Hair cell regeneration in vestibular epithelia -A study in an in vitro model

Mimmi Werner

Department of Clinical Sciences Otorhinolaryngology, Umeå University, Umeå, Sweden 2016

Publisher responsible under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-524-7 ISSN: 0346-6612, new series no 1804 Coverimage: Transitional cells by Vera Werner Elektronic version available at http://umu.diva-portal.org/ Printed by: Print och media Umeå, Sweden 2016

To Mårten, Vera, Lukas, Alvar and Sonja

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Table of Contents

Table of Contents iAbstract iiiAbbreviations vSammanfattning på svenska vi

Bakgrund viMetod viResultat viSlutsatser vii

List of Papers viiiIntroduction 1

1. Our senses for hearing and balance 12. Anatomy of the mammalian ear 13. Auditory and vestibular function 44. Morphology of the utricular sensory epithelia 45. Intercellular junctions in the utricular sensory epithelia 56. Development of the hair cell sensory epithelia 67. Regeneration of hair cells 68. Aminoglycoside ototoxicity 79. Hair cell marker 810. Proliferation marker 811. Choice of method: Organ culture of mammalian utricle 8

Aims of the thesis 10Materials and methods 11

Animals 11Organ culture technique and paradigms 11Light microscopy (LM) - Papers 1 and 2 12Transmission electron microscopy (TEM) - Papers 1, 2 and 4 13Cell counts - Papers 1 and 2 13Calculations of volume - Papers 1 and 2 14Immunohistochemistry - Paper 3 14Comparison of cell counts of BrdU+ and myosin 7a+ cells with morphometric cell counts of SCs and HCs - Paper 3 15Evaluation of HC characteristics and intercellular junctions - Paper 4 16

Results 18Morphology 18Paper 1 18Paper 2 18Paper 3 19Paper 4 20Cell counts 22

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Papers 1 and 2 22Paper 3 23Volume 23Papers 1 and 2 23

Discussion 24Techniques used for cell counting 24Findings that support renewal of vestibular HCs through direct transdifferentiation of SCs 25Repair of HCs 26Cells with two nuclei 27Diverse functions of the SCs 27Clinical implications 28Future research 29

Conclusions 31Acknowledgements 32References 35

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Abstract

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

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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.

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Abbreviations

AIC - Akaike´s information criterion ANOVA - analysis of variance Atho1 – atonal homologue 1 BrdU - 5-bromo 2`-deoxyuridine Group C - control group DIV - days in vitro Group G - gentamicin-exposed group HC - hair cell LM - light microscopy PBS - phosphate buffered saline SC - supporting cell TEM - transmission electron microscopy Wnt – “Wingless-related integration site” Wnt signalling pathways through cell surface receptors

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Sammanfattning på svenska

Bakgrund Hårceller är sensoriska receptorer för både hörsel och balans. Stödjeceller är icke sensoriska celler som omsluter hårcellerna. Skador på hårcellerna på grund av hög ålder, kraftiga ljud eller vissa läkemedel kan ge bestående förlust av hörsel och balans. Fåglar och groddjur kan återskapa sina skadade hårceller genom celldelning av stödjeceller eller genom att en stödjecell direkt omvandlas, transdifferentieras, till en hårcell. Hos däggdjur är förmågan att återskapa hårceller begränsad och sker spontant endast i balansorganen. Utrikeln är ett av innerörats fem balansorgan och dess hårceller registrerar linjär acceleration och lutning av huvudet.

Syftet med detta avhandlingsarbete var att utvärdera morfologiska förändringar under spontan återkomst av hårceller i odlingar av utriklar från råttor.

Metod Den metod för odling av sinnesepitel från utriklar som användes är stabil och reproducerbar i upp till 28 dagar. Hårcellsskada inducerades med hjälp av gentamicin som är ett antibiotikum kännt för att kunna orsaka hörsel- och balanspåverkan. Efter två dagar i odling indelades utriklarna i en kontrollgrupp och en grupp som exponerades för gentamycin. Utrikel odlingarna exponerades för gentamycin under 48 timmar dag 2-3 och fick därefter återhämta sig. De tekniker som använts för utvärdering är ljusmikroskopi, inklusive immunohistokemi, och elektronmikroskopi.

Resultat I kontrollgruppen var sinnesepitelet välbevarat med en liten minskning av hårcells densitet efter 14 dagar i odling. I den gentamycin exponerade gruppen sågs initialt en omfattande minskning av hårcells densitet. Därefter sågs en signifikant proportionell ökning av andelen hårceller i förhållande till stödjeceller. När BrdU användes som en markör för celldelning och myosin 7a som hårcells markör sågs inga dubbelt inmärkta celler.

Epitelets yta förblev obruten under hela odlingsperioden både i kontroll gruppen och i den grupp som exponerats för gentamicin. Celler som morfologiskt verkar utgöra ett mellanstadium mellan stödjeceller och hårceller identifierades. Dessa celler kan ha utskott av sin cytoplasma basalt, ett sk fotutskott, en ansamling av mitokondrier i cellens basala del och de är ofta täckta av utskott från angränsande stödjeceller vid sin övre yta. Celler som klassats som övergångsceller mellan hårceller och stödjeceller hade ofta flera kontakter med angränsande stödjeceller än vanliga hårceller.

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Slutsatser I odlingskulturer av hårcellsepitel från utrikel fann vi:

- Att den morfologiska strukturen av det sensoriska epitelet var

välbevarad under långtids odling. - Att det var en nytillkomst av hårceller efter gentamicin exponering

genom att stödjeceller direkt omvandlades till hårceller. - Att det förekom en celldelning av stödjeceller efter gentamycin

exponering men denna delning av stödjeceller gav inte upphov till nya hårceller.

- Att det fanns celler som under omvandlingen från stödjeceller till hårceller uppvisade en karaktäristisk morfologi.

- Att förslutningen av epitelet med så kallade tight junctions var morfologiskt intakt även efter gentamicin exponering.

- Kommunicerande förbindelser så kallade gap junctions mellan stödjeceller men inte mellan hårceller och stödjeceller och heller inte mellan transitional cells och stödjeceller.

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List of Papers

This thesis is based on the following papers:

1. Werner, M., Van De Water, T.R., Andersson, T., Arnoldsson, G., Berggren, D., 2012. Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants. Hear. Res. 283. 107-116.

2. Werner, M., Van De Water, T.R., Hammarsten, P., Arnoldsson G., Berggren D., 2015. Morphological and morphometric characterization of direct transdifferentiation of support cells into hair cells in ototoxin-exposed neonatal utricular explants. Hear. Res. 321 C, 1-11.

3. Werner, M., Van De Water, T.R., Canlon, B., Viberg, A., Stenlund, H., Berggren, D. Investigation of the relationship between direct transdifferentiation and proliferation in spontaneous regenerated hair cells. (Manuscript)

4. Werner, M., Van De Water, T.R., Stenlund, H., Berggren D. Hair cell characteristics after hair cell renewal - An ultrastructural study of gentamicin exposed rat utricular explants. (Manuscript)

The published papers have been reprinted with the permission of the respective publishers

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Introduction

1. Our senses for hearing and balance Three hundred years BC Aristotle created the concept of the five senses- sight, smell, taste, touch and hearing. Hearing and balance and their corresponding sense organs, the cochlea and the five vestibular organs, are situated in the inner ear. Each sense organ has specialized receptor cells to detect their adequate stimuli.

Hearing and balance disorders are very common disabilities. About 2 to 3 out of every 1000 children are born with a detectable level of hearing loss. The rate of hearing loss increases with age and approximately 2% of adults aged 45 to 54 and 50% of those who are 75 and older have a disabling hearing loss. More than 1 of 20 children between the ages of 3 and 17 suffer from dizziness or balance dysfunction (National Institute on Deafness and Other Communication Disorders, U.S.A.). Among older adults vertigo, unsteadiness and other balance-related symptoms are huge problems (Barin et al., 2011).

