| Background:Epilepsy is a common neurological disease, which is characterized by abnormal neuronal excitability and hypersynchronization. Acute seizure, especially the Status epilepticus (SE) is a life-threatening neurological emergency. Recurrent seizure activities with control may cause permanent neurologic damage, which contributes to severe cognitive and behavior impairment. The effects of anticonvulsant drugs used in clinic are not satisfactroy in the treatment of refractory epilepsy, so the new therapeutic target is needed.Serotonin.(5-hydroxytryptamine,5-HT).is.an.influenced.monoamineneurotransmitter in the central nerve system. The possibility of the link between serotonin and inhibition of epilepsy was first proposed by Bonnycastle in1957. A number of evidence has suggested that the anticonvulsant effects of serotonin might be via serotonin1A receptor (5-HT1A receptor). The effect of5-HT1A receptor in acute and chronic seizure is still controversial. The difference may be due to the different mechanism of epileptic model. Compared with kainic acid model, Li-pilocarpine model are more appropariate to study the various aspects of SE and mimic the severe neuronal impairment induced by long-term SE. Kindling is a good model to imitate the human complex partial and secondary generalized temporal lobe epilepsy. Therefore, this study intends to research effect of5-HT1A receptor on Li-pilocarpine induced SE and amygdaloid kindling modelof epileptogenesis.5-HT1A receptors locate both presynaptically in the raphe nuclei, and postsynaptically abundant in frontal cortex and limbic area[5,14]. It is still unclear that which types of5-HT1A receptor have anti-or proconvulsant effect in SE and epileptogenesis. Hippocampus is a candidate site for its key role in seizure generation[4,15,16]and the high density of5-HT1A postsynaptic receptor distribution{Polter,2010#1}. There is no evidence on effect of hippocampal5-HT1A receptor in systemic intervention induced SE models and kindling model. This study is aimed to figue out whether activation of5-HT1A receptor presents a anticonvulsant or an antiepileptogenesis effect in SE and to reveal whether5-HT1A receptor in hippocampus or other regions are involved in the effect.The density of5-HT1A receptor in epileptic zone and limbic area decreases in both temporal lobe epilepsy (TLE) patients and chronic epilepsy animals. The dynamic changes of5-HT1A receptor may indicating its mechanism. The expression of5-HT1A receptor during the process of SE have not be reported. Extracellular signal regulated kinases (ERKs) is a subfamily of Mitogen-activated protein kinases (MAPK) family, and is involved in the seizure and SE. The activation of ERK pathway will phosphorylates Kv4.2channels, leading to the inhibition of this potassium channel and substantially alters excitability of CA1neurons. Activation of5-HT1A receptor can inhibit the phosphorylation of ERK in normal rats, but whether it has the similar effects in SE rats is unknown. The pathway of ERK might be a possible mechanism for the antiepileptic effect induced by5-HT1A receptor. Therefore, this study will further observe the dynamic changes5-HT1A receptor expression in the process of SE and investigate the effect of5-HT1A receptor on the inhibition of ERK phosphorylation in SE.Part I The anticon vulsant effect of serotonin1A receptor on lithium pilocarpine induced status epilepticusPurpose:To investigate whether5-HT1A receptor has anticonvulsant effect in the lithium pilocarpine induced epileptics status (SE).Methods:1.280-300g male Sprague-Dawley were were implanted electrodes for EEG recording with a10-day period for recovery. To assess the effects of systemic agonist8-OH-DPAT administration, rats were matched for weight and divided into seven groups and received one of the following treatments1h prior to the pilocarpine injection subcutaneously (s.c.):PBS,8-OH-DPAT0.01,0.1,0.5,1.0mg/kg, antagonist WAY-1006351.0mg/kg and WAY-1006351.0mg/kg immediately after8-OH-DPAT1.0mg/kg.2. Epileptic rats were developed by LiCl (3mmol/kg, i.p.)22-24h prior to pilocarpine (25mg/kg, i.p.). The SE incidence, acute mortality rate, GS latency, GS duration, latency to the first epileptiform spikes and onset of electrographic SE, and total time of seizure activity in EEG were recorded. Rats received diazepam (5mg/kg, ip)90min after SE onset.3. For intrahippocampal infusion of drugs, the rats (initially weighted280-300g) were implanted with injection guide cannula (AP=-3.5mm, L=±2.5mm, and V=-2.9mm). In8-OH-DPAT group, the drug was dissolved in PBS and infused15min before pilocarpine injection,while the other group received PBS (1μl) instead.Results:1. In the subcutaneous administration of agonist, no significant difference were found for SE incidence and acute mortality rate between groups.2. In contrast to the PBS group,8-OH-DPAT induced a dose-dependent effect.8-OH-DPAT also increased the latency to the first GS and the GS duration at0.5and1.0mg/kg.3.8-OH-DPAT increased the latency to the first epileptiform spikes, to the onset of electrographic SEand decreased the total time of seizure in EEGat0.5and1.0mg/kg.4. The effects of8-OH-DPAT1.Omg/kg on the EEG and GS activites were inhibited when the antagonist WAY-100635was added. However, WAY-100635administration alone showed no significant changes.5. Rats pretreated with0.01and0.1mg/kg of8-OH-DPAT showed no significant difference on all parameters comparing to the control. 