Menopause: The Journal of The North American Menopause Society Vol. 23, No. 1, pp. 18-26 DOI: 10.1097/GME.0000000000000486 ß 2015 by The North American Menopause Society
Combined exercise ameliorates ovariectomy-induced cognitive impairment by enhancing cell proliferation and suppressing apoptosis Tae-Woon Kim, PhD,1 Chang-Sun Kim, PhD,2 Ji-Yeon Kim, MS,2 Chang-Ju Kim, MD, PhD,1 and Jin-Hee Seo, PhD 3 Abstract Objective: Estrogen plays an important role in cognitive function, including attention, learning, and memory, and affects the structure and function of brain areas. We investigated the effects of combined exercise on memory deficits induced by ovariectomy (OVX) in relation to cell proliferation and apoptosis in the hippocampus. Methods: Rats were randomly divided into four groups: sham, sham and exercise, OVX, and OVX and exercise. Rats in combined exercise groups were subjected to 3 days of resistance training and 3 days of running (for a total of 6 d/wk) for eight consecutive weeks. Rats were tested in step-down avoidance task and Morris water maze task to verify the effects of OVX on short-term and spatial working memory. Results: In the present study, the number of BrdU-positive and doublecortin-positive cells and expression of brain-derived neurotrophic factor, TrkB, and Bcl-2 decreased; expression of Bax and the number of caspase-3– positive and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling–positive cells increased; and short-term and spatial working memory decreased in the OVX group compared with the sham group. Conversely, when the combined exercise group was compared with the OVX group, the number of BrdU-positive and doublecortin-positive cells and expression of brain-derived neurotrophic factor, TrkB, and Bcl-2 increased; expression of Bax and the number of caspase-3–positive and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling–positive cells decreased; and short-term and spatial working memory increased. Conclusions: Combined exercise increases cell proliferation and inhibits apoptosis in the hippocampus and improves cognitive function despite estrogen deficiency. Key Words: Ovariectomy – Cognitive function – Cell proliferation – Apoptosis – Short-term and spatial working memory.
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strogen reduction after menopause influences various organ systems. For example, estrogen deficiency is known to cause harmful effects, including bone and cardiovascular diseases.1,2 In addition, in the past 35 years, it has been clearly shown that one of the major target organs of estrogen is the brain.3 Estrogen in the brain participates in gonadal regulation, physiological homeostasis, and reproduction. Furthermore, estrogen plays an important role in cognitive function, including attention, learning, and memory.4 Estrogen affects the structure and function of brain areas that participate in learning and memory.5 In particular, estrogen contributes to maintaining plasticity and cell survival in the adult brain, and a postmenopausal decrease in estrogen Received January 12, 2015; revised and accepted April 6, 2015. From the 1Department of Physiology, College of Medicine, KyungHee University, Seoul, Republic of Korea; 2Department of Physical Education, College of Natural Science, DongDuk Women’s University, Seoul, Republic of Korea; and 3Division of Sports Science, Baekseok University, Cheonan, Republic of Korea. Funding/support: None. Financial disclosure/conflicts of interest: None reported. Address correspondence to: Jin-Hee Seo, PhD, Division of Sports Science, Baekseok University, 330-704, 76 Munam-ro, Dongnam-gu, Cheonan-si, Chungcheongnam-do, Republic of Korea. E-mail: [email protected]
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induces cognitive dysfunction and increases the risk of various neurodegenerative diseases.6 In young adult female rodents and primates, a difference in cognitive capacity, based on estrogen levels during the natural estrous cycle or the menstrual cycle, was demonstrated. Compared with low estrogen levels, high estrogen levels led to improved cognitive function.7 Among brain areas associated with such cognitive function, the hippocampus, in particular, is strongly influenced by estrogen. Estrogen has been reported to have an important effect on the structure and function of the hippocampus. The hippocampus is one of the brain areas responsible for cognitive function, including learning and memory. In a previous study, estrogen was shown to promote spine maturation in the hippocampus; as a result, hippocampusdependent memory was improved.8 Several studies have shown that brain diseases involving cognitive dysfunction occur because of impaired hippocampal function. Furthermore, the hippocampus plays a key role in storing new memories and is associated with declarative and spatial memory.9 Hippocampal activation leads to increase in neurogenesis, activation of neurotrophic factors, and inhibition of apoptosis; consequently, activation of the hippocampus leads to increase in cognitive function. Neurogenesis occurs through a four-step proliferation process, including migration,
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survival, differentiation and maturation of neural progenitor cells originating from neural stem cells.10 Doublecortin (DCX) is a protein expressed in the brain during neuronal migration and differentiation in neurogenesis.11 Furthermore, neurogenesis is promoted by a variety of neurotrophic factors, and many areas of the brain work in tandem to regulate proliferation and differentiation of neural stem cells or progenitor cells.12-14 In particular, brain-derived neurotrophic factor (BDNF) binds to the TrkB receptor, causing an increase in hippocampal neurogenesis. Activation of neuronal apoptosis and abnormal apoptotic regulatory protein expression cause neurodegenerative disorders such as Parkinson disease and Alzheimer disease.15,16 Apoptosis is regulated via a signaling pathway that includes the antiapoptotic factor Bcl-2 and the proapoptotic factor Bax (a member of the Bcl-2 family) upstream and members of the caspase protein family (such as caspase-3) downstream.17 However, physiological estrogen also plays a role in apoptosis by regulating programmed cell death.18 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay detects features of apoptotic cell death. Neuronal turnover resulting from neuronal death and neurogenesis in the brain is a special form of neuroplasticity that is related to diverse factors such as stress responses, cognitive function, and treatment of mental disorders.19 In previous studies, increased cell death and decreased cell proliferation in the hippocampus during aging, stress, or traumatic brain injury led to deficits in cognitive function such as learning and memory.20,21 In particular, hippocampal neurogenesis has been shown to play an important role in learning and memory.22 As such, estrogen deficiency inhibits hippocampal neurogenesis and induces apoptosis, which may potentially cause cognitive disorders. On the other hand, exercise has been suggested to prevent and relieve various symptoms of menopause.23 Exercise affects physiological and biochemical changes in the brain24 and gene transcription associated with neural activity, plasticity, and regeneration.25 In addition, exercise increases neurogenic nutrient expression26 and inhibits neuronal cell death to improve cognitive function.27 Several previous studies have reported that exercise influences the expression of neurotransmitters and provides support against anxiety, stress, depression, insomnia, and miscellaneous psychological disorders. In particular, neurotransmitters are associated with emotional regulation, awareness, behavior and motor control, memory and learning, temperature regulation, pain transmission, food intake, stress responses, and brain development and maturation.28,29 However, no studies have focused on the effects of exercise on the improvement of hippocampal and cognitive function caused by postmenopausal estrogen deficiency. Consequently, the present study sought to investigate whether a combination of aerobic and anaerobic exercise influences the cognitive function of ovariectomized rats. Therefore, we measured short-term and spatial working memory, performance in step-down avoidance task and
Morris water maze task, cell proliferation, BDNF levels, and apoptosis in the hippocampus. METHODS Animals Eight-week-old Sprague-Dawley rats weighing 230.9 (5.5) g were obtained from a commercial breeder (Orient Co, Seoul, Korea) for the experiment. Experimental procedures were performed in accordance with the animal care guidelines of the National Institutes of Health and the Korean Academy of Medical Sciences. The rats were housed under controlled temperature (mean [SEM], 208C [28C]) and lighting (07:0019:00 h) conditions, with food and water available ad libitum. The rats were randomly divided into four groups (n ¼ 10 per group): sham, sham and exercise, ovariectomy (OVX), and OVX and exercise. Surgical operation For OVX, Zoletil 50 was injected into the abdominal cavity for general anesthesia, and hair was removed from both sides of the back. Surgical operation was performed with the rat in prone position, and the surgical area was sterilized with 10% Betadine solution. A 1.5-cm incision at the midsection of the spine next to the rearmost rib was made, the peritoneum was incised on the left and right, and the ovaries were excised. Absorbable floss was used to suture, after which the ovaries were removed. The peritoneum and muscle layer were simultaneously sutured, whereas the epidermal layer was separately sutured using nonabsorbable suture. In sham rats, the same approach was used up to the step of peritoneum incision; however, these animals were sutured without excising the ovaries. Combined exercise protocol The combined exercise program includes 3 days of resistance training and 3 days of running, for a total of 6 days of exercise. Tower-climbing exercise A ladder with a height of 135 cm, intervals of 2.5 cm, and inclination of 808 was climbed with a weight load near the tail region, as previously described.30 For the adjustment period, a weight load weighing 50% of the rat’s weight was used for 5 days/week for 2 weeks. After measurement of one repetition maximum, exercise began at 50% of the intensity of one repetition maximum, and 50 g of weight was added every 2 weeks to constitute a four-stage tower-climbing exercise. The exercise was performed once a day and 3 days/week for eight consecutive weeks. Treadmill exercise The exercise group began exercise on a treadmill made for use of small animals. Five minutes of warm-up at 08 inclination at 3 meters/minute, 30 minutes of main exercise at 10 meters/minute, and 5 minutes of cool-down at 3 meters/ minute were performed for the first 2 weeks. After this, Menopause, Vol. 23, No. 1, 2016
