Fig. 3

Overexpression of MLKL increases phosphorylated MLKL and reverses the hypoxic preconditioning-induced neuroprotection in CA1 after tGCI. A Design of experiments in which rats were stereotaxically injected bilaterally with MLKL lentiviral vectors in the dorsal CA1 pyramidal layer and subjected to either Sham or tGCI with hypoxia. B Phase contrast and fluorescent images from coronal sections of CA1 following injection of MLKL lentiviral vectors in Sham animals. C Representative photomicrographs show the co-localization of GFP (green), MLKL (red), and DAPI (blue) in CA1 from Sham animals with Lenti-MLKL injection. D Cresyl violet stained and NeuN immunostained hippocampal sections from rats administered bilaterally with either Lenti-control or Lenti-MLKL at 7 days after reperfusion of tGCI with hypoxia. Boxes indicate that the magnified regions displayed in the right panel. E, F Quantitative analyses of surviving cells and NeuN-positive cells in CA1. Each bar represents the mean±S.D. ^*p<0.05 vs. Sham+Lenti-con animals, and ^&p<0.05 vs. HPC group with Lenti-con. G, H Representative immunoblots of MLKL and p-MLKL (Ser345) expression in CA1 after hypoxic preconditioning with or without MLKL lentiviral vector administration. The histogram presents the quantitative analyses of MLKL or p-MLKL protein. Data are expressed as percentage of value of Sham animals. Each bar represents the mean±S.D. ^*p<0.05 vs. Sham animals, and ^&p<0.05 vs. Sham or HPC group with Lenti-con. Lenti-con, Lenti-Conrtol, scrambled lentivirus vector; Lenti-MLKL MLKL-carried lentivirus; HPC hypoxic preconditioning