Behavioural Brain Research 288 (2015) 1–10
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Research report
Flupirtine attenuates chronic restraint stress-induced cognitive deficits and hippocampal apoptosis in male mice Pengcheng Huang a , Cai Li a , Tianli Fu a , Dan Zhao a , Zhen Yi a , Qing Lu a,b,c , Lianjun Guo a,b,c , Xulin Xu a,b,c,∗ a
Department of Pharmacology, School of Basic Medicine, Tongji medical college, Huazhong University of Science and Technology, Wuhan 430030, China The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China c The Institute of Brain Research, Huazhong University of Science and Technology, Wuhan, 430030, China b
h i g h l i g h t s • • • •
Flupirtine rescues spatial learning and memory impairments in stressed mice. Flupirtine alleviates neuronal apoptosis in the hippocampus CA1 of stressed mice. Flupirtine attenuates synaptic loss in the hippocampus CA1 of stressed mice. Flupirtine activates Akt/GSK-3 signaling pathway in the hippocampus of stressed mice.
a r t i c l e
i n f o
Article history: Received 25 November 2014 Received in revised form 1 April 2015 Accepted 4 April 2015 Available online 11 April 2015 Keywords: Chronic restraint stress Flupirtine Cognitive impairment Akt (protein kinase B) Glycogen synthase kinase 3 beta
a b s t r a c t Chronic restraint stress (CRS) causes hippocampal neurodegeneration and hippocampus-dependent cognitive deficits. Flupirtine represents neuroprotective effects and we have previously shown that flupirtine can protect against memory impairment induced by acute stress. The present study aimed to investigate whether flupirtine could alleviate spatial learning and memory impairment and hippocampal apoptosis induced by CRS. CRS mice were restrained in well-ventilated Plexiglass tubes for 6 h daily beginning from 10:00 to 16:00 for 21 consecutive days. Mice were injected with flupirtine (10 mg/kg and 25 mg/kg) or vehicle (10% DMSO) 30 min before restraint stress for 21 days. After stressor cessation, the spatial learning and memory, dendritic spine density, injured neurons and the levels of Bcl-2, Bax, p-Akt, p-GSK-3, p-Erk1/2 and synaptophysin of hippocampal tissues were examined. Our results showed that flupirtine significantly prevented spatial learning and memory impairment induced by CRS in the Morris water maze. In addition, flupirtine (10 mg/kg and 25 mg/kg) treatment alleviated neuronal apoptosis and the reduction of dendritic spine density and synaptophysin expression in the hippocampal CA1 region of CRS mice. Furthermore, flupirtine (10 mg/kg and 25 mg/kg) treatment significantly decreased the expression of Bax and increased the p-Akt and p-GSK-3, and flupirtine (25 mg/kg) treatment up-regulated the p-Erk1/2 in the hippocampus of CRS mice. These results suggested that flupirtine exerted protective effects on the CRS-induced cognitive impairment and hippocampal neuronal apoptosis, which is possibly associated with the activation of Akt/GSK-3 and Erk1/2 signaling pathways. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: CRS, chronic restraint stress; Con, control; Flu, flupirtine; DMSO, dimethyl sulfoxide; NS, normal saline; PBS, phosphate-buffered saline; ANOVA, analysis of variance; Akt, protein kinase B; GSK-3, glycogen synthase kinase 3 beta; Erk1/2, extracellular signal-regulated kinase 1/2; PTEN, phosphatase and tensin homologue deleted on chromosome 10. ∗ Corresponding author at: Department of Pharmacology, School of Basic Medicine, Tongji medical college, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China. Tel./fax: +86 27 83691762. E-mail address: [email protected] (X. Xu). http://dx.doi.org/10.1016/j.bbr.2015.04.004 0166-4328/© 2015 Elsevier B.V. All rights reserved.
Substantial evidence implicates stress is an important factor in the vulnerability to depression and other behavioral disorders [1]. Chronic restraint stress (CRS) can exacerbate neurodegeneration and cognitive deficits [2–4]. Growing evidences have shown that chronic stress causes atrophy and functional impairment in several key brain areas such as the frontal cortex and hippocampus [5,6]. The hippocampus is a region that plays a crucial role in learning and memory and is an area also particularly susceptible to chronic stress [7,8]. Extensive researches have proved that CRS disrupts the hippocampus-dependent cognitive function [4,9,10].
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Akt (Protein kinase B) has been thought to be involved in neuronal survival, and activation of the kinase confers neuroprotection in several models of apoptosis [11–13]. Glycogen synthase kinase3 (GSK-3), a ubiquitous cellular serine/threonine protein kinase, plays a role in various essential physiological processes in the mammalian brain, such as development, cell cycle, or apoptosis [14]. GSK-3 serving as an essential downstream effector of Akt, its activity is inhibited by Akt-mediated phosphorylation at serine 9 [15]. It has been shown that GSK-3 is activated in the hippocampus of chronic stressed animals [9,16], and inhibition of GSK-3 restores chronic stress-induced memory deficit [17] and neuronal apoptosis [18]. Flupirtine is clinically used as a non-opioid analgesic with muscle relaxant [19,20]. In addition to its well-characterized as a Kv7 channel activator, flupirtine also acts like an NMDA receptor antagonist and has GABAA receptor-agonistic properties [21,22]. Several studies have demonstrated that flupirtine has significant powerful anti-oxidative and anti-apoptosis effects either in vitro or in vivo [23–25]. Flupirtine alleviates neuronal degeneration and cognitive impairment induced by repetitive hyperthermic seizures [25]. However, the molecular mechanisms for neuroprotective effects of flupirtine have not yet been fully understood. Furthermore, we have previously shown that flupirtine can prevent impairment of acute stress on spatial memory retrieval via inactivation of GSK-3 [26]. In the present study, we investigated the effects and underlying mechanisms of flupirtine on cognitive deficits and hippocampal apoptosis induced by CRS. 2. Materials and methods 2.1. Animals Adult male Kunming (KM) mice, weighing 20–25 g, were obtained from the Animal Center of Tongji Medical College. Five mice were kept in a cage and were allowed free access to water and food. The mice were maintained at a constant temperature of 23 ± 1 ◦ C, humidity at 55 ± 5% and under a 12:12 light/dark cycle (lights on at 7:00 a.m). The mice were allowed to acclimatize for 7 days before experiments. All experimental protocols were approved by the Review Committee for the Use of Human or Animal Subjects of Huazhong University of Science and Technology in accordance with the NIH guidelines for the care and use of laboratory animals (approval number: S400/5/1/2011). All efforts were made to minimize animal suffering and number of animals necessary. 2.2. CRS model and animals treatment Adult mice were subjected to CRS as previously described [27,28] with minor modifications. The mice were restrained for 6 h (from 10:00 to 16:00) daily in well-ventilated Plexiglass tubes (3.3 cm diameter, 10.5 cm length) for 21 consecutive days. During restraint stress, animals were not physically compressed, but food and water were deprived. The same handle was carried on the unstressed mice. Animals were divided into six groups randomly: 1. Control group: mice were treated with vehicle (normal saline containing 10% DMSO) for 21 days (Con, n = 15); 2. Flupirtine group: mice were injected with flupirtine (10 mg/kg or 25 mg/kg) for 21 days (Flu10, n = 13; Flu25, n = 15); 3. CRS group: mice were injected with vehicle 30 min before restraint stress for 21 days (CRS, n = 16); 4. Flupirtine treatment group: mice were treated with flupirtine (10 mg/kg or 25 mg/kg) 30 min before restraint stress daily for 21 days (CRS + Flu10, n = 14; CRS + Flu25, n = 14).
