data_sheet_2_High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+–Calmodulin-Dependent Protein Kinase II–cAMP-Response Element-Binding Protein Pathway.xlsx

<p>Repetitive transcranial magnetic stimulation (rTMS) can be used in various neurological disorders. However, neurobiological mechanism of rTMS is not well known. Therefore, in this study, we examined the global gene expression patterns depending on different frequencies of repetitive magnetic stimulation (rMS) in both undifferentiated and differentiated Neuro-2a cells to generate a comprehensive view of the biological mechanisms. The Neuro-2a cells were randomly divided into three groups—the sham (no active stimulation) group, the low-frequency (0.5 Hz stimulation) group, and high-frequency (10 Hz stimulation) group—and were stimulated 10 min for 3 days. The low- and high-frequency groups of rMS on Neuro-2a cells were characterized by transcriptome array. Differentially expressed genes were analyzed using the Database of Annotation Visualization and Integrated Discovery program, which yielded a Kyoto Encyclopedia of Genes and Genomes pathway. Amphetamine addiction pathway, circadian entrainment pathway, long-term potentiation (LTP) pathway, neurotrophin signaling pathway, prolactin signaling pathway, and cholinergic synapse pathway were significantly enriched in high-frequency group compared with low-frequency group. Among these pathways, LTP pathway is relevant to rMS, thus the genes that were involved in LTP pathway were validated by quantitative real-time polymerase chain reaction and western blotting. The expression of glutamate ionotropic receptor N-methyl d-aspartate 1, calmodulin-dependent protein kinase II (CaMKII) δ, and CaMKIIα was increased, and the expression of CaMKIIγ was decreased in high-frequency group. These genes can activate the calcium (Ca²⁺)–CaMKII–cAMP-response element-binding protein (CREB) pathway. Furthermore, high-frequency rMS induced phosphorylation of CREB, brain-derived neurotrophic factor (BDNF) transcription via activation of Ca²⁺–CaMKII–CREB pathway. In conclusion, high-frequency rMS enhances the expression of BDNF by activating Ca²⁺–CaMKII–CREB pathway in the Neuro-2a cells. These findings may help clarify further therapeutic mechanisms of rTMS.</p

data_sheet_1_High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+–Calmodulin-Dependent Protein Kinase II–cAMP-Response Element-Binding Protein Pathway.xlsx

<p>Repetitive transcranial magnetic stimulation (rTMS) can be used in various neurological disorders. However, neurobiological mechanism of rTMS is not well known. Therefore, in this study, we examined the global gene expression patterns depending on different frequencies of repetitive magnetic stimulation (rMS) in both undifferentiated and differentiated Neuro-2a cells to generate a comprehensive view of the biological mechanisms. The Neuro-2a cells were randomly divided into three groups—the sham (no active stimulation) group, the low-frequency (0.5 Hz stimulation) group, and high-frequency (10 Hz stimulation) group—and were stimulated 10 min for 3 days. The low- and high-frequency groups of rMS on Neuro-2a cells were characterized by transcriptome array. Differentially expressed genes were analyzed using the Database of Annotation Visualization and Integrated Discovery program, which yielded a Kyoto Encyclopedia of Genes and Genomes pathway. Amphetamine addiction pathway, circadian entrainment pathway, long-term potentiation (LTP) pathway, neurotrophin signaling pathway, prolactin signaling pathway, and cholinergic synapse pathway were significantly enriched in high-frequency group compared with low-frequency group. Among these pathways, LTP pathway is relevant to rMS, thus the genes that were involved in LTP pathway were validated by quantitative real-time polymerase chain reaction and western blotting. The expression of glutamate ionotropic receptor N-methyl d-aspartate 1, calmodulin-dependent protein kinase II (CaMKII) δ, and CaMKIIα was increased, and the expression of CaMKIIγ was decreased in high-frequency group. These genes can activate the calcium (Ca²⁺)–CaMKII–cAMP-response element-binding protein (CREB) pathway. Furthermore, high-frequency rMS induced phosphorylation of CREB, brain-derived neurotrophic factor (BDNF) transcription via activation of Ca²⁺–CaMKII–CREB pathway. In conclusion, high-frequency rMS enhances the expression of BDNF by activating Ca²⁺–CaMKII–CREB pathway in the Neuro-2a cells. These findings may help clarify further therapeutic mechanisms of rTMS.</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 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

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

Up-regulation of ICAM-1 expression and leukocyte adhesion on pulmonary endothelium by <i>E. coli</i> OMVs. (A-C)

<p>C57BL/6 wild-type mice were intraperitoneally administered with PBS or <i>E. coli</i> OMVs (15 µg in total protein/mouse; n  =  5). <b>A.</b> The number of neutrophils in BAL fluid after OMVs injection. <b>(B and C)</b> Six hours after the administration, the lungs were harvested. <b>B.</b> Immunohistochemistry with confocal microscopy of ICAM-1 (green), endothelial cell marker CD31 (red), and nuclei (blue) in the lungs (scale bars, 100 µm). White arrows indicate ICAM-1 positive endothelial cells. <b>C.</b> Hematoxylin and eosin staining of the lungs (scale bars, 100 µm). Black arrows indicate leukocytes on the pulmonary endothelium. <b>(D and E)</b> C57BL/6 wild-type and ICAM-1^-/- mice were intraperitoneally administered with PBS or <i>E. coli</i> OMVs (15 µg in total protein/mouse). Six hours after the administration, the lungs were harvested (n  =  3). <b>D.</b> Immunohistochemistry with confocal microscopy of a neutrophil marker NIMP-R14 (green) and nuclei (blue) in the lungs (scale bars, 50 µm). <b>E.</b> The number of neutrophils per field. *<i>P</i><0.05; **P<0.01; ***<i>P</i><0.001. Results are represented as means ± SEM.</p