Manganese-induced Damage and Apoptosis of Cochlear organotypic cultures

LI Dan; WANG Weilong; LU Lianjun; ZHANG Jianbin; MI Wenjuan; QIU Yang; CHEN Jun; SONG Yongli; TIAN Keyong; TANG Xiaoxu

doi:10.3969/j.issn.1672-2922.2016.02.028

Abstract：To study relations between manganese toxicity the damage and apoptosis of cochlear hair cells. Meth-ods Cochlear organotypic cultures were prepared using postnatal day 3 Sprague–Dawley rats. Mn chloride stock solu-tion was prepared at a stock concentration of 100 mM in serum-free medium and diluted to final concentrations of 1, 3 and 5 mM. Cochlear basilar membrane explants were incubated in the presence of the prepared Mn chloride solution. Ste-reocilia bundles of hair cells were immunolabeled with FITC phalloidin while nerve fibers were labeled with an antibody against theβ–tubulin so that hair cells and nerve fibers in the cochlear basilar membrane were respectively observed un-der a fluorescent micro-scope. TUNEL kit and Cleaved caspase-3 antibody were used to label apoptosis of basilar mem-brane cells. Results Cochlear organotypic cultures treated for 24 h with 1mM Mn showed no obvious damage to the hair cells and auditory nerve fibers in the cochlear basilar membrane compared to the control group (P>0.05). Mn treatment at 5 mM for 24 h caused noticeable damage to nerve fibers compared to the control (P<0.05). Damage by Mn treatment at 2 mM was more obvious in the basal turn than in the middle and apical turns. At 24 h of Mn treatment, the number of TU-NEL (as well as cleaved caspase-3) labeling cells increased along with increasing Mn concentrations. Conclusion In this in vitro organotypic culture of rat cochlea, Mn can cause missing of hair cells and reduced number and size of nerve fi-bers, which may be related to the mechanisms of cellular apoptosis.