Flupirtine maleate (Targsense scientific Co. Ltd, Shanghai, China) was dissolved in normal saline containing 10% DMSO in concentrations of 1 mg/ml and 2.5 mg/ml. Flupirtine or vehicle was administered intraperitoneally (i.p.) in a volume of 10 ml/kg body weight 30 min prior to the onset of restraint stress. 2.3. Morris water maze The spatial learning and memory performance was determined using the Morris water maze (MWM) test as previously described [29] with minor modifications. The Morris water maze consisted of a stainless-steel circular pool (90 cm diameter, 45 cm height) and a Plexiglass platform (15 cm diameter). The container was filled with water (23 ± 2 ◦ C) that was dyed by using non-toxic paint. The Plexiglass platform was submerged approximately 2 cm below the surface of the water and placed in center of second quadrant during the training session. Mice were given four trials per day for four consecutive days after CRS. Mice were placed into the tank facing the wall of the pool, and were allowed to swim and find the hidden platform. The time to reach the platform (escape latency) was recorded in each trial. During each trial, each mouse was given 60 s to find the hidden platform. If the mice failed to find the platform within 60 s, they would be guided to find the platform and stayed on it for 20 s. On 5th day after stress, the platform was removed and mice were tested on a spatial probe trial for 60 s. The time of mice spent in the target quadrant was recorded. An automatic tracking system MT-200 (Chengdu instrument Co., Chengdu, Sichuan province, China) was used to monitor swimming activities. After the Morris water maze test, mice were sacrificed for further histological and biochemical analysis. 2.4. Hematoxylin and eosin staining Mice (n = 4–5) were deeply anesthetized with 10% chloral hydrate (i.p., 0.35 ml/100 g) and perfused with normal saline solution, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). After perfusion, the brains tissues were removed carefully and kept in 4% paraformaldehyde at 4 ◦ C overnight. After fixation and dehydration with gradient ethanol, the brain tissues were embedded in paraffin and sliced into in the coronal plane at 5 m thickness using a section cutter (Leica, Germany). The sections (2 sections/mouse) were stained with hematoxylin and eosin (H&E). Morphology of hippocampus was observed by a light microscope (Olympus Corporation, Japan). Damaged neurons were identified by their acidophilic (eosinophilic) cytoplasm and pyknotic nuclei, which are suggestive of necrotic morphology [30,31]. Cell counting of injured cells was performed in the pyramidal layer of the hippocampus CA1 of the mice. 2.5. Golgi silver staining Dendritic spine of pyramidal neurons in the hippocampus was observed by Golgi silver staining as previous study [32]. Mice (n = 4–5) were anesthetized with 10% chloral hydrate and perfused with normal saline solution. The brain tissues were removed and stored in Golgi-Cox solution for 14 days, then kept in 30% sucrose solution. Brain tissues were sectioned at 50 m thickness in the coronal plane using a vibratome (Camden Instrument, MA752, Leicester, UK). Dendritic images were acquired and analyzed with a microscope of OLYMPUS BX51 and software of Image-Pro Express. The numbers of dendritic spine in the hippocampus CA1 neurons (50 m segment, 70 dendritic segments on 35 neurons from each group) were counted by the observers who were blind to experimental conditions.
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2.6. Immunohistochemical staining
3. Results
Brain tissues of mice (n = 5) were routinely processed in paraffin blocks and later sliced into 5 m in the coronal plane using a section cutter (Leica, Germany). Immunohistochemical staining was performed as previous study [33]. Briefly, after deparaffinization and hydration, endogenous peroxidase was quenched with 0.3% H2 O2 in ultrapure water for 15 min. Nonspecific immunoglobulin binding sites were blocked with 5% normal goat serum for 1 h, then the sections (2 sections/mouse) were incubated with rabbit synaptophysin (1:400, Abcam, Cambridge, MA, USA) at 4 ◦ C overnight followed by incubated for 1 h with secondary antibody at room temperature. Immunoglobulin complexes were visualized on incubation with DAB. PBS was used instead of primary or secondary antibody or ABC reagent as negative controls. Results were captured with a light microscope (Olympus Corporation, Japan) and quantified by mean optical intensity in five random microscopic fields of per-section. To evaluate synaptophysin immunoreactivity (IR), the average optical density of positive in images was quantified with Image-Pro Plus 6.0 software. Analysis was done by observers who were blind to experimental conditions.
3.1. Flupirtine prevented CRS-induced spatial learning and memory impairment
2.7. Western blot In order to alleviate the suffering of the animals, mice were sacrificed by decapitation under urethane anesthesia. The hippocampus was quickly removed from the brain and stored at −80 ◦ C until further use. Hippocampal tissues were homogenized in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. The tissue homogenates were then incubated for 30 min on ice and centrifuged at 16,000 g for 15 min at 4 ◦ C, then the supernatants were collected carefully. Protein concentrations were determined using a BCA protein assay kit (Thermo scientific pierce, Rockford, IL, USA). Equal amount of protein from different group was subjected to SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in Tris-buffered Saline + 0.1% Tween (TBST) buffer (pH = 7.6) for 1 h at room temperature, then the membranes were incubated overnight at 4 ◦ C with primary antibodies. Primary antibodies were against: anti-Phospho-Akt(ser473), anti-Akt, anit-PhosphoGSK-3(ser9), anti-GSK-3, anti--Catenin or anti-Synaptophysin (1:1000, Cell Signaling Technology, Danvers, MA, USA); anit-P-Erk (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Erk2 (1:1000, Proteintech Group Inc, Chicago, IL,USA); anti-Bax, antiBcl-2 or anti- PTEN (1:1000, ImmunoWay, Newark, DE, USA); anti-GAPDH (1:5000, CWBIO, Beijing, China). Following washed in TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoblots were revealed with an enhanced chemiluminescence system (ECL) (Millipore, Billerica, MA, US) and visualized by DNR Bio-Imaging systems (CAT.NO: 70-25-00; MicroChemi, Israel). Optical densities of the bands were scanned and quantified with NIH Image J software. The data from the bands were normalized to GAPDH. Values of p- protein were normalized to total protein. The experiments of western blot were repeated 3–4 times. 2.8. Statistical analyses All the data are expressed as the mean ± standard error of the mean (S.E.M). Differences in the escape latencies in the Morris water maze test were analyzed with two-way ANOVA with repeated measures followed by the Tukey HSD post hoc test for multiple comparisons among different group. The other data were analyzed by one-way ANOVA followed by Tukey HSD post hoc test. P < 0.05 was considered as statistically significant.
