Effect Of Edaravone On Brain Protection And Mechanism Of Pentotetrazole Induced Epileptic Rats

Epilepsy is one of neurological disease characterized by paroxysmal brain dysfunction caused by repeated abnormal discharge of brain neurons.Epileptic seizures can lead to the necrosis or apoptosis of neurons,secondary proliferation of central glial cells,resulting in the establishment of abnormal excitatory neural networks,contributing to recurrent epilepsy or status epilepticus.About 30% of epilepsy patients are accompanied by cognitive impairment.In recent years,cognitive impairment in epilepsy has become a research hotspot.The etiology and pathogenesis of epilepsy are complication and has not been clarified yet.The possible pathogenesis includes oxidative stress,excitatory amino acid cytotoxicity,intracellular calcium overload and so on.Cascade oxygen free radical production and damage induced by oxidative stress are considered to be the important pathological basis for brain neuron damage in epilepsy.Through free radical chain reaction,lipids,organelles and nucleoproteins in cell membranes are oxidized excessively,and the structure and function of mitochondria are changed.The oxidative phosphorylation of respiratory chain of mitochondria damaged results in insufficient energy production of cells,leading to cell death or apoptosis.Therefore,the way of antioxidant stress and of excessive free radical elimination may be an important strategy in the treatment of epilepsy.Edaravone(EDA),as a free radical scavenger,plays a role in anti-oxidative stress and inhibits lipid peroxidation reaction.It has been proved that edaravone can protect tissues by inhibiting the activity of cyclooxygenase(COX-1,COX-2).COX-2 a key enzyme and rate-limiting enzyme,mainly expressed in hippocampal and cortical glutamatergic neurons and regulated the metabolism of prostaglandin E2 and nitric oxide.Prostaglandin E2 and nitric oxide can participate in inflammation,oxidative stress,apoptosis and other pathological processes.Therefore,edaravone may become a new drug for the treatment of epilepsy via antioxidant role.In this study,through intraperitoneal injection of pentylenetetrazol(PTZ)-induced chronic epileptic rat model,and the edaravone intervention with different doses,we investigated the effects of different doses of edaravone on the levels of epileptic seizures,latency of generalized tonic-clonic seizures,latency of minimal clonic seizures,cognitive function,oxidative stress parameters in hippocampus tissues of PTZ-induced epileptic rats,including detection of the COX-2 expression for exploring whether edaravone has cerebral protective role.And its possible mechanism will provide a new stratgy and experimental basis for the choice of antiepileptic drugs.This study is divided into three parts,the contents of each part are as follows.Part one Effect of edaravone on epileptic seizures and cognitive function in PTZ-induced epilepsy ratsObjective: Establishment of epileptic model of pentylenetetrazol chronic kindling in male albino rats.To observe the changes of epileptic seizure grade,generalized tonic clonic seizure latency(GTCS),minimal clonic seizures(MCS)and cognitive function in epileptic rats,and the intervention effect of edaravone on epileptic rats.Methods: Sixty healthy male albino Wistar rats(200-220 g)aged 8-12 weeks were randomly divided into four groups: Sham group,Control group,PTZ+Edaravone 5 mg/kg group and PTZ+Edaravone 10 mg/kg group(n=15for each group).Sham group received intraperitoneal injection of 0.9% Na Cl every other day for 15 times.In control group,PTZ(90 mg/kg)was injected intraperitoneally every other day for 15 times,0.9% Na Cl was injected intraperitoneally every day,and 30 minutes later PTZ(90 mg/kg)was injected intraperitoneally.PTZ + Edaravone 5 mg/kg group: PTZ(90 mg/kg)was injected intraperitoneally every other day for 15 times,5 mg/kg Edaravonewas injected intraperitoneally every day,and 30 minutes later PTZ(90 mg/kg)was injected intraperitoneally.PTZ + Edaravone 10 mg/kg group: PTZ(90mg/kg)was injected intraperitoneally every other day for 15 times,10 mg/kg Edaravone was injected intraperitoneally every day,and 30 minutes later PTZ(90 mg/kg)was injected intraperitoneally.After