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40 minutes of main exercise at 10 meters/minute on weeks 3 and 4, 30 minutes of main exercise at 15 meters/minute on weeks 4 to 6, and 40 minutes of main exercise at 15 meters/ minute for the final weeks 6 to 8 were performed. The exercise was performed once a day and 3 days/week for eight consecutive weeks. During treadmill running, electrical stimulation was removed to minimize stress. Step-down avoidance task Latency time of the step-down avoidance task was determined to evaluate short-term memory. The rats were trained in a step-down avoidance task on the 38th day after the start of the treadmill exercise. Latency time (in seconds) in each group was measured 2 hours after training. The rats were placed on a platform (7 25 cm, 2.5 cm in height). The platform faced a grid (42 25 cm) of parallel stainless steel bars 0.1 cm in caliber and spaced 1 cm apart. During training sessions, the animals received a 0.5-mA scramble foot shock for 2 seconds immediately upon stepping down. The interval that elapsed between the rats stepping down and the rats placing all four paws on the grid was defined as latency time. A latency time longer than 180 seconds was counted as 180 seconds. Morris water maze task Spatial working memory was evaluated with Morris water maze task. This task requires rats to learn the spatial location of a hidden platform in a black circular pool (180 cm in diameter and 50 cm in height) filled with clear water (mean [SEM], 258C [18C]). The hidden platform (15 cm in diameter and 40 cm in height) was placed 2 cm below the surface of the water in the middle of the north quadrant and was camouflaged by transparency against a black background. Distal visual cues were placed on the walls around the pool. The position of the cues remained unchanged throughout the task. One day before training, the rats were habituated to swimming for 60 seconds in the pool without a platform. All rats were trained three times a day for four consecutive days before killing, and probe trail was conducted 24 hours after the last training. When finding the platform, the rats were allowed to remain for 30 seconds. If the rats did not find the platform within 60 seconds, they were guided by hand to the platform. The rats were given a 60-second retention probe test, and the platform was removed from the pool. Data were automatically collected via the Smart Video Tracking System (Smart version 2.5; Panlab, Barcelona, Spain). Tissue preparation The animals were killed immediately after determination of Morris water maze task. For preparation of brain slices, the animals were fully anesthesized with Zoletil 50 (10 mg/kg IP; Vibac, Carros, France), after which the rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS) and fixed with a freshly prepared solution of 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). The brains were removed, postfixed in the same fixative overnight, and
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transferred into a 30% sucrose solution for cryoprotection. Coronal sections 40 mm thick were made using a freezing microtome (Leica, Nussloch, Germany).31 Immunohistochemistry for BrdU To detect newly generated cells in the dentate gyrus, we performed BrdU-specific immunohistochemistry. The sections were permeabilized by incubation in 0.5% Triton X100 in PBS for 20 minutes, pretreated in 50% formamide– 2 standard saline citrate at 658C for 2 hours, denatured in 2N HCl at 378C for 30 minutes, and rinsed twice in 100 mM sodium borate (pH 8.5). Afterward, the sections were incubated overnight at 48C with BrdU-specific mouse monoclonal antibody (1:600; Roche, Mannheim, Germany). The sections were washed with PBS three times and incubated with biotinylated mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA) for 1 hour. The sections were incubated with avidin-peroxidase complex (1:100; Vector Laboratories) for another hour. For visualization, the sections were incubated in 50 mM Tris-HCl (pH 7.6) containing 0.03% 3,30 -diaminobenzidine (DAB), 40 mg/mL nickel chloride, and 0.03% hydrogen peroxide for 5 minutes. After BrdU staining, differentiation of BrdU-positive cells was determined on the same section using mouse anti– neuronal nucleic antibody (1:1000; Chemicon International, Temecula, CA). The sections were washed with PBS three times and incubated with biotinylated anti-mouse secondary antibody for 1 hour. For staining, the sections were incubated in a reaction mixture consisting of 0.03% DAB and 0.03% hydrogen peroxide for 5 minutes. The sections were mounted onto gelatin-coated slides and air-dried overnight at room temperature. Coverslips were mounted using Permount. TUNEL staining To visualize DNA fragmentation (a marker of apoptosis), we performed TUNEL staining using an In Situ Cell Death Detection Kit (Roche), according to the manufacturer’s protocol. The sections were postfixed in ethanol–acetic acid (2:1) and rinsed. The sections were incubated with proteinase K (100 mg/mL), rinsed, incubated in 3% hydrogen peroxide, permeabilized with 0.5% Triton X-100, rinsed again, and incubated in the TUNEL reaction mixture. The sections were rinsed and visualized using Converter-POD with 0.03% DAB. Mayer hematoxylin (DAKO, Glostrup, Denmark) was used as counterstain, and the sections were mounted onto gelatincoated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount. Immunohistochemistry for DCX and caspase-3 To visualize DCX expression, we performed immunohistochemistry for DCX and caspase-3 in the dentate gyrus of the hippocampus. The sections were incubated in PBS for 10 minutes, washed in the same buffer three times, and incubated in 1% hydrogen peroxide for 30 minutes. The sections were selected from each brain and incubated overnight with goat anti-DCX antibody (1:1000; Oncogene Research Product, ß 2015 The North American Menopause Society
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Cambridge, UK) and anti–caspase-3 antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and then with biotinylated rabbit secondary antibody (1:200; Vector Laboratories) for another hour. The secondary antibody was amplified with the Vector Elite ABC Kit (1:100; Vector Laboratories). Antibody-biotin-avidin-peroxidase complexes were visualized using 0.03% DAB, and the sections were mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount. Western blot for BDNF, TrkB, Bax, and Bcl-2 Collected hippocampal tissues were immediately frozen at 708C until Western blot for BDNF, TrkB, Bax, and Bcl-2. The hippocampal tissues were homogenized on ice and lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, and 100 mg/mL leupeptin. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA). Thirty micrograms of protein was separated on sodium dodecyl sulfate–polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse b-actin (1:1000; Santa Cruz Biotechnology), rabbit BDNF and TrkB (1:1000; Santa Cruz Biotechnology), and mouse Bax and Bcl-2 (1:1000; Santa Cruz Biotechnology) antibodies. Horseradish peroxidase– conjugated rabbit BDNF and TrkB antibodies and mouse Bax
and Bcl-2 antibodies were used as secondary antibodies. The experiment was performed under normal laboratory conditions at room temperature, except for transferred membrane. Data analysis To confirm the expressions of BDNF, TrkB, Bax, and Bcl-2, we calculated the detected bands densitometrically using Molecular Analyst version 1.4.1. The numbers of BrdU-positive, DCX-positive, caspase-3–positive, and TUNEL-positive cells in the dentate gyrus were counted hemilaterally under a light microscope (Olympus, Tokyo, Japan) and expressed as the number of cells in the dentate gyrus per square millimeter. The area of the dentate gyrus was measured by Image-Pro Plus image analysis system (Media Cyberbetics Inc, Silver Spring, MD). The relationship between the number of BrdU-positive cells and behavior performance was evaluated using Pearson correlation test. Data were analyzed with one-way analysis of variance and Duncan post hoc test. All values are expressed as mean (SEM). P < 0.05 was considered significant. RESULTS Effects of combined exercise on memory We used step-down avoidance task and Morris water maze task to test the groups’ short-term and spatial working memory. Performance in step-down avoidance task and Morris water maze task is presented in Figure 1. Mean (SEM) latency to
FIG. 1. Effects of combined exercise on memory in step-down avoidance task and Morris water maze task. Left: Short-term memory in step-down avoidance task. Right: Spatial working memory in Morris water maze task. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group. Menopause, Vol. 23, No. 1, 2016
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step-down from the platform for each group was recorded as follows: sham group, 132.44 (13.92) seconds; sham and exercise group, 177.56 (2.44) seconds; OVX group, 35.44 (10.34) seconds; OVX and exercise group, 100.67 (20.3.22) seconds. To investigate spatial memory, we assessed the performance of the groups on Morris water maze task. The mean (SEM) percentage of distance reached in the probe quadrant was as follows: sham group, 34.50% (2.27%); sham and exercise group, 35.96% (1.00%); OVX group, 21.03% (2.90%); OVX and exercise group, 33.64% (1.65%). Our results showed that there was a significant between-group difference in step-down avoidance task and Morris water maze task (both P < 0.001). Post hoc analysis revealed that step-down avoidance task and Morris water maze task were reduced after OVX (both P < 0.001) and significantly increased after 8 weeks of combined exercise (P ¼ 0.017 and P ¼ 0.006, respectively). Effects of combined exercise on cell proliferation and DCX expression in the hippocampal dentate gyrus We used BrdU and DCX immunohistochemical analyses to assess cell proliferation and differentiation. Photomicrographs of BrdU-positive and DCX-positive cells in the dentate gyrus
are presented in Figure 2. The mean (SEM) number of BrdUpositive cells in each group was as follows: sham group, 91.09 (10.59); sham and exercise group, 114.89 (9.06); OVX group, 37.94 (8.01); OVX and exercise group, 65.81 (5.98). The mean (SEM) number of DCX-positive cells in each group was as follows: sham group, 256.89 (20.49); sham and exercise group, 310.39 (14.18); OVX group, 110.41 (11.77); OVX and exercise group, 164.36 (12.37). Our results showed that there was a significant between-group difference in BrdU-positive and DCX-positive cells (both P < 0.001). Post hoc analysis revealed that counts of BrdU-positive and DCX-positive cells decreased after OVX (both P < 0.001) and 8 weeks of combined exercise after OVX resulted in an increase in BrdU-positive and DCXpositive cells (P ¼ 0.004 and P ¼ 0.002, respectively). Effects of combined exercise on BDNF and TrkB expression in the hippocampus BDNF (14 kDa) and TrkB (95 kDa) protein expressions were quantified using Western blot. BDNF and TrkB protein expressions in the hippocampus are presented in Figure 3. BDNF and TrkB expression levels were set to 1.00 in the sham group. Analyses in the other groups showed that mean
FIG. 2. Effects of combined exercise on cell proliferation and differentiation in the dentate gyrus. Left: BrdU-positive cells in the dentate gyrus. Right: Doublecortin (DCX)–positive cells in the dentate gyrus. Top: Photomicrograph of BrdU-positive and DCX-positive cells. Scale bar represents 50 mm. Bottom: Number of BrdU-positive and doublecortin (DCX)–positive cells in each group. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group.
