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Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
Umeå University, Faculty of Social Sciences, Umeå School of Business and Economics (USBE), Statistics.
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2012 (English)In: Hearing Research, ISSN 0378-5955, E-ISSN 1878-5891, Vol. 283, no 1-2, 107-116 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Amsterdam: Elsevier, 2012. Vol. 283, no 1-2, 107-116 p.
National Category
Otorhinolaryngology Neurology
URN: urn:nbn:se:umu:diva-50805DOI: 10.1016/j.heares.2011.11.003ISI: 000301475300011PubMedID: 22127330OAI: diva2:469007
Available from: 2011-12-22 Created: 2011-12-22 Last updated: 2016-08-02Bibliographically approved
In thesis
1. Hair cell regeneration in vestibular epithelia: a study in an in vitro model
Open this publication in new window or tab >>Hair cell regeneration in vestibular epithelia: a study in an in vitro model
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]


Hair cells (HCs) are the sensory receptors in both the auditory and the vestibular organs of the inner ear. Supporting cells (SCs) are non-sensory cells embracing the HCs. Injuries of the HCs by aging, acoustic trauma or ototoxic drugs (mainly aminoglycosides, e.g. gentamicin) and cisplatin, often cause permanent impairment of hearing and balance. Birds and amphibians can regenerate their auditory and vestibular HCs after injury through proliferation of SCs or direct transdifferentiation of a SC into a HC. For mammals this ability is limited and spontaneous HC regeneration occurs only in the vestibular sensory epithelia. The utricle is one of the five vestibular organs and contributes to our balance by registering linear acceleration and head tilts. The aim of this PhD thesis was to investigate morphological and morphometric events during spontaneous HC regeneration following gentamicin exposure in neonatal rat utricular explants.


Long-term organ culture of macula utriculi, which is stable and reproducible for up to 28 days in vitro (DIV), was used in all papers in the thesis. HC damage was induced by gentamicin. On 2 DIV the explanted utricular maculae were divided into two groups, a control group and a gentamicin-exposed group. In the latter group macular explants were exposed to gentamicin for 48 hours during 2-3 DIV and then allowed to recover. Morphologic and morphometric evaluations were done from utricles harvested at various time points during 28 DIV. Imaging techniques used were light microscopy, including immunohistochemistry, and transmission electron microscopy.


In the control group the epithelia were well preserved with a slight decline in HC density after 14 DIV. In the gentamicin-exposed group there was an initial substantial decline in HC density and thereafter the proportion of HCs in relation to SCs increased significantly. Using BrdU as a proliferation marker and myosin 7a as a HC marker, we found no cells that were double marked. At the ultrastructural level, the apical occlusion of the explanted epithelia was intact in both the control and the gentamicin exposed group during the entire in vitro period. Cells that seemed to be in a transitional state, transforming from SCs into HCs were observed in the gentamicin-exposed group. These cells had cytoplasmic extensions basally i.e. foot processes, an assembly of mitochondria basally in the cell or in these foot processes, and often apical SC extensions covering the HC. HCs classified as transitional cells had an increased number of SC connections to their basal parts compared to mature HCs.


In these neonatal rat utricular explants:

- The morphological structure of the sensory epithelia was well preserved during long-term culture.

- The renewal of hair cells after gentamicin exposure occurred through direct transdifferentiation of supporting cells into hair cells.

- There was also a proliferative response by the supporting cells, but this supporting cell proliferation did not contribute to the generation of new hair cells.

- Cells in a transitional state, showing a characteristic morphology, were observed during the process of transdifferentiation from supporting cells into hair cells.

- The tight junctional seal of the epithelia stayed morphologically intact also after gentamicin exposure.

- Gap junctions were observed in between supporting cells but not found in between hair cells and supporting cells or between transitional cells and supporting cells.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2016. 46 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1804
Vestibular hair cell, regeneration, transdifferentiation, proliferation, utricle, rat
National Category
Research subject
urn:nbn:se:umu:diva-124077 (URN)978-91-7601-524-7 (ISBN)
Public defence
2016-09-09, Hörsal D, Undervisningsnod T9, byggnad 1D, plan 9, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Available from: 2016-08-19 Created: 2016-07-13 Last updated: 2016-08-22Bibliographically approved

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