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The effect of the anti-diabetic drug metformin on behavioral manifestations associated with ovariectomy in mice
Neuroscience Letters 690 (2019) 95–98
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
The effect of the anti-diabetic drug metformin on behavioral manifestations associated with ovariectomy in mice
T
Iman Fatemia,b, Fatemeh Delrobaeec, Maryam Bahmanic, Ali Shamsizadeha,b, ⁎ Mohammad Allahtavakolia,b, a
Physiology-Pharmacology Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Physiology and Pharmacology, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran c Student Research Committee, Rafsanjan University of Medical Sciences, Rafsanjan, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovariectomy Metformin Anxiety Cognition
The aim of this study was to evaluate the anti-diabetic drug metformin (Met) effects on the anxiety and cognitive impairment in ovariectomized mice. Thirty-two female adult mice were distributed into four groups: control, sham ovariectomy, ovariectomy + Met 7 mg/kg and ovariectomy + Met 15 mg/kg. The vaginal cytology was used to confirm the ovariectomy surgery. Anxiety was monitored using elevated plus maze test and cognitive function was assessed by novel object recognition task. Animal’s brains were analyzed for the brain-derived neurotrophic factor (BDNF). Our results demonstrated that ovariectomy caused cognitive impairments and anxiety, as well as decreased BDNF levels. Moreover, administration of Met improves ovariectomy-related disorders such as cognitive impairments and anxiety, as well as increased BDNF levels. The results of the present study suggest that Met could be used as a novel therapeutic strategy for the treatment of ovariectomy-related conditions.
1. Introduction It is expected that the global population of menopausal women will increase over 1.2 million by the year 2030 [5]. Menopause is defined by the loss of ovarian follicular activity and decline in gonadal hormones in the females [1,18]. After menopause women usually suffer from menopausal symptom including tender breasts, low bone density, hot flushes, cognitive decline and anxiety [32]. It has been demonstrated that gonadal hormones play an important role in mood and cognition; and sex hormones deficiency is related with anxiety and cognition impairment in menopause [7]. It is well demonstrated that Brain-derived neurotrophic factor (BDNF) levels decrease in the ovariectomized rat which contributed to cognition impairment [10]. Hormone replacement therapy is considered as a therapeutic intervention in anxiety and cognitive impairments, particularly after menopause in women [34]. Nevertheless, clinically administration of these hormones especially estrogen has controversy due to the fact that long-term consumption of it may increase the risk of endometrial and breast cancer development in women [39]. Metformin (Met) is recommended as the first-line drug in patients with type 2 diabetes which reduces hyperglycemia via improving glucose utilization and synthesis [12,23]. This drug has also been reported ⁎
that has pleiotropic effects including anti-inflammatory, antioxidant and neuroprotective effects which may extend its clinical indications [2,25]. It is well established that Met passes the blood-brain barrier and distributes within CNS [27]. Furthermore, several studies have reported the positive effects of this agent on behavioral disorders such as anxiety and cognitive impairment [1,3,13,14]. Due to behavioral manifestations associated with ovariectomy and the effects of Met on behavioral disorders, we hypothesized that Met may has positive effects on anxiety-like behavior and cognitive impairment in ovariectomized (OVX) mice. For this purpose, we compared the anxiety-like behavior, cognitive impairment and BDNF levels of sham and OVX mice with or without Met treatment by elevated plusmaze (EPM) and the novel object recognition task (NORT). 2. Materials and methods 2.1. Animals In this study 32 female mice (22–27 g) were kept under standard laboratory conditions with food and water ad libitum and housed 8 per cage on a reversed 12-h light: 12-h dark cycle. The experiments were done base on the Animal Ethics Committee Guidelines for the use of
Corresponding author at: Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, 7717684884, Iran. E-mail address: [email protected] (M. Allahtavakoli).
https://doi.org/10.1016/j.neulet.2018.10.024 Received 1 July 2018; Received in revised form 6 October 2018; Accepted 11 October 2018 Available online 12 October 2018 0304-3940/ © 2018 Published by Elsevier B.V.
Neuroscience Letters 690 (2019) 95–98
I. Fatemi et al.
2.5. The novel object recognition task (NORT)
experimental animals.
