| Part One Establishment of modified status epilepticus model induced by pilocarpine and its electroencephalographic features in ratsObjective:To establish a stable and reliable rat model of temporal lobe epilepsy (TLE), monitor a normal electrocorticography (ECoG) and the ECoG changes of status epilepticus (SE) and explore and prove the rules of this method.Methods:(1) Establishment of modified lithium-pilocarpine-induced epilepsy models: Healthy Sprague-Dawley (SD) rats (n= 16) were used in this study, and were randomly divided into 2 groups:normal control group (n= 6) and experimental group. Pretreated by lithium chloride at a dose of 125 mg/kg, the rats were injected with pilocaripine 100 mg/kg as first dose after 20 h. Then they were administered with low dose pilocarpine (25 mg/kg) every 30 min until SE broke up, but the maximal dose was 150 mg/kg. It was regarded as failure if the rats didn't reach to IV level. Only induced SE rats were drawn into the next step of the experiment. The behaviors of the rats were observed, and the incubation period of SE, success rate of induction and mortality were recorded. The rats in normal control group were injected with phosphate buffer saline (PBS) to replace pilocarpine, while other steps were the same.(2) Monitoring of electrocorticography (ECoG):According to the duration of SE, the rats in experimental group were randomly divided into experimental subgroup A (n= 6, SE for 15 min) and experimental subgroup B (n= 6, SE for 30 min), which were all anesthetized with urethane to record ECoG.Results:(1) In experimental group,12 rats were successfully kindled by pilocarpine,1 rat died, and 3 rats failed to be kindled. The total success rate of modeling was 75%, among which the success rate for the first dose of pilocarpine was 75%,16.7% for the second dose and 8.3% for the third; the failure rate was 18.8% in this experiment. The average incubation period (min) of SE was 29.7 ±15.1 (13~65) and the average dose of pilocarpine (mg/kg) was 112.5 ± 16.3. The normal control group rats were absent ofSE.(2) ECoG in normal control group was normal, which was similar to that in human beings. Compared with normal control group, ECoG in experimental group A showed higher voltage fast activity appeared in cortex, slow activity under 6 Hz increased gradually. The ECoG was monitored for 90 min after SE, continuously, and none of spikes or sharp waves, spike or wave discharges were observed. In experimental group B, the spikes or sharp waves, spike or wave discharges were noticed. Epileptic discharge was stable continuous for more than 2 h.Conclusions:(1) A modified pilocarpine kindling protocol, which consists of modest dose pilocarpine as the first administration and at most three timed repeated low dose injections in addition, is a simple and effective method to induce epilepsy rat model with a low mortality and short incubation period. The modified nursing method could reduce the mortality significantly. Above all, it is worth to develop and promote this method.(2) The ECoG of lithium-pilocarpine rat model is similar to the changes of SE and TLE in human, which can be conducted in the researches of mechanism and pharmacology study of epilepsy.(3) The fact that temporal variability exists between the originating in the firing of neurons and propagating to cortex is further confirmed by our experiments, thus, it is more meaningful to monitor ECoG 30 min after the onset of SE in SD rats.Part Two Effect of amiloride and acidic liquids on rats with lithium pilocarpine-induced epilepsyObjective:To study the effects of amiloride and acid liquids on the epileptic discharge in ECoG of epilepsy rat model induced by lithium-pilocarpine.Methods:(1) Establishment of modified lithium-pilocarpine induced epilepsy models: Healthy SD rats were induced by administration of pilocarpine hydrochloride 100 mg/kg as first dose 20 h after lithium chloride (127 mg/kg, i.p), and 30 min after atropina (1 mg/kg, i.p), which was injected to limit the peripheral effects of the convulsant pilocaripine. Then they were administered with low dose pilocarpine (25 mg/kg) every 30 min until SE broke up, but the maximal dose was 150 mg/kg. According to Racine classification, the behavior of rats were divided into I-V levels. It was regarded as failure if the rats didn't reach to IV level. There were 24 SE rats drawn into the next step of the experiment.(2) The rats were divided into four groups randomly:the PBS group (PBS, i.p.), the ASICs inhibitor group (amiloride, i.p), the positive control group (levetiracetam, i.p.) and the acidic liquids group (acidic liquids, pH= 1.57, i.p). All of the rats were anesthetized and placed the cortex electrodes after SE for 30 min, and were monitored for 30 min as base line, followed by administrating intraperitoneally different drugs (PBS, amiloride, levetiracetam or acidic liquids) respectively.