Cytokine expressions of spinal cord injury treated by neurotropin and nafamostat mesylate

Zhou

et al. 2021

Preprint

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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.

Section: Discussionsupporting

confidence: 65%

“…There is an expanded effect in other diseases, such as COVID-19 [33]. We have previously reported that NM has bene cial effect on SCI [34,35]. However, its administration time window and speci c mechanism remain unclear.…”

Section: Discussionmentioning

confidence: 99%

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

Zhou

et al. 2021

Preprint

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“…Indeed, NM inhibits lipopolysaccharide‐induced nitric oxide production, apoptosis, and interleukins 6 and 8 levels in cultured human trophoblasts. NM has also antioxidant effects in TNF‐α‐induced ROS production 2, 17 and a neuroprotective role during ischemia‐induced brain or spinal cord injury has also been suggested 18,42 . The cross‐talk between coagulation and inflammation is, indeed, a key issue of the interaction between patients' blood and the external surfaces and circuit, which fuels a detrimental vicious circuit.…”

Section: Discussionmentioning

confidence: 99%

Use of nafamostat mesilate for anticoagulation during extracorporeal membrane oxygenation: A systematic review

et al. 2022

Background Extracorporeal membrane oxygenation (ECMO) represents an advanced option for supporting refractory respiratory and/or cardiac failure. Systemic anticoagulation with unfractionated heparin (UFH) is routinely used. However, patients with bleeding risk and/or heparin‐related side effects may necessitate alternative strategies: among these, nafamostat mesilate (NM) has been reported. Methods We conducted a systematic literature search (PubMed and EMBASE, updated 12/08/2021), including all studies reporting NM anticoagulation for ECMO. We focused on reasons for starting NM, its dose and the anticoagulation monitoring approach, the incidence of bleeding/thrombosis complications, the NM‐related side effects, ECMO weaning, and mortality. Results The search revealed 11 relevant findings, all with retrospective design. Of these, three large studies reported a control group receiving UFH, the other were case series (n = 3) or case reports (n = 5). The main reason reported for NM use was an ongoing or high risk of bleeding. The NM dose varied largely as did the anticoagulation monitoring approach. The average NM dose ranged from 0.46 to 0.67 mg/kg/h, but two groups of authors reported larger doses when monitoring anticoagulation with ACT. Conflicting findings were found on bleeding and thrombosis. The only NM‐related side effect was hyperkalemia (n = 2 studies) with an incidence of 15%–18% in patients anticoagulated with NM. Weaning and survival varied across studies. Conclusion Anticoagulation with NM in ECMO has not been prospectively studied. While several centers have experience with this approach in high‐risk patients, prospective studies are warranted to establish the optimal space of this approach in ECMO.

“…Nafamostat is an FDA-approved drug to treat pancreatitis and is being explored as a treatment for other diseases, such as COVID-19 [ 35 ]. We have previously reported that nafamostat has a beneficial effect on recovery from SCI [ 9 , 36 ]. However, its administration time window and specific mechanism were unclear.…”

Section: Discussionmentioning

confidence: 99%

“…In our previous study, the result of inflammation ChIP sequencing showed that nafamostat could reduce many chemokines expression in local site of SCI [ 36 ]. Here, we also proved that administration of nafamostat after SCI reduced neutrophil and macrophage infiltration significantly, which we believed that the protection of BSCB was a considerable mechanism of the anti-neuroinflammation effect induced by nafamostat.…”

Section: Discussionmentioning

confidence: 99%

Delayed administration of nafamostat mesylate inhibits thrombin-mediated blood–spinal cord barrier breakdown during acute spinal cord injury in rats

Zhou

et al. 2022

J Neuroinflammation

Self Cite

Background Nafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action. Methods Time to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood–spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence. Results The optimal administration time window of nafamostat was 2–12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2–12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site. Conclusions Our study provides preclinical information of nafamostat about the administration time window of 2–12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration.