Post-traumatic stress disorder (PTSD) is a condition which occurs after a person has experienced unusual stress. The neurons in the hippocampus are especially vulnerable to the PTSD. In the present study, the effect of treadmill exercise on spatial learning memory and cell proliferation in the hippocampus of rats with PTSD. Radial 8-arm maze test and immunohistochemistr for 5-bromo-2′-deoxyridine (BrdU) and double-cortin (DCX) were conducted for this experiment. For the inducing PTSD, the rats were exposure to 0.2 mA electric foot shock for 7 consecutive days. Electric foot shock continued 6 seconds, repeated 10 times with a 30 sec interval per one trial, and repeated 3 trials per day. The rats in the exercise groups were forced to run on a motorized treadmill for 30 min once a day for 4 weeks, stating one day after finishing last electric food shock. Presently, the PTSD rats showed longer time of successful performance, higher error number, and lower correct number in the radial-8-arm maze test. Cell proliferation and DCX expression in the hippocampal dentate gyrus were suppressed in the PTSD rats. In contrast, treadmill exercise alleviated PTSD-induced impairment of spatial learning memory. The rats performed treadmill exercise showed longer time of successful performance, higher error number, and lower correct number in the radial-8-arm maze test. Treadmill exercise also enhanced cell proliferation and DCX expression in the hippocampal dentate gyrus of PTSD rats. The present study demonstrated that treadmill exercise ameliorated PTSD-induced memory impairment through enhancing cell proliferation in the hippocampus.
Post-traumatic stress disorder (PTSD) is a condition which occurs after a person has experienced unusual stress. People with PTSD become very anxious whenever reminded of the incident which has caused the initial distress. Frequently they have nightmares or become fearful, depressed and irritable and function less well at work or in social situations (
The hippocampus plays an important role in learning ability and memory capability (
The beneficial effects of regular exercise on brain function and plasticity have been observed in many studies. Regular exercise attenuates motor deficits (
Enhancing effect of exercise on neurogenesis has been well documented, however, the effect of treadmill exercise on PTSD-related neurogenesis has not been elucidated. In the present study, we investigated the effects of treadmill exercise on spatial learning memory and cell proliferation in the hippocampus of rats with PTSD. Radial 8-arm maze test and immunohistochemistr for 5-bromo-2′-deoxyridine (BrdU) and DCX were conducted for this experiment.
The experimental procedures were performed in accordance with the animal care guidelines of the National Institutes of Health and the Korean Academy of Medical Sciences. Male Sprague-Dawley rats, weighing 102.5±10 g (5 weeks old), were used in this experiment (Orient Co., Seoul, Korea). Each animal was housed under controlled temperature (20±2°C) and lighting (07:00–19:00 h) conditions with food and water made available
In order to induce PTSD in rats, the rats were exposure to the repeated inescapable electric foot shock, according to the previously described method (
The rats in the exercise groups were forced to run on a motorized treadmill for 30 min once a day for 4 weeks, stating one day after finishing last electric food shock. The exercise load consisted of running at a speed of 5 meters/min for the first 5 min, 8 meters/min for the next 5 min, and 15 meters/min for the last 20 min, with a 0° inclination. The rats in the control group and in the PTSD-induced group were left on the treadmill without running for the same period as the exercise groups. BrdU (50 mg/kg; Sigma Chemical Co., St. Louis, MO, USA) was given intraperitoneally to all animals 1 h before the starting of treadmill running once a day for 3 consecutive days from the starting of treadmill exercise.
Spatial learning memory was evaluated using the radial 8-arm maze apparatus, as the previously described method (
The animals were sacrificed immediately after finishing radial 8-arm maze test. The animals were anesthetized using Zoletil 50® (10 mg/kg, i.p.; Vibac Laboratories, Carros, France), transcardially perfused with 50 mM phosphate-buffered saline (PBS), and fixed with a freshly prepared solution consisting of 4% paraformaldehyde in 100 mM phosphate buffer (PB, pH 7.4). The brains were dissected and postfixed in the same fixative overnight and transferred into a 30% sucrose solution for cryoprotection. Coronal sections of 40 μm thickness were made with a freezing microtome (Leica, Nussloch, Germany).
