Electroacupuncture Preconditioning Research Articles

Effect of electroacupuncture preconditioning of "Neiguan"(PC6) on myocardial LKB1/AMPK/PFK2 pathway in myocardial ischemia rats

To observe the effect of electroacupuncture (EA) preconditioning on the expression of liver protein kinase 1 (LKB1), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and 6-phosphofructo-2-kinase (PFK2) in cardiomyocytes of rats with acute myocardial ischemia (AMI), so as to explore its mechanisms underlying cardioprotective effect. Thirty male Wistar rats were randomly divided into sham-operation, model and EA pretreatment groups (n＝10 rats per group). The AMI model was established by ligation of the left anterior descending branch of coronary artery. Before modeling, EA preconditioning (2 Hz/15 Hz, 1 mA) was applied to bilateral "Neiguan"(PC6) for 30 min, once daily for 14 days. Histopathological changes of myocardium was observed by microscope after H．E. staining. The level of lactate dehydrogenase (LDH) in serum was detected by ELISA. The expression of autophagy-associated proteins and mRNAs as LKB1, AMPKa1, AMPKa2 and PFK2 were detected by Western blot and real-time PCR, respectively. Compared with the sham-operation group, serum LDH content, and expression levels of myocardial AMPKa2 and PFK2 proteins and mRNAs were significantly up-regulated (P<0.01), and those of LKB1 and AMPKa1 proteins and mRNAs were increased in the model group (P<0.05). Following the intervention, serum LDH were apparently down-regulated (P<0.01), and expression levels of myocardial LKB1, AMPKa1 and PFK2 proteins and mRNAs were apparently up-regulated (P<0.01), but that of AMPKa2 protein and mRNA was remarkably down-regulated in the EA group (P<0.01). H．E. staining showed cell swelling, disordered arrangement of myocardial fibers with obvious rupture, interstitial bleeding and inflammatory infiltration, which was relatively milder in the EA preconditioning group. EA pretreatment can trigger LKB1/AMPK/PFK2 signaling pathway in AMI rats, which may contribute to its cardioprotective effect against ischemic myocardial injury by activating autophagy of cardiomyocytes. .

Development of research on mechanisms of electroacupuncture intervention underlying improvement of cerebral ischemia rats in recent five years

In the present paper, we summarized the relevant research literature on the underlying mechanism of electroacupuncture (EA) intervention in improving cerebral ischemia (CI) in middle cerebral artery occlusion (MCAO) model rats in recent 5 years from preconditioning and regular intervention. Outcomes showed that both EA preconditioning and regular intervention could relieve CI by reducing toxicity of excitatory amino acids (glutamate, aspartate) and TLR4/NF-κB-mediated inflammatory cascade reaction to relieve inflammatory injury. Moreover, EA preconditioning also could suppress the expression of pro-apoptosis genes and proteins to relieve apoptosis, regulate activation of microglia, and down-regulate the expression of blood brain barrier integrity-related matrix metalloprotein 9. Regular EA intervention also could promote angiogenesis to increase supply of blood and oxygen, facilitate regeneration, proliferation and differentiation of neural stem cells via triggering activation of Notch and Shh signaling pathways, and promote the secretion of neurotrophin by up-regulating the expression of brain derived neurotrophic factor and nerve growth factor /Trk A receptor, and promote expression of axon growth and synaptic remodeling-related factor (Ephrin B2/Eph B2) and sr-GTPase activating protein 1 and cell division cycle 42 (Cdc42, axon growth and orientated down-stream molecules). However, up to now, the conclusion is still fragmentary and one-sided, and can not explain the specific mechanism of electroacupuncture in MCAO rats, needing further in-depth study.

