Abstract: BACKGROUND: Eye acupuncture can effectively alleviate the symptoms of patients with cerebral ischemia in clinical practice, but its mechanism of action is less studied. miR-124-3p may be a regulator to improve ischemic brain injury and promote angiogenesis. Lithium chloride is a common activator of the Wnt/β-catenin pathway with the ability to resist nerve damage.
OBJECTIVE: To observe the effect and possible mechanism of eye acupuncture on angiogenesis in the cerebral cortex of ischemic penumbra in rats with cerebral ischemia injury.
METHODS: 108 Sprague-Dawley rats were randomly divided into sham operation group, model group, eye acupuncture group, body acupuncture group, mir-124-3p agonist group and lithium chloride group, with 18 rats in each group. The middle cerebral artery ischemia-reperfusion injury model was established by using the modified thread occlusion method in all groups except for the sham operation group. The eye acupuncture group needled the “liver area,” “kidney area,” “upper focal area” and “lower focal area” around the eyes of rats, the body acupuncture group needled the “Baihui” point, the mir-124-3p agonist group was given the miR-124-3p agonist (20 nmol/mL) in the left ventricle, and the lithium chloride group was intraperitoneally injected with lithium chloride solution 
(4 mmol/kg). The miR-124-3p group was administered once after modeling, and other groups were intervened once a day for 14 days. The pathological changes of the ischemic penumbra were observed. The expression levels of Wnt3a, β-catenin, vascular endothelial growth factor, angiopoietin 2 and miR-124-3p mRNA in rat brain were detected by real-time fluorescence quantitative PCR. The relative expression levels of Wnt3a, β-catenin, vascular endothelial growth factor, and angiopoietin 2 in rat brain were detected by western blot. 
RESULTS AND CONCLUSION: On the 1st day after modeling and the 14th day after intervention, there was no neurological dysfunction in the sham operation group but obvious neurological dysfunction in the model group (P < 0.05). There was no cerebral ischemia in the sham operation group, but rats in the model group had obvious cerebral ischemia injury. After eye acupuncture intervention, the cerebral ischemia area of the rats was significantly reduced (P < 0.05). Compared with the model group, the expression levels of Wnt3a, β-catenin proteins and miR-124-3p, Wnt3a, β-catenin genes related to the cortical pathway in the ischemic penumbra of rats were up-regulated in the eye acupuncture group, body acupuncture group, miR-124-3p agonist group and lithium chloride group (P < 0.05), while the expression levels of vascular endothelial growth factor and angiopoietin 2 proteins and genes were up-regulated (P < 0.05). And the above indexes in the eye acupuncture group were improved more obviously (P < 0.05). These findings suggest that eye acupuncture can promote angiogenesis and improve the neurological function of cerebral ischemic rats by stimulating the expression of angiogenic factors in the cortex of the ischemic penumbra. The mechanism of action may be that eye acupuncture affects Wnt/β-catenin signaling pathway by regulating miR-124-3p, regulates the expression of angiogenic factors and promotes angiogenesis, thereby improving cerebral ischemia injury.

Key words: eye acupuncture, miR-124-3p, Wnt/β-catenin signal pathway, cerebral ischemia, angiogenesis