Isolation And Identification Of Greater Epithelial Ridge In Rat Cochlea

The most common reason for sensorineural hearing loss is the degeneration of cochlear hair cells, resulting from noise overstimulation, ototoxic drugs, aging and other causes. Previous studies indicate that hair cells cannot regenerate after injury because they are terminally differentiated cells, and therefore hair cell loss often leads to permanent hearing problems. Over the past years, scientists have made great efforts on the study about mechanism of hair cell development and regeneration. How to induce the hair cell regeneration and functional restoration in the damaged auditory and vestibular systems is the main research direction all along.Hair cell regeneration is considered as the reappearance of embryonic hair cell development. Therefore, the understanding of hair cell differentiation may be invaluable for regeneration study. The vestibular hair cells of mammalian derive from the progenitors and supporting cells in sensory epithelium in a manner similar to lower vertebrates, while the exact origin of cochlea hair cell in mammal is still not clear up to now. Histological demonstration shows that IHCs and OHCs in the process of embryonic development may derive respectively from GER and LER.Recently, the greater epithelial ridge (GER) of mammalian cochlea has attracted researchers' attention. There are some evidences that the GER may be one of the progenitor cells pools of hair cells. Overexpression of Mathl, a positiveregulator gene for hair cell differentiation, in postnatal rat cochlear explant cultures resulted in new hair cells in GER region. Some of the important genes and growth factor receptors that participate in hair cell differentiation and regeneration such as P27, Hesl, Mathl and FGFR1 are all expressed in different period of GER. These data indicate that GER cells may play important role in hair cell differentiation and regeneration and serve as the progenitor cells of hair cells. So far we know little about the GER. To investigate how to isolate active GER cells, we employed a combinatorial approach of enzymatic digestion and mechanical separation. The isolated GER cells were identified by Morphologic comparison, immunohistochemical staining and RT-PCR analysis and their activity was verified by primary culture.Part one: The isolation of GER in rat cochleaCochlea sensory epithelial sheets were prepared by incubation of basilar membrane dissected from postnatal day 0(P0) SD rats in thermolysin and DNase in D-Hanks' solution for 25 min at 37℃. The GER cells were isolated using mechanical separation technique under high power microscope. The junction between epithelium and connective tissue of cochlea became loosened after incubated in thermolysin, thus the CES including GER, LER, IHCs and OHCs could be easily trimmed down from the basilar membrane using fine tungsten knife. The GER sheets were cut away from the inner of CES.Part two: The identification of rat cochlear GER1. MorphologyThe dissociated CES was embedded in Epon for semithin section and stained with borax toluidine blue. Postnatal rat cochlea was embedded in OCT for frozen section and stained with hematoxylin and eosin. Observationally, CES only...