Effect of "Jiaji" (EX-B2)-electroacupuncture preconditioning on expression of myocardial cytochrome P450 enzyme in rats with acute myocardial ischemia-reperfusion injury

To investigate the effect of "Jiaji"(EX-B2)-electroacupuncture(EA) preconditioning on structural changes of myocardium and expression of cytochrome P450 (CYP450) enzymes in acute myocardial ischemia-reperfusion injury (MIRI) rats. Sixty male Wistar rats were randomly divided into sham operation, model, EX-B2, Neiguan (PC6), Yanglingquan(GB34) and Quchi (LI11) groups (n＝10 in each group). The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min, followed by reperfusion for 30 min. EA preconditioning (2 Hz/100 Hz, 1 mA) was respectively applied to EX-B2, PC6, GB34, and LI11 for 30 min, once daily for 7 days. Morphological changes of myocardium were observed by H．E. staining. The ultrastructure of myocardial cells was observed by transmission electron microscope (TEM). The protein expression levels of myocardial CYP450 (CYP1A1, CYP2B2, CYP2C11 and CYP4A2) were detected by Western blot. In MIRI rats, myocardial pathological changes as swollen, disorganized arrangement, and partially ruptured myocardial fibers with unclear limit and necrosis and infiltration of lots of inflammatory cells under microscope, and focal myofilament degeneration, dissolution and necrosis with interstitial edema, vague mitochondria with swelling and vacuolation, ruptured and disorganized arrangement of crests under TEM were found in the ischemic area, which was obviously milder in rats of both EX-B2 and PC6 groups. After modeling, the expression of myocardial CYP1A1 and CYP2B2 protein were significantly up-regulated in the model group relevant to the sham operation group (P<0.05). In EA preconditioning groups, the expression levels of CYP1A1 and CYP4A2 proteins in the EX-B2, PC6 and GB34 groups were obviously down-regulated (P<0.01), and those of CYP2B2 and CYP2C11 proteins in both EX-B2 and PC6 groups were markedly up-regulated relevant to the model group (P<0.01). Compared with the EX-B2 group, the expression levels of CYP1A1, CYP4A2 proteins in the LI11 group were significantly up-regulated (P<0.05), and CYP2B2, CYP2C11 proteins were significantly down-regulated (P<0.05). No significant differences were found between the model and sham operation groups in the expression levels of myocardial CYP2C11 and CYP4A2 proteins, between the LI11 and model groups in the expression of CYP1A1, CYP2B2, CYP2C11 and CYP4A2, between the GB34 and model groups in the expression of CYP2B2, CYP2C11 (all P>0.05)．. EA preconditioning at EX-B2 and PC6 can suppress MIRI induced up-regulation of myocardial CYP450 ω-hydroxylase CYP1A1 and CYP4A2 expression and further up-regulate the expression of myocardial epoxygenase CYP2B2 and CYP2C11 proteins in acute MIRI rats, thus playing a role in myocardial protection.

Serum metabolic profile involving protective effect of "Neiguan"(PC6)-electroacupuncture preconditioning in rats with myocardial ischemia reperfusion injury

To observe the effect of "Neiguan" (PC6)-electroacupuncture (EA) preconditioning on serum metabolites in myocardial ischemia-reperfusion injury (MIRI) rats, so as to reveal its mechanism underlying improvement of ischemic myocardium from metabonomics. A total of 48 male SD rats were randomly divided into control, model, EA "Neiguan"(PC6) and EA "Hegu"(LI4) groups (n＝12 rats/ group). Rats of the control group were just banded on animal boards for 30 min, once daily for 7 days. The MIRI model was established by occlusion of the left anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 1 h, and rats of the model group were also banded as those in the control group. Before modeling, EA (10 Hz/50 Hz, 1 mA) was applied to bilateral "Neiguan"(PC6) and "Hegu"(LI4) for 30 min, once daily for 7 successive days. After the treatment, serum samples were collected to be analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. The orthogonal partial least squares discriminate analysis (PLS-DA) was employed to distinguish the serum differential metabolic profile of rats in different groups and identify potential biomarkers. After modeling, the ECG of model group and electroacupuncture groups showed T wave towering, and there was no obvious ST segment between R wave and T wave. The T wave decreased more than 0.2 mV after reperfusion, and there was no obvious ST segment. Compared with the control group, MIRI induced significant changes of metabolites in the serum including increase of acetoacetate acid, lectic acid, creatine, glycerol and glucose, and decrease of alanine, glutamine, glycerophosphoryl choline and phosphorylcholine. In comparison with the model group, PC6-EA preconditioning induced significant changes, including an increase of glucose, and a decrease of leucine，isoleucine, valine，3-hydroxybutyric acid，lactate，acetate，acetone，acetoacetate acid，pyruvic acid，glutamine，creatine and glycerol. There is no significant difference in metabolic patterns between "Hegu" group and model group. Metabolic pathway enrichment analysis indicated that the protective effect of PC6-EA pretreatment was realized mainly by regulating pathways of glycolysis, gluconeogenesis, citric acid metabolism, pyruvate metabolism, ketone body metabolism, etc. PC6-EA pretreatment has a role in regulating gluconeogenesis, pyruvate metabolism, amino metabolism, ketone body metabolism and energy metabolism in rats with MIRI, which maybe contribute to its protective effect on ischemic myocardium, but the specific metabolic pathways and mechanisms need being studied further.

