Delayed Administration of Nafamostat Mesylate Inhibits Thrombin-Mediated Blood-Spinal Cord Barrier Breakdown During Acute Spinal Cord Injury in Rats

Abstract Background: Nafamostat mesylate (NM), an FDA-approved serine protease inhibitor, exerts anti-neuroinflammation and neuroprotective effect on rat spinal cord injury (SCI). However, the time window for NM administration after SCI as well as its underlying mechanism remains unclear. Methods: A series of different first administration time points of NM was tested on rat contusive SCI model. The optimal time window of NM was screened by evaluating hindlimb locomotion and electrophysiology. We performed western blot and immunofluorescence to evaluate the drug target thrombin as well as its downstream Protease activated receptor 1 (PAR-1), and matrix metalloproteinase-9 (MMP9). Enzyme activity assay was used to test thrombin activity. The permeability of blood-spinal cord barrier (BSCB) was assessed by Evans Blue leakage. The infiltration of peripheral inflammatory cell was observed by immunofluorescence.Results: The optimal administration time window of NM was 2-12 h. The thrombin specific inhibitor, Argatroban, had similar pattern. The temporal expression pattern of thrombin peaked at 12 hours and returned to normal level at 7 days post SCI. PAR-1, the thrombin receptor, was observed a significant upregulation after SCI. MMP9, downstream of PAR-1, was also increased along with thrombin and PAR1. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). NM significantly downregulated the thrombin and MMP9 expression as well as thrombin activity in the spinal cord, especially in ECs. NM administration at 2-12 h after SCI could inhibit the leakage of Evans blue in the epicenter and upregulate tight junction proteins (TJPs) expression. 8 h administration of NM effectively inhibited the infiltration of peripheral macrophages in the acute SCI. Conclusions: Our study provided preclinical data of NM administration time window in SCI model, which is clinically relevant in the acute SCI. We elucidated the protective mechanism of NM through BSCB protection and anti-neuroinflammation via thrombin intervention.