癫痫损伤机制的探讨及依达拉奉对海人酸致痫大鼠海马神经元的保护作用
|
摘要
癫痫(epilepsy)是一组由已知或未知病因所引起,脑部神经元高度同步化,且常具自限性的异常放电所导致,以反复发作性、短暂性、通常为刻板性的中枢神经系统功能失常为特征的综合征。癫痫反复发作,尤其是长时间全面强直-阵挛发作时,脑细胞严重缺氧,随着病程的延长、发作次数的增加,大脑神经元,尤其海马神经元,可发生变性、坏死,患者逐渐出现脑功能受损症状,如记忆力减退、学习能力下降等多种认知功能障碍,有学者研究发现30%～40%癫痫患者存在认知功能障碍,严重影响患者的生存质量。部分患者病情迁延成慢性癫痫。
目前癫痫的治疗主要靠药物控制痫性发作,抑制致痫区神经元的异常放电,从而减轻神经元的继发性损伤。另外,对于每次不可避免的痫性发作或癫痫持续状态而言,于癫痫发作后尽快采取有效的脑保护措施,将癫痫发作导致的神经元损伤程度降至最低,以延缓或抑制疾病的进展也是非常重要的。
大量的科学实验发现自由基与癫痫的发生、发展与治疗有着十分密切的关系。无论动物实验还是临床观察均表明:癫痫时伴有非常活跃的自由基活动。自由基通过膜的脂质过氧化以及减少致痫灶的GSH - PX水平,引起神经元变性。动物研究发现,癫痫发作后,其海马、皮层、下丘脑及垂体内超氧化物岐化酶(SOD)活性明显下降,而丙二醛(MDA)含量却明显升高,随着癫痫发作次数的增加,这种变化更加显著。
以往的研究发现,在癫痫发作后的脑组织中,与凋亡有关的各种基因表达增加,表明癫痫发作后神经元损伤与细胞凋亡密切相关。在诸多凋亡的诱因中,体内代谢或外源性因素产生的自由基均被证实可诱导细胞凋亡。可见,自由基在癫痫损伤的病理生理机制中发挥着重要的作用。因此清除自由基,阻断自由基反应可以抑制由于癫痫发作导致的细胞毒性反应,抑制细胞的凋亡和死亡,保护神经元,从而抑制病情的进展。
依达拉奉(edaravone)是一种新型自由基清除剂,它能够清除脑梗塞周围缺血半暗带羟自由基、抑制脂质过氧化,从而抑制脑细胞的损伤和凋亡,对脑血管病缺血缺氧导致的神经元损伤具有明显的保护作用。目前依达拉奉对癫痫损伤的保护作用尚未见报道。本实验制作海人酸(kainic acid,KA)癫痫模型,观察依达拉奉对癫痫大鼠海马组织形态学、神经元凋亡、自由基和脂质过氧化物的生成量、以及对慢性癫痫大鼠认知功能的影响,初步探讨依达拉奉的神经保护机制,同时为临床上癫痫的治疗提供新的途径和方法。
第一部分海人酸癫痫模型的制作
方法:
1动物模型
成年健康雄性Wistar大鼠18只,体重260±20g,实验动物随机分为2大组:⑴假手术组(sham)(n= 6)右侧海马CA3区注入与药物注射组等量的生理盐水;⑵模型组(KA)(n=12):依据发作级别和注射药量又进一步将其分为2个亚组:①K1组(n=6):右侧海马CA3区注入KA 2μg/kg(0.4μg/μl);②K2组(n=6):右侧海马CA3区注入KA 4μg/kg(0.4μg/μl)。K1组大鼠表现为II～III级者选6只,7天后行对侧海马组织学观察;K2组大鼠出现Ⅳ～Ⅴ级发作,持续时间约1.5h～2h者选6只,7天后亦行对侧海马组织学观察,模型制作过程中大鼠出现死亡或不符合模型要求者予以剔除并补充,以保证实验动物总数18只。
2脑电图
大鼠麻醉后固定于立体定向仪上,放置深部电极,待大鼠清醒后,按上述分组制作模型,描记脑电图。
3大鼠行为学观察
大鼠海马腹后部CA3区注射KA后,观察癫痫发作情况,并依据Racine六级癫痫发作评价标准,对KA致痫大鼠发作进行分级,按实验要求将大鼠分入K1和K2组。
4大鼠海马CA1、CA3区及门区(CA4)组织形态学观察
大鼠于注药或假手术后7天断头取脑,石蜡切片进行硫堇染色,光学显微镜下观察注药对侧(左侧)海马CA1、CA3区及门区组织形态,并对其组织学改变进行分级(0级:无神经元死亡;I级:散在神经元死亡;II级:成片的神经元死亡;III级:几乎全部的神经元死亡),取左侧等级作为统计值。高倍镜下计数海马CA1、CA3区每1mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目,每张切片取左侧海马计数3个区段平均数为神经元密度(Neuronaldensity,ND)。本实验取注射对侧海马作为研究对象。
结果
1大鼠行为学表现
sham组大鼠行为无异常。K1组共10只Wistar大鼠,右侧海马CA3区注入KA后其中有6只发作表现为Racine II～III级,3只为I级发作,1只为IV级发作,予以剔除。K2组共12只大鼠,均有癫痫发作,其中10只大鼠出现IV～V级发作,其中发作持续时间超过2h者,给予安定腹腔注射,发作终止2只,死亡4只,2只大鼠出现III级以下发作,不符合要求予以剔除。该组死亡率33%。
2脑电图大鼠发作期脑电图显示明显放电,为棘波、尖波、棘-慢综合波、尖-慢综合波,sham组大鼠脑电图无明显异常。
3大鼠海马CA1、CA3及门区(CA4)组织形态学改变
结果显示,硫堇染色后sham组和K1组(小剂量海人酸注射组)对侧海马无明显损伤,组织学分级多为0～I级,海马CA1、CA3区及门区细胞无缺失,排列整体,形态完整,胞核饱满,核仁清晰,尼氏体丰富,细胞有突起。ND值sham组CA1和CA3分别为198±20.62和212±30.14;K1组CA1和CA3分别为186±19.45和208±27.73。K2组大鼠海马神经元可见明显损伤,表现为CA1、CA3及门区细胞缺失、排列紊乱,胞质浓缩、深染,细胞形态有典型的多角形变为三角形或不规则型,残存的锥体细胞周围可见较多的细胞碎片,伴有胶质细胞浸润,齿状回无明显的神经元缺失。K2组组织学分级II～III级,CA1、CA3区ND值为79±13.72和90±14.98,与sham组、K1组相比,组织学分级显著升高(P<0.05),ND值显著降低(P<0.05)。表明癫痫发作越严重,脑组织损伤越严重。在本实验中,只有当癫痫发作程度达到一定的水平,才能引起对侧海马神经元的损伤。为了排除KA本身造成的神经元损伤,我们选择K2组KA注射对侧海马作为研究对象,观察KA致痫大鼠给予依达拉奉干预后其一系列的病理生理学变化。
第二部分癫痫损伤机制的探讨及依达拉奉对海人酸致痫大鼠海马神经元的保护作用
2.1依达拉奉对海人酸致痫大鼠海马神经元保护作用的组织学观察
方法
1动物模型
成年健康雄性Wistar大鼠18只,体重260±20g,实验动物随机分为3组:①假手术组(sham)(n= 6)右侧海马CA3区注入与药物注射组等量的生理盐水;②KA模型组(n=6):右侧海马CA3区注入KA 4μg/kg(0.4μg/μl)。③依达拉奉组(n=6):右侧海马CA3区注入KA 4μg/kg(0.4μg/μl),术后即刻给予依达拉奉8mg/kg腹腔注射,每日一次,直至处死的时间点为止。模型制作过程中大鼠出现死亡或不符合模型要求者予以剔除并补充,以保证实验动物总数18只。注药或假手术后观察各组大鼠的行为学表现,于7天断头取脑,石蜡切片进行硫堇染色,光学显微镜下观察注药对侧(左侧)海马CA1、CA3区及门区组织形态学特征。其余步骤同第一部分。
2大鼠海马CA1、CA3区及门区(CA4)组织形态学观察(方法同第一部分)
结果
1大鼠行为学表现
sham组大鼠无发作表现;KA模型组和依达拉奉注射组发作级别及潜伏期无明显差别(数据略)。
2大鼠海马CA1、CA3及门区(CA4)组织形态学改变