Preference for novelty is an important component of learning and memory. The NORT is commonly used for assessing learning and memory in rodents and this method was describe in details somewhere [33]. Briefly, NORT was done in a Plexiglas chamber (50 × 50 × 50 cm). During the familiar phase, animals were put in the chamber to accommodate for 5 min 24 h later, mice were placed in the box with two identical objects for 5 min. The test phase was done 4 h after accommodation. A familiar object was changed by a novel object and mice were placed in the box for 5 min. The exploration times of novel (A) and familiar (B) objects were recorded and the discrimination ratio was reported as follow: (A − B)/(A + B) [4]. At the end of trials for each mouse, the apparatus was cleaned with ethanol (50%).
2.2. Ovariectomy surgery After 2 weeks of acclimatization period, the animals were anesthetized by ketamine/xylazine (60/4 mg/kg; i.p.) and underwent either a sham or bilateral ovariectomization surgery. Mice were treated with penicillin (22,000 i.u/kg) for two days after ovariectomy. The smears of vagina were collected from mice three days after ovariectomy to confirm the model. The vaginal smears observed under the light microscope for five days and if cornified epithelial cells were not observed in the vaginal smear, the ovariectomy was confirmed [20,26]. The mice in the sham group underwent the same procedure without removal of the ovaries.
2.6. Evaluation of BDNF level 2.3. Drug and treatment 24 h after behavioral tests, mice decapitated and the brains were quickly withdrawn under aseptic condition and homogenized in phosphate-buffered saline (1:10 w/v). The tissue homogenates were centrifuged (Eppendorf, Germany) for 20 min at 16,000 × rpm and the supernatants were collected and stored at −80 °C until further analysis as described below. BDNF levels were assessed by an ELISA kit (Zellbio, Germany) according to the company protocol guideline. Briefly, samples were mixed in the kit wells with the antibody labeled by an enzyme named conjugated. In the next stage, these mixtures were incubated for 45 min by a shaker in laboratory temperature. After that, free antibodies or/and antigens were washed and the substrate was added. After incubation for 15 min, the enzyme-substrate reaction was stopped by an interrupter solution and the light absorption was read by the ELISA Microplate Reader device (RT-2100C, Rayto, China) at the wavelength of 450 nm. All samples determined twice. The concentration of each sample was calculated by plotting the absorbance values on a standard curve (R2 = 0.99) with known concentrations generated by the assay. Results were presented as the percentage of control group. The detection limit of the assay was 0.066 ng/ml and the detection range of the assay was 0.312–20 ng/ml. Total protein in homogenates was measured by Bradford's method using bovine serum albumin as a standard [6].
Met was obtained from Merck Pharmaceuticals (Glucophage®, France, Sante). Met was dissolved in water (10 ml/kg) and administered orally. As we studied the preventive effects of Met on behavioral manifestations associated with ovariectomy, we utilized the lowest (7 mg/kg) and the most applicable (15 mg/kg) dose of Met [21]. The solution was freshly dissolved and the administered doses were based on daily body weight. Mice were randomly assigned as follows: (1) Sham group: sham surgery mice that received the standard drinking water (10 ml/kg) for 21 days; (2) OVX group: the OVX mice that received the standard drinking water (10 ml/kg) for 21 days; (3) Met 7 group: the OVX mice that received 7 mg/kg/day Met for 21 days; and (4) Met 15 group: OVX mice that received 15 mg/kg/day Met for 21 days. 24 h after the last application of Met, the behavioral tests were performed (at the same time of the day).
2.4. The elevated plus-maze test (EPMT) The EPMT is a standard method to assess the anxiety-like behavior in rodents [29]. The EPMT apparatus was made of wood and consisted of open and closed arms (50 cm in length × 10 cm in width). The two closed arms were enclosed by 40 cm high walls. The arms were attached by a central square platform (10 × 10 cm). The apparatus was 50 cm above the floor. Each animal was placed in the center of the apparatus facing a closed arm and allowed to move freely for 5 min. The percent of time spent on the open arms [OAT% (the ratio of times spent in the open arms to total times spent in any arms × 100)] was used as a measure of anxiety. The percent of entries into the open arms [OAE% (the ratio of entries into open arms to the total entries × 100)]. The number of total arm entries was used as a measure of spontaneous locomotor activity. Entry was defined as all four paws in the arm. The experiments were done in a room lit by a light bulb (60 W) placed above the center of the apparatus.