Effect of Electroacupuncture at "Shenmen" (HT 7)- "Tongli" (HT 5) of Heart Meridian on Neuronal Activities in Paraventricular Nucleus of Hypothalamus in Myocardial Ischemia Rats

To observe the effect of electroacupuncture (EA) stimulation of "Shenmen" (HT 7) - "Tongli" (HT 5) segment of the Heart Meridian on neuronal electrical activities of hypothalamic paraventricular nucleus (PVN) in rats with myocardial ischemia (MI), so as to investigate its possible mechanism underlying improvement of MI. Thirty-two SD rats were randomly divided into sham control, model, HT 7-HT 5 and "Taiyuan" (LU 9)- "Lieque" (LU 7) groups (n=8 in each group). EA preconditioning (2 Hz, 1 V, 20 min) was applied to bilateral HT 7-HT 5 and bilateral LU 9-LU 7, respectively, once everyday for 7 days. The electrical activities of the right PVN region were recorded by the implanted microelectrode array(2×4)and Plexon multi-channel acquisition system. Cluster analysis of neuronal signals was carried out by Offline Sorter software. The discharge waveforms, autocorrelation and cross-correlation of neuronal activities were analyzed by using Neuro Explorer software. Cluster analysis of neuronal signals showed that 2, 2, 1 and 1 interneuron in the sham, model, HT 7-HT 5, and LU 9-LU 7 groups, and 3 pyramidal neurons in the HT 7-HT 5 were acquired. Cross correlation analysis showed that the SPK 02 a and SPK 02 b neurons of the HT 7-HT 5 group had an inhibitory relationship. The total discharge frequency was significantly increased in the model group relevant to the sham group (P<0.01), and was markedly lower in the HT 7-HT 5 group than in the model group and LU 9-LU 7 group (P<0.01). Real-time spectrum analysis showed that the local field potential spectrum energy of the HT 7-HT 5 group was significantly lower than that of the model group and the LU 9-LU 7 group. EA of HT 7-HT 5 segment of the Heart Meridian can inhibit the electrical activity of interneuron and activate the electrical activity of pyramidal neuron in PVN region, and an inhibitory relationship exists between the interneuron and pyramidal neuron in MI rats, which may be a mechanism of EA in regulating activities of the ischemic heart.

Effect of Electroacupuncture Preconditioning on Myocardial Ischemia and Expression of TLR 4, MyD 88 and NF-κB mRNAs in "Neiguan" (PC 6) Area in Rats with Myocardial Ischemia-reperfusion Injury

To observe the effect of electroacupuncture (EA) preconditioning on acute myocardial ischemia (MI) and expression of Toll-like receptor 4 (TLR 4), myeloid differentiation factor 88 (MyD 88) and nuclear factor-κB (NF-κB) mRNAs in the "Neiguan" (PC 6) area of rats with MI-reperfusion injury (MIRI), so as to explore its mechanisms underlying improvement of MIRI. Forty-eight male Wistar rats were randomly divided into control, control ＋ EA, sham operation, model, EA and sham EA groups (n＝8). The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min, followed by reperfusion. Before modeling, EA preconditioning was respectively applied to PC 6 or sham PC 6 for 30 min, once daily for 5 days. Electrocardiogram (ECG) of the standard limb-lead Ⅱ was recorded before and after the modeling, and 0.25 h after reperfusion. The expression levels of TLR 4, MyD 88 and NF-κB genes in the PC 6 tissue were detected by Real-time PCR. Compared with the control group, the height of ECG J-point was significantly increased in the model group (P<0.01), suggesting an acute MI, and the height of J-point was significantly lower in the EA group than in the model and sham EA groups (P<0.01). In comparison with the control group, the expression levels of TLR 4, MyD 88 and NF-κB mRNAs were markedly up-regulated in the area of PC 6 in the model group (P<0.01). Compared with the model group, the increased expression levels of TLR 4, MyD 88 and NF-κB mRNAs in the EA preconditioning group were significantly inhibited (P<0.05). No significant differences were found among the control, control＋EA and sham operation groups and between the model and sham EA groups in the expression levels of TLR 4, MyD 88 and NF-κB mRNAs (P>0.05)．. EA preconditioning can improve myocardial ischemia and reduce the expression of TLR 4, MyD 88 and NF-κB mRNAs in PC 6 area of MIRI rats, suggesting an involvement of the regional cutaneous TLR 4/MyD 88/NF-κB signaling suppression in the initiation-transfer-amplification of EA stimulating signal inputs for relieving MIRI.

