 s on day 7 (Fig. 2b), much shorter than that of Veh-treated rats in the learning test (p < 0.05, Fig. 2b) and exhibited no difference compared to Sham rats (Fig. 2b). In the memory test, PPT-treated rats found the hidden platform as quickly and directly as on the last day of learning (Fig. 2b, c). The rats in the Veh-treated group exhibited a longer latency to find the hidden platform in the memory test relative to the last day of the learning test (p < 0.05, Fig. 2b, c), indicating a possible memory deficit. Taken together, the results indicate that prompt ERα treatment rescued spatial cognitive deficits in rats induced by ovariectomy. Ovx rats showed a decreased serum oestradiol level (34.7 ± 1.72 pg/ml, p < 0.01, Fig. 1d), decreased uterus weight (200 ± 30 mg, p < 0.01, Fig. 2e) and increased body weight (357 ± 7.5 g, p < 0.01, Fig. 2f) compared to Sham rats. The serum oestradiol level in PPT-treated rats was 43.3 ± 7.3 pg/ml (Fig. 2d), while it was 30.2 ± 2.3 pg/ml in Veh-treated rats (Fig. 2d). Compared to Ovx rats and Veh-treated rats, PPT-treated rats maintained uterus weight (1000 ± 130 mg, p < 0.01, Fig. 2e) equivalent to that of Sham rats and completely prevented the ovariectomy-induced body weight gain. (324 ± 7.1 g, p < 0.05, Fig. 2f).

PPT increased hippocampal ERα level and neuron number by inhibiting apoptosis

Previous studies have shown that oestrogen is strongly associated with neuronal survival (Dubal et al.2001; Moriarty et al.2006; Tiwari-Woodruff et al.2007; Vegeto et al.2003, 2006) and ovariectomy induces neuron loss in old rats (Bethea et al.2011). In order to detect the quantity of neurons as per above, Nissl staining was used at week 8 after the memory test. We found that, compared to Sham animals, the number of neurons in CA1 and CA3 regions was significantly reduced in Ovx rats (p < 0.05, Fig. 3a, b) and neuron number remained constant in the dentate gyrus region. In addition, we did not find any alternations in the number of cortex neurons in Ovx rats (data not shown). In PPT-treated rats, the number of neurons in CA1 and CA3 regions were not different from Sham (p < 0.05, Fig. 3a, b). It was previously reported that ovariectomy induced a decrease in hippocampal ERα levels starting the first week after ovariectomy (Fig. 1b, c). In PPT-treated rats, the hippocampal ERα level did not decline, in contrast to the Veh-treated rats, examined at week 8 after ovariectomy (p < 0.01, Fig. 3c, d). Moreover, PPT treatment rescued the ovariectomy-induced decreased expression of Bclxl, a key anti-apoptosis factor, in the hippocampus (p < 0.01, Fig. 3c, e). These results indicate the importance of ERα for neuron survival after ovariectomy.

PPT rescued ovariectomy-induced synapse loss in hippocampus

Accumulated data indicate that oestrogen affects synaptic communications in brain regions related to cognitive processing, including the hippocampus (Mukai et al.2010; Smith & McMahon, 2005; Yang et al.2010). The synaptic communication within the hippocampus is particularly important to the spatial memory in the context of the menopause (Foster, 2012; Smith et al.2010). Most hippocampal synapses are located on dendritic spines. Therefore, we examined the effect of ovariectomy and PPT treatment on dendritic spines and the expression of synaptic proteins. By Golgi staining, we found that the number of dendritic spines was significantly decreased in the CA1 region of Ovx rats and Veh-treated rats, excepting PPT-treated rats, compared to the Sham rats (p < 0.01, Fig. 4a, b). These data indicate that PPT treatment hindered the loss of spines such that PPT-treated rats exhibited more spines than other Ovx groups (p < 0.01, Fig. 4a, b). The presynaptic protein, synapsin I, and post-synaptic proteins, NR2A and GluR1, were significantly decreased in the hippocampus in Ovx rats that were not treated with PPT (Fig. 4c, d). Furthermore, in the absence of PPT treatment, Ovx rats exhibited significantly reduced phosphorylation of CaMK Π , ERK and Akt, indicating the down-regulating of their activities by ovariectomy (Fig. 4e, f). The enhanced phosphorylation of CaMK Π , ERK and Akt in PPT-treated rats (Fig. 4e, f) suggests that spatial cognition deficits in young Ovx rats may be due to impaired synapse function as a result of the loss of ERα activity.

Discussion

ERα and ERβ might have different actions in the pathological changes induced by oestrogen deficit. Oestrogen has unequivocally been shown to have many beneficial effects on the brain, including reduced neuronal loss after cardiac arrest and stroke, increased neuronal connectivity, improved cognitive performance and preventing or slowing age-related cognitive decline through its receptors (Correia et al.2010; Pompili et al.2012). Oestrogen deficit because of menopause or ovariectomy induced many pathological changes in the brain, including neuronal cell death, synaptic damage, inflammatory activation and accelerated lesion progression (Bethea et al.2011; Bonomo et al.2009; Turgeon et al.2006). Epidemiological studies indicate a higher prevalence of AD in women and evidence points to a link between oestrogen levels at menopause and deficits in hippocampal-dependent episodic memory, one of the early clinical symptoms of AD (Andersen et al.1999; Pompili et al.2012). Studies assessing spatial memory in rats have revealed a strong correlation between oestrogen and hippocampal-dependent memory tasks. In this study, Ovx rats showed decreased expression of hippocampal ERα, but not ERβ, with spatial learning and memory deficits in the Morris water maze, which could be rescued by prompt PPT treatment. These data suggest the specific role of hippocampal ERα in ovariectomy-induced spatial cognition impairment.

