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Abstract
Objective: To investigate the effect of electroacupuncture (EA) promoting hippocampal neural stem cells (NSCs) proliferation in rats with focal cerebral ischemia- reperfusion (I/R) via Notch pathway and to clarify the possible mechanism of EA in treatment of cerebral ischemia. Method: Fifty- four male adult SD rats were randomly divided into the sham operation control group (SC group), the ischemia model group (IC group) and the electroacupuncture group (EA group). Middle cerebral artery occlusion (MCAO) was performed to establish the focal cerebral I/R injury model. Proliferation of hippocampal NSCs in cerebral I/R injured rats was determined by nestin immunohistochemical staining. The expressions of Notch1 and intracellular domain of Notch(NICD) were detected by Western blotting and RT- PCR. The serum level of vascular endothelial growth factor (VEGF) was measured using enzyme-linked immunosorbent assay (ELISA). Result: EA at Quchi (LI11) and Zusanli (ST36) acupoints alleviated neurological deficits significantly and promoted the proliferation of hippocampal NSCs (IC vs EA: 173.40±38.76 vs 246.80±47.73, P=0.028) in cerebral I/R injured rats; enhanced the expressions of Notch1 and NICD, crucial signaling molecules in Notch signaling pathway, and increased the secretion of VEGF. Conclusion: The up-regulatory effect of EA on Notch signaling pathway and neurotrophic factor secretion may result in the promotion of NSCs proliferation and consequently provide the therapeutic effect on cerebral ischemia.
... Acupuncture points Quchi (LI11) and Zusanli (ST36) are most commonly used in the treatment of post-stroke hemiplegia. Our previous studies suggested that acupuncture at these sites promoted proliferation and differentiation of endogenous neural stem cells in rat models of middle cerebral artery ischemia reperfusion (Chen et al., 2014). Additionally, acupuncture at Quchi and Zusanli enhanced expression of brain derived neurotrophic factor (BDNF) and glial cellderived neurotrophic factor (GDNF) (Ye et al., 2014) and inhibited apoptosis (He et al., 2015), thereby promoting the recovery of motor function through the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) (He et al., 2015), Tolllike receptor 4/nuclear factor kappa B (TLR4/NF-κB) (He et al., 2016), extracellular regulated protein kinases (ERK) (He et al., 2015), and Notch signaling pathways (Chen et al., 2014). ...
... Our previous studies suggested that acupuncture at these sites promoted proliferation and differentiation of endogenous neural stem cells in rat models of middle cerebral artery ischemia reperfusion (Chen et al., 2014). Additionally, acupuncture at Quchi and Zusanli enhanced expression of brain derived neurotrophic factor (BDNF) and glial cellderived neurotrophic factor (GDNF) (Ye et al., 2014) and inhibited apoptosis (He et al., 2015), thereby promoting the recovery of motor function through the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) (He et al., 2015), Tolllike receptor 4/nuclear factor kappa B (TLR4/NF-κB) (He et al., 2016), extracellular regulated protein kinases (ERK) (He et al., 2015), and Notch signaling pathways (Chen et al., 2014). Other studies have reported that acupoint stimulation increases functional recovery in the hemiplegic limb by regulating the excitability of the cerebral cortex (Maioli et al., 2006;Zunhammer et al., 2012). ...
Background and objectives: Effect of conventional rehabilitation methods is still not satisfactory to patients with post-stroke hemiplegia. Studies have demonstrated that acupuncture can improve motor function in stroke patients. Quchi (LI11) and Zusanli (ST36) are most commonly used in the treatment of post-stroke hemiplegia. We will observe the effects of acupuncture on neural plasticity in patients with post-stroke hemiplegia after acupuncture at Quchi and Zusanli.
Design: This is a prospective multicenter randomized parallel controlled trial.
Methods: The trial will be conducted in Shiyan Taihe Hospital, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, and the Rehabilitation Hospital of Fujian University of Traditional Chinese Medicine, China. A total of 210 patients with post-stroke hemiplegia will be randomized into an acupuncture priority group, acupuncture delay group, sham acupuncture priority group, and sham acupuncture delay group (n = 45). On the basis of basic treatment and conventional rehabilitation training, acupuncture (acupuncture priority group and acupuncture delay group) or sham acupuncture (sham acupuncture priority group and sham acupuncture delay group) at Quchi and Zusanli will be performed 15 minutes before conventional rehabilitation training (priority) or 15 minutes after conventional rehabilitation training (delay), once a day for 30 minutes, 5 days per week, for 8 consecutive weeks. Patients will undergo one follow-up assessment for 4 months.