2. Anatomy of the mammalian ear The ear conch and the external auditory canal that ends towards the tympanic membrane comprise the outer ear. The gaseous-filled cavity medial to the tympanic membrane, the middle ear, contains the ossicular chain (the malleus, the incus and the stapes). The footplate of the stapes rests onto the oval window that connects the middle ear to the inner ear. The sensory organs for hearing and balance are well protected in the inner ear and are surrounded by bony structures within the temporal bone at the base of the skull (Fig. 1). The inner ear is filled with two extracellular fluids - perilymph and endolymph, with different ion content (Fig. 2). These two liquid compartments are separated by epithelium that contains the sensory organs of the inner ear. The receptor cells for hearing and balance, the hair cells (HCs), have their apical surfaces exposed to the endolymph. The cochlea is a coiled bony tube divided into three fluid-filled compartments - scala media, scala vestibuli and scala tympani. The organ of Corti (the organ for hearing) is situated in the middle compartment of the cochlea, scala media. The vestibular organs consist of the three semi-circular canals, and the two otolith organs, the saccule and the utricle. The HCs in the vestibular organs are embedded in a gelatinous mass. In the utricle and saccule this gelatinous mass contains small crystals called otoliths that provide inertia making the mass sensitive to gravitation (Fig. 3).

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Fig. 1. Anatomy of the outer, middle and inner ear. The utricle is sited in the vestibular part of the inner ear.

Fig. 2. Schematic picture of the inner ear with the perilymphatic and endolymphatic compartments.

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Fig. 3. Schematic picture of the organisation of the utricular epithelia.

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3. Auditory and vestibular function Sound waves are directed, with help from the pinna, through the external auditory canal to the tympanic membrane. The movements of the tympanic membrane are transmitted and amplified by the ossicular chain (the malleus, incus and stapes) in the gaseous-filled middle ear. The sound vibrations, which reach the footplate of the stapes in the oval window, are converted to pressure waves in the cochlear fluids that cause the basilar membrane at the base of the sensory epithelia to vibrate. This in turn makes the stereocilia on the apical surfaces of the HCs to bend against the covering tectorial membrane. Bending of the stereocilia results in opening of ion transduction channels (Flock, 1965; Russell et al., 1986) causing depolarization of the endocochlear-potential. This depolarization finally results in release of neurotransmitters that send signals via afferent nerve fibres to the central auditory pathways (Ruel et al., 1999).

The three semi-circular canals, the anterior, posterior and horizontal, are located in three dimensions and detect rotational acceleration and deceleration. The semi-circular canals are connected to the utricle. HCs, located in the ampullae at the base of each semi-circular canal, detect movement of the endolymph through bending of the stereocilia (Dohlman, 1969; Dohlman, 1981; Takumida, 2001). The saccule and the utricle are endolymphatic-filled sacs and the sensory epithelia are covered by the otolithic membrane with the otoconia, calcium carbonate crystals, on the surface (Kachar et al., 1990). The saccule is directly connected with the cochlear duct. The weight of the otoconia exerts gravitational forces between the otolithic membrane and the sensory epithelia resulting in bending of the stereocilia of the HCs. The HCs in the saccule and the utricle register linear acceleration and head tilts.

4. Morphology of the utricular sensory epithelia The sensory vestibular epithelia are composed of two basic cell types that include sensory HCs surrounded by non-sensory supporting cells (SCs). The HCs have highly specialized bundles of stereocilia on their apical surfaces. The stereocilia bundles are arranged in a staircase manner with one tall kinocilia followed by stereocilia of descending length. The kinocilia of the HCs are oriented towards a region in the centre of the utricle called the striola. This means that the response of a HC, i.e. bending of the stereocilia bundle toward or away from the kinocilia, to a movement depends on its location in relation to the striola.

Morphologically two types of HCs, type I and type II, have been identified in the vestibular organs (Wersall, 1961). They differ in shape, neural connection and location (Desai et al., 2005; Popper, 2000; Ross, 2000). The type I HCs are flask-shaped, whereas type II HCs, considered to be the phylogenetically older type of HC, are irregular but generally cylindrical in

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shape and vary considerably in length (Popper, 2000). Type I HCs synapse only with afferent calyces, while type II HCs communicate almost entirely with branches of primary vestibular afferent endings in the form of synaptic boutons (Ross, 2000). Efferent nerve endings synapse on the calyx terminals that surround the type I HCs and directly with the cell bodies of the type II HCs. In the striolar region the majority of HCs are type I (Desai et al., 2005). The HCs do not contact the basal membrane and are isolated from one another by the interspaced SC projections. The basal part of the SCs is located on the basal membrane and they span through the entire epithelium with microvilli on their luminar surfaces. The nuclei of the SCs are lined up close to the basal membrane whereas the HC nuclei are located more apical in the luminar two - thirds of the epithelia (Fig. 3).

5. Intercellular junctions in the utricular sensory epithelia Four types of intercellular junctions have been characterized in the epithelial tissue - tight junctions, adherent junctions, desmosomes (Farquhar et al., 1963) and gap junctions (Franke, 2009). Tight junctions line the entire luminal surface of the epithelium and act as a diffusion barrier. Adherent junctions, located beneath the tight junctions, have adhesion functions. Actin filaments associated with adherent junctions are important in contraction of the apical surface of the cell. Desmosomes are dispersed spot like buttons of adhesion located beneath the adherent junctions. Gap junctions act as electronic couplers between cells allowing small metabolites and ions to pass through protein channels, connexons, from one cell to the other.

At the apical surface of the utricular epithelium, the SCs form tight and adherent junctions with each other, and with the HCs (Bagger-Sjoback et al., 1984). Numerous gap junctions are located on the lateral surface of the SCs below the level of the tight junctions in both the auditory and the vestibular part of the inner ear in several different species (Forge et al., 2003). Gap junctionss occur between all progenitor cells in the immature otocyst but during differentiation of HCs an uncoupling occurs so that morphologically mature HCs are lacking gap junctions (Bagger-Sjoback et al., 1984).

In specialized epithelial tissue, such as the inner ear sensory epithelium, intercellular junctions play an essential role for the function. Keeping the apical tight junctional seal intact is prerequisite for maintaining the ionic gradient between the endolymph and perilymph, which is essential for HCs to function. Mutations in genes encoding the gap-junction proteins connexion 26 and connexion 30 are the most common cause of autosomal recessive non-syndromic deafness (Nickel et al., 2008). Gap junctions in the vestibular part of the inner ear are probably also crucial for the function but less is known concerning clinical correlation.

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6. Development of the hair cell sensory epithelia In general HCs and SCs are derived from a common pool of progenitor cells (Fekete et al., 1998; Jiang et al., 2013; Torres et al., 1998). Through lateral inhibition differentiating HCs inhibit neighbouring progenitor cells from developing into HCs, thus creating the mosaic HC-SC pattern characteristic for the mature sensory epithelium (Fig. 3)(Lanford et al., 1999; Lewis et al., 1998). Activation of the Notch signalling pathway and expression of the transcription factor Sox2 are necessary for formation of the pool of progenitor cells (Kiernan et al., 2005; Yamamoto et al., 2011). Activation of the Notch signalling pathway and expression of the transcription factor Atoh 1 are required to make some of the progenitor cells to proceede and develop into HCs (Bermingham et al., 1999; Kelley, 2002; Kelley, 2007; Lanford et al., 1999; Woods et al., 2004).

7. Regeneration of hair cells The process of HC regeneration seems to include similar steps as early development of HCs from progenitor cells (Cotanche et al., 2010; Denman-Johnson et al., 1999; Levic et al., 2007; Mbiene et al., 1984; Stone et al., 2007). The capacity for HC renewal differs between species, between auditory and vestibular HCs, and also depends on the degree of maturation of the epithelia.

