- Email: [email protected]
3 induced by transient cerebral ischemia in the aged gerbil hippocampus
Accepted Manuscript Long-term treadmill exercise improves memory impairment through restoration of decreased synaptic adhesion molecule 1/ 2/3 induced by transient cerebral ischemia in the aged gerbil hippocampus
Ji Hyeon Ahn, Joon Ha Park, Jinseu Park, Myoung Cheol Shin, Jun Hwi Cho, In Hye Kim, Jeong-Hwi Cho, Tae-Kyeong Lee, Jae-Chul Lee, Bich Na Shin, Young-Myeong Kim, Choong Hyun Lee, In Koo Hwang, Il Jun Kang, Bai Hui Chen, Bing Chun Yan, Young Joo Lee, Moo-Ho Won, Soo Young Choi PII: DOI: Reference:
S0531-5565(17)30584-3 https://doi.org/10.1016/j.exger.2018.01.015 EXG 10259
To appear in:
Experimental Gerontology
Received date: Revised date: Accepted date:
2 August 2017 5 January 2018 12 January 2018
Please cite this article as: Ji Hyeon Ahn, Joon Ha Park, Jinseu Park, Myoung Cheol Shin, Jun Hwi Cho, In Hye Kim, Jeong-Hwi Cho, Tae-Kyeong Lee, Jae-Chul Lee, Bich Na Shin, Young-Myeong Kim, Choong Hyun Lee, In Koo Hwang, Il Jun Kang, Bai Hui Chen, Bing Chun Yan, Young Joo Lee, Moo-Ho Won, Soo Young Choi , Long-term treadmill exercise improves memory impairment through restoration of decreased synaptic adhesion molecule 1/2/3 induced by transient cerebral ischemia in the aged gerbil hippocampus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Exg(2017), https://doi.org/10.1016/j.exger.2018.01.015
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Long-term treadmill exercise improves memory impairment through restoration of decreased synaptic adhesion molecule 1/2/3 induced by transient cerebral ischemia in the aged gerbil hippocampus
Ji Hyeon Ahn1, Joon Ha Park1, Jinseu Park1, Myoung Cheol Shin2, Jun Hwi Cho2, In Hye Kim3, Jeong-Hwi Cho3, Tae-Kyeong Lee3, Jae-Chul Lee3, Bich Na Shin4, Young-Myeong Kim5, Choong Hyun Lee6, In Koo
Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University,
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Hwang7, Il Jun Kang8, Bai Hui Chen9, Bing Chun Yan10, Young Joo Lee11, Moo-Ho Won3*, Soo Young Choi1*
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Chuncheon 24252, South Korea
Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 24341,
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South Korea
Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South
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Department of Physiology, College of Medicine, Hallym University, Chuncheon 24252, South Korea
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Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University,
Chuncheon 24341, South Korea
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Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan 31116, South Korea
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Department of Anatomy and Cell Biology, College of Veterinary Medicine, and Research Institute for
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Veterinary Science, Seoul National University, Seoul 08826, South Korea Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, South Korea
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Department of Histology and Embryology, Institute of Neuroscience, Wenzhou Medical University, Wenzhou,
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Zhejiang 325035, P.R. China 10
Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and
Treatment of Senile Diseases, Yangzhou 225001, People’s Republic of China 11
Department of Emergency Medicine, Seoul Hospital, College of Medicine, Sooncheonhyang University, Seoul
04401, South Korea.
- Co-firsts: Ji Hyeon Ahn1 and Joon Ha Park1 have contributed equally to this article. 1
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* Corresponding authors
Professor Moo-Ho Won, DVM, PhD: Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea. TEL: +82-33-250-8891; FAX: +82-33-256-1614. E-mail:
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[email protected]
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Professor Soo Young Choi, PhD: Department of Biomedical Science and Research Institute of Bioscience and
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Biotechnology, Hallym University, Chuncheon 24252, South Korea. TEL: +82-33-248-2112; FAX: +82-33-241-
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1463. E-mail: [email protected]
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Abstract
Exercise improves cognitive impairments induced by transient cerebral ischemia and modulates synaptic adhesion molecules. In this study, we investigated effects of long-term treadmill exercise on cognitive impairments and its relation to changes of synaptic cell adhesion molecule (SynCAM) 1/2/3 in the hippocampus
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after 5 min of transient cerebral ischemia in aged gerbils. Animals were assigned to sedentary and exercised groups, given treadmill exercise for 4 consecutive weeks from 5 days after transient ischemia and evaluated
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cognitive function through passive avoidance test and Morris water maze test. SynCAM 2 protein levels were
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determined in the hippocampus by western blot. In addition, neuronal and synaptic changes were examined by NeuN immunohistochemistry, and SynCAM 1/2/3 and MAP2 double immunofluorescence, respectively. We
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found that transient cerebral ischemia led to neuronal death in the CA1 area and dentate gyrus, and impaired conflictive function; however, treadmill exercise improved ischemia-induced memory impairment. In addition,
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SynCAM 1/2/3 expression in the hippocampus was significantly decreased in the sedentary group after transient cerebral ischemia; however, SynCAM 2 protein level was significantly increased in the ischemic group with exercise. These results suggest that long-term treadmill exercise improves memory impairment through the
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restoration of decreased SynCAM 1/2/3 expression in the hippocampus induced by transient cerebral ischemia
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in the aged gerbil.
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Treadmill exercise
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Keywords: Aging; Cerebral ischemia; Learning and memory; Rehabilitation; Synaptic adhesion molecules;
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1. Introduction
It is well known that transient cerebral ischemia (TCI) induces delayed neuronal death in pyramidal neurons in the CA1 area of the hippocampus proper (CA1-3 areas) about 4 days after 5 min of TCI, while pyramidal neurons in the CA3 area remain relatively intact (Kirino, 1982). Furthermore, the synapse is highly susceptible
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to TCI that affects structural and biochemical alterations in the damaged areas (Costain et al., 2008; Kirino, 1982; Martone et al., 1999), and the synaptic failure occurs even in the absence of neuronal damage after
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ischemic insults (Hofmeijer and van Putten, 2012). These aberrant synaptic changes could affect the function of
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synapses including synaptic signaling, transmission, and plasticity and stability. In this regard, neuronal and synapse alterations after ischemic insults are closely related to impaired neurological function (Dahlqvist et al.,
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2004).
Transsynaptic interaction is formed by a group of cell adhesion molecules (CAMs) that interact in a homo- or
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heterophilic fashion across the synaptic cleft as well as neurotransmitter release (Dalva et al., 2007). Interactions of CAMs can control functions of existing synapses or lead to the formation of new synapses via various signaling pathways (Dalva et al., 2007). In addition, CAMs not only modulate adhesion at synapses, but also
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regulate the recruitment of synaptic vesicles and neurotransmitters (Hoy et al., 2009). Among CAMs, synaptic
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CAMs (SynCAMs) 1-3 are four immunoglobulin proteins containing CAMs that are expressed predominantly at pre- and postsynaptic plasma membranes in the brain (Biederer et al., 2002), and are required for maintaining normal synapse numbers (Robbins et al., 2010).
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It is necessary to study mechanisms underlying effects of long-term exercise on stroke rehabilitation (Macko
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et al., 2015). It is well known that kinds and quality of exercises affects synaptic plasticity and gene expression in normal brain (Farmer et al., 2004; Molteni et al., 2002). Furthermore, physical exercise has been shown to help to recover and improve neurological functions in humans (Duncan et al., 2003; Macko et al., 2005) and animals (Ahn et al., 2016a; Sakakima et al., 2012) after stroke. The hippocampus is a major location of critical aspects of neuronal plasticity (Neves et al., 2008b) and dynamic changes of synapses in the hippocampus display an important role in learning and memory (Neves et al., 2008a). To the best of our knowledge, long-term (chronic) change in SynCAMs expression has not been reported in the aged hippocampus after cerebral ischemia. Therefore, we examined the effect of long-term 4
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treadmill exercise on memory function after TCI in the aged gerbil, which is a good animal for study on TCI (Kirino, 1982). In addition, we investigated whether changes in SynCAM 1/2/3 expressions were related with the improvement in TCI-induced memory impairment or not in the ischemic aged hippocampus.