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FIG. 3. Effects of combined exercise on brain-derived neurotrophic factor (BDNF) and TrkB protein expression in the hippocampus. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group. OD, optical density.
(SEM) BDNF expression was as follows: sham and exercise group, 1.17 (0.02); OVX group, 0.40 (0.03); OVX and exercise group, 0.76 (0.04). Mean (SEM) TrkB expression was as follows: sham and exercise group, 1.51 (0.04); OVX group, 0.50 (0.01); OVX and exercise group, 0.77 (0.02). Our results clearly showed that there was a significant difference in BDNF and TrkB protein expressions between groups (both P < 0.001). Post hoc analysis revealed that BDNF and TrkB protein levels were reduced after OVX (both P < 0.001); nevertheless, they were significantly increased after 8 weeks of combined exercise (both P < 0.001).
Effects of combined exercise on Bax and Bcl-2 expression in the hippocampus Bax (23 kDa) and Bcl-2 (26 kDa) protein expressions were quantified using Western blot. Bax and Bcl-2 protein expressions in the hippocampus are presented in Figure 4. Bax and Bcl-2 were set to 1.00 in the sham group. Our data showed that mean (SEM) Bax protein expression was as follows: sham and exercise group, 0.78 (0.07); OVX group, 2.25 (0.13); OVX and exercise group, 1.77 (0.16). Similarly, mean (SEM) Bcl-2 protein expression was as follows: sham and exercise group, 1.06 (0.05); OVX group, 0.46 (0.04);
FIG. 4. Effects of combined exercise on Bax and Bcl-2 expression in the hippocampus. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group. OD, optical density. Menopause, Vol. 23, No. 1, 2016
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KIM ET AL
OVX and exercise group, 0.74 (0.08). There was a significant difference in Bax and Bcl-2 between groups (both P < 0.001). Post hoc analysis revealed that Bax expression increased after OVX (P < 0.001) and significantly decreased after 8 weeks of combined exercise (P ¼ 0.019). On the contrary, Bcl-2 protein expression was reduced after OVX (P < 0.001); however, it showed a significant increase after 8 weeks of combined exercise (P ¼ 0.001). Effects of combined exercise on caspase-3 and TUNEL expression in the hippocampal dentate gyrus We used caspase-3 and TUNEL immunohistochemical analyses to assess apoptosis. Photomicrographs of caspase3–positive and TUNEL-positive cells in the dentate gyrus are presented in Figure 5. The mean (SEM) number of caspase-3– positive cells was as follows: sham group, 52.16 (9.58); sham and exercise group, 57.46 (11.85); OVX group, 176.30 (14.37); OVX and exercise group, 100.67 (12.65). The mean (SEM) number of TUNEL-positive cells was as follows: sham group, 21.21 (2.03); sham and exercise group, 19.71 (1.59); OVX group, 107.58 (8.66); OVX and exercise group, 79.47 (7.91). Our results showed that there was a significant difference in caspase-3–positive and TUNEL-positive cells between groups (both P < 0.001). Post hoc analysis revealed that counts of caspase-3–positive and TUNEL-positive cells increased after OVX (both P < 0.001) and that 8 weeks of combined exercise after OVX resulted in an increase in caspase-3–positive and TUNEL-positive cells (P ¼ 0.009 and P ¼ 0.011, respectively).
Correlation between hippocampal cell proliferation and cognitive function To examine the correlation between cell loss and cognitive function, we performed a correlation analysis between two variables: number of BrdU-positive cells in the hippocampal dentate gyrus and cognitive behavior (Fig. 6). There was a statistically significant positive correlation between the number of BrdU-positive cells and performance in Morris water maze task (r ¼ 0.662, P < 0.001) and step-down avoidance task (r ¼ 0.617, P < 0.001). DISCUSSION In numerous studies, estrogen has been shown to protect individuals from brain injury, neurodegeneration, and cognitive decline. Hypoestrogenic or postmenopausal conditions in women decrease cognitive function and increase the risk of various neurodegenerative diseases.6 Postmenopausal women have been reported to be at relatively higher risk for developing Alzheimer dementia compared with men.32 Based on behavioral analysis in the present study, shortterm and spatial working memory decreased in ovariectomized rats. Reports on memory deficits and impairment of cognitive function have been associated with estrogen level changes during pregnancy and perimenopause.33 Cognitive decline in ovariectomized rats seems to have a close association with hippocampal function. In the present study, cognitive decline was accompanied by a decrease in BrdUpositive and DCX-positive cells and expression levels of
FIG. 5. Effects of combined exercise on cell death in the dentate gyrus. Left: Caspase-3–positive cells in the dentate gyrus. Right: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)–positive cells in the dentate gyrus. Top: Photomicrograph of caspase-3– positive and TUNEL-positive cells. Scale bar represents 50 mm. Bottom: Number of caspase-3–positive and TUNEL-positive cells in each group. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group.
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EXERCISE AMELIORATES COGNITIVE IMPAIRMENT
FIG. 6. Correlation between hippocampal cell proliferation and cognitive function. Left: Correlation between number of BrdU-positive cells and Morris water maze task. Right: Correlation between number of BrdU-positive cells and step-down avoidance task.
