Antiepileptogenic Effect Of Ketogenic Diet On Amygdaloid Kindling Seizure And Its Potential Mechanisms

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 â…  The antiepileptogenic effect of ketogenic diet to amygdaloid kindling seizurePurpose: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 â…¡ The potential mechanism of antiepileptogenic effect of ketogenic diet on amygdaloid kindling seizurePurpose: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.