结果显示:sham组大鼠注射对侧海马CA1、CA3及门区无明显组织损伤(P>0.05),组织学分级多为0～I级,CA1、CA3区ND值分别为198±20.62和212±30.14;KA模型组大鼠CA1、CA3及门区可见明显的组织损伤,组织学分级多为II～III级,CA1、CA3区ND值为79±13.72和90±14.98,与sham组相比,组织学分级显著升高(P<0.05),ND值显著降低(P<0.01)。依达拉奉组大鼠海马CA1、CA3及门区可见少量、散在性神经元坏死,组织学分级多I～II级,CA1、CA3区ND值分别为101±16.85和135±22.17。与模型组相比,组织学分级显著降低(P<0.05),ND值显著升高(P<0.05),表明依达拉奉能够减轻KA致痫大鼠海马神经元的损伤,对神经元具有保护作用。
2.2依达拉奉抑制海人酸致痫大鼠海马神经元的凋亡过程
方法
成年健康雄性Wistar大鼠66只,体重260±20g,随机分为3组:①假手术组(sham)(n=6):方法同本部分2.1;②KA模型组(n=30):方法同本部分2.1;③依达拉奉组(n=30):方法同本部分2.1。模型组和依达拉奉组分别设4h、1d、3d、7d、14d 5个时间点,每个时间点6只大鼠,于预订的时间点断头取脑,硫堇染色,观察海马组织学分级及ND值,TUNEL染色观察海马神经元凋亡情况。
结果
1大鼠行为学表现及海马组织学特征:同本部分2.1节。
2 TUNEL染色结果
sham组大鼠注射对侧海马CA1、CA3及门区TUNEL染色未见或偶见阳性细胞(P>0.05),KA模型组TUNEL染色结果显示阳性细胞从术后1d开始增多,3d达高峰,7d回落,14d后阳性细胞消失,模型组大鼠癫痫发作后1～7天内TUNEL阳性细胞数与sham组相比明显增多,有显著性差异(P<0.05);依达拉奉组在这一时间内TUNEL阳性细胞数明显少于模型组(P<0.05),但仍高于sham组(P<0.05 )。以上结果提示:癫痫发作时可引起以凋亡为主的神经元损伤,依达拉奉能够部分抑制癫痫发作后的神经元凋亡。
2.3依达拉奉抑制海人酸致痫大鼠海马神经元脂质过氧化过程
方法
成年健康雄性Wistar大鼠66只,体重260±20g,随机分为3组:①假手术组(sham)(n=6):方法同本部分2.1;②模型组(n=30):方法同本部分2.1;③依达拉奉组(n=30):方法同本部分2.1,模型组和依达拉奉组分别设5min、6h、24h、72h、7d 5个时间点,每个时间点6只大鼠,于预订的时间点断头取脑,分离左侧海马,检测左侧海马组织中SOD活性和MDA含量。
结果
1大鼠行为学表现及海马组织学特征:同本部分2.1节。
2大鼠注药对侧海马SOD活性和MDA含量的测定结果结果显示:sham组各个时间点SOD活性及MDA含量均无明显变化(P>0.05);模型组于KA注射后6h可见SOD活性下降,MDA含量升高,于3d时SOD活性降至最低(P<0.01),MDA含量升至最高(P<0.01),随后SOD活性开始回升,MDA生成量开始下降,于7d时SOD活性恢复至基线水平;依达拉奉组于6h～72h时间段内SOD活性明显高于模型组(P<0.05),MDA含量明显低于模型组(P<0.05)。以上结果提示,依达拉奉可有效的提高SOD活性并降低MDA生成,从而提高机体的抗氧化能力,起到保护神经元的作用。
2.4依达拉奉对海人酸致痫大鼠认知功能的影响
方法
成年健康雄性Wistar大鼠30只,体重260±20g,随机分为3组:①假手术组(sham)(n=6):方法同本部分2.1;②模型组(n=12):方法同本部分2.1;③依达拉奉(n=12):方法同本部分2.1。以上三组大鼠分别于术后24h和8w行Morris水迷宫试验,进行认知功能和运动能力的检测。
结果
1定位航行试验(Place navigation test,PNT)逃避潜伏期各实验组大鼠于24h开始的PNT中,各时段逃避潜伏期无显著性差异(P>0.05),模型组、依达拉奉组在8w时开始的PNT中,与sham组相比大鼠各个时段的逃避潜伏期均明显延长,有显著性差异(P<0.05);依达拉奉组与模型组相比,大鼠逃避潜伏期明显缩短,且有显著性差异(P<0.05)。
2空间探索试验(Spatial probe test, SPT):在原平台象限中游泳时间的百分比和穿环数
各组大鼠在原有平台象限内游泳的时间明显高于其他象限;模型组和依达拉奉组在造模成功后24h开始的STP中,与sham组相比原平台象限内的游泳时间、穿环数均无显著性差异(P>0.05)。随着发作时间的延长,模型组和依达拉奉组大鼠于造模成功后8w开始的STP,与sham组相比原平台象限内的游泳时间明显缩短,穿环数明显减少,有显著性差异(P<0.05)。依达拉奉组与模型组相比,大鼠在原平台象限内的游泳时间明显延长,穿环数明显增多,有显著性差异(P<0.05)。
以上结果提示:长期、反复的慢性痫性发作可引起认知功能减退、学习记忆能力下降等表现,依达拉奉可部分改善由于癫痫发作引起的认知功能障碍。
结论
1在KA大鼠致痫模型中,观察癫痫发作本身所致的神经元损伤以及干预后的变化情况,较为理想的参数为4μg/kg (0.4μg/μl)KA一侧海马CA3区注射,将对侧海马作为研究对象。
2在KA大鼠致痫模型中,KA致痫大鼠癫痫发作严重程度与KA用量成正相关,大鼠海马神经元损伤程度与癫痫发作严重程度成正相关。
3严重的癫痫发作导致的神经元损伤可能与神经元凋亡及自由基生成量的增加有关,依达拉奉可部分抑制以上现象的发生。
4长期反复的癫痫发作可导致大鼠认知功能障碍,而依达拉奉可有效改善癫痫大鼠的认知功能。
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 rats
Eighteen 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 EEG
EEGs 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 rats
The 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 rats
All 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.
Results
1 The feature of behavior of rats
Sham-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 EEG
Rats 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 hippocampus
The 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 acid
2.1 The histological changes of protective effect of edaravone on hippocampal neurons in kainic acid-induced epileptic rats
Methods:
1 The establishment of kainic acid-induced epileptic model in rats
Eighteen 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)
Results
1 The feature of behavior of rats
Sham-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 hippocampus
The 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 rat
Sixty-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.