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2. Materials and Methods
2.1. Experimental animals
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Aged male Mongolian gerbils (Meriones unguiculatus, 22 to 24 months) weighing 80 to 90 g were supplied
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by the Experimental Animal Center, Kangwon National University (Chuncheon, South Korea). Animal handling and care followed the guidelines of current international laws and policies (NIH Guide for the Care and Use of
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Laboratory Animals, The National Academies Press, 8th Ed., 2011) (Council, 2011) and experimental protocol was approved by Institutional Animal Care and Use Committee (IACUC) of Kangwon National University
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(approval no. KW-160802-1).
Animals were randomly divided into three groups (n = 63): 1) sham group (n = 21) had no ischemia operation and treadmill (TR) exercise after sham operation, 2) SD4 group (n = 21) had ischemia operation and sedentary
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(SD) for 4 weeks from 5 days post-ischemia, 3) TR4 group (n = 21) had ischemia operation and TR exercise for
2.2. Induction of TCI
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finished in the TR4 group.
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4 weeks from 5 days post-ischemia. All animals were sacrificed 31 days after TCI when TR exercise was
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As previously described (Lee et al., 2015), in brief, the animals were anesthetized with a mixture of 2.5 % isoflurane in 33 % oxygen and 67 % nitrous oxide. Blood flow to the brain was completely interrupted by a 5min occlusion of bilateral common carotid arteries, which was confirmed by observing the retinal central artery under an ophthalmoscope. The body (rectal) temperature was controlled under normothermia (37 ± 0.5 ºC) before, during and after the surgery. Sham operated animals were subjected to the same surgical procedure except that the common carotid arteries were not occluded.
2.3. TR exercise 5
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For TR exercise, running speed and duration was determined according to previous studies (Lee et al., 2003; Sim et al., 2005b; Sim et al., 2004) with modification. Briefly, animals were familiarized with the TR running on a motorized treadmill (Exer3/6, Columbus Instruments) for 15 min/day for 3 consecutive days. From 5 days after TCI, the animals were forced to run on motorized TR for 30 min/day and 5 days/week for 1 or 4 consecutive weeks. The TR exercise workload consisted of running at the speed 5 m/min for the first 5 min, 7
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m/min for the next 5 min, and then 10 m/min for the last 20 min with 0 degree inclination. Animals in the SD
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group were left on the TR for 30 min and were not made to run.
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2.4. Passive avoidance test
Passive avoidance test was done for memory impairment at 1 day before TCI, 5 and 31 days after TCI
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according to a previous study (Horisawa et al., 2011). In brief, the Gemini Avoidance System (GEM 392, San Diego Instruments), which consisted of two compartments (light and dark), was used. In the training session,
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animals were allowed to explore environments of A and B dark compartments for 2 min. Light was turned on only in the A compartment and animals were allowed to explore environments in the A and B compartments for 2 min. When animals entered the B-compartment, the floor was closed an inescapable foot-shock (0.3 mA for 3
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s). The test session was performed 20 min after the training session, namely, each animal was placed in the A
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compartment, the light was turned on, and the floor was opened. Latency time for each animal to enter the B compartment was recorded within 180 s.
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2.5. Morris water maze test
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Morris water maze test was performed for spatial memory from 22 to 31 days after TCI according to published methods (Corbett et al., 1992; Wiard et al., 1995). In short, 3 different visual cues were placed around the inside wall of the tank at a level that would be visible to the animals. Each animal was given 3 daily trials with a 5-min intertrial interval for 10 days and individually placed in the apparatus at one of 3 preselected locations and allowed 50 seconds to escape to the hidden platform. The animals not finding the platform after 50 seconds were guided to it by the experimenter. The animals were allowed to remain on the platform for 10 seconds and then returned to their holding cage until the next trial. The escape time of the gerbil to find the platform was recorded within 50 seconds (escape latency), and the whole process was monitored by a digital camera and a computer 6
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system.
2.6. Western blot analysis As previously described (Ahn et al., 2011), in brief, hippocampi of animals (n = 7 in each group) were dissected. After the tissues were homogenized and centrifuged, and the supernatants were subjected to western
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blot analysis. Rabbit anti-SynCAM 2 (1:1000, Synaptic system, Göttingen, Germany) was used as primary antibody. The result of western blot analysis was scanned, and densitometric analysis for the quantification of
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the bands was done using Image J 1.46 (National Institutes of Health), which was used to count relative optical
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density (ROD): A ratio of the ROD was calibrated as %, with sham group designated as 100 %.
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2.7. Immunohistochemistry
Immunohistochemistry was performed according to our published method (Lee et al., 2012). For tissue
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preparation, animals (n = 7 each group) were anesthetized with sodium pentobarbital (JW Pharm. Co., Ltd., Korea, 40 mg/kg, i.p) and perfused transcardially with 4% paraformaldehyde in solution buffer (PB, pH 7.4). The brain tissues were serially sectioned into 30-μm coronal sections in a cryostat (Leica, Wetzlar, Germany).
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For immunohistochemistry, mouse anti-neuronal nuclei (NeuN, a marker for neurons) (1:1000, Chemicon
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International, Temecula, CA) as primary antibodies, and biotinylated horse anti-mouse IgG (Vector, Burlingame, CA) and streptavidin peroxidase complex (1:200, Vector) as secondary antibodies. For the specificity of the immunostaining, a negative control was tested and resulted in the absence of immunoreactivity.
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For double immunofluorescence staining for SynCAM 1/2/3 and MAP2 (a maker for dendrites), sections
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were incubated with the mixture of rabbit anti-SynCAM 1/2/3 (1:100, Synaptic system, Göttingen, Germany) and mouse anti-MAP2 (1:400, Chemicon International) and incubated in a mixture of both Cy3-conjugated goat anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA) and FITC-conjugated goat anti-mouse IgG (1:200, Jackson ImmunoResearch). The immunoreactions were observed under the confocal MS (LSM510 META NLO, Carl Zeiss, Germany). In order to quantitatively analyze NeuN-immunoreactive neurons, digital images from 6 sections per animal were taken using AxioM1 light microscope (Carl Zeiss, Germany) equipped with digital camera (Axiocam, Carl Zeiss, Germany) connected to a PC monitor. NeuN-immunoreactive neurons were counted in a 250 × 250 μm 7
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square of the stratum pyramidale in the CA1 area and polymorphic layer of the dentate gyrus using an image analyzing system (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ). Cell counts were obtained by averaging the counts from each animal. To measure fluorescence intensities of SynCAM 1/2/3 and MAP2 immunoreactions, digital images from 6 sections per animal were captured with a confocal MS (LSM510 META NLO, Carl Zeiss, Germany). The fluorescence intensities of SynCAM 1/2/3 and MAP2 immunoreactive
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structures were analyzed using Image J 1.46 software (National Institutes of Health, Bethesda, MD). The mean fluorescence intensity of the sham group was designated as 100%, and the relative fluorescence intensity of each
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group was calibrated and expressed as % of the sham group.
2.8. Statistical analysis
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Data are expressed as the mean ± SEM. The latency of the short-term memory ability was analyzed with a two-way repeated ANOVA, followed by post hoc Bonferroni-Dunn Test. Mean numbers and ROD of
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immunoreactive structures were analyzed with a one-way ANOVA, followed by post hoc Bonferroni-Dunn Test. All comparisons were tested using SPSS 17.0 software (IBM, New York, USA). Statistical significance was
3. Result
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3.1. Passive avoidance test
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considered at P < 0.05.
