Experimental Study On Melatonin's Antiepileptic Effect And Its Molecular Mechanisms

Epilepsy is a common chronic clinical syndrome in nerve system. There were about 50,000,000 patients suffered from epilepsy in the world and the incidence of epilepsy was 0.5%o-1%oper year. Temporal lobe epilepsy (TLE) is the most frequent type of human epilepsy, characterized by developing spontaneous recurrent epileptic seizures (SRS) and hippocampal sclerosis. These seizures are difficult-to-treat because of their resistant to common antiepileptic treatment. Approximately 10-30% of patients with epilepsy continue to have seizures despite medical treatment. Some of these patients show pharmaceutic toxicosis because of taking a great deal of medicaments for a long time; some of these patients cann't insist on taking medicaments because of the strong side effect of medicaments; some of these patients' mentality decline because of frequent seizures and(or) taking medicatmets for a long time. Although some of these patients can be improved by surgery to removal epileptogenic focus or interrupt the spread of epileptiform discharge, the surgery is traumatic and some of them are not good candidates for epilepsy surgery. Therefore, it is this group of patients for whom alternative treatments may be helpful.Melatonin (MT) is a hormone synthesized and secreted by the pineal gland during the night, synchronizing circadian systems to the environmental light/dark cycle. A number of neuropharmacological effects (including keeping circadian rhythm, improving sleep, enhancing immunofunction, etc.) of melatonin have been observed. Recently, other functions of melatonin have been suggested, including scavenging of free radicals, neuroprotection, and antiepileptic efficacy in various animal models of epilepsy. Most scholars have attached importance to antiepileptic action of melatonin. Datas showed that melatonin exerted antiepileptic effect in various animal models of epilepsy induced by pentylene tetrazole, quinolate, kainite, glutamate, N-methyl-D-aspartate, L-cysteme, cyanide, maximal electroshock and electrically kindled stimulation of amygdala. Moreover, melatonin could not only significantly reduce the dose of antiepileptic drugs, but alsoreduce the side effect of antiepileptic drugs. There was not obvious side effect when taking melatonin for a long time.Although the antiepileptic effect of melatonin has been reported in numerous reports, little is known about its antiepileptic properties and the mechanisms underlined antiepileptic action. The objectives of the present work were to study melatonin's antiepileptic properties on pilocarpine-induced seizures, and to probe the mechanisms of melatonin's antiepileptic role by observing the effect of melatonin on GABAergic system, the protective action on neuron in hippocampus and the influence on structural plasticity of hippocampus.Main methods and techniquesThe study was composed of four parts:1. Animal model of epilepsy was induced by pilocarpine (PILO) injected intraperitoneally (i.p.) at the dose of 300mg/kg. Electroencepalogram (EEG) and HE staining have been usd to observe the effect of melatonin on epileptiform discharge, changes of behavior and hippocampal morphology in epileptic rats at different dose and time of administration. The effect of melatonin on SRS has also been observed when rats were injected melatonin chronically.2. Immunohistochemistry and RT-PCR were used to detect the changes of GABA, Glu content, GAD67mRNA expression, GABA-immunoreactivity and expression of GABAa receptor a 5 subunit in hippocampus in epileptic rats induced by pilocarpine, and the effect of melatonin on these changes.3. Nissl staining, TdT-mediated dUTP Nick End Labeling (TUNEL) and colormetric method were used to detect neuronal apoptosis, expression of Caspase-3mRNA and protein, the content of malondialdehyde (MDA), glutathione(GSH) and the activity of glutathione reductase(GR) in hippocampus in epileptic rats administered with pilocarpine or with pilocarpine and melatonin, in order to reveal the mechanisms through which melatonin exerts its antiepileptic action.4. Timm staining, immunohistochemistry mothed and in situ hybridization were used to observed the changes of neurogenesis, mossy fiber sprouting (MFS), brain-derived neurotrophic factor(BDNF) mRNA expression in hippocampus in epileptic rats administered with pilocarpine or with pilocarpine and melatonin, and the relationshipbetween these