Otolaryngology - Theses
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Imaging the cochlear response to injury
There are many sites and methods of therapeutic interventions in the ear, but common to most, if not all of them is the risk of degradation of cochlear or vestibular function. This is despite the fact that these interventions are designed particularly to aid function of these organs. The principal aim of this thesis is to explore non-destructive methods of examining the cochlea’s response to attempted therapeutic interventions. Conventional histopathological techniques that have formed the basis of cochlear research over past decades involve potential disruption of anatomical relationships, distortion of fine structures and a lack of a ‘whole cochlear’ picture of the pathological response. Three imaging modalities were selected that were either novel to hearing research or had previously been limited only to descriptive anatomical studies. These imaging techniques were combined with therapeutic interventions that in the clinical setting have the potential to improve or augment hearing. The first experiment of this thesis demonstrated that while continuous drug delivery to the round window membrane may achieve its primary endpoint (BDNF drug effect in the cochlea, in this case), the technique itself involves a risk of cochlear injury which may limit the safety and thus utility of this delivery technique. A combination of destructive and non-destructive imaging techniques was utilised to explore the pathophysiology of this injury. The second experiment investigated the capabilities of heavy-metal augmented MicroCT in assessing the cochlea’s response to implant insertion through characterisation of cochlear endolymphatic hydrops, intrascalar tissue response, changes in vestibular end organ volumes and three-dimensional assessment of implant position. A number of techniques were developed that have been utilised in already published research as well as forming the basis of future study. The third experiment investigated the capability of an emerging imaging technique, TSLIM, to provide more than just anatomical information. With the idea that changes in vascular permeability in the lateral wall of the cochlea may be responsible for changes in scala media observed in Experiment 2, we developed a technique to visualise the permeability of the blood-labyrinthine barrier with spatial resolution sufficient to demonstrate differential vascular permeability in the fibrocyte subregions of the lateral cochlear wall. Furthermore, this technique could be applied to image these changes in the whole cochlea in one instance. The results and techniques presented in this thesis will inform hearing and other researchers of the applicability of these imaging techniques to their research questions to push hearing research beyond histopathology and the mid-modiolar section.
Local and systemic steroids in hearing preservation after cochlear implant surgery: are they equivalent?
Since the conception of the cochlear implant, the criteria for evaluating potential candidates has greatly expanded. We have seen a move to implanting a greater range of ages, including young children, implanting simultaneous bilateral ears, implantation in single sided deafness, and implanting those with a significant level of residual low-frequency hearing. The later permits a combination of electric stimulation from the implant, and natural acoustic stimulation, termed “electroacoustic stimulation”, that greatly enhances an individual’s speech recognition and music appreciation when compared to implant stimulation alone. Unfortunately, implantation often results in either early or delayed loss of residual hearing in the post-operative period. This is speculated to be the result of implantation trauma and delayed inflammatory events. In order for cochlear implantation to progress further, we now find ourselves with the predicament of how to best preserve this hearing. Despite a large body of research into hearing preservation strategies, we are still faced with unacceptable rates of hearing preservation. Corticosteroids are widely used in the peri-operative setting in an attempt to preserve hearing. It is widely believed that that they exert their effects by modulating the inflammatory reaction to an implant. Currently, little is known about the precise mechanism by which corticosteroids preserve hearing after cochlear implantation, or about the optimal administration regimes. The investigations in this thesis aim to explore the mechanism of action of corticosteroids in the context of hearing preservation after cochlear implantation, as well as compare the various routes of administration. Specifically, these investigations will compare local and systemically administered glucocorticoids, pharmacokinetics and pharmacodynamics within the inner ear, and provide a rationale for their administration regimes. Additionally, investigations in this thesis will explore the role of the mineralocorticoid receptor in the pathologic events following cochlear implantation.