Bcl-2, BDNF, and TrkB, whereas Bax and caspase-3 levels increased in the hippocampus. In a previous study, estrogen was shown to regulate BDNF synthesis in the hippocampus. Other reports have shown that OVX caused a decrease in BDNF messenger RNA and cell proliferation in the female rat hippocampus.34,35 In another study, Bax and caspase-3 expressions increased and Bcl-2 and Bcl-x levels decreased in the hippocampus of ovariectomized rats.18,36,37 Estrogen deficiency inhibits cell proliferation and induces apoptosis in the hippocampus, ultimately causing memory decline. Notably, our study showed positive correlations between cell proliferation and cognitive function. A clinical experiment in a previous study reported a correlation between low cell proliferation and differentiation in the hippocampus and memory loss38; this finding is consistent with our results. On the other hand, in the present study, combined exercise in ovariectomized rats increased short-term and spatial working memory. The exercise program in this study consisted of a combined aerobic and anaerobic program that possessed the advantages of aerobic and resistance exercise, thereby facilitating a greater increase in physical strength compared with a single type of exercise. Investigation of the relationship between physical strength and cognitive processes in human studies showed that fitness training influenced various cognitive processes. In particular, fitness training exhibited the strongest effect on executive control processes, including planning, scheduling, working memory, inhibitory processes, and multitasking. Such effects were magnified when fitness training involved a combination of aerobic exercise and strength and flexibility training.39 The combination of treatment protocols may also exhibit a more diverse set of changes in the brain.40 Exercise has been proven to change brain structure and function.41 Aerobic activity improves learning and task acquisition and increases the secretion of important neurochemicals associated with promoting synaptic plasticity and neuronal architecture.42 The results of the present study showed that combined exercise in ovariectomized rats increased the expression of BrdU-positive cells (marker of proliferation), DCX-positive cells (marker of differentiation), BDNF, and TrkB receptor in the hippocampus. Improvement in the cognitive function of ovariectomized rats through exercise is considered the result of restored hippocampal function. Exercise has been
known to increase hippocampal neurogenesis, improve cognitive function, and increase synaptic plasticity. In a previous study, treadmill exercises in middle-aged mice led to increased BrdUpositive and DCX-positive cells, BDNF levels, and TrkB levels.43 Reduced neurogenesis caused by aging was increased, through exercise, to 50% of the levels exhibited by the young group, along with improvements in cognitive function. There was no difference in the morphology of new cells between the young group and the old exercising group.44 Furthermore, voluntary running led to an increase in cell proliferation in the hippocampus of ovariectomized mice.45 BDNF plays an important role in regular hippocampal neurogenesis. In particular, BDNF is known to promote the survival of newborn granule cells.46,47 Exercise is associated not only with cell differentiation and survival but also with cell apoptosis. In the present study, combined exercise inhibited Bax and caspase-3 action and activated Bcl-2 in the hippocampus of ovariectomized rats. In a previous study, exercise inhibited apoptosis in various brain diseases, including Alzheimer disease and traumatic brain injury, by inhibiting Bax and caspase3 and by activating Bcl-2.21,27 Exercise activates various neurotrophins that promote cell survival and inhibit apoptosis.48 In particular, apoptotic stimulation induces caspase-3 activation, but such caspase-3 activation can be blocked by BDNF.49 Ovariectomized rats that were subjected to 8 weeks of combined exercise exhibited an increase in hippocampal BDNF and TrkB expression, which suggests that BDNF-TrkB signaling may be related to inhibition of apoptosis induced by combined exercises. As mentioned earlier, estrogen exhibits a strong neuroprotective effect on plasticity and cell survival in the adult brain. However, postmenopausal women may be less neuroprotected under estrogen deficiency. CONCLUSIONS Ovariectomized rats show deficits in cognitive function (such as short-term and spatial working memory)—resulting from suppression of cell proliferation and BDNF expression—and increased apoptosis in the hippocampus. These deficits in cognitive function are prevented by increased hippocampal cell proliferation and BDNF expression and decreased apoptosis resulting from exercise. The present data suggest that complex exercise increases cell proliferation and BDNF expression and Menopause, Vol. 23, No. 1, 2016
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KIM ET AL
suppresses apoptosis in the hippocampus of rats in a state of estrogen depletion. The data suggest that exercise might be an effective strategy for minimizing cognitive function deficits in postmenopausal women. REFERENCES 1. Weitzmann MN, Pacifici R. Estrogen deficiency and bone loss: an inflammatory tale. J Clin Invest 2006;116:1186-1194. 2. Antonicelli R, Olivieri F, Morichi V, Urbani E, Mais V. Prevention of cardiovascular events in early menopause: a possible role for hormone replacement therapy. Int J Cardiol 2008;130:140-146. 3. McEwen B. Estrogen actions throughout the brain. Recent Prog Horm Res 2002;57:357-384. 4. Gibbs RB, Gabor R. Estrogen and cognition: applying preclinical findings to clinical perspectives. J Neurosci Res 2003;74:637-643. 5. Daniel JM. Effects of oestrogen on cognition: what have we learned from basic research? J Neuroendocrinol 2006;18:787-795. 6. Wise PM. Estrogens and neuroprotection. Trends Endocrinol Metab 2002;13:229-230. 7. Spencer JL, Waters EM, Romeo RD, Wood GE, Milner TA, McEwen BS. Uncovering the mechanisms of estrogen effects on hippocampal function. Front Neuroendocrinol 2008;29:219-237. 8. Li C, Brake WG, Romeo RD, et al. Estrogen alters hippocampal dendritic spine shape and enhances synaptic protein immunoreactivity and spatial memory in female mice. Proc Natl Acad Sci U S A 2004;101:2185-2190. 9. Biegler R, McGregor A, Krebs JR, Healy SD. A larger hippocampus is associated with longer-lasting spatial memory. Proc Natl Acad Sci U S A 2001;98:6941-6944. 10. Kempermann G, Kuhn HG, Gage FH. Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci U S A 1997;94:10409-10414. 11. Francis F, Koulakoff A, Boucher D, et al. Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron 1999;23:247-256. 12. Pencea V, Bingaman KD, Wiegand SJ, Luskin MB. Infusion of brainderived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J Neurosci 2001;21:6706-6717. 13. Barnabe´-Heider F, Miller FD. Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J Neurosci 2003;23:5149-5160. 14. Chmielnicki E, Benraiss A, Economides AN, Goldman SA. Adenovirally expressed noggin and brain-derived neurotrophic factor cooperate to induce new medium spiny neurons from resident progenitor cells in the adult striatal ventricular zone. J Neurosci 2004;24:2133-2142. 15. Su JH, Deng G, Cotman CW. Bax protein expression is increased in Alzheimer’s brain: correlations with DNA damage, Bcl-2 expression, and brain pathology. J Neuropathol Exp Neurol 1997;56:86-93. 16. Hartmann A, Hunot S, Michel PP, et al. Caspase-3: a vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc Natl Acad Sci U S A 2000;97:2875-2880. 17. Jarskog LF, Selinger ES, Lieberman JA, Gilmore JH. Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation. Am J Psychiatry 2004;161:109-115. 18. Stoltzner SE, Berchtold NC, Cotman CW, Pike CJ. Estrogen regulates bcl-x expression in rat hippocampus. Neuroreport 2001;12:2797-2800. 19. Chambers RA, Potenza MN, Hoffman RE, Miranker W. Simulated apoptosis/neurogenesis regulates learning and memory capabilities of adaptive neural networks. Neuropsychopharmacology 2004;29:747-758. 20. Kim SE, Ko IG, Kim BK, et al. Treadmill exercise prevents aginginduced failure of memory through an increase in neurogenesis and suppression of apoptosis in rat hippocampus. Exp Gerontol 2010; 45:357-365. 21. Kim DH, Ko IG, Kim BK, et al. Treadmill exercise inhibits traumatic brain injury–induced hippocampal apoptosis. Physiol Behav 2010;101:660-665. 22. Deng W, Aimone JB, Gage FH. New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 2010;11:339-350. 23. Asikainen TM, Kukkonen-Harjula K, Miilunpalo S. Exercise for health for early postmenopausal women: a systematic review of randomised controlled trials. Sports Med 2004;34:753-778.
