Research ArticleNEUROSCIENCE

Neutrophil extracellular traps exacerbate neurological deficits after traumatic brain injury

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Science Advances  29 May 2020:
Vol. 6, no. 22, eaax8847
DOI: 10.1126/sciadv.aax8847
  • Fig. 1 Generation of NETs following experimental TBI.

    Mixed-sex adult CD-1 mice were subjected to TBI, and pericontusional tissue was prepared for electron microscopy at 24 hours after injury. (A) Representative scanning electron micrograph showing NET-like structures adjacent to an injured blood vessel in the pericontusional cortex after TBI. Data are representative of n = 5 mice. Scale bar, 10 μm. (B) Immunogold labeling showing the extranuclear presence of citrullinated histone H3 (Cit-H3; green arrows), a marker of early-stage NET generation, in a pericontusional neutrophil at 24 hours after TBI. Scale bar, 1 μm. (C) Immunogold labeling of the neutrophil granule enzyme, neutrophil elastase (NE), showing a thread-like localization (blue arrows). Scale bar, 0.5 μm. (D and E) Dual immunogold labeling of the vascular marker, laminin (small spots), and NE (large spots, blue arrows). Red dotted lines demarcate the location of blood vessels. Note the extravascular and clustered appearance of NE, indicative of NET formation. Scale bar, 1 μm.

  • Fig. 2 Increased NET formation in patients with severe neurotrauma.

    (A) Serum DNase-I activity and (B) myeloperoxidase (MPO)–DNA binding, a sensitive measure of NET formation, were quantified by EIA in blood collected from control patients (n = 10) or patients with severe TBI undergoing CSF diversion due to elevated ICP (n = 10). Data are presented as means ± SEM and analyzed using a Student’s t test (**P < 0.01 versus control). (C) Axial computed tomography (CT) scan without contrast and magnetic resonance imaging (MRI) axial diffusion-weighted image from a representative patient (19-year-old male, GCS = 7) used in blood collection. Note the effacement of the gray-white delineation and partial effacement of the right lateral ventricle, consistent with diffuse cerebral edema on the CT scan. Arrow indicates a hyperdense area at the gray-white junction in the right frontal lobe, consistent with diffuse axonal injury/tissue tear hemorrhage at the gray-white junctions. On the MRI image, yellow arrows indicate hyperintense areas, consistent with diffuse axonal injury. The more anterior right-sided hyperintensity is cortical and represents a microcontusion with associated diffuse axonal injury (see blue arrow). The hypointense area coincides with the tract of the ventricular catheter that was placed for both CSF diversion and ICP monitoring (white arrowhead). Correlation analysis between patient serum DNase activity and (D) ICP or (E) Glasgow Coma Scale (GCS) score. Pearson’s correlation coefficient (R) and P value are shown as insets.

  • Fig. 3 TLR4 activation promotes NETs and neurovascular injury after TBI.

    (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).

  • Fig. 4 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. 5 Degradation of NETs using rhDNase improves neurological outcomes after TBI.

    (A) Administration of rhDNase (5 mg/kg i.v.) at 1 hour after TBI reduced the extracellular expression of MPO and NE on Ly6G+TLR4+ neutrophils in both blood and brain tissue after TBI. (B) Quantification of data from (A). Scatterplots are representative of n = 8 mice per group and depict the % TLR4+ neutrophils exhibiting extracellular expression of NE and MPO. (C) Administration of rhDNase (5 mg/kg i.v.) at 1 hour after TBI reduced cerebral edema by MRI (top) and improved cerebral perfusion by MRI (middle) and LSCI (bottom) at 24 hours after injury. Representative images are provided from n = 8 mice per group. (D) Quantification of edema, absolute CBF, and area of slow flow/reduced reflow from (C). Data were analyzed using a 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). (E) Administration of rhDNase (5 mg/kg i.v.) at 1 hour after TBI improved chronic neurological function, as assessed at 2 months after TBI, as compared to placebo-treated mice. Top panels depict representative heat maps obtained in the open-field test, which demonstrated an increased time spent within the center zone (a measure of decreased anxiety) after rhDNase treatment following TBI (see middle panel, left). Representative heat maps obtained following the NOR test show that rhDNase increased the time spent exploring a novel object. The increased discrimination index (middle, right) suggests that rhDNase improves recognition memory at 8 weeks after TBI. Bottom panels demonstrate improvements in chronic, posttraumatic motor function following rhDNase treatment. Administration of rhDNase at 1 hour after TBI reduced the time required to traverse a narrow beam and decreased the number of slips, indicative of better motor function and coordination (bottom left and middle). Treatment with rhDNase also improved grip strength after TBI, as compared to placebo-treated mice, suggestive of better neuromuscular function. Data are means ± SEM from n = 9 to 13 mice per group and were analyzed by one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Supplementary Materials

  • Supplementary Materials

    Neutrophil extracellular traps exacerbate neurological deficits after traumatic brain injury

    Kumar Vaibhav, Molly Braun, Katelyn Alverson, Hesam Khodadadi, Ammar Kutiyanawalla, Ayobami Ward, Christopher Banerjee, Tyler Sparks, Aneeq Malik, Mohammad H. Rashid, Mohammad Badruzzaman Khan, Michael F. Waters, David C. Hess, Ali S. Arbab, John R. Vender, Nasrul Hoda, Babak Baban, Krishnan M. Dhandapani

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