2.7. Statistical analysis Statistical analysis was performed using GraphPad Prism version 6.01 (GraphPad Software, USA). Data were presented as mean ± standard deviation (SD) and compared by one-way ANOVA followed by Tukey’s post hoc test. p value was considered statistically significant when was less than 0.05.
Fig. 1. (A, B) The effect of metformin (7 and 15 mg/kg) on anxiety-like behaviors and (C) locomotor activity of OVX mice. Values are expressed as mean ± SD. In each group n = 8. ** p < 0.05 and *** p < 0.001 compared to the control group. # p < 0.05 and ### p < 0.001 compared to the OVX group. 96
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4. Discussion Hormone replacement therapy has proven to be effective in declining postmenopausal symptoms such an anxiety and cognitive impairment in women [38]. However, the usage of estrogens leads to increasing the risk of breast and endometrial cancer which undoubtedly limited the use of estrogen [9]. Therefore, the search for a safe and effective estrogen substitute gets more attention. In the present study, we investigated the effects of Met on anxiety and cognitive impairment in OVX female mice. Our results showed that Met (7 and 15 mg/kg) improves the cognitive impairments and has anxiolytic effect in ovariectomized mice. It is well-known that during the menopause, reduction of ovarian hormones influences cognitive functions [16]. This may be partially due to the reduction of neurons in specific brain regions such as neurons in CA1, dorsal hippocampus and medial prefrontal cortex [30,37]. In addition, previous studies showed that ovariectomy impairs learning and memory in rodents [11]. In line with these studies, our results showed that ovariectomy impaired the novelty-induced exploratory behaviors in mice. We also observed that Met (7 and 15 mg/kg) improves the preference for novelty in OVX mice. Previous studies have reported that Met improves learning and memory in different pathological conditions. For example, Oliveira et al., showed that Met improved the spatial memory scores of diabetic animals by decreasing neuroinflammation and loss of neurons in the hippocampus [28]. In another study, Ashabi et al., declared that pretreatment with Met had a protective role against memory impairment in transient forebrain global ischemia [3]. Moreover, Ge et al., found that Met attenuated cognitive impairment in Morris water maze after ischemia/reperfusion induced brain damage in rats and survived the hippocampal CA1 pyramidal neurons [17]. Hence, Met might possibly improve cognition, which could be observed by object recognition performance in OVX mice, via the neuroprotective properties. Our results showed that the brain BDNF levels were decreased in OVX mice in comparison with sham mice. Our results also indicated that Met at both doses (7 or 15 mg/kg) increased BDNF levels which is consistent with its improvement in cognitive function. The reduction of BDNF, as an important neurotrophin, is associated with neuronal atrophy and death [36]. Also, it is well established that this factor has an important role in the regulation of neurocognitive functions like memory, synaptic transmission and plasticity [24]. On the other hand, ovarian hormones have regulatory effects on BDNF [8]. It has been reported that Met elevates the BDNF level [8]. For example, Smieszek et al., reported that Met has antioxidant and anti-aging effects on mouse olfactory ensheathing cells through reducing the amount of reactive oxygen species and rising the mRNA expression of BDNF [35]. Ghadernezhadet al., showed that administration of Met for 2 weeks ameliorated neurological functions and increased BDNF following brain ischemia in rats [19]. Therefore, it is possible that some restorative effects of Met on cognitive function may act by increasing the BDNF level. Anxiety is one of the most common disorders in menopausal women and is associated with reduced the quality of life [22]. Rats with absence of ovarian hormones induced by ovariectomy reflect mood disturbances typical of human menopause [31]. In line with previous studies, our results showed that anxiety-like behavior increased in OVX mice. Our results showed that Met (7 and 15 mg/kg) decreased anxietylike behavior in aged animals. The anxiolytic effect of Met is well-established in several studies. Ge et al., found that Met ameliorates anxiety-like behavioral in ischemia/reperfusion via antiapoptotic effects [17]. In another study, Garabadu et al., showed that Met exhibited anxiolytic-like activity in diabetic rats that subjected to repeated stress through increasing the antioxidant defense such as superoxide dismutase and catalase [15]. Therefore, it seems that Met has anxiolytic effects in OVX mice via antioxidative and/or neuroprotective effects. To conclude, the results of our study have shown that Met improves
Fig. 2. The effect of metformin (7 and 15 mg/kg) on preference for novelty. Values are expressed as mean ± SD. In each group n = 8. *** p < 0.001 compared to the control group. # p < 0.05 compared to the OVX group.
