Diazoxide Protects Against Cell Death Of Hippocampal Neurons Of Rats With Seizures Induced By Lithium-Pilocarpine

Epilepsy is a common nervous system disease that seriously endangers human health. It brings a heavy burden to social, family and patients, affecting more than 9 million patients in china. About 25-30% of patients suffer with intractable epilepsy. Temporal lobe epilepsy (TLE) presents the most prevalent refractory epilepsy. In recent years, the research of etiology, pathology and pathogenesis of TLE has made great progress, but pathogenesis of TLE has not yet been fully elucidated, and the usual administration of antiepileptic drugs (AEDs) and surgical therapy is often difficult to obtain significant curative effects. The alteration of behavior, electroencephalogram (EEG) and the hippocampal neuronal injury in pilocarpine-induced seizures in rats is similar to that in TLE patients, so pilocarpine-induced seizures have been one of the most frequently used models to research status epilepticus (SE) and TLE. Recurrent seizures or SE can cause neuronal injury has been widely recognized. However, the specific molecular mechanisms that repeated seizures cause neuronal damage are still not very clear, and so far have not been found particularly effective neuroprotective drugs. To further explore the etiology and pathogenesis of epilepsy, and search for new drugs with neuroprotective effects, has important theoretical and practical significance.Mitochondrial ATP-sensitive potassium channel (mitoKATP channel) is a potassium channel existing in the mitochondrial inner membrane. It has a dual physiological role, the first is to maintain K+ balance in mitochondria, which control the mitochondrial matrix volume changes; the second is in the mitochondrial oxidative phosphorylation to generate energy in the process, through reuptake of K+ can partially compensate for the charge transfer produced by the proton pump to maintain the transmembrane potential difference and the steady-state pH gradient. The channel is opened by GTP, GDP, diazoxide (DZ), cromakalin, and other channel openers. The mitoKATP channel is inhibited by ATP, ADP, 5-hydroxydecanoate (5-HD), glyburide and long-chain CoA esters. When mitoKATP channel opened, K+ could influx and reduce membrane depolarization, mitochondrial calcium overload, increasing matrix volume, which can increase the ATP synthesis, and promote mitochondrial respiration. MitoKATP channel has become a new research spot in the protective effect of ischemia-reperfusion injury, research objects including heart, brain, kidney, and so on. In animal models of ischemia and reperfusion, mitoKATP channel opener such as DZ, Pinacidil that can be simulated to protective effect of ischemic preconditioning, mitoKATP channel blocker 5-HD could block this effect. These indicated that mitoKATP channel play an important role in the ischemia and reperfusion models, and the protective effect mechanism similar to ischemic preconditioning.The pathophysiological process of seizures and ischemia-reperfusion is very different but there are much in common. Whether the mitoKATP channel opener has protective effects on brain injury caused by epileptic seizures has not yet report. At present study, we established epilepy model of rats induced by lithium-pilocarpine, to observe the pathology and molecular characteristics of hippocampal neurons damage in seizure; to observe the effects of DZ on neuronal ultrastructure and free radicals of hippocampus in epileptic rats induced by lithium-pilocarpine; and to investigate the effect of DZ on the mitochondrial apoptosis-related protein in hippocampus, so as to explore whether the mitoKATP channel opener DZ has neuroprotective effects on the hippocampal neuronal injury caused by epileptic seizures and further explore the potential mechanisms. PARTⅠA study of behavior, electroencephalogram and pathobiology in lithium-pilocarpine model of temporal lobe epilepsy in ratsObjectiveTo study the alteration of behavior, electrophysiology and pathology in lithium-pilocarpine-induced seizures in rats, and to explore the hippocampal neuronal injury of rats after SE.MethodsAdult male Wistar rats were given lithium-pilocarpine intraperitoneally to induce SE. The change of behavior in rats was observed. Seizures were allowed to last for 60 min and then were terminated by administration of diazepam. Rats were monitored by video recordings to assure development of seizures. EEG was recorded at 4 h after SE. HE and Nissl staining was used to study the pathological changes at 48 h after SE induced by pilocarpine.Results1.85.41% of the rats were induced to develop SE after administration of lithium and pilocarpine (according to Racine, the rats showing stage IV or V convulsive seizures were considered to develop SE successfully). The time from pilocarpine injection to the first onset of stageⅣSE was 37.5±17.8 min, and the death rate within 72 h (in acute phase) was 31.7%. The latency phase is about 13.6±6.4 days. In chronic phase, SRS were observed,78% of rats showed seizures at least once a week.2. At 4 h after SE (in acute phase), EEG in cortex and hippocampus of rats showed accumulated spike waves.3. HE staining and Nissl staining showed the neuronal damage in hippocampal CA1 regions and CA3 regions at 48 h after pilocarpine-induced seizures. The surviving neurons showed round and palely stained nuclei, meanwhile, the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body.ConclusionsLithium-pilocarpine could induce acute seizures (SE) and chronic seizures (SRSs) according to the alteration of behavior and EEG in rats. Seizures induced by lithium-pilocarpine could cause hippocampal neuronal damage in rats.PARTⅡEffects of diazoxide on neuronal ultrastructure and free radicals of hippocampus in