Regulation Of Utricle Hair Cells Regeneration Through Inhibition FGF Signaling Pathway

OBJECTIVE The toxic effects of inner ear caused by gentamicin and other aminoglycosides can be manifested through hearing loss and balance dysfunction because of destruction of hair cells. But the extent of damage to utricular sensory epithelium caused by gentamicin and the regulation of inhibition of FGF signaling pathway in hair cell regeneration caused by overexpress Atoh1/Math1gene remains to be discussed in details. The objective of our study is to answer the questions go as follows:1. Make use of statistical methods to explore the degree of injury to hair cells and the proliferation of cells in different time periods and different regions (striolar region and extrastriolar region) in utricle cultured after gentamicin treatment.2. The effect of inhibition of FGF signaling pathway to the proliferation of cells and the formation of hair cell-like cells induced by Ad-Mathl in sensory region and nonsensory region of utricle cultured in vitro.3. Whether the hair cell-like cells induced by Ad-Mathl have the planer cell polarity (PCP) and study the mechanism of it.METHOD To establish an injury model of utriclar hair cells through the use of gentamicin in vitro. In this study, we explore the extent of damage caused by gentamicin through observing the destruction of the cilium of hair cell labeled by Phalloidin and the hair cells labeled by the specific antibody Myosinâ…¦a in different time periods and different regions (striolar region and extrastriolar region). To observe the cell proliferation labeled by generative cell-specific staining EdU in sensory region and nonsensory region. Immunofluorescence technique and SPSS were used to prove and analysis the ototoxic effects of gentamicin and evaluate the proliferation activity after gentamicin treatment. Following the Ad-Mathl-transfering tissues fixation and immunohistochemical staining in different time, laser scanning confocal microscope were used to vertify the regulation of Atohl/Mathl gene in the proliferation of cells and the regeneration of new hair cell-like cells in sensory region and nonsensory region. At the same time, when the utriclar tissues were cultured in the nutrient solution fixed with FGF signaling pathway blockers, Ad-Math1and/or gentamicin, the regulatin of inhibition of FGF signaling pathway to hair cell regeneration should be discussed intensively. In the end, take advantage of PCP specific antibodies, spectrin and r-tubulin, to observe the regular pattern of PCP in hair cell-like cells.RESULTS The morphological and statistical studies have shown that after the treatment of gentamicin, stereocilia bundle of cultured utricle was damaged at first, especially in striolar region. MyosinVIIa, the hair cell specific marker, positive cells was reduced with time. The sensory precursor cells labeled by Sox2were increased. But the proliferation of cells gradually decreased with the increase of gentamicin dose. The number of proliferating cells co-cultured in Ad-Math1and gentamicin was more than a simple addition of Ad-Math1or gentamicin in sensory region, especially in striolar region. When mixed with SU5402, the FGF signaling pathway blocker, in the meantime, there was no significant increase in the proliferating cells. But there is an increase in the number of hair cell-like cells which is more than a simple joined Ad-Math1group. The kinocilia of newbore hair cell-like cells migrate from the center of the cell surface to the side. The PCP specific antibodies, spectrin and r-tubulin, were utilized to demonstrate the PCP of newborn hair cell-like cells is consistent with the hair cells, which the stereociliary bundles are coordinately oriented along the mediolateral axis of the epithelia and the hair cells on either side of a line of polarity have opposite orientations. However, hair cell-like cells in nonsensory region oriented inconsistently.CONCLUSION The destruction of utricular hair cells after gentamicin treatment was began with stereocilia bundle and gradually spreaded to cell body, especially in striolar region. The transcription factor Sox2expressed an increase in striolar region. At the same time, the capacity of proliferation in sensory epithelial cells was improving. Over-expression of Atohl/Mathl can increase the proliferation of cells in sensory region and produce new generated hair cell-like cells in sensory region and nonsensory region. The regulatin of inhibition of FGF signaling pathway to hair cell regeneration induced by Ad-Mathl is positive. The planer cell polarity of new generated hair cell-like cells is consistent with the original hair cells, which was consisting in the formation of kinocilia in the embryonic period. While the PCP in hair cell-like cells in nonsensory region is in consonant.