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Firstly, short-term memory ability was assessed between the groups at the same point in time using passive avoidance test (Fig. 1A). At 5 days after ischemia-reperfusion, the latency was significantly decreased in the SD4 and TR4 groups compared with the sham group. Thirty-one days after ischemia-reperfusion, treadmill exercise significantly increased the latency in the TR4 group compared with the SD4 group, although the latency was significantly different from that in the sham group (Fig. 1A). Secondly, short-term memory ability was examined according to times after ischemia in each group using passive avoidance test (Fig. 1B). In the sham group, the latency was not significantly changed with time. On the other hand, the latency in the SD4 group was significantly decreased 5 days after ischemia-reperfusion and the 8
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decreased memory function was maintained until 31 days after ischemia-reperfusion. However, in the TR4 group, the decreased latency at 5 days post-ischemia (by 76.4% compared to that at 1 day before ischemia) was significantly increased with time and recovered by 39.8% at 31 days post-ischemia compared to that at 5 days post-ischemia.
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3.2. Morris water maze test At 22 days after ischemia-reperfusion, escape latency was not significantly different among the sham, SD4,
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and TR4 groups. Escape latency at 31 days after ischemia-reperfusion was significantly reduced compared to
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the first trial in the sham, SD4 and TR4 groups. However, no significant differences were found in the escape latency in Morris water maze test among the groups, although the escape latency was slower in the SD4 group
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compared to that in the sham and TR4 groups (Fig. 1C).
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3.3. SynCAM 2 protein level
In the SD4 group, SynCAM 2 protein level in the hippocampus was significantly decreased compared with that in the sham group; however, SynCAM 2 protein level in the TR4 group was significantly increased (P <
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0.05) compared with that in the SD4 group (Fig. 2)
3.4. NeuN-immunoreactive neurons
NeuN-immunoreactive neurons were shown in the stratum pyramidale of the hippocampus proper (CA1-3
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areas) and in the granule cell layer of the hippocampal dentate gyrus in the sham group (Fig. 3A1-A4). Thirty-
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one days after ischemia-reperfusion, the mean number of NeuN-immunoreactive neurons in the SD4 group was significantly decreased (P < 0.05) in the stratum pyramidale of the CA1 area and in the polymorphic layer of the dentate gyrus (DG), but not in the other subregions (Fig. 3B1-B4 and 3D). At this point in time, the distribution pattern of NeuN-immunoreactive neurons in the hippocampus of the TR4 group was similar to that in the SD4 group (Fig. 3C1-C4 and 3D).
3.5. SynCAM 1/2/3 and MAP2 immunoreactivities 3.5.1. CA1 area 9
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In the sham group, SynCAM 1/2/3 immunoreactivity was highly enriched in MAP2-immunoreactive pyramidal cell membranes in the stratum pyramidale and in cytoplasmic processes, which were dendrites and axons, in strata oriens and radiatum (Fig 4A1-A3). Thirty-one days after ischemia-reperfusion, the density of SynCAM 1/2/3-immunoreactive structures was not found in the pyramidal cell membranes due to the death of the CA1 pyramidal cells and markedly decreased in strata oriens and radiatum in the SD4 group compared with the sham
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group (Fig. 4B1-B3 and 4D). However, the TR4 group, the density of SynCAM 1/2/3-immunoreactive structures was significantly increased in strata oriens and radiatum compared with the SD4 group, in addition,
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the density of MAP2-immunoreactive structures was increased in all layers in this group (Fig. 4C1-C3 and 4D).
3.5.2. CA2/3 area
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In the sham group, the distribution pattern of the SynCAM 1/2/3- and MAP2-immunoreactive structures in the CA3 area was similar to that in the CA1 area; in this area, SynCAM 1/2/3-immunoreactive processes were
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abundant in the stratum lucidum which contains the mossy fiber pathway projecting from the dentate gyrus (Fig. 5A1-A3). In the SD4 group, SynCAM 1/2/3- and MAP2-immunoreactive structures in the stratum pyramidale were similar to those in the sham group; however, in strata lucidum and radiatum, SynCAM 1/2/3-
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immunoreactive structures were markedly decreased, and MAP2-immunoreactive processes were decreased
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compared with the sham group (Fig. 5B1-B3 and 5D). In the TR4 group, densities of SynCAM 1/2/3- and MAP2-immunoreactive structures were significantly increased in strata lucidum and radiatum compared with
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3.5.3. Dentate gyrus
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the SD4 group (Fig. 5C1-C3 and 5D).
In the sham group, SynCAM 1/2/3 immunoreactivity was easily detected in MAP2-immunoreactive granule cell membranes in the granule cell layer and in processes in the molecular layer and polymorphic layer (Fig 6A1A3). At 31 days after ischemia-reperfusion, the density of SynCAM 1/2/3-immunoreactive structures was markedly decreased in the molecular and polymorphic cell layers in the SD4 group compared with the sham group, although MAP2 immunoreactivity was decreased only in the polymorphic cell layer (Fig. 6B1-B3 and 6D). In the TR4 group, the density of SynCAM 1/2/3-immunoreactive structures was increased in the molecular and polymorphic cell layers compared with the SD4 group, in addition, the density of the MAP210
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immunoreactive structures in the polymorphic layer was increased (Fig. 6C1-C3 and 6D).
4. Discussion
In the present study, we investigated effects of long-term TR exercise on cognitive function using passive
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avoidance test and Morris water maze test, and the relation of SynCAM 1/2/3 expression in the hippocampus to memory using western blot analysis and immunohistochemistry in the aged gerbil following 5 min of transient
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cerebral ischemia.
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Short-term learning and memory through passive avoidance test was significantly decreased after transient cerebral ischemia; however, 4-week TR exercise significantly improved learning and memory impairment 31
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days after ischemia-reperfusion compared with that in the sedentary group. This result is consistent with previous studies (Ahn et al., 2016a; Sim et al., 2005a) that reflect that long-term exercise improves ischemia-
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induced short-term memory impairment during chronic stage. On the other hand, spatial learning and memory assessed by Morris water maze test was not significantly impaired 31 days after ischemia-reperfusion. This result is in line with previous studies that show that deficits in spatial learning and memory appear to be
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transient during subacute stage (about 3 days) after 5 min of transient cerebral ischemia, although spatial
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learning and memory impairs after 10 or 15 min of transient cerebral ischemia which leads to damage/death in pyramidal neurons in the CA2/3 area that is resistant to 5 min of transient cerebral ischemia in the gerbil (Corbett et al., 1992; Wiard et al., 1995). These results indicate that spatial learning and memory is closely
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associated with severer neuronal damage in the hippocampus.
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We, in this study, found that neuronal death occurred in neurons in the stratum pyramidale of the CA1 area (Lee et al., 2010) and in the polymorphic layer of the dentate gyrus (Ahn et al., 2016b) after 5 min of transient cerebral ischemia and that numbers of neurons in the CA1 area and dentate gyrus observed at 31 days postischemia was not increased in the TR4 group, which was given long-term therapeutic TR exercise. This finding indicates that long-term exercise after neuronal damage/death could not affect neuronal population in the ischemic hippocampus although long-term exercise could improve learning and memory impairment induced by ischemic insults.
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It has been reported that TR exercise increases spatial memory via increasing synapse related protein, such as synapsin-1 and postsynaptic density protein-95 levels after ischemic insults (Shih et al., 2013). In the present study, we found that SynCAM 1/2/3-immunoreactive structures were enriched in pre- and post-synaptic sites in the hippocampus (Biederer et al., 2002; Robbins et al., 2010) and that ischemia-induced decreased SynCAM 1/2/3 immunoreactivity was significantly improved in the TR group, although SynCAM 1/2/3-immunoreactive
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structures were significantly decreased in all hippocampal subregions in the SD group, interestingly, including the CA3 area where neuronal loss was not observed after ischemia-reperfusion. Robbins et al. (2010) reporetd
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that the overexpression of SynCAM 1 in adult mouse brain improved spatial learning using Morris water maze
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task (Robbins et al., 2010). It has been demonstrated that SynCAMs directly impacts neuronal plasticity through restricting long-term depression which results in the weakening of synaptic connection (Fogel et al., 2007;
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Robbins et al., 2010) and that the knockdown of SynCAM 1 induces a reduction of post-synaptic density length, but the overexpression of SynCAM 1 increases the number of functional excitatory synapses in the
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hippocampus of a transgenic mouse model (Robbins et al., 2010). For the role of SynCAM 1, Jennifer et al. (2009) have suggested that SynCAM 1 might be associated with postsynaptic specialization via recruiting both NMDA- and AMPA-type glutamate receptors to sites of synaptic adhesion (Hoy et al., 2009). Based on the
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above-mentioned and our present findings, it is likely that chronic exercise could promote synapse formation
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and improve synaptic plasticity via increasing SynCAM 1/2/3 expression in ischemic hippocampus.