Effect of Electroacupuncture and Moxibustion Pretreatment on Expression of Autophagy Related Proteins LC 3 and Beclin 1 in Rats with Myocardial Ischemia-reperfusion Injury

To observe the effect of electroacupuncture (EA) and moxibustion (Moxi) pretreatment on myocardial pathological and structural changes and expression of autophagy related protein LC 3 Ⅰ/Ⅱ and Beclin 1 in rats with myocardial ischemia-reperfusion injury (MI/RI), so as to explore their mechanisms underlying improving MI/RI. Forty SD rats were randomly divided into sham operation, model, ischemic preconditioning (IP), EA and Moxi groups (n＝8 in each group). EA (10 Hz/50 Hz，1 mA) or Moxi (ignited moxa stick) was respectively applied to bilateral "Neiguan"(PC 6) for 20 min, once daily for 7 days. The MI/RI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min. The left ventricular (LV) tissue samples were collected and analyzed for pathological (H.E. staining) and ultrastructural changes, for myocardial apoptosis (apoptotic index＝ number of apoptotic cells/total number of cardiomyocytes×100%) with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, and for the expression of LC 3 and Beclin 1 in myocardial cells with Western blot. Following MI/RI, H．E. staining revealed a disorder of arrangement of cardiomyocytes with vague border, inflammatory cell infiltration, intracellular swelling with bleeding, necrosis and dissolution of partial striated muscles of the left ventricle under light microscope, and dual staining of Uranyl acetate and leadnitrate showed atrophy, arrangement disorder, dissolution, necrosis, and interstitial edema of partial myocardial fibers, mitochondrial structural disorder, vacolation, and large body of autophagosomes with bilayers, etc. in ultrastructure, which was relatively lighter in both EA and Moxi groups. The apoptosis index, expression levels of myocardial LC 3 Ⅱ and Beclin 1 and the ratio of LC 3 Ⅱ/LC 3 Ⅰ were significantly higher in the model group than those in the sham operation group (P<0.01), but the expression level of LC 3 Ⅰ was considerably down-regulated in the model group relevant to the sham operation group (P<0.01). Following the intervention and MI preconditioning, the increased apoptosis index and expression levels of LC 3Ⅱ and Beclin 1 proteins and the ratio of LC 3Ⅱ/LC 3 Ⅰ were obviously down-regulated in the IP, EA and Moxi groups relevant to the model group (P<0.01), and the decreased expression of LC 3 Ⅰ protein was up-regulated obviously in the 3 treatment groups (P<0.05，P<0.01). The effects of EA were significantly superior to those of IP and Moxi groups in down-regulating apoptosis index and expression of LC 3 Ⅱ and Beclin 1 and the ratio of LC 3 Ⅱ/LC 3 Ⅰ and in up-regulating expression of LC 3 Ⅰ (P<0.05, P<0.01). Both EA and Moxi preconditioning of PC 6 have a protective effect on ischemic myocardium in MI/RI rats, which is probably related to their effects in regulating expression of myocardial autophagy proteins as LC 3 Ⅰ/Ⅱ and Beclin 1.

