生酮饮食对杏仁核点燃癫痫形成的作用及相关机制研究
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摘要
研究背景：
癫痫(Epilepsy)是一种慢性神经系统常见病,其特点是神经元的异常放电。临床上大多数癫痫患者可以接受药物治疗控制癫痫发作,但仍有20%～30%的患者经药物治疗后难以得到有效控制。虽然抗癫痫药不断研发,但对于难治性癫痫,新型抗癫痫药治疗的疗效仍然有限,需要寻找其它的治疗手段和方法。生酮饮食(Ketogenic diet, KD)成为了研究的热点。
KD是一种高脂肪、低碳水化合物、低蛋白的饮食,目前认为KD是一种治疗药物难治性癫痫的安全有效的治疗手段,它对多种癫痫和癫痫综合症有效。尽管目前临床证实KD治疗癫痫有效,但KD的机制并不明确。研究KD的机制能让KD的过程更简化、更合理,减少潜在的严重副作用,提高对正常脑和癫痫活动时的代谢及生理基础的认识。进一步验证KD,也能为今后其它的治疗手段提供参考。
动物实验是研究KD机制的重要手段,点燃模型能很好模拟人类复杂部分性及继发全面性颞叶癫痫,除了能根据实验需要选择多个刺激点以外,它还能用于研究点燃建立过程中的各个时间点的变化,在研究癫痫的发生过程中有其它模型无法比拟的优势,但目前尚无KD作用于杏仁核点燃过程的研究。本研究拟选用杏仁核电点燃癫痫的模型来研究KD在抗癫痫形成过程中的作用,并观察KD对癫痫形成过程的神经保护作用及对苔藓纤维发芽的作用。
癫痫放电会导致大量的激活后效应,包括各种神经递质、受体、离子通道、细胞内信使和神经因子的mRNA和蛋白的改变。目前认为BDNF参与及调节了与癫痫的病理生理有关的许多机制,包括神经生长、神经突触的形成或调节兴奋性及抑制性的递质。糖酵解抑制剂2DG和FDP都能影响BDNF的基因表达,我们仍不清楚KD是否直接通过抑制BDNF的表达来发挥抗癫痫形成的作用。GABAA受体主要位于突触后膜上,介导快速突触后抑制。在癫痫持续状态模型中,BDNF调节JAK/STAT3通路影响GAB AAαl亚单位表达的下降。STAT3通路是颞叶癫痫的形成过程中一条重要的细胞传导途径,参与颞叶癫痫突触可塑性的重建机制。我们推测BDNF可能在KD抑制杏仁核点燃及KD对海马神经元的保护和对苔藓纤维发芽的抑制过程中发挥了调控作用,并有可能是通过STAT3通路影响GABAA受体起作用的。
第一部分生酮饮食对杏仁核电点燃模型的作用
目的：
观察生酮饮食在杏仁核慢性点燃过程中对癫痫形成的作用,同时,观察生酮饮食对杏仁核点燃模型海马及旁海马区神经元丢失和对海马苔藓纤维发芽的影响。
方法：
1.P28天SD大鼠按体重配对后分入KD组及ND组,喂食Ketocal及正常食物各4周,每隔2天测定体重,每周测定血p-羟丁酸。P56天手术,电极植入基底旁杏仁核,术后休息10天,进入电点燃程序。
2.两组大鼠测定初始后放电阈值(ADT),每天1次予以各自的初始ADT为刺激强度的电流刺激,诱发出癫痫发作,共20天。每天记录行为、根据Racine分级判定刺激后发作等级、后放电时程(ADD)、大发作潜伏期(GSL)等参数,每隔5天测定一次ADT。
3.经麻醉灌注后,两组大鼠各取刺激前(StiO)和刺激20次(Sti20)后两小时脑组织标本,处理后冰冻切片,行Nissl染色和Timm染色,观察海马及旁海马区神经元丢失情况及海马内分子层苔藓纤维发芽情况。
结果：
1.两组大鼠喂养过程中体重均增加,但KD组大鼠体重明显低于ND组。KD组大鼠除了皮毛油性多、脱毛外,其它生物学及行为学特征上和ND组比较并无显著区别。血β羟丁酸比较显示KD大鼠进食生酮饮食后血酮体水平很快明显增高,1周后达到持续酮血症的稳定状态,而ND组大鼠基本保持在一个相对比较低的酮体状态。
2.随着刺激次数的累加,两组大鼠行为学上均出现一系列的变化,发作等级总体上不断增加,ADD不断延长。生酮饮食显著延缓杏仁核点燃过程发作等级和ADD的增加。KD组停留在0级、2级的天数长于ND组。KD组达到2、3、4、5级所需要的刺激数均多于ND组。KD组比ND组在部分性癫痫阶段(1～3级)停留更长的时间,KD组发生大发作的发生率比ND组低。一旦被点燃,两组大发作的潜伏期无差异。随着刺激次数的增加,两组大鼠均出现ADT逐渐降低,KD对第5天的ADT降低有减缓作用,而这种作用在第10天和第15天消失。
3.点燃前神经元密度KD组双侧海马CA1区和刺激同侧DG区的神经元密度比ND组对应区域要小,但两组内嗅皮层和梨状皮层神经元密度无差异。
