Nap Protects Hippocampal Neurons From Cell Death In Epileptic Rats Induced By Lithium-pilocarpine

Epilepsy is a common neurological disease that seriously endangers human beings’s health. It brings a heavy burden to the society, families and patients themselves. There are more than9million patients with epilepsy in china and about25-30%of them suffer with intractable epilepsy. Temporal lobe epilepsy (TLE) presents the most prevalent refractory epilepsy. The pathogenesis of TLE has not yet been fully elucidated although the research on the etiology, pathology and pathogenesis of TLE has made great progress in recent years, and it is difficult to gain significant curative effects through administration of antiepileptic drugs (AEDs) and operating. 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. As we all know, recurrent seizures or SE can cause neuronal injury, however, the specific molecular mechanisms that repeated seizures cause neuronal damage are still not very clear, and so far we have not found particularly effective neuroprotective drugs. To further explore the etiology and pathogenesis of epilepsy, and explore new neuroprotective drugs has important theoretical and practical significance.NAP (NAPVSIPQ) is an8-amino acid peptide derved from activity-dependent neurotective protein (ADNP),was recently discovered. The finding of NAP originated from studies on the peptidergic neurotransmitter vasoactive intestinal peptide (VIP). VIP binds to glial cells and activates a myriad of secondary effects including enhanced synthesis of neuroprotective proteins, such as the NAP containing ADNP. NAP (microgram per kilogram doses) exhibited neuroprotection in vivo and in vitro. It protects neuron to counter the damage caused by amyloid β peptide (Aβ), N-methyl-D-aspartate, the electric blockade, the envelope protein of HIV (GP120), dopamine, H2O2, and zinc overload cells; NAP can also protect the ApoE knockout mice, traumatic head injury, fetal alcohol syndrome and stroke. In recent years, there are more and more studies on the neuroprotective effects of NAP. such as in the irreversible focal cerebral ischemia model it can significantly reduce neural apoptosis. It is reported that NAP play its neuroprotective effect through the mitochondrial appoptic pathway.It has been widely recognized that NAP has anti-apoptotic and neuroprotective effects, but whether it 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; and explored the neuroprotective effects of NAP and its potential mechanisms.PARTIA study of behavior 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.To detect the alteration of the apoptosis-related protein(Bax, Bcl-2and activated caspase-3)levels of hippocampus in pilocarpine-induced seizures in rats, So to explore the molecular mechanisms of hippocampal neuronal damage in seizures. MethodsAdult male Wistar rats were divided randomly into control group,2h,6h、16h,、24h and72h after seizures groups. The seizures group were given lithium-pilocarpine intraperitoneally to induce SE. The change of behavior in rats was observed. Seizures were allowed to last for60min and then were terminated by administration of diazepam. HE and Nissl staining was used to study the pathological changes at24h after SE induced by pilocarpine. Neurons apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technology. The levels of the Bax, Bcl-2and the activated caspase-3in hippocampus in each group were detected by using western blot, and the level of ROS in hippocampus in each group were detected by using microplate reader.Results1.89.3%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 stage IV SE was39.5±18.6min.2. HE staining and Nissl staining showed the neuronal damage in hippocampal CA1regions and CA3regions at24h 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.3. Bax showed a significantly higher expression in pilocarpine groups than in control(p<0.05). In contrast, immunoreactivity of Bcl-2in the hippocampus was decreased after seizures(p<0.05). Moreover, the active cleaved-caspase-3appeared at24h after seizures.(p<0.05)4. The positive cells of TUNEL staining in SE24h group were significantly increased (p<0.05).5. The level of ROS was dramatically increased after SE, and reach the highest point at the6h group after SE.. ConclusionsLithium-pilocarpine could induce acute seizures (SE) according to the alteration of behavior in rats. Seizures induced by lithium-pilocarpine could cause hippocampal neuronal damage in rats.Our results also showed that pilocarpine-induced seizures regulated the mitochondrial apoptosis pathway. Caspase-3was activated finally to lead to neuronal death. We verified the molecular mechanisms of hippocampal neuronal injury induced by seizures, which provided theoretical evidence for the neuroprotective therapy in epilepsy. Part ⅡNAPVSIPQ Protects Hippocampal Neurons From Apoptosis in Epileptic Rats induced by lithium-pilocarpineObjectiveTo study the protective effects of NAP on hippocampal neurons after seizures induced by pilocarpine, and to investigate the effect of NAP on the the mitochondrial apoptosis related protein(Bax and Bcl-2and activated caspase-3) and the level of ROS in nerrons, So to explore the underlying mechanisms of neuroprotective effects of NAP in pilocarpine-induced seizures in rats.MethodsAdult male Wistar rats were randomly divided into4groups for treatment: control, pilocarpine, pilocarpine+saline and piloearpine+NAP. We performed Nissl staining at24h after seizures to examine the number of surviving neurons in hippocampal CA1and CA3regions in rats. The levels of Bax, Bcl-2and the activated caspase-3in hippocampus were detected by western blot and the level of ROS in the neurons were detected by microplate reader.Results1. Nissl staining showed the neuronal damage in hippocampal CA1and CA3regions at24h 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. Few cells showed the characters of dead neuron after NAP adminiatration.2. The number of surviving neurons at24h after seizures was decreased significantly as compared with the control group (p<0.05), moreover, NAP treatment significantly attenuated the neuronal loss induced by seizures(p<0.05). There was no significant difference in neuronal loss between the pilocarpine and pilocarpine+saline groups.3. NAP reversed the increased Bax and activated caspase-3levels at24h after pilocarpine treatment. There was no significant difference between pilocarpine group and pilocarpine+saline group.4. The level of ROS when pretreatment with NAP was dramtically decreased compared with the pilocarpine group.(p<0.05)ConclusionsOur results implied that NAP had neuroprotective effects which were exerted through inhibiting the mitochondrial apoptosis pathway and the oxidative stress. NAP could be a potential benefit treatment for the hippocampal neuron demise caused by pilocarpine-induced prolonged seizures. The study of the neuroprotective effects of NAP may provide novel insights into the therapy of epilepsy. SigniifcanceIn this study, model of SE was induced by lithium-pilocarpine throughintraperitoneal injection. To observe the effect of the NAPVSIPQ, an8-amino acidpeptid on oxidative stress and the mitochondiral apoptosis-related proteins inhippocampus in epileptic rats induced by lithium-pilocarpine, results showed thatNAP can decrease the level of ROS in neurons, improve the response of anti-oxidantsystems, reduce oxidative stress atfer SE, and can inhibit the mitochondiral apoptosispathway so reduce neuronal injury and play an important role in neuroprotection afterSE. Although the mechanism of the neuroprotective effect of NAP on neuronal injuryneeds further study, the present study illuminated the molecular biologicalmechanisms of hippocampal neuronal injury by seizures and the neuroprotectivemechanisms of NAP, which provided a possible new clue for neuroprotectivetreatment in epilepsy.