Alleviation of secondary brain injury, posttraumatic inflammation, and brain edema formation by inhibition of factor XIIa

Figures

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  Fig. 1. Genetic deficiency and pharmacologic inhibition of factor XIIa provides protection from tissue damage. Lesion volumetry shows less necrotic brain tissue in FXII−/− mice and mice treated with rHA-Infestin-4 in comparison with control groups after 1 day (d1) and 3 days (d3), respectively (n = 8, **P < 0.01, *P < 0.05, unpaired, two-tailed Student’s t test)

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  Fig. 2. Factor XII (FXII) deficiency and pharmacologic inhibition of activated FXII displays blood-brain barrier stabilizing and antiedematous effects 1 and 3 days after induction of focal trauma. a Fluorometric measurement of Evans Blue extravasation into the brain parenchyma after 2 h, 12 h, 1 day (d1), and 3 days (d3) in mice treated with rHA-Infestin-4 or vehicle (n = 5–6 per group, *P < 0.05, unpaired, two-tailed Student’s t test). b Fluorometric measurement of Evans Blue extravasation into the brain parenchyma after 1 (d1) and 3 days (d3) in wildtype (WT) mice and FXII-deficient (FXII−/−) mice (n = 5–6 per group, ***P < 0.001, *P < 0.05, unpaired, two-tailed Student’s t test). Right panel shows representative brain slices of Evans Blue extravasation into the tissue. c Antiedematous effect in FXII deficiency and FXIIa inhibition shown by determination of brain water content in the lesioned hemisphere 24 h after trauma (n = 8–9, *P < 0.05, unpaired, two-tailed Student’s t test)

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  Fig. 3. Factor XII (FXII)-deficiency and pharmacologic inhibition of activated FXII inhibits degradation of tight junction protein occludin. Quantification of occludin by Western blot analysis on day 1 (d1, upper panel) and on day 3 (d3, lower panel) after injury in sham-operated animals, WT mice and FXII−/− mice (left), and sham-operated mice and mice treated with rHA-Infestin-4 or vehicle (right), respectively (n = 5 per group, **P < 0.01, *P < 0.05, one-way ANOVA followed by Bonferroni multiple comparison test compared with WT mice, AU arbitrary units)

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  Fig. 4. Factor XII enhances inflammatory processes after traumatic brain injury. a Determination of CCL2 protein concentrations in the brain tissue of sham-operated mice and mice treated with rHA-Infestin-4 or vehicle 2, 6, and 12 h, and 1 day (d1) after injury (n = 4–5 per group, ***P < 0.001, one-way ANOVA followed by Bonferroni multiple comparison test). b Quantification of CCL2 concentrations in the brain tissue of FXII−/− mice, sham-operated mice, and wildtype controls 12 h and 1 day (d1) after injury (n = 4–5 per group, ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA followed by Bonferroni multiple comparison test compared with WT mice)−/−. c Relative gene expression of CCL2 in the injured cortices of FXII-deficient mice and wildtype controls, and rHA-Infestin-4- and vehicle-treated mice on day 1 after trauma induction (n = 5 per group, *P < 0.05, unpaired, two-tailed Student’s t test, amount of gene expression normalized to sham-operated animals (not shown)). d Macrophages and activated microglia were quantified in the lesioned hemispheres after 1 (d1) and 3 days (d3) after brain trauma (left). Representative immunohistochemical stainings of CD11b-positive macrophages/microglia…

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  Fig. 5. a Determination of interleukin-1β (IL-1β) concentrations in the brain tissue of sham-operated mice, FXII−/− mice, and mice treated with rHA-Infestin-4 and their respective controls on day 1 after injury (n = 4–5 per group, ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA followed by Bonferroni multiple comparison test compared with WT mice). b Relative gene expression of IL-1β (lower panel) and tumor necrosis factor α (TNF-α, upper panel) in the injured cortices of rHA-Infestin-4- and vehicle-treated mice on day 1 (d1) and day 3 (d3) after trauma induction (n = 5 per group, **P < 0.01, *P < 0.05, unpaired, two-tailed Student’s t test, amount of gene expression normalized to sham-operated animals (not shown))

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  Fig. 6. Factor XII enhances inflammatory processes after traumatic brain injury. a Neutrophils were quantified in the lesioned hemispheres after 1 (d1) and 3 days (d3) after brain trauma (left). Representative immunohistochemical stainings of neutrophils in FXII-deficient animals and wildtype mice on d1 (right). Scale bar depicts 50 μm (n = 4, **P < 0.01, *P < 0.05, unpaired, two-tailed Student’s t test). b Determination of ICAM-1 plasma concentrations of sham-operated mice, FXII−/− mice and mice treated with rHA-Infestin-4 and their respective controls on day 3 after injury (n = 4–5 per group, *P < 0.05, one-way ANOVA followed by Bonferroni multiple comparison test compared with WT mice). c Relative gene expression of ICAM-1 in FXII-deficient mice (left) and rHA-Infestin-4-treated mice (right) in comparison to their respective control groups 3 days after trauma induction (n = 5, ***P < 0.001, **P < 0.01, unpaired, two-tailed Student’s t test, amount of gene expression normalized to sham-operated animals (not shown))