4.随着电刺激次数的逐渐增加,ND组大鼠海马及旁海马区神经元密度显著降低,在旁海马区,ND组大鼠刺激同侧梨状皮层神经元丢失,而刺激前后对侧梨状皮层神经元无明显丢失。刺激同侧和刺激对侧的内嗅皮层神经元均有不同程度丢失。电流刺激20次后,KD组相比于ND组能减少同侧海马CA1区的神经元丢失。但在CA、DG区或海马旁区,KD没有显示出这种保护作用。
5.两组大鼠刺激前同侧或刺激对侧海马CA3区Timm评分组间比较无显著差异,同一组内刺激同侧与刺激对侧也无差异。电流刺激20次后,ND组刺激同侧Timm分值有增高。KD组刺激同侧或对侧刺激前后无变化。
结论：
1.KD在杏仁核点燃模型癫痫的形成过程中有抗癫痫发生作用,可能是通过抑制后放电的形成起作用,这种作用主要发生在癫痫的部分性阶段。
2.KD能减少杏仁核点燃模型中轻-中度的海马区神经元的丢失,但对旁海马皮层无保护作用。
3.KD可能通过抑制海马的MFS而起到抗癫痫发生的作用。
第二部分生酮饮食抗癫痫发生机制的初步探讨
目的：
揭示KD在杏仁核点燃癫痫形成过程中通过对BDNF/STAT3/GABAA受体的影响来发挥抗癫痫形成或神经保护的作用。
方法：
1.通过Real-time qPCR测定喂食ND及KD两组大鼠海马杏仁核点燃模型各个时间点(电刺激0次、6次、12次、20次)BDNF、STAT3、GABAAα1受体(Gabra1)和GABAAa4受体(Gabra4)基因mRNA表达的变化；
2.通过Western blot方法半定量检测两组大鼠海马杏仁核点燃各个时间点(电刺激0次、6次、12次、20次)BDNF、总STAT3蛋白及磷酸化的pSTAT3的蛋白表达变化。
结果：
1.喂食正常食物大鼠BDNF mRNA随着杏仁核点燃过程的发展增高,在电刺激12次和20次相比电刺激前出现表达量的明显增高；KD组大鼠BDNF的增高不明显,在电刺激12次和电刺激20次,BDNF的mRNA表达量均低于ND组。
2.随着点燃次数的增加,正常对照大鼠的BDNF蛋白表达量增加,在KD组大鼠中,BDNF也出现随点燃过程的发展而表达增高的趋势,相比于ND大鼠,KD组在点燃第6次BDNF的蛋白量较少。
3.随着电刺激的次数的增加,ND组大鼠STAT3mRNA表达量基本无变化；KD组大鼠在电刺激6次、12次和20次STAT3的mRNA表达量均高于同一时间点ND组。
4.ND组大鼠总的STAT3蛋白量基本无变化,而pSTAT3的表达量随着时间逐渐增加；KD组大鼠pSTAT3在点燃早期就处于相对较高的表达量,随着点燃的发展,pSTAT3的表达量基本保持不变。
5.两组大鼠的Gabra1和Gabra4基因表达在杏仁核点燃各个时间点变化均无统计学差异。
结论：
1.杏仁核点燃过程中ND大鼠BDNF的基因表达和蛋白翻译都呈现动态增高改变,但两者并不平行：KD对BDNF mRNA表达的影响主要发生在点燃过程的中后期,而KD对BDNF蛋白表达的影响主要发生在点燃的早期。
2.ND大鼠杏仁核点燃过程中STAT3通路被激活。KD促进了点燃过程STAT3mRNA的基因转录及STAT3蛋白早期磷酸化,且这种磷酸化持续发生在整个点燃过程中。
3.杏仁核点燃过程各个时间点大鼠Gabra1和Gabra4表达变化不明显。KD组对这两个GABAA受体的亚型的表达也无影响。
Background:
Epilepsy is a chronic neurological disease, which is characterized by abnormal discharge of neurons. Most of the patients with epilepsy could be controlled by drugs, but there are still20%to30%patients hardly be effectively controlled. Although anti-epileptic drugs continuous research and development, but for refractory epilepsy, the new antiepileptic drug treatment appears to be limited. There is need to find other means and methods of treatment. Study on the ketogenic diet has become a hot spot.
