Effects of electroacupuncture on the functionality of NG2-expressing cells in perilesional brain tissue of mice following ischemic stroke

Abstract

Neural/glial antigen 2 (NG2)-expressing cells has multipotent stem cell activity under cerebral ischemia. Our study examined the effects of electroacupuncture (EA) therapy (2 Hz, 1 or 3 mA, 20 minutes) at the Sishencong acupoint on motor function after ischemic insult in the brain by investigating the rehabilitative potential of NG2-derived cells in a mouse model of ischemic stroke. EA stimulation alleviated motor deficits caused by ischemic stroke, and 1 mA EA stimulation was more efficacious than 3 mA EA stimulation or positive control treatment with edaravone, a free radical scavenger. The properties of NG2-expressing cells were altered with 1 mA EA stimulation, enhancing their survival in perilesional brain tissue via reduction of tumor necrosis factor alpha expression. EA stimulation robustly activated signaling pathways related to proliferation and survival of NG2-expressing cells and increased the expression of neurotrophic factors such as brain-derived neurotrophic factor, tumor growth factor beta, and neurotrophin 3. In the perilesional striatum, EA stimulation greatly increased the number of NG2-expressing cells double-positive for oligodendrocyte, endothelial cell, and microglia/macrophage markers (CC1, CD31, and CD68). EA therapy also greatly activated brain-derived neurotrophic factor/tropomyosin receptor kinase B and glycogen synthase kinase 3 beta signaling. Our results indicate that EA therapy may prevent functional loss at the perilesional site by enhancing survival and differentiation of NG2-expressing cells via the activation of brain-derived neurotrophic factor -induced signaling, subsequently ameliorating motor dysfunction. The animal experiments were approved by the Animal Ethics Committee of Pusan National University (approval Nos. PNU2019-2199 and PNU2019-2884) on April 8, 2019 and June 19, 2019.

Keywords: brain-derived neurotrophic factor; differentiation; electroacupuncture; motor function; neural/glial antigen 2; perilesional striatum; stroke; survival.

Figure 1

Experimental schematic diagram and the effects of EA stimulation on the improvement of motor functions ofmice following cerebral ischemia. The Sishencong acupoint (A). Experimental timeline of EA treatment, BrdU labeling, edaravone administration, and behavioral testing at 10 and 17 days after MCAO (B). Quantification of the motor function test results of the corner test (C), wire grip test (D), and rotarod test (E). 1 mA EA stimulation was more efficacious than 3 mA EA or edaravone treatment in attenuating motor dysfunction in MCAO mice. n = 6. All data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, vs. control group; #P < 0.05, ###P < 0.001, vs. MCAO group; &P < 0.05, &&P < 0.01, vs. MCAO + EA1 group (one-way analysis of variance with Tukey's post hoc test). EA1: Electroacupuncture at 1 mA; EA3: electroacupuncture at 3 mA; MCAO: middle cerebral artery occlusion.

Figure 2

EA stimulation on the proliferation of NG2-expressing cells in the perilesional striatum and corpus callosum of mice at 21 days after MCAO. Photomicrographs (A) and histograms showing the labeling and quantification of NG2- (green), BrdU- (red) and Ki67 (red) -positive cells in the perilesional striatum (B) and corpus callosum (C). Photomicrographs (D) and histogram showing the labeling and quantification of NG2 (green) and PDGFRα (red) double-positive cells in the perilesional striatum (E). EA stimulation significantly decreased the number of newly generated NG2-expressing cells in the perilesional striatum, relative to the number in the MCAO control group (without EA1 stimulation or edaravone treatment). All data are shown as mean ± SEM (n = 6). #P < 0.05, ##P < 0.01, ###P < 0.001, vs. MCAO group; &P < 0.05, &&P < 0.01, &&&P < 0.001, vs. MCAO + EA1 group (one-way analysis of variance with Tukey's post hoc test). Scale bars in A and D: 20 μm. BrdU: Bromodeoxyuridine; EA1: electroacupuncture at 1 mA; MCAO: middle cerebral artery occlusion; NG2: neural/glial antigen 2; PDGFRα: platelet-derived growth factor receptor alpha.