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The spatial learning and memory performance was assessed with the Morris water maze test. The analysis of escape latency by using two-way ANOVA showed significant differences among groups (groups effect: F(5,81) = 6.367, P = 0.000; training day effect: F(3 , 243) = 43.029, P = 0.000; n = 13–16 per group). Compared with control group, CRS mice exhibited significantly longer escape latencies on day 2, 3 and 4 (P < 0.05, P < 0.01, P < 0.01 respectively, Fig. 1A). Compared with CRS group, flupirtine (10 mg/kg) treatment decreased the escape latencies on day 4 (P < 0.05), and flupirtine (25 mg/kg) treatment decreased the escape latencies on days 3 and 4 (P < 0.001, P < 0.01 respectively, Fig. 1A). In addition, the analysis of the swimming speed by using two-way ANOVA showed no significant differences among groups (groups effect: F(5 , 81) = 1.118, P = 0.3500; training day effect: F(3,243) = 1.447, P = 0.229; Fig. 1B). The probe test was performed for 60 s on the 24 h after the last training test. During the probe test, the analysis of time spent in the target quadrant by using one-way ANOVA showed significant differences among groups (F(5,81) = 4.866, P = 0.001; Fig. 1C, D). CRS mice spent significant less time in target quadrant than control group (P < 0.01). Compared with CRS group, flupirtine (25 mg/kg) treatment increased the time spent in target quadrant (P < 0.05). Otherwise, compared with control group, unstressed mice by treatment with flupirtine (25 mg/kg) spent less time in target quadrant (P < 0.05). Additionally, there were no significant differences of swimming speed among the six groups on day of probe test (F(5,81) = 0.768, P = 0.575; data not shown). These results indicated that flupirtine prevented the impairment of spatial learning and memory induced by CRS. 3.2. Flupirtine alleviated CRS-induced neuronal apoptosis in the hippocampus CA1 The neuronal histopathological changes were observed by hematoxylin and eosin (H&E) staining. The analysis of injured neurons by using one-way ANOVA showed significant differences among groups (F(5,20) = 22.160, P = 0.000). In control group, there were no significant neuronal abnormalities in the hippocampus CA1. However, in CRS group, many more degenerated neurons (eosinophilic pyknotic neuron) were observed in the hippocampus CA1 (P < 0.01, Fig. 2B). Compared with CRS group, flupirtine (10 mg/kg and 25 mg/kg) treatment significantly alleviated the neuronal degeneration (P < 0.01, Fig. 2B). To observe the effect of flupirtine on apoptosis-related proteins, the protein levels of Bax and Bcl-2 were assessed by western blot. The analysis of Bax levels by using one-way ANOVA showed significant differences among groups (F(5,30) = 9.078, P = 0.000). In comparison with control group, the expression of Bax in the hippocampus of CRS mice was significantly increased (P < 0.01; Fig. 2D). Compared with CRS group, flupirtine (10 mg/kg and 25 mg/kg) treatment reduced the increase of Bax in the hippocampus (P < 0.05, P < 0.001, respectively). However, as shown in Fig. 2E, CRS or flupirtine treatment did not influence the levels of Bcl-2 in the hippocampus (F(5,30) = 0.717, P = 0.616). These results revealed that flupirtine protected against CRS-induced neuronal apoptosis in the hippocampus. 3.3. Flupirtine increased CRS-induced reduction of dendritic spine density and synaptophysin expression in the hippocampus CA1 To further investigate the effect of flupirtine on dendritic spine density and synaptophysin expression, the dendritic spine density
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Fig. 1. Flupirtine prevented CRS-induced learning and memory impairment. (A) Escape latencies to find the hidden platform during days 1–4 of training. (B) Swimming speed during days 1–4 of training. (C) Representative swimming paths of mice in different groups during the probe test. (D) Summary of time spent in target quadrant of mice in different groups during the probe test. Data are expressed as mean ± SEM. n = 13-16 per group, * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
was assessed by Golgi Silver Staining and the synaptophysin expression was observed by histochemical staining and western blot. The analysis of spine density by using one-way ANOVA showed significant differences among groups (F(3,14) = 47.204, P = 0.000). Compared with control group, the spinal density in hippocampus CA1 of CRS mice was markedly decreased (P < 0.01; Fig. 3A). Compared with CRS group, flupirtine (25 mg/kg) treatment reversed the CRS-induced reduction of the spinal density in hippocampus CA1 (P < 0.01; Fig. 3A). In immunohistochemical staining, the analysis of synaptophysin with one-way ANOVA demonstrated conspicuous differences among four groups (F(3,15) = 32.545, P = 0.000). Compared with control group, the mean density of synaptophysin immunoreactivity in hippocampus CA1 of CRS mice was dramatically decreased (P < 0.01; Fig. 3B). Compared with CRS group, flupirtine (25 mg/kg) treatment reversed the reduction of synaptophysin expression in hippocampus CA1 (P < 0.01). Additionally, the results of western blot showed that CRS or flupirtine treatment did not influence the expression of synaptophysin in the hippocampus (F(3,12) = 0.540, P =0.664; Fig. 3C). These results suggested that flupirtine reversed CRS-induced dendritic spine density and synaptophysin expression in the hippocampus CA1.
3.4. Flupirtine activated Akt/GSK-3ˇ and Erk1/2 signaling pathways in the hippocampus of CRS mice In order to further explore the effects of flupirtine on Akt/GSK3 and Erk1/2 signaling pathways, the p-Akt, p-GSK-3 and pErk1/2 in the hippocampus were assessed by immunoblot. Analyses
with one-way ANOVA revealed a significant main effect of drug (pAkt: F(5,18) = 9.332, P = 0.000; p-GSK-3: F(5,24) = 11.860, P = 0.000; p-Erk1/2: F(5,27) = 9.084, P = 0.00; Figs. 4–6). Compared with control group, the levels of p-Akt, p-GSK-3 and p-Erk1/2 in the hippocampus of CRS mice were significantly decreased (P < 0.01, P < 0.001, P < 0.01, respectively). Compared with CRS group, flupirtine (10 mg/kg and 25 mg/kg) treatment strongly prevented the reduction of p-Akt and p-GSK-3 (P < 0.05, P < 0.01, respectively), and flupirtine (25 mg/kg) treatment prevented the reduction of p-Erk1/2 (P < 0.01). Analyses with one-way ANOVA showed no statistical differences of total Akt, GSK-3 and Erk2 between groups (Akt: F(5,18) = 0.302, P = 0.905; GSK-3: F(5,18) = 1.103, P = 0.393; Erk2: F(5,24) = 0.246, P =0.938; Fig. 4-6). Additionally, CRS or flupirtine treatment had no effect on the expression of PTEN and -catenin in the hippocampus (PTEN: F(5,36) = 0.613, P = 0.691, Fig. 4D; -catenin: F(5,18) = 0.62, P = 0.687, Fig. 5D). These finding showed that flupirtine activated Akt/GSK-3 and Erk1/2 signaling pathways in the hippocampus of CRS mice.
4. Discussion The data of the present study revealed that flupirtine improved CRS-induced spatial learning and memory impairment. Flupirtine alleviated the neuronal apoptosis and reduction of dendritic spine density and synaptophysin expression in the hippocampus CA1 of CRS mice. Furthermore, flupirtine reduced the expression of Bax and increased the levels of p-Akt, p-GSK-3 and p-Erk1/2 in the hippocampus of CRS mice.
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Fig. 2. Flupirtine alleviated CRS-induced apoptosis in the CA1 region of hippocampus. (A) Hematoxylin- and eosin-stained in hippocampus CA1 neurons of mice in different groups. The arrows indicated injured neurons (cells with pyknotic nuclei, acidophilic cytoplasm and fragmented nuclei) (HE × 400, inset HE × 1000). (B) The number of eosinophilic pyknotic neurons in the hippocampus CA1 was counted. Significant degenerated neurons were observed in the hippocampus CA1 of CRS mice. Treatment with flupirtine (10 mg/kg and 25 mg/kg) significantly alleviated the CRS-induced neuronal degeneration. (C) Representative western blot of Bax and Bcl-2 in the hippocampus. (D) Statistics for western blot results of Bax. Western blot analysis showed that flupirtine (10 mg/kg and 25 mg/kg) treatment reversed the CRS-induced up-regulation of Bax in the hippocampus. (E) Statistics for western blot results of Bcl-2. Bcl-2 showed no change between groups. Data are presented as mean ± SEM. n = 4–6 per group, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
Chronic stress is associated with the development of psychiatric disorders such as depression and post-traumatic stress disorder [2,34,35], which leads to cognitive and emotional impairment. Consistent with previous studies [2,3], our results revealed that CRS impaired the spatial learning and memory functions, CRS mice spent longer time to locate the platform in the training sessions and sojourned shorter in the target quadrant in MWM test. Previous study has shown that flupirtine alleviates spatial learning and memory impairment induced by repetitive hyperthermic seizures [25]. A double-blind study in patients suffering from Creutzfeldt-Jakob’s disease has revealed that oral flupirtine application significantly reduces the deterioration of cognition [36]. Moreover, our previous study has demonstrated that flupirtine protects against memory deficit induced by acute stress [26]. We explored whether flupirtine exerted a protective effect against
CRS-induced learning and memory impairment. Our results showed that flupirtine (10 mg/kg and 25 mg/kg) treatment alleviated the impairment of learning acquisition induced by CRS, and flupirtine (25 mg/kg) treatment prevented memory retrieval impairment induced by CRS. These results suggested that flupirtine could prevent CRS-induced spatial learning and memory impairment. Chronic stress has been known to impair brain function and increase the susceptibility of neurons to injury, especially in the hippocampus [37,38]. Stress induces significant neuronal apoptosis in the hippocampus, and apoptosis has been proposed to be an important mechanism contributing to stress-related cognitive deficits [39,40]. Neuroprotective effects of flupirtine have been previously demonstrated in both in vitro and in vivo experimental models [41–43]. In the present study, we investigated
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Fig. 3. Flupirtine increased the dendritic spine density and synaptophysin expression in the hippocampus CA1 of CRS mice. (A) Representative photomicrographs of Golgi staining from each group (upper). Spine numbers of dendrites (50 m segment) were counted (70 dendritic segments on 35 neurons from each group). The summary data (bottom) showed that treatment with flupirtine (25 mg/kg) reversed CRS-induced the decrease in the dendritic spine density of hippocampus CA1. (B) The photomicrographs (upper) showed synaptophysin expression in hippocampus CA1 pyramidal neurons of mice. The histogram (bottom) showed the change of mean density labeled synaptophysin. (C) Representative western blot (upper) and corresponding densitometric analysis of synaptophysin (bottom) in the hippocampus of mice. Synaptophysin showed no change between groups. Data are presented as mean ± SEM. n = 4–5 per group ** P < 0.01 compared with control group; ## P < 0.01 compared with CRS group.
