| BackgroundEpilepsy is one of the most common neurological disorders characterized by recurrent spontaneous seizures caused by aberrant discharge of neurons. An approximately 1 % of the world population suffer from epilepsy, and there are at least 6 million patients with epilepsy in China. Although epilepsy can be caused by genetic facters, which is called idiopathic epilepsy; it is estimated that up to 50% of all epilepsy cases are initiated by neurological insults and are called acquired epilepsy (AE). AE develops in 3 phases: (1) central nervous system (CNS) injury (acute insult phase), (2) epileptogenesis (latency), and (3) the chronic epileptic phases (spontaneous recurrent seizure).Status epilepticus (SE), stroke, and traumatic brain injury are 3 major examples of common brain injuries that can lead to the development of AE. The epileptogenesis is the process by which an injury such as SE, stroke, or traumatic brain injury produces long-term plasticity change in neurons, resulting in spontaneous recurrent seizures (acquired epilepsy). It is especially important to understand the molecular mechanisms that cause AE because it may lead to innovative strategies to prevent or cure this common condition. Recent studies have offered new insights into the cause of AE and indicate that injury-induced alterations in intracellular calcium concentration levels [Ca2+]i and calcium homeostatic mechanisms play a role in thedevelopment and maintenance of AE, and the Ca2+ hypothesis of epileptogenesis is proposed. The injuries that cause AE are different, but they share a common molecular mechanism for producing brain damage—an increase in extra cellular glutamate concentration that causes increased intracellular neuronal calcium, leading to neuronal injury and/or death. Neurons that survive the injury induced by glutamate and are exposed to increased [Ca2+]i are the cellular substrates to develop epilepsy because dead cells do not seize. The neurons that survive injury sustain permanent long-term plasticity changes in [Ca2+]i and calcium homeostatic mechanisms that are permanent and are a prominent feature of the epileptic phenotype. Although some researches studied the acute changes of neuron [Ca2+]i caused by seizures, the long-term alteration of calcium homeostatic mechanisms in chronic epileptic neurons is less reported. The study of the prolonged alteration in neuronal calcium dynamics can prove that the Ca2+ hypothesis plays an important role in the induction and maintenance of the prolonged neuroplasticity changed underlying the epileptic phenotype.The mechanisms of glutamate receptors, especially NMDA receptor, has been well emphasized in epileptogenesis research, Voltage-dependent Ca2+ channels(VDCCs) are also critical for nerve function, which represent the primary route for translating electrical signal into the biochemical events underlying key processes such as neurotransmitter release, cell excitability, gene expression, and long-term potential. Research evidence has accumulated indicates that VDCCs might play a role in epilepsy. Some VDCCs, such as P/Q-type VDCC, which modulating the release of neurotransmitters, maybe have more effect on epileptogenesis, because the mutation of the gene CACNAIA which code the α 1A subunit of P/ Q-type VDCC may cause epilepsy and episodic ataxia. However, molecular cloning, expression and biochemical studies indicate that VDCCs have high diversity in the discrete brain regions and even within individual neurons. Thus, VDCC diversity is thought go confer neurons with the ability to tailor Ca2+ influx to the demands of discrete functional compartments. A little was studied about the changes of VDCC in the epileptogenesis and the relations between the VDCC and long-term calciumhomeostasis and epileptogenesis.Because of the significant regulatory and signaling role of Ca2+ in neurons as a important second messenger, basal elevations in [Ca2+]i, although slight in magnitude, may sustain alterations in Ca -dependent enzyme systems, calcium/calmodulin kinase Ⅱ (Ca2+/CaMK Ⅱ) is a major Ca2+ messenger component signal transduction, and an important part in Ca2+/CaM dependent protein kinase family that regulates many Ca2+ dependent Processes in neurons. Ca2+/CaMKⅡ is abundant in CNS, and constitutes 2% of total hippocampus protein. It is predominantly expressed in neurons rather than glial cells. The α -subunit is homologous to the major postsynaptic density (PSD) protein which constitutes up to 50% of the total PSD protein. Ca2+/CaMK Ⅱ, as a neuronally