EA improved cerebral perfusion, the neurological function and motor function in moderate ischemic injury.

<p>(A) Effect of EA on perfusion measured by laser Doppler flowmetry in moderate ischemic injury (B) Neurological deficit and motor deficit were assessed 24 h after ischemia. Neurological function was assessed by neurological score, and vestibule-motor function was assessed by the wire grip test. Acute EA treatment after ischemia significantly improved the neurological function and motor function. The results are expressed as means ± SEM. <b>*</b>, <i>P</i><0.05 and <b>**</b>, <i>P</i><0.01 vs. control group unpaired t-test, N = 6.</p

EA increased cerebral perfusion via the muscarinic acetylcholine receptor.

<p>Propranolol (2 mg/kg), a β-adrenergic receptor blocker, phentolamine (10 mg/kg), an α-adrenergic receptor blocker, mecamylamine (2 mg/kg), a blood brain barrier permeable nicotinic acetylcholine receptor blocker, or atropine (5 mg/kg), a blood brain barrier permeable muscarinic acetylcholine receptor blocker were administered intravenously 30 min prior to EA stimulation (arrow). The vehicle groups received saline intravenously in the same volume as the blockers. The horizontal bar represents the EA stimulation period. CBF measurement was conducted for 5 min before EA stimulation, 20 min during EA and 20 min after EA, lasting a total of 45 min. The perfusion responses elicited by EA were almost abolished by atropine (<b>**</b>, <i>P</i><0.01 vs. vehicle group, two-way ANOVA for repeated measures, N = 4) not propranolol, phentolamine or mecamylamine. Vertical error bars indicate ± SEM.</p

Cerebral perfusion responses elicited by EA abolished in eNOS KO.

<p>(A) Cerebral blood flow (CBF) time course after EA (arrow) from a representative experiment from the EA group of C57BL/6J and from the EA group of eNOS KO, and (B) the average of ten experiments. The control groups received the same electrical stimulation at non-acupuncture points. The horizontal bar represents the EA stimulation period. CBF measurement was conducted for 5 min before EA stimulation, 20 min during EA and 20 min after EA, lasting a total of 45 min. The cerebral perfusion response elicited by EA was significantly attenuated in eNOS KO (<b>**</b>, <i>P</i><0.01 vs. control group; ^##, <i>P</i><0.01 vs. EA group of C57BL/6J, two-way ANOVA for repeated measures). Vertical error bars indicate ± SEM.</p

EA improved tissue outcome in moderate ischemic injury, but not severe ischemic injury.

<p>Mice were subjected to 60 min and 90 min MCA occlusion followed by 24-h reperfusion. The mice received 20 min-EA stimulation immediately after the onset of occlusion. (A) Representative photographs of coronal brain sections following infarction stained with 2,3,5-triphenyltetrazolium chloride. The red area is healthy tissue and the white area is infarct tissue. (B) Quantification of indirect infarct volume at 24 h after ischemia. Infarct volume was calculated by integrating the infarct area in 2-mm-thick coronal slices. Results are expressed as means ± SEM. <b>*</b>, <i>P</i><0.05 vs. control group, unpaired t-test, N = 6.</p

EA at Baihui (GV20) and Dazhui (GV14) increased cerebral perfusion in the cerebral cortex, not blood pressure.

<p>(A) Mean arterial blood pressure (MABP, red) and cerebral blood flow (CBF, blue) time course after EA (arrow) from a control group and EA group, and (B) the average of ten experiments. The control groups received the same electrical stimulation at non-acupuncture points. The horizontal bar represents the EA stimulation period. MABP and CBF measurement were conducted for 5 min before EA stimulation, 20 min during EA and 20 min after EA, lasting a total of 45 min. EA significantly increased cerebral perfusion (<b>**</b>, <i>P</i><0.01 vs. control group, two-way ANOVA for repeated measures). Vertical error bars indicate ± SEM.</p

Physiological parameters.

<p>Values are the means ± SEM. MABP (mean arterial blood pressure), pO₂, and pCO₂ are expressed in mmHg.</p

EA increased acetylcholine production and muscarinic acetylcholine receptor expression in the cerebral cortex.

<p>(A) Acetylcholine levels in the cerebral cortex 20 min after the end of EA stimulation were analyzed by ELISA. EA significantly increased acetylcholine (<b>**</b>, <i>P</i><0.01 vs. control group, unpaired t-test, N = 4). Vertical error bars indicate ± SEM. (B) Representative immunohistochemical staining photographs showed muscarinic acetylcholine receptor (mAChR) M3-positive cell expression 20 min after the end of EA stimulation in the cerebral cortex of mice. The red rectangle represents the imaging field. EA stimulation increased mAChR M3 expression in the cerebral cortex. Scale bar = 100 µm.</p

Schematic diagram for experimental procedures.

<p>All experiments were performed on the indicated day.</p

Effects of EA treatment on neurogenesis at 14 days after MCAO.

<p>Schematic diagram shows the regions of the hippocampus (★, in and around the hippocampus), SVZ (*, including the striatum) and cortex (#, the whole cortex) of the brain for immunohistochemical and further Western blot analysis (A). Photomicrograph (B) and its histogram for BrdU/Dcx double-positive cells in the hippocampus (C), SVZ (D) and cortex (E) of the brain. BrdU/Dcx double-positive cells were significantly increased by EA treatment in the hippocampus and SVZ of the ipsilateral hemisphere. ^#<i>P</i><0.05, ^##<i>P</i><0.01 and ^###<i>P</i><0.001 versus the contralateral hemisphere; *<i>P</i><0.05 and **<i>P</i><0.01 versus MCAO mice. Scale bars = 100 µm.</p

RT-PCR analysis for growth and neurotropic factors at 14 days after MCAO.

<p>Expression of each mRNA is expressed as a percentage of contralateral hemisphere of MCAO. Panel (A) its densitometric analysis (B). Panel represents a typical result from three independent experiments. EA treatment significantly increased the mRNA level of BDNF and VEGF in the ipsilateral hemisphere. *<i>P</i><0.05 and ***<i>P</i><0.001 versus MCAO mice.</p