Inhibition Of Propofol On Pilocarpine-Induced Status Epilepticus And Its Possible Mechanisms

Part 1 Inhibition of Propofol on Lithium-Pilocarpine Induced Status Epilepticus in ratsObjective To observe the inhibition of propofol in different dose on status epilepticus(SE) induced by pilocarpine,and explore the therapeutic effect of propofol in SE 30min after admistration.Methods 80 SD rats were divided into 10 groups randomly,include blank(BLK),SE,propofol at dose of 12.5,25,50,75 and 100 mg/kg,diazepam(DZP,mg/kg),scoplamine(SCOP,10 mg/kg) and MK801(2 mg/kg) group respectively.Following establishing SE model in rats induced by pilocarpine,every agent was injected i.p.after 30min when the SE is occurred(equal volume normal saline was injected in the blank group),the EEG was recorded continuously for 5 hours and the survival rates of SE rats after 24 hours.At the same time,efficacy of the diazepam,scopolamine and MK801 on SE were also observed.And evaluating the pathologic changes in rats cortex and hippocampus with HE stain.Observing the effects of propofol on cortical and hippocampal pathologic changes in rats.Results 1.15min after injection of pilocarpine produced a sequence ofbehavioural alterations such as initial akinesia, tremor of the whole body and/or incomplete limbic gustatory automatisms. Gustatory automatism is considered to be a type of preconvulsive behaviour, characterized by myoclonic twitching which is restricted to the head and face and is accompanied with salivation.Those changes were followed by episodes of motor limbic seizures,with or without loss of the righting reflex and persistent general tonic seizure.The EEG showed that the waves from baseline wave to high-frequency,high-amplitude spikes wave continuously in cortex and hippocampus about 10-20 minutes after the injection of pilocarpine.2. Behavioral and EEG analysis revealed that propofol significantly reversing the seizure,decreasing the mortality of the SE rats in 24 hours,meanwhile,diazepam and MK-801 showed the same effects.But the scopolamine 10mg/kg i.p.seizure onset after 30min is hardly effective on SE.The EEG records showed that propofol at dose of 50,75 and 100 mg/kg can effectively decrease the high-frequency,high-amplitude spikes induced by SE.Propofol at 12.5 and 25mg/kg is nearly ineffective on the SE,the amplitudes and frequency of EEG were hardly improved during this time.Although sharp waves were still showed occasionally at 50,75 and 100mg/kg,the epileptic seizure of ethology was not happened.Meanwhile,the EEG records showed that Mk801 and diazepam were effective on the SE induced by pilocarpine,whereas the anticholinergic drug scopolamine was ineffective on EEG after 30 minutes when the SE was occurred. 3.The HE stain showed that cells were decreased significantly in cortex and hippocampus CA1 region when SE onset after 24h,and cellular swelling, plasmatorrhexis,dislimn were observed.However,Compared with SE group,the cells were increased in propofol,diazepam and MK801 group obviously. Conclusions 1.Lithium-pilocarpine can induced typical SE in rats,seizure onset after 30min,the developmental mechanism of SE maybe have no connection with the central cholinergic system.2.Our results show that propofol can effectively inhibit the SE induced by lithium-pilocarpine,improving the EEG changes of SE rats,increasing the SE rats' survival rates,and improving the impared degree of cortical and hippocampal cell.These results also implied that propofol maybe as potential agent for treatment of SE in clinical. Part 2 Protection of Propofol on Toxic Mouse Induced by Lethal dose of N-methyl-D-aspartateObjective:To observe propofol's protective effect on ethology and survival rates of mice treated by N-methyl-D-aspartate(NMDA) at lethal dose,and to investigate the anticonvulsive mechanism of propofol.Methods:Following establishing lethal model in mice induced by NMDA at dose of 175 mg/kg, propofol at dose of 12.5,25,50,75 and 100 mg/kg was injected intraperitoneally (i.p.) just before administration of NMDA in different groups of mice.The positive control