ABSTRACT
Objective To observe the effect of electroacupuncture on the formation of glial scars in the cerebral cortex of rats with focal cerebral ischemia/reperfusion( I /R) . Methods The focal cerebral ischemia/reperfusion model was established by thread method, and "Baihui" ( GV 20) and left "Siguan" (Hegu LI 4/Taichong LR 3) were selected as acupoints points. Seventy-eight male SD rats were randomly divided into sham group ( sham) , focal cerebral ischemia/ reperfusion ( I/R) , focal cerebral ischemia/reperfusion + electroacupuncture group ( I/R + EA ) . Seven days after reperfusion, modified neurological severity score ( mNSS) was used to evaluate neurological deficit, HE staining was used to observe the degree of ischemic cerebral cortex. Immunofluorescencte was used to observe glial fibrillary acidic protein (GFAP) /neurocan, GFAP/phosphacan positive cells in the ischemic cerebral cortex. The average immunofluorescent intensity of GFAP, neurocan and phosphacan, and the levels of GFAP, neurocan and phosphacan mRNA in the ischemic cerebral cortex were analyzed by Real-time PCR, respectively. Results Compared with the I/R group, mNSS score in I/R+EA group decreased significantly (P<0. 01) , and the degree of brain tissue damage reduced obviously. There were very few GFAP+/neurocan+ cells and GFAP+/phosphacan+ cells in the sham group. The astrocytes in I/R group were Irypertroplry, increasing and thickened protrusions, more GFAP+/neurocan+ cells and GFAP+/phosphacan+ cells weredectected as well. While the count of GFAPVneurocan+ cells and GFAP+/phosphacan+ cells in I/R + EA group were significantly lower than those in I /R group. In addition, the average immunofluorescence intensity of GFAP, neurocan and phosphacan in I/R+EA group, and mRNA content of above mentioned indicators were significantly lower than those in I/R group ( P < 0. 05 ) . Conclusion Electroacupuncture promotes the recovery of neurological function in rats with focal cerebral I /R and reduce brain tissue damage, which may be related to the inhibition of glial scar fonnation in cerebral cortex.
ABSTRACT
Objective To investigate the mechanism of TREM2 modulating the polarization of M2 microglia treated by oxygen-glucose deprivation/reoxygenation (OGD/R). Methods Mouse N9 microglial cells were cultured in vitro. N9 cells were transfected with lenti virus for TREM-2-overexpression (LV-TREM2), and LV-scramble acted as control group. OGD/R model was established. The OGD/R cells were randomly divided into OGD/R, OGD/R+LV-scramble and OGD/R+LV-TREM2 groups. Real-time PCR was used to detect the expression of TREM2 mRNA in OGD/R N9 cells within 72 hours after re-oxygenation. Immunofluorescence was applied to observe transfection of lentivirus LV-scramble and LV-TREM2 for normal N9 microglia, and Real-time PCR and Western blotting were used to verify the efficiency of lentivirus transfection. The mRNA and protein contents of Ml microglial markers tumor necrosis factor-a (TNF-α), interleukin-lβ (IL-lβ) and inducible nitric oxide synthase (iNOS), M2 microglial markers arginase-1 (Arg-1) and interleukin-10 (IL-10) were detected by Real-time PCR and ELISA. The expressions of phosphorylated-phosphatidylinositol 3-kinases (p-PI3K), PI3K, phosphorylated protein kinase B (p-Akt), Akt, phosphorylated inhibitor of nuclear factor-κB ( NF-κB)α (p-lκBα) and IκBα protein were detected by Western blotting. The distribution of NF-κB P65 (NF-κB P65) protein in N9 cells was analyzed by immunofluorescence method. Results TREM2 mRNA content in the OGD/R group cells increased significantly within 72 hours after re-oxygenation, and peaked at hour 24 and hour 48. Lenti virus LV- TREM2 effectively promoted the expression of TREM2 mRNA and protein of N9 cells in OGD/R model (P<0.001, P<0.01). Compared with the OGD/R group, the mRNA and protein content of TNF-α, IL-lβ and iNOS decreased significantly, while Arg-1 and IL-10 in OGD/R+LV-TREM2 group increased significantly(P<0.05). Besides, the ratios of P-PI3K/PI3K and p-Akt/Akt increased obviously (P<0.05), the ratio of p-IκBα/IκBα decreased significantly in OGD/ R+ LV-TREM2 group (P<0.001), and the nuclear translocation of NF-κB P65 protein was obviously weakened. Conclusion TREM-2 overexpression exerts anti-inflammatory effect by modulating the polarization of microglia from Ml to M2 type, which is associated with PI3K/Akt and NF-κB signaling pathways regulated by TREM2 in N9 microglia with OGD/R model.