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5. Conclusions
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In conclusion, this study shows that 5 min of transient cerebral ischemia resulted in severe damages in shortterm memory and SynCAM 1/2/3 immunoreactive structures in the aged gerbil hippocampus; however, longterm TR exercise improved ischemic-induced short-term memory impairment by restoring SynCAMs. These results suggest that long-term TR exercise could be an effective treatment for the restoration of synaptic plasticity during chronic stage after ischemic insults in the elderly.
Acknowledgments
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This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT & Future Planning (NRF-2014R1A1A3051721), by the BioSynergy Research Project (NRF-2015M3A9C4076322) of the Ministry of Science, ICT and Future Planning through the National Research Foundation, by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9B6066835), and by a Priority Research Centers
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Program grant (NRF-2009-0093812) through the National Research Foundation of Korea funded by the
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Ministry of Science, ICT and Future Planning.
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The authors declare that they have no conflict of interest.
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Conflict of interests
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Kirino, T., 1982. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57-69. Lee, C.H., Ahn, J.H., Won, M.H., 2015. New expression of 5-HT1A receptor in astrocytes in the gerbil hippocampal CA1 region following transient global cerebral ischemia. Neurol Sci. 36, 383-389.
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Neuronal damage is much delayed and microgliosis is more severe in the aged hippocampus induced by transient cerebral ischemia compared to the adult hippocampus. J Neurol Sci. 294, 1-6. Lee, M.H., Kim, H., Kim, S.S., Lee, T.H., Lim, B.V., Chang, H.K., Jang, M.H., Shin, M.C., Shin, M.S., Kim, C.J., 2003. Treadmill exercise suppresses ischemia-induced increment in apoptosis and cell proliferation in hippocampal dentate gyrus of gerbils. Life Sci. 73, 2455-2465. Lee, T.H., Lee, C.H., Kim, I.H., Yan, B.C., Park, J.H., Kwon, S.H., Park, O.K., Ahn, J.H., Cho, J.H., Won, M.H., Kim, S.K., 2012. Effects of ADHD therapeutic agents, methylphenidate and atomoxetine, on hippocampal neurogenesis in the adolescent mouse dentate gyrus. Neurosci Lett. 524, 84-88. 15
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Macko, R., Ivey, F., Forrester, L., 2015. Task-oriented aerobic exercise in chronic hemiparetic stroke: training protocols and treatment effects. Topics in stroke rehabilitation. 12,45-57 Macko, R.F., Ivey, F.M., Forrester, L.W., Hanley, D., Sorkin, J.D., Katzel, L.I., Silver, K.H., Goldberg, A.P., 2005. Treadmill exercise rehabilitation improves ambulatory function and cardiovascular fitness in patients with chronic stroke a randomized, controlled trial. Stroke. 36, 2206-2211.
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Martone, M.E., Jones, Y.Z., Young, S.J., Ellisman, M.H., Zivin, J.A., Hu, B.-R., 1999. Modification of postsynaptic densities after transient cerebral ischemia: a quantitative and three-dimensional ultrastructural
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Molteni, R., Ying, Z., Gómez‐Pinilla, F., 2002. Differential effects of acute and chronic exercise on plasticity‐ related genes in the rat hippocampus revealed by microarray. European Journal of Neuroscience. 16, 1107-
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Neves, G., Cooke, S.F., Bliss, T.V., 2008b. Synaptic plasticity, memory and the hippocampus: a neural network approach to causality. Nature Reviews Neuroscience. 9, 65-75.
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Robbins, E.M., Krupp, A.J., Perez de Arce, K., Ghosh, A.K., Fogel, A.I., Boucard, A., Sudhof, T.C., Stein, V.,
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Biederer, T., 2010. SynCAM 1 adhesion dynamically regulates synapse number and impacts plasticity and learning. Neuron. 68, 894-906.
Sakakima, H., Khan, M., Dhammu, T.S., Shunmugavel, A., Yoshida, Y., Singh, I., Singh, A.K., 2012.
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Stimulation of functional recovery via the mechanisms of neurorepair by S-nitrosoglutathione and motor
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exercise in a rat model of transient cerebral ischemia and reperfusion. Restorative neurology and neuroscience. 30, 383-396. Shih, P.-C., Yang, Y.-R., Wang, R.-Y., 2013. Effects of exercise intensity on spatial memory performance and hippocampal synaptic plasticity in transient brain ischemic rats. PloS one. 8, e78163. Sim, Y.-J., Kim, H., Kim, J.-Y., Yoon, S.-J., Kim, S.-S., Chang, H.-K., Lee, T.-H., Lee, H.-H., Shin, M.-C., Shin, M.-S., 2005a. Long-term treadmill exercise overcomes ischemia-induced apoptotic neuronal cell death in gerbils. Physiology & behavior. 84, 733-738. Sim, Y.J., Kim, H., Kim, J.Y., Yoon, S.J., Kim, S.S., Chang, H.K., Lee, T.H., Lee, H.H., Shin, M.C., Shin, M.S., 16
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Kim, C.J., 2005b. Long-term treadmill exercise overcomes ischemia-induced apoptotic neuronal cell death in gerbils. Physiol Behav. 84, 733-738. Sim, Y.J., Kim, S.S., Kim, J.Y., Shin, M.S., Kim, C.J., 2004. Treadmill exercise improves short-term memory by suppressing ischemia-induced apoptosis of neuronal cells in gerbils. Neurosci Lett. 372, 256-261. Wiard, R.P., Dickerson, M.C., Beek, O., Norton, R., Cooper, B.R., 1995. Neuroprotective properties of the novel
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antiepileptic lamotrigine in a gerbil model of global cerebral ischemia. Stroke. 26, 466-472.
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Figure legends
Fig. 1. A and B: The latency of the passive avoidance test in the sham, SD4 and TR4 groups: “A” is assessment between the groups at each point in time, and “B” is assessment according to time in each group (n = 7 per group; *P< 0.05, significantly different from the sham group, †P < 0.05, significantly different from the SD4
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group). C: The escape latency of the Morris Water Maze test in the sham, SD4 and TR4 groups. Data are defined
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as the means ± SEM.
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Fig. 2. Western blot analysis of SynCAM 2 in the hippocampus of the sham, SD4 and TR4 groups (n = 7 per group; *P < 0.05, significantly different from the sham group, †P < 0.05, significantly different from the SD4
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group). The bars indicate the means ± SEM.
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Fig. 3. NeuN immunohistochemistry in the sham (A), SD4 (B) and TR4 (C) groups. In the SD4 and TR4 groups, the mean number of NeuN-immunoreactive neurons is significantly decreased in the stratum pyramidale (SP) of the CA1 region and in the polymorphic layer (PoL) of the dentate gyrus (DG). GCL, granule cell layer; MoL,
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molecular layer; SO, stratum oriens; SR, stratum radiatum. Scale bars = 400 μm (A1-C1), 40 μm (A2-C2), and
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100 μm (A3-C4). F: The mean number of NeuN-immunoreactive neurons in the sham, SD4 and TR4 groups (n = 7 per group; *P < 0.05, significantly different from the sham group). The bars indicate the means ± SEM.
