Fig. 4

Efferocytosis of macrophages is impaired in a high salinity environment. a, b ND and HSD mice were subjected to 60min of tMCAO. Brain sections were collected at 3 days after cerebral ischemia. a Confocal microscopy analysis of NeuN (blue), TUNEL (green), and Iba1 (red) triple-staining in vivo. White arrows indicate microglia/macrophages that engulfed dead/dying neurons (Iba1⁺NeuN⁺TUNEL⁺). b Quantification of the total number of Iba1⁺ microglia/macrophages, Iba1⁺NeuN⁺ cells (microglia/macrophages that have engulfed neurons), Iba1⁺NeuN⁺TUNEL⁺ cells (microglia/macrophages that engulfed dead/dying neurons), Iba1⁺NeuN⁺TUNEL^- cells (microglia/macrophages that colocalize with TUNEL^- neurons), Iba1^-NeuN⁺TUNEL⁺ cells (not engulfed dead neurons), and phagocytic index (the proportion of dead/dying neurons engulfed by microglia/macrophages) in ischemic areas in vivo. N = 3 mice per group. *P < 0.05 and **P < 0.01, versus ND group in t test. c Macrophage viability before and after 4h of neuron-efferocytosis was quantified by LDH-assay in vitro. Data were collected from 3 independent experiments. d, e Phagocytosis of PI⁺ dead/dying neurons by macrophages was evaluated in vitro with immunostaining (d) and flow cytometry (e). Engulfed dead/dying neurons (PI⁺) per macrophage (Phalloidin labeled) or the proportion of phagocytic macrophages (PI⁺) was quantified at the indicated time points. The right images enlarged the boxed area. Data were collected from three independent experiments. **P < 0.01 and ***P < 0.001 versus PBS group in t test. f Dead neurons were treated to macrophages at a ratio of neuron: macrophage = 5:1 in vitro. After 6h, expression of proinflammatory (Tnfα, Il6, and Il1β) and inflammatory resolving molecules (Arg1, CD206, TGFβ) in the efferocytic macrophages was analyzed with RT-PCR. Data represent three independent experiments performed in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus PBS group in t test. STR striatum and CTX cortex