Outcome measures: The primary outcome measure will be the efficacy of the treatment after the 8-week period, stated as a percentage calculated by taking the difference between the final simplified Fugl-Meyer motor function score and the baseline score, and dividing this value by the baseline score. Brunnstrom stage classification, Barthel Index score, and electrophysiological changes will serve as secondary outcome measures.
Discussion: We hope to produce new information leading to improved timing for the combined use of acupuncture and modern rehabilitation therapy, and identify the effects of acupuncture at Quchi and Zusanli on cortical excitability and plasticity.
Ethics and dissemination: The protocols have been approved by the Ethics Committee of Shiyan Taihe Hospital (Affiliated Hospital of Hubei University of Medicine) of China (approval No. 2014001-2) on August 26th, 2013. We will submit the trial's results for presentation at international scientific meetings and to peer-reviewed journals. The study design was completed in July 2013. Ethical approval was received in August 2013. Clinical registration was conducted in January–February 2017. Patient recruitment began in March 2017. The follow-up will be completed in September 2018. Data analysis will be conducted in June 2019.
Trial registration: This trial was registered in the Chinese Clinical Trial Registry (registration No. ChiCTR-IPR-17010490) on January 20th, 2017, and in the Acupuncture-Moxibustion Clinical Trial Registry (www.acmctr.org/index.aspx; identifier: AMCTR-IPR-17000038) on February 7th, 2017.
Mouse long-term hematopoietic reconstituting cells exist in the c-Kit+Sca-1+Lin- (KSL) cell population; among them, CD34(low/-) cells represent the most highly purified population of hematopoietic stem cells in the adult bone marrow. Here, we demonstrate that retrovirus-mediated transduction of CD34(low/-)c-Kit+Sca-1+Lin- (34-KSL) cells with the HES-1 gene, which encodes a basic helix-loop-helix transcription factor functioning downstream of the Notch receptor, and is a key molecule for the growth phase of neural stem cells in the embryo, preserves the long-term reconstituting activity of these cells in vitro. We also show that cells derived from the HES-1-transduced 34-KSL population produce progenies characterized by negative Hoechst dye staining, which defines the side population, and by CD34(low/-) profile in the bone marrow KSL population in each recipient mouse at ratios 3.5- and 7.8-fold those produced by nontransduced 34-KSL-derived competitor cells. We conclude that HES-1 preserves the long-term reconstituting hematopoietic activity of 34-KSL stem cells ex vivo. Up-regulation of HES-1 protein in the 34-KSL population before unnecessary cell division, that is, without retrovirus transduction, may represent a potent approach to absolute expansion of hematopoietic stem cells.
To observe the effects of electroacupuncture (EA) at the Conception Vessel on proliferation and differentiation of the nerve stem cells in the inferior zone of the lateral ventricle in cerebral ischemia rats.
The model rats were prepared by occlusion of the middle cerebral artery for 2 hours and then by reperfusion. They were randomly divided into two groups: a control group and an EA group. Changes in differentiation and proliferation of the nerve stem cells were observed 7, 14 and 28 days after successful modeling.
As compared with the 7-day control group (C-7d group), there was no significant difference (P > 0.05) in the numbers of 5-bromodeoxyuridine (Brdu) positive cells, Brdu/GFAP, Brdu/Nestin and Brdu/Nse double-labeled cells in the inferior zone of the lateral ventricle in the EA group 7 days after modeling. However, in the 14-day EA group (R-14d group) and the 28-day EA group (R-28d group), the numbers of Brdu positive cells and Brdu/GFAP, Brdu/Nestin, Brdu/Nse double-labeled cells significantly increased as compared respectively with the 14-day control (C-14d group) and the 28-day control (C-28d) group (P < 0.05 or P < 0.01).
EA at the Conception Vessel promotes differentiation and proliferation of the nerve stem cells in the inferior zone of the lateral ventricle in the cerebral ischemia rats, and may stimulate differentiation of the proliferous nerve stem cells towards the astrocytes.