Effects of electroacupuncture preconditioning on cerebral infarct volume and contents of TNF-α, IL-10 in serum of rats after cerebral ischemia-reperfusion injury

To explore the effect of electroacupuncture(EA) preconditioning on cerebral infarct volume and the contents of TNF-α, IL-10 in serum of rats with cerebral ischemia-reperfusion injury. Thirty-six rats were randomly divided into a sham operation group, a model group and an EA preconditioning group, 12 rats in each group, which were further divided into 12 h and 24 h after reperfusion subgroups, 6 rats in each one. EA was used before model establishment for 2 weeks in the EA preconditioning group. The model of cerebral ischemia-reperfusion injury in rats was established with modified Longa suture method. 12 h and 24 h after reperfusion, the degree of neurological deficit was assessed by the modified behavioral scoring scale; the cerebral infarct volume was measured by TTC method and the contents of TNF-α, IL-10 in serum were detected by ELISA method. Compared with the model group, the neurological severity scores in the EA preconditioning group significantly reduced 12 h and 24 h after reperfusion (both P<0.05), the cerebral infarct volume in the EA preconditioning group significantly reduced 12 h and 24 h after reperfusion (both P<0.05). Compared with the sham operation group, the serum TNF-α, IL-10 contents in the model group increased 12 h and 24 h after reperfusion (both P<0.05). Compared with the model group, the serum TNF-α content reduced, while the serum IL-10 content increased in the EA preconditioning group 12 h after reperfusion (both P<0.05). Compared with the model group, the serum TNF-α, IL-10 contents reduced in the EA preconditioning group 24 h after reperfusion (both P<0.05). EA preconditioning can improve neurological deficit, reduce cerebral infarct volume after cerebral ischemia-reperfusion injury in rats. The mechanism may be related to the regulation of EA on the dynamic balance between pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 in peripheral blood of cerebral ischemia-reperfusion injury in acute phase, thus alleviate acute cerebral ischemia-reperfusion inflammatory response.

Electroacupuncture preconditioning and postconditioning inhibit apoptosis and neuroinflammation induced by spinal cord ischemia reperfusion injury through enhancing autophagy in rats

Electroacupuncture (EA) has beneficial effects on spinal cord ischemia reperfusion (I/R) injury, but the underlying mechanisms are not fully understood. This study aimed to investigate the role of autophagy in the protection of EA preconditioning and postconditioning against spinal cord I/R injury. For this, spinal cord I/R injury was induced by 14min occlusion of the aortic arch, and rats were treated with EA for 20min before or after the surgery. The expression of autophagy components, light chain 3 and Beclin 1, was assessed by Western blot. The hind-limb motor function was assessed using the Basso-Beattie-Bresnahan (BBB) criteria, and motor neurons in the ventral gray matter were counted by histological examination. The apoptosis of neurocyte was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and matrix metalloproteinase-9 (MMP-9) was also measured using Western blot or enzyme-linked immunosorbent assay (ELISA). Either EA preconditioning or postconditioning enhanced autophagy, and minimized the neuromotor dysfunction and histopathological deficits after spinal cord I/R injury. In addition, EA suppressed I/R-induced apoptosis and increased in the expression of TNF-α, IL-1β, and MMP-9. In contrast, the autophagic inhibitor (3-methyladenine, 3-MA) inhibited the neuroprotective effects of EA. Moreover, 3-MA increased the apoptosis and the expression of TNF-α, IL-1β, and MMP-9. In summary, these findings suggested that EA preconditioning and postconditioning could alleviate spinal cord I/R injury, which was partly mediated by autophagy upregulation-induced inhibition of apoptosis and neuroinflammation.

Electroacupuncture preconditioning reduces ROS generation with NOX4 down-regulation and ameliorates blood-brain barrier disruption after ischemic stroke.