Results
1 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 staining
The 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 rats
Sixty-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 rats
The 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 rats
Methods
1 The establishment of kainic acid-induced epilepticmodel in rats
Thirty 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.
Results
1. Place navigation test(PNT)escape latency
All 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.
Conlusions
1 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.
目前癫痫的治疗主要靠药物控制痫性发作,抑制致痫区神经元的异常放电,从而减轻神经元的继发性损伤。另外,对于每次不可避免的痫性发作或癫痫持续状态而言,于癫痫发作后尽快采取有效的脑保护措施,将癫痫发作导致的神经元损伤程度降至最低,以延缓或抑制疾病的进展也是非常重要的。
大量的科学实验发现自由基与癫痫的发生、发展与治疗有着十分密切的关系。无论动物实验还是临床观察均表明:癫痫时伴有非常活跃的自由基活动。自由基通过膜的脂质过氧化以及减少致痫灶的GSH - PX水平,引起神经元变性。动物研究发现,癫痫发作后,其海马、皮层、下丘脑及垂体内超氧化物岐化酶(SOD)活性明显下降,而丙二醛(MDA)含量却明显升高,随着癫痫发作次数的增加,这种变化更加显著。
以往的研究发现,在癫痫发作后的脑组织中,与凋亡有关的各种基因表达增加,表明癫痫发作后神经元损伤与细胞凋亡密切相关。在诸多凋亡的诱因中,体内代谢或外源性因素产生的自由基均被证实可诱导细胞凋亡。可见,自由基在癫痫损伤的病理生理机制中发挥着重要的作用。因此清除自由基,阻断自由基反应可以抑制由于癫痫发作导致的细胞毒性反应,抑制细胞的凋亡和死亡,保护神经元,从而抑制病情的进展。
依达拉奉(edaravone)是一种新型自由基清除剂,它能够清除脑梗塞周围缺血半暗带羟自由基、抑制脂质过氧化,从而抑制脑细胞的损伤和凋亡,对脑血管病缺血缺氧导致的神经元损伤具有明显的保护作用。目前依达拉奉对癫痫损伤的保护作用尚未见报道。本实验制作海人酸(kainic acid,KA)癫痫模型,观察依达拉奉对癫痫大鼠海马组织形态学、神经元凋亡、自由基和脂质过氧化物的生成量、以及对慢性癫痫大鼠认知功能的影响,初步探讨依达拉奉的神经保护机制,同时为临床上癫痫的治疗提供新的途径和方法。
第一部分海人酸癫痫模型的制作
方法:
1动物模型
成年健康雄性Wistar大鼠18只,体重260±20g,实验动物随机分为2大组:⑴假手术组(sham)(n= 6)右侧海马CA3区注入与药物注射组等量的生理盐水;⑵模型组(KA)(n=12):依据发作级别和注射药量又进一步将其分为2个亚组:①K1组(n=6):右侧海马CA3区注入KA 2μg/kg(0.4μg/μl);②K2组(n=6):右侧海马CA3区注入KA 4μg/kg(0.4μg/μl)。K1组大鼠表现为II～III级者选6只,7天后行对侧海马组织学观察;K2组大鼠出现Ⅳ～Ⅴ级发作,持续时间约1.5h～2h者选6只,7天后亦行对侧海马组织学观察,模型制作过程中大鼠出现死亡或不符合模型要求者予以剔除并补充,以保证实验动物总数18只。
2脑电图
大鼠麻醉后固定于立体定向仪上,放置深部电极,待大鼠清醒后,按上述分组制作模型,描记脑电图。
3大鼠行为学观察
大鼠海马腹后部CA3区注射KA后,观察癫痫发作情况,并依据Racine六级癫痫发作评价标准,对KA致痫大鼠发作进行分级,按实验要求将大鼠分入K1和K2组。
4大鼠海马CA1、CA3区及门区(CA4)组织形态学观察
大鼠于注药或假手术后7天断头取脑,石蜡切片进行硫堇染色,光学显微镜下观察注药对侧(左侧)海马CA1、CA3区及门区组织形态,并对其组织学改变进行分级(0级:无神经元死亡;I级:散在神经元死亡;II级:成片的神经元死亡;III级:几乎全部的神经元死亡),取左侧等级作为统计值。高倍镜下计数海马CA1、CA3区每1mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目,每张切片取左侧海马计数3个区段平均数为神经元密度(Neuronaldensity,ND)。本实验取注射对侧海马作为研究对象。
结果
1大鼠行为学表现
sham组大鼠行为无异常。K1组共10只Wistar大鼠,右侧海马CA3区注入KA后其中有6只发作表现为Racine II～III级,3只为I级发作,1只为IV级发作,予以剔除。K2组共12只大鼠,均有癫痫发作,其中10只大鼠出现IV～V级发作,其中发作持续时间超过2h者,给予安定腹腔注射,发作终止2只,死亡4只,2只大鼠出现III级以下发作,不符合要求予以剔除。该组死亡率33%。
2脑电图大鼠发作期脑电图显示明显放电,为棘波、尖波、棘-慢综合波、尖-慢综合波,sham组大鼠脑电图无明显异常。
3大鼠海马CA1、CA3及门区(CA4)组织形态学改变
结果显示,硫堇染色后sham组和K1组(小剂量海人酸注射组)对侧海马无明显损伤,组织学分级多为0～I级,海马CA1、CA3区及门区细胞无缺失,排列整体,形态完整,胞核饱满,核仁清晰,尼氏体丰富,细胞有突起。ND值sham组CA1和CA3分别为198±20.62和212±30.14;K1组CA1和CA3分别为186±19.45和208±27.73。K2组大鼠海马神经元可见明显损伤,表现为CA1、CA3及门区细胞缺失、排列紊乱,胞质浓缩、深染,细胞形态有典型的多角形变为三角形或不规则型,残存的锥体细胞周围可见较多的细胞碎片,伴有胶质细胞浸润,齿状回无明显的神经元缺失。K2组组织学分级II～III级,CA1、CA3区ND值为79±13.72和90±14.98,与sham组、K1组相比,组织学分级显著升高(P<0.05),ND值显著降低(P<0.05)。表明癫痫发作越严重,脑组织损伤越严重。在本实验中,只有当癫痫发作程度达到一定的水平,才能引起对侧海马神经元的损伤。为了排除KA本身造成的神经元损伤,我们选择K2组KA注射对侧海马作为研究对象,观察KA致痫大鼠给予依达拉奉干预后其一系列的病理生理学变化。
第二部分癫痫损伤机制的探讨及依达拉奉对海人酸致痫大鼠海马神经元的保护作用
2.1依达拉奉对海人酸致痫大鼠海马神经元保护作用的组织学观察
方法
1动物模型
成年健康雄性Wistar大鼠18只,体重260±20g,实验动物随机分为3组:①假手术组(sham)(n= 6)右侧海马CA3区注入与药物注射组等量的生理盐水;②KA模型组(n=6):右侧海马CA3区注入KA 4μg/kg(0.4μg/μl)。③依达拉奉组(n=6):右侧海马CA3区注入KA 4μg/kg(0.4μg/μl),术后即刻给予依达拉奉8mg/kg腹腔注射,每日一次,直至处死的时间点为止。模型制作过程中大鼠出现死亡或不符合模型要求者予以剔除并补充,以保证实验动物总数18只。注药或假手术后观察各组大鼠的行为学表现,于7天断头取脑,石蜡切片进行硫堇染色,光学显微镜下观察注药对侧(左侧)海马CA1、CA3区及门区组织形态学特征。其余步骤同第一部分。
2大鼠海马CA1、CA3区及门区(CA4)组织形态学观察(方法同第一部分)
结果
1大鼠行为学表现
sham组大鼠无发作表现;KA模型组和依达拉奉注射组发作级别及潜伏期无明显差别(数据略)。
2大鼠海马CA1、CA3及门区(CA4)组织形态学改变
结果显示:sham组大鼠注射对侧海马CA1、CA3及门区无明显组织损伤(P>0.05),组织学分级多为0～I级,CA1、CA3区ND值分别为198±20.62和212±30.14;KA模型组大鼠CA1、CA3及门区可见明显的组织损伤,组织学分级多为II～III级,CA1、CA3区ND值为79±13.72和90±14.98,与sham组相比,组织学分级显著升高(P<0.05),ND值显著降低(P<0.01)。依达拉奉组大鼠海马CA1、CA3及门区可见少量、散在性神经元坏死,组织学分级多I～II级,CA1、CA3区ND值分别为101±16.85和135±22.17。与模型组相比,组织学分级显著降低(P<0.05),ND值显著升高(P<0.05),表明依达拉奉能够减轻KA致痫大鼠海马神经元的损伤,对神经元具有保护作用。
