Abstract: BACKGROUND: Basic research has confirmed that electroacupuncture can improve the learning and memory ability of rats with middle cerebral artery occlusion/reperfusion. However, its underlying mechanism remains unclear.  
OBJECTIVE: To observe the effects of electroacupuncture at “Shenting” and “Baihui” on the expression of adenosine monophosphate-activated protein kinase (AMPK)/dynamin-related protein 1 (Drp1) pathway-related proteins and synaptic proteins (SYN, PSD95, and GAP43) in rats with learning and memory impairment after middle cerebral artery occlusion/reperfusion.  
METHODS: A total of 48 male Sprague-Dawley rats were randomly assigned into four groups: control group, sham group, model group, and electroacupuncture group. The middle cerebral artery occlusion/reperfusion injury model was established by thread occlusion in the model group and the electroacupuncture group. At 24 hours after modeling, the rats in the electroacupuncture group were treated at “Shenting” and “Baihui” with disperse-dense wave, 2 Hz/10 Hz in frequency and 1 mA in current intensity, and the needles were retained for 30 minutes, once a day for 14 days. The neurological deficit scores were observed after modeling and on the 7th and 14th days of intervention. The learning and memory abilities of the rats were assessed using the Morris water maze test. Neuronal morphology in the rat hippocampal CA1 region was observed by hematoxylin-eosin staining. Adenosine triphosphate was measured by a kit to evaluate mitochondrial function. Transmission electron microscopy was used to examine the ultrastructure of neuronal mitochondria in the hippocampal region. The protein expressions of AMPK, p-AMPK, Drp1, OPA1, Mfn1, Mfn2, SYN, PSD95 and GAP-43 in the hippocampus were detected by western blot assay.  
RESULTS AND CONCLUSION: (1) Compared with the control group and sham group, the neurological deficit scores were increased in the model group and the electroacupuncture group (P < 0.01). After 7 and 14 days of intervention, the neurological deficit scores in the electroacupuncture group were lower than those in the model group (P < 0.05, P < 0.01). (2) The results of Morris water maze test: Compared with the control group and sham group, the model group and the electroacupuncture group showed increased escape latency (P < 0.01) and decreased platform crossings (P < 0.01). Furthermore, electroacupuncture treatment obviously decreased escape latency (P < 0.05) and increased platform crossings (P < 0.01) compared with the model groups. (3) Hematoxylin-eosin staining results showed that compared with the model group, the electroacupuncture group exhibited an increased number of neurons with a more regular arrangement in the hippocampal CA1 region. The structure appeared more normal, with relatively uniform neuronal cell size and morphology. Under the transmission electron microscopy, mitochondrial morphology was improved, characterized by reduced structural damage and an increased number of mitochondrial cristae. (4) Results from the adenosine triphosphate assay kit revealed a significant decrease in adenosine triphosphate content in the hippocampal tissue of the model group relative to the sham group. Meanwhile, the electroacupuncture group exhibited significantly elevated adenosine triphosphate levels compared with the model group (P < 0.01). (5) As revealed by western blot assay, the model group exhibited a significant increase in the protein level of Drp1 (P < 0.05), while showing significant decreases in the protein expressions of p-AMPK/AMPK, OPA1, MFN1, MFN2, SYN, PSD95 and GAP43 in the hippocampal tissue compared with the sham group (P < 0.01). In contrast, the electroacupuncture group displayed an opposite pattern compared with the model group, with decreased Drp1 level and increased expression of p-AMPK/AMPK, OPA1, MFN1, MFN2, SYN, PSD95 and GAP43 (P < 0.05). (6) To conclude, electroacupuncture treatment mitigates brain injury and enhances learning and memory ability in rats subjected to middle cerebral artery occlusion/reperfusion, likely through modulating mitochondrial dynamics mediated by the AMPK/Drp1 pathway.  

Key words: cerebral ischemia/reperfusion injury, learning and memory disorders, electroacupuncture, adenosine monophosphate-activated protein kinase/dynamic-related protein 1, mitochondrial dynamics, signaling pathway