Electroacupuncture promotes post-stroke functional recovery via enhancing endogenous neurogenesis in mouse focal cerebral ischemia

Abstract

To investigate the question of whether electroacupuncture (EA) promotes functional recovery via enhancement of proliferation and differentiation of neuronal stem cells (NSCs) in ischemic stroke, EA stimulation with 2 Hz was applied at bilateral acupoints to Baihui (GV20) and Dazhui (GV14) in middle cerebral artery occlusion (MCAO) mice. EA stimulation improved neuromotor function and cognitive ability after ischemic stroke. EA stimulation resulted in an increase in the number of proliferated cells, especially in the subventricular zone (SVZ) of the ipsilateral hemisphere. Although a very limited number of NSCs survived and differentiated into neurons or astrocytes, EA treatment resulted in a significant increase in the number of proliferative cells and differentiated cells in the hippocampus and SVZ of the ipsilateral hemisphere compared to MCAO mice. EA stimulation resulted in significantly increased mRNA expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Protein levels of these factors were confirmed in the ipsilateral hippocampus and SVZ by immunohistochemical and Western blotting analyses. Expression of phosphorylated phosphatidylinositol-3-kinase, BDNF, and VEGF-mediated down-stream were enhanced by EA stimulation in newly formed neuroblasts. These results indicate that EA treatment after ischemic stroke may promote post-stroke functional recovery by enhancement of proliferation and differentiation of NSCs via the BDNF and VEGF signaling pathway.

Figure 1. Schematic diagram for experimental procedures.

All experiments were performed on the indicated day.

Figure 2. Behavioral assessment.

Rotarod (A) and Morris water maze tests (B) on post-stroke behavior. EA treatment significantly improved neuromotor and cognitional function of MCAO mice at the late phase of the experiment. Mean±SEM. *P<0.05 and **P<0.01 versus MCAO mice.

Figure 3. Effects of EA treatment on neurogenesis in the whole brain at 26 days after MCAO.

Serial sections of the whole brain showing the regions for immunohistochemical analysis (A, cresyl violet stain) and its analysis for double-positive cells BrdU with neuroblast marker Dcx (B) or neuronal marker NeuN (C). Photomicrograph represents a typical result in the cortex. The number of proliferated NSCs indicated by BrdU positive was increased in both hemispheres of MCAO mice and EA treatment significantly increased the number of these cells in the posterior region of the brain. Total number of BrdU and Dcx or NeuN double-positive cells was significantly increased by EA stimulation. *P<0.05, **P<0.01 and ***P<0.001 versus MCAO mice. Scale bars = 100 µm.

Figure 4. Effects of EA treatment on neurogenesis at 14 days after MCAO.

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. ^# P<0.05, ^## P<0.01 and ^### P<0.001 versus the contralateral hemisphere; *P<0.05 and **P<0.01 versus MCAO mice. Scale bars = 100 µm.

Figure 5. Effects of EA treatment on neurogenesis at 47 days after MCAO.

Photomicrograph (B,E) and its histogram for BrdU/Dcx or NeuN double-positive cells in the hippocampus (B,F), SVZ (C,G) and cortex (D,H) of the brain. Both BrdU/NeuN and BrdU/GFAP double-positive cells were significantly increased by EA treatment in the ipsilateral hippocampus and SVZ. *P<0.05, **P<0.01 and ***P<0.001 versus MCAO mice. Scale bars = 100 µm.

Figure 6. RT-PCR analysis for growth and neurotropic factors at 14 days after MCAO.

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. *P<0.05 and ***P<0.001 versus MCAO mice.

Figure 7. Western blot and immunohistochemical analysis for mBDNF at 14 days after MCAO.

Western blot (A) and its densitometric analysis (B) showed that EA treatment significantly improved the expression of mBDNF in the ipsilateral hippocampus. Photomicrograph (C) and its histogram (D) showed that mBDNF positive cells were significantly increased by EA treatment in the ipsilateral hippocampus and SVZ. ^# P<0.05 versus the contralateral hemisphere; *P<0.05 and **P<0.01 versus MCAO mice. Scale bars = 100 µm.

Figure 8. Western blot and immunohistochemical analysis for VEGF at 14 days after MCAO.

Western blot (A) and its densitometric analysis (B) showed that EA treatment significantly improved the expression of VEGF in the ipsilateral hippocampus and cortex. Photomicrograph (C) and its histogram (D) showed that VEGF positive cells were significantly increased by EA treatment in the hippocampus and ipsilateral SVZ. ^# P<0.05 and ^## P<0.01 versus contralateral hemisphere; *P<0.05 and ***P<0.001 versus MCAO mice. Scale bars = 100 µm.

Figure 9. Immunohistochemical analysis for pPI3K in newborn cells at 14 days after MCAO.

Number of pPI3K/BrdU double-positive cells was significantly increased by EA treatment in all regions examined. ^# P<0.05 versus contralateral hemisphere; *P<0.05 versus MCAO mice. Scale bars = 100 µm.