2.2依达拉奉抑制海人酸致痫大鼠海马神经元的凋亡过程
方法
成年健康雄性Wistar大鼠66只,体重260±20g,随机分为3组:①假手术组(sham)(n=6):方法同本部分2.1;②KA模型组(n=30):方法同本部分2.1;③依达拉奉组(n=30):方法同本部分2.1。模型组和依达拉奉组分别设4h、1d、3d、7d、14d 5个时间点,每个时间点6只大鼠,于预订的时间点断头取脑,硫堇染色,观察海马组织学分级及ND值,TUNEL染色观察海马神经元凋亡情况。
结果
1大鼠行为学表现及海马组织学特征:同本部分2.1节。
2 TUNEL染色结果
sham组大鼠注射对侧海马CA1、CA3及门区TUNEL染色未见或偶见阳性细胞(P>0.05),KA模型组TUNEL染色结果显示阳性细胞从术后1d开始增多,3d达高峰,7d回落,14d后阳性细胞消失,模型组大鼠癫痫发作后1～7天内TUNEL阳性细胞数与sham组相比明显增多,有显著性差异(P<0.05);依达拉奉组在这一时间内TUNEL阳性细胞数明显少于模型组(P<0.05),但仍高于sham组(P<0.05 )。以上结果提示:癫痫发作时可引起以凋亡为主的神经元损伤,依达拉奉能够部分抑制癫痫发作后的神经元凋亡。
2.3依达拉奉抑制海人酸致痫大鼠海马神经元脂质过氧化过程
方法
成年健康雄性Wistar大鼠66只,体重260±20g,随机分为3组:①假手术组(sham)(n=6):方法同本部分2.1;②模型组(n=30):方法同本部分2.1;③依达拉奉组(n=30):方法同本部分2.1,模型组和依达拉奉组分别设5min、6h、24h、72h、7d 5个时间点,每个时间点6只大鼠,于预订的时间点断头取脑,分离左侧海马,检测左侧海马组织中SOD活性和MDA含量。
结果
1大鼠行为学表现及海马组织学特征:同本部分2.1节。
2大鼠注药对侧海马SOD活性和MDA含量的测定结果结果显示:sham组各个时间点SOD活性及MDA含量均无明显变化(P>0.05);模型组于KA注射后6h可见SOD活性下降,MDA含量升高,于3d时SOD活性降至最低(P<0.01),MDA含量升至最高(P<0.01),随后SOD活性开始回升,MDA生成量开始下降,于7d时SOD活性恢复至基线水平;依达拉奉组于6h～72h时间段内SOD活性明显高于模型组(P<0.05),MDA含量明显低于模型组(P<0.05)。以上结果提示,依达拉奉可有效的提高SOD活性并降低MDA生成,从而提高机体的抗氧化能力,起到保护神经元的作用。
2.4依达拉奉对海人酸致痫大鼠认知功能的影响
方法
成年健康雄性Wistar大鼠30只,体重260±20g,随机分为3组:①假手术组(sham)(n=6):方法同本部分2.1;②模型组(n=12):方法同本部分2.1;③依达拉奉(n=12):方法同本部分2.1。以上三组大鼠分别于术后24h和8w行Morris水迷宫试验,进行认知功能和运动能力的检测。
结果
1定位航行试验(Place navigation test,PNT)逃避潜伏期各实验组大鼠于24h开始的PNT中,各时段逃避潜伏期无显著性差异(P>0.05),模型组、依达拉奉组在8w时开始的PNT中,与sham组相比大鼠各个时段的逃避潜伏期均明显延长,有显著性差异(P<0.05);依达拉奉组与模型组相比,大鼠逃避潜伏期明显缩短,且有显著性差异(P<0.05)。
2空间探索试验(Spatial probe test, SPT):在原平台象限中游泳时间的百分比和穿环数
各组大鼠在原有平台象限内游泳的时间明显高于其他象限;模型组和依达拉奉组在造模成功后24h开始的STP中,与sham组相比原平台象限内的游泳时间、穿环数均无显著性差异(P>0.05)。随着发作时间的延长,模型组和依达拉奉组大鼠于造模成功后8w开始的STP,与sham组相比原平台象限内的游泳时间明显缩短,穿环数明显减少,有显著性差异(P<0.05)。依达拉奉组与模型组相比,大鼠在原平台象限内的游泳时间明显延长,穿环数明显增多,有显著性差异(P<0.05)。
以上结果提示:长期、反复的慢性痫性发作可引起认知功能减退、学习记忆能力下降等表现,依达拉奉可部分改善由于癫痫发作引起的认知功能障碍。
结论
1在KA大鼠致痫模型中,观察癫痫发作本身所致的神经元损伤以及干预后的变化情况,较为理想的参数为4μg/kg (0.4μg/μl)KA一侧海马CA3区注射,将对侧海马作为研究对象。
2在KA大鼠致痫模型中,KA致痫大鼠癫痫发作严重程度与KA用量成正相关,大鼠海马神经元损伤程度与癫痫发作严重程度成正相关。
3严重的癫痫发作导致的神经元损伤可能与神经元凋亡及自由基生成量的增加有关,依达拉奉可部分抑制以上现象的发生。
4长期反复的癫痫发作可导致大鼠认知功能障碍,而依达拉奉可有效改善癫痫大鼠的认知功能。
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 rats
Eighteen 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 EEG
EEGs 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 rats
The 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 rats
All 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.
Results
1 The feature of behavior of rats
Sham-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 EEG
Rats 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 hippocampus
The 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 acid
2.1 The histological changes of protective effect of edaravone on hippocampal neurons in kainic acid-induced epileptic rats
Methods:
1 The establishment of kainic acid-induced epileptic model in rats
Eighteen 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)
Results
1 The feature of behavior of rats
Sham-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 hippocampus
The 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 rat
Sixty-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.
Results
1 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 staining
The 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 rats
Sixty-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 rats
The 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 rats
Methods
1 The establishment of kainic acid-induced epilepticmodel in rats
Thirty 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.
Results
1. Place navigation test(PNT)escape latency
All 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.
Conlusions
1 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.