whether flupirtine prevented CRS-induced hippocampal apoptosis. We found that CRS caused neuronal apoptosis in the hippocampus CA1 as previous study [3,44], and flupirtine (10 mg/kg and 25 mg/kg) treatment alleviated CRS-induced hippocampal apoptosis (Fig. 2). Furthermore, CRS increased the expression of Bax, and flupirtine (10 mg/kg and 25 mg/kg) treatment reversed the increment of Bax. In addition, our results showed that levels of Bcl-2 in hippocampus of mice in six groups had no obvious differences. The data concerning the Bcl-2 protein levels in the hippocampus after chronic restraint stress are contradictory. Some report that chronic restraint stress (1 h twice/day for 10 days, 2 h/day for 9 days) reduce the Bcl-2 expression [45–47]. On the other hand, some report that mild restraint (1 h/day for 7days, 2.5 h/day for 14 days) elevate the
Bcl-2 expression, and the stress-induced increase in Bcl-2 may correspond to a neuroprotective response [48,49]. Luo C. et al. have found that levels of Bcl-2 are unaltered in the hippocampus after restraint for 21 days [50], and their observations support our results that restraint for 21 days does no affect the Bcl-2 expression in the hippocampus. These discrepancies might be attributed to different stress protocols. Dendritic spines and synapses are known to play a central role in the CNS by forming the complex circuitry, which are critical for neuronal connectivity and brain function including cognitive processes and mood [51,52]. Chronic restraint stress leads to neuronal remodeling of hippocampal neurons, loss of dendritic spines and reduction of synaptic density have been demonstrated in the
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Fig. 4. Flupirtine increased p-Akt in the hippocampus of CRS mice. (A) Representative western blot of p-Akt, total Akt and PTEN. (B) Statistics for western blot results of p-Akt. Flupirtine (10 mg/kg and 25 mg/kg) treatment reversed the CRS-induced reduction of p-Akt. (C) Statistics for western blot results of total Akt. Total Akt showed no change between groups. (D) Statistics for western blot results of PTEN. PTEN showed no change between groups. Data are presented as mean ± SEM. n = 4–6 per group, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
Fig. 5. Flupirtine increased p-GSK-3 in the hippocampus of CRS mice. (A) Representative western blot of p-GSK-3, total GSK-3 and -catenin. (B) Statistics for western blot results of p-GSK-3. Treatment with flupirtine (10 mg/kg and 25 mg/kg) reversed the CRS-induced reduction of p-GSK-3. (C) Statistics for western blot results of total GSK-3. Total GSK-3 showed no change between groups. (D) Statistics for western blot results of -catenin. -catenin showed no change between groups. Data are presented as mean ± SEM. n = 4–7 per group ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
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Fig. 6. Flupirtine increased p-Erk1/2 in the hippocampus of CRS mice. (A) Representative western blot of p-Erk1/2, total Erk2. (B) Statistics for western blot results of p-Erk1/2. Flupirtine (25 mg/kg) treatment reversed the CRS-induced reduction of p-Erk1/2. (C) Statistics for western blot results of total Erk2. Total Erk2 showed no change between groups. Data are presented as mean ± SEM. n = 4–7 per group ** P < 0.01 compared with control group; ## P < 0.01 compared with CRS group.
hippocampus of chronic restraint stressed animals [9,53,54]. To determine whether the protective effects of flupirtine on cognition were accompanied by an increase in dendritic spine and synaptic density in the hippocampus, we investigated the number of dendritic spines and expression of synaptophysin, the most widely used markers for the determination of synaptic density in the hippocampus. As previous studies [27,55], CRS induces a significant reduction of dendritic spine density in the hippocampal CA1 pyramidal neurons, here, we found that flupirtine (10 mg/kg or 25 mg/kg) treatment alleviate the reduction. Western blot results showed that CRS and flupirtine treatment had no influences on synaptophysin expression in the whole hippocampus tissue. Western blot analysis cannot reveal localized changes of synaptic density. Therefore, we used immunohistochemical analysis to observe synaptophysine expression in hippocampal CA1. The results showed that CRS decreased synaptophysin expression, and flupirtine treatment elevated the levels of synaptophysin in hippocampus CA1. Thus, we postulate that the spatial learning and memory improvement effects of flupirtine are due, at least in part, to its enhancement effects on dendritic spine and synapse formation. The Akt/GSK-3 signaling pathway plays an important role in regulating learning and memory, synaptic plasticity and cell survival [56,57]. Previous studies have demonstrated the inactivation of Akt/GSK-3 pathway in the hippocampus of chronic stressed animals [17,58]. Increased activation of the Akt/GSK-3 provides protective effects against neuronal injury [56,57]. 1,2-dilinoleoyl-sn-glycero- 3-phosphocholine upregulates the Akt/GSK-3 pathway and rescues the memory deficits in chronic restraint stressed mice [17]. DL-/PO-phosphatidylcholine restores restraint stress-induced depression-related behaviors and spatial memory impairment via inactivation of GSK-3 [17]. The present study revealed that flupirtine (10 mg/kg and 25 mg/kg) reversed the reduction of p-Akt and p-GSK-3 induced by CRS (Figs. 4 and 5), suggesting that flupirtine presented neuroprotective and cognitive improvement effects might be through activation of Akt/GSK-3 signaling pathway. However, CRS did not cause upregulation of PTEN, one of upstream proteins of Akt, implicating that
the inhibition of Akt activity might be not regulated by PTEN in the hippocampus of CRS mice. We also observed the expression of -catenin, a primary target of GSK-3. Here, we found that the expression of -catenin had no change in the hippocampus of CRS mice. In addition, ERK1/2 signaling plays a crucial role in modulation of hippocampus-dependent cognitive processes. Inhibition of Erk1/2 results in spatial learning and memory impairments [59], activation of Erk1/2 has been found to confer neuroprotection in several models of apoptosis [12,60]. Consistent with published article [61], CRS down-regulated levels of p-Erk1/2 in hippocampus, and flupirtine (25 mg/kg) treatment reversed the reduction of pErk1/2 induced by CRS (Fig. 6). The modulatory action of flupirtine on Akt/GSK-3 and Erk1/2 signaling pathways might contribute to its protective effect on chronic stress-induced spatial learning and memory impairment, neuronal apoptosis. In the present study, flupirtine (10 mg/kg and 25 mg/kg) did not affect spatial learning, neuronal apoptosis, expression of Bcl-2, Bax, p-Akt, p-GSK-3 and p-Erk1/2, but flupirtine (25 mg/kg) impaired the spatial memory in unstressed mice. Besides activating Kv7 channels, flupirtine increases GABA-induced currents and exerts blocking effects on NMDAR [21,22]. In the hippocampal neurons, lower concentration of flupirtine (
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Research report
Flupirtine attenuates chronic restraint stress-induced cognitive deficits and hippocampal apoptosis in male mice Pengcheng Huang a , Cai Li a , Tianli Fu a , Dan Zhao a , Zhen Yi a , Qing Lu a,b,c , Lianjun Guo a,b,c , Xulin Xu a,b,c,∗ a
Department of Pharmacology, School of Basic Medicine, Tongji medical college, Huazhong University of Science and Technology, Wuhan 430030, China The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China c The Institute of Brain Research, Huazhong University of Science and Technology, Wuhan, 430030, China b
h i g h l i g h t s • • • •
Flupirtine rescues spatial learning and memory impairments in stressed mice. Flupirtine alleviates neuronal apoptosis in the hippocampus CA1 of stressed mice. Flupirtine attenuates synaptic loss in the hippocampus CA1 of stressed mice. Flupirtine activates Akt/GSK-3 signaling pathway in the hippocampus of stressed mice.
a r t i c l e
i n f o
Article history: Received 25 November 2014 Received in revised form 1 April 2015 Accepted 4 April 2015 Available online 11 April 2015 Keywords: Chronic restraint stress Flupirtine Cognitive impairment Akt (protein kinase B) Glycogen synthase kinase 3 beta
a b s t r a c t Chronic restraint stress (CRS) causes hippocampal neurodegeneration and hippocampus-dependent cognitive deficits. Flupirtine represents neuroprotective effects and we have previously shown that flupirtine can protect against memory impairment induced by acute stress. The present study aimed to investigate whether flupirtine could alleviate spatial learning and memory impairment and hippocampal apoptosis induced by CRS. CRS mice were restrained in well-ventilated Plexiglass tubes for 6 h daily beginning from 10:00 to 16:00 for 21 consecutive days. Mice were injected with flupirtine (10 mg/kg and 25 mg/kg) or vehicle (10% DMSO) 30 min before restraint stress for 21 days. After stressor cessation, the spatial learning and memory, dendritic spine density, injured neurons and the levels of Bcl-2, Bax, p-Akt, p-GSK-3, p-Erk1/2 and synaptophysin of hippocampal tissues were examined. Our results showed that flupirtine significantly prevented spatial learning and memory impairment induced by CRS in the Morris water maze. In addition, flupirtine (10 mg/kg and 25 mg/kg) treatment alleviated neuronal apoptosis and the reduction of dendritic spine density and synaptophysin expression in the hippocampal CA1 region of CRS mice. Furthermore, flupirtine (10 mg/kg and 25 mg/kg) treatment significantly decreased the expression of Bax and increased the p-Akt and p-GSK-3, and flupirtine (25 mg/kg) treatment up-regulated the p-Erk1/2 in the hippocampus of CRS mice. These results suggested that flupirtine exerted protective effects on the CRS-induced cognitive impairment and hippocampal neuronal apoptosis, which is possibly associated with the activation of Akt/GSK-3 and Erk1/2 signaling pathways. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: CRS, chronic restraint stress; Con, control; Flu, flupirtine; DMSO, dimethyl sulfoxide; NS, normal saline; PBS, phosphate-buffered saline; ANOVA, analysis of variance; Akt, protein kinase B; GSK-3, glycogen synthase kinase 3 beta; Erk1/2, extracellular signal-regulated kinase 1/2; PTEN, phosphatase and tensin homologue deleted on chromosome 10. ∗ Corresponding author at: Department of Pharmacology, School of Basic Medicine, Tongji medical college, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China. Tel./fax: +86 27 83691762. E-mail address: [email protected] (X. Xu). http://dx.doi.org/10.1016/j.bbr.2015.04.004 0166-4328/© 2015 Elsevier B.V. All rights reserved.
Substantial evidence implicates stress is an important factor in the vulnerability to depression and other behavioral disorders [1]. Chronic restraint stress (CRS) can exacerbate neurodegeneration and cognitive deficits [2–4]. Growing evidences have shown that chronic stress causes atrophy and functional impairment in several key brain areas such as the frontal cortex and hippocampus [5,6]. The hippocampus is a region that plays a crucial role in learning and memory and is an area also particularly susceptible to chronic stress [7,8]. Extensive researches have proved that CRS disrupts the hippocampus-dependent cognitive function [4,9,10].
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Akt (Protein kinase B) has been thought to be involved in neuronal survival, and activation of the kinase confers neuroprotection in several models of apoptosis [11–13]. Glycogen synthase kinase3 (GSK-3), a ubiquitous cellular serine/threonine protein kinase, plays a role in various essential physiological processes in the mammalian brain, such as development, cell cycle, or apoptosis [14]. GSK-3 serving as an essential downstream effector of Akt, its activity is inhibited by Akt-mediated phosphorylation at serine 9 [15]. It has been shown that GSK-3 is activated in the hippocampus of chronic stressed animals [9,16], and inhibition of GSK-3 restores chronic stress-induced memory deficit [17] and neuronal apoptosis [18]. Flupirtine is clinically used as a non-opioid analgesic with muscle relaxant [19,20]. In addition to its well-characterized as a Kv7 channel activator, flupirtine also acts like an NMDA receptor antagonist and has GABAA receptor-agonistic properties [21,22]. Several studies have demonstrated that flupirtine has significant powerful anti-oxidative and anti-apoptosis effects either in vitro or in vivo [23–25]. Flupirtine alleviates neuronal degeneration and cognitive impairment induced by repetitive hyperthermic seizures [25]. However, the molecular mechanisms for neuroprotective effects of flupirtine have not yet been fully understood. Furthermore, we have previously shown that flupirtine can prevent impairment of acute stress on spatial memory retrieval via inactivation of GSK-3 [26]. In the present study, we investigated the effects and underlying mechanisms of flupirtine on cognitive deficits and hippocampal apoptosis induced by CRS. 2. Materials and methods 2.1. Animals Adult male Kunming (KM) mice, weighing 20–25 g, were obtained from the Animal Center of Tongji Medical College. Five mice were kept in a cage and were allowed free access to water and food. The mice were maintained at a constant temperature of 23 ± 1 ◦ C, humidity at 55 ± 5% and under a 12:12 light/dark cycle (lights on at 7:00 a.m). The mice were allowed to acclimatize for 7 days before experiments. All experimental protocols were approved by the Review Committee for the Use of Human or Animal Subjects of Huazhong University of Science and Technology in accordance with the NIH guidelines for the care and use of laboratory animals (approval number: S400/5/1/2011). All efforts were made to minimize animal suffering and number of animals necessary. 2.2. CRS model and animals treatment Adult mice were subjected to CRS as previously described [27,28] with minor modifications. The mice were restrained for 6 h (from 10:00 to 16:00) daily in well-ventilated Plexiglass tubes (3.3 cm diameter, 10.5 cm length) for 21 consecutive days. During restraint stress, animals were not physically compressed, but food and water were deprived. The same handle was carried on the unstressed mice. Animals were divided into six groups randomly: 1. Control group: mice were treated with vehicle (normal saline containing 10% DMSO) for 21 days (Con, n = 15); 2. Flupirtine group: mice were injected with flupirtine (10 mg/kg or 25 mg/kg) for 21 days (Flu10, n = 13; Flu25, n = 15); 3. CRS group: mice were injected with vehicle 30 min before restraint stress for 21 days (CRS, n = 16); 4. Flupirtine treatment group: mice were treated with flupirtine (10 mg/kg or 25 mg/kg) 30 min before restraint stress daily for 21 days (CRS + Flu10, n = 14; CRS + Flu25, n = 14).
