Fig. 8: Knockout of ACLY in microglia-derived extracellular vesicles (EVsM1-ACLY KO) alleviated astrocytic inflammation, neuronal damage, cognitive deficits, and synaptic dysfunction in POCD.
A Western blot analysis verifying ACLY knockout in EVs derived from microglia. EV markers CD63 and CD81, and negative marker GM130 are included. B Representative TEM images illustrating the morphology of EVsM1-ACLY siRNA NC and EVsM1-ACLY KO. Scale bar: 100 nm. C Nanoparticle tracking analysis showing particle size distribution profiles of EVsM1-ACLY siRNA NC and EVsM1-ACLY KO. D Immunofluorescence staining for GFAP (astrocyte marker, green), EAAT1 (red), acetylated p65 (yellow), and DAPI (nuclei, blue) in brain tissues from POCD mice treated with EVsM1-ACLY siRNA NC or EVsM1-ACLY KO(5 µg in 2 μl PBS). Corresponding intensity profiles are presented. Scale bar: 20 μm. n = 5 mice per group. *p < 0.05, **p < 0.01, all values represent mean ± standard deviation. independent two-tailed Student’s t tests. E Fluoro-Jade B (FJB) staining assessing neuronal damage in the hippocampus of POCD mice receiving EVsM1-ACLY siRNA NC or EVsM1-ACLY KO treatment. Quantitative analysis presented. Scale bar, indicated. **p < 0.05, n = 5 mice per group; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. F Morris water maze test depicting cognitive performance through representative swim paths, number of crossings over the previous platform location, and escape latency in POCD mice treated with EVsM1-ACLY siRNA NC or EVsM1-ACLY KO. *p < 0.05, n = 15 mice in each group; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. G Time course of glutamate release (% baseline) measured in hippocampal slices following stimulation in POCD mice treated with EVsM1-ACLY siRNA NC or EVsM1-ACLY KO. *p < 0.05, **p < 0.01, n = 5 mice in each group/time point, all values represent mean ± standard deviation; two-way ANOVA with Newman–Keuls multiple comparison test. H, I Representative field excitatory postsynaptic potentials (fEPSP) recorded from hippocampal slices and quantification of normalized fEPSP slopes in POCD mice treated with EVsM1-ACLY siRNA NC or EVsM1-ACLY KO. **p < 0.01, n = 10; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. “1” = baseline fEPSP trace before HFS (pre-LTP induction); “2” = fEPSP trace recorded at the late phase after HFS.