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Fig. 4. Double immunofluorescence staining for SynCAM 1/2/3, MAP2 and merged images in the CA1 area of
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the sham (A), SD4 (B) and TR4 (C) groups. SynCAM 1/2/3- and MAP2-immunoreactive structures are markedly decreased in strata oriens (SO) and radiatum (SR) in the SD4 group. However, SynCAM 1/2/3- and MAP2-immunoreactive structures are increased in the TR4 group. SP; stratum pyramidale. Scale bar = 40 μm. D: Relative immunofluorescence intensity expressed as a percentage of SynCAM 1/2/3- and MAP2immunoreactive structures (n = 7 per group; *P < 0.05, significantly different from the sham group and †P < 0.05, significantly different from the SD4 group). The bars indicate the means ± SEM.
Fig. 5. Double immunofluorescence staining for SynCAM 1/2/3, MAP2 and merged images in the CA3 area of 18
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the sham (A), SD4 (B) and TR4 (C) groups. In the SD4 group, SynCAM 1/2/3- and MAP2- immunoreactive structures are decreased in strata radiatum (SR) and lucidum (SL); however, in the TR4 group, SynCAM 1/2/3and MAP2-immunoreactive structures are increased in the layers. SP; stratum pyramidale. Scale bar = 40 μm. D: Relative immunofluorescence intensity expressed as a percentage of SynCAM 1/2/3- and MAP2immunoreactive structures (n = 7 per group; *P < 0.05, significantly different from the sham group and †P < 0.05,
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significantly different from the SD4 group). The bars indicate the means ± SEM.
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Fig. 6. Double immunofluorescence staining for SynCAM 1/2/3, MAP2 and merged images in the dentate gyrus
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of the sham (A), SD4 (B) and TR4 (C) groups. In the SD4 group, the density of SynCAM 1/2/3-immunoreactive structures is decreased in the molecular (MoL) and polymorphic (PoL) layers compared with the sham group;
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however, in the TR4 group, its density is increased compared with the SD4 group. GCL, granule cell layer. Scale bar = 40 μm. D: Relative immunofluorescence intensity expressed as a percentage of SynCAM 1/2/3- and
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P < 0.05, significantly different from the SD4 group). The bars indicate the means ± SEM.
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†
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MAP2-immunoreactive structures (n = 7 per group; *P < 0.05, significantly different from the sham group and
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Highlights
* Memory was impaired following transient cerebral ischemia
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* Long-term treadmill exercise improved ischemia-induced memory impairment.
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* Transient cerebral ischemia induced neuronal loss in the CA1 area of the hippocampus.
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* Transient cerebral ischemia induced SynCAM reduction in the all hippocampal subregions.
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*Treadmill exercise restored ischemia-induced memory deficits by improving SynCAM
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Figure 1
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Ji Hyeon Ahn, Joon Ha Park, Jinseu Park, Myoung Cheol Shin, Jun Hwi Cho, In Hye Kim, Jeong-Hwi Cho, Tae-Kyeong Lee, Jae-Chul Lee, Bich Na Shin, Young-Myeong Kim, Choong Hyun Lee, In Koo Hwang, Il Jun Kang, Bai Hui Chen, Bing Chun Yan, Young Joo Lee, Moo-Ho Won, Soo Young Choi PII: DOI: Reference:
S0531-5565(17)30584-3 https://doi.org/10.1016/j.exger.2018.01.015 EXG 10259
To appear in:
Experimental Gerontology
Received date: Revised date: Accepted date:
2 August 2017 5 January 2018 12 January 2018
Please cite this article as: Ji Hyeon Ahn, Joon Ha Park, Jinseu Park, Myoung Cheol Shin, Jun Hwi Cho, In Hye Kim, Jeong-Hwi Cho, Tae-Kyeong Lee, Jae-Chul Lee, Bich Na Shin, Young-Myeong Kim, Choong Hyun Lee, In Koo Hwang, Il Jun Kang, Bai Hui Chen, Bing Chun Yan, Young Joo Lee, Moo-Ho Won, Soo Young Choi , Long-term treadmill exercise improves memory impairment through restoration of decreased synaptic adhesion molecule 1/2/3 induced by transient cerebral ischemia in the aged gerbil hippocampus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Exg(2017), https://doi.org/10.1016/j.exger.2018.01.015
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Long-term treadmill exercise improves memory impairment through restoration of decreased synaptic adhesion molecule 1/2/3 induced by transient cerebral ischemia in the aged gerbil hippocampus
Ji Hyeon Ahn1, Joon Ha Park1, Jinseu Park1, Myoung Cheol Shin2, Jun Hwi Cho2, In Hye Kim3, Jeong-Hwi Cho3, Tae-Kyeong Lee3, Jae-Chul Lee3, Bich Na Shin4, Young-Myeong Kim5, Choong Hyun Lee6, In Koo
Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University,
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Hwang7, Il Jun Kang8, Bai Hui Chen9, Bing Chun Yan10, Young Joo Lee11, Moo-Ho Won3*, Soo Young Choi1*
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Chuncheon 24252, South Korea
Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 24341,
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South Korea
Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South
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Korea
Department of Physiology, College of Medicine, Hallym University, Chuncheon 24252, South Korea
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Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University,
Chuncheon 24341, South Korea
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Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan 31116, South Korea
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Department of Anatomy and Cell Biology, College of Veterinary Medicine, and Research Institute for
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Veterinary Science, Seoul National University, Seoul 08826, South Korea Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, South Korea
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Department of Histology and Embryology, Institute of Neuroscience, Wenzhou Medical University, Wenzhou,
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Zhejiang 325035, P.R. China 10
Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and
Treatment of Senile Diseases, Yangzhou 225001, People’s Republic of China 11
Department of Emergency Medicine, Seoul Hospital, College of Medicine, Sooncheonhyang University, Seoul
04401, South Korea.
- Co-firsts: Ji Hyeon Ahn1 and Joon Ha Park1 have contributed equally to this article. 1
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* Corresponding authors
Professor Moo-Ho Won, DVM, PhD: Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea. TEL: +82-33-250-8891; FAX: +82-33-256-1614. E-mail:
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[email protected]
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Professor Soo Young Choi, PhD: Department of Biomedical Science and Research Institute of Bioscience and
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Biotechnology, Hallym University, Chuncheon 24252, South Korea. TEL: +82-33-248-2112; FAX: +82-33-241-
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1463. E-mail: [email protected]
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Abstract
Exercise improves cognitive impairments induced by transient cerebral ischemia and modulates synaptic adhesion molecules. In this study, we investigated effects of long-term treadmill exercise on cognitive impairments and its relation to changes of synaptic cell adhesion molecule (SynCAM) 1/2/3 in the hippocampus
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after 5 min of transient cerebral ischemia in aged gerbils. Animals were assigned to sedentary and exercised groups, given treadmill exercise for 4 consecutive weeks from 5 days after transient ischemia and evaluated
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cognitive function through passive avoidance test and Morris water maze test. SynCAM 2 protein levels were
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determined in the hippocampus by western blot. In addition, neuronal and synaptic changes were examined by NeuN immunohistochemistry, and SynCAM 1/2/3 and MAP2 double immunofluorescence, respectively. We
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found that transient cerebral ischemia led to neuronal death in the CA1 area and dentate gyrus, and impaired conflictive function; however, treadmill exercise improved ischemia-induced memory impairment. In addition,
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SynCAM 1/2/3 expression in the hippocampus was significantly decreased in the sedentary group after transient cerebral ischemia; however, SynCAM 2 protein level was significantly increased in the ischemic group with exercise. These results suggest that long-term treadmill exercise improves memory impairment through the
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restoration of decreased SynCAM 1/2/3 expression in the hippocampus induced by transient cerebral ischemia
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in the aged gerbil.
