Mechanism of electroacupuncture at “Neiguan” (PC6) and “Jianshi” (PC5) in ameliorating blood-brain barrier damage in APP/PS1 mice based on the nucleus tractus solitarius-locus coeruleus neural circuit

To observe the role of tyrosine hydroxylase (TH)-positive neurons in the nucleus tractus solitarius (NTS) and locus coeruleus (LC) in electroacupuncture (EA)-mediated improvement of blood-brain barrier (BBB) damage in APP/PS1 mice, so as to explore the mechanism of the NTS^TH-LC neural circuit underlying the effect of EA on prevention and treatment of Alzheimer’s disease (AD).

(1) Eight 4-month-old male C57BL/6 mice served as the control group, and 16 age-matched male APP/PS1 mice were randomly divided into the model and EA groups (n=8). The EA group received EA stimulation at “Neiguan” (PC6) and “Jianshi” (PC5) once every other day for 4 weeks. After intervention, Morris water maze and novel object recognition tests were used to evaluate learning and memory abilities. Western blot was performed to detect the expression levels of hippocampal tight junction proteins, including Occludin, Claudin-5, and zonula occludens-1 (ZO-1). Immunofluorescence was used to assess the co-localization of TH/c-Fos in NTS and LC, as well as TH/norepinephrine (NE) co-localization in LC. (2) Five TH-cre mice received retrograde tracing virus injection into LC to observe whether TH-positive neurons in NTS project to LC. (3) Twenty-one 7-month-old APP/PS1 mice were injected with chemogenetic activation virus (AAV2/9-hSyn-DIO-hM3D-mCherry-WPRE-hGH-pA) or empty virus (AAV2/9-Ef1α-DIO-mCherry-WPRE-hGH-pA) into the NTS, and AAVretro-TH-CRE-WPRE-hGH-pA virus into the LC. After 21 d of virus expression, 6 mice injected with empty virus were taken as mCherry+CNO+EA group; 3 mice from the activation virus group were randomly selected for brain slice patch-clamp to verify virus functionality. The remaining mice were randomly divided into 2 groups (n=6): hM3D+CNO+EA, and hM3D+saline+EA groups. EA was applied to PC6 and PC5 once daily for 15 d. After intervention, learning and memory abilities were evaluated by Morris water maze and novel object recognition tests. BBB permeability was detected by Evans blue (EB) staining. Immunofluorescence was used to measure TH/c-Fos and TH/NE co-localization in the LC.

(1) Compared with the control group, the model group showed significantly impaired learning and memory abilities (P<0.01), decreased expressions of hippocampal Occludin, Claudin-5, and ZO-1 (P<0.01), and increased TH/c-Fos co-localization (c-Fos expression in TH-positive neurons) in NTS and LC, as well as TH/NE co-localization in LC (P<0.01). Compared with the model group, the EA group exhibited improved learning and memory abilities (P<0.05, P<0.01), increased expressions of hippocampal tight junction proteins (P<0.01), and reduced TH/c-Fos and TH/NE co-localization (P<0.01, P<0.05). (2) Retrograde tracing confirmed that TH-positive neurons in NTS project to LC. (3) Compared with the mCherry+CNO+EA group and hM3D+saline+EA group, the hM3D+CNO+EA group showed significantly impaired learning and memory (P<0.01), increased EB content in brain tissue, and elevated TH/c-Fos and TH/NE co-localization in the LC (P<0.01).

EA at PC6 and PC5 can ameliorate BBB damage and learning/memory deficits in AD mice, and its mechanism may be related to inhibiting the activation of the NTS^TH-LC neural circuit.