Intraoperative monitoring during cochlear implantation and correlations to preservation of hearing
Cochlear implants having been widely used over the past few decades to restore or provide hearing to profoundly deaf individuals. Recently the benefit of combining electrical stimulation from a cochlear implant with low frequency natural hearing or acoustically amplified hearing have been recognised. Benefits include better speech understanding in noise and better appreciation of music (Gantz et al., 2005). Unfortunately inserting a cochlear implant presents significant risk to a patient’s natural hearing therefore, although there are great potential benefits, there is also risk. There are numerous causes of hearing loss after cochlear implant surgery (Rowe et al., 2016). One of those causes is physical damage to the fine structures in the cochlear during array insertion. Electrophysiological monitoring is used to maintain the integrity of important structures for many operations such as facial nerve monitoring in many major ear or parotid gland operations and recurrent laryngeal nerve monitoring in thyroid gland surgery. Electrophysiological monitoring of hearing has been used when resecting tumours near the auditory nerve (Cueva, 2012). Monitoring hearing during cochlear implantation seems a logical extension of a surgeon’s arsenal of monitoring tools. This thesis explores the role of intraoperative monitoring of hearing during cochlear implantation with a goal to reduce postoperative hearing loss by avoiding implant related trauma at the time of surgery. This thesis takes a pragmatic approach focussing only on measurement modalities and methodologies which would be feasible to be use in a day-to-day clinical setting. This work involved: 1. Developing a novel electrophysiological recording system leveraging the telemetry capabilities of commercial cochlear implants. 2. A small animal study to technically validate the function of the system. 3. A small human study to validate the practical use of the system and define stimulus parameters and expected response patterns. 4. A larger human study demonstrating the correlation between adverse intraoperative measurements and failure to preserve operative hearing.
Vestibular migraine: a challenging illness to live with and to manage: a qualitative study of patients’ and specialists’ perspectives
Vestibular migraine (VM) is one of the most common causes of recurrent vertigo with significant effects on patients’ health-related quality of life measures and psychological well-being. It is widely accepted that VM is associated with a range of diagnostic and management challenges for practitioners, predominantly due to the lack of a gold standard diagnostic test and absence of a preferred drug of choice. Consequently, the existing literature is mostly focused on optimising the diagnostics and treatment protocols. Yet, little is known about the therapeutic needs of those who suffer from VM and health professionals’ strategies in providing care beyond medication. This doctoral thesis addresses these gaps in knowledge in two ways: first, by exploring the experiences of vestibular migraineurs in their daily lives and in the management of their condition; second, by juxtaposing these experiences with an exploration of vestibular specialists’ (VSs) perspectives on the management and care of people with VM. This thesis entailed three phases. In Phase A, a preliminary observation of several vestibular migraineurs’ appointments with VSs was conducted to inform the overall direction of the thesis and to fine-tune the research questions. Phase B adopted a qualitative descriptive method across two consecutive studies. In Study 1, 11 adults with VM were interviewed. This study was informed by the preliminary observation in Phase A and explored two aims: 1a) vestibular migraineurs’ lived experiences and therapeutic needs, 1b) vestibular migraineurs’ experiences with healthcare professionals (HCPs). The findings are visually presented in two models: The first model describes participants’ perceptions of living with VM. In this model, VM is depicted as a ‘wall’ to represent participants’ perception of living with extensive functional and psychosocial restrictions. The individual is presented at the centre, surrounded by six aspects of life (i.e., lifestyle, occupation, personal life, self-sufficiency, social network and self-image) and encircled by VM. Hopelessness and isolation are presented adjacent to the VM ‘wall’ to highlight the psychological effects of a restricted life. The second model is a map of vestibular migraineurs’ journeys from symptom presentation to diagnosis/symptom control. This map illustrates the experiences of vestibular migraineurs with different HCPs and represents the convoluted, long and often frustrating journey that patients embark on to find a diagnosis and, possibly, some level of symptom control. This model shows that vestibular migraineurs’ journeys often entail multiple misdiagnoses, dismissals and irrelevant referrals, particularly when patients seek medical advice from general practitioners (GPs). This experience often leads to frustration and loss of trust towards primary care physicians. By contrast, participants depicted a more positive, trusting experience when seeing VSs. Compared to GPs, patients often perceived VSs as more knowledgeable and competent in managing their illness, largely because VSs acknowledged the severity of their illness and did not dismiss them. Vestibular migraineurs’ satisfaction with VSs provided a rationale for exploring these practitioners’ perspectives. The purpose was to elucidate the source of patients’ higher levels of satisfaction and to explore the clinical strategies that VSs adopted in managing VM. Study 2 explored the perspectives of 10 VSs on two topics: 2a) the complexities of VM management and 2b) the features of optimum VM care. VSs were recruited from several fields of balance disorder management including neuro-otology, vestibular audiology, vestibular physiotherapy, and ear, nose and throat surgeons. The outcomes are depicted in two models. The first model illustrates the multidimensionality of complexities posed by VM, indicating that VM is a complex illness to manage due to three factors: 1) scientific ambiguities, 2) patient-related challenges and 3) professional challenges. The second model proposes a framework for VM quality management: ‘SAIPHE’. SAIPHE stands for the six elements that were considered essential for VM quality care: self-efficacy, access, individualisation, wholistic, and education. Each of these elements are described in detail in the thesis. Study 2 concludes Phase B of this thesis. In Phase C, the data obtained in this body of work was triangulated. The outcomes of Study 1 and Study 2 were juxtaposed to examine their consistency. Patients’ accounts of the challenges of living with VM were compared with VSs’ perspectives and elements of SAIPHE were juxtaposed with Study 1 (vestibular migraineurs’ lived experience and expectations of their practitioners). Finally, VSs’ perspectives on features of VM patients’ education/consultation were integrated with vestibular migraineurs’ articulated educational needs and were developed into a VM-specific consultation framework. Overall, the outcomes of this thesis indicate that VM is a complex illness to live with and to manage as its effects extend beyond its immediate symptoms. Patients suffer from functional and psychological implications that, if unattended, could have catastrophic sequelae. Despite advances in evidence-based knowledge, and despite the consistency of VSs’ perspectives, vestibular migraineurs’ needs and expectations are not reflected in non-specialised clinical care, particularly general practice, and frequent misdiagnosis and dismissals create a barrier to timely management of patients’ illnesses. This thesis supports the clinical recommendation of promoting a care plan that extends beyond symptom management and incorporates patient education and management of functional and psychological effects of VM.
Trauma and residual hearing loss after cochlear implantation surgery
For the last 30 years, cochlear implantation (CI) surgery has been successfully used for the treatment of severe to profound deafness. With improvements in cochlear implant technology, the surgical criteria have expanded to include patients with residual low frequency hearing. These patients can benefit from simultaneous use of the implant and a hearing aid in the same ear, termed electro-acoustic stimulation (EAS). EAS has been shown to be particularly beneficial for perceptual tasks relying on pitch resolution, such as music appreciation and speech perception in background noise (Gantz et al., 2005, Santa Maria et al., 2013). Unfortunately, residual hearing loss, termed post-implantation hearing loss, occurs in half of all patients, which degrades many of the benefits of EAS (Carlson et al., 2011). Post-implantation hearing loss may arise from direct surgical trauma and indirect damage to the cochlea (Eshraghi and Van de Water, 2006). The causes of direct trauma include surgical access to the cochlea and electrode insertion trauma. This results in several types of tissue injury, which may cause further hearing loss through a biological response, also termed indirect damage. However, the precise contributions of direct and indirect damage to post-implantation hearing loss remain indeterminate, in part, because of the lack of a standardised animal implantation model. Animal experimentations have been partly hampered by the difficult access to the mammalian inner ear and the absence of a consistent implantation technique (Rowe et al., 2016, Smeds et al., 2015, James et al., 2008) that has led to marked differences in the level of trauma and hearing loss (O'Leary et al., 2013, Farhadi et al., 2013, Honeder et al., 2015). The principal aim of this thesis was to examine the different types of cochlear trauma, how they relate to hearing loss and how hearing loss can be prevented. The issues that have been addressed here include cochlear anatomy and imaging techniques, pathology, pharmacology, electrophysiology and force recordings. This thesis begins by establishing a reproducible animal model of cochlear implantation surgery, followed by systematically examination the relevant contributors to surgical trauma and post-implantation hearing loss. From these findings, pharmacological therapy targeting the indirect damage and recording techniques to reduce post-implantation hearing loss have been investigated. An anatomical study of the guinea pig cochlea was initially performed using novel imaging and processing techniques. This study quantitatively described the soft and bony tissue relationships in the complex hook region of the unoperated guinea pig cochlea. A reproducible technique for implanting in an animal was established in this study for the remaining in vivo studies, including the use of a cochleostomy in an anteroinferior plane (highest predictability and smallest risk of trauma) and closer to the round window (lowest force profile). The first in vivo experiment of this thesis investigated the relative