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Treadmill exercise
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Keywords: Aging; Cerebral ischemia; Learning and memory; Rehabilitation; Synaptic adhesion molecules;
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1. Introduction
It is well known that transient cerebral ischemia (TCI) induces delayed neuronal death in pyramidal neurons in the CA1 area of the hippocampus proper (CA1-3 areas) about 4 days after 5 min of TCI, while pyramidal neurons in the CA3 area remain relatively intact (Kirino, 1982). Furthermore, the synapse is highly susceptible
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to TCI that affects structural and biochemical alterations in the damaged areas (Costain et al., 2008; Kirino, 1982; Martone et al., 1999), and the synaptic failure occurs even in the absence of neuronal damage after
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ischemic insults (Hofmeijer and van Putten, 2012). These aberrant synaptic changes could affect the function of
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synapses including synaptic signaling, transmission, and plasticity and stability. In this regard, neuronal and synapse alterations after ischemic insults are closely related to impaired neurological function (Dahlqvist et al.,
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2004).
Transsynaptic interaction is formed by a group of cell adhesion molecules (CAMs) that interact in a homo- or
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heterophilic fashion across the synaptic cleft as well as neurotransmitter release (Dalva et al., 2007). Interactions of CAMs can control functions of existing synapses or lead to the formation of new synapses via various signaling pathways (Dalva et al., 2007). In addition, CAMs not only modulate adhesion at synapses, but also
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regulate the recruitment of synaptic vesicles and neurotransmitters (Hoy et al., 2009). Among CAMs, synaptic
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CAMs (SynCAMs) 1-3 are four immunoglobulin proteins containing CAMs that are expressed predominantly at pre- and postsynaptic plasma membranes in the brain (Biederer et al., 2002), and are required for maintaining normal synapse numbers (Robbins et al., 2010).
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It is necessary to study mechanisms underlying effects of long-term exercise on stroke rehabilitation (Macko
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et al., 2015). It is well known that kinds and quality of exercises affects synaptic plasticity and gene expression in normal brain (Farmer et al., 2004; Molteni et al., 2002). Furthermore, physical exercise has been shown to help to recover and improve neurological functions in humans (Duncan et al., 2003; Macko et al., 2005) and animals (Ahn et al., 2016a; Sakakima et al., 2012) after stroke. The hippocampus is a major location of critical aspects of neuronal plasticity (Neves et al., 2008b) and dynamic changes of synapses in the hippocampus display an important role in learning and memory (Neves et al., 2008a). To the best of our knowledge, long-term (chronic) change in SynCAMs expression has not been reported in the aged hippocampus after cerebral ischemia. Therefore, we examined the effect of long-term 4
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treadmill exercise on memory function after TCI in the aged gerbil, which is a good animal for study on TCI (Kirino, 1982). In addition, we investigated whether changes in SynCAM 1/2/3 expressions were related with the improvement in TCI-induced memory impairment or not in the ischemic aged hippocampus.
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2. Materials and Methods
2.1. Experimental animals
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Aged male Mongolian gerbils (Meriones unguiculatus, 22 to 24 months) weighing 80 to 90 g were supplied
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by the Experimental Animal Center, Kangwon National University (Chuncheon, South Korea). Animal handling and care followed the guidelines of current international laws and policies (NIH Guide for the Care and Use of
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Laboratory Animals, The National Academies Press, 8th Ed., 2011) (Council, 2011) and experimental protocol was approved by Institutional Animal Care and Use Committee (IACUC) of Kangwon National University
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(approval no. KW-160802-1).
Animals were randomly divided into three groups (n = 63): 1) sham group (n = 21) had no ischemia operation and treadmill (TR) exercise after sham operation, 2) SD4 group (n = 21) had ischemia operation and sedentary
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(SD) for 4 weeks from 5 days post-ischemia, 3) TR4 group (n = 21) had ischemia operation and TR exercise for
2.2. Induction of TCI
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finished in the TR4 group.
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4 weeks from 5 days post-ischemia. All animals were sacrificed 31 days after TCI when TR exercise was
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As previously described (Lee et al., 2015), in brief, the animals were anesthetized with a mixture of 2.5 % isoflurane in 33 % oxygen and 67 % nitrous oxide. Blood flow to the brain was completely interrupted by a 5min occlusion of bilateral common carotid arteries, which was confirmed by observing the retinal central artery under an ophthalmoscope. The body (rectal) temperature was controlled under normothermia (37 ± 0.5 ºC) before, during and after the surgery. Sham operated animals were subjected to the same surgical procedure except that the common carotid arteries were not occluded.
2.3. TR exercise 5
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For TR exercise, running speed and duration was determined according to previous studies (Lee et al., 2003; Sim et al., 2005b; Sim et al., 2004) with modification. Briefly, animals were familiarized with the TR running on a motorized treadmill (Exer3/6, Columbus Instruments) for 15 min/day for 3 consecutive days. From 5 days after TCI, the animals were forced to run on motorized TR for 30 min/day and 5 days/week for 1 or 4 consecutive weeks. The TR exercise workload consisted of running at the speed 5 m/min for the first 5 min, 7
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m/min for the next 5 min, and then 10 m/min for the last 20 min with 0 degree inclination. Animals in the SD
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group were left on the TR for 30 min and were not made to run.
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2.4. Passive avoidance test
Passive avoidance test was done for memory impairment at 1 day before TCI, 5 and 31 days after TCI
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according to a previous study (Horisawa et al., 2011). In brief, the Gemini Avoidance System (GEM 392, San Diego Instruments), which consisted of two compartments (light and dark), was used. In the training session,
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animals were allowed to explore environments of A and B dark compartments for 2 min. Light was turned on only in the A compartment and animals were allowed to explore environments in the A and B compartments for 2 min. When animals entered the B-compartment, the floor was closed an inescapable foot-shock (0.3 mA for 3
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s). The test session was performed 20 min after the training session, namely, each animal was placed in the A
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compartment, the light was turned on, and the floor was opened. Latency time for each animal to enter the B compartment was recorded within 180 s.
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2.5. Morris water maze test
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Morris water maze test was performed for spatial memory from 22 to 31 days after TCI according to published methods (Corbett et al., 1992; Wiard et al., 1995). In short, 3 different visual cues were placed around the inside wall of the tank at a level that would be visible to the animals. Each animal was given 3 daily trials with a 5-min intertrial interval for 10 days and individually placed in the apparatus at one of 3 preselected locations and allowed 50 seconds to escape to the hidden platform. The animals not finding the platform after 50 seconds were guided to it by the experimenter. The animals were allowed to remain on the platform for 10 seconds and then returned to their holding cage until the next trial. The escape time of the gerbil to find the platform was recorded within 50 seconds (escape latency), and the whole process was monitored by a digital camera and a computer 6
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system.
2.6. Western blot analysis As previously described (Ahn et al., 2011), in brief, hippocampi of animals (n = 7 in each group) were dissected. After the tissues were homogenized and centrifuged, and the supernatants were subjected to western
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blot analysis. Rabbit anti-SynCAM 2 (1:1000, Synaptic system, Göttingen, Germany) was used as primary antibody. The result of western blot analysis was scanned, and densitometric analysis for the quantification of
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the bands was done using Image J 1.46 (National Institutes of Health), which was used to count relative optical
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density (ROD): A ratio of the ROD was calibrated as %, with sham group designated as 100 %.
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2.7. Immunohistochemistry
Immunohistochemistry was performed according to our published method (Lee et al., 2012). For tissue
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preparation, animals (n = 7 each group) were anesthetized with sodium pentobarbital (JW Pharm. Co., Ltd., Korea, 40 mg/kg, i.p) and perfused transcardially with 4% paraformaldehyde in solution buffer (PB, pH 7.4). The brain tissues were serially sectioned into 30-μm coronal sections in a cryostat (Leica, Wetzlar, Germany).
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For immunohistochemistry, mouse anti-neuronal nuclei (NeuN, a marker for neurons) (1:1000, Chemicon
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International, Temecula, CA) as primary antibodies, and biotinylated horse anti-mouse IgG (Vector, Burlingame, CA) and streptavidin peroxidase complex (1:200, Vector) as secondary antibodies. For the specificity of the immunostaining, a negative control was tested and resulted in the absence of immunoreactivity.
