The purpose of this study was to examine whether acupuncture improves spatial cognitive impairment induced by repeated corticosterone (CORT) administration in rats. The effect of acupuncture on the acetylcholinergic system was also investigated in the hippocampus. Male rats were subcutaneously injected with CORT (5 mg/kg) once daily for 21 days. Acupuncture stimulation was performed at the HT7 (Sinmun) acupoint for 5 min before CORT injection. HT7 acupoint is located at the end of transverse crease of ulnar wrist of forepaw. In CORT-treated rats, reduced spatial cognitive function was associated with significant increases in plasma CORT level (+36%) and hippocampal CORT level (+204%) compared with saline-treated rats. Acupuncture stimulation improved the escape latency for finding the platform in the Morris water maze. Consistently, the acupuncture significantly alleviated memory-associated decreases in cholinergic immunoreactivity and mRNA expression of BDNF and CREB in the hippocampus. These findings demonstrate that stimulation of HT7 acupoint produced significant neuroprotective activity against the neuronal impairment and memory dysfunction.
Acupuncture has long been known to modulate the biochemical balances in the central nervous system (CNS) and to maintain homeostasis [
Chronic stress causes dysregulation of the HPA axis in the neuroendocrine system, as evidenced by observations that the elevation of circulating CORT levels disrupts circadian regulation of CORT secretion as well as the glucocorticoid (GC) receptor-negative feedback circuit [
The mammalian hippocampus has the highest density of GC receptors and participates in GC-mediated negative feedback of the HPA axis [
The aim of the present study was to explore the efficacy of acupuncture therapy for healing chronic CORT-induced spatial memory impairment in an animal model using behavioral and neurobiological methodologies. To this end, acupuncture stimulation of HT7 (Sinmun) was evaluated for its efficacy in alleviating spatial learning and memory deficits in rats repeatedly exposed to exogenous CORT using the Morris water maze (MWM) test, and its underlying mechanism was elucidated by analyzing cholinergic markers in the hippocampus.
Adult male Sprague-Dawley (SD) rats weighing 260–280 g were obtained from Sam taco Animal Co. (Seoul, Korea). The rats were housed in a limited-access rodent facility with up to five rats per polycarbonate cage. The room controls were set to maintain the temperature at 22°C ± 2°C and the relative humidity at
This study was designed to compare the CORT concentration in rat blood and brain tissue, immediately after CORT injection or immobilization stress for 21 days. The rats were randomly divided into three groups of four individuals each as follows: nontreated normal group (NOR group,
Also, the other experiment was designed to compare the effects of acupuncture stimulation to enhanced CORT concentration in the blood of chronic CORT-injected rats for 21 days. The rats were randomly divided into five groups of four individuals each as follows: vehicle saline-injected group, instead of CORT (0.9% NaCl, s.c., CON group,
This study was designed to explore the efficacy of acupuncture therapy for healing chronic CORT-induced spatial memory impairment in an animal model using behavioral and neurobiological methodologies. The rats were randomly assigned to divided into five groups of seven individuals each as follows; vehicle saline-injected group (CON group,
The experimental schedule of CORT injection, acupuncture treatment, behavioral test, and tissue and blood sampling are shown in Figure
Experimental schedules of corticosterone-induced impairment of spatial memory in the rat. Black arrows indicate acupuncture treatments. Experiment 1 was designed to compare the CORT concentration in rat blood and brain tissue, immediately after CORT injection or immobilization stress for 21 days. It also was designed to compare the effects of acupuncture stimulation to enhanced CORT concentration in the blood of chronic CORT-injected rats for 21 days. Experiment 2 was designed to explore the efficacy of acupuncture therapy for healing chronic CORT-induced spatial memory impairment in an animal model using behavioral and neurobiological methodologies. IHC; Immunohistochemistry.