BrdU-specific immunohistochemistry was performed, according to the previously described method (
After BrdU-specific staining, differentiation of BrdU-positive cells was determined on the same section using a mouse anti-neuronal nucleic (NeuN) antibody (1:300; Chemicon International, Temecula, CA, USA). The sections were washed three times with PBS and incubated for l h with a biotinylated anti-mouse secondary antibody. For staining, the sections were incubated in a reaction mixture consisting of 0.02% DAB and 0.03% hydrogen peroxide for 5 min. The sections were finally mounted onto gelatin-coated slides. The slides were air dried overnight at room temperature, and coverslips were mounted with Permount®.
For visualization of the DCX expression, DCX immunohistochemistry was performed, as the previously described method (
The numbers of BrdU-positive and DCX-positive cells in the hippocampal dentate gyrus were counted using Image-Pro® Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA) attached to a light microscope (Olympus, Tokyo, Japan). The numbers of BrdU-positive and DCX-positive cells were expressed as the numbers of cells per mm2 of cross-sectional area of hippocampal dentate gyrus. Statistical analysis was performed using the one-way ANOVA followed by Duncan’s post-hoc test, and the results are expressed as the mean± standard error of the mean (SEM). Significances were accepted to be present at
Spatial learning memory was evaluated using the radial-8-arm maze test. Time spent for completed eight successful performances was 134.90±11.25 sec in the control group, 106.70±6.26 sec in the control and exercise group, 250.60±22.56 sec in the PTSD-induced group, and 171.90±14.54 sec in the PTSD-induced and exercise group (
The number of correct choices before the first error was 7.50± 0.22 in the control group, 7.70±0.15 in the control and exercise group, 4.60±0.34 in the PTSD-induced group, and 7.00±0.21 in the PTSD-induced and exercise group (
The number of errors made before eight successful performances was 1.20±0.53 in the control group, 0.70±1.16 in the control and exercise group, 11.30±1.34 in the PTSD-induced group, and 2.50±0.54 in the PTSD-induced and exercise group (
The rats in the PTSD-induced group showed longer time for the successful performances, lower number of correct choices, and higher number of errors compared to the rats in the control group. However, treadmill exercise reduced time of the successful performance, increased correct number, and reduced error number in the rats with PTSD.
Photomicrographs of BrdU-positive cells in the hippocampal dentate gyrus are presented in
The present results show that inducing of PTSD decreased cell proliferation in the hippocampal dentate gyrus and that treadmill exercise increased cell proliferation in the rats with PTSD.
Photomicrographs of DCX-positive cells in the hippocampal dentate gyrus are presented in
The present results show that inducing of PTSD decreased DCX expression in the hippocampal dentate gyrus and that treadmill exercise significantly increased DCX expression in the rats with PTSD.
PTSD is induced by exposure to the severe traumatic events that occur during war, after violent personal assault, and following natural disasters. Inescapable electric foot shock also induces behavioral and biological changes similar to PTSD (
Atrophy of the hippocampus was observed who experienced severe, long-lasting traumatic stress (
Exercise is known to ameliorate memory impairment and exerts protective effects against various neuropsychiatric diseases (
Exercise is one of the best-known stimulators of adult hippocampal neurogenesis (
The present study demonstrated that treadmill exercise ameliorated PTSD-induced memory impairment through enhancing cell proliferation in the hippocampus. Based on the present result, treadmill exercise may be useful strategy for the symptom relief on stress-induced neurpsychiatric disorders including PTSD.
This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012S1 A5A8022111).
No potential conflict of interest relevant to this article was reported.
The effect of treadmill exercise on spatial learning memory. (Upper) The time for eight successful performances. (Middle) The number of correct choices made before the first error. (Lower) The number of errors made before eight successful performances. (A) Control group, (B) control and exercise group, (C) PTSD-induced group, (D) PTSD-induced and exercise group. The data are represented as the mean± standard error of the mean (SEM). *Represents
Effect of exercise on cell proliferation in the hippocampus dentate gyrus. Upper: Photomicrographs of 5-bromo-2′-deoxyridine (BrdU)-positive cells. The scale bar represents 50 μm. Lower: The number of BrdU-positive cells. (A) Control group, (B) control and exercise group, (C) PTSD-induced group, (D) PTSD-induced and exercise group. The data are represented as the mean±standard error of the mean (SEM). *Represents
Effect of exercise on doublecortin (DCX) expression in the hippocampal dentate gyrus. Upper: Photomicrographs of DCX-positive cells. The scale bar represents 50 μm. Lower: The number of DCX-positive cells. (A) Control group, (B) control and exercise group, (C) PTSD-induced group, (D) PTSD-induced and exercise group. The data are represented as the mean± standard error of the mean (SEM). *Represents