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A method for long term culture of utricular macula explants is demonstrated to be stable and reproducible over a period of 28 days in vitro (DIV). This culture system for four-day-old rat utricular maculae is potentially suitable for studies of hair cell loss, repair and regeneration processes as they occur in post-natal mammalian inner ear sensory epithelia. The cellular events that occur within utricular macula hair cell epithelia during 28 days of culture are documented from serial sections. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) and the assistance of morphometric computer software. Ultrastructural observations were made with transmission electron microscopy (TEM) for describing the changes in the fine detailed morphological characteristics that occurred in the explants related to time in vitro. After 2 DIV the density of HCs was 77%, at 21 DIV it was 69%, and at 28 DIV it was 52% of HCs present at explantation. Between 2 DIV and 28 DIV there was a 1.7% decrease of the vestibular macula HC density per DIV. The corresponding decrease of SC density within the utricular explants was less than 1% per DIV. The overall morphology of the epithelia, i.e. relationship of HCs to SCs, was well preserved during the first two weeks in culture. After this time a slight deterioration of the epithelia was observed and although type I and type II HCs were identified by TEM observations, these two HC types could no longer be distinguished from one another by LM observations. In preparations cultured for 21 DIV, SC nuclei were located more apical and further away from the basal membrane compared to their position in macula explants fixed immediately after dissection. The loss of cells that occurred was probably due to expulsion from the apical (i.e. luminal) surface of the sensory epithelia, but no lesions of the apical lining or ruptures of the basal membrane were observed. There were no significant changes in the volume of the vestibular HC comprising macular epithelium during the observation period of 28 DIV.

We have studied aminoglycoside-induced vestibular hair-cell renewal using long-term culture of utricular macula explants from 4-day-old rats. Explanted utricles were exposed to 1 mM of gentamicin for 48 h, during 2nd and 3rd days in vitro (DIV), and then recovering in unsupplemented medium. Utricles were harvested at specified time points from the 2nd through the 28th DIV. The cellular events that occurred within hair cell epithelia during the culture period were documented from serial sectioned specimens. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) with the assistance of morphometric software. Ultrastructural observations were made from selected specimens with transmission electron microscopy (TEM). After 7 DIV, i.e. four days after gentamicin exposure, the density of HCs was 11% of the number of HCs observed in non-gentamicin-exposed control explants. At 28 DIV the HC density was 61% of the number of HCs observed in the control group explant specimens. Simultaneously with this increase in HCs there was a corresponding decline in the number of SCs within the epithelium. The proportion of HCs in relation to SCs increased significantly in the gentamicin-exposed explant group during the 5th to the 28th DIV period of culture. There were no significant differences in the volume estimations of the gentamicin-exposed and the control group explants during the observed period of culture. Morphological observations showed that gentamicin exposure induced extensive loss of HCs within the epithelial layer, which retained their intact apical and basal linings. At 7 to 14 DIV (i.e. 3-11 days after gentamicin exposure) a pseudostratified epithelium with multiple layers of disorganized cells was observed. At 21 DIV new HCs were observed that also possessed features resembling SCs. After 28 DIV a new luminal layer of HCs with several layers of SCs located more basally characterized the gentamicin-exposed epithelium. No mitoses were observed within the epithelial layer of any explants. Our conclusion is that direct transdifferentiation of SCs into HCs was the only process contributing to the renewal of HCs after gentamicin exposure in these explants of vestibular inner ear epithelia obtained from the labyrinths of 4-day-old rats.