Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants

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.