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24. Chaouloff F. Effects of acute physical exercise on central serotonergic systems. Med Sci Sports Exerc 1997;29:58-62. 25. Tong L, Shen H, Perreau VM, Balazs R, Cotman CW. Effects of exercise on gene-expression profile in the rat hippocampus. Neurobiol Dis 2001;8:1046-1056. 26. Adlard PA, Perreau VM, Cotman CW. The exercise-induced expression of BDNF within the hippocampus varies across life-span. Neurobiol Aging 2005;26:511-520. 27. Jin JJ, Ko IG, Kim SE, Shin MS, Kim SH, Jee YS. Swimming exercise ameliorates multiple sclerosis–induced impairment of short-term memory by suppressing apoptosis in the hippocampus of rats. J Exerc Rehabil 2014;10:69-74. 28. Dishman RK. Brain monoamines, exercise, and behavioral stress: animal models. Med Sci Sports Exerc 1997;29:63-74. 29. Zhou FC, Sari Y, Zhang JK, Goodlett CR, Li T. Prenatal alcohol exposure retards the migration and development of serotonin neurons in fetal C57BL mice. Brain Res Dev Brain Res 2001;126:147-155. 30. Lee S, Barton ER, Sweeney HL, Farrar RP. Viral expression of insulinlike growth factor-I enhances muscle hypertrophy in resistance-trained rats. J Appl Physiol 2004;96:1097-1104. 31. Kim BK, Seo JH. Treadmill exercise alleviates post-traumatic stress disorder–induced impairment of spatial learning memory in rats. J Exerc Rehabil 2013;9:413-419. 32. Zandi PP, Carlson MC, Plassman BL, et al; Cache County Memory Study Investigators. Hormone replacement therapy and incidence of Alzheimer disease in older women: the Cache County Study. JAMA 2002;288:21232129. 33. Sullivan Mitchell E, Fugate Woods N. Midlife women’s attributions about perceived memory changes: observations from the Seattle Midlife Women’s Health Study. J Womens Health Gend Based Med 2001;10:351-362. 34. Tanapat P, Hastings NB, Reeves AJ, Gould E. Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat. J Neurosci 1999;19:5792-5801. 35. Berchtold NC, Kesslak JP, Pike CJ, Adlard PA, Cotman CW. Estrogen and exercise interact to regulate brain-derived neurotrophic factor mRNA and protein expression in the hippocampus. Eur J Neurosci 2001;14:1992-2002. 36. Sharma K, Mehra RD. Long-term administration of estrogen or tamoxifen to ovariectomized rats affords neuroprotection to hippocampal neurons by modulating the expression of Bcl-2 and Bax. Brain Res 2008;1204:1-15. 37. Li L, Xiao N, Yang X, et al. A high cholesterol diet ameliorates hippocampus-related cognitive and pathological deficits in ovariectomized mice. Behav. Brain Res 2012;230:251-258. 38. Coras R, Siebzehnrubl FA, Pauli E, et al. Low proliferation and differentiation capacities of adult hippocampal stem cells correlate with memory dysfunction in humans. Brain 2010;133:3359-3372. 39. Kramer AF, Erickson KI, Colcombe SJ. Exercise, cognition, and the aging brain. J Appl Physiol 2006;101:1237-1242. 40. Black JE, Isaacs KR, Anderson BJ, Alcantara AA, Greenough WT. Learning causes synaptogenesis, whereas motor activity causes angiogenesis, in cerebellar cortex of adult rats. Proc Natl Acad Sci U S A 1990;87:5568-5572. 41. Booth FW, Lees SJ. Physically active subjects should be the control group. Med Sci Sports Exerc 2006;38:405-406. 42. Hillman CH, Erickson KI, Kramer AF. Be smart, exercise your heart: exercise effects on brain and cognition. Nat Rev Neurosci 2008;9:58-65. 43. Wu CW, Chang YT, Yu L, et al. Exercise enhances the proliferation of neural stem cells and neurite growth and survival of neuronal progenitor cells in dentate gyrus of middle-aged mice. J Appl Physiol 2008;105:1585-1594. 44. van Praag H, Shubert T, Zhao C, Gage FH. Exercise enhances learning and hippocampal neurogenesis in aged mice. J Neurosci 2005;25:8680-8685. 45. Jin J, Jing H, Choi G, et al. Voluntary exercise increases the new cell formation in the hippocampus of ovariectomized mice. Neurosci Lett 2008;439:260-263. 46. Larsson E, Mandel RJ, Klein RL, Muzyczka N, Lindvall O, Kokaia Z. Suppression of insult-induced neurogenesis in adult rat brain by brainderived neurotrophic factor. Exp Neurol 2002;177:1-8. 47. Sairanen M, Lucas G, Ernfors P, Castre´n M, Castre´n E. Brain-derived neurotrophic factor and antidepressant drugs have different but coordinated effects on neuronal turnover, proliferation, and survival in the adult dentate gyrus. J Neurosci 2005;25:1089-1094. 48. Hetman M, Xia Z. Signaling pathways mediating anti-apoptotic action of neurotrophins. Acta Neurobiol Exp 2000;60:531-545. 49. Han BH, D’Costa A, Back SA, et al. BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia. Neurobiol Dis 2000;7:38-53. ß 2015 The North American Menopause Society
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Combined exercise ameliorates ovariectomy-induced cognitive impairment by enhancing cell proliferation and suppressing apoptosis Tae-Woon Kim, PhD,1 Chang-Sun Kim, PhD,2 Ji-Yeon Kim, MS,2 Chang-Ju Kim, MD, PhD,1 and Jin-Hee Seo, PhD 3 Abstract Objective: Estrogen plays an important role in cognitive function, including attention, learning, and memory, and affects the structure and function of brain areas. We investigated the effects of combined exercise on memory deficits induced by ovariectomy (OVX) in relation to cell proliferation and apoptosis in the hippocampus. Methods: Rats were randomly divided into four groups: sham, sham and exercise, OVX, and OVX and exercise. Rats in combined exercise groups were subjected to 3 days of resistance training and 3 days of running (for a total of 6 d/wk) for eight consecutive weeks. Rats were tested in step-down avoidance task and Morris water maze task to verify the effects of OVX on short-term and spatial working memory. Results: In the present study, the number of BrdU-positive and doublecortin-positive cells and expression of brain-derived neurotrophic factor, TrkB, and Bcl-2 decreased; expression of Bax and the number of caspase-3– positive and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling–positive cells increased; and short-term and spatial working memory decreased in the OVX group compared with the sham group. Conversely, when the combined exercise group was compared with the OVX group, the number of BrdU-positive and doublecortin-positive cells and expression of brain-derived neurotrophic factor, TrkB, and Bcl-2 increased; expression of Bax and the number of caspase-3–positive and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling–positive cells decreased; and short-term and spatial working memory increased. Conclusions: Combined exercise increases cell proliferation and inhibits apoptosis in the hippocampus and improves cognitive function despite estrogen deficiency. Key Words: Ovariectomy – Cognitive function – Cell proliferation – Apoptosis – Short-term and spatial working memory.
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strogen reduction after menopause influences various organ systems. For example, estrogen deficiency is known to cause harmful effects, including bone and cardiovascular diseases.1,2 In addition, in the past 35 years, it has been clearly shown that one of the major target organs of estrogen is the brain.3 Estrogen in the brain participates in gonadal regulation, physiological homeostasis, and reproduction. Furthermore, estrogen plays an important role in cognitive function, including attention, learning, and memory.4 Estrogen affects the structure and function of brain areas that participate in learning and memory.5 In particular, estrogen contributes to maintaining plasticity and cell survival in the adult brain, and a postmenopausal decrease in estrogen Received January 12, 2015; revised and accepted April 6, 2015. From the 1Department of Physiology, College of Medicine, KyungHee University, Seoul, Republic of Korea; 2Department of Physical Education, College of Natural Science, DongDuk Women’s University, Seoul, Republic of Korea; and 3Division of Sports Science, Baekseok University, Cheonan, Republic of Korea. Funding/support: None. Financial disclosure/conflicts of interest: None reported. Address correspondence to: Jin-Hee Seo, PhD, Division of Sports Science, Baekseok University, 330-704, 76 Munam-ro, Dongnam-gu, Cheonan-si, Chungcheongnam-do, Republic of Korea. E-mail: [email protected]
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induces cognitive dysfunction and increases the risk of various neurodegenerative diseases.6 In young adult female rodents and primates, a difference in cognitive capacity, based on estrogen levels during the natural estrous cycle or the menstrual cycle, was demonstrated. Compared with low estrogen levels, high estrogen levels led to improved cognitive function.7 Among brain areas associated with such cognitive function, the hippocampus, in particular, is strongly influenced by estrogen. Estrogen has been reported to have an important effect on the structure and function of the hippocampus. The hippocampus is one of the brain areas responsible for cognitive function, including learning and memory. In a previous study, estrogen was shown to promote spine maturation in the hippocampus; as a result, hippocampusdependent memory was improved.8 Several studies have shown that brain diseases involving cognitive dysfunction occur because of impaired hippocampal function. Furthermore, the hippocampus plays a key role in storing new memories and is associated with declarative and spatial memory.9 Hippocampal activation leads to increase in neurogenesis, activation of neurotrophic factors, and inhibition of apoptosis; consequently, activation of the hippocampus leads to increase in cognitive function. Neurogenesis occurs through a four-step proliferation process, including migration,
