Adult neuronal cells which can regenerate have been reported. The present study investigated whether acupuncture enhances neuronal regeneration in ischemic stroke rats. We established an ischemic stroke rat model by occluding the cerebral blood flow of the right middle cerebral artery for 15 minutes and then allowing reperfusion in Sprague–Dawley rats. The results indicated that, in these rats, 2 Hz electroacupuncture (EA) at both Zusanli (ST36) and Shangjuxu (ST37) acupoints reduced the infarction/hemisphere ratio 8 days after reperfusion and reduced the modified neurological severity score (mNSS) and increased the rotarod test time 4 and 8 days after reperfusion, respectively. In addition, 2 Hz reduced nestin immunoreactive cells in the penumbra area and the ischemic core area; 2 Hz EA also reduced Ki67 immunoreactive cells and increased glial fibrillary acidic protein immunoreactive cells in the penumbra area. These findings suggest that 2 Hz EA at the ST36 and ST37 acupoints has a neuroprotective role. However, additional studies are needed to further investigate these preliminary results.
Ischemic stroke is a common type of stroke and a main leading cause of death, disability, and dependency worldwide [
In Taiwan, acupuncture is a traditional Chinese medicine therapeutic strategy with a higher utilization rate in patients with stroke than in people without stroke [
Zhang et al. demonstrated that EA treatment improves the motor functions of the limbs and the activities of daily living in patients with hemiplegia caused by acute cerebral infarction; they also noted that this effect was associated with reduced serum levels of neuron-specific enolase, soluble protein-100B, and endothelin [
Acupuncture at the Zusanli acupoint can increase cell proliferation in the dentate gyrus after transient global ischemia and suppress the ischemia-induced increase in c-Fos expression and apoptosis of the hippocampal CA1 region in gerbils [
Some studies have investigated the effects of EA intervention in the MCAo model. Notably, EA at the Baihui acupoint may facilitate the recovery of motor function and stimulate brain-derived neurotrophic factor (BDNF) and receptor tyrosine kinase B expression in rats with cerebral ischemia [
Adult male Sprague–Dawley (SD) rats weighing between 250 and 350 g were purchased from BioLASCO Taiwan Co., Ltd., and raised in the Animal Center of China Medical University (CMU) in a 12-12-hour light-dark cycle environment. The room temperature was controlled at 25°C, and adequate food and water were provided. Animal use was approved by the Institutional Animal Care and Use Committee of CMU, and all animal protocols were performed in accordance with the
Before the establishment of the MCAo model, the right MCA was exposed through a cranial burr hole that was 2.5 mm lateral and 2.0 mm posterior to the bregma. The MCA blood flow was monitored using Laser Doppler flowmetry (DRT4, Moor Instruments Inc., Wilmington, USA), and was found to be more than 500 min/div before MCAo. Thereafter, the right common carotid artery (CCA) and internal carotid artery (ICA) were exposed through a neck midline incision, followed by the ligation of the pterygopalatine artery proximal to its branch under isoflurane anesthesia. A 3-0 nylon filament suture blunted at the tip by a flame and coated with poly-L-lysine (Sigma, USA) was inserted into the right external carotid artery through the CCA and advanced up to the ICA at a distance of 20–25 mm to block the origin of the right MCA; subsequently, the MCA blood flow was found to decrease to less than 100 min/div. After 15 minutes of blocked blood flow from the right MCA, the suture was removed slowly and reperfusion was allowed for 24 hours.
At 24 hours after reperfusion, the modified neurological severity score (mNSS) was assessed, and the rotarod test (RRT) was performed. The present study only included rats with mNSS ≥ 7, except for the normal group. A total of 60 male SD rats were randomly divided into the following five groups (12 rats each): (1) normal group, (2) control group, (3) sham group, (4) EA1 group, and (5) EA2 group.
In the normal group, the trachea and right carotid artery of the rats were exposed for 15 minutes after a neck midline incision. The incision was then sutured under isoflurane anesthesia, which was subsequently reinduced in the rats for 15 minutes on the first (24 hours), third, fifth, and seventh days postoperation. Prior to this second anesthesia induction, mNSSs were assessed and RRTs were performed on the first day (24 hours after operation) as well as 4th and 8th days postoperation.
