Electroacupuncture treatment improves neurological function associated with regulation of tight junction proteins in rats with cerebral ischemia reperfusion injury

Abstract

Strategies to develop effective neuroprotective therapy to reduce brain damage and related behavioral deficits in stroke patients are of great significance. Electroacupuncture (EA), which derives from traditional Chinese medicine, may be effective as a complementary and alternative method for promoting recovery of neurological function and quality of life. Adult Sprague-Dawley rats were randomly divided into 3 groups: (1) sham, (2) middle cerebral artery occlusion (MCAO) model groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion, and (3) EA groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion. EA groups received EA therapy by needling at GV20 and left ST36. The results show that EA therapy improved the neurological function and reduced infarct volume, confirmed by modified neurological severity scores and TTC staining. Real-time PCR, immunohistochemistry, and western blot assay verified that EA upregulated the expression of tight junction (TJ) claudin-5, occludin, and zonula occluding-1 from 1 to 7 d after reperfusion. Our findings suggest that EA reduces brain damage and related behavioral deficits via upregulation of the TJ proteins.

Figure 1

Neurological functional outcome. Measurement performed at 1, 3, 5, and 7 d after reperfusion using the mNSS. The score for the sham group on each day was 0. Rats receiving EA treatment showed significant improvement in neurological function compared with the MCAO model groups at the same time points. Data (n = 9) is represented as mean ± SD. ^Δ P < 0.05 versus the sham group, ^★ P < 0.05 versus the MCAO group at the same time points, and *P < 0.05 versus the EA groups at the same time points.

Figure 2

TTC staining of brain slices. Ischemic area is white; intact area stained red. Representative coronal sections of sham group, MCAO groups at 2 h MCAO followed by 7 d of reperfusion, and the EA groups of 2 h MCAO followed by 7 d of reperfusion.

Figure 3

Quantification from TTC staining of infarct volume in focal cerebral ischemia rats of 7 d after reperfusion. The ischemic infarct volume was quantified using image analysis software. The infarct volume of the sham group was 0. The infarction induced by MCAO was significantly reduced by EA at GV 20 and ST 36 acupoints. Data (n = 3) is represented as mean ± SD. ^★ P < 0.05 versus the MCAO groups at the same time points. *P < 0.05 versus the EA groups at the same time points.

Figure 4

Effects of EA on the distribution and expression of ZO-1, claudin-5, and occludin on ischemic cerebral microvessels. (a) Representative immunohistochemistry stained tissue of the following groups: sham; M1–M7: MCAO groups after 1, 3, 5, and 7 d of reperfusion; and E1–E7: EA groups after 1, 3, 5, and 7 d of reperfusion. The integrated optical density of ZO-1 (b), claudin-5 (c), and occluding (d). Data (n = 6) are represented as mean ± SD. ^Δ P < 0.05 versus the sham group, ^★ P < 0.05 versus the MCAO group at the same time points, and *P < 0.05 versus the EA groups at the same time points. Arrows show the immunoreactive positive area. Scale bar in A = 50 μm (×400).

Figure 5

mRNA expression of ZO-1, claudin-5, and occludin analyzed by qPCR after EA. GAPDH served as endogenous reference. Experimental groups are sham; MCAO groups after 1, 3, 5, and 7 d of reperfusion; and EA groups after 1, 3, 5, and 7 d of reperfusion, respectively. The mRNA expression levels of ZO-1 (a), claudin-5 (b), and occluding (c) are shown. Data (n = 3) are represented as mean ± SD. ^Δ P < 0.05 versus the sham group, ^★ P < 0.05 versus the MCAO group at the same time points, and *P < 0.05 versus the EA groups at the same time points.

Figure 6

Protein expression levels of ZO-1, claudin-5, and occludin on ischemic cerebral microvessels after cerebral ischemia reperfusion. Western blots of (a) ZO-1, (c) claudin-5, and (e) occludin. From left to right: sham; M1–M7: MCAO groups after 1, 3, 5, and 7 d of reperfusion; and E1–E7: EA groups after 1, 3, 5, and 7 d of reperfusion. β-Actin served as a loading control. IDV of ZO-1 (b), claudin-5 (d), and occluding (f) are shown. Data (n = 3) is represented as mean ± SD. ^Δ P < 0.05 versus the sham group, ^★ P < 0.05 versus the MCAO group at the same time points, and *P < 0.05 versus the EA groups at the same time points.