KD is a high fat, low carbohydrates and low protein diet. KD is considered a safe and effective treatment for refractory epilepsy and it is valid for a variety of epilepsy and epileptic syndromes. Although it is clinically proved effective in the treatment of epilepsy, KD's mechanism is not clear. Study on mechanism of KD can make the administration more convenient and more palatable. Otherwise, we also can reduce potentially serious adverse effects, as well as provide insights into the metabolic and physiological basis of normal brain function and seizure activity. It is also further validate the diet and provide a template for future therapeutic improvements.
Animal experiments are important tools to study the mechanisms of KD. The antiepileptic effect of KD appears to be model-dependent. Kindling is a good model to imitate the human complex partial and secondary generalized temporal lobe epilepsy. Except for the advantage of choosing more stimulating points according to the experiments needs, it can also be used to study the exact changes each point in the kindling building process, which has advantages than other models during the study of epilepsy. However, there is no study of KD on epileptogenesis during amygdaloid kindling process. This study intends to research KD on amygdaloid kindling model in the formation of epilepsy, and observe the effect of KD on neuroprotection, as well as the effect on mossy fiber sprouting in rats.
Epileptic discharges can lead to activation change in the central nervous system, include various neurotransmitters, receptors, ion channels, intracellular messengers and neural factor mRNA and protein change. Now it is widely recognized that BDNF regulate many of the mechanisms of the pathophysiology of epilepsy, including nerve growth, formation of synapses or regulation of excitatory and inhibitory neurotransmitter.2DG and FDP, which are glycolysis inhibitors, can affect the expression of BDNF gene. We still don't know whether KD directly inhibits the expression of BDNF to play the role of antiepileptic formation. GABAA receptors, which mediate fast synaptic inhibition, mainly localized on the postsynaptic membrane. BDNF regulated GABAA receptors by protein phosphorylation. In the status epilepticus model, BDNF affects the expression of GABAA Alpha1subunit by JAK/STAT3pathway. STAT3pathway is an important cell pathway during the epileptogenesis of temporal lobe epilepsy, which affect the reconstruction of synaptic plasticity mechanisms involved in temporal lobe epilepsy. We speculate that BDNF has played an important role in the inhibition process of amygdaloid kindling, as well as protection and mossy fiber sprouting in hippocampus neuron. BDNF also probably regulate the GABAA receptor function through STAT3pathway.
Part Ⅰ The antiepileptogenic effect of ketogenic diet to amygdaloid kindling seizure
Purpose:
To observe the role of ketogenic diet plays to the epileptogenic process in the chronic amygdaloid kindling model. At the same time, to observe the effect of ketogenic diet to the hippocampus and hippocampal neuron loss, as well as the effects of hippocampal mossy fiber sprouting on amygdaloid kindling model.
Methods:
1. Postnatal day28male Sprague-Dawley were matched for weight and divided into two groups. They were feeded by Ketocal or normal food for4weeks. Body weights were measured every three days. The serum concentrations of beta-hydroxybutyrate were taken as a measure of ketonemia. The rats were implanted electrodes to the basolateral amygdale on P56. After a10-day period of recover, the rats were come into the procedure for kindling and threshold measurement.
2. After the determination of the initial after discharge of threshold (ADT), each subject was given their initial ADT to stimulus intensity current stimulation once a day for a total of20days. Seizure severity, discharge duration (ADD), generalized seizure latency(GSL) were determined daily. ADT were measured every5days.
3. After the anesthetic infusion, rats were killed before stimulation (StiO) and2hours after stimulate for20times (Sti20). Brain tissue was frozen, Nissl and Timm staining were observed for the loss of hippocampal neurons and hippocampal mossy fiber sprouting.