引文
1 Douglas AC, Dan CM, Wolfgang L Animal Models of Limbic Epilepsies: What Can They Us? Brain Pathology 2002: 12: 240~56
2 孙艺平 ,张万琴.见:王尧 ,杜子威主编.神经生物化学与 分子生物学.北京:人民卫生出版社 ,1997:415425
3 Loscher W, Animal models of intractable epilepsy. Prog Neurobiol, 1997; 53: 239 ~ 258
4 包新民,舒斯云.大鼠脑立体定位图谱 [M ].第 1 版. 北京:人民卫生出版社,1991. 36
5 RacineIU. Modification of seizure activity by electrieal stirnulation-11, Motor seizures. Electroencephalog Clin Nerophysiol, 1972, 32~281
6 Kato H, Liu Y, Araki T, et al. Temporal profile of the effects of pretreatment with brief cerebral ischemic on the neuronal damage following secondary ischemic insult in the gerbil:cumulative damage and protective effects.Brain Res, 1991, 553: 238~242
7 Thomas PS, Joshuaa H, Aslab P. Do epileptic seizures damage the brain[J]? CurrentOpinion Neurology, 2003, 16,189
8 Dawson RJr, Wallace DR. Kainic acid-induced seizures in aged rats: neurochemical correlates [J]. Brain Res Bull, 1992, 29 (3 - 4): 459~ 468
9 Magloczky Z, Freund TF. Delayed cell death in the contralateral hippocampus following kainite injection into the CA3 subfield. Neuroscience 1995; 66: 847~60
10 Berger M, Ben Ari Y. Auto radiographic visualization of 3H kainic acid receptor subtypes in the rat hippocampus. Neuro sciL ett, 1983, 39: 237
11 CavalheiroEA,LeiteJP Bortolotto ZA,et al.Long- term effects of pilocarpine in rats:struetural damage of the brain triggers kindlingand spontaneous recurrent seizures. Epilepsia, 1991, 32: 778~782
12 E.A.Cavalheiro,D.A. Riche,G. LE GAL Salle. Long-term effects of intrhippocampal kainic acid injection in rats:a method for inducing spontaneous recurrent seizures. Electroencephalography and Clinical Neurophysiology, 1982, 53: 581~589
13 Sloviter RS. "Epileptic”brain damage in rats induced by sustained electrical stimulation of the perforant pathway:electrophysiological and light microscopic studies. Brain Res, 1983, 10:675
14 Nadler JV. Kainci acid as a tool for the study of temperal lobe epilepsy. Life Sci, 1991, 29: 2031
15 Represa A, T remblay E, Nen-A ri Y.Kainate binding sites in the hippocampal mossy fibers: localization and plasticity.Neuro sci, 1987, 20: 739
16 Olney JW, De Gubraeff T. "Epileptic”brain damage in rats induced by sustained electrical stimulation of the perforant path.II. Ultrastructural analysis of acute hippocampal pathology. Brain Res Bull, 1983, 10: 69
17 Gary WM, J ames KP, Thomas LB. Quantified pat terns of mossy fiber sprouting and neuron densites in hippocampal and lesional seizures[J] . Neurosurg , 1995, 82: 211
18 Cavazos JE, Zhang P, Qazi R, et al. Ultrastructural features of sprouted mossy fiber synapses in kindled and kainic acid-treated rats [J]. CompNeurol, 2003, 458(3): 272~292.
19 Sutula TP, Golarai G, Cavazos J. Asessing the functional significance of mossy fiber sprouting in the dentate gyrus and its role in seizures. (Epilepsy Res.) Supp l. 1992: 251~259
20 Okazaki MM, Evenson DA, Nadler JV. Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats:Visualization after retrograde transport of biocytin. [J]. Comp Neurol, 1993, 352: 51530
21 Berger ML. Lassmann H, Homykiewicz O. Limbic seizures without brain damage after injection of low doses kainic acid into amygdala of freely moving rats. Brain Res, 1989, 489:261
22 Nadler JV, Okazaki MM. Protective effect s of mossy fiber lesions against kainic acid - induced seizures and neuronal degeneration [J] . Neuroscience, 1988, 26: 763
23 Olney JW, Fuller T, deGubareff T, et al. Acute dendro toxic changes in the hippocampus of kainite-treated rats. Brain Res, 1979, 176: 91
1 Frantseva M V, Perez Velazquez J L, Hwang P A, et al. Free radical production correlates with death-in an vitro model of epilepsy[J].Eur Neurosci, 2000, 12 (4): 1431~1439
2 Costello D J, Delanty N. Oxidative injury in epilepsy: potential for and tioxidant therapy? [J] . Expert Rev Neurother , 2004, 4( 3 ) : 541~553
3 Ilhan A, Aladag M A, Kocer A, et al. Erdosteine ameliorates PTZ induced oxidative stress in mice seizure model [J] . Brain Res Bull, 2005, 65 (6) :495~499
4 Wojtal K, Borowicz K K, Blaszczyk B, et al. Interactions of excitatory amino acid receptor antagonists with antiepileptic drugs in three basic models of experimental epilepsy[J] .Pharmacol Rep, 2006, 58 (5) :587~598
5 Buttke TM,Sandstrom PA. Oxidative Stress as a mediator of apoptosis. Immunol Today, 1994, 15:7
6 Olney JW. Science, 1969, 164:719~721
7 Takei N, Endo Y. J Brain Res. 1994, 652:65~70
8 Miyashita T, Reed JC. Cell 1995, 80: 293~299
9 Miyashita JC, Green DG. Nat. Ren. Mol. Cell Biol. 2001,2:63~67
10 Zamzami N, Kroemer G, Rev.Mol. Cell Biol. 2002, 2 (1):67~71
11 Miyashit T, Reed JC, Cell 1995, 80:293~299
12 Slee EA, Keogh SA, Martin SJ. Cleavage of BID during cytotoxic drug and UV radiation induced apoptosis occurs downstream of the point of Bcl -2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated J. Cell-Death Differ, 2000, 7(6): 556~ 565
13 GrahamSH, ChenJun, ClarkR5. Bel- 2 family gene cerebral ischemia and traumatic brain injury [J]. JNeurot rauma 2000; 17(10): 831~841
14 Migita K, Yamasaki S, Kita M, et al. Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3 [J] . Immunology, 2001, 1032(3) : 362~367
15 LeeMC, RhoJL, Kim MK, et al. e-JUN expression and apoptotic eell death in kainite-induced temporal lobeepilepsy. [J]. Korean Med Sci. 2001 Oct;16(5): 649~56
16 Harris MH, Thompson CB. The role of the Bel-2 family in theregulation of outer mitochondrial membrane permeability. Cell Death Differ, 2000, 7: 1182~1191
17 Costello DJ, Delanty N. Oxidative injury in epilepsy: potential for antioxidant therapy? [J]. Expert Rev Neurother, 2004, 4 (3) : 541 ~553
18 Martinez- Ballesteros C, Pita - Calandre E, Sanchez - Gonzalez Y, et al. Lipid peroxidation in adult epileptic patients treated with valproic acid [J ]. Rev Neurol, 2004, 38 (2) :101 ~106
19 rantseva M V, Perez Velazquez J L, Hwang P A, et al. Free Radical production correlates with death- in an vitro model of epilepsy [J]. Eur Neurosci, 2000, 12(4) :1431 ~1439
20 Frantseva M V, Perez Velazquez J L, Tsoraklidis G, et al. Oxidative stress is involved in seizure-induced neurodegeneration in the kindling model of epilepsy [J] . Neuroscience, 2000, 97 (3) : 431 ~435