The methods applied to the control group were identical to those applied to the normal group; however, transient MCAo was performed. The methods applied to the sham group were identical to those applied to the control group; however, the acupuncture needles were inserted into the subcutaneous layer at both the Zusanli (ST36) and Shangjuxu (ST37) acupoints. Furthermore, the needles were connected to a stimulator (Trio 300, ITO Co., Ltd., Tokyo, Japan) without electrical discharge. The methods applied to the EA1 group were identical to those applied to the sham group, but the needles were inserted into the muscle layer at both the ST36 and ST37 acupoints. For this group, the needles were connected to the stimulator and received electrical stimulation at a frequency of 2 Hz; this produced muscle contractions that were visible slightly to the naked eye. Finally, the methods applied to the EA2 group were identical to those applied to the EA1 group; however, the electrical stimulation was applied at a frequency of 15 Hz.
All of the rats were sacrificed after completing mNSS and RRT on the 8th day after operation or reperfusion. Six rats from each group were used for the cerebral infarction size study, and the remaining six rats from each group were used for the immunohistochemistry (IHC) study.
mNSS was assessed and RRT was performed on the first, fourth, and eighth day postoperation or reperfusion. mNSS was assessed by a well-trained investigator who was blinded to the groups. The scale provides a behavior deficit score, after reviewing motor, sensory, balance, and reflex functions; the total neurological deficit score is 18 [
The RRT apparatus (Rotamex, Columbus Instrument, Ohio, USA; picture 3.4) was composed of a striated rod (diameter: 8 cm) divided in five lanes (width: 5 cm) and located 8 cm above the ground. Each rat was trained for 3 days on a rotarod cylinder that accelerated from 4 to 40 rpm in 5 minutes, three times per day, and for an additional 5 minutes once before grouping. If a rat fell before 160 seconds, it was returned to the rotating rod for another training session until it reached the criterion [
The mNSS from the first day was used as the baseline. The rats were usually weak for the first 24 hours after reperfusion, which created a potential bias in the RRT time on the first day. Therefore, only the RRT times from the fourth and eighth days were used in the study.
The rat brains were placed in a plastic rat brain model and were sectioned into six slices with 2 mm thickness from the frontal pole. The slices were then stained with 2% 2,3,5-triphenyl tetrazolium chloride (Merk, Germany) for 15 minutes. The area stained white was the ischemic infarction area, and the area stained purple–red was the nonischemic infarction area. After images of these areas were obtained, the third slice from the frontal pole was used to calculate the ratio of the ischemic area to the ipsilateral hemisphere area (I/H ratio) using Image J (USA).
Under deep isoflurane anesthesia, the rats were sacrificed after completing mNSS assessment and RRT on the eighth day after reperfusion. Subsequently, the rats were perfused transcardially with 0.9% sodium chloride and 4% paraformaldehyde for fixation. Their brains were removed and further fixed for 3 days in 4% paraformaldehyde at 4°C, followed by cryoprotection in 30% sucrose for 4 days at 4°C. Next, the brains were sliced into 300
For the IHC staining, the brain slices were stained with antibodies against Ki67 (1 : 300; Millipore, USA), glial fibrillary acidic protein (GFAP; 1 : 200; Calbiochem, USA), and nestin (1 : 200; Millipore). The tissue was washed with phosphate buffered saline (PBS) and was then cocultured with 3% H2O2/methanol for 15 minutes. Next, the tissue was rewashed with PBS and was then cocultured with 10% normal blood serum for 20 minutes (LsAB kit, Zymed, San Francisco, CA, USA). Subsequently, blood serum was wiped away, and the tissue was cocultured with the primary antibody overnight, before being rewashed again with PBS three times and cocultured with the secondary antibody for 10 minutes. Thereafter, the tissue was washed with PBS three times. Finally, the tissue was cocultured with the Ls-AB-peroxidase complex for 10 minutes and then cocultured with DAB for 2 minutes (Liquid DAB substrate kit, Zymed). After hematoxylin staining, the glass slide was mounted for further observation. The stained slices were sealed under the coverslips and examined for the presence of immunoreactive cells using a microscope (Olympus, BX-51, Japan). The number of immunoreactive cells was quantified using National Institutes of Health ImageJ software (Bethesda, MD, USA).
The data are presented as mean ± standard deviation. Between-group comparisons were performed using one-way analysis of variance, followed by Tukey’s test. A
The I/H ratios were higher in the control, sham, EA1, and EA2 groups than in the normal group on the eighth day after reperfusion (all
Effect of electroacupuncture (EA) on cerebral infarct and neurological deficits in ischemia–reperfusion-injured rats.