Acupuncture stimulation was bilaterally performed every second day for 5 min before the CORT injection during the CORT-injection period. The acupoint on tail and the Waiguan acupoint were selected as a nonacupoint and a comparison acupoint, respectively. The acupuncture stimulation was performed as previously described [
After immobilization stress or CORT injection for 21 days, CORT concentration in blood and brain tissues was determined. For this, the unanesthetized rats were rapidly decapitated, and blood was quickly collected via the abdominal aorta. The hippocampus was rapidly removed from the rat brains in randomized order. Special care was taken to avoid predecapitation stress—while rats were rapidly decapitated, the other animals were left outside the room and handled for a few minutes prior to sampling. The blood and tissue samples were stored at −80°C until use. Hippocampus were homogenized in a lysis buffer containing 137 mM NaCl, 20 mM Tris (pH 8.0), 1% NP40, 10% glycerol, 1 mM PMSF, 10 mg/mL aprotinin, 1 mg/mL leupeptin and 0.5 mM sodium vanadate. Homogenization was carried out on ice using a tissue homogenizer and incubated for 1 min at 4°C with shaking. Homogenates were centrifuged and supernatants were collected. Protein concentrations were estimated by the procedure of Lowry et al. [
The MWM test was performed using a polypropylene circular pool (painted white internally, 2.0 m in diameter and 0.35 m high). The pool contained water maintained at a temperature of
The MWM task was performed on 22st day after the CORT injection and acupuncture treatment were commenced. The animals received three trials per day. The rats were trained to find the hidden platform, which remained in a fixed location throughout the test. The trials lasted for a maximum of 180 s, and the time it took to find the submerged platform was recorded each time. The animals were tested in this way three per day for 6 consecutive days, and they received a 60-s probe trial on the seventh day. Finding the platform was defined as staying on it for at least 4 s before the acquisition time of 180 s ended. If the rat failed to find the platform in the allotted time, it was placed onto the platform for 20 s and assigned a latency of 180 s. Between one trail and the next, water was stirred to erase olfactory traces of previous swim patterns. The entire procedure took seven consecutive days, and each animal had three training trials per day, with a 30- to 40-min intertrial interval.
The cued trial (three trials per rat) was performed on the first day (on 22st day) to assess the rats’ motivation to escape from the water and to evaluate their sensor-motor integrity. The platform was placed in the north quadrant and had a visible black cue. The animal was placed in the pool and given 90 s to reach the platform, which was identified by a visible black platform above the surface of the water. Latency to reach the visible platform was automatically calculated.
For the probe trial, each rat was placed into the water diagonally from the target quadrant (north), and for 60 s, was allowed to search the water, from which the platform had been removed. The time (% for total time) spent searching for the platform in the former platform quadrant (north) and in the other three quadrants was measured for each rat.
Prior to water maze testing, the rats were individually housed in a rectangular container made of dark polyethylene (
For immunohistochemical studies, four rats in each groups were deeply anesthetized with sodium pentobarbital (80 mg/kg, by intraperitoneal injection) and perfused through the ascending aorta with normal saline (0.9%) followed by 300 mL (per rat) of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The brains were removed, postfixed overnight, and cryoprotected with 20% sucrose in 0.1 M PBS at 4°C. Coronal sections 30