21 Wojtal K, Borowicz K K, Blaszczyk B, et al. Interactionsof excitatory amino acid receptor antagonists with antiepileptic drugs in three basic models of experimental epilepsy [J] . Pharmacol Rep, 2006, 58(5): 58~ 598
22 Grieb P, Stelmasiak Z, Re jdak K, et al. The mechanisms of neuronal death and advances in the neural protection in epilepsy [J] . Neurol Neurochir Pol, 2000, 34: 85 ~93
23 关中晖, 关汉明.认知, 精神.见关汉明主编.医学神经生物学[M].第一版.北京:人民卫生出版社, 2002: 348~361
24 Beck H, Goussakov IV, Lie A,et al. Synaptic plasticity in the human dentategyurs [J]. Neurosci, 2000, 20(18): 7080~7086
25 Renaud J, Emond M, Meillcur S, et al. AIDA, a class metabotropic glutamate-receptor antagonist limits kainite-induced hippocampal dysufnetion [J]. Epilepsia,2002, 43(11): 1306~1317
26 Niikura Y,Abe K, Misawa M. involvement of-Ltype Ca2+ channels in the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway of anestetized rats [J]. Brain Res, 2004, 1017(1-2): 218~221
27 王怀星, 姚志彬, 顾耀铭等. 老年性记忆减退大鼠空间记忆和搜索策略的改变 [J]. 神经解剖学杂, 2000,16(1): 57~60
28 Lamberty Y, Kitgaard H.Consequences of pentylenetetrazole kinding on spatial memory and emotional responding in the rat [J]. Epilepsy&Behav, 2000, (1): 256~261
1 Douglas AC, Dan CM, Wolfgang L Animal Models of Limbic Epilepsies: What Can They Us? Brain Pathology 2002: 12: 240~256
2 Loscher W, Animal models of intractable epilepsy. Prog Neurobiol, 1997, 53: 239 ~258
3 Ishikawa, -M; Mizukami, -K; Iwakiri, -M; et al. Alterations of heterogeneous nuclear RNP A2 and B1 in the hippocampus of the rat after perforant pathway lesion. Acta-Neuropathol-(Berl). 2004 Feb; 107(2): 144~148
4 van-der-Beek,-E-M;Wiegant,-V-M;Schouten,-W-G;etal.Neuronal number, volume,and apoptosis of the left dentate gyrus of chronically stressed pigs correlate negatively with basal saliva cortisol levels. Hippocampus. 2004; 14(6): 688~700
5 Siew,-S.TI:Scanning electron microscopy of the human hippocampus. Scan-Electron-Microsc.1983; (Pt 1):171~81
6 Engber,-T-M; Dennis,-S-A; Jones,-B-E; et al. Brain regional substrates for the actions of the novel wake-promoting agent modafinil in the rat: comparison with amphetamine. Neuroscience. 1998 Dec; 87(4): 905~911
7 Naber,-P-A; Witter, -M-P.TI: Subicular efferents are organized mostly as parallel projections: a double-labeling, retrograde-tracing study in the rat. J-Comp-Neurol. 1998 Apr 13; 393(3): 284~297
8 Y. Ben-Ari. Limbic seizure and brian damage produced bykainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience,1985,14 (2) :375~405
9 E.A. Cavalheiro,D.A. Riche, G. LE GAL Salle. Long- term effects of intrahippocampal kainic acid injection in rats: a method for inducing spontaneous recurrent seizures. Electroencephalography and Clinical NeuroPhysiology,1982, 53:581~589
10 Gary W. Mathern, Fredi Cifuentes,Joao P. Leite,et al. Hippocampal EEG excitability and chronic spontaneous seizures are associated with aberrant synaptic reorganization in the rat intrahippocampal kainite model. Electroencephalography and Clinical Neurophsiology,1993,87:326~339
11 RacineIU. Modification of seizure activity by electrieal stirnulation-11 , Motor seizures. Electroencephalog Clin Nerophysiol ,1972, 232~281
12 李冬冬, 隋鸿锦, 宫瑾, 等. 海马硬化对癫痫敏感性的影响.中国应用生理学杂志, 2000; 16 (2) :107
13 Okazaki MM, Molnar P, Nadler J Y. Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currnts evoked in presence and absence of seizure induced growth. J Neuro-physiol ,1999; 81(4) :1645~ 1660
14 Ben A Y. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy [J] . Neuroscience ,1985 ,14(2): 375
15 Nadler JV, Okazaki MM. Protective effect s of mossy fiberlesions against kainic acid - induced seizures and neuronal degeneration [J] . Neuroscience , 1988, 26: 763
16 Gary WM, J ames KP, Thomas LB. Quantified pat terns of mossy fiber sprouting and neuron densites in hippocampal and lesional seizures [J] . J Neurosurg , 1995 ,82: 211
17 Yin HZ, Sensi SL, Ogoshi F, et al. Blockade of Ca2 + - permeable AMPA/ kainite channels decreases oxygen - glucose deprivation -induced Zn2 + accumulation and neuronal loss in hippocampal pyramidal neurons [J] . J Neurosci , 2002 ,22(4) :1273
18 Lee MC, Rho JL, Woo YJ, et al. c - J UN expression and apoptotic cell death in kainite - induced temporal lobe epilepsy [J ] . J Korean Med Sci , 2001 ,16(5) :649
19 Pollard H, Charriaut C, Cantagel S, et al. Kainate - induced apoptotic cell deat h in hippocampal neurons [J] . Neuroscience ,1994 ,63: 7
20 David C, Henshall, J un C, et al . Involvement of caspase - 3 -like protease in the mechanism of cell deat h following focally evoked limbic seizures[J] . J Neurochemistry, 2000, 74(3) :1215
21 Gary WM, Cif uentes F, Joao PL, et al. Hippocampal EEG excitability and chronic spontaneous seizures are associated wit haberrant synaptic reorganization in the rat int -rahippocampal kainite model [J].Electroencephalography and Clinical Neurophysiology, 1993 ,87: 326
22 Proper EA, Oest reicher AB, J ansen GH, et al .Immunohisto-chemical characterization of mossy fibre sprouting in the hippocampus of patients with pharmaco- resistant temporal lobe epilepsy[J] . Brain , 2000 ,123 :19
23 Okazaki MM , Molnar P, Nadler JV. Recurrent mossy fiber pathway in rat dentate gyrus : synaptic currents evoked in prsence and absence of seizure induced growth[J] . J Neurophysiol , 1999 ,81(4) :1645
24 Represa A, Pollard H, Moreau J, et al. Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of alpha - tubulin[J] . Neurosci Let t , 1993 ,156(1 - 2) :149
25 Pollard H, Khrestchatisky M, Moreau J, et al . Correlation between reactive sprouting and micro tubule protein expression in epileptic hippocampus[J] . Neuroscience,1994, 61(4) :773
26 Reeben M, Lauurikainen A, Hilt unen JO, et al. The messen-gener RNAs for both glial cell line derived neurot rophic factor receptors. C - ret and GDNFRα are induced in the rat brain in response to kainite - induced excitation[J] . Neuroscience ,1998 ,83(1) :151
27 朱长庚,李龄,魏瑛,等.癫痫病人手术切除标本的组织学、免 疫 组 织 化 学 和 超 微 结 构 研 究 . 解 剖 学 报 ,1997 ,28(3):225~229
28 Bertolino M, Llinas RR. The central role of voltage-activited and receptor operated calcium channels in neuronal cells. Annu Rev pharmacol Toxicol,1992, 32:399~421
29 Simatov R,Crispino M,Hoe W,et al. Changes in expression of neuronal and glial gluta mate transporters in rat hippocampus following kainite induced seisure activity. Brain Res, 1999, 65(l):112~116