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Day 8 |
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RRT | |||||
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Data represent mean ± standard deviation. Normal: normal group; Control: control group; Sham: sham group; EA1: 2 Hz electroacupuncture treatment group; EA2: 15 Hz electroacupuncture group; I/H: infarction/hemisphere ratio; mNSS: modified neurological severity score; RRT: rotarod test;
The mNSSs were higher in the control, sham, EA1, and EA2 groups than in the normal group on the first day after reperfusion (all
The mNSSs were also higher in the control, sham, EA1, and EA2 groups than in the normal group on the fourth day after reperfusion (all
On the eighth day after reperfusion, the mNSSs were still higher in the control, sham, EA1, and EA2 groups than in the normal group (all
The RRT times were higher in the normal group than in the control, sham, EA1, and EA2 groups on the fourth day after reperfusion (all
The RRT times were higher in the normal group than in the control, sham, EA1, and EA2 groups on the eighth day after reperfusion (all
In the penumbra area, the number of Ki67 immunoreactive cells in the control, sham, EA1, and EA2 groups was higher than that in the normal group (all
Effect of EA on Ki67, GFAP, and nestin immunoreactive cells in ischemia–reperfusion-injured rats.
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Ki67 (P) |
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47.0 ± 7.7 |
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GFAP (P) |
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Nestin (P) |
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Nestin (IC) |
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Data represent mean ± standard deviation. Normal: normal group; Control: control group; Sham: sham group; EA1: 2-Hz electroacupuncture treatment group; EA2: 15-Hz electroacupuncture group; Ki67: Ki67 immunoreactive cells; GFAP: GFAP immunoreactive cells; Nestin: nestin immunoreactive cells; P: penumbra area; IC: ischemic core area;
In the penumbra area, the number of GFAP immunoreactive cells in the control, sham, EA1, and EA2 groups was higher than that in the normal group (all
In the penumbra area, no prominent nestin immunoreactive cells were noted in the normal group (Figure
In the ischemic core area, no prominent nestin immunoreactive cells were noted in the normal group (Figure
In the present study, the I/H ratios were higher in the control, sham, and EA2 groups than in the EA1 group on the eighth day after reperfusion. The mNSSs were higher in the control and sham groups than in the EA1 and EA2 groups on the fourth and eighth days after reperfusion. The RRT times were higher in the EA1 and EA2 groups than in the control and sham groups on the fourth and eighth day after reperfusion. Based on the aforementioned results, it was determined that 2 Hz EA could reduce cerebral infarction size and neurological deficit, whereas 15 Hz EA only improved neurological deficit in ischemia–reperfusion-injured rats. This suggests that 2 and 15 Hz EA have differential effects on cerebral infarct.
One previous study found that EA at the GV26 and PC6 acupoints could reduce cerebral infarction size in ischemia–reperfusion-injured rats [
In the present study, 2 Hz EA also reduced the number of Ki67 immunoreactive cells and increased the number of GFAP immunoreactive cells in the penumbra region. However, 15 Hz EA could not alter immunoreactivity in the ischemia–reperfusion-injured rats. Ki67 is a nuclear protein that is used as a mitotic marker. Ki67 is also used as a proliferation marker and is expressed in the initial stage of mitosis during adult neurogenesis [
Nestin is not only a cytoplasmic intermediate filament (IF) protein associated with IF polymerization and macromolecule stability, but also identified in multipotent stem/progenitor cells in the CNS. In addition, nestin is normally expressed during CNS development and is reactivated after minor stresses to the nervous system [
CNS injuries first induce nestin expression in reactive astrocyte glial cells near the wound and then lead to hyperplasia, deformation, transition, and scarring of reactive astrocyte glial cells, sequentially [
The present study has some limitations to acknowledge. The baseline was set only according to the mNSSs at 24 hours after reperfusion, which could not explain whether the cerebral infarction size was similar among the control, sham, EA1, and EA2 groups. Instead, imaging studies involving computer tomography or magnetic resonance imaging should be used to detect the infarction size in live animals.
Treatment with 2 Hz EA at ST36 and ST37 acupoints was determined to reduce cerebral infarction size and neurological deficit scores, as well as increase RRT times. In addition, 2 Hz EA reduced Ki67 and nestin immunoreactive cells and increased GFAP immunoreactive cells in ischemia–reperfusion-injured cerebral infarction rats. These findings suggest that EA plays a neuroprotective role in ischemia–reperfusion-injured rats, although the underlying mechanisms require further study.
The authors declare that they have no conflicts of interest.
This study was supported by the Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW106-TDU-B-212-113004) and by the Chinese Medicine Research Center, China Medical University (Ministry of Education, Aim for the Top University Plan).