Figure 3

EA stimulation on the cell death of NG2-expressing cells in the perilesional striatum of mice at 21 days after MCAO. Photomicrographs (A) and histograms showing the labeling and quantification of TNFα (red; B and C), TUNEL (red; D), and cleaved caspase 3 (cCaspase-3, red; E)-positive cells in the perilesional striatum. EA stimulation significantly decreased the cell death of NG2 (green) -expressing cells. All data are shown as mean ± SEM (n = 6). #P < 0.05, ##P < 0.01, ###P < 0.01, vs. MCAO group; &P < 0.05, &&P < 0.01, &&&P < 0.001, vs. MCAO + EA1 group (one-way analysis of variance with Tukey's post hoc test). Scale bar in A = 20 μm. DAPI: 4′,6-Diamidino-2-phenylindole; EA1: electroacupuncture at 1 mA; IOD: integrated optical density; MCAO: middle cerebral artery occlusion; NG2: neural/glial antigen 2; TNFα: tumor necrosis factor alpha; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 4

Effects of EA stimulation on the expression of signaling markers related to the activation of NG2-expressing cells and on the upregulation of neurotrophic factors in the perilesional striatum of mice at 21 days after MCAO. Experimental timeline of MCAO generation, EA treatment, and sampling (A). Western blots of signaling markers related to NG2 activation (B) and neurotrophic factor expression (D), and their quantification by densitometric analysis (C and E). EA stimulation upregulated signaling pathways related to NG2 cell activation and neurotrophic factor expression involving BDNF, TGFβ, and NT3. All data are shown as mean ± SEM (n = 6). #P < 0.05 and ###P < 0.001, vs. MCAO (independent samples t-test). EA1: Electroacupuncture at 1 mA; GDNF: glial cell-derived neurotrophic factor; MCAO: middle cerebral artery occlusion; NG2: neural/glial antigen 2; NGF: nerve growth factor; NT-3: neurotrophin-3; pAKt: phosphorylated Akt; pERK: phosphorylated ERK; pGSK3β: phosphorylated GSK3β; TGFβ: transforming growth factor-β. Uncropped images of western blot are shown in Additional Figure 1.

Figure 5

Effects of EA stimulation on NG2-expressing cell types in the perilesional striatum and the corpus callosum of mice at 21 days after MCAO. Photomicrographs (A) and histograms (B and C) showing labeling and quantification of NG2 (green) -, CC1 (red) -, CD31 (red) - and CD68 (red) -positive cells in the perilesional striatum and the corpus callosum of MCAO mice. EA stimulation significantly increased the NG2 and CD68double positive cells in the perilesional striatum. n = 6. All data are shown as mean ± SEM. ##P < 0.01, vs. MCAO group; &P < 0.05, vs. MCAO + EA1 group (one-way analysis of variance with Tukey's post hoc tests). Scale bar in A: 20 μm. DAPI: 4′,6-Diamidino-2-phenylindole; EA1: electroacupuncture at 1 mA; MCAO: middle cerebral artery occlusion; NG2: neural/glial antigen 2.

Figure 6

Effects of EA stimulation on the expression of BDNF, pTrkB, and GSK3β in the perilesional striatum of mice at 21 days after MCAO. Photomicrograph (A) and histograms (B and C) showing labeling of BDNF (red) -, pTrkB (red) - and GSK3β(red) -positive cells in the perilesional striatum of MCAO mice. The number of NG2⁺/BDNF⁺ and NG2⁺/pTrkB⁺ cells was significantly increased after EA1 stimulation. n = 6. All data are shown as mean ± SEM. #P < 0.05 and ##P < 0.01, vs. MCAO group; &P < 0.05, vs. MCAO + EA1 group (one-way analysis of variance with Tukey's post hoc test). Scale bar in A: 20 μm. BDNF: Brain-derived neurotrophic factor; DAPI: 4′,6-Diamidino-2-phenylindole; EA1: electroacupuncture at 1 mA; MCAO: middle cerebral artery occlusion; NG2: neural/glial antigen 2; (p)GSK3β: (phosphorylated) glycogen synthase kinase 3 beta; pTrkB: phosphorylated tyrosine receptor kinase B.

Figure 7

Effects of EA stimulation on cellular phenotype and the expression of BDNF and GSK3β in GFP⁺/DAPI⁺ NG2-expressing cells, or whole cells in ipsilateral perilesional striatum of NG2-mEGFP mice at 21 days after MCAO. Quantitative analysis of the flow cytometry data (A and C) and histograms showing the percentage of GFP⁺/DAPI⁺ cells also expressing BrdU, CC1, CD31, CD68, and GSK3β, as well as the percentage of DAPI⁺ cells also expressing BDNF (B and D, respectively). The numbers of GFP⁺ cells that also express BrdU, CC1, CD31and GSKβ were significantly increased after EA1 stimulation. n = 5. All data are shown as mean ± SEM. #P < 0.05, ##P < 0.01, and ###P < 0.01, vs. MCAO group (independent samples t-test). BDNF: Brain-derived neurotrophic factor; BrdU: bromodeoxyuridine; DAPI: 4′,6-Diamidino-2-phenylindole; EA1: Electroacupuncture at 1 mAEA1: electroacupuncture at 1 mA; GFP: green fluorescent protein; GSK3β: glycogen synthase kinase 3 beta; MCAO: middle cerebral artery occlusion; mEGFP: mutated enhanced green fluorescent protein; NG2: neural/glial antigen 2.