drug was the nonspecific NMDA receptor antagonist MK801(2mg/kg i.p.).The ethological changes and survival rates of mice were recorded in these groups.Results:NMDA at dose of 175 mg/kg resulted in general seizure,and made all mice die 10 min after convulsion.When propofol at dose of 12.5,25,50,75 and 100 mg/kg was administrated intraperitoneally to mice 10 min before NMDA injection,the rates of convulsion were decreased and the survival rates were increased at the dose dependent manner.Conclusions:1. NMDA 175mg/kg can result in generalized convulsion,and causing all the mice died.2.Propofol can dose-dependent protect mice against the toxicity of NMDA at lethal dose.These results suggesting that anticonvulsive effect of propofol maybe result from its action on the NMDA receptors.Part 3 The study of NMDA receptors mechanism during Inhibition of Propofol on Lithium-Pilocarpine Induced Status Epilepticus in ratsObjective Establishing status epilepticus(SE) model in rats induced by Lithium- pilocarpine(Li-pilo),to observe the effects of propofol on N-methyl-D-aspartate receptor subunit 2A(NR2A) and 2B(NR2B) in SE rats cortical and hippocampal expression.Methods 60 SD rats were divided into 6 groups randomly,include blank,SE,propofol(50mg/kg),diazepam(10mg/kg), scoplamine(10mg/kg) and MK801(2mg/kg) group respectively.Following establishing SE model in rats induced by Li-pilo except blank group.Convulsion intensity was quantified according to the Racine rating scale.Only the rats received pilocaroine were observed stage 4 and 5 behaviors onset and recurrent attacks can be put into the experiments.Every agent was injected intraperitoneally 30min after the SE is occurred,equal volume normal saline was injected in the blank group.The rats were sacrificed 24h following pilocarpine administration. Immunohistochemistry and Western Blot were employed to determine the cortical and hippocampal expression of NR2A and NR2B in SE rats.Results Immunohistochemistry and Western Blot analysis showed that the expression level of NR2A and NR2B subunits were significantly upregulated in cortices and hippocampus when SE occurred after 24 hours,compared with blank group, P＜0.05.However,propofol at dose of 50mg/kg,which is anesthetic dose,can decrease the NR2B subunit expression significantly,compared with SE group, P＜0.05,whereas the NR2A subunit expression was nearly unchanged.MK801 at dose 2mg/kg can antagonize the NR2A and NR2B subunits expression,compared with SE group,P＜0.05.But the diazepam and scopolamine were not modified the expression level of NR2A and NR2B subunits.Conclusions Our results show that the inhibitive mechanism of propofol on SE rats induced by Li-pilo maybe, at least in part,contribute to its effect of downregulating the expression level of NR2B subunit.These results imply that propofol maybe as a potential agent for treatment of status epilepticus in clinical. Part 4 The study of GABA_A receptors mechanism of propofol on Lithium-Pilocarpine Induced Status Epilepticus in ratsObjective Establishing status epilepticus(SE) model in rats induced by Lithiumpilocarpine (Li-pilo),to observe the effects of propofol on cortical and hippocampal GABA_A receptorα₁ subunit expression in SE rats induced by Li-pilo.Methods 50 SD rats were divided into 5 groups randomly,include blank,SE,propofol(50mg/kg),diazepam(10mg/kg) and scoplamine(10mg/kg) group respectively.Following establishing SE model in rats induced by Li-pilo except blank group.Convulsion intensity was quantified according to the Racine rating scale.Only the rats received pilocaroine were observed stage 4 and 5 behaviors onset and recurrent attacks can be put into the experiments.Every agent was injected intraperitoneally 30min after the SE is occurred,equal volume normal saline was injected in the blank group.The rats were sacrificed 24h following pilocarpine administration.Immunohistochemistry and Western Blot were employed to determine the cortical and hippocampal expression of NR2A and NR2B in SE rats.Results Immunohistochemistry and Western Blot analysis showed that the expression of GABAA receptorα₁ subunit were significantly decreased in cortices and hippocampus when SE occurred after 24 hours. Expression level of GABA_A receptorα₁ subunit were significantly downregulated in cortices and hippocampus when SE occurred after 24 hours,compared with blank group,P＜0.05.Propofol at dose of 50mg/kg,which is anesthetic dose,can significantly upregulate the GABAA receptorα₁ subunit expression in cortices and hippocampus,compared with SE group,P＜0.05.The diazepam at dose of 10mg/kg can also increase the GABA_A receptorα₁ subunit expression.But scopolamine were not modified the expression level of GABA_A receptorα₁ subunit expression.Conclusions Our results show that the inhibitive mechanism of propofol on SE rats induced by Li-pilo maybecontribute to its upregulate the expression level of GABA_A receptorα₁ subunit.These results also imply that propofol maybe as a good agent for treatment of status epilepticus in clinical.Part 5 Study of antioxidant effects of propofol on Lithium-Pilocarpine Induced Status Epilepticus in ratsObjective Establishing status epilepticus(SE) model in rats induced by Lithiumpilocarpine (Li-pilo),to observe the effects of propofol on serum,cortical and hippocampal superoxide dismutase(SOD),glutathione(GSH) and malondialdehyde(MDA) in SE rats induced by Li-pilo.Methods 72 SD rats were divided into 6 groups randomly,include blank,SE,propofol(50mg/kg), diazepam(10mg/kg),scoplamine(10mg/kg) and MK801(2mg/kg) group respectively.Following establishing SE model in rats induced by Li-pilo except blank group.Convulsion intensity was quantified according to the Racine rating scale.Only the rats received pilocaroine were observed stage 4 and 5 behaviors onset and recurrent attacks can be put into the experiments.Every agent was injected intraperitoneally 30min after the SE is occurred,equal volume normal saline was injected in the blank group.2 hours after the agents were given,4 rats of every group were exsanguinated from fossa orbitalis,and centrifugated(1500rpm,15min),drown the supernatant into Epidoff tube to measure the protein level and optical density(OD).Then the 4 rats were sacrificed and the frontal cortex and hippocampus were rapidly and carefully removed. Frontal cortex and hippocampi of the same group were put into a Epidoff tube, and weighted,homogenized at 4℃in distilled water,centrifugated and(12000 rpm,20 min at 4℃),drown the supernatant into different Epidoff tube to measure the protein level and OD.After 24 hours when the agents were given,the same procedure were taken as the time of 2 hours when the agents were given.The OD of SOD,GSH and MDA were measured according to the procedure of kits description Results After 2 hours when the agents were given,SOD activity and MDA in serum,frontal cortex and hippocampus were increased significantly, GSH in these areas were decreased remarkably,compared with the blank group, P＜0.01.The GSH was increased and MDA was decreased notably in propofol group after 2 hours when the agents were given,compared with SE group,P＜0.01. But propofol was not changed the SOD activity augmentation in SE rats. Diazepam,scoplamine and MK801 were not affected on SOD,GSH and MDA both serum and cortex and hippocampus.After 24 hours when the agents were given,SOD activity and MDA in serum,frontal cortex and hippocampus were increased significantly also,GSH in these areas were decreased remarkably, compared with the blank group,P＜0.01.propofol was not changed the SOD activity augmentation in SE rats either.Diazepam,scoplamine and MK801 were not affected on SOD,GSH and MDA in these areas.Conclusions 1.SOD activity in serum,frontal cortex and hippocampus were increased significantly after 2 hours and 24 hours in SE rats,the MDA was increased also,whereas the GSH was decreased remarkable.2.Propofol can increase GSH and decrease MDA level depression either serum or cortex and hippocampus in SE rats.Our results imply that the inhibition of propofol on Li-pilo induced SE in rats is contribute to its antioxidant properties though down-regulating the MDA and up-regulating GSH level.