6. Intrahippocampal injection of8-OH-DPAT1μg decreased the GS duration during the SE, however, did not induce any significant difference on EEG and behavior latency and total seizure time in contrast with control group.Conclusions:1. Subcutaneous administration of8-OH-DPAT presented a dose-dependant anticonvulsant effect in the Li-pilocarpine induced SE process.2. Subcutaneous administration of8-OH-DPAT at0.01and0.1mg/kg did not altered seizures in SE.3. Intrahippocampal administration of8-OH-DAPT induced inhibited the severity of seizure in SE, but had no effect on the generation of SE.Part II The antiepileptic effect of serotonin1A receptor on the epileptogesis of amygdaloid kindling modelPurpose:To investigate whether5-HT1A receptor has antiepileptic effect on the epileptogesis of amygdaloid kindling modelMethods:1. Electrodes were implanted to the basolateral amygdale of rats. To assess the effects of systemic agonist8-OH-DPAT administration, rats were matched for weight and divided into six groups and received one of the following treatments1h prior to the pilocarpine injection subcutaneously (s.c.):PBS,8-OH-DPAT0.01,0.1,0.5,1.0mg/kg, antagonist WAY-1006351.0mg/kg immediately after8-OH-DPAT1.0mg/kg.2. The initial after discharge of threshold (ADT) was determined at the first day. Then the rats were given a daily stimulus for a total16days. The stimulus intensity is same with the initial ADT. The seizure severity and discharge duration (ADD) were recorded.3. For intrahippocampal infusion of drugs, the rats were implanted with injection guide cannula (AP=-3.5mm, L=-2.5mm, and V=-2.9mm). In8-OH-DPAT group, the drug was dissolved in PBS and infused15min before the daily kindlig, while the other group received PBS (1μl) instead.Results:1. Subcutaneous administration of8-OH-DPAT at0.5and1.0mg/kg significantly delayed the progression of seizure stages, and shortened the corresponding ADD at1.0mg/kg compared with PBS group. This effect can be inhibited by pre-treatment of antagonist WAY-100635.2. In8-OH-DPAT1.0mg/kg group, rats stayed in stage1for a longer durations and stayed in stage5for a shorter durations. The number of stimulations required to reach stages2-5is more than PBS group. Also rat of8-OH-DPAT group stayed in stage1-3for longer and stayed in stage4-5shorter than PBS group.3. In antagonist WAY-100635group, rats stayed longer in stage4than8-OH-DPAT1.0mg/kg group, and prevented the effect of shorten number of stimulations required to stage3and4. 4. Rats pretreated with0.01and0.1mg/kg of8-OH-DPAT showed no significant difference on all parameters comparing to the PBS group.5. Intrahippocampal8-OH-DAPT administration induced no significant changes on all parameters comparing to the PBS group.Conclusions:1. Subcutaneous administration of8-OH-DPAT presented an antiepileptic effect in amygdaloid kindling model.2. Subcutaneous administration of8-OH-DPAT at0.01and0.1mg/kg had no effect of in amygdaloid kindling model.3. Intrahippocampal administration of8-OH-DAPT induced had no effect in amygdaloid kindling model.Part Ⅲ The expression of serotonin1A receptor and its possible effect through the ERK pathway in the status epilepticus induced by lithium pilocarpinePurpose:To observe the alternations of5-HT1A receptor in raphe nucleus and hippocampus in SE induced by lithium pilocarpine, and to investigate the effect of5-HT1A receptor on the inhibition of ERK phosphorylation in SE. Methods:1. Epileptic rats were developed by Li-pilocarpine on male SD rats (250-300g). Rats were sacrificed at Oh,1h,2h,6h,24h after pilocarpine injection. The brain tissue were prepared for immuohistochemistry and western blot.2. Paraffin section of4μm thickness were used for immuohistochemistry. All picture got from microscope were analysed with Image-Pro Plus6.0. the positive size, positive cells and the mean intensity were calculated.3. The protein of5-HT1A receptor in hippocampus and raphe nucleus was detected through Western blot.4. To assess the expression of pERK, rats were divided into four groups:Control (without pilocarpine injection), Li-pilocarpine group,8-OH-DPAT1.0mg/kg, and antagonist WAY-1006351.0mg/kg+8-OH-DPAT1.0mg/kg. Li-pilocarpine model were induced and rats were sacrificed at0h,1h,2h,6h,24h after pilocarpine injection. The expression of ERK1/2and pERK1/2in hippocampus were detected through western blot.Results:1.5-HT1A receptor in raphe nucleus was mainly seen in the DRN and MRN. It was mainly expressed in the membrane of soma and dendrate, while less in the cytoplasma. During the early onset of Li-pilocarpine induced SE, expression of5-HT1A receptor significantly increased at1h after pilocarpine injection, while decreased in the6h and24h. The change was greater in DRN than MRN. 2. Hippocampal5-HT1A receptor was distributed in CA1, CA3and DG. The expression on the pyramidal cell was realtively week, mainly on the membrane. During the development of SE, expression of5-HT1A receptor was prominent at2h after pilocarpine injection, while decreased in6h and24h.3. In contrast with control group, pERK/ERK increased in Li-pilocarpine group. The change is significant in pERK2/ERK2while pERK1/ERK1only increased at6h and24h after pilocarpine injection.8-OH-DPAT inhibited the expression of pERKl and pERK2during all the process of SE, while this effect can be inhibited by WAY-100635.Conclusion:1. The expression of5-HT1A receptor in raphe neuclus increased at1h after pilocarpine injection, and decreased in late phase of SE. The change greater in DRN than MRN.2. The expression of5-HT1A receptor in hippocampus increased at2h after pilocarpine injection, and decreased in late phase of SE. The althernation of5-HT1A receptor is not entirely associated with neuronal loss.3. The phospholation of ERK1/2in SE induced by lithium pilocarpine can be inhibited by activation of5-HT1A receptor... |
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