1，6-二磷酸果糖对电点燃模型的作用及相关机制研究
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摘要
研究背景
癫痫是一种常见疾病,据流行病学调查,人群年患病率为7‰,严重危害人类健康和生活质量。目前癫痫的治疗虽然取得了一些进展,如各种新型抗癫痫药物、迷走神经刺激术和不断进步的外科手术治疗,但仍有20%-40%患者的癫痫发作不能得到有效控制,成为难治性癫痫。
生酮饮食(ketogenic diet, KD)是一种高脂肪、低碳水化合物和适量蛋白质的饮食方案。自1921年Wilder等报道了用“生酮”来治疗难治性癫痫以来,KD的疗效已经被大部分学者认同,并且在一些临床中心应用。但是,KD起效周期较长,需要长期改变患者饮食习惯,并可能导致高血脂,低血糖和肾结石等并发症,临床依从性较差。因此,研究者们不断在寻找与KD具有相似的抗癫痫机制、可替代KD,并且副作用小、依从性好、能应用于临床的药物。
1,6-二磷酸果糖(fructose-1,6-diphosphate, FDP)是细胞内天然存在的一种化合物,已经广泛应用于心、肾、肝、脑缺血等的临床治疗,未发现明显副作用。FDP与KD类似,具有抑制糖酵解的作用,并且能增加细胞对抗氧化应激的能力。
在难治性癫痫中,以逐渐发展的癫痫反复发作和海马硬化为特点的颞叶癫痫最为常见。而电点燃模型是用一定强度的电流反复刺激特定脑区,导致癫痫发作活性不断增强,最终形成稳定的癫痫发作状态,是研究颞叶癫痫的理想模型。海马快速电点燃模型建立周期短,干扰因素少,在药物筛选上敏感性较高；而杏仁核慢性电点燃模型发作情况更易于控制,重复性好,对刺激反应的耐受性好,死亡率低,除了可以更细致地评价抗癫痫药物的作用,还能较好地研究抗癫痫药物的副作用和药物机制。因此,我们选用这两种模型进行本实验的研究。
突触结构和功能的变化是颞叶癫痫的主要病理基础,其在电点燃模型中扮演很重要的角色。脑源性神经生长因子(brain derived neurotrophic factor, BDNF)在中枢神经系统许多区域表达并参与突触重塑和神经递质的传递功能,与癫痫的形成关系密切。研究发现,糖酵解过程中的产物还原型烟酰胺腺嘌呤二核苷酸可在转录因子神经元限制性沉默因子的协助下与活性升高的C-端结合蛋白结合。因此,抑制糖酵解可降低与癫痫密切相关的基因的表达,这些基因中就包括与突触可塑性密切相关的基因BDNF.而另外有研究发现BDNF通过激活酪氨酸激酶B受体(tyrosine kinase receptor B, TrkB)调节钾-氯离子共转运体2(K+-Cl- cotransporter 2,KCC2)和钠-钾-氯离子共转运体1(Na+-K+-2Cl- cotransporter 1, NKCC1)的表达,从而调节γ-氨基丁酸A型受体的功能向兴奋性转化,并参与了神经兴奋毒性。但是到目前为止,国内未见关于BDNF介导的FDP抑制癫痫作用的相关机制研究。
因此本研究从电点燃模型入手,观察不同剂量FDP对急性和慢性电点燃模型的行为学和脑电发放的作用。并探讨BDNF和TrkB对电点燃及FDP抑制电点燃模型中的影响,同时,我们也观察了KCC2和NKCC1在其中的作用。
第一部分电点燃模型的建立,行为、脑电及病理学观察
目的：
建立海马快速点燃模型和杏仁核慢性点燃模型,观察癫痫急性和慢性形成过程中大鼠的行为学和深部脑电变化；并从组织学方面研究电点燃模型的海马神经元损伤和星形胶质细胞增生的变化,探讨电点燃模型的致痫机制。
方法：
1.应用立体定位仪对腹侧海马和基底旁侧杏仁核进行定位,准确插入电极。测定点燃阈值后,通过每次10 sec,间隔30 min,12次／天,连续4天的阈上刺激建立大鼠海马快速点燃模型；通过每次2 sec,1次／天,连续16天的阈刺激建立大鼠杏仁核慢性点燃模型。
2.根据Racine分级标准,观察癫痫行为学变化,记录癫痫发作等级、后放电阈值(afterdicharge threshold, ADT )、后放电持续时间(afterdishcharge duration,ADD)、大发作潜伏期(generalized seizure latency, GSL )、大发作持续时间(generalized seizure duration, GSD )、后放电峰值(afterdischarge peak, ADP)等指标。
3.实验结束后,Nissl染色法对电极插入的部位进行确认。
4. Nissl染色法观察杏仁核慢性点燃模型刺激前,刺激6次,16次后海马神经元的形态及数目的改变。
5.免疫组化法观察杏仁核慢性点燃模型刺激前,刺激6次,16次后海马星形胶质细胞的形态及数目的改变。
结果：
1.海马快速点燃模型：随着刺激次数的增加,发作程度逐渐加重；第2天部分动物被点燃(第1次4级发作),第4天,所有动物点燃稳定引出(3次以上5级发作)；随着刺激次数增加,ADD延长,但动物被点燃后,ADD逐渐趋于稳定。
2.杏仁核慢性点燃模型：随着刺激次数的增加,发作程度逐渐加重；点燃稳定引出的平均刺激次数为9.6±1.2次；刺激前ADT在100.0-250.0μA之间,而刺激第16天ADT在60.0-140.0μA之间。
3.海马CA1区,CA3区和齿状回(dentategyrus, DG)神经元在刺激前后没有明显变化；星形胶质细胞在第6次刺激后就开始出现弥漫性增生、胞体增大、发出突起粗长等变化。
结论：
1.通过连续4天,12次／天,每次10 sec的阈上刺激,能够成功建立腹侧海马快速点燃模型。
2.通过连续16天,1次／天,每次2 sec的阈刺激,能够成功建立基底旁侧杏仁核慢性点燃模型。
3.在电刺激后,海马CA1, CA3和DG区没有明显的神经元缺失；但CA1和CA3区发现星形胶质细胞增生。
第二部分1,6-二磷酸果糖对电点燃模型的作用
目的：
观察不同药物浓度FDP (0.5 g/kg和1.0 g／kg)在海马快速点燃模型和杏仁核慢性点燃模型中对颞叶癫痫形成的作用；同时,还将观察FDP对完全点燃后癫痫发作易感性的作用,以及在点燃过程中对后放电阈值的影响。
方法：
1.腹侧海马电极植入术后休息10天,按ADT配对将动物分为对照组、低剂量组(0.5 g/kg FDP)和高剂量组(1.0 g/kg FDP).每天刺激前1h分别穿插给药,根据Racine评分标准记录每次刺激后的癫痫发作等级,ADD, GSL, GSD,ADP等指标的变化。
2.海马快速点燃模型建立后,将动物随机分为对照组、低剂量组和高剂量组。再给予5天,6次／天,与点燃模型相同参数的电刺激,每天刺激前1 h分别穿插给药。根据Racine评分标准记录每次刺激后癫痫发作等级和ADD。
3.基底旁侧杏仁核电极植入术后休息10天,按ADT配对将动物分为对照组、低剂量组和高剂量组。在每天ADT测定0.5 h后,电刺激前1 h,按照组别穿插给药。记录并比较每天刺激前的ADT,根据Racine评分标准记录每次刺激后癫痫发作等级和ADD。
结果：
1.动物腹腔注射FDP后无腹部痉挛等异常反应。从刺激第2天起高剂量FDP显著减少海马快速点燃的每天平均发作等级,而低剂量FDP仅减少第3天的平均发作等级；高剂量FDP缩短第2天和第4天的累积ADD,而低剂量FDP对累积ADD没有显著性作用。
