Journal: Journal of Extracellular Vesicles

Article Title: C1qc Mediates Blood–Brain Barrier Disruption in a Mouse Model of Intracerebral Haemorrhage Through Microglia‐Derived Migrasomes

doi: 10.1002/jev2.70377

Figure Lengend Snippet: C1qc mediates the effects of migrasomes on the BBB via the Nlrp3–Caspase 1–GSDMD pathway. (A) Immunoblots revealed C1qc, Nlrp3, cleaved caspase‐1, and GSDMD‐N protein expression levels in C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Densitometric quantification of protein levels; the data are presented as the mean SD; ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Immunofluorescence staining for GSDMD–N and the endothelial cell marker CD31 in the perihaematomal area of C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Normalized quantitative measurement of GSDMD‐N fluorescence intensity relative to CD31 area. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) Immunofluorescence staining for cleaved caspase‐1 and the endothelial cell marker CD31 in the perihaematomal area of C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Fluorescence signal density from cleaved caspase‐1, normalized against the CD31 area, was analysed quantitatively. The data are expressed as ± SD; *** p < 0.001, **** p < 0.0001. (D) GSDMD‐N staining of endothelial cells that were treated as described in Figure ( n = 3). Quantification of the fluorescence intensity of GSDMD‐N of endothelial cells in the vehicle + microglia‐secreting migrasome group, the hemin + microglia‐secreting migrasome group, and the hemin + siC1qc microglia‐secreting migrasome group were also quantified. Data are presented as the mean ± SD with the indicated significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Cleaved caspase‐1 staining of endothelial cells that were treated as described previously in Figure ( n = 3). Quantification of the fluorescence intensity of Cleaved caspase‐1 of endothelial cells in the vehicle + microglial migrasome groups, hemin + microglial migrasome group, and hemin + siC1qc microglial migrasome group were also quantified. The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01. (F) Immunoblots showing the levels of the Nlrp3 and GAPDH proteins in endothelial cells that were treated as described in Figure ( n = 6). Densitometric quantification of protein levels. The data are presented as the mean ± SD; *** p < 0.001. (G) Claudin‐5 staining of endothelial cells that were treated with MCC950 and microglial migrasomes without C1qc ( n = 5). Quantification of the fluorescence intensity of Claudin‐5. The data are presented as the mean ± SD; ns, no significant difference; ** p < 0.01, *** p < 0.001. (H) GSDMD‐N immunofluorescence in endothelial cells treated with migrasomes, C1qc‐deficient migrasomes, or C1qc‐deficient migrasomes plus Nigericin. Quantification of the fluorescence intensity of GSDMD‐N. The data are presented as the mean ± SD; ** p < 0.01, *** p < 0.001. (I) Claudin‐5 immunofluorescence in endothelial cells treated with migrasomes, C1qc‐deficient migrasomes, or C1qc‐deficient migrasomes plus Nigericin. Quantification of the fluorescence intensity of Claudin‐5. The data are presented as the mean ± SD; * p < 0.05, *** p < 0.001. (J) Immunoblots showing the levels of the GSDMD‐N, Claudin‐5 and GAPDH proteins in endothelial cells that were treated with migrasomes at different time points ( n = 6). Densitometric quantification of protein levels. The data are presented as the mean ± SD; ns = no significant difference, ** p < 0.01, *** p < 0.001. (K) Cell viability dramatically decreased endothelial cells that were treated with migrasomes at different time points ( n = 3). Data are shown as the means ± SD with significance levels; ns = no significant difference, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Ten milligrams of the inflammasome inhibitor MCC950 (Nlrp3 inhibitor)(Selleck Chemicals, Houston, TX, USA) was dissolved in 1 mL of 1× phosphate‐buffered saline (PBS).

Techniques: Western Blot, Expressing, Injection, Immunofluorescence, Staining, Marker, Fluorescence