C1-Inhibitor protects from focal brain trauma in a cortical cryolesion mice model by reducing thrombo-inflammation

Abstract

Traumatic brain injury (TBI) induces a strong inflammatory response which includes blood-brain barrier damage, edema formation and infiltration of different immune cell subsets. More recently, microvascular thrombosis has been identified as another pathophysiological feature of TBI. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is activated in different neurological diseases. C1-Inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-Inhibitor in a model of TBI. Male and female C57BL/6 mice were subjected to cortical cryolesion and treated with C1-Inhibitor after 1 h. Lesion volumes were assessed between day 1 and day 5 and blood-brain barrier damage, thrombus formation as well as the local inflammatory response were determined post TBI. Treatment of male mice with 15.0 IU C1-Inhibitor, but not 7.5 IU, 1 h after cryolesion reduced lesion volumes by ~75% on day 1. This protective effect was preserved in female mice and at later stages of trauma. Mechanistically, C1-Inhibitor stabilized the blood-brain barrier and decreased the invasion of immune cells into the brain parenchyma. Moreover, C1-Inhibitor had strong antithrombotic effects. C1-Inhibitor represents a multifaceted anti-inflammatory and antithrombotic compound that prevents traumatic neurodegeneration in clinically meaningful settings.

Keywords: C1-inhibitor; blood-brain barrier; contact-kinin system; edema; inflammation; thrombosis; traumatic brain injury.

Figure 1

C1-Inhibitor (C1-Inh) protects against traumatic brain injury in mice of both sexes. (A) Left panel shows representative 2,3,5-triphenyltetrazolium chloride (TTC) staining of 5 coronal brain sections of 6-week-old male control mice (Ctrl) and 6-week-old male mice treated with 7.5 IU or 15.0 IU C1-Inh. C1-Inh was always applied 1 h after cortical cryolesion and the lesion volume was assessed from TTC staining at day 1. The lesion volume was reduced in a dose-dependent manner with a significant reduction after treatment with 15.0 IU (n = 11–13, * P < 0.05, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test). (B) Representative TTC staining and lesion volume of 6-week-old female control and 15.0 IU C1-Inh treated mice, showing a significant reduction in lesion volume after treatment with 15.0 IU at day 1 (n = 10–11, ** P = 0.0019, Unpaired t-test). (C) A significant reduction of lesion volume was detectable up to 5 days after treatment with 15.0 IU C1-Inh in 6-week-old male mice (n = 10, * P < 0.05, Unpaired t-test).

Figure 2

C1-inhibitor (C1-Inh) treatment results in stabilization of the blood brain barrier. (A) Vascular leakage on day 1 after cryolesion was significantly decreased after treatment with 15.0 IU of C1-Inh as confirmed by the concentration of Evans Blue detectable in the brain parenchyma (n = 6, * P < 0.05, ns P > 0.05, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test, ipsi: ipsilateral hemisphere, contra: contralateral hemisphere). (B) Edema formation as reflected by the brain water content in the ipsi- and contralateral hemispheres of control and 15.0 IU treated mice on day 1 after cryolesion (n = 6, * P < 0.05, ns P > 0.05, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test). (C) Relative gene expression of occludin in the ipsilateral brain parenchyma of control and 15.0 IU treated mice 24 h after cryolesion or sham operation (n = 7 or 3 for sham operated, * P < 0.05, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test). (D) Relative gene expression of claudin-5 in the ipsilateral brain parenchyma of control and 15.0 IU treated mice 24 h after cryolesion or sham operation (n = 7 or 3 for sham operated, ns P > 0.05, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test).

Figure 3

C1-inhibitor (C1-Inh) treatment results in attenuation of the inflammatory response. (A) Representative immunohistochemical stainings of cerebral CD11b positive cells on day 1 after cryolesion. Quantification of cell number in control and C1-Inh treated mice revealed less CD11b positive cells per mm² in the lesion site of C1-Inh treated mice (n = 5, * P < 0.05, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test, Scale bar 50 μm). (B) Relative gene expression of the genes encoding the proinflammatory cytokines ccl2, ccl3, interleukin (IL)-1β, and tumor necrosis factor (Tnf)α in control and C1-Inh treated mice on day 1 after cryolesion. Gene expression of all proinflammatory cytokines were significantly reduced in C1-Inh treated mice (n = 5, * P < 0.05, ** P < 0.01, *** P < 0.001, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test).

Figure 4

C1-inhibitor (C1-Inh) treatment results in inhibition of thrombus formation. (A) Accumulation of fibrin(ogen) in the ipsilateral (ipsi) and contralateral (contra) brain hemispheres of control and 15.0 IU C1-Inh treated mice was analyzed by immunoblotting 24 h after cryolesion, and bands were quantified by densitometry. The representative immunoblot and the quantification shows a significant reduction of fibrin(ogen) in the 15.0 C1-Inh treated mice (n = 5–6, * P < 0.05, One-way analysis of variance with post hoc Bonferroni’s Multiple Comparison Test; AU = arbitrary units). (B) Representative H8E staining from traumatic hemispheres of vehicle-treated and 15.0 IU C1-Inh treated mice on day 1 after cryolesion. Occluded vessels (arrowhead in the left panel) were more abundant in control mice when compared to vessels of 15.0 IU C1-Inh treated mice (arrowheads in the right panel). This finding was confirmed by the calculation of the thrombosis index showing a highly significant reduction of occluded vessels in 15.0 IU C1-Inh treated mice on day 1 after cryolesion (n = 4, *** P < 0.001, Unpaired t-test; Scale bar 50 μm).