For AchE histochemistry, the sections were washed in PBS and incubated in a solution with 25 mg of acetylthiocholine iodine for 1 h. The solution was composed of 32.5 mL of 0.1 M sodium hydrogen phosphate buffer (NaH2PO4·H2O, pH 6.0), 2.5 mL of 0.1 M sodium citrate, 5 mL of 30 mM copper sulfate, 5 mL of 5 mM potassium ferricyanide, and 5 mL of distilled water. The color of the mixing solution was green. The densities of stained nuclei of the hippocampal cells were measured using a Scion image program (Scion Co., Frederick, Md, USA). The sections were viewed at 200x
The hippocampus from three rats in each group was isolated. After decapitation, the brain was quickly removed and stored at −80°C until use. Total RNA was isolated from the brain samples using TRIzol reagent (Invitrogen Co., Carlsbad, Calif, USA) and was used to extract RNA according to the supplier’s instruction. Complementary DNA was synthesized from total RNA with reverse transcriptase (Takara Co., Shiga, Japan). Brain-derived neurotrophic factor (BDNF) and cAMP-response element-binding protein (CREB) mRNA expression levels were determined by the reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR was performed using a PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, Mass, USA). The operating conditions were as follows: for glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 30 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 30 sec; for BDNF, 27 cycles of denaturation at 95°C for 30 sec, annealing at 57°C for 30 sec, and extension at 72°C for 30 sec; for CREB, 27 cycles of denaturation at 95°C for 30 sec, annealing at 51°C for 30 sec, and extension at 72°C for 30 sec. All primers were designed using published mRNA sequences and primer design software (Primer 3; The Whitehead Institute for Biomedical Research, Cambridge, Mass, USA;
All measurements were performed by an independent investigator blinded to the experimental conditions. Results in figures are expressed as mean ± standard error of means (SE). Differences within or between normally distributed data were analyzed by analysis of variance (ANOVA) using SPSS (Version 13.0; SPSS, Inc., Chicago, Ill, USA) followed by Tukey’s
For statistical analysis of water maze data, the effect of training on the acquisition of the water escape task was assessed using a one-way ANOVA with a repeated-measure factor of sessions (number of days) followed by the appropriate Tukey’s
The ELISA analysis demonstrated that restraint-stress exposure for 21 days and CORT administration for 21 days significantly increased the plasma CORT concentration in the rats by 19.2% (
Corticosterone (CORT) concentrations in rat plasma and hippocampus, immediately after CORT injection or immobilization stress for 21 days. The experimental groups were pretreated with nontreated normal (NOR,
Also, CORT concentration differed among the five groups after acupuncture stimulation [
Effect of acupuncture on plasma CORT concentrations induced by repeated CORT injection in the rats. The experimental groups were pretreated with vehicle saline-injected group, instead of CORT (0.9% NaCl, s.c., CON group,
To exclude the possibility of impairing the animals’ vision and changing the motivation to escape the water due to the acupuncture stimulation, a cued version of the MWM test was performed, and the swimming time to reach the visible platform was measured as illustrated in Figure
Time to escape (latency) during acquisition trials of visible platform (a), hidden platform (b), probe trial (c), and swim speed (d), during the Morris water maze test. The experimental group were pretreated with vehicle saline, instead of CORT (0.9% NaCl, s.c., CON group,
The rats in the CON group rapidly learned the location of the submerged hidden platform and reached it within 20 s on day 6 of the trials (Figure
To examine the spatial memory of rats, we analyzed their performance in the probe test on day 7 by comparing the percentage of time spent swimming to the expected position of the platform (Figure
In an analysis of open-field test results by a parametric one-way ANOVA, no significant differences were observed between groups in terms of memory deficit-related locomotor activity or the total number of line crossings in the open-field test (Figure
Activity counts of locomotor activity (a) and the total number of line crossings (b) in the open-field test.
