The Neuroprotective Effect And Mechanism Of 2-(2-Benzofuranyl)-2-imidazoline In A Rat Model Of Acute Spinal Cord Injury

Part ? 2-(2-benzofuranyl)-2-imidazoline Provides Neuroprotection in a Rat Model of Spinal Cord InjuryObjective:Previous reports showed that 2-(-2-benzofuranyl)-2-imidazoline(2-BFI)has antioxidant,anti-inflammatory and anti-apoptotic effects on neuroprotection in numerous disorders.However,the precise mechanisms of 2-BFI in spinal cord injury(SCI)still remain elusive.The purpose of this study was to investigate the potential neuroprotective effects of 2-BFI in a rat model of SCI established by clip-compression injury method.Methods:The adult male Sprague Dawley rats were divided into 3 groups:(1)sham group;(2)SCI+vehicle group;(3)SCI+2-BFI group.The experimental SCI model was established as described previously.After deep general anesthesia,the rat was placed in a prone position,dorsal midline incision was made and a laminectomy was performed from the T10 to the T12.Rats in the SCI+vehicle group and SCI+2-BFI group received compression for 60 s by a 70-g closing force Yasargil aneurysm clip,while in the remaining rats,the Th10-Th12 laminas were removed until the spinal cord was exposed without causing SCI(the sham group).Indicators of successful injury included the red and swollen appearance of the local spinal cord,fluttering of both hindlimbs immediately after compression,and bilateral hindlimb paralysis when the rats were awake.Drug administration(intraperitoneal injection of 2-BFI at 3 mg/kg or 0.9%saline twice daily)was carried on by an investigator who was blind to the drugs.Rats of each group were randomly selected to perform a locomotion recovery test at day 1,2,3,7,14,21 day after SCI.The locomotion was assessed using the Basso,Beattie and Bresnahan(BBB)locomotor rating scale,which is used to assign scores ranging from 0 points to 21 points.Results:The locomotor recovery test of rats after SCI was evaluated by the BBB locomotor rating scale.Normal scores(21 points)were observed in the sham group.One day after SCI,the BBB locomotor rating scale score was decreased promptly in both the SCI+vehicle group and the SCI+2-BFI group comparing with the score in sham group(0.17±0.41 vs 0.17±0.41,p<0.05,respectively).However,there was no significant difference between these two groups(P>0.05).Furthermore,the BBB scores were still higher in the SCI+2-BFI group than in the SCI+vehicle group at 3 days after SCI,but the difference was not significant(2.00±1.09 vs 1.33±0.82,P>0.05).The average BBB score was significantly improved in rats treated with 2-BFI at both 7 days and 14 days after SCI(3.33±0.52 vs 4.33±0.52,P<0.05;4.67±0.82 vs 6.17±0.98,P<0.05,respectively).Moreover,these differences became more and more remarkable at 21days after SCI(6.17±0.98 vs 8.00±1.10,P<0.01).Conclusion:After treatment of 2-BFI,BBB scores of the SCI rats were significantly higher than those of the SCI+vehicle group,indicating that intraperitoneal injection of 2-BFI(3mg/kg)may provide neuroprotective effect in SCI rats.Part ? Treatment with 2-BFI promotes antioxidant and antiapoptotic effects in a Rat Model of Spinal Cord InjuryObjective:To confirm the neuroprotective effects of 2-BFI in a rat model of SCI established by clip-compression injury method and to investigate potential mechanism of 2-BFI in neuroprotection.Methods:SCI model was established by an aneurysm clip compression injury.The adult male Sprague Dawley rats were divided into 3 groups:(1)sham group;(2)SCI+vehicle group;(3)SCI+2-BFI group.Drug administration was carried on by an investigator who was blind to the drugs.Rats in SCI+2-BFI group received an intraperitoneal injection of 2-BFI at 3 mg/kg twice daily while the rats in SCI+vehicle group received 0.9%NaCl twice daily.The T10-T12 laminas were removed until the spinal cord was exposed without causing SCI in the sham group.The rats were deeply anaesthetized with intraperitoneal sodium pentobarbital(100 mg/kg)and sacrificed at day 3 after the operation.The spinal cord tissue surrounding the damage site was immediately collected on ice and perfused with 200 ml of 4? 