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survival, differentiation and maturation of neural progenitor cells originating from neural stem cells.10 Doublecortin (DCX) is a protein expressed in the brain during neuronal migration and differentiation in neurogenesis.11 Furthermore, neurogenesis is promoted by a variety of neurotrophic factors, and many areas of the brain work in tandem to regulate proliferation and differentiation of neural stem cells or progenitor cells.12-14 In particular, brain-derived neurotrophic factor (BDNF) binds to the TrkB receptor, causing an increase in hippocampal neurogenesis. Activation of neuronal apoptosis and abnormal apoptotic regulatory protein expression cause neurodegenerative disorders such as Parkinson disease and Alzheimer disease.15,16 Apoptosis is regulated via a signaling pathway that includes the antiapoptotic factor Bcl-2 and the proapoptotic factor Bax (a member of the Bcl-2 family) upstream and members of the caspase protein family (such as caspase-3) downstream.17 However, physiological estrogen also plays a role in apoptosis by regulating programmed cell death.18 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay detects features of apoptotic cell death. Neuronal turnover resulting from neuronal death and neurogenesis in the brain is a special form of neuroplasticity that is related to diverse factors such as stress responses, cognitive function, and treatment of mental disorders.19 In previous studies, increased cell death and decreased cell proliferation in the hippocampus during aging, stress, or traumatic brain injury led to deficits in cognitive function such as learning and memory.20,21 In particular, hippocampal neurogenesis has been shown to play an important role in learning and memory.22 As such, estrogen deficiency inhibits hippocampal neurogenesis and induces apoptosis, which may potentially cause cognitive disorders. On the other hand, exercise has been suggested to prevent and relieve various symptoms of menopause.23 Exercise affects physiological and biochemical changes in the brain24 and gene transcription associated with neural activity, plasticity, and regeneration.25 In addition, exercise increases neurogenic nutrient expression26 and inhibits neuronal cell death to improve cognitive function.27 Several previous studies have reported that exercise influences the expression of neurotransmitters and provides support against anxiety, stress, depression, insomnia, and miscellaneous psychological disorders. In particular, neurotransmitters are associated with emotional regulation, awareness, behavior and motor control, memory and learning, temperature regulation, pain transmission, food intake, stress responses, and brain development and maturation.28,29 However, no studies have focused on the effects of exercise on the improvement of hippocampal and cognitive function caused by postmenopausal estrogen deficiency. Consequently, the present study sought to investigate whether a combination of aerobic and anaerobic exercise influences the cognitive function of ovariectomized rats. Therefore, we measured short-term and spatial working memory, performance in step-down avoidance task and
Morris water maze task, cell proliferation, BDNF levels, and apoptosis in the hippocampus. METHODS Animals Eight-week-old Sprague-Dawley rats weighing 230.9 (5.5) g were obtained from a commercial breeder (Orient Co, Seoul, Korea) for the experiment. Experimental procedures were performed in accordance with the animal care guidelines of the National Institutes of Health and the Korean Academy of Medical Sciences. The rats were housed under controlled temperature (mean [SEM], 208C [28C]) and lighting (07:0019:00 h) conditions, with food and water available ad libitum. The rats were randomly divided into four groups (n ¼ 10 per group): sham, sham and exercise, ovariectomy (OVX), and OVX and exercise. Surgical operation For OVX, Zoletil 50 was injected into the abdominal cavity for general anesthesia, and hair was removed from both sides of the back. Surgical operation was performed with the rat in prone position, and the surgical area was sterilized with 10% Betadine solution. A 1.5-cm incision at the midsection of the spine next to the rearmost rib was made, the peritoneum was incised on the left and right, and the ovaries were excised. Absorbable floss was used to suture, after which the ovaries were removed. The peritoneum and muscle layer were simultaneously sutured, whereas the epidermal layer was separately sutured using nonabsorbable suture. In sham rats, the same approach was used up to the step of peritoneum incision; however, these animals were sutured without excising the ovaries. Combined exercise protocol The combined exercise program includes 3 days of resistance training and 3 days of running, for a total of 6 days of exercise. Tower-climbing exercise A ladder with a height of 135 cm, intervals of 2.5 cm, and inclination of 808 was climbed with a weight load near the tail region, as previously described.30 For the adjustment period, a weight load weighing 50% of the rat’s weight was used for 5 days/week for 2 weeks. After measurement of one repetition maximum, exercise began at 50% of the intensity of one repetition maximum, and 50 g of weight was added every 2 weeks to constitute a four-stage tower-climbing exercise. The exercise was performed once a day and 3 days/week for eight consecutive weeks. Treadmill exercise The exercise group began exercise on a treadmill made for use of small animals. Five minutes of warm-up at 08 inclination at 3 meters/minute, 30 minutes of main exercise at 10 meters/minute, and 5 minutes of cool-down at 3 meters/ minute were performed for the first 2 weeks. After this, Menopause, Vol. 23, No. 1, 2016
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40 minutes of main exercise at 10 meters/minute on weeks 3 and 4, 30 minutes of main exercise at 15 meters/minute on weeks 4 to 6, and 40 minutes of main exercise at 15 meters/ minute for the final weeks 6 to 8 were performed. The exercise was performed once a day and 3 days/week for eight consecutive weeks. During treadmill running, electrical stimulation was removed to minimize stress. Step-down avoidance task Latency time of the step-down avoidance task was determined to evaluate short-term memory. The rats were trained in a step-down avoidance task on the 38th day after the start of the treadmill exercise. Latency time (in seconds) in each group was measured 2 hours after training. The rats were placed on a platform (7 25 cm, 2.5 cm in height). The platform faced a grid (42 25 cm) of parallel stainless steel bars 0.1 cm in caliber and spaced 1 cm apart. During training sessions, the animals received a 0.5-mA scramble foot shock for 2 seconds immediately upon stepping down. The interval that elapsed between the rats stepping down and the rats placing all four paws on the grid was defined as latency time. A latency time longer than 180 seconds was counted as 180 seconds. Morris water maze task Spatial working memory was evaluated with Morris water maze task. This task requires rats to learn the spatial location of a hidden platform in a black circular pool (180 cm in diameter and 50 cm in height) filled with clear water (mean [SEM], 258C [18C]). The hidden platform (15 cm in diameter and 40 cm in height) was placed 2 cm below the surface of the water in the middle of the north quadrant and was camouflaged by transparency against a black background. Distal visual cues were placed on the walls around the pool. The position of the cues remained unchanged throughout the task. One day before training, the rats were habituated to swimming for 60 seconds in the pool without a platform. All rats were trained three times a day for four consecutive days before killing, and probe trail was conducted 24 hours after the last training. When finding the platform, the rats were allowed to remain for 30 seconds. If the rats did not find the platform within 60 seconds, they were guided by hand to the platform. The rats were given a 60-second retention probe test, and the platform was removed from the pool. Data were automatically collected via the Smart Video Tracking System (Smart version 2.5; Panlab, Barcelona, Spain). Tissue preparation The animals were killed immediately after determination of Morris water maze task. For preparation of brain slices, the animals were fully anesthesized with Zoletil 50 (10 mg/kg IP; Vibac, Carros, France), after which the rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS) and fixed with a freshly prepared solution of 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). The brains were removed, postfixed in the same fixative overnight, and
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transferred into a 30% sucrose solution for cryoprotection. Coronal sections 40 mm thick were made using a freezing microtome (Leica, Nussloch, Germany).31 Immunohistochemistry for BrdU To detect newly generated cells in the dentate gyrus, we performed BrdU-specific immunohistochemistry. The sections were permeabilized by incubation in 0.5% Triton X100 in PBS for 20 minutes, pretreated in 50% formamide– 2 standard saline citrate at 658C for 2 hours, denatured in 2N HCl at 378C for 30 minutes, and rinsed twice in 100 mM sodium borate (pH 8.5). Afterward, the sections were incubated overnight at 48C with BrdU-specific mouse monoclonal antibody (1:600; Roche, Mannheim, Germany). The sections were washed with PBS three times and incubated with biotinylated mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA) for 1 hour. The sections were incubated with avidin-peroxidase complex (1:100; Vector Laboratories) for another hour. For visualization, the sections were incubated in 50 mM Tris-HCl (pH 7.6) containing 0.03% 3,30 -diaminobenzidine (DAB), 40 mg/mL nickel chloride, and 0.03% hydrogen peroxide for 5 minutes. After BrdU staining, differentiation of BrdU-positive cells was determined on the same section using mouse anti– neuronal nucleic antibody (1:1000; Chemicon International, Temecula, CA). The sections were washed with PBS three times and incubated with biotinylated anti-mouse secondary antibody for 1 hour. For staining, the sections were incubated in a reaction mixture consisting of 0.03% DAB and 0.03% hydrogen peroxide for 5 minutes. The sections were mounted onto gelatin-coated slides and air-dried overnight at room temperature. Coverslips were mounted using Permount. TUNEL staining To visualize DNA fragmentation (a marker of apoptosis), we performed TUNEL staining using an In Situ Cell Death Detection Kit (Roche), according to the manufacturer’s protocol. The sections were postfixed in ethanol–acetic acid (2:1) and rinsed. The sections were incubated with proteinase K (100 mg/mL), rinsed, incubated in 3% hydrogen peroxide, permeabilized with 0.5% Triton X-100, rinsed again, and incubated in the TUNEL reaction mixture. The sections were rinsed and visualized using Converter-POD with 0.03% DAB. Mayer hematoxylin (DAKO, Glostrup, Denmark) was used as counterstain, and the sections were mounted onto gelatincoated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount. Immunohistochemistry for DCX and caspase-3 To visualize DCX expression, we performed immunohistochemistry for DCX and caspase-3 in the dentate gyrus of the hippocampus. The sections were incubated in PBS for 10 minutes, washed in the same buffer three times, and incubated in 1% hydrogen peroxide for 30 minutes. The sections were selected from each brain and incubated overnight with goat anti-DCX antibody (1:1000; Oncogene Research Product, ß 2015 The North American Menopause Society
