Aspect Of Mechanism Of Epileptic Insults And The Protective Effect Of Edaravone On The Hippocampal Neurons In Rats Induced By Kainic Acid

Epliepsy is a syndrome of the dysfunction of central nervous system, which is caused by a group of know or unknown etiologies. The Electrophysiological mechamism is characterized by a abnormaly highly synchronized and self-limited discharge of the neurons. The clinical features present recurrent, short-term, usually stereotype. The patients with epilepsy underwent a severe neuronal anoxic condition due to being exposed to prolonged and repeated seizures, especially to a long duration of generalized tonic-clonic seizures. The patients might developed a degeneration of neurons or a neuronal death in the brain, especially in the hippocampus which generally developed histological sclerosis. On the other hand, hippocampal sclerosis has further led to repeated seizures. The patients with epilepsy always presented the deficiency of learning and memory. It was reported that there are thirty to forty percent of them have a disorder of cognintive function. Thus epilepsy has become an important reason of affecting the life quality of epileptic sufferers, some of which became the chronic suffers.Up to now, the epileptic therapy mainly has been being depended on drugs which took part in the effects of inhibiting abnormal discharge, controlling the seizures, thereby reducing neurons secondary injury. On the other hand , for the inevitable seizures or status epilepticus, we should take effective measures to protect the brain as soon as possible after the seizures .It is very important that alleviating the epileptic insults and inhibiting epileptic seizures.It was found that the free radicals had a very close relationship to the development of epilepsy. It was show that the epilepstic seizure was acompanied by the activities of free radicals. The increase of radicals caused neuronal degeneration through the membrane lipid peroxidation and the reduction of GSH - PX at the epileptic foci. The SOD activity was significantly decreased and the content of MDA has increased significantly in the hippocampus , cerebral cortex, hypothalamus and pituitary after seizures in animal experiments and the changes were even more significant with seizurs becoming frequent.It was shown that in previous studies, apoptosis-related gene expression, such as p53 gene, immediate early genes, bcl-2 gene family and myc genes, increased in various brain tissues after seizures. So that the neuronal damage was involved in mechanism of neronal apoptosis. Among the factors inducing neuronal apoptosis, the free radical inducing apoptosis can be confirmed either in vivo metabolism or exogenous factors. Clearly, free radicals play an important role in the pathophysiological mechanism of epileptic insult. Therefore remove free radicals and free radical reactions can block cytotoxic response caused by seizures, inhibit neuronal apoptosis and death, protect neurons and inhibit the progress of epilepsy.Edaravone is a novel free radical scavenger,which can remove hydroxyl radical of surrounding Penumbra, inhibit lipid peroxidation, thereby inhibited the damage and apoptosis of neurons. Edaravone can protect neurons effectively from ischemic disease.But the protective effect of edaravone of neurons has not been reported. The present study was undertaken to explore the mechanisms of protective effect of edaravone on the hippocampal nuerons in rats exposed severe epileptic seizures or status epilepticus. For the aim, the hippocampal morphology, neuronal apoptosis, free radicals and lipid peroxidation changes, as well as the cognitive function were investigated in kainic acid-induced (Kainic acid, KA) rats epilepsy model.At the same time the effect of edaravone, on the events mentioned above were observed.Part one : Kainic acid-induced epileptic model in rats Methods:1 The establishment of kainic acid-induced epileptic model in ratsEighteen adult male Wistar rats (weight 260±20g) rats were randomly divided into two major groups:⑴sham-operated group(n= 6) :adminicstrated 0.9% saline the same volume with model group;⑵model group (KA) (n=12): According to attack level and volume of injection drug will be further divided into two subgroups:①K1 group (n = 6): administrated kainic acid (4μg/kg) in the right hippocampus CA3 region vis sereotactic technique.;②K2 group (n = 6): administrated kainic acid (4μg/kg) in the right hippocampus CA3 region vis sereotactic technique.K1 group rats were performance for the grade II~III, electied 6; K2 group rats appearedⅣ~Ⅴlevel attack, the duration of about 1.5h~2h, elelected 6, observed the contralateral hippocampus on the seven day after injection KA and saline.The rats happened to death or not meet with the standard were eliminated and the alternative ones were recruited to ensure there were 18 rats in experimental groups.2 EEGEEGs were recorded before the establishment of models. Rats were implanted with a electrode after being anaesthetized. when the rats waked ,we monitored EEG.3 Characteristic of behavior of ratsThe changes of behavior of rats were observed after the administration of kainic acid in the right hippocampus CA3 region.The 4 Histological changes of the CA1, CA3 and CA4 regions of the hippocampus in ratsAll rats were sacrificed on the seven day after the