influence of implant insertion depth on trauma and hearing loss. This study also explored the use of glucocorticosteroids (‘steroids’) to preserve hearing and target the biological response to the implant. Key findings were that implants deeply inserted or in the presence of an osseous spiral lamina fracture caused the greatest low-frequency hearing loss. Steroids reduced the biological response in the most apical regions but had no effect on hearing thresholds. A second in vivo experiment was performed to further investigate the efficacy of steroids and to explore if trauma and hearing loss could be predicted at the time of surgery through force or electrocochleography recordings. Preserving the compound action potential of the electrocochleography recording was found to be predictive of an atraumatic insertion and reduced post-implantation hearing loss. Conversely, force was correlated with trauma but not hearing loss. Pre-operative steroids were effective in reversing the loss of hearing amplitudes evoked by lower frequency tones during implantation with hard electrodes. The results presented in this thesis will help inform the cochlear implant community of potential techniques to improve hearing preservation surgery. Specifically, these results provide a validated experimental model of hearing preservation surgery. These results also suggest a role for steroids in reducing tissue response and synaptopathy, and for electrocochleography as an intraoperative recording paradigm to reduce trauma and improve post-implantation hearing loss.
A study of electrical stimulation levels over 10 years for adults using Nucleus cochlear implants
Cochlear implants have improved sound perception for thousands of people with severe and profound sensorineural hearing loss. To ensure a good quality sound signal, the implant must be individually programmed throughout the user’s lifetime. Programming determines the electrical stimulation level requirements for each electrode of the implant. The frequency with which programming occurs has so far been based primarily on clinical experience and resources available to the clinic for programming purposes. To develop an evidence-based schedule for the frequency of programming, a comprehensive investigation of the change in electrical stimulation level requirements over time for a large group of adults is necessary. The overall purpose of this retrospective study was therefore to investigate the change in electrical stimulation levels up to 10 years postimplantation for a large group of adults using Nucleus cochlear implants. In addition, this study also aimed to investigate whether demographic characteristics and electrode array segments were predictive of the change in electrical stimulation levels. The Cochlear Implant Clinic of the Royal Victorian Eye and Ear Hospital, Victoria, Australia, has been providing programming services for cochlear implant users for over 25 years. The electrical stimulation data obtained at the programming sessions were extracted for 680 participants who used a Nucleus® cochlear implant over the course of 10 years. For each implant user, programming data for the following time points were extracted from Cochlear Limited’s TM Custom Sound 3.2® fitting software: 2, 3, 6, 9 months postimplantation and biennial time points from 1 year up to 10 years postimplantation. For each time point, the mean T level, C level, and dynamic range (DR) were calculated separately for four electrode array segments: apical (mean of electrodes 22, 20, 18), medial (16, 14, and 10) upper-basal (7, 6), and lower-basal (4, 3). The degree of change (DC) in levels between consecutive time points was also calculated. Long-term changes in electrical stimulation levels were also investigated for 128 participants from the same group who had adequate programming data up to 10 years postimplantation. The trends and amount of change in programming levels were investigated separately for the four electrode array segments using regression analyses. The effects of several demographic characteristics (e.g., aetiology and onset of hearing loss) were also investigated. For the 680 participants, the average T levels and C levels were consistent from 3 months and 6 months postimplantation, respectively. In terms of the degree of change between time points, the majority of participants showed an average of ≤ 20% change in levels as a function of DR after the 3- to 6-month time point comparison postimplantation. Long-term patterns of change for 128 individual participants showed that, for 42% of these participants, the electrical stimulation levels remained relatively stable without any significant trends for either the T levels or C levels, from 6 months up to 10 years postimplantation. Some significant changes in levels were evident up to 10 years postimplantation; however, changes were small, equating to less than 6% per year as a function of DR for 75% of the participants and a maximum of 10% change in levels for the remaining 25% of participants. Additional noteworthy findings include the following: 1. For the participants who showed more change in levels in the first 3 to 6 months postimplantation, approximately 70% of these participants showed a non-significant long-term trend in levels up to 10 years postimplantation, 2. The mean T levels and C levels were found to differ significantly for the four electrode array segments in the first 2 years postimplantation, but were relatively consistent in the long-term, and 3. The mean degree of change in levels as a function of DR was significantly greater for the