Influence of Ren and Du meridian electro-acupuncture on neural stem cell proliferation and extracellular signal-regulated kinase pathway in a rat model of focal cerebral ischemia injury

BACKGROUND： Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE： To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Human Anatomy, Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS： Mouse anti-rat bromodeoxyuridine （BrdU） monoclonal antibody was provided by Sigma, USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase （ERK） specific inhibitor PD98059 were provided by Calbiochem, Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies, China. METHODS： A total of 126 rats were randomly assigned to four groups： model （n = 36）, Du meridian （n = 36）, Ren/Du meridian （n = 36）, and Ren/Du meridian ＋ PD98059 （n = 18）. Rats in the Ren/Du meridian ＋ PD98059 group were observed on days 7 （n = 6） and 14 （n = 12） after cerebral ischemia injury. Rats in the model, Du meridian, and Ren/Du meridian groups were observed on days 7, 14, and 28 after cerebral ischemia injury, with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong （DU 26） and Baihui （DU 20） acupoints in the Du meridian group, as well as Chengjiang （RN 24）, Guanyuan （RN 4）, Renzhong, and Baihuiacupoints in the Ren/Du meridian and Ren/Du meridian ＋ PD98059 groups 2 days after model establishment. In addition, electro-acupuncture stimulation with disperse-dense waves was performed, with 30 Hz disperse wave, 100 Hz dense wave, and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian ＋ PD98059 group were treated with 0.2 pg PD98059 injection into the subventricular zone, 2 pL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES： BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 （pERK1/2） expression in the subventricular zone. RESULTS： On days 14 and 28 after cerebral ischemia, there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren/Du meridian group compared with the Du meridian group （P 〈 0.05）. PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the/：ten and Du meridians （P 〈 0.05）. On days 7, 14, and 28 after treatment, pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group （P 〈 0.05）. The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction （P 〈 0.05）. However, PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression （P 〈 0.05）. CONCLUSION： Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells, which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.

Electroacupuncture at Du channel and meridian of foot-Taiyang for hippocampal neurons in rats with depression

BACKGROUND： Long-term anti-depression treatment can promote the regeneration of hippocampal regeneration. Up-regulation of hippocampal regeneration can reverse or prevent against the injury of stress to cerebrum, especially to hippocampal structure and function. Therefore, promoting hippocampal neuronal regeneration may be a new strategy for treating depression and anxiety, OBJECTIVE： To observe the effect of electro-acupuncture at Du channel and meridian of foot-Taiyang on hippocampal neurons from model rats of depression. DESIGN： A randomized controlled animal experiment. SETTING： Department of Acupuncture and Moxibustion, Shenzhen Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine. MATERIALS： Twenty-four Wistar rats, of either gender, aged 2 months old, weighing 200 - 220 g, were provided by the Animal Experimental Center, Guangzhou University of Traditional Chinese Medicine. METHODS： This experiment was carried out in the Clinical Molecular Biochemical Laboratory, Shenzhen Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine between October 2006 and April 2007. ①The involved rats were randomized into 4 groups according to body mass： blank control group, model group, electroacupuncture A group and electroacupuncture B group, with 6 in each. Rats in the blank control group were free to access to water, and were not given any intervention. Rats in the latter 3 groups were developed into rat depression models by chronic stress combined with feeding alone, and received 21-day unpredictable various stresses. Rats in the model group were euthanized at 14 days after modeling, and their brain tissues were harvested. Rats in the electroacupuncture A group were modeled, then points ＂Baihui＂ and ＂Shenting＂ were chosen, and given electroacupuncture, once a day, 20 minutes once. Rats in the electroacupuncture B group were modeled, then points ＂Baihui＂, ＂Shenting＂, ＂Xinshu＂and ＂Ganshu＂ were chosen, and frequency and therapeutic time were the same as those of electroacupuncture A group. Rats in the latter 2 groups were also given electroacupuncture, and their brain tissues were harvested 14 days later. ②Index evaluation： Neurons in hippocampal CA 1 and CA3 regions as well as dentate gyrus [neuron specific enolase （NSE）-positive cells] were counted by immunofluorescent technique. Each rat was weighted on the day of grouping and before execution at 35 days, MAIN OUTCOME MEASURES： Neuron amount in each hippocampal region and body mass of rats in each group. RESULTS： Twenty-four rats were involved in the final analysis.①Neuronal amount in each region of hippocampus： Neurons in the hippocampal CA1 and CA3 regions as well as dentate gyrus of rats in the model group were significantly less than those in the blank control group （P 〈 0.01）. Neurons in hippocampal CAland CA3 regions as well as dentate gyrus of rats in the electroacupuncture A and B groups were more than those in the model group （P 〈 0.05）. ② Change in body mass of rats： Body mass increase of rats in the model group was less than that in the blank control group （P 〈 0.05）, and body mass increase of electroacupuncture A and B groups was significantly more than that in the model group, respectively （P 〈 0.01）. CONCLUSION： Electroacupuncture at Du channel and meridian of foot-Taiyang alleviates hippocampal neuronal injury of rats, and thereby, improves depression and promotes the increase of body mass of rats.

Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

BACKGROUND： The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE： To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN： Randomized controlled study. SETTING： Department of Anatomy, Sun Yat-Sen University. MATERIALS： A total of 60 healthy adult male Wistar rats weighing （250±10） g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS： The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups： normal group （n = 6）, sham operation group （n = 18）, model group （n = 18）, and electroacupuncture group （n = 18）. Middle cerebral artery occlusion （MCAO） was performed in the model group and electroacupuncture group. Zea Longa＇s grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between l and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery （CCA） was isolated, and the external carotid artery （ECA） was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture （People＇s Publishing House; 1997; First Edition）. The Chengjiang, Qihai, and Guanyuan acupoints were labeled and connected to a G6805 electroacupuncture apparatus with sparse-dense waves （sparse waves were 30 Hz, dense waves were 100 Hz）, with a frequency of 6-15 V. The duration was 20 minutes. Two days after surgery, the model and sham operation groups were placed with their backs on the operating table, but they received no acupuncture. However, the normal group received acupuncture. The experimental animals under anesthesia were sacrificed on days 7, 14, and 28 post-surgery. Western blot analysis was used to measure expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall. Expression was measured in the normal group at time points corresponding to the sham operation group. MAIN OUTCOME MEASURES： Expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall at different time points after intervention. RESULTS： All 60 rats were included in the final analysis, without any loss. Seven days after MCAO, there was no significant difference in extracellular signal-regulated kinases 1/2 expression in the electroacupuncture group compared to the model group （P 〉 0.05）. However, extracellular signal-regulated kinases 1/2 expression significantly increased in the model group at 14 and 28 days after treatment （P 〈 0.05）. CONCLUSION： Electroacupuncture at the Ren channel can enhance extracellular signal-regulated kinasesl/2 expression in the inferior region of the lateral cerebral ventricle wall of rats with focal cerebral ischemia. However, this effect is not apparent until 14 days after electroacupuncture intervention.

Electroacupuncture at Ren channel point combined with injection of basic fibroblast growth factors for neural stem cell proliferation in subventricular zone of cerebral ischemic side

