Taylor, Ruth Rebecca; (2003) An In Vitro Study of Hair Cell Regeneration within the Inner Ear of the Newt, Notophthalmus viridescens. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The newt, Notophthalmus viridescens, an Urodele amphibian, is capable of regenerating many body parts and tissues. The newt presented a potential alternative to the avian and mammalian animal models in which to investigate the mechanisms of the hair cell regeneration in the auditory and vestibular organs. As part of the initial study, the morphology of the seven sensory epithelia were examined using several techniques. The main aim of this study was to assess recovery of hair cells and distinguish the means by which this recovery occurred. An in vitro system was established in which to characterize hair cell recovery following ablation with gentamicin. The level of apoptotic bodies in the epithelia following treatment demonstrated that hair cells were lost via programmed cell death. There was no evidence for repair of non-lethally damaged hair cells. Hair cell recovery was found to be robust but slow. Incubation with bromodeoxyuridine revealed a stimulation of proliferation amongst supporting cells, although new hair cells did not arise from these mitotic events. Hes1, p27kip1 and Ath1, known to be expressed during developmental patterning of the sensory epithelia in the mammalian inner ear, were investigated in newt sensory epithelia. All three were shown to be present in the saccule. The presence of Ath1 and p27kip1 was examined in undamaged tissue and during the recovery period following ablation of hair cells by immunohistochemistry. Labelling for p27kip1 remained unaltered throughout the recovery period, but no labelling with antibody to Ath1 was apparent in regenerated hair cells. The effect of the limb blastemal (growth zone) environment, known to induce cell cycle re-entry of other newt cells, was also investigated by the implantation of undamaged saccules. Implanted saccules were found to survive for up to six days within the blastema.

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