3. Results 3.1. OVX-induced anxiety-like behaviors were attenuated by Met The data of the EPMT are illustrated in Fig. 1. the results showed that ovariectomy decreased %OAT [F (3, 28) = 6.454, p < 0.01] and %OAE [F (3, 28) = 21.19, p < 0.01] in comparison with the control group and Met at the doses of 7 and 15 mg/kg increased %OAT [F (3, 28) = 6.454, all p < 0.05] and %OAE [F (3, 28) = 21.19, all p < 0.001] in OVX mice in comparison with the OVX group (Fig. 1A and B). Our results also showed that the locomotor activity of experimental groups did not change statistically significant [F (3, 28) = 1.727, all p > 0.05] (Fig. 1C).
3.2. OVX-induced cognitive impairments were attenuated by Met The OVX mice showed impaired preference for novel object [F (3, 27) = 9.325, p < 0.001], while Met at both doses reversed the deficiency of ovariectomy-induced preference for the novel objects, as shown by a higher discrimination ratio in comparison with OVX animals [F (3, 27) = 9.325, all p < 0.05] (Fig. 2).
3.3. OVX-induced alterations in BDNF levels were attenuated by Met Compared to the control group, BDNF was significantly decreased in OVX animals [F (3, 28) = 11.35, p < 0.05] (Fig. 3). Met at the doses of 7 and 15 mg/kg increased the BDNF levels in OVX mice compared to OVX alone mice [F (3, 28) = 11.35, p < 0.01 and p < 0.001, respectively].
Fig. 3. The effect of metformin (7 and 15 mg/kg) on BDNF level. Values are expressed as mean ± SD. In each group n = 8. * p < 0.05 compared to the control group. ## p < 0.01 and ### p < 0.001 compared to the OVX group. 97
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OVX-related disorders such as cognitive impairments (possibly by a mechanism associated with an increase in BDNF levels) and anxiety. If this translates to humans, administration of Met to menopausal women can be considered as off-label use of Met and a potential therapeutic approach in the treatment of menopausal symptoms. Moreover, in menopausal diabetic women, it is better to use Met to reduce blood glucose levels and treat menopausal symptoms. However, more studies are needed to reveal the underlaying the exact molecular mechanisms of Met.
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Conflict of interest The authors have declared that no conflict of interest exists related to this work. Acknowledgments This paper is derived from Ms. Maryam Bahmani’s M.Sc. thesis and was supported by grant no. 9/3581 from the Vice Chancellor for Research and Technology, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. References [1] M. Allahtavakoli, N. Honari, I. Pourabolli, M. Kazemi Arababadi, H. Ghafarian, A. Roohbakhsh, A. Esmaeili Nadimi, A. Shamsizadeh, Vitex agnus castus extract improves learning and memory and increases the transcription of estrogen receptor alpha in hippocampus of ovariectomized rats, Basic Clin. Neurosci. 6 (2015) 185–192. [2] M. Allahtavakoli, A.P. Shabanzadeh, S.S. Sadr, M. Parviz, B. Djahanguiri, Rosiglitazone, a peroxisome proliferator-activated receptor-gamma ligand, reduces infarction volume and neurological deficits in an embolic model of stroke, Clin. Exp. Pharmacol. Physiol. 33 (2006) 1052–1058. [3] G. Ashabi, A. Sarkaki, F. Khodagholi, S. Zareh Shahamati, M. Goudarzvand, Y. Farbood, M. Badavi, L. Khalaj, Subchronic metformin pretreatment enhances novel object recognition memory task in forebrain ischemia: behavioural, molecular, and electrophysiological studies, Can. J. Physiol. Pharmacol. 