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

Abstract

Background: Traumatic brain injury (TBI) is a devastating neurological condition and a frequent cause of permanent disability. Posttraumatic inflammation and brain edema formation, two pathological key events contributing to secondary brain injury, are mediated by the contact-kinin system. Activation of this pathway in the plasma is triggered by activated factor XII. Hence, we set out to study in detail the influence of activated factor XII on the abovementioned pathophysiological features of TBI.

Methods: Using a cortical cryogenic lesion model in mice, we investigated the impact of genetic deficiency of factor XII and inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused Infestin-4 on the release of bradykinin, the brain lesion size, and contact-kinin system-dependent pathological events. We determined protein levels of bradykinin, intracellular adhesion molecule-1, CC-chemokine ligand 2, and interleukin-1β by enzyme-linked immunosorbent assays and mRNA levels of genes related to inflammation by quantitative real-time PCR. Brain lesion size was determined by tetrazolium chloride staining. Furthermore, protein levels of the tight junction protein occludin, integrity of the blood-brain barrier, and brain water content were assessed by Western blot analysis, extravasated Evans Blue dye, and the wet weight-dry weight method, respectively. Infiltration of neutrophils and microglia/activated macrophages into the injured brain lesions was quantified by immunohistological stainings.

Results: We show that both genetic deficiency of factor XII and inhibition of activated factor XII in mice diminish brain injury-induced bradykinin release by the contact-kinin system and minimize brain lesion size, blood-brain barrier leakage, brain edema formation, and inflammation in our brain injury model.

Conclusions: Stimulation of bradykinin release by activated factor XII probably plays a prominent role in expanding secondary brain damage by promoting brain edema formation and inflammation. Pharmacological blocking of activated factor XII could be a useful therapeutic principle in the treatment of TBI-associated pathologic processes by alleviating posttraumatic inflammation and brain edema formation.

Keywords: Brain edema; Factor XII; Focal brain lesion.

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)

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)

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)

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 in FXII-deficient animals and wildtype mice on d1 (right). Scale bar depicts 100 μm (n = 4, ***P < 0.001, **P < 0.01, *P < 0.05, unpaired, two-tailed Student’s t test)

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))

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))