adminstration of saline or kainic acid. Histological changes of the CA1 and CA3 regions of the hippocampus under thionine staining. Histological changes were divided into the following 4 grades (histological grade, HD) under light microscope: grade 0, no neuron death; grade I, scattered single neuron death; grade II, death of many neurons; grade III, death of almost complete neurons. The average HG of the lefe hippocampus was counted as statistical data. The neuronal density (ND) of the hippocampal CA1 and CA3 subfield was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1, CA3. The average of number of pyramidal neurons in 3 areas of the hippocampal CA1, CA3 subfield was calculated as value of ND.Results1 The feature of behavior of ratsSham-operated rats had normal behavior. K1 group has 10 Wister rats, showed that 6 rats for Racine II~III level epileptic attack, 3 rats for Class I epileptic attack, one rat for Class IV epileptic attack after injection KA , so be moved from goup . K2 group has 12 Wistar rats, 10 rats appeared IV ~V Class epileptic attack, which lasted more than two hours, four rats died, 2 rats which have Class III attack do not meet the requirement, so be removed. The group mortality is 33 percent.2 EEGRats in itcal period presented obvious epileptic discharges characterized by spikes, sharps, polyspike and spike-wave complexes. There is no abnormal existence in sham group.3 Histological changes of the CA1, CA3 and CA4 regions of the hippocampusThe results as followed : no significant neuronal damage was found in contralateral hippocampus of rats in the sham and K1 groups (P<0.05). In the sham group, HD in the CA1 and CA3 was 0～I,and values of ND were 198±20.62 and 212±30.14, In the K1 group , HD in the CA1 and CA3 was 0～I, and values of ND were 186±19.45 and 208±27.73. In K2 group , as compared with the sham and K1 groups, the values of ND were 79±13.72 and 90±14.98,significantly lower (P<0.01). HG(grade II～III) was much higher(P<0.05), suggesting that the more serious epilepsy spontaneously, the more serious damage. In this experimental model of epilepsy, only when the seizure attack reaches a certain level, the contralateral hippocampal neurons can be injury. So we chosed K2 group as research object, observed a series of pathophysiological changes in kainic acid-induced epilepsy rats after administrated edaravone.Part two: Aspect of mechanism of epileptic insults and the protective effect of edaravone on the hippocampal neurons in rats induced by kainic acid2.1 The histological changes of protective effect of edaravone on hippocampal neurons in kainic acid-induced epileptic ratsMethods:1 The establishment of kainic acid-induced epileptic model in ratsEighteen mature male Wistar rats (weight 260±20g) were randomly divided into 3 groups:①sham operation group (n = 6) :the same as the first part;②KA model group (n = 6): administrated kainic acid (4μg/kg) in the right hippocampus CA3 region vis sereotactic technique;③edaravone group (n = 6): administrated kainic acid (4μg/kg) in the right hippocampus CA3 region vis stereotactic technique, and immediately administrated edaravone (8mg/kg) once dayily i.p.. The rats happened to death or not meet with the standard were eliminated and the alternative ones were recruited to ensure there were 18 rats in experimental group.All rats were sacrificed on the seven day after the injection of saline or kainic acid . Histological changes of the CA1, CA3 and CA4 regions of the hippocampus under thionine staining. The remaining steps is the same with the first part.2 Histological changes of the CA1, CA3 and CA4 regions of the hippocampus (the same way with the first part)Results1 The feature of behavior of ratsSham-operated rats had normal performance. The latent period and the duration of seizure graded were no significant difference in model and edaravone groups .(data abbreviated).2 Histological changes of the CA1, CA3 and CA4 regions of the hippocampusThe results showed that: no significant neuronal damage was found in contralateral hippocampus of rats in the sham group. HD in the CA1 and CA3 was 0～I,and values of ND were 198±20.62 and 212±30.14. In model group, as compared with the sham group, the values of ND were 79±13.72 and 90±14.98,significantly lower (P<0.01), HG (grade II～III) was much higher (P<0.05). In edaravone group, as compared with the model group, there were scatted necrosis cells in the CA1 and CA3, and values of ND were 101±16.85 and 135±22.17, significantly higher (P<0.01). HG (gradeI~II) was much lower (P<0.05), indicating edaravone can protect hippocampal neurons from damage.2.2 The anti-apoptosis effect of edaravone on the hippocampal neurons in kainic acid-induced epileptic rats Methods:1 The establishment of kainic acid-induced epileptic model in ratSixty-six adult male Wistar rats (weight 260±20g), were randomly divided into three groups:①sham operation group (n = 6) : the same with the first part ;②KA model group (n = 6): the same with the 2.1 part;③edaravone group (n = 6): the same with the 2.1 part. Model and edaravone groups were set 5 points-in-time, respectively 4h, 1d, 3d, 7d, 14d, each time point has six rats. The rats were sacrficed in the booking time point. Observed the change of HD and the values of ND, TUNEL staining was used to detect the number of apoptotic neurons of hippocampal CA1 and CA3 