2.高剂量组比对照组需要额外将近30%的刺激次数或者约47%的累积ADD才能到达4级；刺激第4天,高剂量组发作5级的次数是对照组的1／3；高剂量FDP显著缩短了GSD。
3.高、低剂量FDP都不能减少海马完全点燃后癫痫反复发作的等级或者缩短ADD。
4.随着刺激的进展,FDP不能阻止点燃引起ADT降低,但可以减缓其降低的趋势
结论：
1.海马快速点燃模型中,FDP能显著减少癫痫发作等级并缩短后放电,但其作用具有剂量依赖性。
2.高剂量FDP能抑制癫痫点燃的形成。
3.动物完全点燃以后,FDP并不能减少癫痫的反复发作和缩短反复发作后放电。
4.杏仁核慢性点燃模型中,FDP能有效地抑制癫痫发作并缩短后放电,且FDP的作用同样具有剂量依赖性。随着刺激的进展,FDP并不能阻止阈值降低的趋势,但可以减缓阈值降低的过程。
第三部分1,6-二磷酸果糖抑制癫痫机制的初步探讨
目的：
研究在海马快速点燃和杏仁核慢性点燃过程中海马BDNF和TrkB的mRNA和蛋白表达情况,并对FDP是否通过BDNF/TrkB途径起作用做初步探讨。同时,我们也观察了电刺激和FDP对海马氯离子通道KCC2与NKCC1的mRNA和蛋白表达的影响,为GABAA受体功能转化是否在FDP抑制癫痫过程中发挥作用提供理论依据。
方法：
1.未刺激大鼠连续1天,2天和4天腹腔注射高剂量FDP,每天注射后1h取海马组织提取RNA进行定量PCR,以GAPDH为参考,测定BDNF,TrkB,KCC2和NKCC1的基因表达情况。
2.以ADT为参考,建立海马快速点燃模型。配对将动物均分为对照组和高剂量组,每天刺激前1 h,按照组别穿插给药,在刺激前、第5、24和48次刺激结束后4个时间点分别取动物的海马组织提取RNA进行定量PCR,以GAPDH为参考,测定不同时间点BDNF,TrkB,KCC2和NKCC1的基因表达的变化.
3.以ADT参考,建立杏仁核慢性点燃模型。配对将动物均分为对照组、低剂量组和高剂量组。每天阈值测定0.5 h后,电刺激前1 h,按照组别穿插给药。第16天刺激结束后,分别石蜡包埋进行免疫组化实验和分离海马组织进行免疫印迹实验。观察海马BDNF,KCC2的蛋白表达的变化。
结果：
1.未刺激大鼠第1天腹腔注射高剂量FDP后海马BDNF mRNA表达即开始降低；而连续4天腹腔注射高剂量FDP对TrkB mRNA表达并没有显著影响。
2.对比刺激前,海马BDNF mRNA表达在第5次刺激后即开始升高,一直持续到48次刺激结束；与对照组比较,高剂量FDP在刺激前和第5次刺激后均降低BDNF mRNA的表达；BDNF的特异性受体TrkB mRNA表达与刺激前比较仅在第24次刺激后显著增加,而高剂量FDP能抑制TrkB mRNA表达的增加。
3.免疫组化实验发现,在杏仁核慢性点燃结束后,与对照组比较,低剂量组CA1、CA3区和DG区的BDNF蛋白表达降低,其中CA1区更为明显；高剂量组3个观察区域BDNF蛋白表达均降低。采用免疫印迹方法,我们也发现FDP能降低海马区BDNF蛋白的表达。
4.未刺激大鼠连续2天注射高剂量FDP后KCC2 mRNA表达开始升高；NKCC1 mRNA在第1天和连续4天注射高剂量FDP后表达降低。
5.与刺激前比较海马KCC2 mRNA在第5次刺激后开始降低,一直持续到第48次刺激结束；而高剂量FDP在第24和48次刺激后均抑制了KCC2 mRNA表达的降低。NKCC1 mRNA与刺激前相比并没有显著改变；但高剂量FDP在刺激前、第5次和第24次刺激后与对照组相比,降低NKCC1 mRNA的表达。
6.脑组织石蜡切片进行KCC2和Hoechst免疫荧光双染,结果提示,KCC2主要位于神经元细胞膜上；KCC2抗体免疫组化染色结果显示,在杏仁核慢性点燃结束后,与对照组相比较,高、低剂量FDP均能增加KCC2蛋白的表达。
结论：
1. BDNF/TrkB途径参与电点燃癫痫的形成。
2.FDP本身对BDNF的表达具有调控作用。
3.细胞内氯离子失衡导致的GABAA受体兴奋性改变很可能参与了电点燃癫痫的形成。
4.FDP可能通过调节GABAA受体兴奋性改变而起到抗癫痫的作用。
Background
Epilepsy is a common disease in China. Base on the recent epidemiology investigation, the morbidity rate for epilepsy is 7‰. It is harmful for people's healthy and life. The current treatment methods of epilepsy have quickly developed including:a variety of new antiepileptic drugs, vagus nerve stimulation, and development in surgical treatment of epilepsy and so on. Despite numerous treatment methods,20%-40% patients with epilepsy are still diagnosed as refractory epilepsy.
The ketogenic diet (KD) is diet of a high-fat, adequate-protein, low-carbohydrate. The original of ketogenic diet for treating refractory epilepsy was dated from 1921 by Dr. Wilder. The KD is used to treat intractable epilepsy by more and more researchers worldwide, and has been used in some clinical epilepsy research centers. The KD is not a benign, holistic or natural treatment for epilepsy; as with any serious medical therapy, there may be complications, including hyperlipoidemia, hypoglycemia, kidney stones and other short-term or long-term side effects. Researchers in the field of the KD have recently embarked on finding and making "a simple pill", with the ultimate aim of reducing the "difficult" KD regimen and minimizing the complications.
