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. 2023 Jul 16;29:196–213. doi: 10.1016/j.bioactmat.2023.07.009

Fig. 6.

Fig. 6

Knockdown FUS reduced neuroprotective effects of HypEVs in vitro and in vivo. (A) FUS expression in HypEV was significantly reduced after specific siRNA knockdown in parent cells. (B) Confocal fluorescence images depict sEV uptake by ischemic neurons after co-culturing FUS siRNA-treated HypEV (FUS siRNA-HypEV; PKH26, red) and HypEV (PKH67, green) from primary cortical neurons for 24 h. Scale bar = 20 μm. Nuclei are stained blue (DAPI). Data are expressed as mean ± SEM (n = 17 neurons), and statistical significance was assessed by Student's t-test with differences between groups were considered significant if p < 0.05. (C) MAP2 staining was used to characterize neuron morphology in vitro for neurons incubated with PBS (OGD/R), FUS siRNA–HypEVs (OGD/R + FUS siRNA–HypEV), or HypEVs (OGD/R + HypEV) (scale bar = 50 μm). The bottom yellow frame outlines single neurons (indicated by yellow arrows using NeuronJ) to show the dendritic branching complexity of neurons with reperfusion (24 h) after OGD (3 h) and under normoxic conditions (control) (scale bar = 50 μm). (E) The dendritic complexity was quantified by Sholl analysis, with the numbers of intersections (as shown in Fig. 6C), total neurite length, and neurite number of single neurons (D) expressed as means ± SEM (n = 42–59 neurons per group from three experiments). Statistical significance was assessed by one-way ANOVA with a value of p < 0.05 was considered significant. The Control, OGD/R and OGD/R + HypEV groups were shared with Fig. 4A. (F) Representative images of the infarct core (upper) and three-dimensional images of brain infarction (bottom: the view of the coronal section, horizontal section, and sagittal section from left to right) captured by MRI (T2 phase) in PT mice treated with PBS, HypEVs, or FUS siRNA-HypEVs. (G) The infarct volume and the corresponding ratio (percentage of whole brain) were expressed as means ± SEM (n = 6). Statistical significance was assessed by one-way ANOVA, and differences between groups were considered significant when p < 0.05. (H) and (I) Western blotting analysis of PSD95, synapsin-1 and synaptophysin (synaptic vesicle proteins of the pre-synapse). Data are presented as means ± SEM, n = 7 from 3 mice after MRI analysis. Statistical significance was assessed by one-way ANOVA with a value of p < 0.05 was considered significant.