Spontaneous hair-cell renewal following gentamicin exposure in postnatal rat utricular explants.
We have established an in vitro model of long-time culture of 4-day-old rat utricular maculae to study aminoglycoside-induced vestibular hair-cell renewal in the mammalian inner ear. The explanted maculae were cultured for up to 28 days on the surface of a membrane insert system. In an initial series of experiments utricles were exposed to 1 mM of gentamicin for 48 h and then allowed to recover in unsupplemented medium or in medium supplemented with the anti-mitotic drug aphidicolin. In a parallel control series, explants were not exposed to gentamicin. Utricles were harvested at specified time points from the second through the 28th day in vitro. Whole-mount utricles were stained with phalloidin-fluorescein isothiocyanate and their stereociliary bundles visualized and counted. In a second experimental series 2'-bromo-5'deoxyuridine labeling was used to confirm the antimitotic efficacy of aphidicolin. Loss of hair-cell stereociliary bundles was nearly complete 3 days after exposure to gentamicin, with the density of stereociliary bundles only 3-4% of their original density. Renewal of hair-cell bundles was abundant (i.e. 15x increase) in cultures in unsupplemented medium, with a peak of stereociliary bundle renewal reached after 21 days in vitro. A limited amount of hair-cell renewal also occurred in the presence of the anti-mitotic drug, aphidicolin. These results suggest that spontaneous renewal of hair-cell stereociliary bundles following gentamicin damage in utricular explants predominantly follows a pathway that includes mitotic events, but that a small portion of the hair-cell stereociliary bundle renewal does not require mitotic activity.
Berggren
Diana
D
Department of Otolaryngology, Albert Einstein College of Medicine, New York, NY, USA.
Liu
Wei
W
Frenz
Dorothy
D
Van De Water
Thomas
T
D008267:Acoustic Maculae / Q000166:cytology / Q000187:drug effects; D000818:Animals; D000831:Animals, Newborn / Q000502:physiology*; D016590:Aphidicolin / Q000494:pharmacology; D005839:Gentamicins / Q000494:pharmacology*; D018069:Hair Cells, Vestibular / Q000187:drug effects* / Q000502:physiology*; D008938:Mitosis / Q000187:drug effects; D009924:Organ Culture Techniques; D051381:Rats; D017208:Rats, Wistar; D012038:Regeneration; D012444:Saccule and Utricle / Q000166:cytology / Q000187:drug effects*; D013997:Time Factors
D005839:Gentamicins; D016590:Aphidicolin