Fructose-1,6-diphosphate (FDP) is a naturally occurring high-energy intermediate in the glycolytic pathway. It has been used for many years in cardiovascular disease, renal injury, liver injury and brain damage without significant side effects. FDP exerts potent feedback inhibition on the activity of phosphofructokinase-1---a rate-limiting enzyme in glycolysis, decreases reactive oxygen species (ROS) production and exerts a neuroprotective effect against excitotoxicity. In theory, it would inhibit the progress of epilepsy; however, few relevant researches have been reported.
Temporal lobe epilepsy (TLE) is the commonest form of refractory epilepsy, which characterized by spontaneous recurrent epileptic seizure (SRS) and hippocampal sclerosis (HS). Kindling is an ideal model of TLE, induced by certain intensity electrical stimulation of the brain, which would increase the neuronal activity and eventually form a stable state of epileptic discharge. The rapid hippocampal kindling model is characterized by its short-period establishment, less interference factors and high sensitivity in drug screening. While chronic amygdala kindling model has many advantages including easy controlling of the epileptic discharge, reproducible, tolerated, with low mortality et al. and making more detailed evaluation of antiepileptic drugs. It is suitable for study of the side effects of antiepileptic drugs and potential mechanisms of the drugs. Therefore, we chose these two classical models to carry out our experiment.
Synaptic plasticity is the main pathological mechanism of TLE, and it also plays an important role in electrical kindling. Brain derived neurotrophic factor (BDNF) is a critical factor in brain development and plasticity, and has been demonstrated as having close relationship with epilepsy. A recent study showed that NADH generated by glycolysis can destabilize the interaction of the co-repressor C-terminal binding protein (CtBP) and neural restrictive silencing factor (NRSF), allowing transcription of NRSF target genes (including BDNF) and maintaining normal neuronal excitability. Inhibition of glycolysis reduces NADH concentrations and recruits CtBP to form the NRSF-CtBP complex on NRSF target genes, thereby reducing their transcription (including BDNF). Lower expression of BDNF and TrkB leads to reduced neuronal excitability, and increase seizure threshold and inhibit progression of kindling. In pathological conditions such as seizures in the CNS, BDNF triggers intracellular signaling cascades that ultimately lead to the downregulation of KCC2. Decreased expression of KCC2 results in a diminution in the capacity of neurons to extrude chloride, thereby increasing intracellular chloride level and causing GABA to become depolarizing, and substantially enhanced the excitability of the cells. Despite the inhibition effects of GABA have been observed, the regulation of the GABAA functions during the electrical stimulation models, as well as inhibitory effects of FDP modulated by BDNF/TrkB have been rarely reported.
In this study, we observed the semiology and electroencephalography (EEG) changes during rapid hippocampal kindling and chronic amygdala kindling with different doses of FDP. To understand the mechanism underlying how FDP works, we investigated the mRNA expression profiles of BDNF, TrkB, KCC2, and NKCC1 in the ipsilateral hippocampus during the rapid hippocampal kindling process, as well as BDNF and KCC2 protein expression after the chronic amygdala kindling process.
PartⅠSemiology, EEG and pathology of electrical kindling models.
Objective
To investigate the alternation of behavior and EEG during rapid hippocampal kindling and chronic amygdala kindling, and observe seizure-dependent changes in neuronal injury and proliferation of astrocytes in rats' hippocampus induced by electrical kindling.
Method
1. Electrodes were implanted into the right ventral hippocampus and basolateral amygdala (BLA). After the after-discharge threshold (ADT) was determined, the animals were subjected to 12 kindling stimulations per day with 10 sec stimulation duration at 30-min intervals for 4 consecutive days, or 2 sec stimulation every day for 16 days for establishing electrical simulation models.
2. Seizures were graded according to an adjusted version of the Racine scale. ADT, afterdischarge duration (ADD), generalized seizure latency (GSL), generalized seizure duration (GSD), and afterdischarge peak (ADP) were recorded and analyzed.
3. At the end of the experiment, the electrode placements were histologically verified by Nissl staining.
4. To evaluate the pathology features of hippocampal neurons of different time points, the rats'brain were removed before, after 6 stimulations and after 16 stimulations and stained by Nissl.
5. To detect GFAP immunoreactive positive cells in hippocampus, the rats'brain were removed before, after 6 stimulations and after 16 stimulations and stained by immuohistochemistry.
Result
1. The mean seizure stage increased gradually with the development of hippocampal stimulation. Some animals began to attain stage 4 after two days'stimulation, and all of the animals attained fully kindled (more than 3 times of stage 5) on day 4. The ADD was extended with the stimulations, however, it gradually stabilized after attaining to stage 4.
2. The mean seizure stage increased gradually with the development of amygdale stimulation. The mean stimulation number of fully kindled was 9.6±1.2, The ADT before the stimulation was ranged from 100.0μA to 250.0μA, however the ADT ranged from 60.0 to 140.0μA after finishing the amygdala kindling (day 16).
3. Nissl staining showed no significant change of neuron damage during hippocampal kindling process in CA1, CA3 and dentategyrus region (DG). Immunohistochemistry showed that electrical stimulation induced GFAP immunoreactivity enhancement in the CA1 and CA3 regions of hippocampus after 6 and 16 stimulations, indicating astroglisis.
Conclusion
1. The rapid hippocampal kindling model was established successfully by 12 kindling stimulations per day with 10 sec stimulation duration at 30-min intervals for 4 consecutive days.
2. The basolateral amygdala kindling model was established successfully by 2 sec stimulation every day for 16 days.
3. No significant change of neuron damage was found in CA1, CA3 and DG regions, however astroglisis was found in the CA1 and CA3 regions after 6 and 16 stimulations
PartⅡAntiepileptogenic action of fructose-1,6-diphosphate on electrical kindling models.
Objective
To investigate the antiepileptogenic effect of different doses (0.5 g/kg or 1.0 g/kg) of FDP in the rapid hippocampal kindling and chronic amygdala kindling models. Moreover, we observed the anticonvulsant effects of FDP on hippocampal fully kindled animals and the change of ADT during chronic amygdala kindling process. Method
1. After 10 days'recovery, the animals with electrode embedded in ventral hippocampus were randomly divided into 3 groups (each group was characterized by similar mean average ADT values):control group, low-dose FDP group (0.5 g/kg) and high-dose FDP group (1.0 g/kg) and were investigated the antiepileptogenic effect of FDP. FDP or saline was administered 1 h (i.p.) before kindling stimulation. Seizures were graded according to an adjusted version of the Racine scale, and other parameters were recorded and analyzed including ADD, GSL,GSD, and ADP.
2. After establishing the rapid hippocampal kindling models, the animals were randomly divided into 3 groups:control group, low-dose group and high-dose group. FDP or saline was administered 1 h (i.p.) before stimulation. To evaluate the anticonvulsant effect of FDP, a retest paradigm (5 days of retest stimulation with 6 stimulations per day) was administrated to the kindled rats. Seizures were graded according to an adjusted version of the Racine scale, and ADD was recorded and analyzed.
