DSpace Angular :: Browsing by Author "Flynn, Brianna"

Browsing by Author "Flynn, Brianna"

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The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection
(Elsevier, 2011-08) Wise, Andrew; Tu, Tian; Atkinson, Patrick; Flynn, Brianna; Sgro, Beatrice; Hume, Cliff; O'Leary, Stephen; Shepherd, Robert; Richardson, Rachael
A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the level of transfection was remarkably lower following longer durations of deafness. There was a significant increase in SGN survival in the entire basal turn for cochleae that received NT gene therapy compared to the untreated contralateral control cochleae for the one week deaf group. In the four week deaf group significant SGN survival was observed in the lower basal turn only. There was no increase in SGN survival for the eight week deaf group in any cochlear region. These findings indicated that the efficacy of NT gene therapy diminished with increasing durations of deafness leading to reduced benefits in terms of SGN protection. Clinically, there remains a window of opportunity in which NT gene therapy can provide ongoing trophic support for SGNs.
Hair Cell Regeneration after ATOH1 Gene Therapy in the Cochlea of Profoundly Deaf Adult Guinea Pigs
(PLoS ONE, 2014-07-18) Atkinson, Patrick; Wise, Andrew; Flynn, Brianna; Nayagam, Bryony; Richardson, Rachael
The degeneration of hair cells in the mammalian cochlea results in permanent sensorineural hearing loss. This study aimed to promote the regeneration of sensory hair cells in the mature cochlea and their reconnection with auditory neurons through the introduction of ATOH1, a transcription factor known to be necessary for hair cell development, and the introduction of neurotrophic factors. Adenoviral vectors containing ATOH1 alone, or with neurotrophin-3 and brain derived neurotrophic factor were injected into the lower basal scala media of guinea pig cochleae four days post ototoxic deafening. Guinea pigs treated with ATOH1 gene therapy, alone, had a significantly greater number of cells expressing hair cell markers compared to the contralateral non-treated cochlea when examined 3 weeks post-treatment. This increase, however, did not result in a commensurate improvement in hearing thresholds, nor was there an increase in synaptic ribbons, as measured by CtBP2 puncta after ATOH1 treatment alone, or when combined with neurotrophins. However, hair cell formation and synaptogenesis after co-treatment with ATOH1 and neurotrophic factors remain inconclusive as viral transduction was reduced due to the halving of viral titres when the samples were combined. Collectively, these data suggest that, whilst ATOH1 alone can drive non-sensory cells towards an immature sensory hair cell phenotype in the mature cochlea, this does not result in functional improvements after aminoglycoside-induced deafness.
Halting the progression of noise-induced hearing loss with gene therapy
(2013) Richardson, Rachael; Atkinson, Patrick; Wise, Andrew; Flynn, Brianna; O'Leary, Stephen; Hume, Clifford; Shepherd, Robert
Progressive hearing loss is often ignored until there is significant loss of cochlear hair cells (HCs) and spiral ganglion neurons (SGNs). It usually begins as a mild high-frequency threshold shift which worsens and also spreads to the lower frequencies. Our previous research indicated that gene therapy is effective for long-term preservation of SGNs when administered shortly after ototoxic hearing loss, but has greater potential to protect residual HCs and SGNs after the onset of progressive hearing loss and even to restore hearing.
Neurotrophin gene therapy for sustained neural preservation after deafness
(PLOS, 2012-12-17) Atkinson, Patrick; Wise, Andrew; Flynn, Brianna; Nayagam, Bryony; Hume, Clifford; O'Leary, Stephen; Shepherd, Robert; Richardson, Rachael
The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.
