Otolaryngology - Theses
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ItemLocal and systemic steroids in hearing preservation after cochlear implant surgery: are they equivalent?( 2019)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.
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ItemThe potential of induced pluripotent stem cells for spiral ganglion neuron replacement( 2014)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.
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ItemFactors affecting inner ear pharmacokinetics( 2013)There is growing interest in treating a variety of inner ear disorders with local drug delivery methodologies due to the lower risk of side effects compared to systemic drug delivery. This includes stand-alone drug therapies such as gentamicin to treat Mèniére’s disease, as well as transition-therapies that augment existing treatments, such as the delivery of steroids or growth factors with cochlear implantation to improve hearing outcomes. Choosing the most appropriate drug treatment protocol is problematic however, due to the limited information available detailing the precise pharmacokinetics of each therapeutic drug and delivery method used clinically. Inner ear pharmacokinetics is a branch of pharmacology concerned with the movement of drugs in the ear. This is influenced by various factors such as the concentration and properties of the drug itself, as well as the duration and location in which it is delivered. Combined with the complexities of the fluidic environment of the inner ear and the limitations of current measurement methods, such as marker injection and sample withdrawal, quantifying the precise pharmacokinetics associated with each drug/delivery combination is very difficult. Without a thorough understanding of the drug distribution however, clinicians are uncertain about which drug delivery method to use, the optimal dosage, and application time in order to effectively treat vital structures whilst avoiding harmful overdosing. In the case of aminoglycoside treatment for Mèniére’s disease, this uncertainty has resulted in different dosages being administered to patients leading to variable therapeutic outcomes and undesirable side effects such as permanent hearing loss. Establishing communication routes from the middle ear into the inner ear, and between different fluidic compartments of the inner ear is complex. The most advanced quantitative technique to monitor drug distribution directly, in real-time and in-vivo involves sealing ion-selective microelectrode(s) into the otic capsule of the cochlea in an animal model to monitor the concentration of an ionic marker diffusing through perilymph (Salt et al., 1991a; Salt et al., 1991b). Limitations of this method however are that marker concentration can only be monitored at the microelectrode site and only a small number of electrodes can be embedded simultaneously. Thus a complex computational model (Plontke et al., 2004) is used to predict drug concentrations throughout the rest of the inner ear. At the beginning of the present research, this computational model did not yet allow drug distribution patterns to be simulated during the surgical events of cochleostomy and electrode insertion. Therefore the effect of cochlear implantation procedures on intracochlear drug distribution could only be speculated. When a prior functional study (Eastwood et al., 2010b) suggested that these surgical procedures may significantly alter intracochlear drug distribution, quantifying the distribution with direct measures became a higher priority in order to optimize the protection of residual hearing with steroids, which is known to improve hearing outcomes for cochlear implant recipients. The aim of this research was therefore to address the clinically significant question, how does cochlear implantation procedures affect intracochlear drug distribution? To investigate this, a series of in-vivo experiments (in guinea pigs) and in-vitro experiments were conducted using a variety of markers, different delivery methods, direct and in-direct drug measurement techniques, semi-quantitative and quantitative analysis, and computational modeling. During these experiments, other novel findings were uncovered with wider clinical ramifications for local drug delivery to the inner ear. This thesis presents the first study known to the author to assess marker distribution during a cochleostomy and electrode insertion using direct, real-time measurements of concentration. This revealed that cochlear implantation procedures can have a substantial and immediate influence on drug levels in the basal turn. The act of drilling a cochleostomy resulted in a substantial rise in marker concentration along the basal turn of scala tympani when a portion of the rotating burr entered the scala, which did not occur when the cochleostomy was performed using soft surgical techniques. Concentration further along the cochlea was not substantially affected. Additionally, inserting the electrode into scala tympani did not push intracochlear drug toward the apex as previously suspected. To the contrary, in the