BackgroundElectroacupuncture (EA) is a modern application based on combination of traditional manual acupuncture and electrotherapy that is frequently recommended as an adjuvant treatment for ischemic stroke. EA preconditioning can ameliorate blood-brain barrier (BBB) dysfunction and brain edema in ischemia-reperfusion injury; however, its mechanism remains unclear. This study investigated the preventive effects of EA preconditioning, particularly on BBB injury, followed by a transient middle cerebral artery occlusion (MCAO) model in mice.ResultsMice were treated with EA (20 min) at Baihui (GV20) and Dazhui (GV14) acupoints once a day for 3 days before ischemic injury. Infarct volume, neurological deficits, oxidative stress, Evans blue leakage and brain edema were evaluated at 24 h after ischemia-reperfusion injury. EA preconditioning significantly decreased infarct volume and improved neurological function even after ischemic injury. In addition, both Evans blue leakage and water content were significantly reduced in EA preconditioned mice. Whereas the expression of tight junction proteins, ZO-1 and claudin-5, were remarkably increased by EA preconditioning. Mice with EA preconditioning showed the reduction of astrocytic aquaporin 4, which is involved in BBB permeabilization. In addition, we found that EA preconditioning decreased reactive oxygen species (ROS) in brain tissues after ischemic injury. The expression of NADPH oxidase 4 (NOX4), not NOX2, was significantly suppressed in EA preconditioned mice.ConclusionsThese results suggest that EA preconditioning improve neural function after ischemic injury through diminishing BBB disruption and brain edema. And, the reduction of ROS generation and NOX4 expression by EA preconditioning might be involved in BBB recovery. Therefore, EA may serve as a potential preventive strategy for patients at high risk of ischemic stroke.

Role of HO-1 in protective effect of electro-acupuncture against endotoxin shock-induced acute lung injury in rabbits

Heme oxygenase (HO)-1 has been reported to play a great role in attenuating lung injury during endotoxic shock in our previous research. Although electro-acupuncture has been explored to reduce oxidative stress and decrease inflammatory reaction in animals with endotoxic shock, the mechanism of this effect is still unclear. The aim of this study was to determine whether HO-1 is involved in the effect of electro-acupuncture on the injured lung during endotoxic shock in rabbits. Sixty New England white rabbits were randomly divided into groups C, Z, ES, EA, AP, and EAZ. Before inducing endotoxic shock, group ES received no electro-acupuncture, while group EA received electro-acupuncture at ST36 (zusanli) and BL13 (feishu) acupoints on both sides for five days and group AP received electro-acupuncture (EA) stimulation at a non-acupoint. Groups ES, AP, EA, and EAZ received LPS to replicate the experimental model of injured lung induced by endotoxic shock, and electro-acupuncture was performed throughout the procedure with the same parameter. Groups EAZ and Z received the HO-1 inhibitor, ZnPP-IX, intraperitoneally. The animals were sacrificed by blood-letting at 6 h after LPS administration. The blood samples were collected for serum examination, and the lungs were removed for pathology examination, detection of alveolaer epithelial cell apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL assay), determination of wet to dry ratio, measurement of Evans blue (EB) contents, and determination of HO-1protein and mRNA expression. According to the results, EA at ST36 and BL13 could increase the expression of HO-1. At the same time, index of quantitative assessment (IQA) score and the number of TUNEL-positive cells decreased, while electro-acupuncture at the other points did not exert this effect, and pretreatment with ZnPP-IX in group EAZ suppressed the efficacy of electro-acupuncture preconditioning. In summary, electro-acupuncture stimulation at ST36 and BL13, while not the non-acupoint, could attenuate the lung injury during the endotoxic shock, and this effect was due to increased expression of HO-1.

Involvement of δ-and μ-opioid receptors in the delayed cerebral ischemic tolerance induced by repeated electroacupuncture preconditioning in rats

Preconditioning with repeated electroacupuncture (EA) could mimic ischemic preconditioning to induce cerebral ischemic tolerance in rats. The present study was designed to investigate whether mu (micro)-, delta (delta)- or kappa (kappa)-opioid receptors are involved in the neuroprotection induced by repeated EA preconditioning. The rats were pretreated with naltrindole (NTI), nor-binaltorphimine (nor-BNI) or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), which is a highly selective delta-, kappa- or micro-opioid receptor antagonist respectively, before each EA preconditioning (30 minutes per day, 5 days). Twenty-four hours after the last EA treatment, the middle cerebral artery occlusion (MCAO) was induced for 120 minutes. The brain infarct volume was determined with 2, 3, 5-triphenyltetrazolium chloride staining at 24 hours after MCAO and compared with that in rats which only received EA preconditioning. In another experiment, the met-enkephalin-like immunoreactivity in rat brain was investigated by immunohistochemistry in both EA preconditioning and control rats. The EA preconditioning reduced brain infarct volume compared with the control rats (P = 0.000). Administration of both NTI and CTOP attenuated the brain infarct volume reduction induced by EA preconditioning, presenting with larger infarct volume than that in the EA preconditioning rats (P < 0.001). But nor-BNI administration did not block the infarct volume reduction induced by EA preconditioning, presenting with smaller infarct volume than the control group rats (P = 0.000). The number of met-enkephalin-like immunoreactivity positive neurons in the EA preconditioning rats was more than that of the control rats (P = 0.000). Repeated EA preconditioning stimulates the release of enkephalins, which may bind delta- and micro-opioid receptors to induce the tolerance against focal cerebral ischemia.