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Cambridge, UK) and anti–caspase-3 antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and then with biotinylated rabbit secondary antibody (1:200; Vector Laboratories) for another hour. The secondary antibody was amplified with the Vector Elite ABC Kit (1:100; Vector Laboratories). Antibody-biotin-avidin-peroxidase complexes were visualized using 0.03% DAB, and the sections were mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount. Western blot for BDNF, TrkB, Bax, and Bcl-2 Collected hippocampal tissues were immediately frozen at 708C until Western blot for BDNF, TrkB, Bax, and Bcl-2. The hippocampal tissues were homogenized on ice and lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, and 100 mg/mL leupeptin. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA). Thirty micrograms of protein was separated on sodium dodecyl sulfate–polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse b-actin (1:1000; Santa Cruz Biotechnology), rabbit BDNF and TrkB (1:1000; Santa Cruz Biotechnology), and mouse Bax and Bcl-2 (1:1000; Santa Cruz Biotechnology) antibodies. Horseradish peroxidase– conjugated rabbit BDNF and TrkB antibodies and mouse Bax
and Bcl-2 antibodies were used as secondary antibodies. The experiment was performed under normal laboratory conditions at room temperature, except for transferred membrane. Data analysis To confirm the expressions of BDNF, TrkB, Bax, and Bcl-2, we calculated the detected bands densitometrically using Molecular Analyst version 1.4.1. The numbers of BrdU-positive, DCX-positive, caspase-3–positive, and TUNEL-positive cells in the dentate gyrus were counted hemilaterally under a light microscope (Olympus, Tokyo, Japan) and expressed as the number of cells in the dentate gyrus per square millimeter. The area of the dentate gyrus was measured by Image-Pro Plus image analysis system (Media Cyberbetics Inc, Silver Spring, MD). The relationship between the number of BrdU-positive cells and behavior performance was evaluated using Pearson correlation test. Data were analyzed with one-way analysis of variance and Duncan post hoc test. All values are expressed as mean (SEM). P < 0.05 was considered significant. RESULTS Effects of combined exercise on memory We used step-down avoidance task and Morris water maze task to test the groups’ short-term and spatial working memory. Performance in step-down avoidance task and Morris water maze task is presented in Figure 1. Mean (SEM) latency to
FIG. 1. Effects of combined exercise on memory in step-down avoidance task and Morris water maze task. Left: Short-term memory in step-down avoidance task. Right: Spatial working memory in Morris water maze task. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group. Menopause, Vol. 23, No. 1, 2016
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KIM ET AL
step-down from the platform for each group was recorded as follows: sham group, 132.44 (13.92) seconds; sham and exercise group, 177.56 (2.44) seconds; OVX group, 35.44 (10.34) seconds; OVX and exercise group, 100.67 (20.3.22) seconds. To investigate spatial memory, we assessed the performance of the groups on Morris water maze task. The mean (SEM) percentage of distance reached in the probe quadrant was as follows: sham group, 34.50% (2.27%); sham and exercise group, 35.96% (1.00%); OVX group, 21.03% (2.90%); OVX and exercise group, 33.64% (1.65%). Our results showed that there was a significant between-group difference in step-down avoidance task and Morris water maze task (both P < 0.001). Post hoc analysis revealed that step-down avoidance task and Morris water maze task were reduced after OVX (both P < 0.001) and significantly increased after 8 weeks of combined exercise (P ¼ 0.017 and P ¼ 0.006, respectively). Effects of combined exercise on cell proliferation and DCX expression in the hippocampal dentate gyrus We used BrdU and DCX immunohistochemical analyses to assess cell proliferation and differentiation. Photomicrographs of BrdU-positive and DCX-positive cells in the dentate gyrus
are presented in Figure 2. The mean (SEM) number of BrdUpositive cells in each group was as follows: sham group, 91.09 (10.59); sham and exercise group, 114.89 (9.06); OVX group, 37.94 (8.01); OVX and exercise group, 65.81 (5.98). The mean (SEM) number of DCX-positive cells in each group was as follows: sham group, 256.89 (20.49); sham and exercise group, 310.39 (14.18); OVX group, 110.41 (11.77); OVX and exercise group, 164.36 (12.37). Our results showed that there was a significant between-group difference in BrdU-positive and DCX-positive cells (both P < 0.001). Post hoc analysis revealed that counts of BrdU-positive and DCX-positive cells decreased after OVX (both P < 0.001) and 8 weeks of combined exercise after OVX resulted in an increase in BrdU-positive and DCXpositive cells (P ¼ 0.004 and P ¼ 0.002, respectively). Effects of combined exercise on BDNF and TrkB expression in the hippocampus BDNF (14 kDa) and TrkB (95 kDa) protein expressions were quantified using Western blot. BDNF and TrkB protein expressions in the hippocampus are presented in Figure 3. BDNF and TrkB expression levels were set to 1.00 in the sham group. Analyses in the other groups showed that mean
FIG. 2. Effects of combined exercise on cell proliferation and differentiation in the dentate gyrus. Left: BrdU-positive cells in the dentate gyrus. Right: Doublecortin (DCX)–positive cells in the dentate gyrus. Top: Photomicrograph of BrdU-positive and DCX-positive cells. Scale bar represents 50 mm. Bottom: Number of BrdU-positive and doublecortin (DCX)–positive cells in each group. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group.
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EXERCISE AMELIORATES COGNITIVE IMPAIRMENT
FIG. 3. Effects of combined exercise on brain-derived neurotrophic factor (BDNF) and TrkB protein expression in the hippocampus. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group. OD, optical density.
(SEM) BDNF expression was as follows: sham and exercise group, 1.17 (0.02); OVX group, 0.40 (0.03); OVX and exercise group, 0.76 (0.04). Mean (SEM) TrkB expression was as follows: sham and exercise group, 1.51 (0.04); OVX group, 0.50 (0.01); OVX and exercise group, 0.77 (0.02). Our results clearly showed that there was a significant difference in BDNF and TrkB protein expressions between groups (both P < 0.001). Post hoc analysis revealed that BDNF and TrkB protein levels were reduced after OVX (both P < 0.001); nevertheless, they were significantly increased after 8 weeks of combined exercise (both P < 0.001).
Effects of combined exercise on Bax and Bcl-2 expression in the hippocampus Bax (23 kDa) and Bcl-2 (26 kDa) protein expressions were quantified using Western blot. Bax and Bcl-2 protein expressions in the hippocampus are presented in Figure 4. Bax and Bcl-2 were set to 1.00 in the sham group. Our data showed that mean (SEM) Bax protein expression was as follows: sham and exercise group, 0.78 (0.07); OVX group, 2.25 (0.13); OVX and exercise group, 1.77 (0.16). Similarly, mean (SEM) Bcl-2 protein expression was as follows: sham and exercise group, 1.06 (0.05); OVX group, 0.46 (0.04);
FIG. 4. Effects of combined exercise on Bax and Bcl-2 expression in the hippocampus. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group. OD, optical density. Menopause, Vol. 23, No. 1, 2016
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KIM ET AL
OVX and exercise group, 0.74 (0.08). There was a significant difference in Bax and Bcl-2 between groups (both P < 0.001). Post hoc analysis revealed that Bax expression increased after OVX (P < 0.001) and significantly decreased after 8 weeks of combined exercise (P ¼ 0.019). On the contrary, Bcl-2 protein expression was reduced after OVX (P < 0.001); however, it showed a significant increase after 8 weeks of combined exercise (P ¼ 0.001). Effects of combined exercise on caspase-3 and TUNEL expression in the hippocampal dentate gyrus We used caspase-3 and TUNEL immunohistochemical analyses to assess apoptosis. Photomicrographs of caspase3–positive and TUNEL-positive cells in the dentate gyrus are presented in Figure 5. The mean (SEM) number of caspase-3– positive cells was as follows: sham group, 52.16 (9.58); sham and exercise group, 57.46 (11.85); OVX group, 176.30 (14.37); OVX and exercise group, 100.67 (12.65). The mean (SEM) number of TUNEL-positive cells was as follows: sham group, 21.21 (2.03); sham and exercise group, 19.71 (1.59); OVX group, 107.58 (8.66); OVX and exercise group, 79.47 (7.91). Our results showed that there was a significant difference in caspase-3–positive and TUNEL-positive cells between groups (both P < 0.001). Post hoc analysis revealed that counts of caspase-3–positive and TUNEL-positive cells increased after OVX (both P < 0.001) and that 8 weeks of combined exercise after OVX resulted in an increase in caspase-3–positive and TUNEL-positive cells (P ¼ 0.009 and P ¼ 0.011, respectively).
Correlation between hippocampal cell proliferation and cognitive function To examine the correlation between cell loss and cognitive function, we performed a correlation analysis between two variables: number of BrdU-positive cells in the hippocampal dentate gyrus and cognitive behavior (Fig. 6). There was a statistically significant positive correlation between the number of BrdU-positive cells and performance in Morris water maze task (r ¼ 0.662, P < 0.001) and step-down avoidance task (r ¼ 0.617, P < 0.001). DISCUSSION In numerous studies, estrogen has been shown to protect individuals from brain injury, neurodegeneration, and cognitive decline. Hypoestrogenic or postmenopausal conditions in women decrease cognitive function and increase the risk of various neurodegenerative diseases.6 Postmenopausal women have been reported to be at relatively higher risk for developing Alzheimer dementia compared with men.32 Based on behavioral analysis in the present study, shortterm and spatial working memory decreased in ovariectomized rats. Reports on memory deficits and impairment of cognitive function have been associated with estrogen level changes during pregnancy and perimenopause.33 Cognitive decline in ovariectomized rats seems to have a close association with hippocampal function. In the present study, cognitive decline was accompanied by a decrease in BrdUpositive and DCX-positive cells and expression levels of
FIG. 5. Effects of combined exercise on cell death in the dentate gyrus. Left: Caspase-3–positive cells in the dentate gyrus. Right: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)–positive cells in the dentate gyrus. Top: Photomicrograph of caspase-3– positive and TUNEL-positive cells. Scale bar represents 50 mm. Bottom: Number of caspase-3–positive and TUNEL-positive cells in each group. (A) Sham group. (B) Sham and exercise group. (C) Ovariectomy group. (D) Ovariectomy and exercise group. Data are expressed as mean (SEM). P < 0.05 compared with the sham group. #P < 0.05 compared with the ovariectomy group.