95 (2016) 388–395. [4] F. Ayoobi, I. Fatemi, A. Roohbakhsh, A. Shamsizadeh, Tactile learning within the early phase of experimental autoimmune encephalomyelitis in mice, Neurophysiology 45 (2013) 306–311. [5] F. Borrelli, E. Ernst, Black cohosh (Cimicifuga racemosa) for menopausal symptoms: a systematic review of its efficacy, Pharmacol. Res. 58 (2008) 8–14. [6] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [7] J.T. Bromberger, H.M. Kravitz, Y. Chang, J.F. Randolph Jr., N.E. Avis, E.B. Gold, K.A. Matthews, Does risk for anxiety increase during the menopausal transition? Study of women’s health across the nation, Menopause 20 (2013) 488–495. [8] D.L. Carbone, R.J. Handa, Sex and stress hormone influences on the expression and activity of brain-derived neurotrophic factor, Neuroscience 239 (2013) 295–303. [9] R.T. Chlebowski, J.E. Manson, G.L. Anderson, J.A. Cauley, A.K. Aragaki, M.L. Stefanick, D.S. Lane, K.C. Johnson, J. Wactawski-Wende, C. Chen, L. Qi, S. Yasmeen, P.A. Newcomb, R.L. Prentice, Estrogen plus progestin and breast cancer incidence and mortality in the Women’s Health Initiative Observational Study, J. Natl. Cancer Inst. 105 (2013) 526–535. [10] S.F. da Silva Moreira, L.F. Medeiros, A. de Souza, C. de Oliveira, V.L. Scarabelot, F. Fregni, W. Caumo, I.L. Torres, Transcranial direct current stimulation (tDCS) neuromodulatory effects on mechanical hyperalgesia and cortical BDNF levels in ovariectomized rats, Life Sci. 145 (2016) 233–239. [11] Y. Dong, A. Jiang, H. Yang, H. Chen, Y. Wang, Phytoestrogen α-zearalanol improves memory impairment and hippocampal neurogenesis in ovariectomized mice, Sci. World J. 2014 (2014). [12] I. Fatemi, M. Amirteimoury, A. Shamsizadeh, A. Kaeidi, The effect of metformin on morphine analgesic tolerance and dependence in rats, Res. Pharm. Sci. 13 (2018) 316–323. [13] I. Fatemi, A. Khaluoi, A. Kaeidi, A. Shamsizadeh, S. Heydari, M.A. Allahtavakoli, Protective effect of metformin on D-galactose-induced aging model in mice, Iran. J. Basic Med. Sci. 21 (2018) 19–25. [14] Y. Gao, Y. Li, J. Xue, Y. Jia, J. Hu, Effect of the anti-diabetic drug metformin on bone mass in ovariectomized rats, Eur. J. Pharmacol. 635 (2010) 231–236.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
The effect of the anti-diabetic drug metformin on behavioral manifestations associated with ovariectomy in mice
T
Iman Fatemia,b, Fatemeh Delrobaeec, Maryam Bahmanic, Ali Shamsizadeha,b, ⁎ Mohammad Allahtavakolia,b, a
Physiology-Pharmacology Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Physiology and Pharmacology, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran c Student Research Committee, Rafsanjan University of Medical Sciences, Rafsanjan, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovariectomy Metformin Anxiety Cognition
The aim of this study was to evaluate the anti-diabetic drug metformin (Met) effects on the anxiety and cognitive impairment in ovariectomized mice. Thirty-two female adult mice were distributed into four groups: control, sham ovariectomy, ovariectomy + Met 7 mg/kg and ovariectomy + Met 15 mg/kg. The vaginal cytology was used to confirm the ovariectomy surgery. Anxiety was monitored using elevated plus maze test and cognitive function was assessed by novel object recognition task. Animal’s brains were analyzed for the brain-derived neurotrophic factor (BDNF). Our results demonstrated that ovariectomy caused cognitive impairments and anxiety, as well as decreased BDNF levels. Moreover, administration of Met improves ovariectomy-related disorders such as cognitive impairments and anxiety, as well as increased BDNF levels. The results of the present study suggest that Met could be used as a novel therapeutic strategy for the treatment of ovariectomy-related conditions.
1. Introduction It is expected that the global population of menopausal women will increase over 1.2 million by the year 2030 [5]. Menopause is defined by the loss of ovarian follicular activity and decline in gonadal hormones in the females [1,18]. After menopause women usually suffer from menopausal symptom including tender breasts, low bone density, hot flushes, cognitive decline and anxiety [32]. It has been demonstrated that gonadal hormones play an important role in mood and cognition; and sex hormones deficiency is related with anxiety and cognition impairment in menopause [7]. It is well demonstrated that Brain-derived neurotrophic factor (BDNF) levels decrease in the ovariectomized rat which contributed to cognition impairment [10]. Hormone replacement therapy is considered as a therapeutic intervention in anxiety and cognitive impairments, particularly after menopause in women [34]. Nevertheless, clinically administration of these hormones especially estrogen has controversy due to the fact that long-term consumption of it may increase the risk of endometrial and breast cancer development in women [39]. Metformin (Met) is recommended as the first-line drug in patients with type 2 diabetes which reduces hyperglycemia via improving glucose utilization and synthesis [12,23]. This drug has also been reported ⁎
that has pleiotropic effects including anti-inflammatory, antioxidant and neuroprotective effects which may extend its clinical indications [2,25]. It is well established that Met passes the blood-brain barrier and distributes within CNS [27]. Furthermore, several studies have reported the positive effects of this agent on behavioral disorders such as anxiety and cognitive impairment [1,3,13,14]. Due to behavioral manifestations associated with ovariectomy and the effects of Met on behavioral disorders, we hypothesized that Met may has positive effects on anxiety-like behavior and cognitive impairment in ovariectomized (OVX) mice. For this purpose, we compared the anxiety-like behavior, cognitive impairment and BDNF levels of sham and OVX mice with or without Met treatment by elevated plusmaze (EPM) and the novel object recognition task (NORT). 2. Materials and methods 2.1. Animals In this study 32 female mice (22–27 g) were kept under standard laboratory conditions with food and water ad libitum and housed 8 per cage on a reversed 12-h light: 12-h dark cycle. The experiments were done base on the Animal Ethics Committee Guidelines for the use of
Corresponding author at: Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, 7717684884, Iran. E-mail address: [email protected] (M. Allahtavakoli).
https://doi.org/10.1016/j.neulet.2018.10.024 Received 1 July 2018; Received in revised form 6 October 2018; Accepted 11 October 2018 Available online 12 October 2018 0304-3940/ © 2018 Published by Elsevier B.V.
Neuroscience Letters 690 (2019) 95–98
I. Fatemi et al.
2.5. The novel object recognition task (NORT)
experimental animals.
Preference for novelty is an important component of learning and memory. The NORT is commonly used for assessing learning and memory in rodents and this method was describe in details somewhere [33]. Briefly, NORT was done in a Plexiglas chamber (50 × 50 × 50 cm). During the familiar phase, animals were put in the chamber to accommodate for 5 min 24 h later, mice were placed in the box with two identical objects for 5 min. The test phase was done 4 h after accommodation. A familiar object was changed by a novel object and mice were placed in the box for 5 min. The exploration times of novel (A) and familiar (B) objects were recorded and the discrimination ratio was reported as follow: (A − B)/(A + B) [4]. At the end of trials for each mouse, the apparatus was cleaned with ethanol (50%).
2.2. Ovariectomy surgery After 2 weeks of acclimatization period, the animals were anesthetized by ketamine/xylazine (60/4 mg/kg; i.p.) and underwent either a sham or bilateral ovariectomization surgery. Mice were treated with penicillin (22,000 i.u/kg) for two days after ovariectomy. The smears of vagina were collected from mice three days after ovariectomy to confirm the model. The vaginal smears observed under the light microscope for five days and if cornified epithelial cells were not observed in the vaginal smear, the ovariectomy was confirmed [20,26]. The mice in the sham group underwent the same procedure without removal of the ovaries.
2.6. Evaluation of BDNF level 2.3. Drug and treatment 24 h after behavioral tests, mice decapitated and the brains were quickly withdrawn under aseptic condition and homogenized in phosphate-buffered saline (1:10 w/v). The tissue homogenates were centrifuged (Eppendorf, Germany) for 20 min at 16,000 × rpm and the supernatants were collected and stored at −80 °C until further analysis as described below. BDNF levels were assessed by an ELISA kit (Zellbio, Germany) according to the company protocol guideline. Briefly, samples were mixed in the kit wells with the antibody labeled by an enzyme named conjugated. In the next stage, these mixtures were incubated for 45 min by a shaker in laboratory temperature. After that, free antibodies or/and antigens were washed and the substrate was added. After incubation for 15 min, the enzyme-substrate reaction was stopped by an interrupter solution and the light absorption was read by the ELISA Microplate Reader device (RT-2100C, Rayto, China) at the wavelength of 450 nm. All samples determined twice. The concentration of each sample was calculated by plotting the absorbance values on a standard curve (R2 = 0.99) with known concentrations generated by the assay. Results were presented as the percentage of control group. The detection limit of the assay was 0.066 ng/ml and the detection range of the assay was 0.312–20 ng/ml. Total protein in homogenates was measured by Bradford's method using bovine serum albumin as a standard [6].
Met was obtained from Merck Pharmaceuticals (Glucophage®, France, Sante). Met was dissolved in water (10 ml/kg) and administered orally. As we studied the preventive effects of Met on behavioral manifestations associated with ovariectomy, we utilized the lowest (7 mg/kg) and the most applicable (15 mg/kg) dose of Met [21]. The solution was freshly dissolved and the administered doses were based on daily body weight. Mice were randomly assigned as follows: (1) Sham group: sham surgery mice that received the standard drinking water (10 ml/kg) for 21 days; (2) OVX group: the OVX mice that received the standard drinking water (10 ml/kg) for 21 days; (3) Met 7 group: the OVX mice that received 7 mg/kg/day Met for 21 days; and (4) Met 15 group: OVX mice that received 15 mg/kg/day Met for 21 days. 24 h after the last application of Met, the behavioral tests were performed (at the same time of the day).
2.4. The elevated plus-maze test (EPMT) The EPMT is a standard method to assess the anxiety-like behavior in rodents [29]. The EPMT apparatus was made of wood and consisted of open and closed arms (50 cm in length × 10 cm in width). The two closed arms were enclosed by 40 cm high walls. The arms were attached by a central square platform (10 × 10 cm). The apparatus was 50 cm above the floor. Each animal was placed in the center of the apparatus facing a closed arm and allowed to move freely for 5 min. The percent of time spent on the open arms [OAT% (the ratio of times spent in the open arms to total times spent in any arms × 100)] was used as a measure of anxiety. The percent of entries into the open arms [OAE% (the ratio of entries into open arms to the total entries × 100)]. The number of total arm entries was used as a measure of spontaneous locomotor activity. Entry was defined as all four paws in the arm. The experiments were done in a room lit by a light bulb (60 W) placed above the center of the apparatus.
2.7. Statistical analysis Statistical analysis was performed using GraphPad Prism version 6.01 (GraphPad Software, USA). Data were presented as mean ± standard deviation (SD) and compared by one-way ANOVA followed by Tukey’s post hoc test. p value was considered statistically significant when was less than 0.05.
Fig. 1. (A, B) The effect of metformin (7 and 15 mg/kg) on anxiety-like behaviors and (C) locomotor activity of OVX mice. Values are expressed as mean ± SD. In each group n = 8. ** p < 0.05 and *** p < 0.001 compared to the control group. # p < 0.05 and ### p < 0.001 compared to the OVX group. 96
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4. Discussion Hormone replacement therapy has proven to be effective in declining postmenopausal symptoms such an anxiety and cognitive impairment in women [38]. However, the usage of estrogens leads to increasing the risk of breast and endometrial cancer which undoubtedly limited the use of estrogen [9]. Therefore, the search for a safe and effective estrogen substitute gets more attention. In the present study, we investigated the effects of Met on anxiety and cognitive impairment in OVX female mice. Our results showed that Met (7 and 15 mg/kg) improves the cognitive impairments and has anxiolytic effect in ovariectomized mice. It is well-known that during the menopause, reduction of ovarian hormones influences cognitive functions [16]. This may be partially due to the reduction of neurons in specific brain regions such as neurons in CA1, dorsal hippocampus and medial prefrontal cortex [30,37]. In addition, previous studies showed that ovariectomy impairs learning and memory in rodents [11]. In line with these studies, our results showed that ovariectomy impaired the novelty-induced exploratory behaviors in mice. We also observed that Met (7 and 15 mg/kg) improves the preference for novelty in OVX mice. Previous studies have reported that Met improves learning and memory in different pathological conditions. For example, Oliveira et al., showed that Met improved the spatial memory scores of diabetic animals by decreasing neuroinflammation and loss of neurons in the hippocampus [28]. In another study, Ashabi et al., declared that pretreatment with Met had a protective role against memory impairment in transient forebrain global ischemia [3]. Moreover, Ge et al., found that Met attenuated cognitive impairment in Morris water maze after ischemia/reperfusion induced brain damage in rats and survived the hippocampal CA1 pyramidal neurons [17]. Hence, Met might possibly improve cognition, which could be observed by object recognition performance in OVX mice, via the neuroprotective properties. Our