3. After recovery for 10 days, the animals with electrode embedded in BLA were randomly divided into 3 groups (each group was characterized by similar mean average ADT values):control group, low-dose group and high-dose group to investigate the antiepileptogenic effect of FDP in chronic epilepsy. FDP or saline was administered 1 h (i.p.) before kindling stimulation each day. Seizures were graded according to an adjusted version of the Racine scale and ADD were recorded and analyzed. Additionally, the ADT was recorded and analyzed before stimulation each day.
Result
1. The animals displayed no signs of abdominal cramps or other abnormal behavior after intraperitoneal injection of FDP. The high-dose FDP significantly inhibited seizures on days 2,3, and 4, and the cumulative ADD on day 2 and day 4 were significantly suppressed; while low-dose FDP only had a significant inhibitory effect on seizure on day 3.
2. The rats pretreated with 1.0 g/kg of FDP needed approximately 30% more kindling stimulations and 47% more cumulative ADD to reach stage 4 than the rats pretreated with saline. Additionally, pretreatment of high-dose FDP reduced the number of stage 5 seizures by 66% on day 4. Moreover, pretreatment of high-dose FDP significantly reduced the GSD.
3. FDP has no anticonvulsant effect on the rapid hippocampal kindling models.
4. The high-dose FDP significantly retarded the kindling acquisition of chronic amygdala kindling and shortened the ADD, while the low-dose FDP had no such effect. High-dose FDP increased the number of days and ADD for which animals stayed in stages 1 and 2, while decreased the number of days and ADD stayed in stages 5.
5. FDP did not change the downward trend of the ADT with the process of kindling, and there is no significant difference of the ADT between the control and high-dose group while they stayed in stage 1-4. However, the high-dose FDP treated rats had a higher ADT than control when they stayed in stage 5.
Conclusion
1. FDP significantly inhibited epilepsy and shortened ADD in the rapid hippocampal kindling, while the effect of FDP was dose-dependent.
2. High-dose FDP has an antiepileptogenic effect.
3. When animals were fully kindled, FDP has effected neither on the mean seizure stage nor on the ADD.
4. The FDP has also had a dose-dependent antiepileptogenic effect on chronic amygdala kindling. The high-dose FDP retard the rats to be kindled. FDP did not change the downward trend of the ADT with the process of kindling; however it slowed down the reduction of ADT.
PartⅢThe potential mechanism of antiepileptogenic effects of fructose-1,6-diphosphate.
Objective
To investigate the BDNF and TrkB mRNA expression during rapid hippocampal kindling and the BDNF protein expression after chronic amygdala kindling, and analyze whether the BDNF/TrkB signaling pathway participate in the antiepileptogenic action of FDP. Moreover, we investigated the KCC2 and NKCC1 mRNA expression during rapid hippocampal kindling and the KCC2 protein expression after chronic amygdala kindling, in order to analyze whether transformation of GABAA function played a role in the antiepileptogenic effects of FDP. Method
1. We had performed a biochemical data analysis (BDNF, TrkB, KCC2 and NKCC1 mRNA expression compared with GAPDH) of 4 continuous days'administration of FDP (1.0 g/kg) in normal rats. Brains were removed and hippocampi were separated to investigate the genes expression by real-time PCR.
2. Before rapid hippocampal kindling, the rats were divided into 2 groups (each group characterized by similar mean average ADT values):control and high-dose group to investigate the BDNF, TrkB, KCC2 and NKCC1 mRNA expression compared with GAPDH in ipsilateral hippocampus. The rats were killed by decapitation before and after 5,24 and 48 stimulations, and the brains were removed within 1 h after the last stimulation, and then the ipsilateral hippocampi were separated immediately for the molecular analyses.
3. Before the chronic amygdala kindling, the rats were divided into 3 groups (each group characterized by similar mean average ADT values):control, low-dose and high-dose group to investigate the BDNF and KCC2 protein expression in ipsilateral hippocampus. The rats were killed by decapitation after accomplishment of the 16 days'stimulation, and the brains were removed within 1 h after the last stimulation, and then the ipsilateral hippocampi were separated immediately for western-blotting or the brains were embedded in paraffin for immunohistochemical expenments.
Result
1. In normal rats, FDP significantly downregulated the BDNF mRNA expression (after 1 day,2 days and 4 days administration). However the TrkB mRNA level did not significantly downregulated.
2. Following the administration of kindling stimulations to the right ventral hippocampus, the relative abundance of BDNF mRNA significantly increased after 5 stimulations. This change continued until the completion of 48 stimulations. Consistent with the increase in BDNF levels, there was a significant upregulation of TrkB mRNA after 24 stimulations. This upregulation was transient, and the expression returned to the baseline level at the end of 48 stimulations. In animals pretreated with FDP, the BDNF mRNA level was further significantly downregulated after 5 kindling stimulations than the control, and TrkB mRNA level was conspicuously downregulated after 24 stimulations.
3. Immunohistochemistry results showed that the BDNF protein expression was downregulated in the rats pretreated with FDP in the CA1, CA3 and DG regions of hippocampus, which was consistent with the results of Western-blotting analysis.
4. FDP had significant upregulation on the KCC2 mRNA expression after 2 and 4 days administration and there was a significant downregulation of FDP alone on the NKCC1 mRNA expression after 1 day and 4 days administration.
5. Without FDP treatment, the relative copies of KCC2 mRNA decreased after 5 stimulations and this change continued until the completion of 48 stimulations. However, the expression levels of NKCC1 mRNA did not change significantly during the kindling process. Compared with the control rats, the KCC2 mRNA level increased significantly after 24 and 48 stimulations in the animals pretreated with FDP. A significant reduce in NKCC1 mRNA expression was observed after 5 and 24 stimulations in the FDP pretreated animals.
6. Immunofluorescence staining of KCC2 antibody and Hoechst showed that the K+-C1-co-transporter protein was localized in the membrane of neurons. Immunohistochemistry showed that the KCC2 protein expression was upregulated in the rats pretreated with FDP in the CA1, CA3 and DG regions of hippocampus
Conclusion
1. Stimulation induced enhancement of BDNF and TrkB expression, indicating that the BDNF/TrkB signaling pathway may participates in the kindling models.
2. In our experiment, FDP can decrease BDNF expression before kindling and reduces BDNF expression in early phase of kindling. This reduce was probably mediated by FDP itself, rather than secondary to reduction of seizure severity.
3. KCC2 was downregulated in late phase of kindling, indicating that chloride homeostasis may modulate rapid kindling and supporting the role of "GABAergic excitation" in epileptogenesis.
4. FDP may also play its antiepileptogenic role by regulating the intracellular chloride level and GABAergic excitation in neurons.
