Research On Chronic Manganism Induced Hearing Loss In Rats

Part one：Effects of Chronic Manganism on Hearing and Cochlear CellsObjective：To study the effects of chronic manganism on hearing、cochlear cells andexpression of Mfn2in rats by using animal model of chronic manganism.Methods：Sixty adult Sprague-Dawley (SD) rats were randomly divided into Mn-exposedand control groups. Rats were treated with MnCl2·4H2O(100mg/kg/d) or deionized waterby gastric perfusion, lasted for12weeks. At4weeks,8weeks and12weeks after treatmentthe Mn concentration in peripheral blood was measured respectively. At12weeks aftertreatment, distortion product otoacoustic emissions(DPOAE) and auditory brainstemresponse (ABR)of SD rats were detected. The hair cells morphology and count wereobserved by stretched preparation of basilar membrane stained with FITC-phalloidin, andthe spiral ganglion cells morphology and count were observed by HE staining, theultrastructure changes of hair cells and spiral ganglion neurons were detected bytransmission electron microscopy. The expression of Mfn2was observed by Western blot.Results①The blood Mn concentration increased gradually with time after treatment.②ABR thresholds at4,8,16,24and32kHz were significantly increased at12weeks after treatment, especially in the high-frequency range.DPOAE amplitudes graduallydecreased from4kHz to40kHz.③Morphological study at12weeks after treatmentshowed loss of outer hair cells, mainly in the basal turn of the cochlea, and decreasednumber of spiral ganglion cells. The ultrastructure changes of outer hair cells and spiralganglion cells include the break-ups, disappearance or vacuolar change of mitochondriacristas.④Western blot showed up-regulated expression of Mfn2on the base membraneand SGN at12weeks after treatment.Conclusion: Our data demonstrate that chronic manganism can cause loss of outer haircells and spiral ganglion cells in cochlear of rats, lead to hearing loss and up-regulatedexpression of Mfn2，but its mechanism needs further study in future. Part two：Research on the Apoptosis of Mn-induced SGNs In VitroObjective: to establish the injury model of manganism on the spiral ganglion cells(SGNs) in vitro、observe the spiral ganglion cells morphology and study the translocationof Bax in the process of manganism induced apoptosis of SGNs.Methods：The primary cultured SGNs were divided into Mn-exposed and controlgroups.Exposure time were6h,12h and24h.Mn-exposed concentrations was5mM(MnCl2·4H2O). The SGNs morphology were observed by inverted microscope.Theapoptosis of SGNs was detected by β-tublin and Tunel immunofluorescence staining.The translocation of Bax was observed by Mitotracker Green FM and Baximmunofluorescent staining. The ultrastructure changes of SGNs were detected bytransmission electron microscopy.Resluts：①Under inverted microscope, manganese induced cell gap increasing、cellshrinkage and shorten cell axons at6h.Cell gap further increased and nuclear pyknosis were detected at12h and24h.②At12h and24h, manganese led to cells apoptosis by β-tublin and Tunel immunofluorescence staining.③At6h, manganese caused Bax focusedon the mitochondria surrounding；At12h and24h，Bax transferred from the cytosol to themitochondrial by the Mitotracker FM Green and Bax immunofluorescence staining.④Under transmission electron microscope,at6h heterochromatin margination andvacuolar change of mitochondria cristas.At12h and24h, apoptosis of SGNs was observed.Conclusionin: In vitro Manganese can induce apoptosis of spiral ganglion cells（SGNs）with Bax translocation and mitochondrial pathological changes.