The possibility that the deficits in septal-hippocampus-dependent learning and memory of the chronic exposure to CORT are associated with cholinergic deficits was examined histologically following completion of the behavioral tasks using ChAT immunohistochemistry. The number of septal-hippocampal cholinergic neurons immunohistochemically stained for ChAT was counted in the septohippocampal fibers, represented by medial septum or hippocampus. The results of number of ChAT-stained septohippocampal cholinergic neurons are shown in (Figure
Representative photographs showing the distribution of choline acetyltransferase (ChAT) immunostaining cells in medial septum and acetylcholinesterase (AchE) reactive cells in hippocampus of CORT-induced memory impairment or CORT-injected and Sinmun (HT7) acupoint-stimulated rats. Lower magnification of the small box in panel (d) and high magnification of the big box in panel (g and j) of the same fields of AchE- stained nuclei of the hippocampus. Scale bar represents 100
The percentage (±SE) values of the mean number of choline acetyltransferase- (ChAT-) stained septohippocampal cholinergic neurons after the Morris water maze task. Immunohistochemical data were analyzed via a separate one-way ANOVA followed by Tukey’s
The density of AchE-immunopositive fibers in the CA1 area of the rat hippocampus was significantly reduced by repeated injections of CORT in the CORT group compared with the saline-injected vehicle group (CON group) (Figure
The percentage (±SE) values of the immune-staining density of acetylcholinesterase in different hippocampal areas after the Morris water maze task. Immunohistochemical data were analyzed via a separate one-way ANOVA followed by Tukey’s
The density of AchE fiber in the CA3 region of the hippocampus was also markedly reduced by repeated injections of CORT in the CORT group compared with the CON group (Figure
The effect of acupuncture stimulation to the HT7 acupoint on BDFN and CREB mRNA expression levels in the rats with CORT-induced hippocampus lesions was investigated by RT-PCR analysis (Figure
The PCR bands and their relative intensities for brain-derived neurotrophic factor (BDNF) and cAMP-response element-binding (CREB) protein in the hippocampus of rats that received chronic CORT administration. Data were analyzed via separate one-way ANOVAs followed by Tukey’s
In the present study, acupuncture stimulation to the HT7 acupoint significantly improved learning and memory retention in the MWM and increased ChAT and AchE immunoreactivities in the hippocampus areas of chronic CORT-induced memory impairment male rats. Interestingly, 5-min acupuncture stimulation prior to CORT administration was enough to modulate CORT-induced neurochemical and behavioral responses (data not shown). In addition, only the acupuncture stimulation to the HT7 acupoint elicited significant responses, compared with another acupoint on a different meridian, TE5, or to a nonacupoint on the tail. These results indicate that stimulation of the acupuncture point spreads throughout the body at a rapid rate, and its effect is highly point specific, at least for modulating CORT-induced memory impairments. Furthermore, acupuncture to HT7 was capable of attenuating a complex behavioral syndrome and protecting the hippocampus from deficits.
In traditional oriental medicine, the Sinmun (HT7) is a specific acupoint located on the heart channel, which is also called the “spirit gate” to the pathway that is used clinically to treat mental, psychosomatic, and cognitive disorders [
Many studies very well recognized that dysregulation of the HPA axis by chronic stress or elevated levels of circulating CORT produces hyperactivity of sympathetic adrenomedullary system, such as CORT, corticosteroid-binding-globulin, ACTH, norepinephrine (NE), and epinephrine (E) [
In the CORT-treated rats, reduced spatial cognitive abilities were associated with a significantly increase in plasma and cerebral CORT levels as compared to control group. In present study, we found that acupuncture at HT7 decreased the CORT release in plasma after chronic CORT administration. Our findings may help to explain that stimulation at HT7 acupoint may affect the hippocampus to received biochemical and behavioral signals induced by reduced CORT level in plasma. Therefore, acupuncture at HT7 may modulate the dysregulation of HPA axis, which means acupuncture could influence secretion of CORT, thereby normalizing behavioral and neurochemical response. The effects of acupuncture stimulation might possess a relative specificity on acupoints. The correlation observed between brain cholinergic neurons and improved memory abilities were associated with interaction between CORT concentrations and acupuncture in the control of cognitive processes linked to cholinergic neurons. Accordingly, our results suggest that rewarding effect of acupuncture stimulation of HT7 on exogenous CORT-induced cognitive deficits might be the restoration of HPA axis hyperactivity through the decrease of endogenous CORT levels in the CNS, as noted in some studies [
To assess spatial learning and memory in rats, the MWM is more advantageous than other conventional mazes such as the T maze and the radial-arm maze. Training for spatial memory can be easily achieved after several acquisition trials, and the task does not require strong motivating traces or conditions such as scent, punishments, and food and water deprivation. The MWM is a hippocampus-dependent memory task, frequently used for demonstrating cognitive deficits and to examine permanent spatial learning capability and reference memory in rodents [
In terms of average swim speed and rest time, which are indices of motor function, the CORT group was not significantly different from HT7-acupuncture group. This indicates that motor impairment was not the main cause of the poor performance of the CORT group in the MWM test. Accordingly, it is evident that acupuncture at the HT7 acupoint significantly improved MWM performance by enhancing spatial working memory.