Otosclerosis pathological group (n = 72) compared to all other groups, and for the prelingual onset of deafness group compared to the postlingual group. This thesis provided strong evidence that electrical stimulation levels change in the first 6 months but expected to remain relatively stable up to 10 years postimplantation for the majority of adults using Nucleus cochlear implants. Based on this evidence, a programming protocol for 10 years postimplantation has been proposed. The proposed protocol recommends more frequent programming sessions in the first 6 months compared to the number of sessions thereafter. The reduced number of sessions over an implant user’s lifetime will assist in managing the increasing caseload with the amount of resources currently available for programming. Given that the levels differed across the segments in the first 2 years postimplantation, but not in the long-term, the proposed protocol recommends measuring levels separately for the four segments in the early postimplantation period. This can then be limited to one or two segments beyond 2 years postimplantation. The protocol also suggests that more programming sessions may be required in the first two years for implant users with Otosclerosis and those with a prelingual onset of hearing loss.
Virtual reality for the training of ear surgery
In current residency surgical training programs, residents learn by observing their mentor and practicing under his/her supervision. For patient safety, the mentor needs to ensure that residents have sufficient practice before they perform surgery on patients. Simulation, ranging from low fidelity (e.g. suture practice on rubber models) to high fidelity (e.g. cadaveric temporal bone dissection), is ideal for this purpose and cadaveric bone dissection is the gold standard of simulated training for temporal bone surgery. However, it has a few setbacks. For example, it is limited by the shortage of cadaveric temporal bones and the bones have to be discarded after drilling and cannot be reused. Also, it is difficult to ensure that residents are exposed to a wide range of temporal bones, specifically ones with rare anatomical anomalies. Another concern is that illnesses may be transmitted through contact with diseased tissues and fluids. Virtual reality surgical simulation is an alternative form of high fidelity simulation that offers risk-free, relatively low cost training environments that can be used repeatedly if necessary at the convenience of the user. Reviews have found that practicing surgical procedures on virtual reality simulators improves trainees' operative performance. One major factor contributing to the success of virtual reality surgical simulation systems is their ability to capture clinical variation. Current simulators in Ear Nose and Throat (ENT) surgery mostly use the core training methodology: starting from easier procedures and moving onto more difficult ones. However, this configuration has traditionally been based on the same anatomical model. To the author’s best knowledge, there do not exist virtual reality simulators in the field of ENT that capture anatomical variation as a basis for surgical education. The aim of this thesis was to determine whether exposure to anatomical variation in a virtual reality simulated environment leads to better surgical outcomes, to establish the foundations of a more efficient model for surgical education. To achieve this goal, several investigations were carried out. Firstly, a systematic review and meta-analyses were conducted to examine the role of virtual reality simulation based training in ENT surgery, and to investigate whether it can improve the acquisition of surgical skills. This has revealed that there is currently no literature available in this field to inform whether anatomical variation is important for improving outcomes in simulation based surgical training. Secondly, methods were developed to identify internal anatomical structure variation of temporal bones by examining the bony surface landmarks in an effort to guide the selection of specimens that exhibit differences in anatomy. To this end, correlations between internal and external landmarks were determined and mathematical models developed to predict internal structure variation given combinations of multiple external landmarks using a library of CT scans. It was observed that these correlations and mathematical models were able to predict some anatomical variations with a high level of accuracy. Thirdly, to standardize the evaluation of competence in cochleostomy, an objective assessment tool was developed and validated. Its construct validity and inter-rater reliability were established to be high through evaluations of resident and expert performances on a standardized virtual temporal bone conducted by expert assessors using the proposed tool. Lastly, a randomised controlled trial of ENT residents performing cochleostomy was conducted to determine whether anatomical variation in virtual reality simulation based training improved surgical performance. Participants were randomly allocated to two groups: the experimental group who were exposed to a six different temporal bone anatomies over a period of three weeks and the control group who were trained on one standardized temporal bone over the same period of time. At the end of the training period participants’ performance on two novel temporal bones was assessed. It was observed that ENT residents who were exposed to a range of anatomical variations performed significantly better than those trained on a single anatomical model.
