| Background:Epilepsy is a chronic neurological disease characterized by abnormal discharge of neurons, of which about30%are intractable. As an effective anti-convulsant therapy, Ketogentic diet(KD) has been successfully used for almost a century for intractable epilepsy. Clinical studies found that KD is successful not only in various syndromes in the pediatric population, but also has been effectively used in adults and adolescents. KD is a diet of high fat, adequate protein and low carbohydrate. Currently antiepileptic properties of KD in the treatment of refractory epilepsy has been widely accepted by most scientists, gradually becoming a safe and effective therapy of drug non-response epilepsy and a variety of epilepsy syndromes.Animal experiments are important means to study the mechanism of KD. PTZ (pentylenetetrazol, PTZ) is the most widely used as the convulsant drug through the systemic administration way, which has been one of the most common used experimently to induce generalized tonic-clonic seizures(GTCS). Rodents are often injected of PTZ to induce generalized seizures for testing antiepileptic drugs(AEDs) broadly. PTZ can be injected by intraperitoneal, subcutaneous, or intravenous infusion. Timed tail intravenous infusion of PTZ is a good choice to explore the efficacy of KD on the thresholds of various seizures. While earlier studies just measured the clinical seizure thresholds in acute epilepsy models. There are seldom studies focusing on the efficacy of KD on the thresholds of epileptiform discharges in brain. Kindling is a great model to simulate the human complex partial and secondary generalized epilepsy by exploring the each phase in seizure building process, and also has an incomparable advantages in the study of epilepsy, including electrical kindling and chemical kindling. PTZ-kindling model is a more classic chemical kindled animal models, similar to the human absence epilepsy, myoclonic, clonic and generalized tonic-clonic seizures and can be used to study the formation and course of epilepsy mechanism. We have investigated the antiepileptogenic effect of KD on amygdaloid kindling seizure and its potentiall mechanisms. Therefore, our study was aimed to research efficacy of KD on acute and chronic PTZ-induced rats to identify a broad range thresholds of clinical seizures and epileptiform EEG discharges and to explore the formation process of epilepsy.KD have shown beneficial effects in terms of anti-epileptogentic properties in several clinical and experimental studies. However, the mechanisms undering KD are not known. Further validation of KD can also provide reference for other treatments in the future. Currently accumulating evidence reveals that a shift in y-aminobutyric acid type-A(GABAA) mediated excitation may be involved in epileptogenesis. The cell membrane chloride ion transporters and intracellular chloride ion concentration. K+-CI-cotransporter2(KCC2) and Na+-K+-2CI-cotransporterl(NKCC1) are two cation-chloride co-transporters(CCTs), controlling electrochemical potential gradient of chloride ions of neuronal membrane and driving them out of and into the cell. In vitro studies have shown that KD might modify the CCTs to regulate excitation of GABAA receptor. To understand the mechanism of KDs action on epilepsy, we explore whether the regulation of KCC2and NKCC1protein expression in cortex participate in epileptogenesis of KD. Methods:1. Postnatal day28male Sprague-Dawley(SD) were divided into two weight-matched groups. They were feeded by Ketocal or normal food for4weeks. Body weights were measured every three days. The rats were implanted four stainless steel crew into the cortex and an electrode to the right ventral hippocampus on P56. After a10-day period of recover, the rats were come into the procedure for threshold measurement.2. The threshold measurement of PTZ-induced seizures. A freshly prepared20mg/ml compound was infused at6ml/h using timed intravenous PTZ infusion, inducing a series of electroclinical seizures. Finally, all thresholds were expressed in miligrams of PTZ infused by kilograms of body weight of the rat.3. Behavioral testing of PTZ-kindlng model. Postnatal day28male SD rats were divided randomly into two weight-matched groups. Following four weeks of diet treatment, the rats were come into the procedure for PTZ-kindling model on P56. And the KD administration was still continued during the whole kindling. A freshly prepared PTZ solution at a sub-convulsive dose(35mg/kg, i.p.) was administered in rats without surgery on alternation days on13occasions until the rats were considered to be fully kindled. After each PTZ injection, convulsive behavior was observed for30min and classified into the stages according to a modified Racine scale, as well as the latency of myoclonic convulsion and clonic convulsion and