An open-field test was also performed to rule out any confounding motor impairments, which can influence outcomes in many behavioral tests of depression. No significant individual differences in locomotor activities were observed between groups in the open-field test, suggesting that acupuncture stimulation to the HT7 acupoint had no effect on sensorimotor performance. Accordingly, the changes in behavioral performance in the MWM task were likely due to improved memory instead of differences in sensorimotor function, motor output, or limb flexibility.
Many reports have verified that sustained elevation of circulating GC concentrations produces a variety of cognitive deficits. For instance, rats exposed to daily CORT injections for 8 weeks demonstrated decreased spontaneous alteration in the T maze [
Chronic CORT administration might increase circulating GC levels in the CA1 region of the rat hippocampus, resulting in impairment of long-term potentiation and spatial learning and memory in the MWM [
Cholinergic neurons originating in the medial septum (MS) project to the cortex and hippocampus, which play key roles in ACh-associated cognition [
The treatment of chronic CORT-induced memory disorders with acupuncture stimulation to the HT7 acupoint might enhances Ach recycling and efficient choline reutilization, and concomitantly induces increases in ChAT and AchE activities. AchE and ChAT belong to a family of enzymatic proteins that are extensively expressed in cholinergic neurons. ChAT is responsible for ACh biosynthesis and is required for cholinergic neurotransmission in the central and peripheral nervous systems. Acetylcholine is rapidly hydrolyzed by AchE; therefore, the duration of ACh action in the synaptic cleft is dependent upon AchE activity [
In the present study, we did not know precisely whether the acupuncture stimulation affects memory improvement through neurochemical changes of cholinergic system or first causes memory improvement leading to the neurochemical changes, because we did not have time-course data of Morris water maze task and cholinergic immunoreactivity in the brain hippocampus. However, CORT levels in the blood were almost restored to the normal level right after the acupuncture stimulation at HT-7 in the CORT-HT group despite little statistical significance (Figure
In the acupuncture treatment against various memory-related disorders, the salvage capacity of ACh is enhanced. It also improves cholinergic neurons in the frontoparietal cortex and CA1 region of the hippocampus and continuously induces increases in ChAT and AchE activities, which eventually results in recovery of the entire cholinergic circulation pathway [
We thus propose that chronic exposure to CORT triggers dysregulation of the HPA axis, which, in turn, elicits a reduction of ChAT and AchE expression in the hippocampus and eventually causes memory and cognitive decline in the rats. Also, recent studies have shown that chronic CORT-induced HPA axis hyperactivity or excessive increase of GC levels regulated expression of BDNF and CREB and impacted function of the BDNF pathway in the hippocampus [
On the other hand, recent experimental evidence strongly supports the role of hippocampal BDNF in learning and memory processes, besides its actions on neuronal cell survival and prevention of neurodegeneration [
In oriental traditional medicine, acupuncture improves reversible malfunctions of the body via direct activation of various brain pathways, and thus contributes to the restoration of normal systemic balance, probably due to regulation of neurotransmitters including Ach [
The present study demonstrated that memory and cognitive deficits induced by exogenous CORT-induced septohippocampal cholinergic neuron loss were closely related to the degeneration of cholinergic neurons in the rat hippocampus and that acupuncture stimulation to the HT7 acupoint significantly ameliorated learning and memory deficits through recovery of the ACh system. Acupuncture improved performance on the spatial memory test and protected septohippocampal cholinergic neurons from exogenous CORT-induced destruction. The attenuation of impairments of memory and cognition by acupuncture stimulation might be due to the restoration of cholinergic neurochemical abnormalities. It is most likely that acupuncture therapy is strongly effective in protecting against memory-related neuronal degeneration in the brain and retards the progression of memory defects in neurodegenerative diseases.
This research was supported by a grant from the Kyung Hee University in 2010 (no. KHU-20100115) and by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (no. 2010-0003678).