Both the auditory and the vestibular HCs are replaced continuously throughout life in fish and amphibians. For birds, HCs in the vestibular organs are replaced continuously (Corwin, 1985; Jorgensen et al., 1988; Kil et al., 1997; Roberson et al., 2004; Shang et al., 2010), whereas for their auditory organ a trauma is needed to induce HC regeneration (Corwin et al., 1988; Cotanche, 1987; Cruz et al., 1987; Lippe et al., 1991; Ryals et al., 1988).

In mammals, regeneration of HCs is very limited and although the process of HC differentiation during development is fast and complete, regeneration is slow and incomplete (Forge et al., 1993; Izumikawa et al., 2005; Sher, 1971; Staecker et al., 2007). The epithelium in the mature mammalian organ of Corti lacks the ability to be spontaneously renewed (Forge et al., 1998; Roberson et al., 1994; Stone et al., 1998). In contrast, within the vestibular organs in mammals, a limited spontaneous regeneration of HCs will occur in response to a trauma (Forge et al., 1998; Forge et al., 1993; Tanyeri et al., 1995; Warchol et al., 1993) and can be increased further by specific methods of gene stimulation (Huang et al., 2009; Izumikawa et al., 2005; Jung et al., 2013; Lin et al., 2011; Staecker et al., 2007; Staecker et al., 2014). After stimulation of the gene Atoh 1, improvements of balance function were demonstrated in ototoxin-treated adult mice (Staecker et al., 2007) and hearing improvements were demonstrated in deafened adult guinea pigs (Izumikawa et al., 2005).

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In the mammalian cochlea the ability to spontaneously generate new HCs is only present for a very limited time during the neonatal period (Chai et al., 2012; Cox et al., 2014; Shi et al., 2012; Shi et al., 2013). In the vestibular part of the inner ear the extent of HC renewal decreases with maturation but lost HCs can still spontaneously be replaced in the mature mammalian utricle (Forge et al., 1998; Golub et al., 2012; Kawamoto et al., 2009; Taylor et al., 2015; Warchol et al., 1993).

The SCs are the source of all HC renewal and they have two mechanisms at their disposal - direct transdifferentiation and mitotic cell division (i.e. proliferation) (Roberson et al., 2004; Taylor et al., 2005). Direct transdifferentiation is a process in which SCs start to express HC genes, eventually changing their phenotype and convert into HCs without cell division. Direct transdifferentiation results in the loss of one SC for each new HC gained. In HC renewal through proliferation a SC re-enters the cell cycle and after mitotic division gives rise to two daughter cells, one that can become a HC and one that can replace the original mother SC.

Both direct transdifferentiation and proliferation contribute to HC renewal in the auditory and vestibular epithelia of non-mammalian vertebrates (Adler et al., 1996; Baird et al., 2000; Corwin et al., 1988; Jones et al., 1996; Ryals et al., 1988; Taylor et al., 2005). In the avian auditory epithelium, the first new HCs, after a gentamicin insult, are generated through direct transdifferentiation and appear in the epithelium as early as 3 days after the gentamicin exposure (Cafaro et al., 2007; Roberson et al., 2004). The first HCs generated through proliferation can be seen 1-2 days later, and 6 days after gentamicin exposure proliferation through mitosis is by far the major source to the new HCs (Duncan et al., 2006; Roberson et al., 2004).

In the mammalian vestibular epithelia, morphological and immunohistochemical evaluations indicate that new HCs emerge from direct transdifferentiation of SCs (Li et al., 1997; Rubel et al., 1995; Staecker et al., 2007). It was recently described in the neonatal mouse for both the cochlea (Bramhall et al., 2014) and the utricle (Lin et al., 2015; Wang et al., 2015) that there is a subgroup of SCs that after a trauma can reactivate the Wnt target gene Lgr5 and act as HC progenitors. There are also indications that mainly the Lgr5+ SCs contribute to the process of HC renewal (Bramhall et al., 2014).

Proliferation occurs among the SCs in the mammalian vestibular inner ear epithelium but only at a very low rate and it has not proven to give rise to any new HCs (Rubel et al., 1995).

8. Aminoglycoside ototoxicity Aminoglycosides is a group of antibiotic drugs that is both oto- and nephrotoxic. The first aminoglycoside, streptomycin, was used in the

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treatment of tuberculosis (Jones et al., 1944). Less toxic, semisynthesised aminoglycosides are used in the clinic today and it is routine to monitor their serum concentration carefully to avoid ototoxic effects.

In experimental research, aminoglycosides have been used to damage the HCs in the inner ear. The aminoglycosides show different patterns of cochleotoxicity and vestibulotoxicity. The vestibular part of the inner ear is especially sensitive to gentamicin (Karasawa et al., 2011). Apoptosis is the major pathway for death of HCs within the mammalian vestibular epithelium after gentamicin exposure (Lang et al., 1997; Nakagawa et al., 1997).

9. Hair cell marker Myosin 7a is important in the development and function of HCs and it has been used as a HC marker (Li et al., 2003). Myosin 7a is present in stereocilia, cuticular plates, and cell bodies in utricular HCs type I and II in the guinea pig and mouse (Hasson et al., 1997). Myosin 7a is located in association with the cross-links between adjacent stereocilia and is probably required for the structural integrity of hair bundles (Hasson et al., 1997). Mice with mutations in the genes encoding myosin 7a lack auditory and vestibular function (Hasson et al., 1997). Usher syndrome type 1B is an autosomal inherited cause of sensorineural hearing loss, vestibular dysfunction and early blindness where a gene encoding myosin 7a is responsible. Myosin 7a is expressed early during development suggesting that it may serve as a useful early marker for HC differentiation (Self et al., 1998).

10. Proliferation marker During the S-phase of cellular proliferation the DNA chains are separated and new nucleosides incorporated. 5-bromo 2`-deoxyuridine (BrdU) is a nucleoside analogue of thymidine used as a marker for cell proliferation.

11. Choice of method: Organ culture of mammalian utricle This thesis exclusively concerns in-vitro circumstances utilizing whole organ cultures of macula utriculi explants from 4-day old rat pups.

The inner ear labyrinth, comprising the utricle, is embedded in the temporal bone, which is considered to be the hardest bone in the body. This location makes direct in vivo observations of the sensory organs of the inner ear hard to achieve, and administration of drugs is complicated with unpredictable concentrations in the target tissue. Various in vitro techniques have therefore often been used in studies of the inner ear. The mechanism of HC death after aminoglycoside exposure, i.e. mainly by apoptosis, is reported to be the same in vitro as it is in vivo (Forge et al., 2000; Lang et al., 1997).

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In this thesis work when utilizing organ culture, a whole organ, the macula utriculi, is explanted to in vitro conditions. Organ culture is thought to mimic in vivo circumstances better than single cell culture. Free dissected organs from the inner ear, the cochlea as well as the vestibular organs are very delicate. If these organs are cultured free floating they will tend to contract, and this is especially true for the macula utriculi. Hence a prerequisite for organ culture of utricular explants for longer periods than a few days, is that the explants can adhere to an underlying surface. Initial research in HC regeneration in the utricle was done in avian where the process of renewal of HCs only takes a couple of days. In mammalian utricle HC renewal spans over weeks and free floating cultures are therefore not adequate.

Since 1996 our research group has used a technique in which the inner ear explant is directly placed on a porous membrane, which facilitates anchorage of the explant (Abstract IEB, Berggren, 1996). The membrane is sealed in a housing and placed in a well, and submerged with the nutrient medium. The microporous membrane allows access for nutrients and other media components also to the anchored basal surface of the explant. At the end of the culturing period the nutrient medium is replaced by a fixative solution. After fixation the explant is still attached to the thin membrane, and the part of the membrane that holds the explant can easily be cut out. Membrane and explant can be processed intact for light and electron microscopy.

