Selective factor XIIa inhibition attenuates silent brain ischemia: application of molecular imaging targeting coagulation pathway

Abstract

Objectives: The purpose of this study was use molecular imaging targeting coagulation pathway and inflammation to better understand the pathophysiology of silent brain ischemia (SBI) and monitor the effects of factor XIIa inhibition.

Background: SBI can be observed in patients who undergo invasive vascular procedures. Unlike acute stroke, the diffuse nature of SBI and its less tangible clinical symptoms make this disease difficult to diagnose and treat.

Methods: We induced SBI in mice by intra-arterial injection of fluorescently labeled microbeads or fractionated clot into the carotid artery. After SBI induction, diffusion-weighted magnetic resonance imaging was performed to confirm the presence of microinfarcts in asymptomatic mice. Molecular imaging targeting the downstream factor XIII activity (single-photon emission computed tomography/computed tomography) at 3 h and myeloperoxidase activity (magnetic resonance imaging) on day 3 after SBI induction were performed, without and with the intravenous administration of a recombinant selective factor XIIa inhibitor derived from the hematophagous insect Triatoma infestans (rHA-Infestin-4). Statistical comparisons between 2 groups were evaluated by the Student t test or Mann-Whitney U test.

Results: In SBI-induced mice, we found abnormal activation of the coagulation cascade (factor XIII activity) and increased inflammation (myeloperoxidase activity) close to where emboli lodge in the brain. rHA-Infestin-4 administration significantly reduced ischemic damage (53% to 85% reduction of infarct volume, p < 0.05) and pathological coagulation (35% to 39% reduction of factor XIII activity, p < 0.05) without increasing hemorrhagic frequency. Myeloperoxidase activity, when normalized to the infarct volume, did not significantly change with rHA-Infestin-4 treatment, suggesting that this treatment does not further decrease inflammation other than that resulting from the reduction in infarct volume.

Conclusions: Focal intracerebral clotting and inflammatory activity are part of the pathophysiology underlying SBI. Inhibiting factor XIIa with rHA-Infestin-4 may present a safe and effective treatment to decrease the morbidity of SBI.

Figure 1. Model of SBI

(A) Fluorescent microbeads and fractionated fluorescent thromboemboli prior to injection. (B) Surgical approach with catheter retrogradely inserted into the external carotid artery (ECA). During injection, the common carotid artery (CCA) was temporarily ligated to force the embolic material into the internal carotid artery (ICA). (C) Ex vivo fluorescence images of the brain surface after injection of either microbeads or thromboemboli, scale bar indicates 200μm. (D) Diffusion weighted MRI was done 4 hours after embolism and shows hallmarks of SBI similar to what is seen in patients. Multiple small lesions (some marked by arrows) are revealed by diffusion weighted imaging (DWI). Decrease in the apparent diffusion coefficient (ADC) in the corresponding areas confirmed restricted diffusion, characteristic of acute infarcts.

Figure 2. rHA-Infestin-4 reduces injury quantified by MRI and TTC on day 3 after SBI

(A, B) Assessment of infarct volume by T2 MRI on day 3 after SBI (Mann-Whitney U test). (C, D) Assessment of tissue damage by TTC staining on day 3 after SBI. Figure shows bar graphs, and representative MRI. Data are presented as mean ± SEM. * p < 0.05.

Figure 3. Secondary hemorrhage is not increased by rHA-Infestin-4

Assessment of secondary hemorrhage on day 3 after injection of microbeads or thromboemboli. One representative brain slice is shown per mouse. Red frames indicate mice in which hemorrhage was detected (arrows).

Figure 4. rHA-Infestin-4 reduces clotting in thromboemboli-induced SBI

(A) Assessment of transglutaminase activity (FXIIIa) 3 hours after application of thromboemboli by SPECT-CT, autoradiography, and fluorescence reflectance imaging (FRI). (B) After rHA-Infestin-4 administration. (C) Quantification of SPECT target to background ratio (TBR), ex vivo scintillation counting of entire brain (Mann-Whitney U test). Data are shown as percent injected dose per gram tissue (%IDGT). (D) quantification of autoradiography (CPM: counts per minute). Data are presented as mean ± SEM. * p < 0.05.

Figure 5. rHA-Infestin-4 reduces clotting in microbead-induced SBI

(A) Assessment of transglutaminase activity (FXIIIa) 3 hours after application of microbeads by SPECT-CT, autoradiography, and fluorescence reflectance imaging (FRI). (B) After rHA-Infestin-4 administration. (C) quantification of SPECT target to background ratio (TBR) and ex vivo scintillation counting of entire brain (Mann-Whitney U test). Data are shown as percent injected dose per gram tissue (%IDGT). (D) quantification of autoradiography (CPM: counts per minute). Data are presented as mean ± SEM. * p < 0.05.

Figure 6. Immunohistochemistry for FXIII

Immunohistochemical staining for FXIII, 3 hours after induction of SBI. Bar indicates 50 μm.

Figure 7. MR imaging of MPO activity on day 3 after thromboemboli-induced SBI

Assessment of MPO activity 3 days after SBI induced by thromboemboli. Yellow arrows point at MPO⁺ lesions, black arrows at ventricle. FRI: Fluorescence reflectance imaging, showing location of thromboemboli. MPO+ volume is normalized to T2+ volume. CNR: Contrast-to-noise-ratio. AU: Arbitrary units. Data are presented as mean ± SEM.

Figure 8. MR imaging of MPO activity on day 3 after microbead-induced SBI

Assessment of MPO activity 3 days after SBI after microbead embolism. Yellow arrows point at MPO⁺ lesions, black arrows at ventricle. FRI: Fluorescence reflectance imaging, showing position of beads. MPO+ volume is normalized to T2+ volume. AU: Arbitrary units. CNR: Contrast-to-noise-ratio. Data are presented as mean ± SEM.