Browsing by Author "Needham, Karina"

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    Challenges for stem cells to functionally repair the damaged auditory nerve.
    (Informa Healthcare, 2013-01) Needham, Karina; Minter, Ricki; Shepherd, Robert; Nayagam, Bryony
    INTRODUCTION: In the auditory system, a specialized subset of sensory neurons are responsible for correctly relaying precise pitch and temporal cues to the brain. In individuals with severe-to-profound sensorineural hearing impairment these sensory auditory neurons can be directly stimulated by a cochlear implant, which restores sound input to the brainstem after the loss of hair cells. This neural prosthesis therefore depends on a residual population of functional neurons in order to function effectively. AREAS COVERED: In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, the benefits derived from a cochlear implant may be minimal. One way in which to restore function to the auditory nerve is to replace these lost neurons using differentiated stem cells, thus re-establishing the neural circuit required for cochlear implant function. Such a therapy relies on producing an appropriate population of electrophysiologically functional neurons from stem cells, and on these cells integrating and reconnecting in an appropriate manner in the deaf cochlea. EXPERT OPINION: Here we review progress in the field to date, including some of the key functional features that stem cell-derived neurons would need to possess and how these might be enhanced using electrical stimulation from a cochlear implant.
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    Challenges for the application of optical stimulation in the cochlea for the study and treatment of hearing loss
    (Taylor and Francis, 2017-02) Richardson, Rachael; Thompson, Alexander; Wise, Andrew; Needham, Karina
    INTRODUCTION: Electrical stimulation has long been the most effective strategy for evoking neural activity from bionic devices and has been used with great success in the cochlear implant to allow deaf people to hear speech and sound. Despite its success, the spread of electrical current stimulates a broad region of neural tissue meaning that contemporary devices have limited precision. Optical stimulation as an alternative has attracted much recent interest for its capacity to provide highly focused stimuli, and therefore, potentially improved sensory perception. Given its specificity of activation, optical stimulation may also provide a useful tool in the study of fundamental neuroanatomy and neurophysiological processes. Areas covered: This review examines the advances in optical stimulation - infrared, nanoparticle-enhanced, and optogenetic-based - and its application in the inner ear for the restoration of auditory function following hearing loss. Expert opinion: Initial outcomes suggest that optogenetic-based approaches hold the greatest potential and viability amongst optical techniques for application in the cochlea. The future success of this approach will be governed by advances in the targeted delivery of opsins to auditory neurons, improvements in channel kinetics, development of optical arrays, and innovation of opsins that activate within the optimal near-infrared therapeutic window.
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    Combined optogenetic and electrical stimulation of auditory neurons increases effective stimulation frequency—an in vitro study
    (IOP Publishing, 2020-01) Hart, William; Richardson, Rachael; Kameneva, Tatiana; Thompson, Alex; Wise, Andrew; Fallon, James; Stoddart, Paul; Needham, Karina
    OBJECTIVE: The performance of neuroprostheses, including cochlear and retinal implants, is currently constrained by the spatial resolution of electrical stimulation. Optogenetics has improved the spatial control of neurons in vivo but lacks the fast-temporal dynamics required for auditory and retinal signalling. The objective of this study is to demonstrate that combining optical and electrical stimulation in vitro could address some of the limitations associated with each of the stimulus modes when used independently. APPROACH: The response of murine auditory neurons expressing ChR2-H134 to combined optical and electrical stimulation was characterised using whole cell patch clamp electrophysiology. MAIN RESULTS: Optogenetic costimulation produces a three-fold increase in peak firing rate compared to optical stimulation alone and allows spikes to be evoked by combined subthreshold optical and electrical inputs. Subthreshold optical depolarisation also facilitated spiking in auditory neurons for periods of up to 30 ms without evidence of wide-scale Na+ inactivation. Significance These findings may contribute to the development of spatially and temporally selective optogenetic-based neuroprosthetics and complement recent developments in "fast opsins".
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    Directing human induced pluripotent stem cells into a neurosensory lineage for auditory neuron replacement
    (Mary Ann Liebert, 2014) Gunewardene, Niliksha; Van Bergen, Nicole; Crombie, Duncan; Needham, Karina; Dottori, Mirella; Nayagam, Bryony
    Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (AN) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol 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 analyze the expression of key markers involved in AN development, at defined time points of differentiation. The hiPSC and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), pro-neurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160) and sensory AN 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. Furthermore, the neurons generated from this assay were found to be electrically active. Whilst all cell lines analyzed produced functional 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. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs towards a neurosensory lineage, but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.
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    An In Vitro Model of Developmental Synaptogenesis Using Cocultures of Human Neural Progenitors and Cochlear Explants
    (Mary Ann Liebert Inc Publishers, 2013-03) Nayagam, Bryony; Edge, Albert; Needham, Karina; Hyakumura, Tomoko; Leung, Jessie; Nayagam, David; Dottori, Mirella
    In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be signifi- cantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of lo- cating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.This is a copy of an article published in the Stem Cells and Development Journal © 2013 [copyright Mary Ann Liebert, Inc.]; Stem Cells and Development is available online at: http://online.liebertpub.com.
