Therapeutic Efficacy And Correlated Mechanism Of Action Study Of Phencynonate Hydrochloride On Experimental Cerebral Ischemia Reperfusion Injury

Part 1 Establishment of the focal cerebral ischemia reperfusion injury modelObjective: To establish a convenient manipulation, stable and reliable model of focal cerebral ischemia in rats. Methods: The focal middle cerebral artery occlusion model in rats was made by the modified suture- occluded method of Zea longa and Koizumi. Two hours and twenty four hours after operation, give a mark to neurological function defect symptom, and then combine with TTC staining to judge whether the MCAO model is successful or nor. Results: All the survival rats appear different extent neurological function defect and cerebral ischemia infarction after operation. Meanwhile, there are problems such as subarachnoid hemorrhage, cervical part angiorrhexis, imcomplete cerebral ischemia during operation. Conclusion: The improved model is simple and reliable with high achievement ratio. It is advisable to use this experimental animal model to study focal cerebral ischemia. There are key points to build a successful model such as rat's weight, anesthetic application, suture diameter, depth of penetration, control of hemorrhage and postoperative care.Part 2 Drugs'therapeutic efficacy and instructional significance on cerebral ischemia reperfusion injury such as NimodipineObjective: To investigate the therapeutic efficacy of drugs with different mechanism of action used single or combinative on cerebral ischemia reperfusion injury in rats. Method:Forty-eight adult male SD rats were randomly allocated into 6 groups: model (I/R, group A), edaravone low-dose (group B), edaravone high-dose (group C), nimodipine ( group D), drug combinationⅠ(group E) and drug combinationⅡ(group F) groups( n =8 in each group). A rat model of middle cerebral artery occlusion (MCAO) was induced by the suture-occluded method; cerebral ischemia kept 2 hours and reperfusion kept 24 hours. At 20 minutes before reperfusion after cerebral ischemia and 12 hours after reperfusion, the medications were injected into the femoral veins of rats. Group A: saline 1.5ml; group B: edaravone 1.5mg/kg; Group C: edaravone 3.0mg/kg; Group D: nimodipine 1.0mg/kg; Group E: nimodipine 0.5mg/kg plus cyclophosphamide 50 mg/kg; and Group F: nimodipine 1.0mg/kg plus cyclophosphamide 50 mg/kg. The neurologic impairment scores and cerebral infarction percentage were measured 24 hours after cerebral ischemia reperfusion.Results: Neurologic impairment scores in groups A, B, C, D, E and F were 3.12±0.83, 2.12±0.64, 2.00±0.53, 2.12±0.83, 2.12±0.64 and 1.37±0.52, respectively. There were significant differences between each medication group and I/R group (P< 0.01); and there were significant differences among groups B, D, E and F ( P< 0.05). Cerebral infarction percentage in rats were 15.89±2.08, 7.24±1.08, 6.89±1.00, 6.97±0.84, 6.37±0.92 and 4.95±0.82, respectively. Cerebral infarction percentage were reduced in each medication group as compared with I/R group, there were significant differences (P< 0.01); There were significant differences between group F and the other 4 medication groups ( P< 0. 05). Conclusion: The neuro-protective effects of nimodipine 1.0mg/kg plus cyclophosphamide 50 mg/kg were significant on focal cerebral ischemia reperfusion injury in rats. Part 3 Neuroprotective effects of phencynonate hydrochloride on focal cerebral ischemia reperfusion injury in ratsObjective: To investigate the neuroprotective effects of phencynonate hydrochloride on cerebral ischemia reperfusion injury in rats. Method: Sixty-four adult male SD rats were randomly allocated into 8 groups: sham operation group(SH), model group(I/R, saline 1.5ml), positive medicine group (nimodipine 1.0mg/kg plus cyclophosphamide 50 mg/kg ), phencynonate high-dose group(PCH 12mg/kg), phencynonate moderate-dose group(PCH 8mg/kg), phencynonate low-dose group(PCH 4mg/kg), phencynonate 8mg/kg plus cyclophosphamide 50 mg/kg group and dizocilpine group(MK-801 2mg/kg) ( n =8 in each group). A rat model of middle cerebral artery occlusion (MCAO) was induced by the suture-occluded method; cerebral ischemia kept 2 hours and reperfusion kept 24 hours. At 20 minutes before reperfusion after cerebral ischemia and 12 hours after reperfusion, the medications were injected into the femoral veins of rats. The neurologic impairment scores, cerebral infarction percentage, SOD activity and MDA contents were measured 24 hours after cerebral ischemia reperfusion. Results:①The neurologic impairment score of each medication group was degrade obviously and had statistical differences compared with I/R group (P< 0.01 or P< 0.05); Compared with positive medicine group, the scores of PCH12 and PCH 4 were higher and had significant differences ( P< 0.01) , the scores of PCH8 and PCH plus Cyc group were equivalent; Compared with PCH plus Cyc group, the scores of PCH12 and PCH 4 were higher and had significant differences ( P< 0.05) , the scores of PCH8 was equivalent.②The cerebral infarction percentage of each medication group was reduced and had significant differences as compared with I/R group (P< 0.01); Compared with positive medicine group, the percentage of other four medication group were higher and had significant differences (P<0.01或P<0.05) except PCH plus Cyc group; Compared with PCH plus Cyc group, the percentage of PCH12 and PCH 4 were higher and had significant differences ( P< 0.01) , the percentage of PCH8 was equivalent.③The SOD activity of each group following MCAO 24 hours was reduced obviously as compared with sham operation group; Compared with I/R group, the activity of each medication group were higher on different degree (P<0.01或P<0.05) except MK-801 group; Compared with positive medicine group, the activity of each phencynonate group were lower and had significant differences (P<0.01或P<0.05); There weren't significant differences between phencynonate applied alone or combinative.④The MDA contents of each group following MCAO 24 hours was heightened obviously as compared with sham operation group; Compared with I/R group, the contents of each medication group were lower on different degree (P<0.01或P<0.05) except MK-801 group; Compared with positive medicine group, the contents of each phencynonate group were higher and had significant differences (P<0.01或P<0.05); There weren't significant differences between phencynonate applied alone or combinative. Conclusion: Phencynonate hydrochloride relieved neurologic impairment symptom, diminished cerebral infarction, elevated SOD activity and degraded MDA contents in cerebral ischemia reperfusion injury rats, so it had neuroprotective effects.Part 4 Influence of phencynonate hydrochloride on NMDA receptors subunit i expression in focal cerebral ischemia reperfusion injury ratsObjective: To investigate the influence of phencynonate hydrochloride on NMDA receptors subunit expression in focal cerebral ischemia rats. Method:Sixy adult male SD rats were randomly allocated into 6 groups: Sham group, MCAO model group, phencynonate high dose group, phencynonate low dose group, nimodipine plus cyclophosphamide group. Ten rats in each group. At 20 minutes before reperfusion after cerebral ischemia and 12 hours after reperfusion, the medications were injected into the femoral veins of rats. Extracted total RNA from the area of infarct in cerebral cortex, and then amplified by reverse transcription of both NR2A and NR2B mRNA. Results: Expression of NR2A and NR2B mRNAs up regulated in cortex after 6h of reperfusion following middle cerebral artery occlusion (MCAO), but then became significantly lower at 24 h of reperfusion. Both high dose and low dose of phencynonate and MK-801 could down regulate the NR2A and NR2B mRNA level at 6 h of reperfusion; This down regulate effects of NR2A and NR2B receptor could be continued to 24 h by MK-801;however, phencynonate could still down regulate NR2B receptor at 24h. Conclusion: Expression of NR2A and NR2B mRNAs up regulated in cortex of reperfusion following middle cerebral artery occlusion (MCAO); MK-801and phencynonate achieve the therapeutic effect of focal cerebral ischemia may though the down regulate effects of NR2A and NR2B receptor.