Beneficial effects of electro-acupuncture treatment in photothrombotic ischemia model rats by remodeling neurovascular unit

BACKGROUND: Electro-acupuncture (EA) treatment at LI11 (Quchi) and ST36 (Zusanli) acupoints promotes functional recovery after ischemia in animal trials. However, the possible mechanism responsible for neurovascular unit (NVU) in ischemic stroke remains elusive. The study aimed to investigate the protective effects of EA on angiogenesis and neurovascular unit in a rat model of photothrombotic ischemia (PTI). METHODS: Ischemic stroke was generated via Rose Bengal - photothrombosis in rat motor cortex and EA treatment started at 24 h after the PTI operation, continued for 7 days until animals were sacrificed. Open field test, rotating rod instrument and 3D gait analysis system were performed to observe the changes in motor behavior of rats. Animals were euthanized for T2- weighted imaging (T2WI), magnetic resonance angiography (MRA), magnetic resonance spectroscopy imaging (MRS) and diffusion kurtosis imaging (DKI) study. Then western blot, immunofluorescence and histopathological techniques were used to analyze angiogenesis associated protein expression, the micro- and ultra-structure of the NVU in the motor cortex. RESULTS: Animal behavior assessments showed that gait, equilibrium and coordination of rats in the EA group were significantly improved compared with those in the model group. EA stimulation significantly alleviated the tendency of cortical ischemia area expansion, reduced cellular inflammatory infiltrationand neuronal necrosis, and increased the compactness and structural complexity of tissue in the ischemic area, thereby restoring the integrity of the blood-brain barrier(BBB). Meanwhile, the levels of N-acetyl aspartate (NAA), choline (Cho), gamma-aminobutyric acid (GABA) and glutamic acid (Glu) in the tissues around cerebral ischemia were significantly decreased. In contrast, the expression of endogenous angiogenesis inhibitors, endostatin (ES) and thrombospondin-1(TSP-1) proteins, was significantly downregulated, while the expression of angiogenesis regulating factors, vascular endothelial growth factor (VEGF) and angiopoietin-1(Ang-1) proteins, was significantly upregulated. Immunofluorescence results demonstrated that the number of zonula occludens-1 (ZO-1), glial fibrillary acidic protein (GFAP), and platelet-derived growth factor receptorβ(PDGFR β) positive cells in the ischemia marginal area of the cerebral cortex was significantly increased in the EA group. Furthermore, the number of cells co-localized with neuronal nuclear antigen (NeuN) and VEGF in the ischemic boundary area of the cerebral cortex was also significantly increased in the EA group, as well as the number of cells co-localized with NeuN and nuclear factor erythroid 2-related factor 2 (Nrf2). CONCLUSIONS: Our findings suggested that EA treatment promoted angiogenesis and NVU remodeling in rats following cerebral ischemia. Its mechanisms may involve the promotion of angiogenesis, the increase in neuron number and enhancement of neuronal activity, and the integrity of damaged neural structural units and the BBB. Our study helps to better understand the mechanism of action of EA in the treatment of ischemic stroke, and it may also enrich the theory about clinical application of EA.