Flupirtine maleate (Targsense scientific Co. Ltd, Shanghai, China) was dissolved in normal saline containing 10% DMSO in concentrations of 1 mg/ml and 2.5 mg/ml. Flupirtine or vehicle was administered intraperitoneally (i.p.) in a volume of 10 ml/kg body weight 30 min prior to the onset of restraint stress. 2.3. Morris water maze The spatial learning and memory performance was determined using the Morris water maze (MWM) test as previously described [29] with minor modifications. The Morris water maze consisted of a stainless-steel circular pool (90 cm diameter, 45 cm height) and a Plexiglass platform (15 cm diameter). The container was filled with water (23 ± 2 ◦ C) that was dyed by using non-toxic paint. The Plexiglass platform was submerged approximately 2 cm below the surface of the water and placed in center of second quadrant during the training session. Mice were given four trials per day for four consecutive days after CRS. Mice were placed into the tank facing the wall of the pool, and were allowed to swim and find the hidden platform. The time to reach the platform (escape latency) was recorded in each trial. During each trial, each mouse was given 60 s to find the hidden platform. If the mice failed to find the platform within 60 s, they would be guided to find the platform and stayed on it for 20 s. On 5th day after stress, the platform was removed and mice were tested on a spatial probe trial for 60 s. The time of mice spent in the target quadrant was recorded. An automatic tracking system MT-200 (Chengdu instrument Co., Chengdu, Sichuan province, China) was used to monitor swimming activities. After the Morris water maze test, mice were sacrificed for further histological and biochemical analysis. 2.4. Hematoxylin and eosin staining Mice (n = 4–5) were deeply anesthetized with 10% chloral hydrate (i.p., 0.35 ml/100 g) and perfused with normal saline solution, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). After perfusion, the brains tissues were removed carefully and kept in 4% paraformaldehyde at 4 ◦ C overnight. After fixation and dehydration with gradient ethanol, the brain tissues were embedded in paraffin and sliced into in the coronal plane at 5 m thickness using a section cutter (Leica, Germany). The sections (2 sections/mouse) were stained with hematoxylin and eosin (H&E). Morphology of hippocampus was observed by a light microscope (Olympus Corporation, Japan). Damaged neurons were identified by their acidophilic (eosinophilic) cytoplasm and pyknotic nuclei, which are suggestive of necrotic morphology [30,31]. Cell counting of injured cells was performed in the pyramidal layer of the hippocampus CA1 of the mice. 2.5. Golgi silver staining Dendritic spine of pyramidal neurons in the hippocampus was observed by Golgi silver staining as previous study [32]. Mice (n = 4–5) were anesthetized with 10% chloral hydrate and perfused with normal saline solution. The brain tissues were removed and stored in Golgi-Cox solution for 14 days, then kept in 30% sucrose solution. Brain tissues were sectioned at 50 m thickness in the coronal plane using a vibratome (Camden Instrument, MA752, Leicester, UK). Dendritic images were acquired and analyzed with a microscope of OLYMPUS BX51 and software of Image-Pro Express. The numbers of dendritic spine in the hippocampus CA1 neurons (50 m segment, 70 dendritic segments on 35 neurons from each group) were counted by the observers who were blind to experimental conditions.
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2.6. Immunohistochemical staining
3. Results
Brain tissues of mice (n = 5) were routinely processed in paraffin blocks and later sliced into 5 m in the coronal plane using a section cutter (Leica, Germany). Immunohistochemical staining was performed as previous study [33]. Briefly, after deparaffinization and hydration, endogenous peroxidase was quenched with 0.3% H2 O2 in ultrapure water for 15 min. Nonspecific immunoglobulin binding sites were blocked with 5% normal goat serum for 1 h, then the sections (2 sections/mouse) were incubated with rabbit synaptophysin (1:400, Abcam, Cambridge, MA, USA) at 4 ◦ C overnight followed by incubated for 1 h with secondary antibody at room temperature. Immunoglobulin complexes were visualized on incubation with DAB. PBS was used instead of primary or secondary antibody or ABC reagent as negative controls. Results were captured with a light microscope (Olympus Corporation, Japan) and quantified by mean optical intensity in five random microscopic fields of per-section. To evaluate synaptophysin immunoreactivity (IR), the average optical density of positive in images was quantified with Image-Pro Plus 6.0 software. Analysis was done by observers who were blind to experimental conditions.
3.1. Flupirtine prevented CRS-induced spatial learning and memory impairment
2.7. Western blot In order to alleviate the suffering of the animals, mice were sacrificed by decapitation under urethane anesthesia. The hippocampus was quickly removed from the brain and stored at −80 ◦ C until further use. Hippocampal tissues were homogenized in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. The tissue homogenates were then incubated for 30 min on ice and centrifuged at 16,000 g for 15 min at 4 ◦ C, then the supernatants were collected carefully. Protein concentrations were determined using a BCA protein assay kit (Thermo scientific pierce, Rockford, IL, USA). Equal amount of protein from different group was subjected to SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in Tris-buffered Saline + 0.1% Tween (TBST) buffer (pH = 7.6) for 1 h at room temperature, then the membranes were incubated overnight at 4 ◦ C with primary antibodies. Primary antibodies were against: anti-Phospho-Akt(ser473), anti-Akt, anit-PhosphoGSK-3(ser9), anti-GSK-3, anti--Catenin or anti-Synaptophysin (1:1000, Cell Signaling Technology, Danvers, MA, USA); anit-P-Erk (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Erk2 (1:1000, Proteintech Group Inc, Chicago, IL,USA); anti-Bax, antiBcl-2 or anti- PTEN (1:1000, ImmunoWay, Newark, DE, USA); anti-GAPDH (1:5000, CWBIO, Beijing, China). Following washed in TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoblots were revealed with an enhanced chemiluminescence system (ECL) (Millipore, Billerica, MA, US) and visualized by DNR Bio-Imaging systems (CAT.NO: 70-25-00; MicroChemi, Israel). Optical densities of the bands were scanned and quantified with NIH Image J software. The data from the bands were normalized to GAPDH. Values of p- protein were normalized to total protein. The experiments of western blot were repeated 3–4 times. 2.8. Statistical analyses All the data are expressed as the mean ± standard error of the mean (S.E.M). Differences in the escape latencies in the Morris water maze test were analyzed with two-way ANOVA with repeated measures followed by the Tukey HSD post hoc test for multiple comparisons among different group. The other data were analyzed by one-way ANOVA followed by Tukey HSD post hoc test. P < 0.05 was considered as statistically significant.
