Fig. 4

Prophylactic atRA treatment inhibited NETs formation. a In situ NETs formation was assessed with immunofluorescent staining of citrullinated histone 3 (CitH3, red) in the stroke lesion at 3 days after tMCAO. Representative images and quantification of NETs were shown. N = 4 mice per group. *P ≤ 0.05, versus PBS-treated group in t test. b Primary culture of bone marrow derived neutrophil was pre-treated with atRA (1 μM for 6 h) then subjected to NETs induction with PMA (20 nM, 3 h). Neutrophil was stained with Sytox green (fluorescent dye of nucleic acids). NETs were identified as the enlarge cloudlike structure (emphasized white arrow) while intact neutrophils were with the morphology of rounded shape (emphasized with yellow arrow head). Representative images and quantification of NETs were presented. Experiments were repeated for three times. *P ≤ 0.05, versus DMSO-treated group in t test. c Schematic diagram showing the collection process of atRA NETs conditioned medium (CM) and the following treatment to ischemic neurons. Primary cultured neutrophils were first pre-treated with atRA (1 μM for 6 h) then subjected to NETs induction with PMA (20 nM, 3 h). After retreating PMA, NETs forming neutrophils were cultured with normal neutrophil medium for another 24 h and CM was collected. Primary cultured neurons were subjected to 6 h of oxygen glucose deprivation (OGD) followed by reperfusion with normal neuron medium. CM from atRA pre-treated NETs forming neutrophil (atRA NETs CM) was applied to the re-perfused neurons (CM: neuron medium = 1:1) for another 24 h. Viability of the ischemic neurons was assessed with immunofluorescent staining of NeuN. d Representative images of neuronal viability and quantification of lived neurons (NeuN⁺ green) were displayed. Experiments were repeated for three times. ***P ≤ 0.001 in one-way ANOVA