changes and SRS so to probe the mechanisms through which melatonin could change structural plasticity of hippocampus and attenuate the susceptibility to epilepsy.Main results and conclusions1. Use model of epilepsy induced by pilocarpine injected intraperitoneally at the dose of 300mg/kg, rats showed first seizure after a latent period of 23.00±4.92min and then seizures developed to repeated generalized clonic seizures and finally to SE within approximately 30min. At the same time, EEG showed typically epileptiform discharge and a lot of neurons swelled and degeneralized especially in CA1-CA3 region of hippocampus. About two weeks after SE, rats showed SRS and the frequency of SRS was steady in 8 weeks' observation. These findings indicated that the model of epilepsy induced by pilocarpine was successful and efficiency.2. Melatonin could prolong the latency to the appearance of first seizure, but the role was faint and was depended on the time and dose of administration. Melatonin's antiepileptic effect appeared to be stronger in the light-dark transition and at the dose of 25 mg/kg. There was significant decrease in the number of SRS in epileptic rats treated with pilocarpine and melatonin compare to rats only treated with pilocarpine, which indicated that melatonin has a good antiepileptic action in epileptic rats induced by pilocarpine.3. Reduced GABAergic inhibition was a key factor for seizure generation and developing in rats post-SE induced by pilocarpine. Melatonin exerted antiepileptic action not only by upregulating expression of GAD67mRNA and enhancing GABA-immunoreactivity, content of GABA in hippocampus, but also by protecting hippocampal neurons against damage and enhancing expression of GABA A receptor a 5 in hippocampus of epileptic rats.4. The neuronal damage in hippocampus post-SE were more severe in rats only treated with pilocarpine than in rats treated with pilocarpine and melatonin. There was significant increase in the number of TUNEL positive cells in hippocampus in rats only treated with pilocarpine compare to control rats and rats treated with pilocarpine and melatonin. The findings indicated that neuron death in hippocampus included necrosis and apoptosis. Melatonin could protect hippocampal neurons against damage post-SE, mainly by inhibiting neuron apoptosis.5. There were significant increases in expression of Caspase-3 mRNA, Caspase-3 protein, content of MDA and significant decreases in content of GSH and GR activity in hippocampus at 6h~72h post-SE in rats treated with pilocarpine compare to control rats. These findings indicated that neuron apoptosis mediated by Caspase-3 and oxidative stress were involved in neuronal damage in hippocampus of rats post-SE.6. There were significant decreases in expression of Caspase-3 mRNA and Caspase-3 protein in hippocampus at 6h~72h post-SE in rats treated with pilocarpine and melatonin compare to rats only treated with pilocarpine, which indicated that melatonin interrupted cell apoptosis by inhibiting expression of Caspase-3 mRNA and Caspase-3 protein. There were signifiant decreases in content of MDA and significant increases in content of GSH and GR activity in hippocampus at 6h~72h post-SE in rats treated with pilocarpine compare to rats only treated with pilocarpine, which indicated that melatonin exerted neuroprotective action by enhancing neuronal antioxidative ability in hippocampus, inhibiting the produce of free radicles, attenuating brain lipid peroxidation damage.7. There was significant increase in the number of Brdu-labeled cells and intensity of supragranular MFS in rats treated with pilocarpine compare to control rats at 6h~28d post-SE. The findings indicated the change of neurogenesis in dentate gyrus and MFS may be a key factor for development of SRS. There was significant increase in BDNFmRNA expression in granule cell layer, pyramid cell layer and some interneuron in hilus in rats treated with pilocarpine compare to control rats. High expression of BDNFmRNA could last till 14d post-SE, which was consistent with the onset of MFS and synaptic reorganization. The findings indicated that change of BDNFmRNA expression might play an important role in onset of MFS and synaptic reorganization in hippocampus.8. Melatonin could inhibit neurogenesis in dentate gyrus and MFS in hippocampus of rats post-SE by regulating BDNFmRNA expression, which may be one of the most important mechanisms through which melatonin could prevent SRS.