Rapid tolerance to focal cerebral ischemia in rats is induced by preconditioning with electroacupuncture: window of protection and the role of adenosine

The aim of the present study was to investigate the first protective window of preconditioning with electroacupuncture (EA) against focal cerebral ischemia, and to explore whether adenosine is involved in the rapid tolerance phenomenon. Sixty-four male Sprague–Dawley rats were randomly assigned to eight groups ( n = 8 in each). Animals in the control group received no treatment, and animals in EA1–EA4 groups received EA at 0.5, 1, 2 and 3 h before induction of focal cerebral ischemia, respectively. Rats in DPCPX group were intraperitoneally injected with 1 mg kg −1 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 3 h before induction of focal cerebral ischemia. Animals in vehicle group and EA + DPCPX group were pretreated with 1 ml kg −1 dimethyl sulfoxide (DMSO, the solvent of DPCPX) and 1 mg kg −1 DPCPX 30 min before preconditioning with EA, respectively. All rats were anesthetized with 40 mg kg −1 pentobarbital sodium intraperitoneally. Animals that required EA preconditioning, received EA with intensity of 1 mA and frequency of 15 Hz at the Baihui acupoint (GV 20) for 30 min. The focal cerebral ischemia was produced by the right middle cerebral artery occlusion (MCAO) for 120 min. The neurologic deficit scores (NDS) and brain infarct volumes were evaluated at 24 h after reperfusion. All rats survived until 24 h after reperfusion. Preconditioning with EA at 2 h before induction of focal cerebral ischemia improved neurologic outcome ( P < 0.05 versus control) and reduced the infarct volume ( P < 0.01 versus control) at 24 h after reperfusion. These beneficial effects were reversed by pretreatment with 1 mg kg −1 DPCPX, whereas this agent itself did not affect the NDS and volume in drug-ischemic controls after ischemia. The results show that preconditioning with single EA session induces rapid tolerance to focal cerebral ischemia. The rapid ischemic tolerance appears at 2 h (but not at 0.5, 1, or 3 h) after preconditioning, and is possibly mediated through an adenosine A1 receptor-related mechanism.

Electro-acupuncture preconditioning abrogates the elevation of c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brains induced by glibenclamide, an ATP-sensitive potassium channel blocker

This study aimed to clarify the neuroprotective mechanism of electro-acupuncture (EA) preconditioning on hypoxic-ischemic brain injury (HIBI). Using Western blot, the expression of c-fos protein (c-Fos) and c-jun protein (c-Jun) induced by glibenclamide, an ATP-sensitive potassium (K ATP) channel blocker was examined from cerebral cortical and hippocampal samples in neonatal hypoxic-ischemic rats, with or without EA preconditioning. EA was performed on Hegu (LI4), a well-known acupoint commonly used in Oriental medicine for the treatment of neuronal injury resulting from hypoxia–ischemia (HI). Preconditioned rats were treated with either diazoxide, a K ATP channel opener, glibenclamide, or sterile saline injected into the left lateral ventricle (i.c.v.), with or without EA administration before HI insult. Interestingly, low c-Fos and c-Jun expressions were found both in diazoxide and EA groups, 24 h after HI. Furthermore, significant differences in relative optical density (ROD) were found between glibenclamide and HI control groups ( P≤0.05), as well as between the group administered glibenclamide after EA and the HI control group ( P≤0.05). However, the level of c-Fos and c-Jun expression in the group administered glibenclamide after EA was significantly lower than in the glibenclamide group ( P≤0.05). The present findings indicate that the effectiveness of EA preconditioning against HIBI may be mediated via the opening of K ATP channels.