each administration of PTZ,the behavioral changes of rats were observed.Racine(1972)behavioral grading method was used to classify epileptic seizures in rats.Morris water maze test was performed for testing the learning and memory ability after the last administration with PTZ after 24 h.Results:1 After continuous intraperitoneal injection of PTZ,epileptic seizures,mainly including the increased initial gaze,nodding,facial washing,the epileptic seizures gradually,and eventually a generalized tonic-clonic seizure occurred in rats.Compared with the control group,different doses of edaravone(5 mg/kg,10 mg/kg)could effectively reduce the epileptic seizure grade of pentylenetetrazol-kindled epilepsy rats(P<0.05).There was no significant difference in reducing epileptic seizures between edaravone(5mg/kg,10 mg/kg)groups(P>0.05).There was no epileptic seizure in Sham group.Compared with control group,edaravone(5 mg/kg,10 mg/kg)significantly prolonged the overall tonic-clonic seizure latency(GTCS)and minimal clonic seizure latency(MCS)in epileptic rats(P<0.05)and the effect of high dose edaravone intervention more effective than low dose group on prolonging latency,the difference was statistically significant(P<0.05).2 Morris water maze test results2.1 results of localization navigation experiment: the mean value of daily escape latency was compared,and the results showed that(1)the escape latency of rats in Sham group,Control group,PTZ+Edaravone 5 mg/kg group,PTZ+Edaravone 10 mg/kg group had no statistical significance on day 1 and 2(P>0.05).(2)Compared with Sham group,the escape latency of rats in Control group was significantly longer at day 3,4 and 5 of the experiment(P< 0.05);the escape latency of rats in PTZ + Edaravone group(5 mg/kg,10mg/kg)was significantly shorter than that in Control group(P<0.05);the escape latency of rats in PTZ + Edaravone group(5 mg/kg,10 mg/kg)was not significantly different from that in Sham group(P>0.05).There was no significant difference in latency between PTZ + Edaravone(5 mg/kg and 10mg/kg)groups(P>0.05).2.2 Spatial exploration test results: The number of rats crossing the platform area in 120 s in control group was significantly lower than that in Sham group(P<0.05),the number of rats crossing the platform area in 120 s in PTZ +Edaravone group(5 mg/kg,10 mg/kg)was significantly higher than that in control group(P<0.05).The number of times of PTZ + Edaravone group(5mg/kg,10 mg/kg)rats crossing the plateau area did not change obviously compared with Sham group(P>0.05).There was no significant difference between the two groups Edaravone(5 mg/kg,10 mg/kg)(P>0.05).Conclusion: Continuous intraperitoneal injection of PTZ can successfully establish chronic kindling rats model.Edaravone intervention can significantly reduce the level of epileptic seizures,prolong the latency of generalized tonic-clonic seizures and minimal clonic seizures,and delay the process of epilepsy in PTZ-induced rats,indicating that Edaravone has anti-epileptic effects.The spatial learning and memory abilities of rats were significantly enhanced compared with control group in Morris water maze experiment,indicating that edaravone could significantly improve the cognitive impairment of PTZ kindled rats.Part two Effect of edaravone on oxidative stress in brain tissue of epileptic rats induced by PTZObjective: To observe the oxidative stress response in brain tissue from epileptic rats kindled by PTZ and the protective effect of edaravone on the antioxidant defense ability in hippocampus from epileptic rats.Methods: Sixty healthy male albino Wistar rats(200-220 g)aged 8-12 weeks were randomly divided into four groups: Sham group,Control group,PTZ+Edaravone 5 mg/kg group and PTZ+Edaravone 10 mg/kg group(n=15for each group).Sham group received intraperitoneal injection of 0.9% Na Cl every other day for 15 times.In control group,PTZ(90 mg/kg)was injected intraperitoneally every other day for 15 times,0.9% Na Cl was injected intraperitoneally every day,and 30 min later PTZ(90 mg/kg)was injected intraperitoneally.PTZ + Edaravone 5 mg/kg group: PTZ(90 mg/kg)was injected intraperitoneally every other day for 15 times,5 mg/kg Edaravone was injected intraperitoneally every day,and then PTZ(90 mg/kg)was