Electroacupuncture treatment improves motor function and neurological outcomes after cerebral ischemia/reperfusion injury

Abstract

Electroacupuncture (EA) has been widely used for functional restoration after stroke. However, its role in post-stroke rehabilitation and the associated regulatory mechanisms remain poorly understood. In this study, we applied EA to the Zusanli (ST36) and Quchi (LI11) acupoints in rats with middle cerebral artery occlusion and reperfusion. We found that EA effectively increased the expression of brain-derived neurotrophic factor and its receptor tyrosine kinase B, synapsin-1, postsynaptic dense protein 95, and microtubule-associated protein 2 in the ischemic penumbra of rats with middle cerebral artery occlusion and reperfusion. Moreover, EA greatly reduced the expression of myelin-related inhibitors Nogo-A and NgR in the ischemic penumbra. Tyrosine kinase B inhibitor ANA-12 weakened the therapeutic effects of EA. These findings suggest that EA can improve neurological function after middle cerebral artery occlusion and reperfusion, possibly through regulating the activity of the brain-derived neurotrophic factor/tyrosine kinase B signal pathway. All procedures and experiments were approved by the Animal Research Committee of Shanghai University of Traditional Chinese Medicine, China (approval No. PZSHUTCM200110002) on January 10, 2020.

Keywords: Nogo receptor; brain-derived neurotrophic factor; dendritic; electroacupuncture; ischemia/reperfusion; motor function; neurite outgrowth inhibitor-A; neurological outcomes; synapse; tyrosine kinase B.

Figure 1

Time course of BDNF expression in the cortical ischemic penumbra and experimental protocol. (A) Flowchart of the experiment. (B) Rat that received EA treatment in the awake state. (C) The locations of the Quchi (LI11) and Zusanli (ST36) acupoints. (D) The mRNA expression of BDNF in the ischemic penumbra cortex was measured using quantitative real-time polymerase chain reactions. Data are presented as the mean ± SD (n = 6). #P < 0.05, ##P < 0.01, vs. sham group (one-way analysis of variance followed by least significant difference post hoc test). The experiments were repeated three times. BDNF: Brain-derived neurotrophic factor; EA: electroacupuncture; MCAO: middle cerebral artery occlusion.

Figure 2

EA improves functional recovery after ischemic stroke in terms of gait. EA treatment was applied at the LI11 and ST36 acupoints in MCAO/R rats. ANA-12 was administered via intraperitoneal injection at 0.5 mg/kg once per day for 7 consecutive days. (A) Schematic of the CatWalk behavioral test. (B) Run duration. (C) Run average speed. (D, E) RF and RH body speed. (F, G) RF and RH stand time. (H, I) RF and RH swing speed. (J, K) RF and RH stride length. Data are presented as the mean ± SD (n = 9). #P < 0.05, ##P < 0.01, vs. sham group; *P < 0.05 vs. MCAO/R group (one-way analysis of variance followed by least significant difference post hoc test). The experiments were repeated three times. ANA-12: TrkB inhibitor; EA: electroacupuncture; LF: left forelimb; LH: left hindlimb; MCAO: middle cerebral artery occlusion and reperfusion; RF: right forelimb; RH: right hindlimb; TrkB: tyrosine kinase B.

Figure 3

Effects of EA on MCAO/R-induced brain injury in rats at 7 days after MCAO/R. EA was applied at the LI11 and ST36 acupoints in MCAO/R-treated rats. ANA-12 was delivered via intraperitoneal injection at 0.5 mg/kg once per day for 7 consecutive days. (A) Effect of EA on neurological deficit scores 7 days after MCAO/R according to the Zea Longa score (n = 9). (B) 2,5-Triphenyltetrazolium chloride staining of brain slices. Normal tissue is shown in red and infarcted tissue in white. The infarct volume in the MCAO/R group was significantly increased compared with that in the sham group, the area of the cerebral infarction was significantly decreased after EA compared with that in the MCAO/R group, and ANA-12 attenuated the effect of EA treatments. (C) Quantitative analyses of infarct size (n = 6). Data are presented as the mean ± SD. #P < 0.05, ##P < 0.01, vs. sham group; *P < 0.05, vs. MCAO/R group; &&P < 0.01, vs. MCAO/R + EA group (one-way analysis of variance followed by least significant difference post hoc test). (D, E) Representative hematoxylin-eosin and Nissl staining performed on sections from ischemic brains (n = 6). The ischemic penumbra histopathological alterations and neuronal loss induced by the MCAO/R were alleviated after treatment with EA. Scale bar: 1000 μm (left) and 100 μm (right). The experiments were repeated three times. ANA-12: TrkB inhibitor; EA: electroacupuncture; MCAO/R: middle cerebral artery occlusion and reperfusion; TrkB: tyrosine kinase B.