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EXERCISE AMELIORATES COGNITIVE IMPAIRMENT
FIG. 6. Correlation between hippocampal cell proliferation and cognitive function. Left: Correlation between number of BrdU-positive cells and Morris water maze task. Right: Correlation between number of BrdU-positive cells and step-down avoidance task.
Bcl-2, BDNF, and TrkB, whereas Bax and caspase-3 levels increased in the hippocampus. In a previous study, estrogen was shown to regulate BDNF synthesis in the hippocampus. Other reports have shown that OVX caused a decrease in BDNF messenger RNA and cell proliferation in the female rat hippocampus.34,35 In another study, Bax and caspase-3 expressions increased and Bcl-2 and Bcl-x levels decreased in the hippocampus of ovariectomized rats.18,36,37 Estrogen deficiency inhibits cell proliferation and induces apoptosis in the hippocampus, ultimately causing memory decline. Notably, our study showed positive correlations between cell proliferation and cognitive function. A clinical experiment in a previous study reported a correlation between low cell proliferation and differentiation in the hippocampus and memory loss38; this finding is consistent with our results. On the other hand, in the present study, combined exercise in ovariectomized rats increased short-term and spatial working memory. The exercise program in this study consisted of a combined aerobic and anaerobic program that possessed the advantages of aerobic and resistance exercise, thereby facilitating a greater increase in physical strength compared with a single type of exercise. Investigation of the relationship between physical strength and cognitive processes in human studies showed that fitness training influenced various cognitive processes. In particular, fitness training exhibited the strongest effect on executive control processes, including planning, scheduling, working memory, inhibitory processes, and multitasking. Such effects were magnified when fitness training involved a combination of aerobic exercise and strength and flexibility training.39 The combination of treatment protocols may also exhibit a more diverse set of changes in the brain.40 Exercise has been proven to change brain structure and function.41 Aerobic activity improves learning and task acquisition and increases the secretion of important neurochemicals associated with promoting synaptic plasticity and neuronal architecture.42 The results of the present study showed that combined exercise in ovariectomized rats increased the expression of BrdU-positive cells (marker of proliferation), DCX-positive cells (marker of differentiation), BDNF, and TrkB receptor in the hippocampus. Improvement in the cognitive function of ovariectomized rats through exercise is considered the result of restored hippocampal function. Exercise has been
known to increase hippocampal neurogenesis, improve cognitive function, and increase synaptic plasticity. In a previous study, treadmill exercises in middle-aged mice led to increased BrdUpositive and DCX-positive cells, BDNF levels, and TrkB levels.43 Reduced neurogenesis caused by aging was increased, through exercise, to 50% of the levels exhibited by the young group, along with improvements in cognitive function. There was no difference in the morphology of new cells between the young group and the old exercising group.44 Furthermore, voluntary running led to an increase in cell proliferation in the hippocampus of ovariectomized mice.45 BDNF plays an important role in regular hippocampal neurogenesis. In particular, BDNF is known to promote the survival of newborn granule cells.46,47 Exercise is associated not only with cell differentiation and survival but also with cell apoptosis. In the present study, combined exercise inhibited Bax and caspase-3 action and activated Bcl-2 in the hippocampus of ovariectomized rats. In a previous study, exercise inhibited apoptosis in various brain diseases, including Alzheimer disease and traumatic brain injury, by inhibiting Bax and caspase3 and by activating Bcl-2.21,27 Exercise activates various neurotrophins that promote cell survival and inhibit apoptosis.48 In particular, apoptotic stimulation induces caspase-3 activation, but such caspase-3 activation can be blocked by BDNF.49 Ovariectomized rats that were subjected to 8 weeks of combined exercise exhibited an increase in hippocampal BDNF and TrkB expression, which suggests that BDNF-TrkB signaling may be related to inhibition of apoptosis induced by combined exercises. As mentioned earlier, estrogen exhibits a strong neuroprotective effect on plasticity and cell survival in the adult brain. However, postmenopausal women may be less neuroprotected under estrogen deficiency. CONCLUSIONS Ovariectomized rats show deficits in cognitive function (such as short-term and spatial working memory)—resulting from suppression of cell proliferation and BDNF expression—and increased apoptosis in the hippocampus. These deficits in cognitive function are prevented by increased hippocampal cell proliferation and BDNF expression and decreased apoptosis resulting from exercise. The present data suggest that complex exercise increases cell proliferation and BDNF expression and Menopause, Vol. 23, No. 1, 2016
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suppresses apoptosis in the hippocampus of rats in a state of estrogen depletion. The data suggest that exercise might be an effective strategy for minimizing cognitive function deficits in postmenopausal women. REFERENCES 1. Weitzmann MN, Pacifici R. Estrogen deficiency and bone loss: an inflammatory tale. J Clin Invest 2006;116:1186-1194. 2. Antonicelli R, Olivieri F, Morichi V, Urbani E, Mais V. Prevention of cardiovascular events in early menopause: a possible role for hormone replacement therapy. Int J Cardiol 2008;130:140-146. 3. McEwen B. Estrogen actions throughout the brain. Recent Prog Horm Res 2002;57:357-384. 4. Gibbs RB, Gabor R. Estrogen and cognition: applying preclinical findings to clinical perspectives. J Neurosci Res 2003;74:637-643. 5. Daniel JM. Effects of oestrogen on cognition: what have we learned from basic research? J Neuroendocrinol 2006;18:787-795. 6. Wise PM. Estrogens and neuroprotection. Trends Endocrinol Metab 2002;13:229-230. 7. Spencer JL, Waters EM, Romeo RD, Wood GE, Milner TA, McEwen BS. Uncovering the mechanisms of estrogen effects on hippocampal function. Front Neuroendocrinol 2008;29:219-237. 8. Li C, Brake WG, Romeo RD, et al. Estrogen alters hippocampal dendritic spine shape and enhances synaptic protein immunoreactivity and spatial memory in female mice. Proc Natl Acad Sci U S A 2004;101:2185-2190. 9. Biegler R, McGregor A, Krebs JR, Healy SD. A larger hippocampus is associated with longer-lasting spatial memory. Proc Natl Acad Sci U S A 2001;98:6941-6944. 10. Kempermann G, Kuhn HG, Gage FH. Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci U S A 1997;94:10409-10414. 11. Francis F, Koulakoff A, Boucher D, et al. Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron 1999;23:247-256. 12. Pencea V, Bingaman KD, Wiegand SJ, Luskin MB. Infusion of brainderived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J Neurosci 2001;21:6706-6717. 13. Barnabe´-Heider F, Miller FD. Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J Neurosci 2003;23:5149-5160. 14. Chmielnicki E, Benraiss A, Economides AN, Goldman SA. Adenovirally expressed noggin and brain-derived neurotrophic factor cooperate to induce new medium spiny neurons from resident progenitor cells in the adult striatal ventricular zone. J Neurosci 2004;24:2133-2142. 15. Su JH, Deng G, Cotman CW. Bax protein expression is increased in Alzheimer’s brain: correlations with DNA damage, Bcl-2 expression, and brain pathology. J Neuropathol Exp Neurol 1997;56:86-93. 16. Hartmann A, Hunot S, Michel PP, et al. Caspase-3: a vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc Natl Acad Sci U S A 2000;97:2875-2880. 17. Jarskog LF, Selinger ES, Lieberman JA, Gilmore JH. Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation. Am J Psychiatry 2004;161:109-115. 18. Stoltzner SE, Berchtold NC, Cotman CW, Pike CJ. Estrogen regulates bcl-x expression in rat hippocampus. Neuroreport 2001;12:2797-2800. 19. Chambers RA, Potenza MN, Hoffman RE, Miranker W. Simulated apoptosis/neurogenesis regulates learning and memory capabilities of adaptive neural networks. Neuropsychopharmacology 2004;29:747-758. 20. Kim SE, Ko IG, Kim BK, et al. Treadmill exercise prevents aginginduced failure of memory through an increase in neurogenesis and suppression of apoptosis in rat hippocampus. Exp Gerontol 2010; 45:357-365. 21. Kim DH, Ko IG, Kim BK, et al. Treadmill exercise inhibits traumatic brain injury–induced hippocampal apoptosis. Physiol Behav 2010;101:660-665. 22. Deng W, Aimone JB, Gage FH. New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 2010;11:339-350. 23. Asikainen TM, Kukkonen-Harjula K, Miilunpalo S. Exercise for health for early postmenopausal women: a systematic review of randomised controlled trials. Sports Med 2004;34:753-778.
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