0.9%saline.The protein of Bcl-2,BAX and caspase 3 were estimated by Western blotting.The activity level of superoxidase dismutase(SOD)and glutathione peroxidase(GPx)in spinal cord tissue samples were estimated by using commercially available ELISA kits specific for rats according to the manufacturer's instructions.Fluoro-Jade B(FJB)staining and TUNEL staining were performed to detect cell necrosis and apoptosis in spinal cord tissues surrounding the damage site.Results:Compared with those in the sham group,the activity levels of SOD and GPx were significantly decreased in the SCI+vehicle group(P<0.01).Meanwhile,the activity levels were significantly enhanced after the administration of 2-BFI(P<0.05).The Bax protein and cleaved caspase-3 protein(p19 caspase-3 and p17 caspase-3)levels were appreciably increased in the spinal cord tissue collected from rats subjected to SCI,while the Bcl-2 protein levels were markedly reduced(P<0.01 and P<0.05,respectively).However,application of 2-BFI significantly increased the Bcl-2 protein levels after SCI(P<0.01).In contrast,the Bax protein and cleaved caspase-3 protein(p19 caspase-3 and p17 caspase-3)levels were downregulated by 2-BFI.Meanwhile,the Bcl-2/Bax ratio was effectively reduced after SCI and increased in the SCI+2-BFI group compared with the SCI+vehicle group(P<0.01).There were seldom FJB-positive or TUNEL-positive cells in the spinal cord of the sham group.Obviously,a number of FJB-positive and TUNEL-positive cells were observed in the SCI+vehicle group.The treatment of 2-BFI significantly reduced the number of FJB-positive and TUNEL-positive cells(P<0.05 and P<0.01,respectively).Conclusion:2-BFI upgraded the activities of SOD and GPx,the expression of Bcl-2 and downregulated the expression of Bax and cleaved caspase 3(both p17 and p19),further confirming the neuroprotective effects.Moreover,the decreased level of necrosis and apoptosis estimated by FJB staining and TUNEL staining showed corroborative evidence of neuroprotection of 2-BFI.Taken together,these findings indicated that 2-BFI may attenuate SCI by inhibiting oxidative stress and neuronal apoptosis to improve functional recovery after SCI.Part ? Treatment with 2-BFI activates the Nrf2/HO-1 signaling pathway in neurons and astrocytesObjective:To determined the neuroprotective role of 2-BFI in a rat model of spinal cord injury(SCI)mediated by activation of Nrf2/HO-1 signaling pathway which inhibiting oxidative stress and neuronal apoptosis.Methods:SCI model that underwent compression of the spinal cord by an aneurysm clip was established.The adult male Sprague Dawley rats were divided into 3 groups:(1)sham group;(2)SCI+vehicle group;(3)SCI+2-BFI group.Drug administration was carried on by an investigator who was blind to the drugs.Rats in SCI+2-BFI group received an intraperitoneal injection of 2-BFI at 3 mg/kg twice daily while the rats in SCI+vehicle group received 0.9%NaCl twice daily.The T10-T12 laminas were removed until the spinal cord was exposed without causing SCI in the sham group.The rats were deeply anaesthetized with intraperitoneal sodium pentobarbital(100 mg/kg)and sacrificed at day 3 after SCI.The spinal cord tissue surrounding the damage site was immediately collected on ice and perfused with 200 ml of 4? 0.9%saline.The protein of Nrf2?HO-1 and NQO1were estimated by Western blotting.Immunofluorescence staining was used to locate Nrf2 protein and to observe the changes of Nrf2 expression in neurons and astrocytes of spinal cord tissue around the injured site.Results:Both Nrf2 and HO-1 protein levels were increasing significantly after SCI(P<0.01)whereas the NQO1 protein expression was decreased in the SCI+vehicle group compared with the sham group(P<0.01).Additionally,Nrf2,as well as HO-1,was further increased significantly in the SCI+2-BFI group compared with the SCI+vehicle group(P<0.01).However,no significant difference was observed in the expression of NQO1 between the SCI+vehicle group and the SCI+2-BFI group(P>0.05).Immunofluorescence staining showed that the expression of Nrf2 was dramatically higher in both neurons and astrocytes after SCI than that in the sham group(P<0.01).In addition,the level of Nrf2 significantly increased in the SCI+2-BFI group than in the SCI+vehicle group(P<0.01).Conclusion:Treatment with 2-BFI increased the expression of Nrf2 and HO-1,which indicates that activation of Nrf2/HO-1 signaling by 2-BFI protects neurons from damage after SCI in the present study.Collectively,these results showed that 2-BFI may attenuate SCI mediated by activation of the Nrf2/HO-1 signaling pathway.