enriched enzyme, regulates many important cellular functions, Ca2+/CaMK Ⅱ phosphorylates and regulates gene transcription, neuroskeletal elements, neurotransmission, and it is also involved in hippocampal LTP. Significant alteration of Ca2+/CaMK Ⅱ activity has been observed in many models of ischemia, glutamate excitotoxicity, and acute seizure activity, even in some models of neuronal cultures. Therefore the alterations of neuronal [Ca2+]i after neuronal damage, including SE, ischemia, etc. may induce the alteration of Ca2+/CaMK Ⅱ activity, which may be involved in the epileptogenesis. At present, the cellular mechanisms regulating the alteration of Ca2+/CaMK Ⅱ activity is still unclear, moreover, the alteration of Ca2+/CaMK Ⅱ activity in chronic epilepsy models is less reported.Lithium-Pilocarpine can induce SE, which may cause the hippocampus damage, and the damage of hippocampus induce the plastic change of epileptogenesis, at last, the rat represent recurrent spontaneous seizures. The phenotype of Lithium-Pilocarpine induced epilepsy is like the temporal lobe epilepsy of human, and provide a wonderful animal model for the study of human temporal lobe epilepsy - a common refractory epilepsy in human. This study mainly addresses the long-term alteration of calcium homeostatic, the change of P/Q-type calcium channel α 1a subunit, the alteration of Ca2+/CaMK Ⅱ activity and the α subunit expression in chronic Lithium-Pilocarpine model of temporal lobe epilepsy. ObjectiveTo investigate the dynamic changes of [Ca 2+]i levels in the hippocampal neurons, the expression of P/Q-type calcium channel α 1A subunit mRNA and proteins, and the alteration of Ca2+/CaMK Ⅱ activity in the hippocampus of rats during different periods after lithium- pilocarpine injection, we first developed the chronic epileptic model of rats by Lithium-Pilocarpine ip, and investigated the changes of epileptic rats in praxiology, electrophysiology and pathology. Afterwards, we systemically discuss the role of [Ca2+]i changes and calcium homeostatic mechanisms in the development and maintenance of AE, and the effects of P/Q-type calcium channel α 1A subunit, Ca2+/CaMKⅡ on the development of epilepsy. Methods1. Adult rats were injected lithium 3mEq/Kg , Scopolamine methylnitrate 1mg/kg, pilocarpine 30mg/Kg in appropriate times, and were injected diazepam after SE 1.5 hours. Afterwards we investigate the spontaneous seizures every day, record EEG of rat at 6h, 24h, 72h, 7d, 14d, 30d, and study the pathological changes with HE, Nissl, Timm stain.2. Hippocampal neurons were acutely isolated from control and different time after lithium pilocarpine induced SE, and the [Ca 2+]i levels in hippocampal neurons were detected by using laser scanning confocal microscope and Ca2+ fluorescent dyes Fura-2/AM. And the baseline [Ca 2+]i levels and the ability to restore resting [Ca 2+]i levels after a brief exposure to 5 μ M glutamate in control and epileptic neurons were evaluated.3. P/Q-type calcium channel α1A subunit mRNA were detected By using RT-PCR at different time points in the dentate gyrus, hippocampus and cerebrum, and P/Q-type calcium channel α 1A immunohistochemistry were used to analyze the temporal- spatial changes after lithium pilocarpine induced epilepsy.4. Hippocampus and cerebrum were acutely isolated from control and different time after lithium pilocarpine induced SE, Left tissues were used for investigating Ca2+/CaMKⅡ activity, which was expressed as phosphate incorporated into the enzyme per minute (pmol · min-1· mg-1); Right tissues were used for RT-PCR, fordetecting the expression of α -subunit of Ca2+/CaMK Ⅱ in hippocampus and cerebrum at different time points after lithium pilocarpine induced epilepsy. Results1. After injection of lithium and pilocarpine, 91.7% of the rats were induced to SE, and after a latency phase, average about 14 days, spontaneous recurrent seizures could appear. EEG showed accumulated spike waves in acute SE phase, EEG is normal during the latency phase, and EEG may show epileptic waves during the chronic epileptic phase. The pathologic investigation using HE Nissl, Timm staining and electron microscope showed the hippocampus neuron damage and mossy fiber spouting.2. The[Ca2+]i level of acute separated hippocampal neurons from the control rats was 95.4±22.1nM. After injection of lithium pilocarpine, the [Ca2+] i level of acute separated hippocampal neurons increased dramatically to 867.6 ± 35.2nM, and decreased to 292.8+ 18.3nM at 7th day, then was lasting at about this high level, even