The Study Of Protective Effects Of Magnesium Ion On The Inner Ear

Mg is the divalent cation most abundant in cells. It is required by hundreds of enzymes in the body, including any reaction with adenylate cyclase, ATP, Na + /K + - ATPase, and phospholipase C. This is understandable given the complexity of cochlear neurochemistry and neuropharmacology, the influence of Mg deficiency on inner cell function. In additional, magnesium has been called nature's physiological calcium channel blocker. As we known, under the situation of ischemic injury, hypoxia or noise damage et al, glutamate accumulation in the synaptic cleft of afferent neurons of inner ear leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca2+ overload, which initiates a cascade of events such as increasing of reactive oxygen species and cellular membrane permeability, and leads to inner cells death. In this study we utilize the cultured spiral ganglion cells and SD rats to explore the mechanism of protective effects of magnesium on inner ear, so as to provide some novel strategy for treatment of sensorineural deafness.PartⅠThe primary culture of spiral ganglion cells of newborn rats in serum free mediumObjective: To culture the spiral ganglion cells in vitro with serum free medium in order to farther investigate the protective effects of magnesium on spiral ganglion cells.Methods: The spiral ganglion from newborn rats was digested in 0.25% trypsin and 0.001% DNase. The SGCs suspension was plated at a density of 6×106 cells/ml. The cells were kept in serum free medium (DMEM +B27 supplement). 5μM cytosine arabinoside (Ara-C) was added to the medium on the second day and maintained for 48 hours. The morphology and the purity of SGCs was observed under microscope.Result: SGCs cultured by serum free medium (DMEM +B27 supplement) and Ara-C grew well, which were identified with mouse anti neurofilament protein antibody by immunohistochemistry methods. Highly purified neurons can be harvested.Conclusion: The method used in this study is an optimal means to culture and purify SGCs that can meet the needs of further study. PartⅡExcitotoxic effects of glutamate on the in vitro spiral ganglion cellsObjective: To understand the excitotoxic effects of glutamate on the cultured spiral ganglion cells (SGCs) of rat.Methods: Spiral ganglion cells culture in vitro, after exposure to 1mmol/L glutamate, damaged cells double-labeling with Hoechest33258 and PI were observed by fluorescence microscope, fluo-3 and LSCM for measurement of intracellular calcium levels.Results: Most cells were damaged and the intracellular calcium increased and after exposure to 1mmol/L glutamate ( P < 0.05).Conclusions: Glutamate excitotoxicity was associated with free intracellular calcium ion concentration elevation in SGCs.PartⅢProtective effects of magnesium ion on in vitro spiral ganglion cells damaged induced by glutamateObjective: To understand the effects of magnesium on survival of the cultured spiral ganglion cells (SGCs) of rat after exposed to glutamate.Methods: Spiral ganglion cells cultured in vitro, damaged cells double-labeling with Annexin and PI were observed by laser scanning confocal microscope(LSCM), fluo-3 and LSCM for measurement of intracellular calcium levels. Glutamate - induced neurotoxicity and magnesium protection were used in this study.Results: Most cells were damaged and the intracellular calcium increased and after exposure to 1mmol/L glutamate. Pretreatment of the cultured cells with magnesium (1mmol/L) could reduce the damage(P<0.05) and inhibit the increasing of intracelluar calcium.Conclusions: Glutamate excitotoxicity was associated with free intracellular calcium ion concentration elevation in SGCs, and magnesium could protect SGCs against glutamate neurotoxicity by inhibiting the increasing of intracelluar calcium.PartⅣEffect of dietary magnesium variety on hearing loss in acoustic trauma in ratsObjective: To study the influence of different dietary magnesium on acoustic trauma in rats, evaluating the role of magnesium in acoustic trauma.Methods: 24 Sprague Dawley male rats were divided into three groups, 8 each and fed with man-made dietary. The rates in the first group were fed Mg deficient diet (100 Mg mg/kg diet), in the second group were fed Mg adequate diet (507 Mg mg/kg diet), while in the third group fed Mg excess diet (1000 Mg mg/kg diet). After 20 days adaptation to pair-fed, a acoustic trauma experiment was carried on. All rats were exposed to octave band noise with a central frequency of 4 kHz, 105 dB SP L, for 3h. auditory brainstem response(ABR), distortion product otoacoustic emissions (DPOAE) were recorded; the magnesium concentration of serum and perilymph, the total antioxidant capacity ( TAC ) in serum were tested.Results: Comparing the ABR threshold, the change of DPOAE, the magnesium concentration of serum and perilymph, the total antioxidant capacity( TAC ) in serum of the third group with those of the first group and the second group after exposing to noise, the difference between them were all significant statistically.Conclusion: Mg deficiency could increase the susceptibility of rats to noise damage.