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Aims of the thesis

To evaluate the morphological characteristics of surviving HCs and SCs during long term culture of neonatal rat maculae utriculi.

To evaluate if SCs can be transdifferentiated into HCs after gentamicin exposure in cultures of neonatal rat maculae utriculi.

To investigate the occurrence of spontaneous transdifferentiation of SCs into HCs, and proliferation of SCs within gentamicin-exposed neonatal rat utricular explants.

To characterize HCs and the presence of intercellular junctions, at the ultrastructural level, after HC renewal in gentamicin-exposed neonatal rat utricular explants.

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Materials and methods

The experimental model used in all four papers is an in vitro technique in which whole organs of macula utriculi from 4-day-old rats are cultured.

Animals Litters of 4-day-old (P4) Wistar rat pups were obtained from Charles River Laboratories. The P4 rats stayed with their mother until shortly before they were euthanized by rapid cervical dislocation. The care and use of animals was approved by the Regional Care and Use Committee of the University of Umeå (Umeå, A 49-08) and conformed to NIH guidelines for the care and use of laboratory animals.

Organ culture technique and paradigms The heads of the P4 rats were sprayed with 70 % ethanol. The temporal bones were removed and the capsule covering the vestibule was opened. Utricular maculae were excised as a unit and their otoconial membranes gently removed. The explanted preparations were composed of the complete macular epithelium with a minimal amount of sub-epithelial tissue. The dissection was performed in Dulbecco's phosphate buffered saline (PBS) supplemented with glucose (6 g/L) at room temperature. The explants were placed with their epithelial surfaces uppermost on perforated insert membranes (Transwell Clear with pore-size 0.4 µm. Costar, Cambridge, MA). The inserts were placed in 6-well-plates containing Dulbecco's Modified Eagles Medium (DMEM) supplemented with glucose to a final concentration of 6 g/L, 10% foetal bovine serum, N-1 supplement (Sigma), 100 ml to 10 ml of medium, and penicillin (100 U/ml). By reducing the depth of the nutrient medium above the insert membrane the explants became flattened and anchored to the culture insert membrane. The explants were incubated at 37 °C in a humidified 5 % carbon dioxide atmosphere and the medium was renewed every second day.

The day of explantation was denoted as day zero in vitro (0 DIV). Explants were cultured from 0 DIV to 28 DIV. During the initial 48 hrs of incubation all explants were cultured in medium without gentamicin to allow the explants to adhere to the insert membrane and to allow any damage induced during the surgical excision and transfer to stabilize prior to their exposure to gentamicin. On the second day in vitro (2 DIV) the explanted utricular maculae were divided into two groups, a control group (C) and a gentamicin-exposed group (G). Group C explants were cultured in nutrient medium without any drugs added throughout the experiment. Group G explants were exposed to 1 mM gentamicin (Sigma, G-3632) for 48 h (i.e. 2 DIV and 3 DIV), and then allowed to recover in medium without gentamicin.

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Group C explants were harvested for light microscopy (LM) analysis on 0, 2, 5, 7, 11, 14, 21 and 28 DIV and for transmission electron microscopy (TEM) analysis on 0, 2, 5, 7, 14, 21 and 28 DIV. Group G explants were harvested for LM analysis on 5, 7, 11, 14, 17, 21 and 28 DIV and for TEM analysis on 5, 7, 11, 14, 17, 21 and 28 DIV.

Paper 1 comprises results from group C, and paper 2 comprises results from both groups C and G. In paper 3 the explants were exposed to BrdU (Sigma) at a concentration of 10 mM during specified 48 h periods after the gentamicin exposure during 2 DIV and 3 DIV. The periods of BrdU exposure were 4-5 DIV, 6-7 DIV, 8-9 DIV, 10-11 DIV or 12-13 DIV, respectively. All explants exposed to BrdU were harvested on their 21 DIV.

Light microscopy (LM) - Papers 1 and 2 While still attached to the insert membrane the explants were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide, followed by 1% uranyl acetate. Thereafter, samples on cut-out pieces of the culture insert membrane were placed in 70% ethanol and dehydrated in series of graded ethanol followed by propylene, and finally placed in a solution of propylene and Poly/Bed (50:50). The maculae, still attached to a piece of the membrane, were then embedded in Poly/Bed (Polysciences Inc., Warrington, PA, USA).

Each specimen was randomly orientated around the axis orthogonal to the surface of the sensory epithelium, serially sectioned at a thickness of 1 µm, and stained with 1 % toluidine blue.

The sections were analysed with LM (Axiophot, Zeiss, Germany) with an attached camera (MTI 3CCD). The software Image-Pro Plus (version 4.0) was used and the images were displayed on a computer monitor. Panoramic 250 Flash (3D-HISTOTEC, Budapest, Hungary) was used to scan selected slides before they were re-embedded for TEM. The software used was Panoramic Viewer version 1.15.2.

The numbers of obtained preparations from different harvest time points are illustrated in Table 1.

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Transmission electron microscopy (TEM) - Papers 1, 2 and 4 One micron plastic LM sections, chosen for TEM analysis, were photo-documented and then re-embedded for TEM by using the method of King and co-workers (King et al., 1982). In brief, the sections were placed in xylene for 24 hours and then rinsed in 100 % ethanol. They were then placed in acetone for 1 hour. Beem capsules filled with unpolymerized Poly/Bed® were inverted over the sections and polymerized at 60 °C. Ultrathin (600 Å) sections were made from the re-embedded preparations and from the explants prepared directly for TEM. Sections were mounted on formvar-coated grids, stained with uranyl acetate and lead citrate to enhance contrast, and viewed in papers 1 and 2 with a JEOL 1200 EX TEM. In paper 4 the photodocumentation and analysis were done with a JEOL 1230 TEM equipped with a digital micrograph software.

Cell counts - Papers 1 and 2 For each macula the systematic analysis with identification and counting of cells started when a pseudostratified epithelium with vestibular HCs was identified in the serially sectioned material. Every tenth section of the consecutively following sections was analysed until there were no longer any identifiable HCs in the epithelium. The mean number of analysed sections per macula was 78 (range 46-118) for group C and 73 (range 39-125) for group G. At each analysed section measure lines were placed randomly along the basal membrane and the cells were counted along these lines until a minimum length of 150 µm was achieved, making sure that no overlapping of the counted area occurred.

Table1.Cultureparadigmspapers1and2.

DIV

NumberofpreparationsLM;serialsections

TEM;re-embeddedLMsections

TEM;serialsections

C G C G C G0 2 1 2 1 1 1 5 2 2 1 1 17 2 1 2 1 111 2 2 1 114 2 2 3 1 17 2 221 1 2 1 1 2 228 2 2 1 1 1

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The following criteria for counting HCs, SCs and unknown cells were employed: HC - the base of the cell was not in contact with the basal membrane and a visible nucleus was present within the uppermost 2/3 area of the epithelia; SC - the base of the cell was in contact with the basal membrane and a visible nucleus was present within the bottom 1/3 area of the epithelia; unknown cell - those that did not fit the outlined characteristics for either a HC or a SC and had a visible nucleus. The HCs identified as described above that also had visible stereocilia were noted as a subgroup.

Calculations of volume - Papers 1 and 2 Volumes were estimated using systematic sampling (Gundersen et al., 1999). By using the serially sectioned material, the epithelial volume was calculated for each macula using the software program Image Pro Plus version 4.0. With a randomized starting-point, the first section in which sensory HC epithelia could be recognized, the epithelial area in each fiftieth section was manually outlined and the area measured. The area measurements were summarised and multiplied with the distance between them (50 µm) to obtain the volume. The validity of the manual marking of areas was evaluated by repeated measurements of the same area. For the volume measurements the standard deviation (SD), the coefficient of variation (CV %) and the confidence interval were calculated for each preparation.

