Change search
ReferencesLink to record
Permanent link

Direct link
Hair cell regeneration in vestibular epithelia: a study in an in vitro model
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background

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.

Methods

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.

Results

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.

Conclusions 

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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1804
Keyword [en]
Vestibular hair cell, regeneration, transdifferentiation, proliferation, utricle, rat
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
URN: urn:nbn:se:umu:diva-124077ISBN: 978-91-7601-524-7OAI: oai:DiVA.org:umu-124077DiVA: diva2:948823
Public defence
2016-09-09, Hörsal D, Undervisningsnod T9, byggnad 1D, plan 9, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-08-19 Created: 2016-07-13 Last updated: 2016-08-22Bibliographically approved
List of papers
1. Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants
Open this publication in new window or tab >>Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants
Show others...
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
National Category
Otorhinolaryngology Neurology
Identifiers
urn:nbn:se:umu:diva-50805 (URN)10.1016/j.heares.2011.11.003 (DOI)000301475300011 ()22127330 (PubMedID)
Available from: 2011-12-22 Created: 2011-12-22 Last updated: 2016-08-02Bibliographically approved
2. Morphological and morphometric characterization of direct transdifferentiation of support cells into hair cells in ototoxin-exposed neonatal utricular explants
Open this publication in new window or tab >>Morphological and morphometric characterization of direct transdifferentiation of support cells into hair cells in ototoxin-exposed neonatal utricular explants
Show others...
2015 (English)In: Hearing Research, ISSN 0378-5955, E-ISSN 1878-5891, Vol. 321, 1-11 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Otorhinolaryngology
Identifiers
urn:nbn:se:umu:diva-102465 (URN)10.1016/j.heares.2014.12.011 (DOI)000350933600001 ()25576788 (PubMedID)
Available from: 2015-05-19 Created: 2015-04-26 Last updated: 2016-08-18Bibliographically approved
3. Role of Transdifferentiation/Proliferation in Vestibular Hair Cell Renewal within Ototoxin-challenged Utricle Explants
Open this publication in new window or tab >>Role of Transdifferentiation/Proliferation in Vestibular Hair Cell Renewal within Ototoxin-challenged Utricle Explants
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Otorhinolaryngology
Identifiers
urn:nbn:se:umu:diva-124073 (URN)
Available from: 2016-07-12 Created: 2016-07-12 Last updated: 2016-08-02
4. Hair cell characteristics after hair cell renewal-An ultrastructural study of gentamicin exposed rat utricular explants
Open this publication in new window or tab >>Hair cell characteristics after hair cell renewal-An ultrastructural study of gentamicin exposed rat utricular explants
(English)Manuscript (preprint) (Other academic)
National Category
Otorhinolaryngology
Identifiers
urn:nbn:se:umu:diva-124074 (URN)
Available from: 2016-07-12 Created: 2016-07-12 Last updated: 2016-08-02

Open Access in DiVA

fulltext(48871 kB)47 downloads
File information
File name FULLTEXT01.pdfFile size 48871 kBChecksum SHA-512
24933e0acb2795e99f6907e97364a35f678896bf3553051b9fa952643a526fcaa9f56e6eed500b2cde49446e189329c6ac54e9a22d5ca31e0a1145aaafb31ba5
Type fulltextMimetype application/pdf
spikblad(75 kB)3 downloads
File information
File name FULLTEXT02.pdfFile size 75 kBChecksum SHA-512
646e3242f8f6fbd7abf55908e7374263eb005486a8ee178e9722ca3b95e3feea70284fc75453150b7f5a08212210ca10c2a00dcdb54ea7c8c542f45100f8cea3
Type spikbladMimetype application/pdf
cover(2294 kB)4 downloads
File information
File name FULLTEXT03.pdfFile size 2294 kBChecksum SHA-512
ff514e2e5d08ff0f5c031a96905bb52730fb45134e7b76b64e4e4371960a7ba014e3af9f6c8bc56f2b81bde875bc53c6f5b8707546e6d3acbc942439dc25ae14
Type coverMimetype application/pdf

Search in DiVA

By author/editor
Werner, Mimmi
By organisation
Otorhinolaryngology
Otorhinolaryngology

Search outside of DiVA

GoogleGoogle Scholar
Total: 54 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 777 hits
ReferencesLink to record
Permanent link

Direct link