Antiepileptogenic Action Of Fructose-1,6-diphosphate On Electrical Kindling Models And Its Potential Mechanisms

BackgroundEpilepsy 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.ObjectiveTo 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.Method1. 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.Result1. 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.Conclusion1. 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.ObjectiveTo 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. Method1. 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.Result1. 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.Conclusion1. 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.ObjectiveTo 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. Method1. 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.Result1. 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 hippocampusConclusion1. 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.