Immunohistochemistry - Paper 3 BrdU labelling was used as a proliferation marker and myosin 7a immunostaining was used as a HC marker (Li et al., 2003; Self et al., 1998). Observations were obtained from a total of the following 12 utricular explant samples - two utricular explants exposed to BrdU 4-5 DIV, three each exposed to BrdU 6-7, 8-9, 10-11 DIV, and one exposed to BrdU 12-13 DIV. Each specimen was randomly orientated around the axis, orthogonal to the surface of the sensory epithelium, and serially cryo-sectioned at a thickness of 14 µm. Each fifth section was double immunostained with mouse anti-BrdU antibodies (Becton Dickinson) tagged with biotin and visualized with DAB-Ni, and polyclonal rabbit anti-myosin 7a antibodies (Affinity Bio Reagents Inc.) visualized using the Vector VIP substrate kit.

The following criteria for BrdU+ and Myosin 7a+ cells were employed: BrdU+, a brown staining of a nucleus of a cell present within the sensory epithelia; myosin 7a+, a purple staining of the cytoplasm of a cell with a pale area without staining, corresponding to the nucleus in the same cell, present within the sensory epithelia. Double labelled cells, BrdU+/Myosin 7a+ were a brown staining of the nucleus and a purple staining of the cytoplasm of the same cell present within the sensory epithelia.

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The location of the cells with positive immunostaining was noted, i.e. in basal 1/3, middle 1/3, or apical 1/3 of the sensory epithelia. When analysing this cryo-section material, the same method was used for counting BrdU+ and Myosin 7a+ cells and calculation of densities as described above for counting HCs and SCs in papers 1 and 2.

Comparison of cell counts of BrdU+ and myosin 7a+ cells with morphometric cell counts of SCs and HCs - Paper 3

In papers 1 and 2 the number of HCs, SCs, and other cells were estimated from 1 µm plastic sections with serial sections taken every 10 µm.

In paper 3, 14 µm thick frozen sections were used. Exposure to BrdU, a proliferation marker, occurred at time-points after 4-5, 6-7, 8-9, 10-11 and 12-13 DIV and labelled nuclei were counted after 21 DIV. Furthermore, in these sections immunohistochemically staining of the HC cell marker myosin 7a was also done.

In order to compare the findings in paper 3 with earlier observations in paper 2, the difference in thicknesses of sections between the studies must be considered. Also important is that the frozen sections are translucent allowing all immunohistochemically stained cells within the depth of the section to be visualized. A correction factor was developed to allow us to determine how many more nuclei would have been observed in paper 2 if we had used 14 µm thick sections instead of 1 µm thick sections.

The distance between the incisions described in paper 2 was 10 µm. Had 14 µm thick sections been used, this portion would include two (1 µm) thick sections with a probability of 0.4 and one (1 µm) with a probability of 0.6. Such calculations are based on that a 14 µm thick section could entail 10 portions, four of which include two 1 µm thick sections and six including one 1 µm thick section. So if we assume that in a 1 µm thick section N nuclei are observed, the number observed in a 14 µm thick section will be:

0.6 x N + 0.4 x 2 x N = 1.4 x N

This means that the correction factor will be 1.4 (Fig. 4).

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Fig. 4. Schematic illustration of correction factor.

Evaluation of HC characteristics and intercellular junctions - Paper 4

HCs with a visible nucleus and a cell membrane possible to follow from the luminar surface and all the way around the cell, were selected from 18 utricles at various time points in vitro from the control and the gentamicin-exposed groups (groups C and G) (Table 1 in Paper 4). Routine photomicrography was performed as follows: a full-frame survey with single-portrayals of each chosen HC from the luminal surface to the bottom part was taken at a magnification of x 4000. These overview photos were blinded according to time point in vitro and C and G group identity respectively, and analysed by two of the co-authors separately. In cases in which the individual assessment was not consistent regarding an observation, this was discussed until consensus was reached, prior to breaking the code for the entire material. A systematic morphologic evaluation of each individual HC-image was done regarding TJs, foot process, lateral extension, apical SC extensions covering the HC, accumulation of mitochondria basally and number of SCs connecting the HC.

Intercellular junctions in HCs and their SC area were analysed by screening of an extended material that included the 82 HCs and from the same utricles in the following manner: The apical side of the HC border to adjacent SC were photo documented at higher magnifications (x 4000 to 8000). The junctions at each side of the luminary surface of the HC were photodocumented at a magnification of x 60000 to 100000. Thereafter the cell membrane was followed at a magnification of x 20000. When an electron dense area or a narrowing of the cell membranes was identified the area was photodocumented with a magnification of x 60000 to 100000. The

�

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SC area was defined by the next SC-SC tight junctions lateral on each side of the chosen HC and a line from that and down to the basal membrane (Fig. 5). The SC area around each analysed HC was searched for cell membranes with a magnification of x 20000. When an electron dense area or a narrowing of the cell membranes was identified the area was photodocumented at a magnification of x 100000. These photos at higher magnifications of selected HCs and their SC areas were analysed and each junction was classified morphologically as tight juncton, adherent junction, desmosome or gap junction.

Fig. 5. Schematic illustration of the SC area screened for intercellular junctions in paper 4.

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Results

Morphology

Paper 1 In group C the presence of neural elements, i.e. nerve calyces, were observed within the in vivo preparations at 0 DIV. At 2 DIV only remnants of intraepithelial neuronal elements were observed and thereafter no signs of neural elements remained within the epithelium. Except for this finding, the sensory epithelium harvested after 2, 5, 7 and 11 DIV appeared similar to the in vivo epithelium. The SC nuclei lined up along the basal membrane and HCs type I and II showed their nuclei in the upper 2/3 of the epithelia and with bundles of stereocilia at their luminal surface (Fig. 6).

After 14 DIV type I and type II HCs could no longer be distinguished from each other with LM. With TEM however, it was still possible to identify both HC types. In preparations cultured for 21 DIV and 28 DIV SC nuclei were observed to be more apically located, i.e. further away from the basal membrane. Ten cells out of a total of 54,745 cells appeared to be fused cells, i.e. with two nuclei.

Paper 2 In group G after 5 DIV, i.e. 24 hours after a period of 48 hours of gentamicin exposure, only a few HCs remained. In the upper 2/3 of the epithelium, SCs filled out the space at which HCs were lost, keeping the integrity of the epithelium intact. After 5 and 7 DIV, the epithelial appearance was almost the same with a preserved stratification with intact basal and apical linings but with signs of HC degeneration. After 11 DIV, only a few HCs with degenerative changes were observed in the epithelium. After 14 DIV, HCs with small apical surfaces and immature stereociliary bundles were present.

From 14 DIV there was an increasing group of cells that did not fulfil the morphologic criteria of either SCs or HCs. The majority of these cells resembled SCs, but their nuclei were placed in a more apical position in the stratum of the epithelium. Some cells fulfilled the criteria of HCs but had extensions of their cytoplasm towards the basal membrane. After 21 DIV the density of HCs had increased and the apical surfaces of new HCs were partly covered by cytoplasmic extensions from bordering SCs. The SC nuclei were not lined up in close association with the basal membrane, but were instead seen at various levels within the epithelium (Fig. 6). After 28 DIV, numerous HCs had stereocilia that were longer and had a more mature appearance with staircase formations. The apical surface of the HCs was, after 28 DIV, in full contact with the luminal surface and no longer covered by apical

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extensions of neighbouring SCs. Seven cells of a total amount of 32,886 cells appeared to be fused cells.

