Fig. 8
Lactate from PMN-MDSCs significantly reduces brain injury by decreasing microglial activation and inflammatory factor expression. HIF-1αfl/fl S100A8cre mice were divided into two groups: HIF-1αfl/fl S100A8cre mice group, HIF-1αfl/fl S100A8cre mice + Lactate group. In the intervention experiments, lactate at 2 g/kg was injected into mice intraperitoneally for two consecutive days and brain pathology was evaluated at the 48 h after hypoxia. (A) Statistical analysis of the percentage of brain infarct area in lactate supplementation assay (n = 4). (B) Statistical analysis of brain water content results in lactate supplementation assay (n = 4). (C) Statistical analysis of geotaxis reflex time in lactate supplementation assay (n = 4). (D) Representative H&E staining image (original magnification, ×400) in lactate supplementation assay. (E) Representative Nissl staining image (original magnification, ×400) and (F) statistical analysis of the hippocampal neuronal damage scores in lactate supplementation assay (n = 4). (G) Representative images of NeuN (red) and TUNEL staining (green) in the hippocampal region and DAPI staining is shown in blue. Scale bar = 50 μm. (H) Statistical analysis of the percentage of TUNEL cells and the number of NeuN-TUNEL positive mouse hippocampal neurons in lactate supplementation assay (n = 4). (I and J) Representative merged and enlarged images showing CD68, IBA1, and DAPI co-staining in the hippocampus (Scale bar = 50 μm) and statistical analysis of the number of CD68+ IBA1+ double positive microglia cells for lactate supplementation assay (n = 4). (K) Statistical analysis of CD80/CD206 and CD86/CD206 relative ratios after hypoxia is shown (n = 4). (L) Statistical analysis of p-STAT1, pNF-κB Thr254, and pNF-κB Ser529 levels in microglia at 48 h after hypoxia (n = 5). (M-O) Sorted microglia cell derived from neonatal mice brain or microglia cell line (BV2/HMC3 cell) was co-cultured with different concentrations lactate (5mM or 10mM). Subsequently, relative ratios of CD80/CD206 and CD86/CD206 were analyzed by flow cytometry after 72 h. Data are pooled from two independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Bar graphs show the mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t-test (A-C, F, H, and J-O)