癫痫是一种常见疾病,据流行病学调查,人群年患病率为7‰,严重危害人类健康和生活质量。目前癫痫的治疗虽然取得了一些进展,如各种新型抗癫痫药物、迷走神经刺激术和不断进步的外科手术治疗,但仍有20%-40%患者的癫痫发作不能得到有效控制,成为难治性癫痫。
生酮饮食(ketogenic diet, KD)是一种高脂肪、低碳水化合物和适量蛋白质的饮食方案。自1921年Wilder等报道了用“生酮”来治疗难治性癫痫以来,KD的疗效已经被大部分学者认同,并且在一些临床中心应用。但是,KD起效周期较长,需要长期改变患者饮食习惯,并可能导致高血脂,低血糖和肾结石等并发症,临床依从性较差。因此,研究者们不断在寻找与KD具有相似的抗癫痫机制、可替代KD,并且副作用小、依从性好、能应用于临床的药物。
1,6-二磷酸果糖(fructose-1,6-diphosphate, FDP)是细胞内天然存在的一种化合物,已经广泛应用于心、肾、肝、脑缺血等的临床治疗,未发现明显副作用。FDP与KD类似,具有抑制糖酵解的作用,并且能增加细胞对抗氧化应激的能力。
在难治性癫痫中,以逐渐发展的癫痫反复发作和海马硬化为特点的颞叶癫痫最为常见。而电点燃模型是用一定强度的电流反复刺激特定脑区,导致癫痫发作活性不断增强,最终形成稳定的癫痫发作状态,是研究颞叶癫痫的理想模型。海马快速电点燃模型建立周期短,干扰因素少,在药物筛选上敏感性较高；而杏仁核慢性电点燃模型发作情况更易于控制,重复性好,对刺激反应的耐受性好,死亡率低,除了可以更细致地评价抗癫痫药物的作用,还能较好地研究抗癫痫药物的副作用和药物机制。因此,我们选用这两种模型进行本实验的研究。
突触结构和功能的变化是颞叶癫痫的主要病理基础,其在电点燃模型中扮演很重要的角色。脑源性神经生长因子(brain derived neurotrophic factor, BDNF)在中枢神经系统许多区域表达并参与突触重塑和神经递质的传递功能,与癫痫的形成关系密切。研究发现,糖酵解过程中的产物还原型烟酰胺腺嘌呤二核苷酸可在转录因子神经元限制性沉默因子的协助下与活性升高的C-端结合蛋白结合。因此,抑制糖酵解可降低与癫痫密切相关的基因的表达,这些基因中就包括与突触可塑性密切相关的基因BDNF.而另外有研究发现BDNF通过激活酪氨酸激酶B受体(tyrosine kinase receptor B, TrkB)调节钾-氯离子共转运体2(K+-Cl- cotransporter 2,KCC2)和钠-钾-氯离子共转运体1(Na+-K+-2Cl- cotransporter 1, NKCC1)的表达,从而调节γ-氨基丁酸A型受体的功能向兴奋性转化,并参与了神经兴奋毒性。但是到目前为止,国内未见关于BDNF介导的FDP抑制癫痫作用的相关机制研究。
因此本研究从电点燃模型入手,观察不同剂量FDP对急性和慢性电点燃模型的行为学和脑电发放的作用。并探讨BDNF和TrkB对电点燃及FDP抑制电点燃模型中的影响,同时,我们也观察了KCC2和NKCC1在其中的作用。
第一部分电点燃模型的建立,行为、脑电及病理学观察
目的：
建立海马快速点燃模型和杏仁核慢性点燃模型,观察癫痫急性和慢性形成过程中大鼠的行为学和深部脑电变化；并从组织学方面研究电点燃模型的海马神经元损伤和星形胶质细胞增生的变化,探讨电点燃模型的致痫机制。
方法：
1.应用立体定位仪对腹侧海马和基底旁侧杏仁核进行定位,准确插入电极。测定点燃阈值后,通过每次10 sec,间隔30 min,12次／天,连续4天的阈上刺激建立大鼠海马快速点燃模型；通过每次2 sec,1次／天,连续16天的阈刺激建立大鼠杏仁核慢性点燃模型。
2.根据Racine分级标准,观察癫痫行为学变化,记录癫痫发作等级、后放电阈值(afterdicharge threshold, ADT )、后放电持续时间(afterdishcharge duration,ADD)、大发作潜伏期(generalized seizure latency, GSL )、大发作持续时间(generalized seizure duration, GSD )、后放电峰值(afterdischarge peak, ADP)等指标。
3.实验结束后,Nissl染色法对电极插入的部位进行确认。
4. Nissl染色法观察杏仁核慢性点燃模型刺激前,刺激6次,16次后海马神经元的形态及数目的改变。
5.免疫组化法观察杏仁核慢性点燃模型刺激前,刺激6次,16次后海马星形胶质细胞的形态及数目的改变。
结果：
1.海马快速点燃模型：随着刺激次数的增加,发作程度逐渐加重；第2天部分动物被点燃(第1次4级发作),第4天,所有动物点燃稳定引出(3次以上5级发作)；随着刺激次数增加,ADD延长,但动物被点燃后,ADD逐渐趋于稳定。
2.杏仁核慢性点燃模型：随着刺激次数的增加,发作程度逐渐加重；点燃稳定引出的平均刺激次数为9.6±1.2次；刺激前ADT在100.0-250.0μA之间,而刺激第16天ADT在60.0-140.0μA之间。
3.海马CA1区,CA3区和齿状回(dentategyrus, DG)神经元在刺激前后没有明显变化；星形胶质细胞在第6次刺激后就开始出现弥漫性增生、胞体增大、发出突起粗长等变化。
结论：
1.通过连续4天,12次／天,每次10 sec的阈上刺激,能够成功建立腹侧海马快速点燃模型。
2.通过连续16天,1次／天,每次2 sec的阈刺激,能够成功建立基底旁侧杏仁核慢性点燃模型。
3.在电刺激后,海马CA1, CA3和DG区没有明显的神经元缺失；但CA1和CA3区发现星形胶质细胞增生。
第二部分1,6-二磷酸果糖对电点燃模型的作用
目的：
观察不同药物浓度FDP (0.5 g/kg和1.0 g／kg)在海马快速点燃模型和杏仁核慢性点燃模型中对颞叶癫痫形成的作用；同时,还将观察FDP对完全点燃后癫痫发作易感性的作用,以及在点燃过程中对后放电阈值的影响。
方法：
1.腹侧海马电极植入术后休息10天,按ADT配对将动物分为对照组、低剂量组(0.5 g/kg FDP)和高剂量组(1.0 g/kg FDP).每天刺激前1h分别穿插给药,根据Racine评分标准记录每次刺激后的癫痫发作等级,ADD, GSL, GSD,ADP等指标的变化。
2.海马快速点燃模型建立后,将动物随机分为对照组、低剂量组和高剂量组。再给予5天,6次／天,与点燃模型相同参数的电刺激,每天刺激前1 h分别穿插给药。根据Racine评分标准记录每次刺激后癫痫发作等级和ADD。
3.基底旁侧杏仁核电极植入术后休息10天,按ADT配对将动物分为对照组、低剂量组和高剂量组。在每天ADT测定0.5 h后,电刺激前1 h,按照组别穿插给药。记录并比较每天刺激前的ADT,根据Racine评分标准记录每次刺激后癫痫发作等级和ADD。
结果：
1.动物腹腔注射FDP后无腹部痉挛等异常反应。从刺激第2天起高剂量FDP显著减少海马快速点燃的每天平均发作等级,而低剂量FDP仅减少第3天的平均发作等级；高剂量FDP缩短第2天和第4天的累积ADD,而低剂量FDP对累积ADD没有显著性作用。