Results:
1. In both groups, body weight increased through the course of diet treatment. However, the weight of KD rats was significantly lower than the ND group. Except for an oily appearance of their fur, molting and being slightly lower in weight, the KD-fed rats showed no differen in their behavior and health compared with the ND-fed rats. KD induced persistent ketonemia one week later, the BHB levels in KD-fed rats were significantly higher than that the ND-fed group.
2. KD significantly delayed the progression of seizure stages and shortened the corresponding ADD compared with ND group. KD prevented the ADT decrease on day5compared to ND. However, there no group differences in the ADTs on day10or day15. In the KD group, rats stayed in both stage0and stage2for a longer durations for both. Compared with the ND group, KD also increased the number of stimulations required to reach stages2-5. In the KD group, the rats stayed in stages1-3for longer than the ND group. KD decreased the incidence of GS compared to ND, but did not prolong the latency period to the onset of GS in amygdala kindled seizures and ADD increased.
3. After4weeks of diet administration (before kindling), rats in the KD group showed a lower density of neurons than the ND group in the bilateral hippocampal CA1and hilus of the ipsilateral DG.
4. In ND group, the neuron density significantly decreased in the hippocampus and parahippcampal cortices during kindling. In ND group, there is obvious neuronal loss on ipsilateral Piriform cortex, while no obvious neuronal loss in contralateral piriform cortex after0and20stimulations. The neurons on bilateral entorhinal cortex suffered varying degrees of loss after0and20stimulations. After20stimulations, KD attenuated the neuronal loss in the ipsilateral hippocampal CA1region in the neuronal loss in the ipsilateral hippocampal CA1region in the KD group.
5. There was no significant difference of the Timm score on bilateral hippocampal CA3between the two goups of rats before the stimulation. For both groups, there was also no difference of the Timm score between the ipsilateral and contralateral CA3before stimulations. After20stimulations, ipsilateral CA3Timm score increased in ND groups, while there was no difference of bilateral CA3in KD group.
Conclusions:
1. We have demonstrated that KD has anti-epileptogenetic functions in amygdaloid kindling seizures, which are more prominent in the stage of partial epilepsy stages.
2. KD also has neuroprotective effects on hippocampal neurons although the neuronal loss was found to be mild in this model. However, the parahippocampal cortex does not protected by KD.
3. KD may play the role of antiepileptogenesis through inhibition of hippocampal MFS and change the hippocampal synaptic plasticity.
Part Ⅱ The potential mechanism of antiepileptogenic effect of ketogenic diet on amygdaloid kindling seizure
Purpose:
To investigate the BDNF, STAT3, Gabral and Gabra4mRNA expression during amygdale kindling and the translation of BDNF and STAT3, as well as phosphorylated of STAT3protein. We analyze whether BDNF, STAT3pathway and GABAA receptor participate in epileptogenesis and neuroprotection of KD.
Methods:
1. We analysis the rats hippocampal BDNF, STAT3, GABAA Alpha1receptors (Gabra1) and GABAA alpha4receptor (Gabra4) gene mRNA expression by real-time quantity PCR at each time point (day0, day6, day12and day20) during kindling in both ND and KD group;
2. We analysis the rats hippocampal BDNF, STAT3and pSTATt3protein by Western blot at each time point (day0, day6, day12and day20) during kindling in both ND and KD group.
Results:
1. BDNF mRNA increased during the development of amygdaloid kindling process in ND group, which was obvious on day12and day20. There was little increased BDNF in KD group, and it is significant difference between the two groups on day12and day20.
2. BDNF protein increased during kindling in ND group, as well as in KD group. However, the KD group translated little BDNF than ND rats at stimulation6,12and20.
3. There was no change of STAT3mRNA expression during the kindling in ND group. The STAT3mRNA expressions at stimulation12and20of KD group were higher than that in ND group.
4. There was no change in total STAT3protein in ND group but the expression of pSTAT3gradually increased over time. The pSTAT3expression in KD group increased during early stage of kindling, and it remained at a high level.
5. There were no significant differences of the Gabral and Gabra4gene expression between the two groups of rats.
Conclusions:
1. BDNF gene expression and protein translation was changed dynamic during the amygdale kindling in ND group. KD delayed BDNF mRNA expression at the late stages, but the effect on the translation of BDNF protein mainly occurred in the early stages of kindling.
2. STAT3gene expression levels were not changed in different stages of the amygdaloid kindling in ND group, KD promotes the STAT3mRNA transcription and the STAT3phosphorylation, which was persisting during the whole process of kindling.
3. The Gabra1, Gabra4mRNA expression is not obvious changed during the amygdaloid kindling process. KD also didn't affect on the two subtypes of GABAA receptor expression.