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For double immunofluorescence staining for SynCAM 1/2/3 and MAP2 (a maker for dendrites), sections
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were incubated with the mixture of rabbit anti-SynCAM 1/2/3 (1:100, Synaptic system, Göttingen, Germany) and mouse anti-MAP2 (1:400, Chemicon International) and incubated in a mixture of both Cy3-conjugated goat anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA) and FITC-conjugated goat anti-mouse IgG (1:200, Jackson ImmunoResearch). The immunoreactions were observed under the confocal MS (LSM510 META NLO, Carl Zeiss, Germany). In order to quantitatively analyze NeuN-immunoreactive neurons, digital images from 6 sections per animal were taken using AxioM1 light microscope (Carl Zeiss, Germany) equipped with digital camera (Axiocam, Carl Zeiss, Germany) connected to a PC monitor. NeuN-immunoreactive neurons were counted in a 250 × 250 μm 7
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square of the stratum pyramidale in the CA1 area and polymorphic layer of the dentate gyrus using an image analyzing system (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ). Cell counts were obtained by averaging the counts from each animal. To measure fluorescence intensities of SynCAM 1/2/3 and MAP2 immunoreactions, digital images from 6 sections per animal were captured with a confocal MS (LSM510 META NLO, Carl Zeiss, Germany). The fluorescence intensities of SynCAM 1/2/3 and MAP2 immunoreactive
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structures were analyzed using Image J 1.46 software (National Institutes of Health, Bethesda, MD). The mean fluorescence intensity of the sham group was designated as 100%, and the relative fluorescence intensity of each
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group was calibrated and expressed as % of the sham group.
2.8. Statistical analysis
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Data are expressed as the mean ± SEM. The latency of the short-term memory ability was analyzed with a two-way repeated ANOVA, followed by post hoc Bonferroni-Dunn Test. Mean numbers and ROD of
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immunoreactive structures were analyzed with a one-way ANOVA, followed by post hoc Bonferroni-Dunn Test. All comparisons were tested using SPSS 17.0 software (IBM, New York, USA). Statistical significance was
3. Result
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3.1. Passive avoidance test
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considered at P < 0.05.
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Firstly, short-term memory ability was assessed between the groups at the same point in time using passive avoidance test (Fig. 1A). At 5 days after ischemia-reperfusion, the latency was significantly decreased in the SD4 and TR4 groups compared with the sham group. Thirty-one days after ischemia-reperfusion, treadmill exercise significantly increased the latency in the TR4 group compared with the SD4 group, although the latency was significantly different from that in the sham group (Fig. 1A). Secondly, short-term memory ability was examined according to times after ischemia in each group using passive avoidance test (Fig. 1B). In the sham group, the latency was not significantly changed with time. On the other hand, the latency in the SD4 group was significantly decreased 5 days after ischemia-reperfusion and the 8
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decreased memory function was maintained until 31 days after ischemia-reperfusion. However, in the TR4 group, the decreased latency at 5 days post-ischemia (by 76.4% compared to that at 1 day before ischemia) was significantly increased with time and recovered by 39.8% at 31 days post-ischemia compared to that at 5 days post-ischemia.
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3.2. Morris water maze test At 22 days after ischemia-reperfusion, escape latency was not significantly different among the sham, SD4,
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and TR4 groups. Escape latency at 31 days after ischemia-reperfusion was significantly reduced compared to
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the first trial in the sham, SD4 and TR4 groups. However, no significant differences were found in the escape latency in Morris water maze test among the groups, although the escape latency was slower in the SD4 group
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compared to that in the sham and TR4 groups (Fig. 1C).
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3.3. SynCAM 2 protein level
In the SD4 group, SynCAM 2 protein level in the hippocampus was significantly decreased compared with that in the sham group; however, SynCAM 2 protein level in the TR4 group was significantly increased (P <
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0.05) compared with that in the SD4 group (Fig. 2)
3.4. NeuN-immunoreactive neurons
NeuN-immunoreactive neurons were shown in the stratum pyramidale of the hippocampus proper (CA1-3
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areas) and in the granule cell layer of the hippocampal dentate gyrus in the sham group (Fig. 3A1-A4). Thirty-
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one days after ischemia-reperfusion, the mean number of NeuN-immunoreactive neurons in the SD4 group was significantly decreased (P < 0.05) in the stratum pyramidale of the CA1 area and in the polymorphic layer of the dentate gyrus (DG), but not in the other subregions (Fig. 3B1-B4 and 3D). At this point in time, the distribution pattern of NeuN-immunoreactive neurons in the hippocampus of the TR4 group was similar to that in the SD4 group (Fig. 3C1-C4 and 3D).
3.5. SynCAM 1/2/3 and MAP2 immunoreactivities 3.5.1. CA1 area 9
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In the sham group, SynCAM 1/2/3 immunoreactivity was highly enriched in MAP2-immunoreactive pyramidal cell membranes in the stratum pyramidale and in cytoplasmic processes, which were dendrites and axons, in strata oriens and radiatum (Fig 4A1-A3). Thirty-one days after ischemia-reperfusion, the density of SynCAM 1/2/3-immunoreactive structures was not found in the pyramidal cell membranes due to the death of the CA1 pyramidal cells and markedly decreased in strata oriens and radiatum in the SD4 group compared with the sham
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group (Fig. 4B1-B3 and 4D). However, the TR4 group, the density of SynCAM 1/2/3-immunoreactive structures was significantly increased in strata oriens and radiatum compared with the SD4 group, in addition,
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the density of MAP2-immunoreactive structures was increased in all layers in this group (Fig. 4C1-C3 and 4D).
3.5.2. CA2/3 area
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In the sham group, the distribution pattern of the SynCAM 1/2/3- and MAP2-immunoreactive structures in the CA3 area was similar to that in the CA1 area; in this area, SynCAM 1/2/3-immunoreactive processes were
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abundant in the stratum lucidum which contains the mossy fiber pathway projecting from the dentate gyrus (Fig. 5A1-A3). In the SD4 group, SynCAM 1/2/3- and MAP2-immunoreactive structures in the stratum pyramidale were similar to those in the sham group; however, in strata lucidum and radiatum, SynCAM 1/2/3-
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immunoreactive structures were markedly decreased, and MAP2-immunoreactive processes were decreased
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compared with the sham group (Fig. 5B1-B3 and 5D). In the TR4 group, densities of SynCAM 1/2/3- and MAP2-immunoreactive structures were significantly increased in strata lucidum and radiatum compared with
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3.5.3. Dentate gyrus
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the SD4 group (Fig. 5C1-C3 and 5D).
In the sham group, SynCAM 1/2/3 immunoreactivity was easily detected in MAP2-immunoreactive granule cell membranes in the granule cell layer and in processes in the molecular layer and polymorphic layer (Fig 6A1A3). At 31 days after ischemia-reperfusion, the density of SynCAM 1/2/3-immunoreactive structures was markedly decreased in the molecular and polymorphic cell layers in the SD4 group compared with the sham group, although MAP2 immunoreactivity was decreased only in the polymorphic cell layer (Fig. 6B1-B3 and 6D). In the TR4 group, the density of SynCAM 1/2/3-immunoreactive structures was increased in the molecular and polymorphic cell layers compared with the SD4 group, in addition, the density of the MAP210
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immunoreactive structures in the polymorphic layer was increased (Fig. 6C1-C3 and 6D).
4. Discussion
In the present study, we investigated effects of long-term TR exercise on cognitive function using passive
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avoidance test and Morris water maze test, and the relation of SynCAM 1/2/3 expression in the hippocampus to memory using western blot analysis and immunohistochemistry in the aged gerbil following 5 min of transient
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cerebral ischemia.
