Electroacupuncture stimulates the proliferation and differentiation of endogenous neural stem cells in a rat model of ischemic stroke

Abstract

Electroacupuncture (EA) may stimulate neurogenesis in animal models of ischemic stroke; however, the associated mechanisms are not clear. The present study aimed to evaluate the neurogenesis efficacy of EA on ischemic stroke and the underlying associated mechanisms. A model of middle cerebral artery occlusion (MCAO) was employed as the rat model of brain ischemia and reperfusion. EA treatment at the GV20 (Baihui) and GV14 (Dazhui) acupoints was conducted for 30 min daily following MCAO. Immunofluorescence was performed to measure the number of bromodeoxyuridine (BrdU)/nestin- or BrdU/doublecortin (DCX)-positive cells in the sham, MCAO and MCAO + EA groups. Results indicated that EA stimulation significantly decreased the neurological score and neuronal loss in rats in the MCAO group (both P<0.05). Furthermore, immunostaining assays indicated that BrdU/nestin- and BrdU/DCX-positive cells in EA-treated rats were significantly increased (P<0.05) when compared with the rats in the MCAO group, indicating EA may induce the proliferation and differentiation of endogenous neural stem cells (eNSCs) during cerebral ischemia-reperfusion. In addition, EA treatment significantly enhanced the protein expression levels of plasticity-related gene 5 (PRG5), a critical neurogenesis factor, and significantly decreased the protein expression levels of three neurogenesis inhibiting molecules, NogoA, lysophosphatidic acid and RhoA (all P<0.05). These results suggested that EA promotes the proliferation and differentiation of eNSCs, likely through modulating PRG5/RhoA signaling.

Keywords: RhoA; cerebral ischemia; electroacupuncture; neural stem cells; plasticity-related gene 5.

Figure 1.

Neurological scores of rats following focal ischemia-reperfusion injury. Neurological scores of rats in each group at day (A) 1, (B) 7 and (C) 14 after reperfusion. The scores in the MCAO + EA group were significantly decreased compared with those in the MCAO group, which received sham stimulation at day 1, 7 and 14 following reperfusion. Data are represented as the mean ± standard error of the mean (n=6 in each group). ^#P<0.05 vs. Sham group; *P<0.05 vs. MCAO group. MCAO, middle cerebral artery occlusion model; EA, electroacupuncture.

Figure 2.

EA treatment protects hippocampal CA1 neurons against ischemic injury. Representative micro-photographs of Nissl-stained neurons in hippocampal CA1 regions at day 14 following reperfusion in mice. (A) Sham, (B) MCAO with sham stimulation at day 14 and (C) MCAO + EA at day 14. (D) CA1 neurons were counted and analyzed 14 days following reperfusion. The percentage of viable neurons was significantly decreased in the CA1 region in the MCAO group compared with those in the sham group, whereas the percentage of viable neurons was significantly increased the MCAO + EA group compared with MCAO group at day 14 following reperfusion. Data were represented as the mean + standard error of the mean (n=6 in each group). *P<0.05 vs. Sham group; ^#P<0.05 vs. MCAO group. MCAO, middle cerebral artery occlusion model; EA, electroacupuncture.

Figure 3.

EA treatment promotes proliferation of endogenous neural stem cells in rats with MCAO. Representative DG zone from (A) sham, (B) MCAO and (C) MCAO + EA groups were subjected to immunofluorescence labeling with BrdU (red) and nestin (green) staining. Results suggested that the number of BrdU/Nestin positive stained cells (yellow) was attenuated in the MCAO group compared with that in sham group. However, EA treatment increased the number of BrdU/Nestin positive cells compared with that in the MCAO group. Scale bar=20 µm. (D) The quantified data of nestin/BrdU positive cells. Data were represented as the mean + standard error of the mean (n=6 in each group). *P<0.05 vs. Sham group; ^#P<0.05 vs. MCAO group. MCAO, middle cerebral artery occlusion model; EA, electroacupuncture; BrdU, bromodeoxyuridine.

Figure 4.

EA treatment promotes neuron differentiation of endogenous neural stem cells in rats with MCAO. Representative images of BrdU- and DCX-positive cells in the DG Zone of the (A) Sham, (B) MCAO and (C) MCAO + EA groups at day 14. Groups were subjected to immunofluorescence labeling with BrdU (green), DCX (red) staining. Double-stained cells (yellow) represent newly generated immature neurons. White arrows indicate BrdU and DCX stained cells. Scale bar=20 µm. (D) Quantified results of nestin/DCX-positive cells. Data were represented as the mean + standard error of the mean (n=6 in each group). *P<0.05 vs. Sham group; ^#P<0.05 vs. MCAO group. MCAO, middle cerebral artery occlusion model; EA, electroacupuncture; BrdU, bromodeoxyuridine; DCX, doublecortin.

Figure 5.

EA treatment increases PRG5 expression and reduces NogoA-LPA/RhoA signaling in the brain following focal ischemia-reperfusion injury. (A) Protein expression levels of PRG5, RhoA, LPA and NogoA in the ischemic brains of rats at day 7 following reperfusion were determined by western blot analysis. Protien expression levels of (B) PRG5, (C) RhoA, (D) LPA and (E) NogoA were quantified using ImageJ software and normalized to β-actin. Data were represented as the mean + standard error of the mean (n=6 in each group). *P<0.05 vs. Sham group; ^#P<0.05 vs. MCAO group. MCAO, middle cerebral artery occlusion model; EA, electroacupuncture; PRG5, plasticity-related gene 5; LPA, lysophosphatidic acid.