Study On 5-HT Levels Variation In Epileptogenesis Process Of Pentylenetetrazol-induced Kindling Rats

Epilepsy is a common clinical syndrome in nervous system,with chronic, accidental,and repeatedly stereotyped as its primary clinical characteristics.Although the therapy for Epilepsy is advancing,there are still about 20%-30%of all Epilepsy patients,with seizures out of controlled,belonging to refractory epilepsy.So that further study on epileptogenesis mechanisms is the premise of searching new type anti-epileptic measures.In recent years,there has been increasing evidence that serotonergic neurotransmission modulates a wide variety of experimentally induced seizures.Generally,agents that elevate extracellular serotonin(5-HT) levels,such as 5-hydroxytryptophan and serotonin reuptake blockers,inhibit both focal and generalized seizures.Conversely,depletion of brain 5-HT lowers the threshold to audiogenically,chemically and electrically evoked convulsions.We use rats which can analogue human epilepsy better as experimental animals to investigate 5-HT levels variation in the process of pentylenetetrazol-induced kindling seizures and epilepsy,and pursue mechanism of 5-HT in epiletogenesis process.By means of hippocampus electrode EEG and microdialysis technology, systematic observation free-running rats behavior,EEG and hippocampus 5-HT levels in the process of pentylenetetrazol-induced kindling in vivo can avoid the effect of anesthetic and operative procedure trauma,and benefit to accurate results of detection. In our study,main contents and objectives as follow:1.To establish hippocampus electrode EEG and microdialysis model in free-running rats,provides fundament for us to approach epileptogenesis mechanisms.2.To investigate variation of behavior, EEG and 5-HT levels in pentylenetrazol-induced seizures rats,and hippocampus neuron morphologic change after seizure.3.To investigate variation of behavior, EEG and 5-HT levels in PTZ-induced kindling epilepsy rats,and hippocampus neuron morphologic change after kindled.4.To investigate the effect of 5-HT levels increase on the PTZ-induced kindling rats;5.To investigate the effect of 5-HT levels decrease on the PTZ-induced kindling rats.Through observation character of 5-HT levels variation in the process of PTZ-induced kindling epilepsy,to pursue possible mechanism of 5-HT in epiletogenesis process.After then,to pursue new type anti-epileptic measures;to provide experimental and theory basis for study and exploitation new type anti-epileptic drugs.PartⅠThe establishment of hippocampus electrode EEG and microdialysis model in free-running ratsObjective:To establish hippocampus electrode EEG and microdialysis model in free-running rats,provides fundament for us to approach mechanisms of epileptic genesis.Methods:Hippocampus electrode EEG recording and microdialysis taking samples,HPLC-ECD detection Hippocampus 5-HT,5-HIAA concentrations and 5-HT turnover rate(5-HIAA/5-HT) at 8AM,11AM,14PM,17PM,20PM in 12 free-running rats,monitor rats' behavior,EEG and 5-HT levels at the same time.Results:(1)At conditions of alertness,peacefulness,drowsiness,sleeping,waves likeαwaves,βwaves,θwaves andδwaves were recording in EEG.(2)At night,5-HT concentrations increase significantly,and 5-HT turnover rate degrade significantly compare with other conditions,have significant difference(P＜0.05).5-HIAA concentrations variation does not have significant difference.PartⅡ5-HT levels variation in pentylenetetrazol-induced seizures ratsObjective:To investigate variation of EEG and 5-HT levels in pentylenetetrazol (PTZ)-induced seizures rats,and hippocampus neuron morphologic change after seizures.Methods:The microdialysis probe and hippocampus electrode were placed into the dorsal hippocampus of rats,at the time of before,during and after seizures (60mg/kg PTZ IP once),dialysate was collected,concentrations of extracellular 5-HT, and its metabolite 5-hydroxyindolacetic acid(5-HIAA) were assessed by HPLC-ECD. Recording hippocampus EEG at the same time in 24 free-running rats.24h after seizures,to view hippocampus neuron morphologic change by transmission electron microscope.Results:(1) In PTZ group during PTZ-induced seizures,5-HT levels increase significantly and 5-HT turnover(5-HIAA/5-HT) decrease significantly compare with those at the time of before and after seizures,and that of control,have significant difference(P＜0.05);5-HIAA levels change in PTZ group compare with control group have no significant diffenence.(2) EEG show gradual aggravation of epileptic discharge.