2.高剂量组比对照组需要额外将近30%的刺激次数或者约47%的累积ADD才能到达4级；刺激第4天,高剂量组发作5级的次数是对照组的1／3；高剂量FDP显著缩短了GSD。
3.高、低剂量FDP都不能减少海马完全点燃后癫痫反复发作的等级或者缩短ADD。
4.随着刺激的进展,FDP不能阻止点燃引起ADT降低,但可以减缓其降低的趋势
结论：
1.海马快速点燃模型中,FDP能显著减少癫痫发作等级并缩短后放电,但其作用具有剂量依赖性。
2.高剂量FDP能抑制癫痫点燃的形成。
3.动物完全点燃以后,FDP并不能减少癫痫的反复发作和缩短反复发作后放电。
4.杏仁核慢性点燃模型中,FDP能有效地抑制癫痫发作并缩短后放电,且FDP的作用同样具有剂量依赖性。随着刺激的进展,FDP并不能阻止阈值降低的趋势,但可以减缓阈值降低的过程。
第三部分1,6-二磷酸果糖抑制癫痫机制的初步探讨
目的：
研究在海马快速点燃和杏仁核慢性点燃过程中海马BDNF和TrkB的mRNA和蛋白表达情况,并对FDP是否通过BDNF/TrkB途径起作用做初步探讨。同时,我们也观察了电刺激和FDP对海马氯离子通道KCC2与NKCC1的mRNA和蛋白表达的影响,为GABAA受体功能转化是否在FDP抑制癫痫过程中发挥作用提供理论依据。
方法：
1.未刺激大鼠连续1天,2天和4天腹腔注射高剂量FDP,每天注射后1h取海马组织提取RNA进行定量PCR,以GAPDH为参考,测定BDNF,TrkB,KCC2和NKCC1的基因表达情况。
2.以ADT为参考,建立海马快速点燃模型。配对将动物均分为对照组和高剂量组,每天刺激前1 h,按照组别穿插给药,在刺激前、第5、24和48次刺激结束后4个时间点分别取动物的海马组织提取RNA进行定量PCR,以GAPDH为参考,测定不同时间点BDNF,TrkB,KCC2和NKCC1的基因表达的变化.
3.以ADT参考,建立杏仁核慢性点燃模型。配对将动物均分为对照组、低剂量组和高剂量组。每天阈值测定0.5 h后,电刺激前1 h,按照组别穿插给药。第16天刺激结束后,分别石蜡包埋进行免疫组化实验和分离海马组织进行免疫印迹实验。观察海马BDNF,KCC2的蛋白表达的变化。
结果：
1.未刺激大鼠第1天腹腔注射高剂量FDP后海马BDNF mRNA表达即开始降低；而连续4天腹腔注射高剂量FDP对TrkB mRNA表达并没有显著影响。
2.对比刺激前,海马BDNF mRNA表达在第5次刺激后即开始升高,一直持续到48次刺激结束；与对照组比较,高剂量FDP在刺激前和第5次刺激后均降低BDNF mRNA的表达；BDNF的特异性受体TrkB mRNA表达与刺激前比较仅在第24次刺激后显著增加,而高剂量FDP能抑制TrkB mRNA表达的增加。
3.免疫组化实验发现,在杏仁核慢性点燃结束后,与对照组比较,低剂量组CA1、CA3区和DG区的BDNF蛋白表达降低,其中CA1区更为明显；高剂量组3个观察区域BDNF蛋白表达均降低。采用免疫印迹方法,我们也发现FDP能降低海马区BDNF蛋白的表达。
4.未刺激大鼠连续2天注射高剂量FDP后KCC2 mRNA表达开始升高；NKCC1 mRNA在第1天和连续4天注射高剂量FDP后表达降低。
5.与刺激前比较海马KCC2 mRNA在第5次刺激后开始降低,一直持续到第48次刺激结束；而高剂量FDP在第24和48次刺激后均抑制了KCC2 mRNA表达的降低。NKCC1 mRNA与刺激前相比并没有显著改变；但高剂量FDP在刺激前、第5次和第24次刺激后与对照组相比,降低NKCC1 mRNA的表达。
6.脑组织石蜡切片进行KCC2和Hoechst免疫荧光双染,结果提示,KCC2主要位于神经元细胞膜上；KCC2抗体免疫组化染色结果显示,在杏仁核慢性点燃结束后,与对照组相比较,高、低剂量FDP均能增加KCC2蛋白的表达。
结论：
1. BDNF/TrkB途径参与电点燃癫痫的形成。
2.FDP本身对BDNF的表达具有调控作用。
3.细胞内氯离子失衡导致的GABAA受体兴奋性改变很可能参与了电点燃癫痫的形成。
4.FDP可能通过调节GABAA受体兴奋性改变而起到抗癫痫的作用。
Background
Epilepsy is a common disease in China. Base on the recent epidemiology investigation, the morbidity rate for epilepsy is 7‰. It is harmful for people's healthy and life. The current treatment methods of epilepsy have quickly developed including:a variety of new antiepileptic drugs, vagus nerve stimulation, and development in surgical treatment of epilepsy and so on. Despite numerous treatment methods,20%-40% patients with epilepsy are still diagnosed as refractory epilepsy.
The ketogenic diet (KD) is diet of a high-fat, adequate-protein, low-carbohydrate. The original of ketogenic diet for treating refractory epilepsy was dated from 1921 by Dr. Wilder. The KD is used to treat intractable epilepsy by more and more researchers worldwide, and has been used in some clinical epilepsy research centers. The KD is not a benign, holistic or natural treatment for epilepsy; as with any serious medical therapy, there may be complications, including hyperlipoidemia, hypoglycemia, kidney stones and other short-term or long-term side effects. Researchers in the field of the KD have recently embarked on finding and making "a simple pill", with the ultimate aim of reducing the "difficult" KD regimen and minimizing the complications.
