Anticoagulation with warfarin and rivaroxaban ameliorates experimental autoimmune encephalomyelitis

Abstract

Background: In multiple sclerosis, coagulation factors have been shown to modulate inflammation. In this translational study, we investigated whether long-term anticoagulation with warfarin or rivaroxaban has beneficial effects on the course of autoimmune experimental encephalomyelitis (EAE).

Methods: Female SJL/J mice treated with anticoagulants namely warfarin or rivaroxaban were immunized with PLP_139-151. Stable anticoagulation was maintained throughout the entire experiment. Mice without anticoagulation treated with the vehicle only were used as controls. The neurological deficit was recorded during the course of EAE, and histopathological analyses of inflammatory lesions were performed.

Results: In preventive settings, both treatment with warfarin and rivaroxaban reduced the maximum EAE score as compared to the control group and led to a reduction of inflammatory lesions in the spinal cord. In contrast, therapeutic treatment with warfarin had no beneficial effects on the clinical course of EAE. Signs of intraparenchymal hemorrhage at the site of the inflammatory lesions were not observed.

Conclusion: We developed long-term anticoagulation models that allowed exploring the course of EAE under warfarin and rivaroxaban treatment. We found a mild preventive effect of both warfarin and rivaroxaban on neurological deficits and local inflammation, indicating a modulation of the disease induction by anticoagulation.

Keywords: Anticoagulation; Experimental autoimmune encephalomyelitis; Inflammation; Mice; Multiple sclerosis.

Fig. 1

Long-term anticoagulation of SJL/J mice. PLP_139–151-immunized mice were anticoagulated with warfarin via s.c. injections every 12 h during the course of the experiment. INR was measured on a regular basis (a). PLP_139–151-immunized mice were anticoagulated with rivaroxaban. Rivaroxaban plasma concentration and thrombin activity in the plasma were measured on d15 and d27 post immunization (b, c)

Fig. 2

Preventive anticoagulation with warfarin ameliorates neurological deficits of EAE. Forty mice were immunized on d0 with PLP_139–151 and 20 were treated with warfarin, starting on d1. The clinical scores of the mice were measured daily (a). Maximum scores were found decreased in warfarin-treated mice as compared to the control group (b). There was no difference in loss of weight at the end of the experiment between the groups (c)

Fig. 3

Preventive anticoagulation with rivaroxaban ameliorates neurological deficits of EAE. Forty mice were immunized on d0 with PLP_139–151 and 20 were treated with rivaroxaban, starting on d1. The clinical score of the mice were measured daily (a). Maximum scores were found decreased in rivaroxaban-treated mice as compared to the control group (b). Loss of weight was reduced in the anticoagulated group at the end of the experiment (c)

Fig. 4

Preventive anticoagulation with warfarin and rivaroxaban decrease inflammatory lesions in the spinal cord of PLP_139–151-immunized SJL/J mice. Representative images of DAPI stained lumbar spinal cord sections for the respective groups. Inflammatory lesions are marked by asterisk (a–d, ×2.5 magnification, scale bar = 500 μm). Quantitative analysis of the cervical, thoracic, and lumbar spinal cord shows reduced inflammatory lesions on d15 and d27 after immunization in anticoagulated mice compared to controls (e, f)

Fig. 5

Thrombin increases brain endothelial permeability in vitro. PBMECs were seeded on transwell inserts and continuous TEER values were measured by a cellZscope® system. The graph displays a representative experiment that shows reduced TEER values of thrombin-treated PBMECs compared to control-treated ones (a). Quantification of brain endothelial permeability 1 h after thrombin treatment shows a decrease of TEER values in a dose-dependent manner. At least three replicates were averaged for one biological sample (n = 3, b)