Figure 4

EA increases the expression of BDNF and p-TrkB in the cortical ischemic penumbra 7 days after MCAO/R. EA treatment was performed at the LI11 and ST36 acupoints in MCAO/R-treated rats. ANA-12 was delivered by intraperitoneal injection at 0.5 mg/kg once per day for 7 consecutive days. (A) Representative immunofluorescence images indicating BDNF-positive expression (green, Alexa Fluor 488). Blue: DAPI. BDNF expression in the MCAO/R group was decreased compared with that in the sham group, whereas the expression of BDNF in the EA-treated group was dramatically higher than that in the MCAO/R group. Scale bar: 20 μm. (B–D) BDNF and p-TrkB protein expression in the cortical ischemic penumbra determined by western blot analysis. (E) BDNF mRNA expression in the ischemic penumbra was detected by quantitative real-time polymerase chain reaction. Data are presented as the mean ± SD (n = 6). ##P < 0.01, vs. sham group; *P < 0.05, **P < 0.01, vs. MCAO/R group; &&P < 0.01, vs. MCAO/R + EA group (one-way analysis of variance followed by least significant difference post hoc test). The experiments were repeated three times. ANA-12: TrkB inhibitor; BDNF: brain-derived neurotrophic factor; DAPI: 4,6-diamidino-2-phenylindole; EA: electroacupuncture; MCAO/R: middle cerebral artery occlusion and reperfusion; p-TrkB: phosphorylated- tyrosine kinase B; TrkB: tyrosine kinase B.

Figure 5

EA promotes the repair of myelin in the ischemic penumbra and inhibits the expression of Nogo-A and NgR 7 days after MCAO/R. EA treatment was performed at the LI11 and ST36 acupoints in MCAO/R rats. ANA-12 was delivered via intraperitoneal injection at 0.5 mg/kg once per day for 7 consecutive days. (A) Representative images of Luxol fast blue staining at 7 days. There was a loss of myelin in destructive lesions post-MCAO/R, demyelination was significantly reduced after EA intervention, and the outcome in the MCAO/R + ANA-12 group was worse than that in the MCAO/R group. The arrow indicates the myelin in the ischemic penumbra. Scale bars: 1000 μm (upper) and 50 μm (lower). (B) Representative immunofluorescence images of Nogo-A (green, Alexa Fluor 488) in the ischemic penumbra. Nogo-A was significantly increased in the MCAO/R group compared with the sham group, and it could be reversed by EA treatment. Green: Nogo-A; blue: DAPI. Scale bars: 20 μm and 5 μm in the enlarged part. (C) The results of the immunofluorescence examination of NgR (green, Alexa Fluor 488) in the ischemic penumbra. Blue: DAPI. NgR expression was increased in the MCAO/R group, and significantly decreased after the EA intervention. Scale bars: 20 μm and 5 μm in the enlarged part. (D–F) EA inhibited the MCAO/R-induced protein expression of Nogo-A and NgR in the ischemic penumbra cortex, as determined by western blot. (G, H) Nogo-A and NgR mRNA expression levels were detected via quantitative real-time polymerase chain reaction. Data are presented as the mean ± SD (n = 6). ##P < 0.01, vs. sham group; *P < 0.05, **P < 0.01, vs. MCAO/R group; &&P < 0.01, vs. MCAO/R + EA group (one-way analysis of variance followed by least significant difference post hoc test). The experiments were repeated 3 times. ANA-12: TrkB inhibitor; DAPI: 4,6-diamidino-2-phenylindole; EA: electroacupuncture; MCAO/R: middle cerebral artery occlusion and reperfusion; NgR: Nogo receptor; TrkB: tyrosine kinase B.