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The spatial learning and memory performance was assessed with the Morris water maze test. The analysis of escape latency by using two-way ANOVA showed significant differences among groups (groups effect: F(5,81) = 6.367, P = 0.000; training day effect: F(3 , 243) = 43.029, P = 0.000; n = 13–16 per group). Compared with control group, CRS mice exhibited significantly longer escape latencies on day 2, 3 and 4 (P < 0.05, P < 0.01, P < 0.01 respectively, Fig. 1A). Compared with CRS group, flupirtine (10 mg/kg) treatment decreased the escape latencies on day 4 (P < 0.05), and flupirtine (25 mg/kg) treatment decreased the escape latencies on days 3 and 4 (P < 0.001, P < 0.01 respectively, Fig. 1A). In addition, the analysis of the swimming speed by using two-way ANOVA showed no significant differences among groups (groups effect: F(5 , 81) = 1.118, P = 0.3500; training day effect: F(3,243) = 1.447, P = 0.229; Fig. 1B). The probe test was performed for 60 s on the 24 h after the last training test. During the probe test, the analysis of time spent in the target quadrant by using one-way ANOVA showed significant differences among groups (F(5,81) = 4.866, P = 0.001; Fig. 1C, D). CRS mice spent significant less time in target quadrant than control group (P < 0.01). Compared with CRS group, flupirtine (25 mg/kg) treatment increased the time spent in target quadrant (P < 0.05). Otherwise, compared with control group, unstressed mice by treatment with flupirtine (25 mg/kg) spent less time in target quadrant (P < 0.05). Additionally, there were no significant differences of swimming speed among the six groups on day of probe test (F(5,81) = 0.768, P = 0.575; data not shown). These results indicated that flupirtine prevented the impairment of spatial learning and memory induced by CRS. 3.2. Flupirtine alleviated CRS-induced neuronal apoptosis in the hippocampus CA1 The neuronal histopathological changes were observed by hematoxylin and eosin (H&E) staining. The analysis of injured neurons by using one-way ANOVA showed significant differences among groups (F(5,20) = 22.160, P = 0.000). In control group, there were no significant neuronal abnormalities in the hippocampus CA1. However, in CRS group, many more degenerated neurons (eosinophilic pyknotic neuron) were observed in the hippocampus CA1 (P < 0.01, Fig. 2B). Compared with CRS group, flupirtine (10 mg/kg and 25 mg/kg) treatment significantly alleviated the neuronal degeneration (P < 0.01, Fig. 2B). To observe the effect of flupirtine on apoptosis-related proteins, the protein levels of Bax and Bcl-2 were assessed by western blot. The analysis of Bax levels by using one-way ANOVA showed significant differences among groups (F(5,30) = 9.078, P = 0.000). In comparison with control group, the expression of Bax in the hippocampus of CRS mice was significantly increased (P < 0.01; Fig. 2D). Compared with CRS group, flupirtine (10 mg/kg and 25 mg/kg) treatment reduced the increase of Bax in the hippocampus (P < 0.05, P < 0.001, respectively). However, as shown in Fig. 2E, CRS or flupirtine treatment did not influence the levels of Bcl-2 in the hippocampus (F(5,30) = 0.717, P = 0.616). These results revealed that flupirtine protected against CRS-induced neuronal apoptosis in the hippocampus. 3.3. Flupirtine increased CRS-induced reduction of dendritic spine density and synaptophysin expression in the hippocampus CA1 To further investigate the effect of flupirtine on dendritic spine density and synaptophysin expression, the dendritic spine density
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Fig. 1. Flupirtine prevented CRS-induced learning and memory impairment. (A) Escape latencies to find the hidden platform during days 1–4 of training. (B) Swimming speed during days 1–4 of training. (C) Representative swimming paths of mice in different groups during the probe test. (D) Summary of time spent in target quadrant of mice in different groups during the probe test. Data are expressed as mean ± SEM. n = 13-16 per group, * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
was assessed by Golgi Silver Staining and the synaptophysin expression was observed by histochemical staining and western blot. The analysis of spine density by using one-way ANOVA showed significant differences among groups (F(3,14) = 47.204, P = 0.000). Compared with control group, the spinal density in hippocampus CA1 of CRS mice was markedly decreased (P < 0.01; Fig. 3A). Compared with CRS group, flupirtine (25 mg/kg) treatment reversed the CRS-induced reduction of the spinal density in hippocampus CA1 (P < 0.01; Fig. 3A). In immunohistochemical staining, the analysis of synaptophysin with one-way ANOVA demonstrated conspicuous differences among four groups (F(3,15) = 32.545, P = 0.000). Compared with control group, the mean density of synaptophysin immunoreactivity in hippocampus CA1 of CRS mice was dramatically decreased (P < 0.01; Fig. 3B). Compared with CRS group, flupirtine (25 mg/kg) treatment reversed the reduction of synaptophysin expression in hippocampus CA1 (P < 0.01). Additionally, the results of western blot showed that CRS or flupirtine treatment did not influence the expression of synaptophysin in the hippocampus (F(3,12) = 0.540, P =0.664; Fig. 3C). These results suggested that flupirtine reversed CRS-induced dendritic spine density and synaptophysin expression in the hippocampus CA1.
3.4. Flupirtine activated Akt/GSK-3ˇ and Erk1/2 signaling pathways in the hippocampus of CRS mice In order to further explore the effects of flupirtine on Akt/GSK3 and Erk1/2 signaling pathways, the p-Akt, p-GSK-3 and pErk1/2 in the hippocampus were assessed by immunoblot. Analyses
with one-way ANOVA revealed a significant main effect of drug (pAkt: F(5,18) = 9.332, P = 0.000; p-GSK-3: F(5,24) = 11.860, P = 0.000; p-Erk1/2: F(5,27) = 9.084, P = 0.00; Figs. 4–6). Compared with control group, the levels of p-Akt, p-GSK-3 and p-Erk1/2 in the hippocampus of CRS mice were significantly decreased (P < 0.01, P < 0.001, P < 0.01, respectively). Compared with CRS group, flupirtine (10 mg/kg and 25 mg/kg) treatment strongly prevented the reduction of p-Akt and p-GSK-3 (P < 0.05, P < 0.01, respectively), and flupirtine (25 mg/kg) treatment prevented the reduction of p-Erk1/2 (P < 0.01). Analyses with one-way ANOVA showed no statistical differences of total Akt, GSK-3 and Erk2 between groups (Akt: F(5,18) = 0.302, P = 0.905; GSK-3: F(5,18) = 1.103, P = 0.393; Erk2: F(5,24) = 0.246, P =0.938; Fig. 4-6). Additionally, CRS or flupirtine treatment had no effect on the expression of PTEN and -catenin in the hippocampus (PTEN: F(5,36) = 0.613, P = 0.691, Fig. 4D; -catenin: F(5,18) = 0.62, P = 0.687, Fig. 5D). These finding showed that flupirtine activated Akt/GSK-3 and Erk1/2 signaling pathways in the hippocampus of CRS mice.
4. Discussion The data of the present study revealed that flupirtine improved CRS-induced spatial learning and memory impairment. Flupirtine alleviated the neuronal apoptosis and reduction of dendritic spine density and synaptophysin expression in the hippocampus CA1 of CRS mice. Furthermore, flupirtine reduced the expression of Bax and increased the levels of p-Akt, p-GSK-3 and p-Erk1/2 in the hippocampus of CRS mice.
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Fig. 2. Flupirtine alleviated CRS-induced apoptosis in the CA1 region of hippocampus. (A) Hematoxylin- and eosin-stained in hippocampus CA1 neurons of mice in different groups. The arrows indicated injured neurons (cells with pyknotic nuclei, acidophilic cytoplasm and fragmented nuclei) (HE × 400, inset HE × 1000). (B) The number of eosinophilic pyknotic neurons in the hippocampus CA1 was counted. Significant degenerated neurons were observed in the hippocampus CA1 of CRS mice. Treatment with flupirtine (10 mg/kg and 25 mg/kg) significantly alleviated the CRS-induced neuronal degeneration. (C) Representative western blot of Bax and Bcl-2 in the hippocampus. (D) Statistics for western blot results of Bax. Western blot analysis showed that flupirtine (10 mg/kg and 25 mg/kg) treatment reversed the CRS-induced up-regulation of Bax in the hippocampus. (E) Statistics for western blot results of Bcl-2. Bcl-2 showed no change between groups. Data are presented as mean ± SEM. n = 4–6 per group, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
Chronic stress is associated with the development of psychiatric disorders such as depression and post-traumatic stress disorder [2,34,35], which leads to cognitive and emotional impairment. Consistent with previous studies [2,3], our results revealed that CRS impaired the spatial learning and memory functions, CRS mice spent longer time to locate the platform in the training sessions and sojourned shorter in the target quadrant in MWM test. Previous study has shown that flupirtine alleviates spatial learning and memory impairment induced by repetitive hyperthermic seizures [25]. A double-blind study in patients suffering from Creutzfeldt-Jakob’s disease has revealed that oral flupirtine application significantly reduces the deterioration of cognition [36]. Moreover, our previous study has demonstrated that flupirtine protects against memory deficit induced by acute stress [26]. We explored whether flupirtine exerted a protective effect against
CRS-induced learning and memory impairment. Our results showed that flupirtine (10 mg/kg and 25 mg/kg) treatment alleviated the impairment of learning acquisition induced by CRS, and flupirtine (25 mg/kg) treatment prevented memory retrieval impairment induced by CRS. These results suggested that flupirtine could prevent CRS-induced spatial learning and memory impairment. Chronic stress has been known to impair brain function and increase the susceptibility of neurons to injury, especially in the hippocampus [37,38]. Stress induces significant neuronal apoptosis in the hippocampus, and apoptosis has been proposed to be an important mechanism contributing to stress-related cognitive deficits [39,40]. Neuroprotective effects of flupirtine have been previously demonstrated in both in vitro and in vivo experimental models [41–43]. In the present study, we investigated
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Fig. 3. Flupirtine increased the dendritic spine density and synaptophysin expression in the hippocampus CA1 of CRS mice. (A) Representative photomicrographs of Golgi staining from each group (upper). Spine numbers of dendrites (50 m segment) were counted (70 dendritic segments on 35 neurons from each group). The summary data (bottom) showed that treatment with flupirtine (25 mg/kg) reversed CRS-induced the decrease in the dendritic spine density of hippocampus CA1. (B) The photomicrographs (upper) showed synaptophysin expression in hippocampus CA1 pyramidal neurons of mice. The histogram (bottom) showed the change of mean density labeled synaptophysin. (C) Representative western blot (upper) and corresponding densitometric analysis of synaptophysin (bottom) in the hippocampus of mice. Synaptophysin showed no change between groups. Data are presented as mean ± SEM. n = 4–5 per group ** P < 0.01 compared with control group; ## P < 0.01 compared with CRS group.