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Short-term learning and memory through passive avoidance test was significantly decreased after transient cerebral ischemia; however, 4-week TR exercise significantly improved learning and memory impairment 31
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days after ischemia-reperfusion compared with that in the sedentary group. This result is consistent with previous studies (Ahn et al., 2016a; Sim et al., 2005a) that reflect that long-term exercise improves ischemia-
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induced short-term memory impairment during chronic stage. On the other hand, spatial learning and memory assessed by Morris water maze test was not significantly impaired 31 days after ischemia-reperfusion. This result is in line with previous studies that show that deficits in spatial learning and memory appear to be
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transient during subacute stage (about 3 days) after 5 min of transient cerebral ischemia, although spatial
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learning and memory impairs after 10 or 15 min of transient cerebral ischemia which leads to damage/death in pyramidal neurons in the CA2/3 area that is resistant to 5 min of transient cerebral ischemia in the gerbil (Corbett et al., 1992; Wiard et al., 1995). These results indicate that spatial learning and memory is closely
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associated with severer neuronal damage in the hippocampus.
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We, in this study, found that neuronal death occurred in neurons in the stratum pyramidale of the CA1 area (Lee et al., 2010) and in the polymorphic layer of the dentate gyrus (Ahn et al., 2016b) after 5 min of transient cerebral ischemia and that numbers of neurons in the CA1 area and dentate gyrus observed at 31 days postischemia was not increased in the TR4 group, which was given long-term therapeutic TR exercise. This finding indicates that long-term exercise after neuronal damage/death could not affect neuronal population in the ischemic hippocampus although long-term exercise could improve learning and memory impairment induced by ischemic insults.
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It has been reported that TR exercise increases spatial memory via increasing synapse related protein, such as synapsin-1 and postsynaptic density protein-95 levels after ischemic insults (Shih et al., 2013). In the present study, we found that SynCAM 1/2/3-immunoreactive structures were enriched in pre- and post-synaptic sites in the hippocampus (Biederer et al., 2002; Robbins et al., 2010) and that ischemia-induced decreased SynCAM 1/2/3 immunoreactivity was significantly improved in the TR group, although SynCAM 1/2/3-immunoreactive
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structures were significantly decreased in all hippocampal subregions in the SD group, interestingly, including the CA3 area where neuronal loss was not observed after ischemia-reperfusion. Robbins et al. (2010) reporetd
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that the overexpression of SynCAM 1 in adult mouse brain improved spatial learning using Morris water maze
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task (Robbins et al., 2010). It has been demonstrated that SynCAMs directly impacts neuronal plasticity through restricting long-term depression which results in the weakening of synaptic connection (Fogel et al., 2007;
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Robbins et al., 2010) and that the knockdown of SynCAM 1 induces a reduction of post-synaptic density length, but the overexpression of SynCAM 1 increases the number of functional excitatory synapses in the
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hippocampus of a transgenic mouse model (Robbins et al., 2010). For the role of SynCAM 1, Jennifer et al. (2009) have suggested that SynCAM 1 might be associated with postsynaptic specialization via recruiting both NMDA- and AMPA-type glutamate receptors to sites of synaptic adhesion (Hoy et al., 2009). Based on the
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above-mentioned and our present findings, it is likely that chronic exercise could promote synapse formation
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and improve synaptic plasticity via increasing SynCAM 1/2/3 expression in ischemic hippocampus.
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5. Conclusions
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In conclusion, this study shows that 5 min of transient cerebral ischemia resulted in severe damages in shortterm memory and SynCAM 1/2/3 immunoreactive structures in the aged gerbil hippocampus; however, longterm TR exercise improved ischemic-induced short-term memory impairment by restoring SynCAMs. These results suggest that long-term TR exercise could be an effective treatment for the restoration of synaptic plasticity during chronic stage after ischemic insults in the elderly.
Acknowledgments
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This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT & Future Planning (NRF-2014R1A1A3051721), by the BioSynergy Research Project (NRF-2015M3A9C4076322) of the Ministry of Science, ICT and Future Planning through the National Research Foundation, by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9B6066835), and by a Priority Research Centers
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Program grant (NRF-2009-0093812) through the National Research Foundation of Korea funded by the
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Ministry of Science, ICT and Future Planning.
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The authors declare that they have no conflict of interest.
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Conflict of interests
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Figure legends
Fig. 1. A and B: The latency of the passive avoidance test in the sham, SD4 and TR4 groups: “A” is assessment between the groups at each point in time, and “B” is assessment according to time in each group (n = 7 per group; *P< 0.05, significantly different from the sham group, †P < 0.05, significantly different from the SD4
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group). C: The escape latency of the Morris Water Maze test in the sham, SD4 and TR4 groups. Data are defined
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as the means ± SEM.
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Fig. 2. Western blot analysis of SynCAM 2 in the hippocampus of the sham, SD4 and TR4 groups (n = 7 per group; *P < 0.05, significantly different from the sham group, †P < 0.05, significantly different from the SD4
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group). The bars indicate the means ± SEM.
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Fig. 3. NeuN immunohistochemistry in the sham (A), SD4 (B) and TR4 (C) groups. In the SD4 and TR4 groups, the mean number of NeuN-immunoreactive neurons is significantly decreased in the stratum pyramidale (SP) of the CA1 region and in the polymorphic layer (PoL) of the dentate gyrus (DG). GCL, granule cell layer; MoL,
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molecular layer; SO, stratum oriens; SR, stratum radiatum. Scale bars = 400 μm (A1-C1), 40 μm (A2-C2), and
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100 μm (A3-C4). F: The mean number of NeuN-immunoreactive neurons in the sham, SD4 and TR4 groups (n = 7 per group; *P < 0.05, significantly different from the sham group). The bars indicate the means ± SEM.
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Fig. 4. Double immunofluorescence staining for SynCAM 1/2/3, MAP2 and merged images in the CA1 area of
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the sham (A), SD4 (B) and TR4 (C) groups. SynCAM 1/2/3- and MAP2-immunoreactive structures are markedly decreased in strata oriens (SO) and radiatum (SR) in the SD4 group. However, SynCAM 1/2/3- and MAP2-immunoreactive structures are increased in the TR4 group. SP; stratum pyramidale. Scale bar = 40 μm. D: Relative immunofluorescence intensity expressed as a percentage of SynCAM 1/2/3- and MAP2immunoreactive structures (n = 7 per group; *P < 0.05, significantly different from the sham group and †P < 0.05, significantly different from the SD4 group). The bars indicate the means ± SEM.
Fig. 5. Double immunofluorescence staining for SynCAM 1/2/3, MAP2 and merged images in the CA3 area of 18
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the sham (A), SD4 (B) and TR4 (C) groups. In the SD4 group, SynCAM 1/2/3- and MAP2- immunoreactive structures are decreased in strata radiatum (SR) and lucidum (SL); however, in the TR4 group, SynCAM 1/2/3and MAP2-immunoreactive structures are increased in the layers. SP; stratum pyramidale. Scale bar = 40 μm. D: Relative immunofluorescence intensity expressed as a percentage of SynCAM 1/2/3- and MAP2immunoreactive structures (n = 7 per group; *P < 0.05, significantly different from the sham group and †P < 0.05,
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significantly different from the SD4 group). The bars indicate the means ± SEM.
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Fig. 6. Double immunofluorescence staining for SynCAM 1/2/3, MAP2 and merged images in the dentate gyrus
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of the sham (A), SD4 (B) and TR4 (C) groups. In the SD4 group, the density of SynCAM 1/2/3-immunoreactive structures is decreased in the molecular (MoL) and polymorphic (PoL) layers compared with the sham group;
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however, in the TR4 group, its density is increased compared with the SD4 group. GCL, granule cell layer. Scale bar = 40 μm. D: Relative immunofluorescence intensity expressed as a percentage of SynCAM 1/2/3- and
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P < 0.05, significantly different from the SD4 group). The bars indicate the means ± SEM.
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†
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MAP2-immunoreactive structures (n = 7 per group; *P < 0.05, significantly different from the sham group and
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Highlights
* Memory was impaired following transient cerebral ischemia
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* Long-term treadmill exercise improved ischemia-induced memory impairment.
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* Transient cerebral ischemia induced neuronal loss in the CA1 area of the hippocampus.
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* Transient cerebral ischemia induced SynCAM reduction in the all hippocampal subregions.
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*Treadmill exercise restored ischemia-induced memory deficits by improving SynCAM
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Figure 1
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