Figure 6

EA promotes dendritic branching and growth in the ischemic penumbra. EA treatment was performed at the LI11 and ST36 acupoints in MCAO/R rats. ANA-12 was delivered via intraperitoneal injection at 0.5 mg/kg once per day for 7 consecutive days. (A–C) A representative coronal Golgi-Cox staining section illustrating the phenomenon of MCAO/R injury. Scale bars: 2000 μm in A and 50 μm in B. (D) Illustration of the demarcation between the apical and basal dendrites. (E, F) Sholl analysis of the complexity of the dendritic arborization of neurons, represented as dendritic intersections at a given distance from the soma. (G, H) Total number of apical (G) and basal (H) intersections. (I, J) Number of apical (I) and basal (J) dendritic branches. (K, L) Quantification of total apical (K) and basal (L) dendritic length. Data are presented as the mean ± SD (n = 3). #P < 0.05, ##P < 0.01, vs. sham group; *P < 0.05, vs. MCAO/R group; &&P < 0.01, vs. MCAO/R + EA group (one-way analysis of variance followed by least significant difference post hoc test). The experiments were repeated three times. ANA-12: TrkB inhibitor; EA: electroacupuncture; MCAO/R: middle cerebral artery occlusion and reperfusion; TrkB: tyrosine kinase B.

Figure 7

EA reverses impaired spine density and increased levels of synaptic-related proteins in the ischemic penumbra. EA treatment was performed at the LI11 and ST36 acupoints in MCAO/R rats. ANA-12 was delivered via intraperitoneal injection at 0.5 mg/kg once per day for 7 consecutive days. (A) Representative electron microscopy images of the synaptic structures (n = 3). The synaptic ultrastructure revealed a reduced number of synaptic vesicles in the MCAO/R group, markedly improved synaptic structure in the MCAO/R + EA group, and relatively poor performance in the MCAO/R + ANA-12 group. The arrows indicate synapses. Scale bars: 1 μm (upper) and 0.2 μm (lower). (B, C) Density of apical dendritic spines. The densities in the apical dendritic spines were markedly decreased in MCAO/R rats compared with the sham group, and this was dramatically rescued by the administration of EA. Scale bars: 10 μm. (D, E) Density of basal dendritic spines. The densities in the basal dendritic spines were markedly decreased in MCAO/R rats compared with the sham group, and this was dramatically rescued by administration of EA. Scale bars: 10 μm. (F–H) EA increased the protein expression of synapsin-1 and PSD95 in the ischemic penumbra cortex, as determined by western blot. (I, J) Synapsin-1 and PSD95 mRNA expression levels were assessed by quantitative real-time polymerase chain reaction. Data are presented as the mean ± SD (n = 6). ##P < 0.01, vs. sham group; *P < 0.05, **P < 0.01, vs. MCAO/R group; &&P < 0.01, vs. MCAO/R + EA group (one-way analysis of variance followed by least significant difference post hoc test). The experiments were repeated three times. ANA-12: TrkB inhibitor; EA: electroacupuncture; MCAO/R: middle cerebral artery occlusion and reperfusion; PSD95: postsynaptic density protein 95; TrkB: tyrosine kinase B.

Figure 8

EA promotes the expression of MAP-2 and reduces injury-induced neuronal apoptosis in the ischemic penumbra area. EA treatment was performed at the LI11 and ST36 acupoints in MCAO/R rats. ANA-12 was delivered via intraperitoneal injection at 0.5 mg/kg once per day for 7 consecutive days. (A) Representative immunofluorescence images indicating MAP-2 immunopositivity (green, Alexa Fluor 488). Blue: DAPI. MAP-2 expression was significantly increased in the MCAO/R + EA group compared with the MCAO/R group. Scale bars: 20 μm (left three columns) and 10 μm (right most column). (B, C) EA promoted the expression of MAP-2 in the ischemic penumbra, as determined by western blot. (D) Apoptosis in the rat ischemic penumbra cortex in each group was measured by TUNEL. The number of apoptotic cells was increased post-MCAO/R, and EA-treated rats had few apoptotic cells compared with the MCAO/R group. Scale bars: 20 μm. Data are presented as the mean ± SD (n = 6). ##P < 0.01, vs. sham group; *P < 0.05, **P < 0.01, vs. MCAO/R group; &&P < 0.01, vs. MCAO/R + EA group (one-way analysis of variance followed by least significant difference post hoc test). The experiments were repeated three times. ANA-12: TrkB inhibitor; DAPI: 4,6-diamidino-2-phenylindole; EA: electroacupuncture; MAP-2: microtubule-associated protein 2; MCAO/R: middle cerebral artery occlusion and reperfusion; TrkB: tyrosine kinase B; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling.