| Epilepsy,which is characterized by attack,recurrence and rigidity,is a common chronic neurological disease,and can lead to high morbidity or mortality.TLE,the most common type of epilepsy syndrome,is characterized by recurrent epileptic episodes.Repeated epileptic seizures can seriously affect patients’work,life and study,and frequently produce cognitive impairment,further aggravating the patient’s psychological trauma.Currently,two thirds of the patients take antiepileptic drugs(AEDs)to control or reduce seizures.However,AEDs are ineffective in another one third of patients,who developped into refractory epilepsy.Currently,most of the clinical AEDs can only suppress the symptoms of acute seizures,and rarely delay or stop epileptogenesis.Therefore,it is of great significance to find drugs that can effectively prevent epileptogenesis and improve the patients’cognitive impairment.Epileptic seizures can lead to brain damage,which is characterized by neuronal degeneration and necrosis.The studies on the mechanism and protection of neuronal injury associated with epileptic seizure play an important role in the prevention and treatment of epilepsy.The pathogenesis of epilepsy involves the excitatory amino acids toxicity,oxidative stress,calcium overload,energy imbalance,inflammatory cascade and activation of apoptosis related proteins,etc.These mechanisms act on each other,and promoteepileptogenesis.Especially,theoxidativestressinjury,inflammatory cascade and apoptosis exists were found in epileptic brain tissue and distant damaged tissues,causing dysfunction and death of neurons.Therefore,anti-oxidative stresses,inhibition of inflammatory cascade and apoptosis are important aspects to protect the neuronal injury associated with epileptic seizure.Methylene blue(MB)is a water-soluble thiophenazine compound and can exert diverse pharmacological effects such as anti-inflammatory,anti-oxidant,anti-tumor and anti-apoptosis.It can quickly pass through the blood-brain barrier with its strong lipophilic ability,easily accumulating in the central nervous system to play a direct neuroprotective role.It has already been confirmed that MB not only produced a nerve-protective effect in ischaemic brain damage,Leber optic neuropathy,Alzheimer disease and Parkinson disease,but also improve the memory of patients in Alzheimer,showing it is a potential drug for treating mild cognitive impairment.MB has been widely used in clinical for treatment of multitudinous central diseases.However,the neuroprotective effects in epilepsy have not been clearly reported to date.In this experiment,SSSE and chronic BLA-kindling models of epilepsy were induced by electrical stimulation on BLA to investigate the neuroprotective effects of MB during acute epileptic seizure,epileptogenesis and cognitive impairment associated with epileptic seizure.Possible mechanisms were also investigated to supplement the treatment of epilepsy.Part one Establishment of epilepsy model of rats by electric stimulation on BLAObjective:To test the inductive effect of inducing SSSE model and BLA chronic kindling model under different electric stimulus parameters.The afterdischarge threshold,Racine classification and EEG of seizure were recorded during the experiments.Summarize the experience to improve the success rate of odel establishment in our laboratory,laying a foundation for exploring new antiepileptic drugs or nerve protectant.Methods:Male healthy adult Wistar rats were randomly divided into four groups(15 per group).Control group(rats without any treatment);Sham group(rats received electrode implantation but without electrical stimulation);EP group(rats received electrode implantation and chronic electric stimulus);SSSE group(rats received electrode implantation and SSSE electric stimulus).The stimulating electrodes,which were made of twisted stainless-steel Teflon-coated wires,were implanted into the right BLA(AP=-2.2 mm,ML=-4.7 mm,DV=8.5 mm).24h before the kindling,ADT was determined and later BLA chronic kindling model and SSSE model were induced under different electric stimulus parameters.Experiment(1):establishment of a rat model of chronic kindling.Rats that meet the ADT standard were subjected to chronic kindling program with a constant current of 500μA,waviness width1ms,monophasic square-wave pulses,and 60 Hz for 1 s,once every day.The seizure stage was assessed according to the Racine’s classification,and the rats were thought kindled successfully after consecutive 3 times of 5 stage seizures.The events were recorded everyday,including loss of electrode,death of rat,the seizure stage and ADD were recorded.Experiment(2):establishment of a rat model of SSSE.Rats that meet the ADT standard were subjected to SSSE stimulus program.The stimulus program consisted of 100 ms trains of 1 ms biphasic square wave pulses and the trains were given at a frequency of 2/s and the intra-train pulse frequency was 50/s.Peak pulse intensity was 700μA and the stimulation lasted 25 minutes in total.After the cessation of stimulation,EEG of amygdala was recorded through the same electrodes.A prominent feature of SSSE on EEG recording was the appearance of high-amplitude(>2×baseline)and high-frequency(>8 Hz)discharges that lasted for at least 5 s.The end of the electrographic seizure was characterized by a brief(1-3 s)‘flat period’on the EEG.The high-amplitude and high-frequency discharges repeated intermittently for at least 15 minutes,we confirmed the successful establishment of SSSE.The events during 12h EEG recording were recorded,including loss of electrode,death of rat,the number and accumulated time of seizure activities were recorded.Results:1.The rats in the Control and Sham group had no epileptic seizure during the experimental process.2.Chronic BLA kindling models:during the determination of ADT,one rat had no obvious AD,another two rats’ADT exceeded 300μA.Finally,12rats met the standard of ADT and were subject to the subsequent tests.During chronic kindling process,electrode was lost in one rat and another one rat died.Skull infection occurred in another rat,leading to AD disappeared.These three rats failed to be kindled and were excluded from the follow-up experiments.In the end,9 rats were thought kindled successfully during 22 days of kindling.The sensitivity of epileptic seizures was maintained.3.BLA electric stimulated SSSE models:during the determination of ADT,one rat had no obvious AD,another one rat’s ADT exceeded 300μA.Finally,13 rats met the standard of ADT and were subject to the subsequent tests.After 25min electric stimulus on BLA,9 rats were thought induced successfully in the end.The successful rate was 69.23%.One rat failed to be induced SSSE and electrode was lost in another two rats.Another one rat died and the rate of death was 7.69%.The number time of seizure activities was 36.22±2.67,and the accumulated time of seizure activities was53.44±6.26min according to 12h EEG recording.Part two Methylene blue exerts anticonvulsant and neuroprotective effects on SSSE induced by prolonged basolateral amygdala stimulation in Wistar ratsObjective:SSSE models was induce by prolonged BLA electric stimulus to investigate the effect of MB on the number time and accumulated time of acute seizure activities and surviving pyramidal neurons in hippocampus.The possible mechanisms were explored.Methods:Fifty male healthy adult Wistar rats weighted 250-350 grams were randomly divided into four groups.Control group(rats without any treatment);Sham group(rats received electrode implantation but without electrical stimulation);SSSE group(rats received electrode implantation and SSSE electric stimulus).SSSE+MB group(rats received 1 mg/kg MB intraperitoneal injection five minutes after establishment of SSSE).There were 12 rats in each of Control and Sham group and 13 rats in each of SSSE and SSSE+MB group,respectively.The kindling and induction programs were performed as described in partⅠ.EEG recording were continuously recorded for 12h.The severities of SSSE in different groups were assessed by the quantity of separate seizures and the accumulated time of seizures.The levels of MDA/GSH in hippocampus tissue of rat collected 24h after the emergence of SSSE was detected by the enzyme-linked immunosorbent assay.Nissl staining was used to show the surviving pyramidal neurons in hippocampal CA1 and CA3 regions.Western blotting assay was performed for the expression of apoptosis-related markers Caspase3,BCL2 and BAX.Results:1.After 25 minutes prolonged stimulus at BLA,one rat in SSSE+MB group failed to induce SSSE and one rat in SSSE group died during the stimulus process.These rats were excluded from the follow-up experiments.Finally,12 met the standard of ADT and were subject to the subsequent tests.The eventual number and cumulative time of seizure activities in SSSE+MB group were significantly decreased compared to the SSSE group(P<0.01;P<0.05,respectively).The rats in the Control and Sham group had no epileptic seizure during the experimental process.2.SSSE group showed a marked evaluation of the MDA content(P<0.01)and reduction of GSH(P<0.01)verse the Control group.However,treatment with MB significant lowered MDA(P<0.01)and raised the activity of GSH(P<0.01)compared with the SSSE group.There was nonsignificant difference between the Sham group and the Control group regarding the two parameters(P>0.05).3.Compared with the Control group,the surviving pyramidal neurons of SSSE group in both CA1 and CA3 seem sparse and shrunken,and the number of them significantly decreased(P<0.01).Treatment with MB markedly improved the cell morphology and alleviated the neuronal loss compared with the SSSE group(P<0.01).There was no significant difference in the number of surviving neurons between the Sham group and the Control group(P>0.05).4.Compared with the Control group,the level of CASP3 in the SSSE group remarkably increased 24 hours after establishment of SSSE(P<0.05),whereas treatment with MB significantly weakened the increase of CASP3during SSSE(P<0.05).Unlike CASP3,BCL2 activity notably decreased compared with the Control group(P<0.05),and treatment with MB inhibited the declining trend of BCL2 activity(P<0.05).The variation trending of BAX shared the same pattern with CASP3,which increased during SSSE,and treatment with MB could prevent this increasing trend(P<0.05).Part threeEffect of methylene blue on chronic BLA-kindling seizures and cognitive function in ratsObjective:Kindling models was induced by chronic BLA electric stimulus to investigate the influence of MB on epileptogenesis and the cognitive impairment caused by epilepsy.Methods:Forty male healthy adult Wistar rats weighted 250-350 grams were randomly divided into four groups,10 per group.Control group(rats without any treatment);Sham group(rats received electrode implantation but without electrical stimulation);EP group(rats received electrode implantation and chronic electric stimulus).MB+EP group(rats received 1 mg/kg MB intraperitoneal injection 30min before electric stimulus).The kindling and induction programs were performed as described in partⅠ.The rats were thought kindled successfully after consecutive 3 times of 5 stage seizures.Recorded seizure stage and ADD daily until all rats in EP group were kindled successfully.After that,Morris water maze were performed to evaluate learning and memory of rats.Results:1.Repeated ANOVA analysis showed that there were statistical differences in seizure stage between EP and MB+EP group(0.01<P=0.042<0.05),treatment of MB had no interaction with time(P>0.05),which indicated that these two groups shared the same change trends.There were also statistical differences in ADD between these two groups(0.01<P=0.02<0.05),and treatment of MB had interaction with time(P<0.05),which indicated that these two groups shared different change trends.Finally,9 rats were thought kindled successfully during 20 days of kindling.The sensitivity of epileptic seizures was maintained.However,the average of seizure stage in MB+EP group was 4.44 at 20~thh day.2.The results of navigation experiment in Morris water maze showed that there was no statistical difference in escape latency between any two groups at the first two days(P>0.05).At days 3-5,the escape latency of rats in every group was entirely shorter than that at the first two days.Rats in the EP group exhibited prolonged escape latency when compared to the Control group(P<0.05).However,this poor performance was mitigated by pretreatment with MB(P<0.05).3.The number of crossing the plat form in the EP group obviously decreased as compared to the Control group(P<0.01),while pretreatment with MB increased the number of crossing(P<0.05).In the probe trial,the EP group spent significantly less time in the target quadrant than the Control group(P<0.01),while pretreatment with MB improved the performance(P<0.05).Part four Mechanisms of methylene blue on epileptogenesis during chronic BLA-kindling seizures in ratsObjective:Kindling models was induce by chronic BLA electric stimulus to investigate the mechanisms of inflammation in epileptogenesis,and explore the neuroprotective effect of MB during the influence of epileptogenesis.Methods:Thirty-six male healthy adult Wistar rats weighted 250-350grams were randomly divided into four groups,9 per group.Control group(rats without any treatment);Sham group(rats received electrode implantation but without electrical stimulation);EP group(rats received electrode implantation and chronic electric stimulus).MB+EP group(rats received 1mg/kg MB intraperitoneal injection 30min before electric stimulus).The kindling and induction programs were performed as described in partⅠ.The rats were thought kindled successfully after consecutive 3 times of 5 stage seizures.Recorded seizure stage and ADD daily until all rats in EP group were kindled successfully.After that,hippocampus tissue of rat was collected,and Westernblottingassaywasperformedfortheexpressionof inflammation-related markers NF-_ΚB,IL-1βand IL-6.Results:NF-_ΚB expressed normally in the Control and Sham groups,and there was no statistical difference between these two groups(P>0.05).Compared with the Control group,the level of NF-_ΚB in the EP group remarkably increased(P<0.05),whereas treatment with MB significantly weakened the expression of NF-_ΚB during chronic kindling(P<0.05).The variation trending of IL-1βand IL-6 shared the same pattern with NF-_ΚB,which increased during chronic kindling,and treatment with MB could prevent this increasing trend(P<0.05).Conclusions:1.SSSE and chronic rat models were successfully established by electric stimulation on BLA in this study.They were both stable and have a good repeatability,which can provide a stable model for the next experimental studies.The process of epileptic seizure and epileptogenesis can well imitate clinical seizures and the pathophysiological progress of human TLE.So,they were ideal models for studying the pathogenesis of human TLE.2.MB can markedly reduce the severity of the seizure activities and improved the cell morphology and alleviated the neuronal loss in hippocampus.The mechanism might be related to MB’s ability to reducing oxidative stress damage and apoptosis.3.Chronic BLA seizures was associated with increased expression of NF-_ΚB,IL-1βand IL-6,which indicated that inflammation took part in epileptogenesis.MB could delay epileptogenesis to a certain extent and improve the cognitive function of epilepsy.The underlying neuroprotective mechanism might be related to ability of MB to down-regulating expressinon of NF-_ΚB,IL-1βand IL-6,leading to the reduced inflammation in brain. |
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