Fructose-1,6-diphosphate (FDP) is a naturally occurring high-energy intermediate in the glycolytic pathway. It has been used for many years in cardiovascular disease, renal injury, liver injury and brain damage without significant side effects. FDP exerts potent feedback inhibition on the activity of phosphofructokinase-1---a rate-limiting enzyme in glycolysis, decreases reactive oxygen species (ROS) production and exerts a neuroprotective effect against excitotoxicity. In theory, it would inhibit the progress of epilepsy; however, few relevant researches have been reported.
Temporal lobe epilepsy (TLE) is the commonest form of refractory epilepsy, which characterized by spontaneous recurrent epileptic seizure (SRS) and hippocampal sclerosis (HS). Kindling is an ideal model of TLE, induced by certain intensity electrical stimulation of the brain, which would increase the neuronal activity and eventually form a stable state of epileptic discharge. The rapid hippocampal kindling model is characterized by its short-period establishment, less interference factors and high sensitivity in drug screening. While chronic amygdala kindling model has many advantages including easy controlling of the epileptic discharge, reproducible, tolerated, with low mortality et al. and making more detailed evaluation of antiepileptic drugs. It is suitable for study of the side effects of antiepileptic drugs and potential mechanisms of the drugs. Therefore, we chose these two classical models to carry out our experiment.
Synaptic plasticity is the main pathological mechanism of TLE, and it also plays an important role in electrical kindling. Brain derived neurotrophic factor (BDNF) is a critical factor in brain development and plasticity, and has been demonstrated as having close relationship with epilepsy. A recent study showed that NADH generated by glycolysis can destabilize the interaction of the co-repressor C-terminal binding protein (CtBP) and neural restrictive silencing factor (NRSF), allowing transcription of NRSF target genes (including BDNF) and maintaining normal neuronal excitability. Inhibition of glycolysis reduces NADH concentrations and recruits CtBP to form the NRSF-CtBP complex on NRSF target genes, thereby reducing their transcription (including BDNF). Lower expression of BDNF and TrkB leads to reduced neuronal excitability, and increase seizure threshold and inhibit progression of kindling. In pathological conditions such as seizures in the CNS, BDNF triggers intracellular signaling cascades that ultimately lead to the downregulation of KCC2. Decreased expression of KCC2 results in a diminution in the capacity of neurons to extrude chloride, thereby increasing intracellular chloride level and causing GABA to become depolarizing, and substantially enhanced the excitability of the cells. Despite the inhibition effects of GABA have been observed, the regulation of the GABAA functions during the electrical stimulation models, as well as inhibitory effects of FDP modulated by BDNF/TrkB have been rarely reported.
In this study, we observed the semiology and electroencephalography (EEG) changes during rapid hippocampal kindling and chronic amygdala kindling with different doses of FDP. To understand the mechanism underlying how FDP works, we investigated the mRNA expression profiles of BDNF, TrkB, KCC2, and NKCC1 in the ipsilateral hippocampus during the rapid hippocampal kindling process, as well as BDNF and KCC2 protein expression after the chronic amygdala kindling process.
PartⅠSemiology, EEG and pathology of electrical kindling models.
Objective
To investigate the alternation of behavior and EEG during rapid hippocampal kindling and chronic amygdala kindling, and observe seizure-dependent changes in neuronal injury and proliferation of astrocytes in rats' hippocampus induced by electrical kindling.
Method
1. Electrodes were implanted into the right ventral hippocampus and basolateral amygdala (BLA). After the after-discharge threshold (ADT) was determined, the animals were subjected to 12 kindling stimulations per day with 10 sec stimulation duration at 30-min intervals for 4 consecutive days, or 2 sec stimulation every day for 16 days for establishing electrical simulation models.
2. Seizures were graded according to an adjusted version of the Racine scale. ADT, afterdischarge duration (ADD), generalized seizure latency (GSL), generalized seizure duration (GSD), and afterdischarge peak (ADP) were recorded and analyzed.
3. At the end of the experiment, the electrode placements were histologically verified by Nissl staining.
4. To evaluate the pathology features of hippocampal neurons of different time points, the rats'brain were removed before, after 6 stimulations and after 16 stimulations and stained by Nissl.
5. To detect GFAP immunoreactive positive cells in hippocampus, the rats'brain were removed before, after 6 stimulations and after 16 stimulations and stained by immuohistochemistry.
Result
1. The mean seizure stage increased gradually with the development of hippocampal stimulation. Some animals began to attain stage 4 after two days'stimulation, and all of the animals attained fully kindled (more than 3 times of stage 5) on day 4. The ADD was extended with the stimulations, however, it gradually stabilized after attaining to stage 4.
2. The mean seizure stage increased gradually with the development of amygdale stimulation. The mean stimulation number of fully kindled was 9.6±1.2, The ADT before the stimulation was ranged from 100.0μA to 250.0μA, however the ADT ranged from 60.0 to 140.0μA after finishing the amygdala kindling (day 16).
3. Nissl staining showed no significant change of neuron damage during hippocampal kindling process in CA1, CA3 and dentategyrus region (DG). Immunohistochemistry showed that electrical stimulation induced GFAP immunoreactivity enhancement in the CA1 and CA3 regions of hippocampus after 6 and 16 stimulations, indicating astroglisis.
Conclusion
1. The rapid hippocampal kindling model was established successfully by 12 kindling stimulations per day with 10 sec stimulation duration at 30-min intervals for 4 consecutive days.
2. The basolateral amygdala kindling model was established successfully by 2 sec stimulation every day for 16 days.
3. No significant change of neuron damage was found in CA1, CA3 and DG regions, however astroglisis was found in the CA1 and CA3 regions after 6 and 16 stimulations
PartⅡAntiepileptogenic action of fructose-1,6-diphosphate on electrical kindling models.
Objective
To investigate the antiepileptogenic effect of different doses (0.5 g/kg or 1.0 g/kg) of FDP in the rapid hippocampal kindling and chronic amygdala kindling models. Moreover, we observed the anticonvulsant effects of FDP on hippocampal fully kindled animals and the change of ADT during chronic amygdala kindling process. Method
1. After 10 days'recovery, the animals with electrode embedded in ventral hippocampus were randomly divided into 3 groups (each group was characterized by similar mean average ADT values):control group, low-dose FDP group (0.5 g/kg) and high-dose FDP group (1.0 g/kg) and were investigated the antiepileptogenic effect of FDP. FDP or saline was administered 1 h (i.p.) before kindling stimulation. Seizures were graded according to an adjusted version of the Racine scale, and other parameters were recorded and analyzed including ADD, GSL,GSD, and ADP.
2. After establishing the rapid hippocampal kindling models, the animals were randomly divided into 3 groups:control group, low-dose group and high-dose group. FDP or saline was administered 1 h (i.p.) before stimulation. To evaluate the anticonvulsant effect of FDP, a retest paradigm (5 days of retest stimulation with 6 stimulations per day) was administrated to the kindled rats. Seizures were graded according to an adjusted version of the Racine scale, and ADD was recorded and analyzed.