BACKGROUND： Electroacupuncture at Ren channel point can treat stroke and generate some neurodevelopment-related growth factors, which are associated with the proliferation and differentiation of stem cells. OBJECTIVE： To observe the effect of electroacupuncture at Ren channel point combined with injection of basic fibroblast growth factors （bFGF） on the expression of 5-bromo-2-deoxyuridine （BrdU）-Iabeled and BrdU/Neuroepthelial stem cell protein （Nestin）-Iabeled positive cells in subventricular zone of cerebral ischemic rats. DESIGN ： Completely randomized grouping design and controlled trial.SETTING： Department of Acupuncture and Moxibustion, Shenzhen Hospital, Guangzhou University of Traditional Chinese Medicine. MATERIALS ： Eighty-four adult healthy male Wistar rats were involved and divided into 5 groups by a lot： blank group （n =6）, sham-operation group （n =6）, model group （n =26）, injection of bFGF group （bFGF group, n =24） and electroacupuncture at Ren channel combined with injection of bFGF group （bFGF group, n =22）. Three time points were set in each of the latter three groups for observation： 7, 14 and 28 days after ischemia respectively. Mouse BrdU and Nestin monoclonal antibody （analytical pure, China）, G6805 electro-acupuncture apparatus （Huayi Medical Instrument, Shanghai） and Olympus FV 500 laser confocal microscope （Olympus Company） were used. METHODS ： This trial was carried out in the Laboratory of Anatomy, Medical College of Sun Yat-sen University from September 2004 to July 2005. ① Except for blank group and sham-operation group, the rats in the other groups were prepared into rat models of local cerebral ischemia by suture-occluded method. The rats were reperfused, and then they were graded in neurologic deficits based on neurological scoring of Longa. Rat model was successful when the neurological scoring ranged from 1 to 4 points and used for the trial. Blank group was untouched; Right common carotid artery was isolated and right external carotid artery was dissected without embolism in the sham-operation group. On the 2nd day after operation, the rats in the combination group were given acupuncture at Chengjiang, Qihai and Guanyuan point, then, electro-acupuncture apparatus, with sparse wave 30 Hz, dense wave 100 Hz, intensity 6 to 15 V, was used to stimulate these three points. Stimulation standard was based on the slight trill appeared in the corresponding position, and the stimulation lasted 20 minutes. After being acupunctured, the rats were intramuscularly injected with 4 000 U/d, and administration was immediately after reperfusion, after this, bFGF was intramuscularly injected following acupuncture, once a day. In the bFGF group, the rats were intramuscularly injected with bFGF on the 2^nd day after operation. The dosage and time of injection were the same as those in the combination group. The rats in the model group and sham-operation group were mounted on the acupuncture bench for 20 minutes on the 2^nd day after operation, and acupuncture was omitted. ② The expressions of BrdU-labeled positive cells （ reflect cell proliferation） and BrdU/Nestin-labeled positive cells （reflect neural stem cells proliferation） in the lateral subventricular zone were determined by immunofluorescence. Positive cells were counted in five visual fields using 200-fold Olympus FV 500 laser confocal microscope system. ③ One-way analysis of variance was used for multiple comparison of the mean of samples. MAIN OUTCOME MEASURES ： The effect of electroacupuncture combined with injection of bFGF on the number of BrdU and BrdU/Nestin-labeled positive cells in subventricular region of cerebral ischemic side. RESULTS： Altogether 8,6 and 4 rats in model group, combination group and bFGF group died respectively during modeling, finally 6 rats respectively in the normal group and sham-operation group and 18 rats respectively Le., 6 rats at each time point, in the model group, combination group and bFGF group were involved in the result analysis.① 7 days after cerebral ischemia, BrdU and BrdU/Nestin-labeled positive cells in the model group were （272.6±36.9）and （65.3±11.8）/5 visual fields, respectively, which were more than those of normal group [（47.2±6.1） ,（15.6±4.2）/5 visual fields, P 〈 0.01], but less than those in the bFGF group [（365.5±42.7 ）, （82.9±12.2）/5 visual fields] and in the combination group [（427.8±46.9） ,（125.6±18.5）/5 visual fields, P 〈 0.01]. The BrdU and BrdU/Nestin-labeled positive cells in the bFGF group were significantly less than those in the combination group （P 〈 0.05）. ② 14 days after cerebral ischemia, the BrdU and BrdU/Nestin-labeled positive cells in the model group were significantly more than those in the blank group [（184.7±28.1 ）,（46.5±9.6）/5 visual fields, P 〈 0.01], but less than those in the bFGF group [（259.4＋32.8）,（68.1 ± 10.1 ）/5 visual fields] and combination group [（308.6±34.2）,（87.2±16.9）/5 visual fields, P 〈 0.01]. The BrdU and BrdU/Nestin-labeled positive cells in the bFGF group were significantly less than those in the combination group （P〈0.05）. ③ 28. days after cerebral ischemia, the BrdU and BrdU/Nestin-labeled positive cells in the model group were close to those in the normal group [（51.3±8.4）,（17.8±5.3）/5 visual fields, P 〉 0.05], but significantly less than those in the bFGF group [（128.1 ±23.6）,（41.3±11.4）/5 visual fields]and in the combination group [（162.5±26.1）,（62.3±15.8）/5 visual fields, P〈 0.01]. The BrdU and BrdU/Nestin-labeled positive cells in the bFGF group were significantly less than those in the combination group （P〈 0.05）. ④ There were no significant differences in BrdU/Nestin-labeled and Nestin-labeled positive cells between sham-operation group and normal group（P 〉 0.05）. CONCLUSION ： Electroacupuncture at Ren channel point alone or combined with injection of bFGF can significantly promote neural stem cell proliferation of rats after local cerebral ischemia. Electroacupuncture at Ren channel point and injection of bFGF have synergistic effect.