Electrical Stimulation publications

Adeno-associated virus (AAV) unequivocally emerges as one of the most powerful and promising delivery vectors for gene therapy targeting hereditary hearing loss. Following AAV transduction in the inner ear, varying degrees of natural immune responses are triggered, primarily characterized by macrophage activation and the secretion of pro-inflammatory factors. Additionally, the production of neutralizing antibodies may affect the efficacy of gene therapy. To evaluate immune dynamics, we injected AAV1 and AAV-ie (capsids with distinct transfection efficiencies) into murine cochleae and analyzed temporal transcriptomic profiles. Our results demonstrate that both capsids induce immune activity but with critical temporal and intensity differences that AAV1 elicits significantly later and milder immune reactions compared to AAV-ie. These findings establish that dynamic cochlear gene expression profiles directly inform the selection of immunologically optimized AAV vectors to minimize adverse responses in future hereditary hearing loss gene therapies.

Background: Preserving residual hearing after cochlear implant (CI) surgery remains a crucial challenge. The application of dexamethasone (DEX) has been proven to positively affect residual hearing. To deliver DEX in a localized and controlled way, a round window niche implant (RNI), allowing drug diffusion via the round window membrane into the cochlea, may be used. To prove this concept, an RNI for guinea pigs as a CI-trauma model was manufactured by molding and tested for its drug release in vitro and biological effects in vivo. Methods: The RNIs were molded using silicone containing 10% DEX. Release was analyzed over time using high-performance liquid chromatography (HPLC). Fourteen adult guinea pigs were randomly assigned to two groups (CI or CI + RNI group). All animals received a unilateral CI electrode insertion trauma followed by CI insertion. The CI + RNI group was additionally implanted with an RNI containing 10% DEX. Animals were followed up for 4 weeks. Acoustically evoked auditory brainstem response and impedance measurement, micro-computed tomography (µCT) imaging, and histology were performed for evaluation. Results: DEX was released for more than 250 days in vitro, with an initial burst followed by a slower release over time. Comparing the hearing threshold shift (from day 0 to day 28) of the CI and CI + RNI groups, significant differences were observed at 32 and 40 kHz. The impedance shift at basal contacts was lower in the CI + RNI group than in the CI group. Moreover, the fibrosis in the lower basal turn was reduced in the CI + RNI group in contrast to the CI group. Conclusions: The RNI containing 10% DEX has anti-inflammatory potential concerning fibrosis inhibition and has beneficial effects on hearing preservation at high frequencies.

We examined the effect of neonatal deafening on frequency-specific pathways for processing of interaural time differences (ITDs) in cochlear-implant stimuli. Animal studies have demonstrated differences in neural ITD sensitivity in the inferior colliculus (IC) depending on the intracochlear location of intracochlear stimulating electrodes. We used neonatally deafened (ND) rats of both sexes and recorded the responses of single neurons in the IC to electrical stimuli with ITDs delivered to the apical or basal cochlea and compared them with acutely deafened (AD) rats of both sexes with normal hearing (NH) during development. We found that neonatal deafness significantly impacted the ITD sensitivity and the ITD tuning patterns restricted to apically driven IC neurons. In ND rats, the ITD sensitivity of apically driven neurons is reduced to values similar to basally driven neurons. The prevalence of ITD-sensitive apical neurons with a peak-shaped ITD tuning curve, which may reflect predominant input from the medial superior olivary (MSO) complex, in ND rats was diminished compared with that in AD rats (67%, AD vs 40%, ND). Conversely, monotonic-type responses rarely occurred in AD rats (14%) but were approximately equally as prevalent as peak-type tuning curves in ND rats (42%). Nevertheless, in ND rats, the ITD at the maximum slope of the ITD tuning curve was still more concentrated within the physiological ITD range in apically driven than in basally driven neurons. These results indicate that the development of high ITD sensitivity processed by low-frequency pathways depends on normal auditory experience and associated biases in ITD tuning strategies.

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the ‘cochlear amplifier' selectively express the type III intermediate filament peripherin gene ( Prph) . Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the Prph KO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, “contralateral suppression” of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the Prph KO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and Prph KO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24–32 kHz) in Prph KO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the Prph KO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.