injected intraperitoneally after 30 minutes.PTZ + Edaravone 10 mg/kg group:PTZ(90 mg/kg)was injected intraperitoneally every other day for 15 times,10 mg/kg Edaravone was injected intraperitoneally every day,and then PTZ(90 mg/kg)was injected intraperitoneally after 30 minutes.24 h after the last administration of PTZ,NO level was detected in the tail vein of rats in each group.The expression of oxidative stress parameters(MDA,GSH,SOD,Catalase,Gpx,Thiol content)was detected in the hippocampus of rats.The levels of reactive oxygen species(ROS)and mitochondrial transmembrane potential(??m)in hippocampal cells were measured by flow cytometry.Results:1.Oxidative stress parameters Compared with Sham group,the expressions of GSH,SOD,CAT,Gpx and Thiol content in hippocampus from control group and PTZ + Edaravone 5 mg/kg group were decreased(P<0.05)whereas the contents of MDA was increased(P<0.05).Compared with the control group,the content of antioxidant parameters GSH,SOD,CAT,Gpx and Thiol content in hippocampus from rats in PTZ + Edaravone group(5mg/kg,10 mg/kg)were increased significantly(P<0.05),and the level of oxidative stress parameter MDA in hippocampus from rats was decreased significantly(P<0.05).Compared with PTZ+Edaravone 10 mg/kg group and PTZ+Edaravone 5 mg/kg group,the contents of oxidative stress parameters were different(P<0.05),but there was no significant difference between PTZ+Edaravone 10 mg/kg group and Sham group(P>0.05).2.Levels of intracellular ROS and ??m in hippocampus Compared with Sham group,ROS level in control group,PTZ + Edaravone 5 mg/kggroup and PTZ + Edaravone 10 mg/kg group increased(P<0.05),and ??m decreased significantly(P<0.05).Compared with the control group,the ROS level in hippocampal cells of epileptic rats decreased significantly after PTZ +Edaravone(5 mg/kg,10 mg/kg)treatment(P<0.05),and the ??m level increased significantly(P<0.05).There was no significant difference in ROS level and ??m level between PTZ + Edaravone 10 mg/kg group and PTZ +Edaravone 5 mg/kg group(P>0.05).3.NO content detection Compared with Sham group,the levels of NO in control group,PTZ + Edaravone 5 mg/kg group and PTZ + Edaravone 10mg/kg group were all increased(P<0.05).Compared with the control group,the serum NO level of epileptic rats was decreased significantly after PTZ +Edaravone(5 mg/kg,10 mg/kg)intervention(P<0.05).There was no significant difference in NO content between PTZ + Edaravone(5 mg/kg,10mg/kg)groups(P>0.05).Conclusions: PTZ kindled rats had obvious oxidative stress reaction and impaired antioxidant ability in hippocampus.Edaravone can effectively reduce oxidative stress injury,protect the antioxidant defense ability of hippocampus cells and the function of hippocampal neuron mitochondria in epileptic rats.Part three Protective effect of edaravone on hippocampal neurons in pentylenetetrazol-induced epilepsy ratsObjective: To observe the effects of different doses of edaravone on the apoptotic rate of hippocampal cells,the expression of COX-2 gene and protein,and the level of prostaglandin E2(PGE2)production in epileptic rats,and to explore the mechanism by which edaravone plays role on brain protection in epileptic rats induced by PTZ.Methods: Sixty healthy male albino Wistar rats(200-220 g)aged 8-12 weeks were randomly divided into four groups: Sham group,Control group,PTZ+Edaravone 5 mg/kg group and PTZ+Edaravone 10 mg/kg group(n=15for each group).Sham group received intraperitoneal injection of 0.9% Na Cl every other day for 15 times.In control group,PTZ(90 mg/kg)was injectedintraperitoneally every other day for 15 times,0.9% Na Cl was injected intraperitoneally every day,and 30 min later PTZ(90 mg/kg)was injected intraperitoneally.PTZ + Edaravone 5 mg/kg group: PTZ(90 mg/kg)was injected intraperitoneally every other day for 15 times,5 mg/kg Edaravone was injected intraperitoneally every day,and then PTZ(90 mg/kg)was injected intraperitoneally 30 minutes after Edaravone injection.PTZ +Edaravone 10 mg/kg group: PTZ(90 mg/kg)was injected intraperitoneally every other day for 15 times,10 mg/kg Edaravone was injected intraperitoneally every day,and then PTZ(90 mg/kg)was injected intraperitoneally after 30 minutes.After the last PTZ administration for 24 