Pharmacokinetics and tissue distribution of neurotrophin 3 after intracochlear delivery
(Elsevier B.V., 2019-02) Richardson, Rachael; Hu, Qi-Ying; Shi, Fuxin; Nguyen, Trung; Fallon, James; Flynn, Brianna; Wise, Andrew
Neurotrophin therapy has potential to reverse some forms of hearing loss. However, cochlear pharmacokinetic studies are challenging due to small fluid volumes. Here a radioactive tracer was used to determine neurotrophin- 3 retention, distribution and clearance after intracochlear administration. 125I- neurotrophin-3 was injected into guinea pig cochleae using a sealed injection technique comparing dosing volumes, rates and concentrations up to 750 μg/mL. Retention was measured by whole-cochlear gamma counts at five time points while distribution and clearance were assessed by autoradiography. Smaller injection volumes and higher concentrations correlated with higher retention of neurotrophin-3. Distribution of neurotrophin-3 was widespread throughout the cochlear tissue, decreasing in concentration from base to apex. Tissue distribution was nonuniform, with greatest density in cells lining the scala tympani and lower density in neural target tissue. The time constant for clearance of neurotrophin-3 from cochlear tissues was 38 h but neurotrophin-3 remained detectable for at least 2 weeks. Neurotrophin-3 was evident in the semi-circular canals with minor spread to the contralateral cochlea. This study is the first comprehensive evaluation of the disposition profile for a protein therapy in the cochlea. The findings and methods in this study will provide valuable guidance for the development of protein therapies for hearing loss.
A radiolab ele d drug tracing method to study neurotrophin-3 retention and distribution in the cochlea after nano-based local delivery
(Elsevier B.V., 2020-09) Lam, Patrick; Gunewardene, Niliksha; Ma, Yutian; Caruso, Frank; Nguyen, Trung; Flynn, Brianna; Wise, Andrew; Richardson, Rachael
Hearing loss is the most common sensory deficit worldwide with no approved therapeutics for treatment. Local neurotrophin delivery into the cochlea has shown great potential in protecting and repairing the sensory cells important for hearing. However, delivery of these factors into the inner ear at therapeutic levels over a sustained period of time has remained a challenge restricting clinical translation. We have developed a method to test the pharmacokinetics of neurotrophin released from porous silica particles called ‘supraparticles’ that can provide sustained release of neurotrophins to the inner ear. This report describes a radiolabeling method to examine neurotrophin retention and distribution in the cochlea. The neurotrophin was labeled with a radioactive tracer (iodine 125: 125I) and delivered into the cochlea via the supraparticle system. Gamma counts reveal drug levels and clearance in the intact cochlea, as well as accumulation in off-target organs (safety test). Autoradiography analyses using film and emulsion permit quantification and visualization of drug distribution at the cellular level. The method has a detection limit of 0.8 pg of radiolabeled neurotrophin-3 in cochlear sections exposed to film. The tracer 125I with a half-life of 59.4 days can be used to label other drugs/substances with a tyrosine residue and therefore be broadly applicable for long-term pharmacokinetic studies in other systems.
Regeneration of cochlear hair cells with Atoh1 gene therapy after noise-induced hearing loss
(SciTechnol, 2015-06) Wise, Andrew; Flynn, Brianna; Atkinson, Patrick; Fallon, James; Nicholson, Madeline; Richardson, Rachael
Background: Degeneration of hair cells in the mammalian cochlea results in irreversible hearing loss with no current treatment options to regain lost hair cell function. The Atoh1 gene is necessary for hair cell development and recent research has shown that Atoh1 gene therapy promotes new hair cell formation and hearing restoration in adult rodent deafness models. Objective: The aim of this study was to examine new hair cell formation via Atoh1 gene therapy in noise-deafened adult guinea pigs. Methods: Guinea pigs were deafened by noise exposure (130 dB, 11-13 kHz, 2 hours). After two weeks, the left cochleae were injected with an adenoviral vector containing the Atoh1 gene. Control animals were injected with a control adenoviral vector. Three weeks after injection cochleae were assessed for hair cell density, maturity and hair cell synaptogenesis with auditory neurons. Hearing thresholds were assessed throughout. Results: There were significantly more myosinVIIa-positive hair cells in cochleae that received Atoh1 gene therapy compared to contralateral cochleae and compared to cochleae that received control gene therapy (p<0.05 one way ANOVA). However, the number of hair cells in Atoh1-treated animals was far below normal. Expression of Atoh1 had a significant preservation effect on the cytoarchitecture of the sensory epithelium compared to controls (p<0.001 one way ANOVA). Expression of the synaptic protein CtBP2 was present in some transfected cells from Atoh1-injected guinea pigs but at a reduced density compared to normal cochleae. There was evidence of auditory neuron preservation near transfected hair cells in Atoh1-injected cochleae (p<0.05 one way ANOVA), but there were no improvements in hearing thresholds. Conclusion: This study supports growing evidence that new hair cell formation is possible in mature cochleae that have been severely damaged, in this case by noise, and demonstrates a protective influence of Atoh1 gene therapy on the immediate surrounding cellular environment.