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    Laminin coated diamond electrodes for neural stimulation
    (Elsevier B.V., 2020-09) Sikder, Md..Kabir Uddin; Tong, Wei; Pingle, Hitesh; Kingshott, Peter; Needham, Karina; Shivdasani, Mohit; Fallon, James; Seligman, Peter; Ibbotson, Michael; Prawer, Steven; Garrett, David
    The performance of many implantable neural stimulation devices is degraded due to the loss of neurons around the electrodes by the body's natural biological responses to a foreign material. Coating of electrodes with biomolecules such as extracellular matrix proteins is one potential route to suppress the adverse responses that lead to loss of implant functionality. Concurrently, however, the electrochemical performance of the stimulating electrode must remain optimal to continue to safely provide sufficient charge for neural stimulation. We have previously found that oxygen plasma treated nitrogen included ultrananocrystalline diamond coated platinum electrodes exhibit superior charge injection capacity and electrochemical stability for neural stimulation (Sikder et al., 2019). To fabricate bioactive diamond electrodes, in this work, laminin, an extracellular matrix protein known to be involved in inter-neuron adhesion and recognition, was used as an example biomolecule. Here, laminin was covalently coupled to diamond electrodes. Electrochemical analysis found that the covalently coupled films were robust and resulted in minimal change to the charge injection capacity of diamond electrodes. The successful binding of laminin and its biological activity was further confirmed using primary rat cortical neuron cultures, and the coated electrodes showed enhanced cell attachment densities and neurite outgrowth. The method proposed in this work is versatile and adaptable to many other biomolecules for producing bioactive diamond electrodes, which are expected to show reduced the inflammatory responses in vivo.
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    Organotypic Cocultures of Human Pluripotent Stem Cell Derived- Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices
    (Hindawi, 2019-12) Hyakumura, Tomoko; McDougall, Stuart; Finch, Sue; Needham, Karina; Dottori, Mirella; Nayagam, Bryony
    Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.
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    Thermal damage threshold of neurons during infrared stimulation
    (Biomedical Optics Express, 2020-04) Brown, William; Needham, Karina; Begeng, James; Thompson, Alexander; Nayagam, Bryony; Kameneva, Tatiana; Stoddart, Paul
    In infrared neural stimulation (INS), laser-evoked thermal transients are used to generate small depolarising currents in neurons. The laser exposure poses a moderate risk of thermal damage to the target neuron. Indeed, exogenous methods of neural stimulation often place the target neurons under stressful non-physiological conditions, which can hinder ordinary neuronal function and hasten cell death. Therefore, quantifying the exposure-dependent probability of neuronal damage is essential for identifying safe operating limits of INS and other interventions for therapeutic and prosthetic use. Using patch-clamp recordings in isolated spiral ganglion neurons, we describe a method for determining the dose-dependent damage probabilities of individual neurons in response to both acute and cumulative infrared exposure parameters based on changes in injection current. The results identify a local thermal damage threshold at approximately 60 °C, which is in keeping with previous literature and supports the claim that damage during INS is a purely thermal phenomenon. In principle this method can be applied to any potentially injurious stimuli, allowing for the calculation of a wide range of dose-dependent neural damage probabilities. Unlike histological analyses, the technique is well-suited to quantifying gradual neuronal damage, and critical threshold behaviour is not required.
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    Time-dependent activity of primary auditory neurons in the presence of neurotrophins and antibiotics
    (Elsevier, 2017-04) Cai, Helen; Gillespie, Lisa; Wright, Tess; Brown, William; Minter, Ricki; Nayagam, Bryony; O'Leary, Stephen; Needham, Karina
    In vitro cultures provide a valuable tool in studies examining the survival, morphology and function of cells in the auditory system. Primary cultures of primary auditory neurons have most notably provided critical insights into the role of neurotrophins in cell survival and morphology. Functional studies have also utilized in vitro models to study neuronal physiology and the ion channels that dictate these patterns of activity. Here we examine what influence time-in-culture has on the activity of primary auditory neurons, and how this affects our interpretation of neurotrophin and antibiotic-mediated effects in this population. Using dissociated cell culture we analyzed whole-cell patch-clamp recordings of spiral ganglion neurons grown in the presence or absence of neurotrophins and/or penicillin and streptomycin for 1-3 days in vitro. Firing threshold decreased, and both action potential number and latency increased over time regardless of treatment, whilst input resistance was lowest where neurotrophins were present. Differences in firing properties were seen with neurotrophin concentration but were not consistently maintained over the 3 days in vitro. The exclusion of antibiotics from culture media influenced most firing properties at 1 day in vitro in both untreated and neurotrophin-treated conditions. The only difference still present at 3 days was an increase in input resistance in neurotrophin-treated neurons. These results highlight the potential of neurotrophins and antibiotics to influence neural firing patterns in vitro in a time-dependent manner, and advise the careful consideration of their impact on SGN function in future studies.