To explore the mechanism of electroacupuncture for treating focal cerebral ischemia-reperfusion injury.
Seventy-five Wistar rats were randomly divided into a control group, a model group and an electroacupuncture group, 25 cases in each group. The model of focal cerebral ischemia-reperfusion was established by inserting nylon thread into the internal carotid artery except the control group which was only separated of the carotid artery without occlusion. Electroacupuncture group was treated with electroacupuncture at "Baihui (GV 20)" and "Dazhui (GV 14)" and the other groups without electroacupuncture treatment. The number of nestin positive cells expression at 1st, 3rd, 7th, 14th and 21st days after focal cerebral ischemia reperfusion was observed by use of immunohistochemistry method.
The number of nestin positive cells in electroacupuncture group at ischemia side was significantly more than that in the model group at 3rd, 7th, 14th and 21st days (P < 0.05, P < 0.01), and at contralateral ischemia side, the number of nestin positive cells in the electroacupuncture group was significantly more than that in the model group only at 7th day (P < 0.05).
Electroacupuncture at "Baihui (GV 20)" and "Dazhui (GV 14)" in rats can increase the number of nestin positive cells in hippocampus after focal cerebral ischemia-reperfusion, which may be one of the important mechanisms of electroacupuncture in treating acute cerebral ischemic diseases.
Convincing evidence has shown that brain ischemia causes the proliferation of neural stem cells/neural progenitor cells (NSCs/NPCs) in both the subventricular zone (SVZ) and the subgranular zone (SGZ) of adult brain. The role of brain ischemia-induced NSC/NPC proliferation, however, has remained unclear. Here we have determined whether brain ischemia-induced amplification of the NSCs/NPCs in adult brain is required for brain self-protection. The approach of intracerebroventricular (ICV) infusion of cytosine arabinoside (Ara-C), an inhibitor for cell proliferation, for the first 7days after brain ischemia was used to block ischemia-induced NSC/NPC proliferation. We observed that ICV infusion of Ara-C caused a complete blockade of NSC/NPC proliferation in the SVZ and a dramatic reduction of NSC/NPC proliferation in the SGZ. Additionally, as a result of the inhibition of ischemia-induced NSC/NPC pool amplification, the number of neurons in the hippocampal CA1 and CA3 was significantly reduced, the infarction size was significantly enlarged, and neurological deficits were significantly worsened after focal brain ischemia. We also found that an NSC/NPC-conditioned medium showed neuroprotective effects in vitro and that adult NSC/NPC-released brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are required for NSC/NPC-conditioned medium-induced neuroprotection. These data suggest that NSC/NPC-generated trophic factors are neuroprotective and that brain ischemia-triggered NSC/NPC proliferation is crucial for brain protection. This study provides insights into the contribution of endogenous NSCs/NPCs to brain self-protection in adult brain after ischemia injury.
This study was carried out to observe the effect of electroacupuncture (EA) on neurological deficits, proliferation and differentiation of nerve stem cells (NSCs) in adult rats with middle cerebral artery occlusion (MCAO) and to study its possible role in the treatment of cerebral ischemic injury.
A rat model of MCAO was established and interfered with EA. On days 4, 7, 14 and 21 after ischemic injury, neurological deficits were scored. On days 4, 7, 14 and 21 after injury, effect of EA interference on the proliferation and differentiation of rat NSCs was observed with BrdU/NeuN and BrdU/GFAP immunofluorescence double labeling.
A significant difference was found in the scores of rat neurological deficits between the EA and model groups 7, 14 and 21 days after cerebral ischemic injury (p<0.05). BrdU positive cells were found in the subventricular zone (SVZ) 4, 7, 14 and 21 days after ischemic injury. The number of positive BrdU cells in the SVZ reached its peak 7 days after injury and was greater in the EA group than in the model group 7 and 14 days after injury (p<0.05). The number of BrdU/GFAP doubly labeled positive cells in the SVZ was greater in the EA group than in the model group 7 and 14 days after ischemic injury (p=0.012 and p=0.025, respectively). There was no difference in the number of BrdU/NeuN doubly labeled positive cells 4, 7 and 14 days in the striatum, but a significant difference 21 days (p=0.033) after ischemic injury between the two groups.
