Fig 2: PAD4 promotes NET formation and exacerbates neurological injury after TBI.(A) Blood and pericontusional brain tissue were collected at 24 hours after sham/TBI, and a population of TLR4+PAD4+ neutrophils were selected by flow cytometry. This population was further gated for the extracellular expression of NE and MPO and for intracellular expression of interleukin-8 (IL-8; nine- and eightfold increase in blood and brain, respectively, after TBI), an activated neutrophil marker and autocrine inducer of NETs. The % TLR4+PAD+ neutrophils expressing IL-8 and extracellular NE/MPO are shown. (B) Placebo or Cl-amidine (30 to 50 mg/kg i.p.) was administered at 10 min after sham/TBI, and pericontusional brain tissue was collected for flow cytometry at 24 hours after injury. Representative histograms and quantified data indicate that Cl-amidine reduces total neutrophil infiltration and attenuates both extracellular MPO and Cit-H3 expression in Ly6G+CD11b+ neutrophils. (C) Placebo or Cl-amidine (10 to 50 mg/kg i.p.) was administered at 10 min after sham/TBI. Representative LSCI (top) and MRI (bottom) images show a reduction in cerebral edema (24 hours after injury) and improved cerebral perfusion (1 to 24 hours after injury) following Cl-amidine (50 mg/kg) treatment. Data are means ± SEM from n = 8 mice per group. (D) Administration of Cl-amidine (50 mg/kg) at 10 min after TBI reduced the time to cross a narrow beam and improved both revolutions per minute (RPM) attained and time spent on the rotarod, as compared to placebo-treated mice after TBI. Data are means ± SEM from n = 10 to 12 mice per group. For all panels, data were analyzed by one-way 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). SSC, side scatter; FSC, forward scatter.

Fig 3: Overexpression of PAD4 exacerbates BBB breakdown and reduced revascularization.a PAD4 mRNA expression was measured by quantitative real-time PCR in the peri-infarct cortex at 3 days, compared with sham-operated mice (n = 5); Mann–Whitney test was applied with *P = 0.0079. b, c Representative immunoblots (b) and quantification of PAD4 levels (c) in the peri-infarct cortex at 4 days after injection of control (Ad-Con) or PAD4 adenovirus (Ad-PAD4) (n = 5); Mann–Whitney test was applied with *P = 0.0079). d Representative images of recombinant Adeno-PAD4-flag-infected cells in the ischemic cortex. Mice were treated with recombinant Adeno-PAD4-flag (right panel) or empty adenovirus (left panel). At 4 days after injection, brain sections were stained with an antibody against Flag. Bar = 40 µm. e Representative immunoblots of H3Cit levels in the peri-infarct cortex at 3 days. f Quantification of the numbers of H3Cit-positive neutrophils in the peri-infarct cortex at 4 days after injection of control or PAD4 adenovirus (n = 6); unpaired two-tailed Student’s t-test was applied with *P = 0.0230. g In-vivo multiphoton microscopic images of intravenously injected FITC-dextran (MW = 40,000 Da; green) leakage in cortical vessels at 14 days. Bar = 100 µm. h Quantification of the permeability (P) product of FITC-dextran for each group (n = 6); unpaired two-tailed Student’s t-test was applied with *P = 0.0244. i Quantification of vascular branches in mice treated with control or PAD4 adenovirus at 14 days after stroke (n = 6), unpaired two-tailed Student’s t-test was applied with *P = 0.0170. j, k Confocal images of CD31-positive microvessels (j) and quantification of microvascular density (k) in the peri-infarct cortex at 14 days (n = 6), unpaired two-tailed Student’s t-test was applied with *P = 0.0019. Bar = 50 µm. l, m In-vivo multiphoton microscopic images of perfused cortical capillaries with intravenously injected FITC-dextran (l) and quantification of perfused capillary length (m) at 14 days after stroke (n = 6), unpaired two-tailed Student’s t-test was applied with *P = 0.0017. Bar = 100 µm. n–p Overexpression of PAD4 worsened neurological outcomes in beam walking test (n, o) and forelimb force test (p) (n = 10). One-way ANOVA test was applied with *P = 0.001 (n), *P = 0.0015 (o), *P < 0.0001 (p) (Sham and Ad-Con). *P = 0.0015 (n), *P = 0.0015 (o), *P = 0.0279 (p) (Ad-Con and. Ad-PAD4). Data are presented as mean ± SD. Source data underlying graph a–c, e, f, h, i, k, m–p are provided as a Source Data file.