Neuroprotective effects of melatonin on kainic acid-induced oxidative stress in organotypic hippocampal slice cultures

Abstract

[한글]

[영문]The brain is organ with an high oxygen consumption where a lot of free radicals are present there. These free radicals attack proteins, lipids, and DNA that consist of intracellular elements to modify, damage and finally cause various diseases. In order to protect the cells and organs from diseases by this oxidative damage, the importance of antioxidant is getting increased. Melatonin, one of the free radical scavengers, is known as an antioxidant that removes the reactive oxygen species (ROS) effectively in various oxidative damage models. It is regarded as a terminal antioxidant because melatonin makes stabilized end product from melatonin by removing free radicals not through oxidation-reduction cycle.The purpose of this study is to demonstrate the effectiveness of melatonin on kainic acid (KA)-induced oxidative stresses that affect various neurodegenerative diseases. The melatonin by different concentrations and secreted melatonin from cultured pineal gland were used in organotypic hippocampal slice cultures (OHSCs) model that causes oxidative damage by using KA. Postnatal 6~8 day rats were used for OHSCs model, and slices were cultured for 3 weeks. 5 μΜ KA was treated to cultured tissues for 18 hours, and neuronal damage and ROS formation from KA by were observed measuring PI uptake and spectrum of 2’,7’- dichlorohydrofluorescein diacetate (DCFH-DA) .To observe protective effects of melatonin, two experiments were carried out. First, KA- injured OHSCs model was treated with 0.01, 0.1 and 1 mΜ of melatonin to measure the effects of melatonin by different concentrations,. Second, the co-culture method together with KA injured OHSCs was used to observe the effects of melatonin that was secreted from pineal cells.Study Ⅰ demonstrated that 0.1 and 1 mΜ of melatonin protected neuronal cell death effectively at 24 and 48 hours after melatonin treatment after measuring the PI uptake. Besides, by measuring intact cells using cresyl violet staining, the measured data correlated with PI uptake at 48 hours after melatonin treatment. After measuring the DCFH-DA spectrum, ROS formation in 0.01, 0.1 and 1 mM melatonin treated groups after 24 hours were less than KA group. In order to detect the protein expression levels of 5-lipoxigenase (5-LO), caspase-3, calmodulin and superoxide dismutase-2 (SOD-2), western blot was carried out. 5-LO, caspase-3, calmodulin, and SOD-2 expression have similar tendency. All of the expressions increased in KA-induced injury group and melatonin treated groups had reduced expressions compared to the KA-injued group.Study Ⅱ, neuronal cell death decreased at 48 hours after the treatment with melatonin co-culture using fetal and adult pineal cells. PI uptake and cresyl violet staining was carried out from this method to detect the neuronal cell death and intact neuronal cells,. Fetal and adult pineal cells co-cultured groups significantly protected neurons from neuronal cell death by KA-injury. For measuring DCFH-DA, the spectrum was decreased at 24 hours after co-culturing fetal and adult pineal cells. The expression of 5-LO, caspase-3, calmodulin, and SOD-2 significantly decreased by fetal and adult cells compared with KA only-treated group. Therefore, these results demonstrated that melatonin that was secreted from pineal cells was effective for attenuating oxidative damage.Based on the result, it is suggested that melatonin is effective on KA-induced oxidative stress in OHSCs model.