(3) TEM show hydropic degeneration in hippocampus neuron. PartⅢ5-HT levels variation in pentylenetetrazol-induced kindling epilepsy ratsObjective:To investigate variation of behavior,EEG and 5-HT levels in pentylenetrazol(PTZ)-induced kindling epilepsy rats,and hippocampus neuron morphologic change after kindled.Methods:The microdialysis probe and hippocampus electrode were placed into the dorsal hippocampus of rats at the 7d,14d, 21d,28d of PTZ(35mg/kg /d IP) kindling,recording hippocampus EEG,and at seizures interval(three hours after seizures) dialysate were collected,5-HT levels, 5-HIAA levels were assessed by HPLC-ECD in 24 free-running rats.After rat been kindled,to view its hippocampus neuron morphologic change by transmission electron microscope.Results:(1) Accompanying days of kindling passing,latent period of seizures shorten gradually,degree of seizures anabatic.At 10-14 days all rats were kindled,one died during kindling.(2) Accompanying seizures anabatic, EEG shows gradual aggravation of epileptic discharge.Latent period of epileptic discharge was shorten.Spontaneous discharge appears during seizures interval.(3)In earlier period(7d) of kindling,5-HT levels in rats dorsal hippocampus were a little increased and 5-HT turnover(5-HIAA/5-HT) decreased,but not have significant difference.In advanced stage(28d) of kindled,5-HT levels and 5-HIAA levels were decrease significantly compared with that of control have significant difference (P＜0.05),change of 5-HT turnover has no significant difference in comparing with that of control.After rats have been kindled,maintenance efficient kindling(PTZ 20mg/kg/d IP),5-HT levels decrease continuously.5-HT and 5-HIAA levels at the 7d and 14d have significant different from those at the 21d and 28d(P＜0.05).(4) TEM show cellular necrosis in hippocampus neuron.PartⅣEffect of 5-HT levels alteration on pentylenetetrazol-induced kindling ratsObjective:To investigate the effect of citalopram(increase 5-HT levels) and reserpine(decrease 5-HT levels) on the pentylenetetrazol-induced kindling rats,to test the hypothesis that 5-HT levels change plays a role in the modulation of experimental epilepsy.Methods:48 Rats were divided to 4 groups in random(control group,PTZ group,CTP+PTZ group,RSP+PTZ group).Separate intervention by citalopram (0.5mg/kg/d) intragastric administration and reserpine(1mg/kg/d) intragastric administration one week before PTZ(35mg/kg/d) intraperitoneal injection, observation of behavior everyday.At the 7d,14d,21d,28d,of PTZ(35mg/kg/d IP) kindling,recording hippocampus EEG,and at seizures interval(three hours after seizures) dialysate were collected,5-HT levels,5-HIAA levels were assessed by HPLC-ECD in 48 free-running rats.Results:1.During kindling,in CTP group, seizures degree was lighter than PTZ group,latent period of induced seizures and time to be kindled were longer than that of PTZ group(P＜0.05).After kindled,in CTP group has no significant difference in these parameter.During kindling,in CTP group, latent period of epileptic discharge recorded by EEG,was longer than that of PTZ group(P＜0.05).During kindling(7d),in CTP group 5-HT levels were increase and 5-HT turnover(5-HIAA/5-HT) decrease compared with that of PTZ group,have significant difference(P＜0.05).After kindled(28d),in CTP group 5-HT levels were decrease compare with that of PTZ have no difference,compared with that of control, have significant difference(P＜0.05).2.During kindling(7d),in RSP group,seizures degree was severer,latent period of seizures was shorter,time to be kindled was shorter than that of PTZ group,have significant difference(P＜0.05);During kindling, in RSP group,EEG latent period of epileptic discharge was shorter than that of PTZ group(P＜0.05).During kindling,in RSP group,5-HT levels were decrease and 5-HT turnover(5-HIAA/5-HT) increase compared with that of control and PTZ group,have significant difference(P＜0.05).After kindled(28d),in RSP group as well as PTZ group 5-HT levels and 5-HIAA levels were decrease compared with that of control group,have significant difference(P＜0.05).ConclusionsRats 5-HT levels were increase at night,there is existence of circadian change. PTZ-induced seizures result in significant increase of hippocampus 5-HT levels,and 5-HT levels decrease to normal after seizures;The longer of seizures last,the higher of 5-HT levels increase;After seizures,there are hydropic degeneration in rats hippocampus neuronsDuring epileptogenesis development,hippocampus 5-HT levels increase in early stage of kindling;and decrease gradually in advanced stage of kindled.Accompanying the time of kindling passing,levels of hippocampus 5-HT decreased continuously in progress.Hippocampus 5-HT levels significant decreased down to normal levels after rats being kindled,and it exist persistently.After being kindled,cellular necrosis appears in hippocampus neurons. Brain 5-HT levels increase result in significant prolongation time of PTZ-induced kindling rats to be kindled.During period of kindling,increase of 5-HT levels lessen PTZ-induced seizures degree obviously.5-HT levels increase can't block PTZ-induced kindling epileptic genesis.5-HT levels increase can't inhibit epileptic seizures after rats being kindled by PTZ.Brain 5-HT levels decrease result in significant short time of rats to be kindled by PTZ and aggravation PTZ-induced seizures degree.5-HT levels decrease can facilitate PTZ-induced kindling rats epileptic genesis.