Journal: Science Advances
Article Title: Neutrophil extracellular traps exacerbate neurological deficits after traumatic brain injury
Figure Lengend Snippet: ( A ) Increased TLR4 expression in neutrophils from blood or pericontusional brain tissue at 24 hours after sham (blue)/TBI (red). Scatterplots show % TLR4 + neutrophils from n = 5 mice per group. ( B ) Extracellular expression of MPO, NE, and Cit-H3 in CD11b + Ly6G + neutrophils derived from blood or brain from C3H/OuJ (blue) or C3H/HeJ (red) mice after sham/TBI. Representative flow cytometry scatterplots are provided along with quantified data. Brain panels are depicted as % total brain cells, and blood panels are shown as % leukocytes [% white blood cell (WBC)]. ( C ) Cerebral perfusion and cerebral edema were quantified by MRI in C3H/OuJ or C3H/HeJ mice at 24 hours after TBI. Cerebral hypoperfusion, cerebral edema, and the region of slow flow are attenuated in C3H/HeJ mice, indicative of improved cerebrovascular function following TLR4 inhibition. MPO-DNA binding, a quantitative marker of NET formation, was measured in blood from mice immediately following the final imaging session. ( D ) Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled wild-type (WT; C3H/OuJ) or mutant (MUT; C3H/HeJ) neutrophils were administered to WT or MUT mice at the time of TBI. NET formation was elevated in WT > WT and WT > MUT mice, as assessed by flow cytometry, as compared to MUT > MUT and MUT > WT mice. ( E and F ) Representative images showing changes in cerebral edema (top) and CBF (bottom), as assessed by MRI and LSCI, respectively. For all panels, data are means ± SEM from n = 8 mice per group from two independent experiments. Data were analyzed using a Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ns, not statistically significant).
Article Snippet: Cells were incubated with conjugated antibodies against the following markers to detect the extracellular presence of TLR4 (BioLegend, catalog no. 145409), CD11b (BioLegend, catalog no. 101212), F4/80 (BioLegend, catalog no. 123107), Ly6G (BioLegend, catalog no. 127608), NE (Bioss, catalog no. bs-6982R), MPO (Invitrogen, catalog no. PA5-16672), and Cit-H3 (Cell Signaling Technology, catalog no. 9715).
Techniques: Expressing, Derivative Assay, Flow Cytometry, Inhibition, Binding Assay, Marker, Imaging, Labeling, Mutagenesis