Fig. 6. Upper: Epithelia after 2 DIV without gentamicin exposure (paper 1). Lower: Epithelia after 21 DIV, exposed to gentamicin during 48 hours i.e. 2-3 DIV, and thereafter recovered in medium without gentamicin (paper 2).

Paper 3 Immunohistochemical labelling of utricles from group G showed a considerably greater number of cells with BrdU positive (BrdU+) staining in the fibroblast layer adjacent to the sensory epithelium as compared to BrdU+ cells within the sensory epithelium. Forty-seven percent of the BrdU+ cells were located in the basal 1/3 of the epithelium close to the basal membrane. Forty-one percent of the BrdU+ cells were located in the middle 1/3 of the epithelium, and 12% were located in the upper 1/3 of the epithelium, i.e. close to the luminal surface.

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There were no myosin 7a positive (7a+) cells in the fibroblast layer or in the basal 1/3 of the epithelium. Only one myosin 7a+ cell was located in the middle 1/3 of the epithelium. More than 99% of the myosin 7a+ cells were found in the upper 1/3 of the epithelium.

Paper 4 Supporting cells transdifferentiating into hair cells in utricle cultures from rat were characterised by foot processes, various degrees of apical covering by processes from neighbouring supporting cells, basal accumulation of mitochondria and an increased amount of connections with supporting cells (Fig. 8). TEM analysis of intercellular junctions showed HCs with characteristic junctional zonula of high electron density with tight junctions, adherent junctions and desmosomes in the apical lateral borders to the contiguous SCs at all analysed time points. The apical occlusion seemed to be morphologically intact in both groups C and G. Further down along the lateral borders of the HCs occasional desmosomes were found. We did not find any distinct areas with gap junction-like morphology between HCs and adjacent SCs. Between the SCs there were numerous desmosomes as well as distinct sections of the membrane revealing gap junction-like morphology (Fig. 7). These distances with gap junction-like morphology differed a lot in length from short distances to long sheets. Vesicles with double membrane with morphological features of gap junction plaques were found intracellulary in both SCs and HCs. The cell membrane of SCs was often hard to follow since it included a number of various loops and invaginations, especially in the area beneath the HCs.

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Fig. 7. A gap junction between two SCs in group C after 21 DIV (paper 4).

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Fig. 8. Epithelia after 21 DIV, exposed to gentamicin during 48 hours i.e. 2-3 DIV, and thereafter recovered in medium without gentamicin (papers 2 and 4).

Cell counts

Papers 1 and 2 The density of the total amount of epithelial cells in group G was about 75

% of that found in group C at 5 DIV (p<0.001). There was a daily average 0.6% decrease of the density of the total count of cells for both groups between 5 DIV to 28 DIV. In group C there was a slight decrease of the proportion of HCs in relation to SCs during 5 DIV to 28 DIV (p=0.0419). After 7 DIV, i.e. four days after gentamicin exposure, the density of HCs in group G was 11% of the density of HCs observed in group C after 7 DIV. At 28 DIV the HC density in group G was 61% of the HC density observed in group C after 28 DIV. Simultaneously with this increase in HCs there was a

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corresponding decline in the number of SCs within the epithelium. The proportion of HCs increased significantly in group G during the 5 DIV to 28 DIV period of culture (p<0.001). The proportion of HCs with discernible stereocilia among the total HCs did not change substantially with time in vitro in group C (p=0.113). There was an increased proportion of HCs with visible stereocilia among the total HCs in group G during 5 DIV to 28 DIV (p<0.001). The results of the cell counts are illustrated in Fig. 1 and Table 2 in paper 2.

Paper 3 We used our cell counts of the cell densities in group G and adjusted for differences in technique, i.e. multiplied by 1.4 to get expected cell densities in the labelled cryo-sections.

From the findings of an average of 9.6 SCs per 100 µm in group G after 21 DIV in paper 2 there should be approximately 13.5 SCs per 100 µm in the cryo-sections. Of these expected SCs, 3.4 SCs per 100 µm (25%) were observed to be BrdU positive. With the same correction factor applied there should be approximately 4.45 HCs per 100 µm and at least 1.79 (40%) of the HCs present can be considered to be new HCs. Of these expected HCs, 0.89 HCs per 100 µm were identified as myosin 7a+. Hence after 21 DIV and 18 days post-gentamicin exposure, approximately 20% of the HCs that fulfilled the light microscopic criteria for HCs (paper 2), showed myosin 7a positivity (paper 3). No cells with double staining for both myosin 7a and BrdU were observed in any of the BrdU exposed explants.

Volume

Papers 1 and 2 There was no significant change in the volume of the HC comprising epithelium of the macular explants during 0-28 DIV in Group C. There were no significant differences between the volume of the explants of groups C and G during 0-28 DIV.

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Discussion

Using our model of explanted maculae utriculi we have consistently found the same pattern of spontaneous HC renewal within the gentamicin- damaged sensory epithelia (papers 2, 3, 4 and (Berggren et al., 2003)). The lowest HC density is seen after 7 DIV, i.e. 3 days after termination of gentamicin exposure. Thereafter there is a robust renewal of HCs. Morphological and morphometric characterizations show that replacement HCs is the result of direct transformation of an explant´s existing SCs into new HCs. There is also a proliferative response by the SCs but this SC proliferation does not contribute to the generation of new HCs.

The tight junctional seals at the luminal border of HCs are morphologically intact at the various time points in vitro both without and after gentamicin exposure.

Transitional cells in intermediate states of direct transdifferentiation from SCs into HCs have been identified. Characteristics for these intermediate cells are cytoplasmatic extensions from neighbouring SCs apically, basal extensions toward the basal membrane, i.e. foot processes, and an assembly of mitochondria basally in the cell or within these foot processes. These transdifferentiating cells have an intact tight junction seal apically and an increased number of SC connections basally.

Techniques used for cell counting We used relatively thin sections (1 µm) and counted cells from every 10th section throughout the whole utricles in papers 1 and 2. With this method the whole volume of the sensory epithelia is covered evenly, which should be an advantage in preparations with an uneven distribution of cells such as in the utricle. With our technique we get cell counts that are relative to a distance but we cannot estimate the total number of cells within the macula.

A strength in papers 1 and 2 is that volume estimations performed indicate that the changes in cell density are not due to volume change but instead of actual cell alterations.

Another technique for cell counting is stereo-optical methods, i.e. a dissector technique in which cells are followed through a depth in thicker sections (Gundersen et al., 1999; Kirkegaard et al., 2005; Severinsen et al., 2008). This technique makes it possible to estimate the total number of cells in the macula. A disadvantage though is, that in the above referred studies where this technique was used, relatively large areas were not analysed. Thus there is a possibility that the number of HCs is underestimated if there are areas with a high HC density such as in the utricular striola.

Surface visualization methods, i.e. phalloidin-flourescein isothiocyanate staining of the stereociliary bundles, have often been used in inner ear

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regeneration research. Even though it is a delicate method it has been questioned because it does not mirror the actual cell changes and repair of the HCs cannot be excluded.

Findings that support renewal of vestibular HCs through direct transdifferentiation of SCs Our cell counts in paper 2 show that gentamicin exposure evokes an initial substantial reduction of HC density followed by a significant increase of the proportion of HC density in relation to SCs. Parallel to the increase of HC density there is also a significant decrease of SC density. Our interpretation is that there is a substantial HC renewal after gentamicin exposure in these utricular explants. The reduction in SC density is in concordance with the theory that renewed HCs are products of direct transdifferentiation of SCs.

In papers 1 and 2 we analysed a total number of 87,631 cells with distinct visible nuclei from 27 utricles. Only one of these cells was noted as possibly being in a mitotic process. This finding indicates that mitosis is a rare event in these utricular cultures.

In paper 3 we show that some cells are BrdU+ after 21 DIV. These BrdU+ cells are found in the SC layer of the epithelia and none of these cells are myosin 7a+. Our interpretation is that there is a limited proliferation among the SCs but this does not give rise to any new HCs during the observed time period.

