Expression Changes And Meaning Of TREK-2 Potassium Ion Channel In Hippocampus And Entorhinal Cortex Of Rat In Pilocarpin-induced Epilepsy Model

Epilepsy is one of the most ancient diseases in the world and its seizure was first noted by the eminent Greek doctor Hippocrates in the 5 BC. It is a chronic disease caused by the sudden, repetitive and paroxysmal abnormal discharge of the brain neurons, lead to the temporary dysfunction of the central nervous system. Recently, epilepsy has become the second common disease of nervous system after stroke. The characteristic syndrome of epilepsy is repeated and unpredictable convulsion induced by repetitive discharge of the neurons. There are about 50 million epileptics need surgical intervention or medication around the world till now. However, the risk of surgery is high and it cannot eradicate the epilepsy. In addition, approximately 30% epileptics develop to intractable epilepsy because of antiepileptic drug resistance, and 70% of intractable epilepsy is temporal lobe epilepsy. Epilepsy not only reduces the life quality of the patients obviously, but also improves severe physiological and economic burden of family and society. Hence, to seek an effective way to treat epilepsy is a hot point in the research of epileptic treatment.Two-pore-domain potassium channels(K2P) is a newly discovered subtype of potassium channel. There are 15 members of mammalian K2 P channel family, which can be divided into six distinct subfamilies contains TWIK(tandem-pore-domain weakly inward rectifying potassium channel)、TASK(TWIK-related Acid-Sensitive K+ channels)、TREK(TWIK related two-pore-domain potassium channel)and so on. TREK-2 is the most important subtype of K2 P which is widely distributed and expressed among the K2 P. The molecular sequences and regulatory mechanisms of TREK-2 are similar to TREK-1 and TRAAK, hence they all belong to the TREK subfamily. TREK-2 has important physiological and neuro-protective functions. Researches had indicated that TREK-2 was widely expressed on the membrane of the neurons and played an important part in the regulation of the excitation of the neuron. And the electric physiology nature of epilepsy is a variety of mechanisms result in disorder of excitement and inhibition of balance, produce excessive synchronization discharge of neurons. But the research about the timing and spatial expression of TREK-2 in the epileptic rat model was seldom recently.Our research carried out on the lithium-pilocarpine-induced epileptic rat model and focusing on the TREK-2 expression characteristics of TREK-2 in the prone area of epilepsy in Li-Pilo model at different time point using Western-blot and immunofluorescence. Then the TREK-2 was down regulated by si RNA interference to reveal how TREK-2 inference the epileptic seizure. The aim of this study was to demonstrate that the TREK-2 was involved in the development of epilepsy, then provide the new target for the antiepileptic drug development and new therapy strategy of epilepsy. Experiment 1:Establishment of experimental lithium-pilocarpine-induced temporal lobe epileptic rat model.Objective: To view the behavioral, electroencephalographic and histological characteristics of the rats in the lithium-pilocarpine-induced temporal lobe epileptic model. Methods:(1) SPF male Sprague Dawley(SD) rats weighing 150~200g were acclimatized for 1 week before experiment. Rats was pretreated with lithium chloride 180mg/kg intraperitoneally(i.p.), followed by scopolamine 1mg/kg i.p. 18~20h later, then pilocarpine 30 mg/kg was injected i.p. 30 mins after scopolamine treatment. After the drug treatment, the behavioral alterations were observed according to the Racine scale. Status epilepticus(SE)( > Racine 4) was considered as model successfully established. Diazepam 10mg/kg i.p. was administered to stop the status epilepticus to reduce the mortality of the model rat. The rats in the control group was received the same does of saline at the same time point.(2) Electrodes were implanted over the cortex to record the EEG alternations before and after epileptic seizure.(3) HE staining was used to observe the morphological changes of the Hippocampus at the different time point after SE. Results: Rats in model group exhibited different degrees of cholinergic signs such as salivation, diarrhea and lacrimation immediately after pilocarpine injection. Seizure onset was happened 15~20mins around pilocarpine administration, revealed as a sequence of behavioral alternations including vibrissae moment, head bobbing, facial twitching, forelimb and/or tail extension, stiff posture, generalized clonic convulsion followed by generalized tonic-clonic seizures with stand or fall. 89.8% of the rats in the model group exhibited epilepsy-like seizures and the survival rate was 75%. The rats in control group did not see any epilepsy seizures.(2)The paroxysmal epileptiform discharge of EEG was seen in epileptic rat model, displayed as high amplitude cluster of spike waves, sharp waves and spike-sharp waves. However, the EEG of control rats just showing normal waking baseline and none of the three waves above was seen in this group.