results showed that the brain BDNF levels were decreased in OVX mice in comparison with sham mice. Our results also indicated that Met at both doses (7 or 15 mg/kg) increased BDNF levels which is consistent with its improvement in cognitive function. The reduction of BDNF, as an important neurotrophin, is associated with neuronal atrophy and death [36]. Also, it is well established that this factor has an important role in the regulation of neurocognitive functions like memory, synaptic transmission and plasticity [24]. On the other hand, ovarian hormones have regulatory effects on BDNF [8]. It has been reported that Met elevates the BDNF level [8]. For example, Smieszek et al., reported that Met has antioxidant and anti-aging effects on mouse olfactory ensheathing cells through reducing the amount of reactive oxygen species and rising the mRNA expression of BDNF [35]. Ghadernezhadet al., showed that administration of Met for 2 weeks ameliorated neurological functions and increased BDNF following brain ischemia in rats [19]. Therefore, it is possible that some restorative effects of Met on cognitive function may act by increasing the BDNF level. Anxiety is one of the most common disorders in menopausal women and is associated with reduced the quality of life [22]. Rats with absence of ovarian hormones induced by ovariectomy reflect mood disturbances typical of human menopause [31]. In line with previous studies, our results showed that anxiety-like behavior increased in OVX mice. Our results showed that Met (7 and 15 mg/kg) decreased anxietylike behavior in aged animals. The anxiolytic effect of Met is well-established in several studies. Ge et al., found that Met ameliorates anxiety-like behavioral in ischemia/reperfusion via antiapoptotic effects [17]. In another study, Garabadu et al., showed that Met exhibited anxiolytic-like activity in diabetic rats that subjected to repeated stress through increasing the antioxidant defense such as superoxide dismutase and catalase [15]. Therefore, it seems that Met has anxiolytic effects in OVX mice via antioxidative and/or neuroprotective effects. To conclude, the results of our study have shown that Met improves
Fig. 2. The effect of metformin (7 and 15 mg/kg) on preference for novelty. Values are expressed as mean ± SD. In each group n = 8. *** p < 0.001 compared to the control group. # p < 0.05 compared to the OVX group.
3. Results 3.1. OVX-induced anxiety-like behaviors were attenuated by Met The data of the EPMT are illustrated in Fig. 1. the results showed that ovariectomy decreased %OAT [F (3, 28) = 6.454, p < 0.01] and %OAE [F (3, 28) = 21.19, p < 0.01] in comparison with the control group and Met at the doses of 7 and 15 mg/kg increased %OAT [F (3, 28) = 6.454, all p < 0.05] and %OAE [F (3, 28) = 21.19, all p < 0.001] in OVX mice in comparison with the OVX group (Fig. 1A and B). Our results also showed that the locomotor activity of experimental groups did not change statistically significant [F (3, 28) = 1.727, all p > 0.05] (Fig. 1C).
3.2. OVX-induced cognitive impairments were attenuated by Met The OVX mice showed impaired preference for novel object [F (3, 27) = 9.325, p < 0.001], while Met at both doses reversed the deficiency of ovariectomy-induced preference for the novel objects, as shown by a higher discrimination ratio in comparison with OVX animals [F (3, 27) = 9.325, all p < 0.05] (Fig. 2).
3.3. OVX-induced alterations in BDNF levels were attenuated by Met Compared to the control group, BDNF was significantly decreased in OVX animals [F (3, 28) = 11.35, p < 0.05] (Fig. 3). Met at the doses of 7 and 15 mg/kg increased the BDNF levels in OVX mice compared to OVX alone mice [F (3, 28) = 11.35, p < 0.01 and p < 0.001, respectively].
Fig. 3. The effect of metformin (7 and 15 mg/kg) on BDNF level. Values are expressed as mean ± SD. In each group n = 8. * p < 0.05 compared to the control group. ## p < 0.01 and ### p < 0.001 compared to the OVX group. 97
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OVX-related disorders such as cognitive impairments (possibly by a mechanism associated with an increase in BDNF levels) and anxiety. If this translates to humans, administration of Met to menopausal women can be considered as off-label use of Met and a potential therapeutic approach in the treatment of menopausal symptoms. Moreover, in menopausal diabetic women, it is better to use Met to reduce blood glucose levels and treat menopausal symptoms. However, more studies are needed to reveal the underlaying the exact molecular mechanisms of Met.
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