epileptic rats induced by lithium-pilocarpineObjectiveTo observe the effect of the diazoxide (DZ), a mitochondrial ATP-sensitive potassium channel (mitoKATP channel) opener, on neuronal ultrastructure and free radicals in hippocampus in epileptic rats induced by lithium-pilocarpine, and explore the mechanism of the neuroprotective effect of DZ on epilepsy.MethodsAdult male Wistar rats were randomly divided into four groups:the control group, the epilepsy group (PILO group), the diazoxide group (DZ group), the diazoxide and 5-hydroxydecanoate (5-HD) group (DZ+5-HD group). Model of status epilepticus (SE) was induced by lithium-pilocarpine. Before preparation of the model, the DZ group was treated with DZ 5mg/kg, intraperitoneal inject (ip). The DZ+5-HD group was treated with 5-HD 8mg/kg, ip, then DZ 5mg/kg, ip. The change of behavior in rats was observed.4,24,48 hours after SE, the rats were sacrificed and the hippocampus were removed. The contents of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) were measured as the methods described by the commercial assay kits. At 48 h after SE, the neuronal ultrastructure was observed by electron microscope and the pathological changes were observed by Nissl staining. And the number of surviving neurons in hippocampal CA1 and CA3 regions in rats were examined.Results1. The epileptic paroxysm latent period of the DZ group was significantly prolonged. DZ prolonged the latent period of SE and decreased the mortality rate of animals with SE.2. In PILO group, there were obviously neural injury and neuron depletion in hippocampus (P<0.05); DZ significantly decreased the neuron depletion induced by SE (P<0.05).3. Under electron microscopy, the hippocampal neurons in PILO group and DZ+5-HD group were damaged severely after SE. The number of neurons was decreased, the neurons were swelled and some of them ruptured. There were numerous clumps distributed throughout the nucleus. Mitochondrial swelling was accompanied by disappearing of cristae and disruption of membrane integrity. In the more severe cases, mitochondria were vacuolar. However, the damage of neurons in DZ group was fewer severe.4. The level of MDA was dramatically increased and the activities of SOD, GSH-Px were significantly decreased after SE in PILO group (P<0.05). In DZ group, the MDA content was significantly decreased while the activities of SOD, GSH-Px were significantly increased compared to PILO group (P<0.05).5. The effect of DZ may be abolished by 5-HD treatment.ConclusionsThe mitoKATP channel opener DZ can decrease the level of the mitochondrial free radicals, thereby improve the response of anti-oxidant systems, reduce oxidative stress after SE, so reduce neuronal injury and neuron depletion, and thus play an important role in neuroprotection after SE. PART IIIThe Mitochondrial ATP-sensitive Potassium Channel Opener, Diazoxide, Protects Hippocampal Neurons From Apoptosis in Epileptic RatsObjectiveTo investigate the effect of DZ, a mitoKATP channel opener, on the mitochondrial apoptosis-related protein in hippocampus in epileptic rats induced by lithium-pilocarpine, further explore the mechanism of the neuroprotective effect of DZ on epileptic rats.MethodsModel of SE were induced by lithium-pilocarpine through intraperitoneal injection. To detect the time course of apoptosis-related protein levels of hippocampus, rats were divided randomly into control group, SE 24 h,72 h,5 d group. Next, to study the neuroprotective effects of DZ on hippocampus injury, rats were randomly divided into control group, PILO 72 h group (PILO group), PILO+DZ group, PILO+DZ+5-HD group. Neurons apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technology. The express of Bcl-2, Bax, activated caspase-3 proteins in rat hippocampus and release of cytochrome c (cytC) from mitochondria to cytoplasm were detected respectively by Western blotting.Results1. Compared with control group, the expression of Bcl-2 protein in rat hippocampus and cytochrome c in mitochondrial fraction (mito-cytC) in SE 24 h,72 h, 5 d group was markedly decreased (P<0.05). In contrast, the levels of Bax, activated caspase-3 proteins in hippocampus and cytochrome c in cytosolic fraction(cyto-cytC) in SE 24 h,72 h,5 d group were significantly increased (P<0.05). The positive cells of TUNEL staining in SE 24 h,72 h,5 d group were significantly increased (P<0.05).2. At 72 h after SE, compared with PILO group and PILO+DZ+5-HD group, the decrease in Bcl-2 protein levels induced by seizures was reversed markedly in PILO+DZ group (P<0.05), while the increased Bax, activated caspase-3 proteins levels were reversed in PILO+DZ group (P<0.05). The releases of cytC from mitochondria to cytoplasm were suppressed (P<0.05). The positive cells of TUNEL staining were markedly decreased in PILO+DZ group (P<0.05).3. The protective effect of DZ may be abolished by 5-HD treatment.ConclusionsDZ can protect hippocampal neurons from apoptosis in epileptic rats induced by lithium-pilocarpine by up-regulating Bcl-2/Bax, suppressing cytC release and inhibiting the activation of caspase-3, and the mechanism maybe related to inhibiting the mitochondrial apoptosis pathway. These beneficial effects may suggest that mitoKATP openers could be a possible new target for neuroprotective treatment of epilepsy.SignificanceIn this study, model of SE was induced by lithium-pilocarpine through intraperitoneal injection. To observe the effect of the DZ, a mitoKATP channel opener, on neuronal ultrastructure and free radicals and the mitochondrial apoptosis-related protein in hippocampus in epileptic rats induced by lithium-pilocarpine, results showed that DZ can decrease the level of the mitochondrial free radicals, improve the response of anti-oxidant systems, reduce oxidative stress after SE, and can inhibit the...