Oestrogen has been reported to have a neuroprotective effect in many neurodegenerative diseases and improved cognitive ability (Correia et al.2010; Daniel & Bohacek, 2010; LeBlanc et al.2001). The Women's Health Initiative Memory Study and the WHI reported their failure to observe a protective effect of oestrogen replacement therapy on the cardiovascular system and reported a small but significant increase in risk for stroke and dementia (Bonomo et al.2009; Espeland et al.2004; LeBlanc et al.2001; Shumaker et al.2004; Turgeon et al.2006; Zandi et al.2002). Oestrogen replacement therapy has been limited due to the diverse and sometimes opposite effects of oestrogen in basic and clinical research that possibly result from differences in the oestrogenic compounds employed, routes of delivery to women of different ages and health status (Bonomo et al.2009; Turgeon et al.2006). The popular ‘critical period’ hypothesis (Sherwin & Henry, 2008), which attempted to reconcile positive and negative effects of oestrogen on cognition, suggested that oestrogen replacement therapy initiated early, around the time of the menopause and continued for several years, has an enduring neuroprotective effect on cognitive functioning two to three decades later, which, if initiated late, might cause more harm than good (Daniel & Bohacek, 2010; Sherwin, 2009; Suzuki et al.2007). As shown by Brinton and Gleason (Brinton, 2005; Gleason et al.2005), oestrogen replacement therapy prolonged 5 yr beyond the critical post-menopausal period increased the risk of developing dementia. The underlying mechanisms for the critical period hypothesis remain unknown. The timing of treatment with the oestrogen receptor agonist is also important for some neurodegenerative diseases. The ERα agonist has been reported to be neuroprotective at the onset and throughout the disease course in multiple sclerosis and the ERβ agonist had no effect at disease onset but promoted recovery during the chronic phase of the disease (Tiwari-Woodruff et al.2007). In this study we found that prompt PPT treatment maintained the expression of hippocampal ERα and reinstated ovariectomy-induced neuron loss and synapse loss in the hippocampus and prevented ovariectomy-induced cognition and memory deficits. The results suggest that ERα activation has neuroprotective effects following ovariectomy and is an important contributor in determining the ‘critical treatment period’.

PPT displays 400-fold more binding affinity for ERα than ERβ (Carroll & Pike, 2008; Lewis et al.2008; Waters et al.2009) and in some studies much higher dosages of PPT (2.0–15.0 mg/kg) have been used (Gordon et al.2009; Harris et al.2002; Tiwari-Woodruff et al.2007). Previous research has proved that the decrease in ERα sensitivity was tissue-specific; unlike the hippocampal CA1 region, ERα did not decrease in the uterus following long-term oestradiol deprivation and the uterus remained sensitive to the uterotropic action of oestradiol (Zhang et al.2009). In our study, PPT treatment in Ovx rats maintained uterus weight. The effect of long-term PPT supplement on the uterus needs further study.

Previous research concluded that oestrogen up-regulated immunoreactivity for the largest N-methyl-d-aspartate (NMDA) receptor subunit and PPT was shown to modulate both hippocampal NMDA receptors and AMPA receptors in ovariectomized rats and increase the number of hippocampal synapse (Carroll & Pike, 2008; Lewis et al.2008; Waters et al.2009). In comparison, the ERβ agonist, 2, 3-bis(4-hydroxyphenyl)-propionitrile, displaying 100-fold more affinity for ERβ than ERα and no effect on ERα transcriptional activity, did not regulate the activity of the glutamatergic receptor (Morissetteet al.2008a, b). Some research reported that oestrogen can trigger many intracellular signalling cascades, such as the activation of MAPK and phosphatidylinositol 3-kinase (PI3 K)/Akt pathways, induction of ion channel fluxes and G-protein-coupled receptor-mediated second messenger generation (McEwen et al.2001; Moriarty et al.2006; Yildirim et al.2011). Oestrogen mediated neuroprotection against glutamate excitotoxicity and Aβ via MAPK pathways and prevented neurons from ischaemia-induced apoptosis via the Akt pathway (Moriarty et al.2006; Turgeon et al.2006). In addition, oestrogen activates CaMK Π α, ERK and Akt and contributes to the formation of long-term potentiation in mouse hippocampus through ERα (O'Neill et al.2008; Sawai et al.2002). Therefore, we tested the possible synaptic mechanism of ERα in ovariectomy-induced cognitive impairment. The results show that levels of synapse-related proteins and the activities of CaMK Π α, ERK and Akt in the hippocampus of rats are down-regulated after ovariectomy. PPT treatment rescued the levels of presynaptic protein synapsin I and the post-synaptic proteins, NR2A and GluR1 and the activities of CaMK Π α, ERK and Akt. These results indicate that ERα plays an important role in oestrogen-participated synaptic function and the formation of learning and memory.

In summary, we observed that Ovx SD rats, aged 3 months, displayed apparent spatial learning and memory deficits in the Morris water maze as well as the loss of neurons and synapses in the hippocampus after the operation. Together with the rapid decline of serum oestradiol levels, the expression of ERα in the hippocampus, but not ERβ, decreased starting 1 wk after ovariectomy. Prompt PPT treatment improved spatial learning and memory ability of Ovx rats and rescued ovariectomy-induced neuron loss and synapse loss. The present results support the idea that ERα plays an important role in neuroprotection and indicate that prompt ERα rescue is important for hippocampal-dependent cognition after ovariectomy.

Acknowledgments

This work was supported by grants from the Natural Science Foundation of China (30971008, 30971204, 30871035). Thanks for the language editing by Dr Zhen Li from McGill University, Montreal, Canada and Dr Jie Chen from Medical School of the University of North Carolina at Chapel Hill, NC, USA.

Statement of Interest

None.

References