Cerebral ischemic injury induces proliferation of NSCs, some of which will differentiate into both astroglia and neurons. EA may promote cells proliferation, stimulate the proliferating cells to differentiate into astroglia and mature into neurons, which may be one of the important reasons why EA can alleviate neurological deficits.
Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a gamma secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a gamma secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.
The effects of acupuncture on cell proliferation in the dentate gyrus of gerbils after transient global ischemia were investigated in this study. Acupuncture was performed on Zusanli (ST36), which is a well known acupoint in animals and humans. In Oriental medicine, Zusanli has been commonly used for the enhancement of functional recovery in stroke patients. Through 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry, an increase in cell birth in the dentate gyrus of gerbils after ischemic injury was detected. Interestingly, acupunctural treatment in ischemic gerbils resulted in a significant increase in the number of BrdU-positive cells in the dentate gyrus. The present findings indicate that acupuncture may affect cell proliferation in the dentate gyrus of gerbils after ischemic injury.
Accumulating evidence indicates that neurogenesis in the adult brain occurs in restricted brain regions, including the hippocampal dentate gyrus and is promoted by ischemia. The mechanism responsible for ischemia-induced neurogenesis in the adult brain, however, remains unclear. Notch pathway plays a pivotal role in the regulation of the timing for differentiation and determination of the fate of neural progenitor cells in the developing nervous system. To elucidate the mechanism underlying ischemia-induced neurogenesis, we investigated changes in the expression of mRNAs of Hes5, which is a downstream target of Notch, and Mash1, a neurogenic basic helix-loop-helix factor, which is negatively regulated by Hes5, in the adult hippocampal dentate gyrus after transient forebrain ischemia. Transient forebrain ischemia was produced by four-vessel occlusion procedure in rats. The levels of Hes5 mRNA decreased on days 1 and 3 after the start of reperfusion and the decreased levels of the mRNA returned to the basal level by 5 days after ischemia. In contrast, the level of Mash1 mRNA increased on day 1 and then returned to the basal level by 3 days after ischemia. These results suggest that an inhibition of Notch activity and subsequent expression of neurogenic basic helix-loop-helix factors, including Mash1, may, at least in part, contribute to ischemia-induced neurogenesis in the adult dentate gyrus.
This review discusses the potential usefulness of several selected polypeptide growth factors as treatments for stroke. Distinctions between global vs. focal cerebral ischemia, permanent vs. temporary focal ischemia, and acute stroke vs. stroke recovery are first discussed. Potential routes of administration of growth factors are also considered. The growth factors basic fibroblast growth factor (bFGF), osteogenic protein-1 (OP-1), vascular endothelial growth factor (Veg-f), erythropoietin (EPO), and granulocyte colony stimulating factor (G-CSF) all show potential usefulness in animal models of acute stroke and stroke recovery. Two of these factors, bFGF and EPO, have reached human clinical trials for acute stroke, and the data are discussed. Future directions in this field are also discussed.
Senescence-accelerated mouse prone 8 (SAMP8) is an autogenic senile strain characterized by early cognitive impairment and age-related deterioration of learning and memory. To investigate the effect of acupuncture on behavioral changes and brain cell events, male 4-month-old SAMP8 and age-matched homologous normal aging SAMR1 mice were divided into four groups: SAMP8 acupuncture group (Pa), SAMP8 non-acupoint control group (Pn), SAMP8 control group (Pc) and SAMR1 normal control group (Rc). By Morris water maze test, the cognitive deficit of SAMP8 was revealed and significantly improved by "Yiqitiaoxue and Fubenpeiyuan" acupuncture. Meanwhile, by 5'-bromo-2'-deoxyuridine (BrdU) specific immunodetection, the decreased cell proliferation in dentate gyrus (DG) of SAMP8 was greatly enhanced by the therapeutic acupuncture, suggesting acupoint-related specificity. Even though no significant differences were found in ventricular/subventricular zones (VZ/SVZ) of the third ventricle (V3) and lateral ventricle (LV) between groups, we obtained interesting results: a stream-like distribution of newly proliferated cells presented along the dorsum of alveus hippocampi (Alv), extending from LV to corpus callosum (CC), and the therapeutic acupuncture showed a marked effect on this region. Our research suggests that acupuncture can induce different cell proliferation in different brain regions of SAMP8, which brings forth the need to explore further for the mechanism of cognitive deficits and acupuncture intervention in this field.