“Tiaoshen Tongluo” electroacupuncture improves learning-memory ability by regulating microglial polarization via the Nrf2/TREM2 signaling pathway in rats with cerebral ischemia-reperfusion injury

To observe the effect of “Tiaoshen Tongluo” (mind regulation and meridian-collateral dredging) electroacupuncture (EA) on the nuclear factor erythroid 2-related factor 2 (Nrf2)/triggering receptor expressed on myeloid cells 2 (TREM2) signaling pathway and hippocampal microglial polarization in rats with learning-memory impairment induced by middle cerebral artery occlusion/reperfusion (MCAO/R).

Male SD rats were randomly divided into 7 d sham group (n=10), 14 d sham group (n=10), and pre-model group. The MCAO/R model was established by Koizumi intraluminal filament MCAO method. Rats meeting the criteria of learning-memory impairment due to MCAO/R, identified by the Morris water maze tests, were randomly assigned to 7 d model, 7 d EA, 14 d model and 14 d EA groups, with 10 rats in each group. Rats of the EA groups received “Tiaoshen Tongluo” EA stimulation at “Baihui” (GV20), “Yintang” (GV24⁺), and bilateral “Taichong” (LR3) and “Hegu” (LI4) for 10 min per session, once daily, for 7 or 14 consecutive days. The modified neurological severity score (mNSS) was used to assess the neurological deficits before and after the intervention. Morris water maze test was conducted to evaluate learning and memory abilities. HE staining was used to observe histopathological changes, and Nissl staining was applied to examine neuronal morphology and Nissl bodies in the hippocampal CA1 region. Immunofluorescence double staining was employed to determine the proportions of CD68⁺/Iba1⁺ (M1 type) and CD206⁺/Iba1⁺ (M2 type) positive cells in the hippocampal tissue. Western blot was used to measure the relative protein expression levels of Nrf2, TREM2, DNAX activator protein 12 (DAP12), tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in the hippocampal tissue.

Compared with the 7 d and 14 d sham groups, the 7 d and 14 d model groups had a significant increase in the mNSS (P<0.01), and an obvious down-regulation in expression levels of hippocampal Nrf2, TREM2, and DAP12 proteins (P<0.05, P<0.01), and loose arrangement of neurons and reduction in the number of Nissl bodies in the hippocampal CA1 region. In contrast to the 14 d sham group, the 14 d model group had a notable increase in the escape latency, the number of CD68⁺/Iba1⁺ dual labeled cells, and the expression level of TNF-α in the hippocampus (P<0.05, P<0.01), and a striking decrease in the number of original platform crossings and time spent in the target quadrant (P<0.01). Compared with the 7 d and 14 d model groups, the 7 d and 14 d EA groups exhibited reduction of mNSS and TNF-α expressions (P<0.01, P<0.05), up-regulation of expression levels of Nrf2 and TREM2 proteins (P<0.05, P<0.01), alleviated arrangement of CA1 neurons, and increased Nissl bodies. Compared with the 14 d model group, the 14 d EA group showed a decrease in the escape latency and the number of CD68⁺/Iba1⁺ dual labeled cells (P<0.01), and an increase in the number of original platform crossings and time spent in the target quadrant, number of CD206⁺/Iba1⁺ dual labeled cells, and the expression of DAP12 and TGF-β proteins (P<0.05, P< 0.01). Compared with the 7 d EA group, the 14 d EA group showed a further reduction in mNSS score (P<0.05), and a further up-regulation of expression of TREM2 protein (P<0.05), a more compact neuronal arrangement in the CA1 region with the morphological structure being close to the normal.

“Tiaoshen Tongluo” EA can improve learning-memory ability in MCAO/R rats, which may be associated with its functions in activating the Nrf2/TREM2 signaling pathway, inhibiting microglial M1 polarization and promoting M2 polarization. In addition, the therapeutic effect of 14 d EA is better than that of 7 d EA.