after 1 month, [Ca2+] i remained at 220.8 + 17.6nM, which is higher than the control ones. The distribution of neuronal [Ca2+]i showed that 92% neurons of control rats are in normal range [Ca2+] i level (25- 150nM) ; after SE 6 hours, all neurons [Ca2+] i level increased, and 85% of them were higher than 500nM; after SE 7 days, 75% of neurons [Ca2+] i level were higher; after 14 days ,60% were higher; even after SE 30 days, 52% of neurons still manifested elevated [Ca2+] i level, but no one was higher than 500nM (P<0.01). After exposure to 5 μM glutamate treatment for 2min, [Ca2+] i of the normal control neurons can restored to baseline values in 9.5 + 3.4min, whereas the SE group, epileptogenesis and chronic phases all exhibited a statistically significant delay in the returning to baseline values(P<0.01).3. The immunohistochemistry study of P/Q- type calcium channel α1A subunit showed a increasing of α 1A subunit-positive cells and average optical density in hippocampus and dentate gyrus after SE 1d(P<0.05), and reached the peak at the 3rd day(P<0.01), lasted to the 7th day(P<0.05), then decreased to the normal levels after SE 14 days. Semi-quantitive RT-PCR showed, the a1A subunit mRNA expression in hippocampus increased after SE 6h(P<0.05), reach the peak at the 3rd day(P<0.01),lasted to the 7th day, and decreased to normal level after SE 14 days. The expression of α 1A subunit mRNA in cerebellum had no difference during epileptogenesis compared with control group.4. The hippocampal Ca2+/CaMK II values (pmol ·min-1·mg-1) was 243.7± 11 .4, and cerebellar Ca2+/CaMKⅡ values was 105.3 ±8.5 in the control rats. Compared with the controls, the hippocampal Ca2+/CaMK Ⅱ activity decreased significantly (P<0.01) after lithium-pilocarpine induced epilepsy, and lasting to 1 month(P<0.05), while the cerebellar Ca2+/CaMKⅡ activity did not changed during the course of epileptogenesis(P>0.05). Semi-quantitive RT-PCR showed, the expression of Ca2+/CaMK Ⅱ α -subunit mRNA in hippocampus and cerebrum never changed during the course of epileptogenesis(P>0.05). Conclusions1. Lithium-pilocarpine induced epilepsy develops in 3 phases: ①the acute injury phase(status epilepticus); ② the latency phase(epileptogenesis); ③ the chronic epileptic phase(spontaneous recurrent seizure). And lithium pilocarpine can cause neurons damage and mossy fiber spouting.2. Lithium-pilocarpine induced epilepsy causes a long-term alteration of calcium homeostatic mechanisms of hippocampus neurons, which may plays an important role in the development and maintenance of spontaneous recurrent seizures in lithium-pilocarpine model.3. P/Q-type calcium channel α1A subunit expression may increase in hippocampus and dentate gyrus during the early phase of epileptogenesis, which may play a role in the induction of spontaneous recurrent seizures and the damage of neurons in lithium-pilocarpine model.4. There is a significant, long-lasting inhibition of hippocampal Ca2+/CaMKⅡ activity in lithium-pilocarpine chronic epilepsy model, which is not associated with the Ca2+/CaMK Ⅱ α subunit mRNA expression. Lont-lasting inhibition of this enzyme may play a functional role in epilepgenesis and the maintenance of spontaneous recurrent seizures in the temporal lobe epilepsy model.SignificanceOur study using the lithium pilocarpine chronic epilepsy model, investigated the long-term plastic changes of calcium homeostatic mechanisms, P/Q-type calcium channel α 1A subunit expression and Ca2+/CaMK Ⅱ activity in hippocampus, and showed their roles in the development and maintenance of spontaneous recurrent seizures in lithium-pilocarpine model. At precent, as many as 20% — 30% of medicated epilepsy patients have inadequate seizure control, called intractable epilepsy, and many anti-epileptic drugs have serious side effects and lifelong medication may be required. So it has been proposed, recently, that the focus of epilepsy research should be shifted to curing epilepsy as defined by "no seizures and no side effects". To attain this goal requires a more detailed understanding of the mechanism of epileptogenesis. Study targeting alterations in Ca2+ homeostatic mechanisms induced in the development of AE may offer novel strategies for the development on new anticonvulsant, antiepileptogenic, and possibly agents that may cure epilepsy. Thus, understanding the role of Ca2+ in the development and maintenance of AE may provide novel insights into therapeutic advances that will prevent and even cure epilepsy.
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