so4. Finally, relative protein expression levels of KCC2and NKCC1are measured in cortex at pre-kindling (after a monthly diet treatment), myoclonic jerks and GTCS condition respectively using western blot.Results:1. Body weight in two groups increased through the four-week diet treatment. While the weight of KD was significantly lower than the ND group. KD group showed no different in their behavior and health compared with ND group except for an oily appearance of their fur, molting and being slightly lower in weight.2. Electroclinical thresholds of PTZ-induced seizures:during the procedure, the diet did not disturb each step of the seizure. The two groups had similar manifestation in behavior and EEG successively. We observed EEG thresholds in turn:isolate spike; followed by the first spike-and-wave discharge(SWD); subcontinuous SWD, were determined at the onset of the first SWD followed by recurrent SWDs lasting for>50%of the recording time; high-amplitude-spike-and-wave activity with single or double limbs clonic; polyspikes with severe GTCS. Considering behavior thresholds, we discovered from the clinical absence, first jerk, first overt myoclonia to clonic seizures in succession. The KD group experienced higher thresholds than the ND group for the first spike and wave, subcontinuous spike-and-wave, high amplitude spike-and-wave, and polyspikes. There was no difference in isolated spikes between the two groups. All EEG manifestations in the frontal cortex occurred earlier than that in the hippocampus in the two groups, and EEG thresholds in the frontal cortex were lower. EEGs in the right hippocampus had similar electrical manifestations as those described above. The KD group experienced higher thresholds than the ND group for the first spike and wave, subcontinuous spike-and-wave, high amplitude spike-and-wave, and polyspikes except for the isolated spikes. The KD inhibited cortical epileptiform discharges and reduced numbers of cortex polyspikes significantly compared with the ND group. Considering behavior thresholds:the KD group had significantly higher thresholds than the ND group for clinical absence, first jerk, first overt myoclonia, and clonic seizure. However, propagation of discharge of the first spike from the frontal cortex to the right hippocampus between the two groups was not significantly different. 3. Followed with the cumulative kindling, the two groups showed various similar behavior of increasing the severity and prolonging the duration of seuzures. The KD significantly reduced seizure severity during13instances of PTZ injection. The overall proportion of stage-4and stage-5scores was less while the overall proportion of stage-1scores was significantly higher if rats were fed a KD. Next, we analyzed the progression of PTZ-kindling seizures. The KD prolonged the latency of myoclonic convulsions compared with rats fed normal diets. The KD not only delayed the onset of clonic convulsions but also shortened their duration. Even if the rats developed GTCS, a KD could shorten its duration significantly.4. A more significant increase in expression of KCC2protein was induced in the KD group than in the ND group after a monthly of KD treatment before PTZ-kindling; following the PTZ-kindling model, the expression of KCC2protein of the KD group in myoclonic jerks and GTCS periods decresed gradually and remarkably, while compared with that of the ND group without any PTZ injection, there were still no statistical significance. During kindling, expression of KCC2protein in the two groups decreased as more severe seizures developed. However the KD group still showed higher expression of KCC2protein than the ND group irrespective of whether rats developed myoclonic jerks or GTCS. We found that the expression of NKCC1protein did not change after a monthly treatment without any PTZ injections compared with the ND group. With the increasing of PTZ injection number, there was a rising trend of the expression NKCC protein in the KD group, but it is no ststistical difference among each state. However, the expression of NKCC1protein in the ND group increased dynamically during the kindling and was significantly higher than that of the KD group in both myoclonic jerks and GTCS states.5. Conclusions:1. We have demonstrated that KD plays the important role of anti-epileptogenesis in PTZ-induced model, which may elevate various thresholds of behavioral seizures and epileptiform discharges. However the KD does not prevent these occurrence of electroclinical seizures.2. KD may have anti-epileptogenic effects in PTZ-kindling through reducing the severity of epilepsy and delaying the progression.3. KD may play its antiepileptogenic role by regulating the expression level of KCC2and NKCC1to change the excitation of GABAA receptor. |
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