2 孙艺平 ,张万琴.见:王尧 ,杜子威主编.神经生物化学与 分子生物学.北京:人民卫生出版社 ,1997:415425
3 Loscher W, Animal models of intractable epilepsy. Prog Neurobiol, 1997; 53: 239 ~ 258
4 包新民,舒斯云.大鼠脑立体定位图谱 [M ].第 1 版. 北京:人民卫生出版社,1991. 36
5 RacineIU. Modification of seizure activity by electrieal stirnulation-11, Motor seizures. Electroencephalog Clin Nerophysiol, 1972, 32~281
6 Kato H, Liu Y, Araki T, et al. Temporal profile of the effects of pretreatment with brief cerebral ischemic on the neuronal damage following secondary ischemic insult in the gerbil:cumulative damage and protective effects.Brain Res, 1991, 553: 238~242
7 Thomas PS, Joshuaa H, Aslab P. Do epileptic seizures damage the brain[J]? CurrentOpinion Neurology, 2003, 16,189
8 Dawson RJr, Wallace DR. Kainic acid-induced seizures in aged rats: neurochemical correlates [J]. Brain Res Bull, 1992, 29 (3 - 4): 459~ 468
9 Magloczky Z, Freund TF. Delayed cell death in the contralateral hippocampus following kainite injection into the CA3 subfield. Neuroscience 1995; 66: 847~60
10 Berger M, Ben Ari Y. Auto radiographic visualization of 3H kainic acid receptor subtypes in the rat hippocampus. Neuro sciL ett, 1983, 39: 237
11 CavalheiroEA,LeiteJP Bortolotto ZA,et al.Long- term effects of pilocarpine in rats:struetural damage of the brain triggers kindlingand spontaneous recurrent seizures. Epilepsia, 1991, 32: 778~782
12 E.A.Cavalheiro,D.A. Riche,G. LE GAL Salle. Long-term effects of intrhippocampal kainic acid injection in rats:a method for inducing spontaneous recurrent seizures. Electroencephalography and Clinical Neurophysiology, 1982, 53: 581~589
13 Sloviter RS. "Epileptic”brain damage in rats induced by sustained electrical stimulation of the perforant pathway:electrophysiological and light microscopic studies. Brain Res, 1983, 10:675
14 Nadler JV. Kainci acid as a tool for the study of temperal lobe epilepsy. Life Sci, 1991, 29: 2031
15 Represa A, T remblay E, Nen-A ri Y.Kainate binding sites in the hippocampal mossy fibers: localization and plasticity.Neuro sci, 1987, 20: 739
16 Olney JW, De Gubraeff T. "Epileptic”brain damage in rats induced by sustained electrical stimulation of the perforant path.II. Ultrastructural analysis of acute hippocampal pathology. Brain Res Bull, 1983, 10: 69
17 Gary WM, J ames KP, Thomas LB. Quantified pat terns of mossy fiber sprouting and neuron densites in hippocampal and lesional seizures[J] . Neurosurg , 1995, 82: 211
18 Cavazos JE, Zhang P, Qazi R, et al. Ultrastructural features of sprouted mossy fiber synapses in kindled and kainic acid-treated rats [J]. CompNeurol, 2003, 458(3): 272~292.
19 Sutula TP, Golarai G, Cavazos J. Asessing the functional significance of mossy fiber sprouting in the dentate gyrus and its role in seizures. (Epilepsy Res.) Supp l. 1992: 251~259
20 Okazaki MM, Evenson DA, Nadler JV. Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats:Visualization after retrograde transport of biocytin. [J]. Comp Neurol, 1993, 352: 51530
21 Berger ML. Lassmann H, Homykiewicz O. Limbic seizures without brain damage after injection of low doses kainic acid into amygdala of freely moving rats. Brain Res, 1989, 489:261
22 Nadler JV, Okazaki MM. Protective effect s of mossy fiber lesions against kainic acid - induced seizures and neuronal degeneration [J] . Neuroscience, 1988, 26: 763
23 Olney JW, Fuller T, deGubareff T, et al. Acute dendro toxic changes in the hippocampus of kainite-treated rats. Brain Res, 1979, 176: 91
1 Frantseva M V, Perez Velazquez J L, Hwang P A, et al. Free radical production correlates with death-in an vitro model of epilepsy[J].Eur Neurosci, 2000, 12 (4): 1431~1439
2 Costello D J, Delanty N. Oxidative injury in epilepsy: potential for and tioxidant therapy? [J] . Expert Rev Neurother , 2004, 4( 3 ) : 541~553
3 Ilhan A, Aladag M A, Kocer A, et al. Erdosteine ameliorates PTZ induced oxidative stress in mice seizure model [J] . Brain Res Bull, 2005, 65 (6) :495~499
4 Wojtal K, Borowicz K K, Blaszczyk B, et al. Interactions of excitatory amino acid receptor antagonists with antiepileptic drugs in three basic models of experimental epilepsy[J] .Pharmacol Rep, 2006, 58 (5) :587~598
5 Buttke TM,Sandstrom PA. Oxidative Stress as a mediator of apoptosis. Immunol Today, 1994, 15:7
6 Olney JW. Science, 1969, 164:719~721
7 Takei N, Endo Y. J Brain Res. 1994, 652:65~70
8 Miyashita T, Reed JC. Cell 1995, 80: 293~299
9 Miyashita JC, Green DG. Nat. Ren. Mol. Cell Biol. 2001,2:63~67
10 Zamzami N, Kroemer G, Rev.Mol. Cell Biol. 2002, 2 (1):67~71
11 Miyashit T, Reed JC, Cell 1995, 80:293~299
12 Slee EA, Keogh SA, Martin SJ. Cleavage of BID during cytotoxic drug and UV radiation induced apoptosis occurs downstream of the point of Bcl -2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated J. Cell-Death Differ, 2000, 7(6): 556~ 565
13 GrahamSH, ChenJun, ClarkR5. Bel- 2 family gene cerebral ischemia and traumatic brain injury [J]. JNeurot rauma 2000; 17(10): 831~841
14 Migita K, Yamasaki S, Kita M, et al. Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3 [J] . Immunology, 2001, 1032(3) : 362~367
15 LeeMC, RhoJL, Kim MK, et al. e-JUN expression and apoptotic eell death in kainite-induced temporal lobeepilepsy. [J]. Korean Med Sci. 2001 Oct;16(5): 649~56
16 Harris MH, Thompson CB. The role of the Bel-2 family in theregulation of outer mitochondrial membrane permeability. Cell Death Differ, 2000, 7: 1182~1191
17 Costello DJ, Delanty N. Oxidative injury in epilepsy: potential for antioxidant therapy? [J]. Expert Rev Neurother, 2004, 4 (3) : 541 ~553
18 Martinez- Ballesteros C, Pita - Calandre E, Sanchez - Gonzalez Y, et al. Lipid peroxidation in adult epileptic patients treated with valproic acid [J ]. Rev Neurol, 2004, 38 (2) :101 ~106
19 rantseva M V, Perez Velazquez J L, Hwang P A, et al. Free Radical production correlates with death- in an vitro model of epilepsy [J]. Eur Neurosci, 2000, 12(4) :1431 ~1439
20 Frantseva M V, Perez Velazquez J L, Tsoraklidis G, et al. Oxidative stress is involved in seizure-induced neurodegeneration in the kindling model of epilepsy [J] . Neuroscience, 2000, 97 (3) : 431 ~435