The medial olivocochlear (MOC) efferent feedback circuit projecting to the cochlear outer hair cells (OHCs) confers protection from noise-induced hearing loss and is generally thought to be driven by inner hair cell (IHC) - type I spiral ganglion afferent (SGN) input. Knockout of the Prph gene ( Prph KO) encoding the peripherin type III intermediate filament disrupted the OHC - type II SGN innervation and virtually eliminated MOC – mediated contralateral suppression from noise delivered to the opposite ear, measured as a reduction in cubic distortion product otoacoustic emissions. Electrical stimulation of the MOC pathway elicited contralateral suppression indistinguishable between wildtype (WT) and Prph KO mice, indicating that the loss of contralateral suppression was not due to disruption of the efferent arm of the circuit; IHC – type I SGN input was also normal, based on auditory brainstem responses. High-intensity, broadband noise (108 dB SPL, 1 hour) produced permanent hearing loss in Prph KO mice, but not in WT littermates. These findings associate OHC-type II input with MOC efferent - based otoprotection at loud sound levels.

Clinical management of auditory synaptopathies like other genetic hearing disorders is currently limited to the use of hearing aids or cochlear implants. However, future gene therapy promises restoration of hearing in selected forms of monogenic hearing impairment, in which cochlear morphology is preserved over a time window that enables intervention. This includes non-syndromic autosomal recessive hearing impairment DFNB93, caused by defects in the CABP2 gene. Calcium-binding protein 2 (CaBP2) is a potent modulator of inner hair cell (IHC) voltage-gated calcium channels Ca V 1.3. Based on disease modeling in Cabp2 –/– mice, DFNB93 hearing impairment has been ascribed to enhanced steady-state inactivation of IHC Ca V 1.3 channels, effectively limiting their availability to trigger synaptic transmission. This, however, does not seem to interfere with cochlear development and does not cause early degeneration of hair cells or their synapses. Here, we studied the potential of a gene therapeutic approach for the treatment of DFNB93. We used AAV2/1 and AAV-PHP.eB viral vectors to deliver the Cabp2 coding sequence into IHCs of early postnatal Cabp2 –/– mice and assessed the level of restoration of hair cell function and hearing. Combining in vitro and in vivo approaches, we observed high transduction efficiency, and restoration of IHC Ca V 1.3 function resulting in improved hearing of Cabp2 –/– mice. These preclinical results prove the feasibility of DFNB93 gene therapy.

Age-related hearing loss (ARHL) is the most common sensory disorder among older people, and yet, the treatment options are limited to medical devices such as hearing aids and cochlear implants. The high prevalence of ARHL mandates the development of treatment strategies that can prevent or rescue age-related cochlear degeneration. In this study, we investigated a novel pharmacological strategy based on inhibition of the adenosine A2A receptor (A2AR) in middle aged C57BL/6 mice prone to early onset ARHL. C57BL/6J mice were treated with weekly istradefylline (A2AR antagonist; 1 mg/kg) injections from 6 to 12 months of age. Auditory function was assessed using auditory brainstem responses (ABR) to tone pips (4–32 kHz). ABR thresholds and suprathreshold responses (wave I amplitudes and latencies) were evaluated at 6, 9, and 12 months of age. Functional outcomes were correlated with quantitative histological assessments of sensory hair cells. Cognitive function was assessed using the Morris water maze and the novel object recognition test, and the zero maze test was used to assess anxiety-like behaviour. Weekly injections of istradefylline attenuated ABR threshold shifts by approximately 20 dB at mid to high frequencies (16–32 kHz) but did not improve ABR suprathreshold responses. Istradefylline treatment improved hair cell survival in a turn-dependent manner, whilst the cognitive function was unaffected by istradefylline treatment. This study presents the first evidence for the rescue potential of istradefylline in ARHL and highlights the role of A2AR in development of age-related cochlear degeneration.

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene ( OTOF ), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out ( Otof- KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof- KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9—using a single overloaded AAV vector—is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

Currently, there are limited reports available regarding the treatment and prevention of progressive age-related hearing loss. This is because age-related hearing loss is not a critical disease with direct fatalities and has several well-established countermeasures such as hearing aids and cochlear implants. This study evaluated the efficacy of Ninjinyoeito (NYT) in the treatment of age-related hearing loss. C57BL/6J mice were divided into three groups: baseline group, untreated group, and NYT-treated group, with the latter receiving NYT treatment for 2 months. The mice were fed with NYT extract mixed with 4% mouse normal chow. Hearing loss was confirmed by a reduction in intact cell density of the auditory nerve from the age of 5-7 months. The suppression of hearing loss with aging and decrease in the intact cell density of the auditory nerve were significant in mice fed with NYT for 2 months. NYT has been reported to improve blood flow and enhance mitochondrial activity and may exert its protective effects on spiral neurons through these mechanisms. There was no decrease in the size of the stria vascularis from the age of 5-7 months in C57BL/6J mice. The present model failed to reveal the effect of NYT on atrophy of the stria vascularis of the cochlear duct. In conclusion, NYT appears to have a protective effect on the auditory nerve and suppress the progression of age-related hearing loss by reducing age-related auditory nerve degeneration.