regions.Results1 The feature of behavior of rats and histological changes of the CA1, CA3 and CA4 regions of the hippocampus: the same as the part of 2.1 above.2 The result of TUNEL stainingThe results as followed: there were no positive cells in contralateral hippocampal pyramidal CA1, CA3 and CA4 in sham group (P<0.05). In model group the staining showed that the number of TUNEL-positive cells rised from first day, reached the peak on the third day, began to fall after the seventh day, disappeared in the fourteen day. There were significant differences in the number of TUNEL-positive cells, as compared with the sham group, from the first day to the seventh day in model group (P<0.05). In edaravone group, as compared with the sham group, there was significantly less than the model group (P<0.05), but still higher than the sham group(P<0.05). These results indicated that: epilepsy can evoke apopotosis of neurons, edaravone can partly inhibit neurons apoptosis.2.3 Edaravone inhibit the lipid peroxidation process of hippocampal neurons in kainic acid-induced epileptic rats.Methods:1 The establishment of kainic acid-induced epileptic model in ratsSixty-six adult male Wistar rats(weight 260±20g), were randomly divided into three groups①sham operation group (n = 6): the same way with the first part;②model group (n =30): the same way with the 2.1 part;③edaravone group (n =30): the same way with the 2.1 part. Model and edaravone groups were divided into 5 subgroups: 5min, 6h, 24h, 72h, 7d, each subgroup has six rats. The rats were sacrificed in the booking time point. 10% homogenate were made rapidly with the brains in different time point of different groups , and SOD activity and MDA content were detected.Results:1 The feature of behavior of rats: the same as the part of 2.1 above.2 The result of SOD activity and MDA content of contralateral hippocampus of ratsThe results showed that: SOD activity and MDA content were no significant change at each time point in sham-operated group (P<0.05). In model group SOD activity decreased and MDA content increased from 6h, SOD decreased the lowest and MDA reached the peak on the third day, and then SOD activity began to pick up , MDA content begin to decline. SOD activity restore to baseline levels at the seventh day. From 6h to 72h period, in edaravone group, as compared with the sham group SOD activity was significantly higher (P<0.05), and MDA content was obvious lower (P<0.05) . These results suggest that the free radicl play a important role in epileptic injury. Edaravone effectively increased SOD activity and reduced MDA content, enhanced the capacity of body's antioxidant, and protected neurons from injury . 2.4 The effect of edaravone on cognitive function of kainic acid-induced epileptic ratsMethods1 The establishment of kainic acid-induced epilepticmodel in ratsThirty adult male Wistar rats (weight 260±20g), were randomly divided into three groups:①sham operation group (n=6): the same way with the first part;②model group (n=12):the same way with the section 2.1part③edaravone (n = 12): the same way with the section 2.1part. The three groups of rats, respectively, were evaluated Morris water maze test in order to test cognitive function and learning capacity at 24h and 8w after operation.Results1. Place navigation test(PNT)escape latencyAll rats escape latency periods in the beginning of PNT 24h were no significantly extended, as compared with the sham group, there was no obvious difference; model and edaravone groups were prolonged in the PNT 8w which was significantly different from the sham group (P<0.05), and the edaravone group was significantly shorter than that of model group (P<0.05).2. Spatial probe test (SPT):The spending time in the original platform quadrant of allof groups rats was significantly higher than that of other quadrant. The model and edaravone groups in the beginning of 24h STP, as compared with the sham group, were no significant difference (P>0.05). The model and edaravone groups rats at STP 8w, as compared with the sham group, the platform quadrant of swimming time was shortened, the times of wear ring decreased significantly (P <0.05 ).These results suggest that repeated, chronic seizures can cause the declining of cognitive function, learning and memory capacity. Edaravone can partly protect cognitive function.Conlusions1 In the epileptic model rats induced by KA, to observe the neuronal injury caused by the epileptic seizurs themselves and the changes by interventions, the more ideal parameter is that KA 4μg/kg (0.4μg/μl) was injected in one side of the hippocampal CA3 region, and the contralateral hippocampus was taken as the study object.2 In the epileptic model rats induced by KA, the severity of epileptic seizures of rats has a positive correlation with the the amount of KA; the damage degree of hippocampal neurons has a positive correlation with the the severity of epileptic seizures of rats.3 The neuronal injury induced by the severe epileptic seizures might be associated with the neuronal apoptosis and the increase in generation of free radical and the administration of edaravone could inhibit the occurrence of the event metioned above. 4 The long-term and recurring seizures can lead to the cognitive dysfunction in rats, and the application of edaravone could be effective in improving cognitive function in rats.