Local delivery of Dexamethasone for the preservation of residual hearing in an animal cochlear implant model
This thesis uses an experimental model of cochlear implantation to examine the hearing protection afforded by the application of dexamethasone to the round window prior to surgery.
A novel vestibulo-cerebellar ataxia, Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS): clinical phenotype, pathology, imaging, differential diagnoses and a quantitative bedside test
Patients with combined cerebellar and vestibular impairment were first identified in about 1979 and were studied as a pathophysiological model. The syndrome of Cerebellar Ataxia with Bilateral Vestibulopathy (CABV) and it’s characteristic oculomotor abnormality, that is, the abnormal visually-enhanced vestibulo-ocular reflex (VVOR) was then subsequently described in 2005 in 4 patients, 3 of whom had a peripheral sensory deficit. Here I set out to establish whether these three patients actually had a totally new, so far undescribed, neurological disease. The work in this thesis defines this new neurological disease that is now called ‘CANVAS’: an acronym for Cerebellar Ataxia with Neuropathy and bilateral Vestibular Areflexia Syndrome. This thesis details the (A) clinical presentation and evolution, (B) essential oculomotor and vestibular abnormalities, (C) neuropathology, (D) otopathology, (E) anatomical pattern of cerebellar atrophy, (F) the neurophysiological characteristics of the somatosensory impairment, (G) differential diagnoses, (H) apparent genetic basis and (I) a diagnostic quantitative bedside oculomotor test.
Evaluation of the probiotic Streptococcus salivarius fro the inhibition of the pneumococcal colonisation and prevention of otitis media
Otitis media (OM) is one of the commonest infections of infants and young children, being second only to the common cold. Acute OM (AOM) is also the commonest illness of children for which medical attention is sought in developed countries. Indigenous populations such as Inuits, Australian Aborigines and American Indians are regarded to be high-risk populations. Large numbers of pathogenic bacteria which colonise the nasopharynx of Indigenous children from early life are thought to be the main reason for this high incidence. Following an episode of AOM, Indigenous children often suffer from recurrent acute OM, which may progress to chronic otorrhoea and chronic OM, whereas in non-indigenous children, OM usually resolves without specific treatment. In low-risk populations OM is usually amenable to medical or surgical treatment, whereas in high-risk population these interventions are not as effective. Streptococcus pneumoniae, one of the three main bacterial causes of OM, colonises the nasopharynx prior to disease development. Immunisation with pneumococcal conjugate vaccines is effective against invasive infections, but has a limited impact on OM. Serotype replacement after immunisation and poorly effective treatments also contribute to the high prevalence of OM in high-risk populations. Novel strategies aimed at decreasing the load of OM pathogens and/or colonising the nasopharynx with beneficial bacteria may reduce the prevalence of OM, and are worth investigating. Probiotics are live micro-organisms that offer health benefits by modulating the microbial community and enhancing host immunity. The beneficial effects of probiotics in some gastrointestinal tract illness are well documented. By contrast, limited data are available on the effect of probiotics on upper respiratory tract infections. Probiotics isolated from the upper respiratory tract are likely to be most effective against upper respiratory tract pathogens. In this context, more specific and enduring health benefits are postulated to occur by using a novel approach called “bacterial therapy” which uses probiotic strains of species that are indigenous to the target tissue. A strain of Streptococcus. salivarius, isolated from the oral cavity of a healthy child is commercially available as BLIS K12. BLIS is an acronym for bacteriocin-like inhibitory substance and is an early candidate for bacterial therapy. The inhibitory activity of this probiotic against β-haemolytic streptococci is attributed to lantibiotics encoded by a megaplasmid carried by the K12 strain. Preliminary studies have shown that following oral intake, BLIS can persistently colonise the upper respiratory tract, and inhibit pathogenic bacteria. BLIS also has an excellent safety record. The promising benefits of S. salivarius probiotic warrant further investigation, however, as the data available are limited and inconclusive. For this thesis, I explored the potential of S. salivarius to inhibit pneumococcal colonisation and OM using in vitro assays and a mouse model of OM. I also evaluated the capacity of BLIS K12 to colonise the upper respiratory tract of healthy adult volunteers. Colonisation of the respiratory epithelium by S. pneumoniae is the first step in the pathogenesis of OM. Accordingly, I determined the ability of S. salivarius K12 to inhibit