癫痫(Epilepsy)是一种慢性神经系统常见病,其特点是神经元的异常放电。临床上大多数癫痫患者可以接受药物治疗控制癫痫发作,但仍有20%～30%的患者经药物治疗后难以得到有效控制。虽然抗癫痫药不断研发,但对于难治性癫痫,新型抗癫痫药治疗的疗效仍然有限,需要寻找其它的治疗手段和方法。生酮饮食(Ketogenic diet, KD)成为了研究的热点。
KD是一种高脂肪、低碳水化合物、低蛋白的饮食,目前认为KD是一种治疗药物难治性癫痫的安全有效的治疗手段,它对多种癫痫和癫痫综合症有效。尽管目前临床证实KD治疗癫痫有效,但KD的机制并不明确。研究KD的机制能让KD的过程更简化、更合理,减少潜在的严重副作用,提高对正常脑和癫痫活动时的代谢及生理基础的认识。进一步验证KD,也能为今后其它的治疗手段提供参考。
动物实验是研究KD机制的重要手段,点燃模型能很好模拟人类复杂部分性及继发全面性颞叶癫痫,除了能根据实验需要选择多个刺激点以外,它还能用于研究点燃建立过程中的各个时间点的变化,在研究癫痫的发生过程中有其它模型无法比拟的优势,但目前尚无KD作用于杏仁核点燃过程的研究。本研究拟选用杏仁核电点燃癫痫的模型来研究KD在抗癫痫形成过程中的作用,并观察KD对癫痫形成过程的神经保护作用及对苔藓纤维发芽的作用。
癫痫放电会导致大量的激活后效应,包括各种神经递质、受体、离子通道、细胞内信使和神经因子的mRNA和蛋白的改变。目前认为BDNF参与及调节了与癫痫的病理生理有关的许多机制,包括神经生长、神经突触的形成或调节兴奋性及抑制性的递质。糖酵解抑制剂2DG和FDP都能影响BDNF的基因表达,我们仍不清楚KD是否直接通过抑制BDNF的表达来发挥抗癫痫形成的作用。GABAA受体主要位于突触后膜上,介导快速突触后抑制。在癫痫持续状态模型中,BDNF调节JAK/STAT3通路影响GAB AAαl亚单位表达的下降。STAT3通路是颞叶癫痫的形成过程中一条重要的细胞传导途径,参与颞叶癫痫突触可塑性的重建机制。我们推测BDNF可能在KD抑制杏仁核点燃及KD对海马神经元的保护和对苔藓纤维发芽的抑制过程中发挥了调控作用,并有可能是通过STAT3通路影响GABAA受体起作用的。
第一部分生酮饮食对杏仁核电点燃模型的作用
目的：
观察生酮饮食在杏仁核慢性点燃过程中对癫痫形成的作用,同时,观察生酮饮食对杏仁核点燃模型海马及旁海马区神经元丢失和对海马苔藓纤维发芽的影响。
方法：
1.P28天SD大鼠按体重配对后分入KD组及ND组,喂食Ketocal及正常食物各4周,每隔2天测定体重,每周测定血p-羟丁酸。P56天手术,电极植入基底旁杏仁核,术后休息10天,进入电点燃程序。
2.两组大鼠测定初始后放电阈值(ADT),每天1次予以各自的初始ADT为刺激强度的电流刺激,诱发出癫痫发作,共20天。每天记录行为、根据Racine分级判定刺激后发作等级、后放电时程(ADD)、大发作潜伏期(GSL)等参数,每隔5天测定一次ADT。
3.经麻醉灌注后,两组大鼠各取刺激前(StiO)和刺激20次(Sti20)后两小时脑组织标本,处理后冰冻切片,行Nissl染色和Timm染色,观察海马及旁海马区神经元丢失情况及海马内分子层苔藓纤维发芽情况。
结果：
1.两组大鼠喂养过程中体重均增加,但KD组大鼠体重明显低于ND组。KD组大鼠除了皮毛油性多、脱毛外,其它生物学及行为学特征上和ND组比较并无显著区别。血β羟丁酸比较显示KD大鼠进食生酮饮食后血酮体水平很快明显增高,1周后达到持续酮血症的稳定状态,而ND组大鼠基本保持在一个相对比较低的酮体状态。
2.随着刺激次数的累加,两组大鼠行为学上均出现一系列的变化,发作等级总体上不断增加,ADD不断延长。生酮饮食显著延缓杏仁核点燃过程发作等级和ADD的增加。KD组停留在0级、2级的天数长于ND组。KD组达到2、3、4、5级所需要的刺激数均多于ND组。KD组比ND组在部分性癫痫阶段(1～3级)停留更长的时间,KD组发生大发作的发生率比ND组低。一旦被点燃,两组大发作的潜伏期无差异。随着刺激次数的增加,两组大鼠均出现ADT逐渐降低,KD对第5天的ADT降低有减缓作用,而这种作用在第10天和第15天消失。
3.点燃前神经元密度KD组双侧海马CA1区和刺激同侧DG区的神经元密度比ND组对应区域要小,但两组内嗅皮层和梨状皮层神经元密度无差异。