in-vitro experiments, bulk longitudinal flow was observed in the opposite direction to the electrode trajectory, expelling fluorescent marker from the tube. The ramifications of this for protecting residual hearing with steroids during cochlear implantation is that there may be a lower drug concentration in the basal turn than previously anticipated due to local losses from the cochleostomy site. It is not advised to assume that the electrode array will rapidly re-distribute intracochlear drug from the basal turn toward the apex during insertion into the cochlea. To achieve higher drug levels in apical regions, alternative strategies, such as longer drug administration times or delivering drugs from the implant itself, may be required. Delivering drugs intracochlearly also has the benefit of reducing the high variability of drug levels achieved in inner ear fluids compared to when they are administered intratympanically. Up to now, it has been widely accepted that drugs applied to the middle ear enter the inner ear through the round window membrane (RWM) and spread locally to the vestibule through surrounding semi-permeable tissues. This thesis presents a body of evidence that shows in addition to RWM entry, large quantities of drug (up to 90%) rapidly enter the vestibule directly in the vicinity of the oval window, likely through the annular ligament of the stapediovestibular joint, following intratympanic delivery. The ramification of this on wider clinical practice is that greater drug concentrations may be present in the perilymph of the vestibule and semicircular canals following intratympanic delivery than has previously been appreciated. The independent loading of scala tympani (ST) and scala vestibuli (SV) has significant implications for the quantification of drug dispersion in the cochlea. With a lower concentration gradient between ST and SV, higher levels of drug are maintained in ST, allowing drug to spread more quickly along the scala. Hence when the vestibule is pre-loaded, this may act to raise the level of therapeutic drugs available for inner ear protection during cochlear implantation by replacing the local drug losses from the basal turn mentioned above from SV. This finding also has ramifications for the current clinical drug therapy to treat Mèniére’s disease. Currently, vestibulotoxic aminoglycoside gentamicin is injected through the tympanic membrane (ear drum). The variability of drug concentration in inner ear fluids following this delivery method has been problematic in the past, leading to variable therapeutic outcomes and undesirable side effects such as permanent hearing loss. Following the observation that drug levels in the vestibule were heavy influenced by direct entry through the oval window, the effect of targeting gentamicin onto the stapes footplate was investigated. The application of a high concentration of gentamicin to this region resulted in greater hearing loss (significant between 8-32 kHz) and increased vestibulotoxicity (significant in the utricle) in a guinea pig model compared to animals receiving gentamicin on the RWM. This suggested that gentamicin preferentially entered the inner ear through the oval window, a theory supported by computational modeling. Previously it was assumed that gentamicin applied intratympanically suppressed function of the otolithic organs and semicircular canals with little effect on hearing by entering the RWM and spreading locally from ST to SV in the basal turn, and from there into the vestibule. It was assumed that to achieve this, the vestibular hair cells were more sensitive to the toxic influence of gentamicin than cochlear hair cells. However the possibility must now be considered that higher gentamicin concentrations exist in the vestibule than previously appreciated. The impact this may have on clinical practice is that, with optimization of the dosage, targeted drug therapy to the stapes footplate may become a more viable route to deliver ototoxic drugs to the inner ear. It is speculated that delivering a small volume of the optimized dosage directly to the stapes footplate may enable better control of drug concentration in inner ear fluids, minimizing the risk of overdosing leading to permanent hearing loss. For applications where the goal is to maximize drug levels in the cochlea, it is speculated that it may not be necessary to specifically target the RWM in future. Instead, it may be just as effective to flood the middle ear space with a simple injection through the tympanic membrane to allow the drug to be in contact with both the round and oval windows. This may negate the need for patients to remain in a lateral recumbent position for long periods in a clinical setting. Entry of steroids, neurotrophins, and other therapeutic substances through the oval window has not yet been investigated, but appears to be a worthy line of investigation for future research. If such substances permeate the oval window as effectively as some of the markers tested in this thesis, targeted drug delivery to the stapes footplate may become a more efficacious way to deliver drugs locally to the inner ear in future.