(3) The frequency of discharge, interval and amplitude of discharge were selected as the indicators for comparison. The ECoG date was artificially divided into four phases (-30-0 min,0-30 min,30-60 min and 60-90 min), and the earliest phase (-30-0 min) was dated before drug injection. The data of 30 min ECoG per rat before injection were set as baseline. The values were processed as the ratio of the data of different phase to those of 30 min ECoG per rat before injection.Results:(1) ECoG monitored sharp and spike wave, and the epileptic discharges were sustained and stable.(2) Compared with PBS group, at 60~90 min after injection, the frequency of discharge in ASICs inhibitor group were reduced (0.69 ± 0.08 vs0.89 ± 0.07), the interval amplitude were declined (0.70 ±0.16 vs0.90 ± 0.08), while the interval of discharge was increased (1.46 ± 0.18 vs 1.15 ± 0.10) (P< 0.05).(3) Compared with PBS group, at 0-30 min after injection, the frequency of discharge in acidic liquids group increased significantly (1.05 ± 0.07 vs0.9 ± 0.07), the interval were not prolonged (1.00 ± 0.05 vs 1.12 ± 0.09) (P< 0.05), then at 30~90 min after injection, the frequency and amplitude were declined and interval of discharge were prolonged but with no significant difference.(4) At 0-90 min after injection, the positive control group and the ASICs inhibitor group were no significant in the frequency, amplitude and interval of discharge.Conclusion:(1) Amiloride can inhibit epilepsy induced by lithium-pilocarpine;(2)Acidic liquids can promote epileptic seizures transiently, which may be concerned with activation of ASICs channel.Part Three The study for expression of ASICs & NHE and antiepileptic mechanism of amiloride suppressing pilocarpine-induced seizuresObjective:To investigate the effect of acid sensing ion channels (ASICs) and Na+/H+ exchanger (NHE) gene expression in seizure attack and the antiepileptic mechanism of amiloride in pilocarpine induced seizures rats.Methods:(1) Establishment of modified lithium-pilocarpine induced epilepsy models: Healthy SD rats were induced by administration of pilocarpine hydrochloride 100 mg/kg as first dose 20 h after lithium chloride (127 mg/kg, i.p), and 30 min after atropina (1 mg/kg, i.p), which was injected to limit the peripheral effects of the convulsant pilocaripine. Then they were administered with low dose pilocarpine (25 mg/kg) every 30 min until SE broke up, but the maximal dose was 150 mg/kg. According to Racine classification, the behavior of rats were divided into I-V levels. It was regarded as failure if the rats didn't reach to IV level. There were 18 SE rats drawn into the next step of the experiment.(2) We choosed 6 normal rats as normal control group (n= 6), and divided the SE rats into 3 groups randomly:SE 1 h group (PBS, i.p.), SE 2 h group (PBS, i.p.) and amiloride group (amiloride, i.p.), followed by corresponding drug injection, respectively.(3) The rats of SE 1 h group were executed at 1 hour later, and the rats of SE 2 h group, normal control group and amiloride group at 2 hours later, respectively, and the cerebral cortex was separated. Finally, we extracted the total RNA for RT-PCR, detected the mRNA expression of ASICla, ASIC3 and NHE in the four groups.Results:(1) The mRNA of ASIC3 (0.01 ± 0.00) and NHE (0.01 ± 0.00) were seldom expressed in the normal group, while the ASICla mRNA (0.19 ± 0.01) expressed little.(2) Compared with normal control group, the mRNA of ASICla, ASIC3 and NHE in SE 1 h group (0.89 ± 0.01 vs 0.19 ± 0.01,0.29 ± 0.04 vs 0.01 ± 0.00,2.16 ± 0.01vs 0.01 ± 0.00) increased significantly.(3) Compared with normal control group, the mRNA of ASICla, ASIC3 and NHE in SE 2 h group (1.24 ± 0.01 vs 0.19 ± 0.01,0.81 ± 0.04 vs 0.01 ± 0.00,2.73 ± 0.09vs 0.01 ± 0.00) increased significantly; Compared with SE 1 h group, the mRNA of ASICla, ASIC3 and NHE in SE 2 h group (1.24 ± 0.01 vs 0.89 ± 0.01,0.81 ± 0.04 vs 0.29 ± 0.04,2.73 ± 0.09 vs 2.16 ± 0.01) increased significantly.(4) Compared with SE 1 h and SE 2 h group, the mRNA expression of ASICla (0.44 ± 0.01) and ASIC3 (0.09 ± 0.01) in amiloride group declined significantly (P< 0.01). Although the mRNA expression of NHE decreased, but compared with the SE1 h group and SE 2 h group, there was no statistical significance (P> 0.05).Conclusions:(1) The mRNA expression of ASIC3 and NHE are rare in normal rat's cortex, while ASICla mRNA is little.(2) ASICla, ASIC3 and NHE take apart in epileptogenesis.(3) Amiloride suppresses pilocarpine-induced seizures by regulating the mRNA expression of ASICla and ASIC3 in SE rats, but not NHE. |
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