The Study On Spinal Cord Injury Repair By Nafamostat Mesilate

?Objective?Spinal cord injury(SCI)leads to severe neural damage for which there is currently no effective treatment.Owing to the local micro-environment imbalance,SCI can trigger a cascaded inflammatory response.Serine proteases,such as thrombin,tryptas,are significantly enhanced,and activate the PAR1 receptor which can activate a series of downstream signaling pathways to mediate inflammation response and neuralgia.Exploration of the neuroprotective effect among clinically approved drugs will speed up clinical translation of SCI.Nafamostat mesilate(NM)as a synthetic serine protease inhibitor has been used clinically in pancreatitis treatments.However,its effectiveness in SCI is unknown.The aim of this study is to confirm the efficacy of NM in ameliorating SCI.?Methods?1.An SCI model was established using IMPACTOR MODEL-II.Female Wistar rats(240 ± 10 g)were randomly divided into three groups: Group I: Sham(laminectomy only without SCI),Group II: Injury + saline,Group III: Injury + NM.H&E staining and LFB staining were used to assess contusion area,cavity formation and demyelination in different groups.2.Tissue preservation.HE staining and LFB staining were used to assess contusion area,cavity formation and demyelination in different groups.At two time points,three days and 8 weeks after SCI,the rats were anaesthetized and subjected to H&E staining and LFB staining according to the standard procedures.3.Behavioural analysis.Female Wistar rats(240 ± 10 g)were randomly divided into three groups: Group I: Sham(laminectomy only without SCI),Group II: Injury + saline,Group III: Injury + NM.To evaluate the functional recovery,we administered a battery of behavioural tests from day 1 to week 8 following surgery.Two observers,blinded to all group assig NM ents,recorded the behavioural tests.Functional restoration was assessed according to BBB locomotor scores and the inclined plane test.Before evaluation,the rats were allowed to move freely.4.To explore the mechanism of NM in ameliorating SCI.Female Wistar rats(240 ± 10 g)were randomly divided into three groups: Group I: Sham(laminectomy only without SCI),Group II: Injury + saline,Group III: Injury + ?NM.Pro-inflammatory cytokines were detected by ELISA and the expression of thrombin was tested by Western blot.The activation of Microglia/ macrophages was detected by CD68 of Immunofluorescence staining and astrocyte by GFAP of Immunofluorescence staining.Apoptosis was examined by TUNEL staining,Annexin V-FITC/PI,Caspase-3 and Bcl-2 and neurotrophins were tested by Western blot.?Results?1.HE staining and LFB staining both provided histological evidence of improved behavior scores,showing a significant increase in spared tissue in both grey and white matter.Compared with Injury group,H&E staining showed that NM intervention could significantly improve the damaged area at early state of SCI(p < 0.05)and the cavity formation at chronic phase of that(p < 0.01);LFB staining showed that NM treatment group had more LFB positive region,indicating that NM has a effect in alleviating demyelination(p < 0.05).2.BBB locomotor score and inclined plane test were used to evaluate the recovery of hindlimb motor function in rats within 8 weeks after SCI.The results of BBB score and inclined plane test showed that NM treatment could improve the behavior of rats compared with Injury group.The results have statistical difference.3.NM intervention has a beneficial effect on inflammation,apoptosis and neuroprotection after SCI.NM decreased the expression of the proinflammatory cytokines TNF-? and IL-6(p < 0.01).In addition,apoptosis was also significantly decreased,as shown by TUNEL staining,Annexin-V/PI and by Western blot for Caspase-3 and Bcl-2 expression(p < 0.01).Due to the mechanism of action of NM as a serine protease inhibitor,the drug decreased thrombin expression in the damaged spinal cord.Furthermore,NM increased the expression of neurotrophins(p < 0.05).?Conclusion?Upon NM treatment,the functional and histological outcomes were improved and microenviro NM ent upon SCI was modulated.As a clinically approved drug,NM holds promise for clinical use after spinal cord injury.