(3) Compared to the normal group, HE strain revealed that there existed different degrees of neurons deficiency in hippocampus region at the different time points after SE in model rats, the deficiency degree of CA1 and CA3 regions were more severer than CA2 and DG regions. Conclusion: Temporal lobe epileptic rat model was successfully established with relatively high survival rate and can be used in the following experiment. Experiment2: The expression characteristic of TREK-2 in hippocampus and EC region at the different time points after SE in epileptic rat model, and its role in epilepsy. Objective:To investigate the expression characteristic of TREK-2 in hippocampus and EC region at the different time points after SE in epileptic rat model, and its effects on the development of epilepsy. Method:According to the random number table, fifty six epileptic model rats were decapitated at 6h, 1d, 3d, 1w, 2w, 4w, 8w after SE,each time point of eight, and the tissue of right hippocampus and EC region were obtained for Western-blot to assess the expression of TREK-2.The harvested tissue were store at-80℃ before test, the eight rats in each control group were dealt the same as the epileptic model rats. Meanwhile, the other 3 rats in each time point were used for immunofluorescence assay to evaluate the TREK-2 expression. The 3 rats in each control group were dealt the same as the epileptic model rats. Results:(1)The results of Western-blot of hippocampus region indicated that, compared to control group, the expression of TREK-2 began to decrease at the 3d day after the induced SE(P<0.05); it was obvious decreased at the 1w, 2w, 4w(P<0.01); and remain maintained at the low level at 8w(P<0.001).(2) The Western-blot results in EC region demonstrated that, compare to control group, the expression of TREK-2 was obviously reduced at the 1d after the induced SE(P<0.001); but returned to the normal expression normal level at 2w(P>0.05); then continuous to reduce and reached the lowest level at 8w. The results of immunofluorescence assay of both hippocampus and EC region revealed the same tendency as Western-blot of each region. Conclusion:The expression level of TERK-2 was decreased at different extent at each time point after SE demonstrated that TREK-2 was participated in the development of epilepsy. Experiment3: Effects of down-regulation of TREK-2 expression in hippocampus and EC region on the seizure of epilepsy in pilocarpine-induced rat model. Objective: TREK-2 expression in hippocampus and EC region was down regulated by TREK-2 si RNA to investigate its effect on epileptic seizures in rat model. Methods: SPF male SD rat was placed on the stereotaxis instrument after anesthesia(10% chloral hydrate 0.3ml/kg i.p.). Hippocampus was located according to Paxinos and the neddle was inserted based on coordinate of the brain stereotaxic atlas. The injected point was reassured by the slice of brain tissue. Eighteen SD rats were randomized in to normal control group(sham), solvent group(no si RNA), scr RNA group(scr RNA), TREK-2 si RNA A group(5n M), TREK-2 si RNA-B group(5n M), TREK-2 si RNA-B group(10n M), 3 rats in each group. 2μl substrate of each group was administrated through microinjection at the rate of 0.5μl/min. The rats in control group was sham operated and not administrated any substrate. The hippocampus was harvested 72 h after injection to test the expression alternations of TERK-2 using Western-blot.(The sequence of si RNA A: 5’-UUC UCC GAA CGU GUC ACG UTT-3’; The sequence of si RNA B: 5’-TAG GAG GGC GGA AAT ACC AAA-3’).(2) The TREK-2 si RNA B sequence with the optimal interference efficacy was selected to use in the following experiment. SD rats were divided into epilepsy group and interfering epilepsy group. Hippocampus region of the rats in each group was injected with 2μl si RNA B or solvent respectively using the method described above. Epileptic rat model was established 72 h after injection, and the behavioral change of the rats in each group was recorded. Five survival rats in each group were sacrificed to harvest hippocampus for Western-blot to evaluate the expression of TREK-2. The experimental procedure of EC region was the same as Hippocampus. Results:(1) The results of Western-blot demonstrated no differences in the interference efficacy among control group, solvent group and scr RNA group, indicating that the interference of these three groups has no effects on the expression of TREK-2. si RNA B reduced the expression of TREK-2 about 50%(P<0.001),and there was no matter of the dose. Then, 5 n M si RNA B sequence was chosen in the following experiment.(2)Epileptic rat model was established with or without si RNA interference. Behavior observation revealed that the degree of seizures in interfering epilepsy group was severer than epilepsy group. In addition, the expression of TREK-2 in interfering epilepsy 1d group was significant lower than epilepsy group(P<0.01)and interfering group.Conclusion: TERK-2 si RNA interference has further demonstrated that TREK-2 involved in the development of epilepsy. Down-regulate the expression of TREK-2 could aggravate the severity of epilepsy.