pneumococcal adherence to human epithelial CCL-23 cells at varying doses and times of administration in vitro. These studies revealed dose- and time-dependent inhibition of pneumococcal adherence by S. salivarius K12. In view of these findings, I studied the effects of S. salivarius K12 on nasopharyngeal colonisation by pneumococci and the development of OM in a mouse model. Repeated intranasal doses of S. salivarius K12 were administered to infant C57BL/6 and BALB/c mice in the presence or absence of influenza A virus to assess the short- and long-term effects of S. salivarius on pneumococcal colonisation and OM. These studies showed that S. salivarius caused only a short-lived reduction in pneumococcal colonisation, with no sustained effect on long-term pneumococcal colonisation. S. salivarius also did not reduce the number of pneumococci in the ears of mice that developed OM. Because S. salivarius was originally isolated from a human, I hypothesised that this probiotic may not be able to colonise mice sufficiently well to interfere with S. pneumoniae. Accordingly, I evaluated the capacity of S. salivarius to colonise the upper respiratory tract of adult volunteers. This pilot study showed that S. salivarius colonised the oral cavity of adults for up to two weeks following seven days of oral intake of BLIS K12. The effect of probiotic consumption on salivary IgA and IgG in these volunteers was inconclusive. Nevertheless, this pilot study provides useful information on how to design a randomised, double-blind clinical trial to study the effect of S. salivarius on OM.
The potential of induced pluripotent stem cells for spiral ganglion neuron replacement
In mammals, overexposure to loud noise, ototoxic medication or even ageing can incur irreversible damage to the sensory hair cells and spiral ganglion neurons (SGNs) resulting in sensorineural hearing loss (SNHL). Currently, the cochlear implant is the only available treatment for SNHL, but its functionality is dependent on a healthy complement of SGNs. Therefore in cases of severe SNHL, where the numbers of SGNs are significantly depleted, the efficacy of this neural prosthesis may be compromised. Using stem cells to replace damaged SGNs is an emerging therapeutic strategy for deafness. Whilst previous studies have explored the potential for several stem cell types, particularly human embryonic stem cells (hESCs) to replace SGNs, it will eventually be important that transplanted cells are from an autologous source. This thesis therefore aims to explore the potential of human induced pluripotent stem cells (hiPSCs) for SGN replacement. These cells offer the option of transplanting SGNs generated from a patient’s own cells to potentially restore hearing function and/or improve the efficiency of the cochlear implant. To investigate the potential of hiPSCs, it is first necessary to assess their potential to differentiate into a SGN lineage. In the first study of the thesis, an established neural induction protocol was used to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC, H9) towards a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyse the expression of key markers involved in SGN development, at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain and otic placodal markers (PAX7 and PAX2), pro-neurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160) and sensory SGN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC-and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Whilst all cell lines analysed produced neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared to the hESC controls. Thereby, suggesting that hiPSCs have a more variable differentiation potential compared to the hESCs. The functionality of the hiPSC-derived neurons was next assessed using patch clamp electrophysiology and in vitro co-culture assays. It was found that the cells were capable of firing action potentials in response to depolarisation and exhibited a phasic profile of activity, thus indicating that the neurons were physiologically active. Following co-culture of cochlear explants or denervated explants with hiPSC- and hESC-derived neurons, their neural processes were observed to make direct contact and form extensive synaptic connections with inner and outer hair cells in vitro. However the hiPSC-derived neurons were observed to innervate fewer hair cells, compared to hESC-derived neurons. Preliminary data also suggests that hiPSC-derived neurons are able to survive and maintain a neural phenotype two weeks post-transplantation in the mammalian cochlea. Overall, this thesis demonstrates that hiPSCs are capable of differentiating into functional neurosensory-like progenitors and innervating developing hair cells in vitro. However the differentiation and innervation potentials of hiPSC-derived neurons were observed to be less consistent, compared to hESC-derived neurons. While it is important that these variabilities are minimised prior to the clinical translation of this treatment, the use of hiPSCs for SGN replacement in the deaf cochlea holds potential.