In all commercial cochlear implant (CI) devices, the electric stimulation is performed with a rectangular pulse that generally has two phases of opposite polarity. To date, developing new stimulation strategies has relied on the efficacy of this shape. Here, we investigate the potential of a novel stimulation paradigm that uses biophysically-inspired electrical ramped pulses. Using electrically-evoked auditory brainstem response (eABR) recordings in mice, we found that less charge, but higher current level amplitude, is needed to evoke responses with ramped shapes that are similar in amplitude to responses obtained with rectangular shapes. The most charge-efficient pulse shape had a rising ramp over both phases, supporting findings from previous in vitro studies. This was also true for longer phase durations. Our study presents the first physiological data on CI-stimulation with ramped pulse shapes. By reducing charge consumption ramped pulses have the potential to produce more battery-efficient CIs and may open new perspectives for designing other efficient neural implants in the future.

The ability to measure the voltage readout from a sensor implanted inside the living cochlea enables continuous monitoring of intracochlear acoustic pressure locally, which could improve cochlear implants. We developed a piezoelectric intracochlear acoustic transducer (PIAT) designed to sense the acoustic pressure while fully implanted inside a living guinea pig cochlea. The PIAT, fabricated using micro-electro-mechanical systems (MEMS) techniques, consisted of an array of four piezoelectric cantilevers with varying lengths to enhance sensitivity across a wide frequency bandwidth. Prior to implantation, benchtop tests were conducted to characterize the device performance in air and in water. When implanted in the cochlea of an anesthetized guinea pig, the in vivo voltage response from the PIAT was measured in response to 80–95 dB sound pressure level 1–14 kHz sinusoidal acoustic excitation at the entrance of the guinea pig’s ear canal. All sensed signals were above the noise floor and unaffected by crosstalk from the cochlear microphonic or external electrical interference. These results demonstrate that external acoustic stimulus can be sensed via the piezoelectric voltage response of the implanted MEMS transducer inside the living cochlea, providing key steps towards developing intracochlear acoustic sensors to replace external or subcutaneous microphones for auditory prosthetics.

Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

Auditory information relayed by auditory nerve fibers and somatosensory information relayed by granule cell parallel fibers converge on the fusiform cells (FCs) of the dorsal cochlear nucleus, the first brain station of the auditory pathway. In vitro, parallel fiber synapses on FCs exhibit spike-timing-dependent plasticity with Hebbian learning rules, partially mediated by the NMDA receptor (NMDAr). Well-timed bimodal auditory-somatosensory stimulation, in vivo equivalent of spike-timing-dependent plasticity, can induce stimulus-timing-dependent plasticity (StTDP) of the FCs spontaneous and tone-evoked firing rates. In healthy guinea pigs, the resulting distribution of StTDP learning rules across a FC neural population is dominated by a Hebbian profile while anti-Hebbian, suppressive and enhancing LRs are less frequent. In this study, we investigate in vivo, the NMDAr contribution to FC baseline activity and long term plasticity. We find that blocking the NMDAr decreases the synchronization of FC- spontaneous activity and mediates differential modulation of FC rate-level functions such that low, and high threshold units are more likely to increase, and decrease, respectively, their maximum amplitudes. Three significant alterations in mean learning-rule profiles were identified: transitions from an initial Hebbian profile towards (1) an anti-Hebbian; (2) a suppressive profile; and (3) transitions from an anti-Hebbian to a Hebbian profile. FC units preserving their learning rules showed instead, NMDAr-dependent plasticity to unimodal acoustic stimulation, with persistent depression of tone-evoked responses changing to persistent enhancement following the NMDAr antagonist. These results reveal a crucial role of the NMDAr in mediating FC baseline activity and long-term plasticity which have important implications for signal processing and auditory pathologies related to maladaptive plasticity of dorsal cochlear nucleus circuitry.

Dynamin-1 (Dnm1) encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for a novel spontaneous Dnm1 mutation--fitful--experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Fitful is a missense mutation in an exon that defines the DNM1a isoform, leaving intact the alternatively spliced exon that encodes DNM1b. The expression of the corresponding alternate transcripts is developmentally regulated, with DNM1b expression highest during early neuronal development and DNM1a expression increasing postnatally with synaptic maturation. Mutant DNM1a does not efficiently self-assemble into higher order complexes known to be necessary for proper dynamin function, and it also interferes with endocytic recycling in cell culture. In mice, the mutation results in defective synaptic transmission characterized by a slower recovery from depression after trains of stimulation. The DNM1a and DNM1b isoform pair is highly conserved in vertebrate evolution, whereas invertebrates have only one isoform. We speculate that the emergence of more specialized forms of DNM1 may be important in organisms with complex neuronal function.