Morphological observations show a more apical placement of some of the SC nuclei in the epithelia after gentamicin exposure (papers 2 and 4). This finding is in agreement with direct transdifferentiation since a transforming SC needs to reorganize its nucleus position from the basal location of a SC nucleus to placement in the upper 2/3 of the epithelia as a HC.

Some of the HCs appear immature after 14 DIV in the gentamicin-exposed group. These HCs have small apical surfaces and small stereociliary bundles and the kinocilium might not yet be lateralized. We interpret these HCs with an immature appearance as new HCs (paper 2).

In papers 2 and 4 we also identified cells that fulfilled some of the criteria for SCs and some of the criteria for HCs, i.e. cells that could neither be classified as HCs nor SCs. Characteristic for these intermediate cells are various degrees of cytoplasmic extensions from neighbouring SCs covering the apical surface, basal extensions towards the basal membrane, i.e. foot processes, and an assembly of mitochondria located basally in the cell or within these foot processes. At an ultrastructural level these transdifferentiating cells have intact tight junctional seals apically, and an increased number of SC connections basally. Our interpretation is that these cells are cells in intermediate states of direct transdifferentiation from SCs into HCs.

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Our findings strongly suggest that vestibular HCs of a mammalian utricle can regenerate spontaneously through direct transformation of existing SCs into new HCs, after gentamicin-induced trauma in vitro.

An advantage in our studies is that the dissection, the culture, the tissue processing and the exposure to gentamicin is identical in all four papers. This gives a possibility to compare the results from papers 1 and 2 with the results in paper 3. Limitations in paper 3, however, are that no controls without gentamicin exposure are used and that the sample size of each BrdU exposition group is small. In papers 1 and 2 the sample size and the amount of sections analysed is large and controls without gentamicin are used.

Even though there is a potential for HC renewal through direct transdifferentiation in the vestibular part of the mammalian inner ear the amount of new HCs demonstrated in this PhD thesis are limited. This is in concordance with reports from other studies (Forge et al., 1998; Golub et al., 2012; Kawamoto et al., 2009; Tanyeri et al., 1995). The process of direct transdifferentiation seems to span over a long time period and it is uncertain if all new HCs ever mature completely. Since one SC converts into one HC during direct transdifferentiation, HC renewal through direct transdifferentiation will, according to Cotanach et. al., soon lead to disruption of the epithelium (Cotanche, 2008). After gentamicin exposure we can identify a decrease in SC density in spite of the fact that there is also a proliferation of the SCs. This SC decrease is partially due to the consumption of SCs that are converted into HCs through direct transdifferentiation. Why HC renewal is incomplete in mammals is still unclear, but one can speculate that the process of direct transdifferentiation of SCs into HCs is limited due to lack of SCs that possesses the ability to transform into HCs.

Repair of HCs Since damaging or ototoxical methods probably do not kill all original HCs the possibility that repair of HCs contributes to the HC renewal cannot be ruled out in most studies (Sobkowicz et al., 1999; Zheng et al., 1999). To control the degree of HC damage and the following HC renewal the apical stereociliary bundles have often been used for identification (Abdouh et al., 1993; Berggren et al., 2003; Chardin et al., 1995; Lefebvre et al., 1993; Zheng et al., 1996), therefore the relative contribution of cellular repair versus regeneration of the HCs has been unclear.

In paper 2 we counted all the cells that contained a nucleus, i.e. the potential sub-lethal damaged HCs are counted as HCs both at the down peak as well as later after they may have undergone repair and regained their apical structures. Since these cells are counted as HCs at every stage they cannot contribute to the increase in HC density that we observed. Therefore, our increase of HC density does not include repair of damaged HCs but only renewal through direct transdifferentiation of SCs into HCs.

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Cells with two nuclei A few cells with two nuclei were observed when analysing sections of the explanted maculae with LM, and some of these cells were also observed in the TEM re-embedded preparations (papers 1 and 2). We interpret these binucleated cells as fused cells. The phenomenon of fused cells has been reported to be associated with fusion of stem cells and damaged specialized cells in several different tissues (Rizvi et al., 2006; Willenbring, 2005).

Diverse functions of the SCs During our in vitro culture of macula utriculi two important roles governed by the SCs are clear, preservation of the epithelial barrier and renewal of HCs.

The luminar surface of the sensory epithelium is well preserved without lesions or disruptions in the control group as well as in the gentamicin-exposed group (papers 1 and 2). After gentamicin exposure with a distinct reduction in HC density, the SCs reach out with cytoplasmatic extensions covering the space that earlier was occupied by HCs completely. This scarformation by the SCs is fast and effective and 48 hours after the gentamicin exposure no signs of trauma to the epithelia can be observed except for a reduction in HC density. In paper 4 we show that the tight junctional seals at the luminal border of HCs are morphologically intact at various time points in vitro both without and after gentamicin exposure.

Cell counts, morphological observations and immunohistochemical results strongly support HC renewal through direct transdifferentiation of SCs into HCs (papers 1, 2, 3 and 4). Transforming cells are often seen with covering extensions from neighbouring SCs apically (demonstrated in paper 2 and 4). One question is if the transforming SC first withdraws its apical connection or if SCs with the ability to transform into HCs do not have an apical connection before they are activated by the trauma.

Our findings are in concordance with a considerable number of other reports that auditory and vestibular SCs play important roles in support of the HCs through development, function, damage, death and renewal (Golub et al., 2012; Jagger et al., 2006; Lahne et al., 2008; Li et al., 1997; Lin et al., 2015; Tritsch et al., 2007).

The organ of Corti has a high degree of SC heterogeneity and five different types of SCs have been distinguished from one another (Raphael et al., 2003). They differ in morphology and they also exhibit distinct expression patterns of a number of genes (Wan et al., 2013).

The SCs of the vestibular part of the inner ear in mammals have a more uniform morphology than the auditory SCs. However, little is known when it comes to differences in gene expression and function. Recently a subgroup of the SCs was identified that has the ability to express Lgr5 after trauma (Lin et al., 2015; Wang et al., 2015). Lgr5+ cells are the only SCs that seem to

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contribute to the HC renewal both through direct transdifferentiation and proliferation. This finding raises the question of whether there are a larger variety of subgroups of utricular SCs with different functions than has been assumed so far. The SCs in the vestibular part of the mammalian inner ear might not be such a uniform group as previously thought.

In our TEM study (paper 4) we observed that intermediate cells that morphologically appeared to be in the process of direct transdifferentiation seemed to have an increased number of SC connections compared to mature HCs. In the bird cochlea there are three to four SCs for each HC (Goodyear et al., 1997). In our in vivo preparations, i.e. at 0 DIV, there are 1.7 SCs for each HC. After 28 DIV there are 2.2 SCs for each HC in the C group and 2.3 SCs for each HC in the G group. Since there are a limited number of SCs per HC the most probable explanation to the observation of more SC contacts for the transdifferentiating cells is that each SC will increase the number of HCs they are in contact with during direct transdifferentiation. One question is if all SCs have this ability to reach out with multiple extensions towards SCs transforming into HCs, or if this ability is limited to a subgroup of the SCs.

In paper 4 we observed that the intercellular membrane of the SCs was only possible to distinguish and follow at the apical part of the epithelium. At the bottom of the epithelium the cell membrane of SCs seemed to take loops, turns, disappear and turn up at a different location. It has been suggested that the SCs could be organised in a syncytium without clear boundaries between each SC (Kawamoto et al., 2009). This observation, in combination with the increased numbers of SC contacts at the borders of transforming cells, indicates a spider-like morphology of the SCs with multiple extensions. To our knowledge this increasing number of SC contacts during direct transdifferentiation has not been reported earlier. This needs to be further evaluated.