3. After recovery for 10 days, the animals with electrode embedded in BLA were randomly divided into 3 groups (each group was characterized by similar mean average ADT values):control group, low-dose group and high-dose group to investigate the antiepileptogenic effect of FDP in chronic epilepsy. FDP or saline was administered 1 h (i.p.) before kindling stimulation each day. Seizures were graded according to an adjusted version of the Racine scale and ADD were recorded and analyzed. Additionally, the ADT was recorded and analyzed before stimulation each day.
Result
1. The animals displayed no signs of abdominal cramps or other abnormal behavior after intraperitoneal injection of FDP. The high-dose FDP significantly inhibited seizures on days 2,3, and 4, and the cumulative ADD on day 2 and day 4 were significantly suppressed; while low-dose FDP only had a significant inhibitory effect on seizure on day 3.
2. The rats pretreated with 1.0 g/kg of FDP needed approximately 30% more kindling stimulations and 47% more cumulative ADD to reach stage 4 than the rats pretreated with saline. Additionally, pretreatment of high-dose FDP reduced the number of stage 5 seizures by 66% on day 4. Moreover, pretreatment of high-dose FDP significantly reduced the GSD.
3. FDP has no anticonvulsant effect on the rapid hippocampal kindling models.
4. The high-dose FDP significantly retarded the kindling acquisition of chronic amygdala kindling and shortened the ADD, while the low-dose FDP had no such effect. High-dose FDP increased the number of days and ADD for which animals stayed in stages 1 and 2, while decreased the number of days and ADD stayed in stages 5.
5. FDP did not change the downward trend of the ADT with the process of kindling, and there is no significant difference of the ADT between the control and high-dose group while they stayed in stage 1-4. However, the high-dose FDP treated rats had a higher ADT than control when they stayed in stage 5.
Conclusion
1. FDP significantly inhibited epilepsy and shortened ADD in the rapid hippocampal kindling, while the effect of FDP was dose-dependent.
2. High-dose FDP has an antiepileptogenic effect.
3. When animals were fully kindled, FDP has effected neither on the mean seizure stage nor on the ADD.
4. The FDP has also had a dose-dependent antiepileptogenic effect on chronic amygdala kindling. The high-dose FDP retard the rats to be kindled. FDP did not change the downward trend of the ADT with the process of kindling; however it slowed down the reduction of ADT.
PartⅢThe potential mechanism of antiepileptogenic effects of fructose-1,6-diphosphate.
Objective
To investigate the BDNF and TrkB mRNA expression during rapid hippocampal kindling and the BDNF protein expression after chronic amygdala kindling, and analyze whether the BDNF/TrkB signaling pathway participate in the antiepileptogenic action of FDP. Moreover, we investigated the KCC2 and NKCC1 mRNA expression during rapid hippocampal kindling and the KCC2 protein expression after chronic amygdala kindling, in order to analyze whether transformation of GABAA function played a role in the antiepileptogenic effects of FDP. Method
1. We had performed a biochemical data analysis (BDNF, TrkB, KCC2 and NKCC1 mRNA expression compared with GAPDH) of 4 continuous days'administration of FDP (1.0 g/kg) in normal rats. Brains were removed and hippocampi were separated to investigate the genes expression by real-time PCR.
2. Before rapid hippocampal kindling, the rats were divided into 2 groups (each group characterized by similar mean average ADT values):control and high-dose group to investigate the BDNF, TrkB, KCC2 and NKCC1 mRNA expression compared with GAPDH in ipsilateral hippocampus. The rats were killed by decapitation before and after 5,24 and 48 stimulations, and the brains were removed within 1 h after the last stimulation, and then the ipsilateral hippocampi were separated immediately for the molecular analyses.
3. Before the chronic amygdala kindling, the rats were divided into 3 groups (each group characterized by similar mean average ADT values):control, low-dose and high-dose group to investigate the BDNF and KCC2 protein expression in ipsilateral hippocampus. The rats were killed by decapitation after accomplishment of the 16 days'stimulation, and the brains were removed within 1 h after the last stimulation, and then the ipsilateral hippocampi were separated immediately for western-blotting or the brains were embedded in paraffin for immunohistochemical expenments.
Result
1. In normal rats, FDP significantly downregulated the BDNF mRNA expression (after 1 day,2 days and 4 days administration). However the TrkB mRNA level did not significantly downregulated.
2. Following the administration of kindling stimulations to the right ventral hippocampus, the relative abundance of BDNF mRNA significantly increased after 5 stimulations. This change continued until the completion of 48 stimulations. Consistent with the increase in BDNF levels, there was a significant upregulation of TrkB mRNA after 24 stimulations. This upregulation was transient, and the expression returned to the baseline level at the end of 48 stimulations. In animals pretreated with FDP, the BDNF mRNA level was further significantly downregulated after 5 kindling stimulations than the control, and TrkB mRNA level was conspicuously downregulated after 24 stimulations.
3. Immunohistochemistry results showed that the BDNF protein expression was downregulated in the rats pretreated with FDP in the CA1, CA3 and DG regions of hippocampus, which was consistent with the results of Western-blotting analysis.
4. FDP had significant upregulation on the KCC2 mRNA expression after 2 and 4 days administration and there was a significant downregulation of FDP alone on the NKCC1 mRNA expression after 1 day and 4 days administration.
5. Without FDP treatment, the relative copies of KCC2 mRNA decreased after 5 stimulations and this change continued until the completion of 48 stimulations. However, the expression levels of NKCC1 mRNA did not change significantly during the kindling process. Compared with the control rats, the KCC2 mRNA level increased significantly after 24 and 48 stimulations in the animals pretreated with FDP. A significant reduce in NKCC1 mRNA expression was observed after 5 and 24 stimulations in the FDP pretreated animals.
6. Immunofluorescence staining of KCC2 antibody and Hoechst showed that the K+-C1-co-transporter protein was localized in the membrane of neurons. Immunohistochemistry showed that the KCC2 protein expression was upregulated in the rats pretreated with FDP in the CA1, CA3 and DG regions of hippocampus
Conclusion
1. Stimulation induced enhancement of BDNF and TrkB expression, indicating that the BDNF/TrkB signaling pathway may participates in the kindling models.
2. In our experiment, FDP can decrease BDNF expression before kindling and reduces BDNF expression in early phase of kindling. This reduce was probably mediated by FDP itself, rather than secondary to reduction of seizure severity.
3. KCC2 was downregulated in late phase of kindling, indicating that chloride homeostasis may modulate rapid kindling and supporting the role of "GABAergic excitation" in epileptogenesis.
4. FDP may also play its antiepileptogenic role by regulating the intracellular chloride level and GABAergic excitation in neurons.
引文
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