21 Wojtal K, Borowicz K K, Blaszczyk B, et al. Interactionsof excitatory amino acid receptor antagonists with antiepileptic drugs in three basic models of experimental epilepsy [J] . Pharmacol Rep, 2006, 58(5): 58~ 598
22 Grieb P, Stelmasiak Z, Re jdak K, et al. The mechanisms of neuronal death and advances in the neural protection in epilepsy [J] . Neurol Neurochir Pol, 2000, 34: 85 ~93
23 关中晖, 关汉明.认知, 精神.见关汉明主编.医学神经生物学[M].第一版.北京:人民卫生出版社, 2002: 348~361
24 Beck H, Goussakov IV, Lie A,et al. Synaptic plasticity in the human dentategyurs [J]. Neurosci, 2000, 20(18): 7080~7086
25 Renaud J, Emond M, Meillcur S, et al. AIDA, a class metabotropic glutamate-receptor antagonist limits kainite-induced hippocampal dysufnetion [J]. Epilepsia,2002, 43(11): 1306~1317
26 Niikura Y,Abe K, Misawa M. involvement of-Ltype Ca2+ channels in the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway of anestetized rats [J]. Brain Res, 2004, 1017(1-2): 218~221
27 王怀星, 姚志彬, 顾耀铭等. 老年性记忆减退大鼠空间记忆和搜索策略的改变 [J]. 神经解剖学杂, 2000,16(1): 57~60
28 Lamberty Y, Kitgaard H.Consequences of pentylenetetrazole kinding on spatial memory and emotional responding in the rat [J]. Epilepsy&Behav, 2000, (1): 256~261
1 Douglas AC, Dan CM, Wolfgang L Animal Models of Limbic Epilepsies: What Can They Us? Brain Pathology 2002: 12: 240~256
2 Loscher W, Animal models of intractable epilepsy. Prog Neurobiol, 1997, 53: 239 ~258
3 Ishikawa, -M; Mizukami, -K; Iwakiri, -M; et al. Alterations of heterogeneous nuclear RNP A2 and B1 in the hippocampus of the rat after perforant pathway lesion. Acta-Neuropathol-(Berl). 2004 Feb; 107(2): 144~148
4 van-der-Beek,-E-M;Wiegant,-V-M;Schouten,-W-G;etal.Neuronal number, volume,and apoptosis of the left dentate gyrus of chronically stressed pigs correlate negatively with basal saliva cortisol levels. Hippocampus. 2004; 14(6): 688~700
5 Siew,-S.TI:Scanning electron microscopy of the human hippocampus. Scan-Electron-Microsc.1983; (Pt 1):171~81
6 Engber,-T-M; Dennis,-S-A; Jones,-B-E; et al. Brain regional substrates for the actions of the novel wake-promoting agent modafinil in the rat: comparison with amphetamine. Neuroscience. 1998 Dec; 87(4): 905~911
7 Naber,-P-A; Witter, -M-P.TI: Subicular efferents are organized mostly as parallel projections: a double-labeling, retrograde-tracing study in the rat. J-Comp-Neurol. 1998 Apr 13; 393(3): 284~297
8 Y. Ben-Ari. Limbic seizure and brian damage produced bykainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience,1985,14 (2) :375~405
9 E.A. Cavalheiro,D.A. Riche, G. LE GAL Salle. Long- term effects of intrahippocampal kainic acid injection in rats: a method for inducing spontaneous recurrent seizures. Electroencephalography and Clinical NeuroPhysiology,1982, 53:581~589
10 Gary W. Mathern, Fredi Cifuentes,Joao P. Leite,et al. Hippocampal EEG excitability and chronic spontaneous seizures are associated with aberrant synaptic reorganization in the rat intrahippocampal kainite model. Electroencephalography and Clinical Neurophsiology,1993,87:326~339
11 RacineIU. Modification of seizure activity by electrieal stirnulation-11 , Motor seizures. Electroencephalog Clin Nerophysiol ,1972, 232~281
12 李冬冬, 隋鸿锦, 宫瑾, 等. 海马硬化对癫痫敏感性的影响.中国应用生理学杂志, 2000; 16 (2) :107
13 Okazaki MM, Molnar P, Nadler J Y. Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currnts evoked in presence and absence of seizure induced growth. J Neuro-physiol ,1999; 81(4) :1645~ 1660
14 Ben A Y. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy [J] . Neuroscience ,1985 ,14(2): 375
15 Nadler JV, Okazaki MM. Protective effect s of mossy fiberlesions against kainic acid - induced seizures and neuronal degeneration [J] . Neuroscience , 1988, 26: 763
16 Gary WM, J ames KP, Thomas LB. Quantified pat terns of mossy fiber sprouting and neuron densites in hippocampal and lesional seizures [J] . J Neurosurg , 1995 ,82: 211
17 Yin HZ, Sensi SL, Ogoshi F, et al. Blockade of Ca2 + - permeable AMPA/ kainite channels decreases oxygen - glucose deprivation -induced Zn2 + accumulation and neuronal loss in hippocampal pyramidal neurons [J] . J Neurosci , 2002 ,22(4) :1273
18 Lee MC, Rho JL, Woo YJ, et al. c - J UN expression and apoptotic cell death in kainite - induced temporal lobe epilepsy [J ] . J Korean Med Sci , 2001 ,16(5) :649
19 Pollard H, Charriaut C, Cantagel S, et al. Kainate - induced apoptotic cell deat h in hippocampal neurons [J] . Neuroscience ,1994 ,63: 7
20 David C, Henshall, J un C, et al . Involvement of caspase - 3 -like protease in the mechanism of cell deat h following focally evoked limbic seizures[J] . J Neurochemistry, 2000, 74(3) :1215
21 Gary WM, Cif uentes F, Joao PL, et al. Hippocampal EEG excitability and chronic spontaneous seizures are associated wit haberrant synaptic reorganization in the rat int -rahippocampal kainite model [J].Electroencephalography and Clinical Neurophysiology, 1993 ,87: 326
22 Proper EA, Oest reicher AB, J ansen GH, et al .Immunohisto-chemical characterization of mossy fibre sprouting in the hippocampus of patients with pharmaco- resistant temporal lobe epilepsy[J] . Brain , 2000 ,123 :19
23 Okazaki MM , Molnar P, Nadler JV. Recurrent mossy fiber pathway in rat dentate gyrus : synaptic currents evoked in prsence and absence of seizure induced growth[J] . J Neurophysiol , 1999 ,81(4) :1645
24 Represa A, Pollard H, Moreau J, et al. Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of alpha - tubulin[J] . Neurosci Let t , 1993 ,156(1 - 2) :149
25 Pollard H, Khrestchatisky M, Moreau J, et al . Correlation between reactive sprouting and micro tubule protein expression in epileptic hippocampus[J] . Neuroscience,1994, 61(4) :773
26 Reeben M, Lauurikainen A, Hilt unen JO, et al. The messen-gener RNAs for both glial cell line derived neurot rophic factor receptors. C - ret and GDNFRα are induced in the rat brain in response to kainite - induced excitation[J] . Neuroscience ,1998 ,83(1) :151
27 朱长庚,李龄,魏瑛,等.癫痫病人手术切除标本的组织学、免 疫 组 织 化 学 和 超 微 结 构 研 究 . 解 剖 学 报 ,1997 ,28(3):225~229
28 Bertolino M, Llinas RR. The central role of voltage-activited and receptor operated calcium channels in neuronal cells. Annu Rev pharmacol Toxicol,1992, 32:399~421
29 Simatov R,Crispino M,Hoe W,et al. Changes in expression of neuronal and glial gluta mate transporters in rat hippocampus following kainite induced seisure activity. Brain Res, 1999, 65(l):112~116