whether flupirtine prevented CRS-induced hippocampal apoptosis. We found that CRS caused neuronal apoptosis in the hippocampus CA1 as previous study [3,44], and flupirtine (10 mg/kg and 25 mg/kg) treatment alleviated CRS-induced hippocampal apoptosis (Fig. 2). Furthermore, CRS increased the expression of Bax, and flupirtine (10 mg/kg and 25 mg/kg) treatment reversed the increment of Bax. In addition, our results showed that levels of Bcl-2 in hippocampus of mice in six groups had no obvious differences. The data concerning the Bcl-2 protein levels in the hippocampus after chronic restraint stress are contradictory. Some report that chronic restraint stress (1 h twice/day for 10 days, 2 h/day for 9 days) reduce the Bcl-2 expression [45–47]. On the other hand, some report that mild restraint (1 h/day for 7days, 2.5 h/day for 14 days) elevate the
Bcl-2 expression, and the stress-induced increase in Bcl-2 may correspond to a neuroprotective response [48,49]. Luo C. et al. have found that levels of Bcl-2 are unaltered in the hippocampus after restraint for 21 days [50], and their observations support our results that restraint for 21 days does no affect the Bcl-2 expression in the hippocampus. These discrepancies might be attributed to different stress protocols. Dendritic spines and synapses are known to play a central role in the CNS by forming the complex circuitry, which are critical for neuronal connectivity and brain function including cognitive processes and mood [51,52]. Chronic restraint stress leads to neuronal remodeling of hippocampal neurons, loss of dendritic spines and reduction of synaptic density have been demonstrated in the
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Fig. 4. Flupirtine increased p-Akt in the hippocampus of CRS mice. (A) Representative western blot of p-Akt, total Akt and PTEN. (B) Statistics for western blot results of p-Akt. Flupirtine (10 mg/kg and 25 mg/kg) treatment reversed the CRS-induced reduction of p-Akt. (C) Statistics for western blot results of total Akt. Total Akt showed no change between groups. (D) Statistics for western blot results of PTEN. PTEN showed no change between groups. Data are presented as mean ± SEM. n = 4–6 per group, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
Fig. 5. Flupirtine increased p-GSK-3 in the hippocampus of CRS mice. (A) Representative western blot of p-GSK-3, total GSK-3 and -catenin. (B) Statistics for western blot results of p-GSK-3. Treatment with flupirtine (10 mg/kg and 25 mg/kg) reversed the CRS-induced reduction of p-GSK-3. (C) Statistics for western blot results of total GSK-3. Total GSK-3 showed no change between groups. (D) Statistics for western blot results of -catenin. -catenin showed no change between groups. Data are presented as mean ± SEM. n = 4–7 per group ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with CRS group.
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Fig. 6. Flupirtine increased p-Erk1/2 in the hippocampus of CRS mice. (A) Representative western blot of p-Erk1/2, total Erk2. (B) Statistics for western blot results of p-Erk1/2. Flupirtine (25 mg/kg) treatment reversed the CRS-induced reduction of p-Erk1/2. (C) Statistics for western blot results of total Erk2. Total Erk2 showed no change between groups. Data are presented as mean ± SEM. n = 4–7 per group ** P < 0.01 compared with control group; ## P < 0.01 compared with CRS group.
hippocampus of chronic restraint stressed animals [9,53,54]. To determine whether the protective effects of flupirtine on cognition were accompanied by an increase in dendritic spine and synaptic density in the hippocampus, we investigated the number of dendritic spines and expression of synaptophysin, the most widely used markers for the determination of synaptic density in the hippocampus. As previous studies [27,55], CRS induces a significant reduction of dendritic spine density in the hippocampal CA1 pyramidal neurons, here, we found that flupirtine (10 mg/kg or 25 mg/kg) treatment alleviate the reduction. Western blot results showed that CRS and flupirtine treatment had no influences on synaptophysin expression in the whole hippocampus tissue. Western blot analysis cannot reveal localized changes of synaptic density. Therefore, we used immunohistochemical analysis to observe synaptophysine expression in hippocampal CA1. The results showed that CRS decreased synaptophysin expression, and flupirtine treatment elevated the levels of synaptophysin in hippocampus CA1. Thus, we postulate that the spatial learning and memory improvement effects of flupirtine are due, at least in part, to its enhancement effects on dendritic spine and synapse formation. The Akt/GSK-3 signaling pathway plays an important role in regulating learning and memory, synaptic plasticity and cell survival [56,57]. Previous studies have demonstrated the inactivation of Akt/GSK-3 pathway in the hippocampus of chronic stressed animals [17,58]. Increased activation of the Akt/GSK-3 provides protective effects against neuronal injury [56,57]. 1,2-dilinoleoyl-sn-glycero- 3-phosphocholine upregulates the Akt/GSK-3 pathway and rescues the memory deficits in chronic restraint stressed mice [17]. DL-/PO-phosphatidylcholine restores restraint stress-induced depression-related behaviors and spatial memory impairment via inactivation of GSK-3 [17]. The present study revealed that flupirtine (10 mg/kg and 25 mg/kg) reversed the reduction of p-Akt and p-GSK-3 induced by CRS (Figs. 4 and 5), suggesting that flupirtine presented neuroprotective and cognitive improvement effects might be through activation of Akt/GSK-3 signaling pathway. However, CRS did not cause upregulation of PTEN, one of upstream proteins of Akt, implicating that
the inhibition of Akt activity might be not regulated by PTEN in the hippocampus of CRS mice. We also observed the expression of -catenin, a primary target of GSK-3. Here, we found that the expression of -catenin had no change in the hippocampus of CRS mice. In addition, ERK1/2 signaling plays a crucial role in modulation of hippocampus-dependent cognitive processes. Inhibition of Erk1/2 results in spatial learning and memory impairments [59], activation of Erk1/2 has been found to confer neuroprotection in several models of apoptosis [12,60]. Consistent with published article [61], CRS down-regulated levels of p-Erk1/2 in hippocampus, and flupirtine (25 mg/kg) treatment reversed the reduction of pErk1/2 induced by CRS (Fig. 6). The modulatory action of flupirtine on Akt/GSK-3 and Erk1/2 signaling pathways might contribute to its protective effect on chronic stress-induced spatial learning and memory impairment, neuronal apoptosis. In the present study, flupirtine (10 mg/kg and 25 mg/kg) did not affect spatial learning, neuronal apoptosis, expression of Bcl-2, Bax, p-Akt, p-GSK-3 and p-Erk1/2, but flupirtine (25 mg/kg) impaired the spatial memory in unstressed mice. Besides activating Kv7 channels, flupirtine increases GABA-induced currents and exerts blocking effects on NMDAR [21,22]. In the hippocampal neurons, lower concentration of flupirtine (