4.随着电刺激次数的逐渐增加,ND组大鼠海马及旁海马区神经元密度显著降低,在旁海马区,ND组大鼠刺激同侧梨状皮层神经元丢失,而刺激前后对侧梨状皮层神经元无明显丢失。刺激同侧和刺激对侧的内嗅皮层神经元均有不同程度丢失。电流刺激20次后,KD组相比于ND组能减少同侧海马CA1区的神经元丢失。但在CA、DG区或海马旁区,KD没有显示出这种保护作用。
5.两组大鼠刺激前同侧或刺激对侧海马CA3区Timm评分组间比较无显著差异,同一组内刺激同侧与刺激对侧也无差异。电流刺激20次后,ND组刺激同侧Timm分值有增高。KD组刺激同侧或对侧刺激前后无变化。
结论：
1.KD在杏仁核点燃模型癫痫的形成过程中有抗癫痫发生作用,可能是通过抑制后放电的形成起作用,这种作用主要发生在癫痫的部分性阶段。
2.KD能减少杏仁核点燃模型中轻-中度的海马区神经元的丢失,但对旁海马皮层无保护作用。
3.KD可能通过抑制海马的MFS而起到抗癫痫发生的作用。
第二部分生酮饮食抗癫痫发生机制的初步探讨
目的：
揭示KD在杏仁核点燃癫痫形成过程中通过对BDNF/STAT3/GABAA受体的影响来发挥抗癫痫形成或神经保护的作用。
方法：
1.通过Real-time qPCR测定喂食ND及KD两组大鼠海马杏仁核点燃模型各个时间点(电刺激0次、6次、12次、20次)BDNF、STAT3、GABAAα1受体(Gabra1)和GABAAa4受体(Gabra4)基因mRNA表达的变化；
2.通过Western blot方法半定量检测两组大鼠海马杏仁核点燃各个时间点(电刺激0次、6次、12次、20次)BDNF、总STAT3蛋白及磷酸化的pSTAT3的蛋白表达变化。
结果：
1.喂食正常食物大鼠BDNF mRNA随着杏仁核点燃过程的发展增高,在电刺激12次和20次相比电刺激前出现表达量的明显增高；KD组大鼠BDNF的增高不明显,在电刺激12次和电刺激20次,BDNF的mRNA表达量均低于ND组。
2.随着点燃次数的增加,正常对照大鼠的BDNF蛋白表达量增加,在KD组大鼠中,BDNF也出现随点燃过程的发展而表达增高的趋势,相比于ND大鼠,KD组在点燃第6次BDNF的蛋白量较少。
3.随着电刺激的次数的增加,ND组大鼠STAT3mRNA表达量基本无变化；KD组大鼠在电刺激6次、12次和20次STAT3的mRNA表达量均高于同一时间点ND组。
4.ND组大鼠总的STAT3蛋白量基本无变化,而pSTAT3的表达量随着时间逐渐增加；KD组大鼠pSTAT3在点燃早期就处于相对较高的表达量,随着点燃的发展,pSTAT3的表达量基本保持不变。
5.两组大鼠的Gabra1和Gabra4基因表达在杏仁核点燃各个时间点变化均无统计学差异。
结论：
1.杏仁核点燃过程中ND大鼠BDNF的基因表达和蛋白翻译都呈现动态增高改变,但两者并不平行：KD对BDNF mRNA表达的影响主要发生在点燃过程的中后期,而KD对BDNF蛋白表达的影响主要发生在点燃的早期。
2.ND大鼠杏仁核点燃过程中STAT3通路被激活。KD促进了点燃过程STAT3mRNA的基因转录及STAT3蛋白早期磷酸化,且这种磷酸化持续发生在整个点燃过程中。
3.杏仁核点燃过程各个时间点大鼠Gabra1和Gabra4表达变化不明显。KD组对这两个GABAA受体的亚型的表达也无影响。
Background:
Epilepsy is a chronic neurological disease, which is characterized by abnormal discharge of neurons. Most of the patients with epilepsy could be controlled by drugs, but there are still20%to30%patients hardly be effectively controlled. Although anti-epileptic drugs continuous research and development, but for refractory epilepsy, the new antiepileptic drug treatment appears to be limited. There is need to find other means and methods of treatment. Study on the ketogenic diet has become a hot spot.