Shepherd, R. K., P. Carter, Y. L. Enke, A. Thompson, B. Flynn, E. Trang, A. Dalrymple, and J. B. Fallon. 2020. Chronic intracochlear electrical stimulation at high charge densities: Reducing platinum dissolution
(IOP Publishing, 2020-09) Shepherd, Robert; Carter, Paul; Enke, Ya Lang; Thompson, Alex; Flynn, Brianna; Trang, Ella; Dalrymple, Ashley; Fallon, James
OBJECTIVE: Cochleae of long-term cochlear implant users have shown evidence of particulate platinum (Pt) corroded from the surface of Pt electrodes. The pathophysiological effect of Pt within the cochlea has not been extensively investigated. We previously evaluated the effects of Pt corrosion at high charge densities and reported negligible pathophysiological impact. The present study extends this work by examining techniques that may reduce Pt corrosion. APPROACH: Deafened guinea pigs were continuously stimulated for 28 days using biphasic current pulses at extreme charge densities using: (i) electrode shorting; (ii) electrode shorting with capacitive coupling (CC); or (iii) electrode shorting with alternating leading phase (AP). On completion of stimulation, cochleae were examined for corrosion product, tissue response, auditory nerve (AN) survival and trace levels of Pt; and electrodes examined for surface corrosion. MAIN RESULTS: Pt corrosion was evident at > 200 μC/cm2/phase; the amount dependent on charge density (p < 0.01) and charge recovery technique (p < 0.01); reduced corrosion was apparent using CC. Tissue response increased with charge density (p < 0.007); cochleae stimulated at > 200 μC/cm2/phase exhibited a vigorous response including a focal region of necrosis and macrophages. Notably, tissue response was not dependent on the charge recovery technique (p = 0.56). Despite stimulation at high charge densities resulting in significant levels of Pt corrosion, there was no stimulus induced loss of ANs. SIGNIFICANCE: Significant increases in tissue response and Pt corrosion were observed following stimulation at high charge densities. Charge recovery using CC, and to a lesser extent AP, reduced the amount of Pt corrosion but not the tissue response. Stimulation at change densities an order of magnitude higher than those used when programming cochlear implant recipients in the clinic, produced a vigorous tissue response and corrosion products without evidence of neural loss.
Viability of long-term gene therapy in the cochlea
(Nature Publishing Group, 2014-04-22) Atkinson, Patrick; Wise, Andrew; Flynn, Brianna; Nayagam, Bryony; Richardson, Rachael
Gene therapy has been investigated as a way to introduce a variety of genes to treat neurological disorders. An important clinical consideration is its long-term effectiveness. This research aims to study the long-term expression and effectiveness of gene therapy in promoting spiral ganglion neuron survival after deafness. Adenoviral vectors modified to express brain derived neurotrophic factor or neurotrophin-3 were unilaterally injected into the guinea pig cochlea one week post ototoxic deafening. After six months, persistence of gene expression and significantly greater neuronal survival in neurotrophin-treated cochleae compared to the contralateral cochleae were observed. The long-term gene expression observed indicates that gene therapy is potentially viable; however the degeneration of the transduced cells as a result of the original ototoxic insult may limit clinical effectiveness. With further research aimed at transducing stable cochlear cells, gene therapy may be an efficacious way to introduce neurotrophins to promote neuronal survival after hearing loss.

Browsing by Author "Flynn, Brianna"

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