Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1

Abstract

Electroacupuncture preconditioning at acupoint Baihui (GV20) can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 (Drp1) can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 (depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day). Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

Keywords: apoptosis; cytochrome c; death-associated protein kinases; dynamin-related protein 1; electroacupuncture; focal cerebral ischemia/reperfusion injury; mitochondrial dynamics; mitochondrial ultrastructure; nerve regeneration; neural regeneration.

Figure 1

Effects of EA pretreatment on cellular apoptosis and neurological deficit scores in rats with focal cerebral ischemia/reperfusion (IR) injury. (A) Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining of cells (arrows) at 24 hours after IR or sham surgery. Scale bars: 50 μm. (B) Proportion of TUNEL-positive cells in the ischemic penumbra (n = 4 rats per group). (C) Neurological deficit score at 6, 24 and 48 hours after reperfusion or sham surgery (n = 8 per group at 6 and 48 hours after reperfusion; n = 16 per group at 24 hours after reperfusion). Data are presented as the mean ± SD, and analyzed by one-way analysis of variance followed by the post hoc Student-Newman-Keuls test. **P < 0.01, vs. sham group; #P < 0.05, vs. electroacupuncture (EA) group at the same time point. I: Sham; II: EA 6 hours; III: IR 6 hours; IV: EA 24 hours; V: IR 24 hours; VI: EA 48 hours; VII: IR 48 hours.

Figure 2

Effect of electroacupuncture (EA) preconditioning on neuronal histopathological structure in the ischemic penumbra of rats with focal cerebral ischemia/reperfusion (IR) injury (hematoxylin-eosin staining). Representative stained images in the ischemic penumbra at 6, 24 and 48 hours post-reperfusion or sham surgery. Shrunken cell bodies and nuclear pyknosis (arrows) were observed in the EA and IR groups, but not in the sham group. The degree of histopathological changes in the IR group was more obvious than that in the EA group. Scale bars: 50 μm (n = 4 rats)

Figure 3

Effect of electroacupuncture (EA) preconditioning on mitochondrial ultrastructure in the brains of rats with focal cerebral ischemia/reperfusion (IR) injury. Mitochondrial (arrows) morphological structure observed under the transmission electron microscope in the ischemic penumbra at 24 hours post-reperfusion or sham surgery. Scale bars: 0.5 μm. (B) Quantitative parameters for mitochondrial morphological changes, including aspect ratio, vacuolation ratio and mean area density of vacuolated mitochondria. Data are presented as the mean ± SD (n = 4 rats per group), and analyzed by one-way analysis of variance followed by the post hoc Student-Newman-Keuls test. **P < 0.01, vs. sham group; #P < 0.05, vs. EA 24 hour group. I: Sham; II: EA 24 hours; III: IR 24 hours.

Figure 4

Effects of electroacupuncture (EA) preconditioning on dynamin-related protein 1 (Drp1) expression in focal cerebral ischemia/reperfusion (IR) injury rats. Total-Drp1 (A) and Mitochondrial Drp1 (Mito-Drp1) (B) at 6, 24 and 48 hours post-reperfusion or sham surgery, as measured by western blot assay and densitometric analysis, and quantified relative to β-actin (Total-Drp1) or cyclooxygenase (Cox)-IV (Mito-Drp1). The lack of expression of the Rho GDP dissociation inhibitor (RhoGDI; a specific cytosolic [but not mitochondrial] biomarker (Francis et al., 2014)) verified the purity of the mitochondrial fraction. Data are presented as the mean ± SD (n = 4 rats per group at each time point), and analyzed by one-way analysis of variance followed by the post hoc Student-Newman-Keuls test. Data are representative of three independent tests. *P < 0.05, **P < 0.01, vs. sham group; #P < 0.05, vs. EA group at the same time point. I: Sham; II: EA 6 hours; III: IR 6 hours; IV: EA 24 hours; V: IR 24 hours; VI: EA 48 hours; VII: IR 48 hours.

Figure 5

Effects of electroacupuncture (EA) preconditioning on cytochrome (cyto) expression in focal cerebral ischemia/reperfusion (IR) injury rats. Total-cyto c (A) and cytosolic cytochrome c (cyto-cyto c) (B) at 6, 24 and 48 hours post-reperfusion or sham surgery. Data were determined by western blot assay, and are presented as the densitometric ratio relative to β-actin. Data are shown as the mean ± SD (n = 4 rats per group at each time point), and analyzed by one-way analysis of variance followed by the post hoc Student-Newman-Keuls test. Data are representative of three independent tests. **P < 0.01, vs. sham group; #P < 0.05, vs. EA group at the same time point. I: Sham; II: EA 6 hours; III: IR 6 hours; IV: EA 24 hours; V: IR 24 hours; VI: EA 48 hours; VII: IR 48 hours.