KD is a high fat, low carbohydrates and low protein diet. KD is considered a safe and effective treatment for refractory epilepsy and it is valid for a variety of epilepsy and epileptic syndromes. Although it is clinically proved effective in the treatment of epilepsy, KD's mechanism is not clear. Study on mechanism of KD can make the administration more convenient and more palatable. Otherwise, we also can reduce potentially serious adverse effects, as well as provide insights into the metabolic and physiological basis of normal brain function and seizure activity. It is also further validate the diet and provide a template for future therapeutic improvements.
Animal experiments are important tools to study the mechanisms of KD. The antiepileptic effect of KD appears to be model-dependent. Kindling is a good model to imitate the human complex partial and secondary generalized temporal lobe epilepsy. Except for the advantage of choosing more stimulating points according to the experiments needs, it can also be used to study the exact changes each point in the kindling building process, which has advantages than other models during the study of epilepsy. However, there is no study of KD on epileptogenesis during amygdaloid kindling process. This study intends to research KD on amygdaloid kindling model in the formation of epilepsy, and observe the effect of KD on neuroprotection, as well as the effect on mossy fiber sprouting in rats.
Epileptic discharges can lead to activation change in the central nervous system, include various neurotransmitters, receptors, ion channels, intracellular messengers and neural factor mRNA and protein change. Now it is widely recognized that BDNF regulate many of the mechanisms of the pathophysiology of epilepsy, including nerve growth, formation of synapses or regulation of excitatory and inhibitory neurotransmitter.2DG and FDP, which are glycolysis inhibitors, can affect the expression of BDNF gene. We still don't know whether KD directly inhibits the expression of BDNF to play the role of antiepileptic formation. GABAA receptors, which mediate fast synaptic inhibition, mainly localized on the postsynaptic membrane. BDNF regulated GABAA receptors by protein phosphorylation. In the status epilepticus model, BDNF affects the expression of GABAA Alpha1subunit by JAK/STAT3pathway. STAT3pathway is an important cell pathway during the epileptogenesis of temporal lobe epilepsy, which affect the reconstruction of synaptic plasticity mechanisms involved in temporal lobe epilepsy. We speculate that BDNF has played an important role in the inhibition process of amygdaloid kindling, as well as protection and mossy fiber sprouting in hippocampus neuron. BDNF also probably regulate the GABAA receptor function through STAT3pathway.
Part Ⅰ The antiepileptogenic effect of ketogenic diet to amygdaloid kindling seizure
Purpose:
To observe the role of ketogenic diet plays to the epileptogenic process in the chronic amygdaloid kindling model. At the same time, to observe the effect of ketogenic diet to the hippocampus and hippocampal neuron loss, as well as the effects of hippocampal mossy fiber sprouting on amygdaloid kindling model.
Methods:
1. Postnatal day28male Sprague-Dawley were matched for weight and divided into two groups. They were feeded by Ketocal or normal food for4weeks. Body weights were measured every three days. The serum concentrations of beta-hydroxybutyrate were taken as a measure of ketonemia. The rats were implanted electrodes to the basolateral amygdale on P56. After a10-day period of recover, the rats were come into the procedure for kindling and threshold measurement.
2. After the determination of the initial after discharge of threshold (ADT), each subject was given their initial ADT to stimulus intensity current stimulation once a day for a total of20days. Seizure severity, discharge duration (ADD), generalized seizure latency(GSL) were determined daily. ADT were measured every5days.
3. After the anesthetic infusion, rats were killed before stimulation (StiO) and2hours after stimulate for20times (Sti20). Brain tissue was frozen, Nissl and Timm staining were observed for the loss of hippocampal neurons and hippocampal mossy fiber sprouting.
Results:
1. In both groups, body weight increased through the course of diet treatment. However, the weight of KD rats was significantly lower than the ND group. Except for an oily appearance of their fur, molting and being slightly lower in weight, the KD-fed rats showed no differen in their behavior and health compared with the ND-fed rats. KD induced persistent ketonemia one week later, the BHB levels in KD-fed rats were significantly higher than that the ND-fed group.