Signal segregation in bilateral cochlear implant users
Cochlear implant (CI) users can often achieve high levels of speech comprehension in quiet condition but typically face challenges in noisy environments. Under such conditions, the ability to detect, perceive and comprehend a target signal is often degraded by the presence of interfering signals that are said to ‘mask’ the target. Masking can occur when competing signals have overlapping spectral content that compete for neural resources at the auditory periphery (energetic masking). It can also result from confusions at a central processing level (informational masking) that make it difficult to perceptually segregate the target from the interferers (stream segregation). These confusions can be more prevalent, for example, when attending speech in the presence of interfering speech signals rather than non-speech noise. However in both scenarios, energetic masking can apply. Listeners with normal unimpaired hearing can often alleviate informational masking by exploiting differences that exist between competing signals to ‘cue’ stream segregation. Differences in the relative level (interaural level differences, ILDs) and timing (interaural timing differences, ITD) of signals at the two ears are two such segregation cues. They are processed binaurally and are also often referred to as ‘spatial cues’ as they can provide information about the spatial location of the signal source. Very few studies that have examined the impacts of masking on CI users have explicitly considered the underlying mechanisms involved (i.e. informational versus energetic). Even fewer have investigated the potential benefits of binaural segregation cues in providing informational masking release to bilateral implantees. For these reasons, the broad objectives of the current research were to (1) investigate the impact of informational masking on BiCI users while energetic masking was controlled, and (2) explore the potential benefits of binaural cues in eliciting release from informational masking. Two approaches were employed to address those broad objectives. The first involved the examination of informational masking effects on speech comprehension and on concurrent two-talker localization, and the potential informational masking release provided by binaural spatial segregation cues, when listening bilaterally in the sound-field with current sound processing systems. Informational masking was controlled by varying the masker to comprise either speech or noise, which were matched energetically to each other, while the target was fixed as speech. Energetic masking was controlled by manipulating the relative timings of competing signals such that their tokens were either synchronized (full temporal overlap) or interleaved (no temporal overlap) with each other. The second approach employed pulse train stimuli presented via direction stimulation to investigate the potential of binaural cues to improve the detectability of a target signal in the presence of synchronized interferers, and to elicit stream segregation when competing stimuli were temporally synchronized (simultaneous segregation) and interleaved (sequential segregation). Target and masker were presented to tonotopically distant electrodes so as to minimize energetic masking. As such, masking effects observed were most likely due to informational considerations The first key finding of the current research was that BiCI users can be substantially impacted by informational masking. This was demonstrated by the significantly poorer speech comprehension and concurrent source localization performances when interfering signals were speech rather than noise, and by the observed degradation of target detectability by simultaneous maskers presented via direct stimulation. The second key finding was that binaural segregation cues can provide strong informational unmasking benefits when signals are temporally interleaved, but are weak when signals are synchronized. This was demonstrated in the speech comprehension task where energetic factors may have degraded the salience of binaural cues of synchronized signals. Similar findings were however also observed with direct stimulation where energetic masking was minimal, which suggests that strong (informational) grouping factors also likely diminished the influence of binaural segregation cues. Such factors, both energetic and informational, were minimal when signals temporally interleaved.