h,the apoptosis of hippocampal neurons was quantitatively analyzed by flow cytometry in the brain tissues from each group.The morphological changes of hippocampal neurons in epileptic rats were observed by nishi staining.The content of PGE2 in hippocampus of each group was detected.Western blot and Real-time PCR were used to quantify the expression of COX-2 m RNA and protein in hippocampal tissues.Results:1.Nissl staining results In the CA1 and CA3 regions of hippocampal tissue,the neurons in the Sham group had complete morphology,clear nucleolus,abundant Nissl bodies in the cytoplasm,regular and compact pyramidal cells,and no obvious loss of neurons was found.Compared with Sham group,the loss and disarrangement of hippocampal neurons in Control group was obvious,cells shrunk,nucleus pyknosis and the number of Nissl bodies were decreased.Compared with the control group,after PTZ +Edaravone(5 mg/kg,10 mg/kg)intervention,the hippocampal neurons of epileptic rats had clear margins,the number of Nissl bodies was significantly increased,and the loss of neurons was significantly ameliorated.Number of hippocampal neurons survived: Compared with Sham group,the number of neurons in CA1 and CA3 areas of hippocampus were decreased significantlty in Control group,PTZ + Edaravone 5 mg/kg group and PTZ +Edaravone 10 mg/kg group(P<0.05).Compared with Control group,PTZ +Edaravone group(5 mg/kg,10 mg/kg)was significantly increased in the number of surviving neurons(P<0.05).Compared with PTZ + Edaravone 5mg/kg group,the survival number of hippocampal neurons in PTZ +Edaravone 10 mg/kg group had significant increased(P<0.05).2.Cell apoptosis rate Compared with Sham group,control group,PTZ+Edaravone 5 mg/kg group and PTZ+Edaravone 10 mg/kg group increased the apoptotic rate of hippocampal cells(P<0.05).Compared with the Control group,the apoptotic rate of hippocampus from the PTZ + Edaravone group(5 mg/kg,10 mg/kg)was decreased significantly(P<0.05).Compared with PTZ+Edaravone 10 mg/kg group and PTZ+Edaravone 5 mg/kg group,the apoptotic rate of hippocampal tissues from various treated rats had significant difference(P<0.05).The changed apoptosis rate in hippocampal cells was consistent with results of Nissl staining.3.PGE2 content detection Compared with Sham group,the level of PGE2 in Control group was increased significantly(P<0.05).Compared with the control group,the level of PGE2 decreased significantly after PTZ +Edaravone(5 mg/kg,10 mg/kg)treatment(P<0.05).4.Real-time PCR detection for the expression of COX-2 m RNA in the hippocampal tissuesCompared with Sham group,the m RNA level of COX-2 in hippocampus from Control group,PTZ+Edaravone 5 mg/kg group and PTZ+Edaravone 10mg/kg group were increased significantly(P<0.05).Compared with the control group,the expression of COX-2 in hippocampus was significantly decreased after PTZ + Edaravone(5 mg/kg,10 mg/kg)(P<0.05).There was significant difference between PTZ + Edaravone 10 mg/kg group and PTZ +Edaravone 5 mg/kg group(P<0.05).5.Western blot detection of the expression of COX-2 in rat hippocampal tissuesCompared with Sham group,the expression level of COX-2 protein in hippocampus in Control group,PTZ+Edaravone 5 mg/kg group and PTZ+Edaravone 10 mg/kg group increased significantly(P<0.05).Comparedwith the control group,the expression of COX-2 protein in hippocampus was significantly reduced after PTZ + Edaravone(5 mg/kg,10 mg/kg)treatment(P<0.05).There was a significant difference between PTZ + Edaravone 10mg/kg group and PTZ + Edaravone 5 mg/kg group(P<0.05).Conclusions : The hippocampal neurons in pentylenetetrazol-kindled epilepsy rats were damaged and apoptotic.The content of prostaglandin E2 and the expression of COX-2 were also increased significantly.Edaravone can down-regulate the COX-2 m RNA and protein level,inhibit the production of prostaglandin E2,protect the structure and function of hippocampal neurons from the apoptosis of hippocampal cells.Therefore,it is speculated that edaravone may alleviate oxidative stress injury and hippocampal neuron apoptosis induced by epileptic seizures via regulating the abnormal expression of COX-2/PGE2,and improve cognitive impairment in epileptic rats,which may be the mechanism of edaravone's brain protection.