2. KD significantly delayed the progression of seizure stages and shortened the corresponding ADD compared with ND group. KD prevented the ADT decrease on day5compared to ND. However, there no group differences in the ADTs on day10or day15. In the KD group, rats stayed in both stage0and stage2for a longer durations for both. Compared with the ND group, KD also increased the number of stimulations required to reach stages2-5. In the KD group, the rats stayed in stages1-3for longer than the ND group. KD decreased the incidence of GS compared to ND, but did not prolong the latency period to the onset of GS in amygdala kindled seizures and ADD increased.
3. After4weeks of diet administration (before kindling), rats in the KD group showed a lower density of neurons than the ND group in the bilateral hippocampal CA1and hilus of the ipsilateral DG.
4. In ND group, the neuron density significantly decreased in the hippocampus and parahippcampal cortices during kindling. In ND group, there is obvious neuronal loss on ipsilateral Piriform cortex, while no obvious neuronal loss in contralateral piriform cortex after0and20stimulations. The neurons on bilateral entorhinal cortex suffered varying degrees of loss after0and20stimulations. After20stimulations, KD attenuated the neuronal loss in the ipsilateral hippocampal CA1region in the neuronal loss in the ipsilateral hippocampal CA1region in the KD group.
5. There was no significant difference of the Timm score on bilateral hippocampal CA3between the two goups of rats before the stimulation. For both groups, there was also no difference of the Timm score between the ipsilateral and contralateral CA3before stimulations. After20stimulations, ipsilateral CA3Timm score increased in ND groups, while there was no difference of bilateral CA3in KD group.
Conclusions:
1. We have demonstrated that KD has anti-epileptogenetic functions in amygdaloid kindling seizures, which are more prominent in the stage of partial epilepsy stages.
2. KD also has neuroprotective effects on hippocampal neurons although the neuronal loss was found to be mild in this model. However, the parahippocampal cortex does not protected by KD.
3. KD may play the role of antiepileptogenesis through inhibition of hippocampal MFS and change the hippocampal synaptic plasticity.
Part Ⅱ The potential mechanism of antiepileptogenic effect of ketogenic diet on amygdaloid kindling seizure
Purpose:
To investigate the BDNF, STAT3, Gabral and Gabra4mRNA expression during amygdale kindling and the translation of BDNF and STAT3, as well as phosphorylated of STAT3protein. We analyze whether BDNF, STAT3pathway and GABAA receptor participate in epileptogenesis and neuroprotection of KD.
Methods:
1. We analysis the rats hippocampal BDNF, STAT3, GABAA Alpha1receptors (Gabra1) and GABAA alpha4receptor (Gabra4) gene mRNA expression by real-time quantity PCR at each time point (day0, day6, day12and day20) during kindling in both ND and KD group;
2. We analysis the rats hippocampal BDNF, STAT3and pSTATt3protein by Western blot at each time point (day0, day6, day12and day20) during kindling in both ND and KD group.
Results:
1. BDNF mRNA increased during the development of amygdaloid kindling process in ND group, which was obvious on day12and day20. There was little increased BDNF in KD group, and it is significant difference between the two groups on day12and day20.
2. BDNF protein increased during kindling in ND group, as well as in KD group. However, the KD group translated little BDNF than ND rats at stimulation6,12and20.
3. There was no change of STAT3mRNA expression during the kindling in ND group. The STAT3mRNA expressions at stimulation12and20of KD group were higher than that in ND group.
4. There was no change in total STAT3protein in ND group but the expression of pSTAT3gradually increased over time. The pSTAT3expression in KD group increased during early stage of kindling, and it remained at a high level.
5. There were no significant differences of the Gabral and Gabra4gene expression between the two groups of rats.
Conclusions:
1. BDNF gene expression and protein translation was changed dynamic during the amygdale kindling in ND group. KD delayed BDNF mRNA expression at the late stages, but the effect on the translation of BDNF protein mainly occurred in the early stages of kindling.
2. STAT3gene expression levels were not changed in different stages of the amygdaloid kindling in ND group, KD promotes the STAT3mRNA transcription and the STAT3phosphorylation, which was persisting during the whole process of kindling.
3. The Gabra1, Gabra4mRNA expression is not obvious changed during the amygdaloid kindling process. KD also didn't affect on the two subtypes of GABAA receptor expression.
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