It is no doubt that SCs do have a lot of various functions and show plasticity in reacting to changed circumstances. The pluripotency of SCs make them a promising target to stimulate to gain renewal of HCs and hopefully possibilities to restore hearing and balance impairments.

Clinical implications Hearing and balance disorders are very common disabilities. One of the most frequent causes of sensorineural hearing loss and peripheral vestibular disorders is a loss of the mechanoreceptive HCs and/or primary sensory neurons of the inner ear (Nadol, 1993; Van Eyken et al., 2007). Viral infections, ototoxic drugs, noise exposure, inner ear diseases and aging can cause HC damage in both the vestibular and auditory receptor cells of the inner ear and may result in chronic balance dysfunction, oscillopsia and hearing disabilities (Baker et al., 2009; Barin et al., 2011; Friedman et al.,

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2003; Li-Korotky, 2012; Monzack et al., 2013; Proctor et al., 1979; Staecker et al., 2007).

Cochlear implants have substantially changed life for many deaf borne children providing them hearing properties and speaking and language abilities. Also adults with an acquisitioned severe to profound bilateral hearing disorder due to HC damage can regain hearing through a cochlear implant. The criteria for implantation have expanded because of improved outcomes. Hearing preserving surgery and shorter electrodes have made patients with residual hearing into candidates for cochlear implantation. Worldwide about 150000-200000 people have a cochlear implant. Around 300 cochlear implants are implanted annually in Sweden. Deaf borne children are implanted bilateral and represent about half of the yearly implants.

At present there is no equivalent technical device available for the treatment of patients with a balance disorder that is due to vestibular HC damage. The majority of children with permanent hearing loss also have abnormalities of the vestibular portion of their inner ears (Buchman et al., 2004; Cushing et al., 2008; Wolter et al., 2015). Cochlear implant failure is more common among children with vestibular and balance impairment, probably due to an increased risk of falling resulting in an associated head injury (Wolter et al., 2015). This emphasizes the need to investigate the vestibular inner ear and to hopefully find treatments to replace the information input that is missing from the damaged vestibular system.

Even though cochlear implant is a great advancement there are strict indications and also financial limitations. Approximations indicate that less than 10% of candidate adults receive a cochlear implant in USA (Sorkin, 2013). Worldwide this number is certainly even less.

To tackle the huge worldwide problems of hearing disabilities and balance disorders we need an arsenal of various treatment options. Today cochlear implantation is a well-established method and also combinations of electrical and acoustic stimulation (EAS). In the future combining electrical and acoustic stimulation with substances of benefit for hearing and balance might be a reality. Substances of benefit for hearing and balance could include stem cells, pharmacological protection as well as gene stimulating factors.

Future research Although deleterious changes occur in adjacent neurons and at higher

levels within the central nervous system following HC loss, it is generally believed that regeneration of new HCs will provide alleviation of hearing and balance impairments (Stone et al., 2007). The recent finding of a pluripotent subgroup of the SCs that can be activated by trauma to generate HCs might be a more direct route to HC regeneration than implantation of stem cells.

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Atoh1 is a promising candidate for gene stimulation of HC regeneration through both proliferation and direct transdifferentiation of the SCs. Major questions are why regeneration of HCs is incomplete, and what the limiting factors are for a more substantial renewal of HCs in the mammalian inner ears. We need to know more about the mechanisms of trauma-initiated HC renewal and to understand why this ability to regenerate replacement of HCs has been lost or diminished in mammals if an effective HC regenerating therapy is to be developed.

There is ongoing research with the aim to increase hearing gain for patients after cochlear and brainstem implantation by simultaneously implantation of modified endogenous nerve cells (Jiao et al., 2014; Novozhilova et al., 2013). There are however still considerable hurdles that need to be overcome regarding stem-cell-based regenerative medicine such as strategies to generate functional specific neurons from stem cells (Low et al., 2015).

In future we might have in our treatment arsenal combined cochlear-vestibular implants that are implanted through hearing and balance preserving surgery and a simultaneous administration of substances stimulating endogen HC renewal and adequate neuronal attraction.

One possible way to gain more knowledge about the mechanisms behind gene regulation in the inner ear is to study phenotypes with specific mutations in genes involved in HC development. The transcription factor gene LHX3 is expressed specifically in auditory and vestibular HCs soon after terminal mitosis and persists into adulthood in vestibular HCs (Hertzano et al., 2007; Hume et al., 2007). A mutation in the LHX3 gene has been described in six patients in Northern Sweden with congenital combined pituitary hormone deficiency and a sensorineural hearing defect (Kristrom et al., 2009). We are now planning collaboration with Kriström et. al. to follow up the hearing of these patients, and also to investigate if these patients have vestibular dysfunctions.

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Conclusions

In these neonatal rat utricular explants:

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

- 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 during hair cell renewal through direct transdifferentiation from supporting cells have a characteristic morphology.

- 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.

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Acknowledgements

Finally my gratitude to all that made this thesis possible. In particular I want to thank: Diana Berggren, my supervisor, for your enthusiasm, for always being optimistic, for all agreements and the disagreements that we overcame, and for showing me that almost everything is possible. Thomas R. Van De Water, for helping me to state what I actually mean in all the papers. Sten Hellström, for your extremely valuable comments in writing the thesis and hints like ”Do you really think this is a good way to start the discussion?” Göran Laurell, my co-supervisor, for your availability, for your prompt reply and for your excellent suggestions in writing the thesis. Barbara Canlon, my co-supervisor, for helping in writing even though you have been busy. Anders Asplund, for always helping me out with the pictures and especially for your useful quote ” Du kan få va fan du vill, Mimmi”. Hans Stenlund and Göran Arnoldsson for illumination in the world of statistics. My co-authors Therese Andersson, Peter Hammarsten and Agneta Viberg for helping me with the papers. Kristina Forsgren and Cathrin Johansson, for skillful technical assistance Mats Aadde, for being my friend, college and brother in arms. You are almost always literally right (except about the laundry). Eleonor Koro, for your contagious enthusiasm. All my co-workers at the ENT department in Örnsköldsvik for working as a team and, making work a nice place to go to. Joakim Grendin, for your enthusiasm to always develop and improve and for letting me be a part of it.

33

Henrik Smeds, because of you and not only because of your excellent guiding in otosurgery and for the comforting statement “ This is as hard as it gets, Mimmi”. Anders Kinnefors, for guiding me in otosurgery in the most humble way. You are always welcome to Ö-vik. Claude Laurent, for always supporting me, for your generosity, for good times in Cape Town and wonderful wine tasting. James Look (Bond) alias Daniel Craig, for your generousity, for your valuable clinical and surgical guiding in Cape Town and for interesting conversations. Katarina Olofsson, my dear friend for all good times and for all therapeutic running. Per, Ida, Elma and Fanny, for lending me a part of Katarina and for all delicious dinners and sauna baths at Blågrundet. The Nilsson, Lambertson-Strinholm, Mjörnheim, Ardnor, Väppling families for all nice moments we have shared, without your distraction this thesis had never been possible. Anna, Carola, Matilda and Malena, my oldest friends, you are so extremely important to me even though distance at times keeps us apart, and don´t forget that we will unite as it all started in a common corridor. Elsa Liljeholm, for explaining basic maths in times of despair. Sonja, it took me one year of persuasion to get you and ever since your love and enjoy daily prove how right I was. Alvar, for always provoking, pushing everything to its edge, immediate action, for your joy, to love and to hate and nothing in between. Lukas, for being such a nice person, for interesting discussions, for your always right on target comments and for encouragements as “ Get shit done candy”. Vera, for everything and especially for letting me be a part of your life when you are grown up, it´s like you are giving me the youth once again, and for the nice illustrations in my thesis. Mårten, for better or for worse forever and for making all times into good times, we were made for each other.

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