Multidrug combination therapy in the inner ear faces diverse challenges due to the distinct physicochemical properties of drugs and the difficulties of overcoming the oto-biologic barrier. Although nanomedicine platforms offer potential solutions to multidrug delivery, the access of drugs to the inner ear remains limited. Micro/nanomachines, capable of delivering cargo actively, are promising tools for overcoming bio-barriers. Herein, a novel microrobot-based strategy to penetrate the round window membrane (RWM) is presented and multidrug in on-demand manner is delivered. The tube-type microrobot (TTMR) is constructed using the template-assisted layer-by-layer (LbL) assembly of chitosan/ferroferric oxide/silicon dioxide (CS/Fe3O4/SiO2) and loaded with anti-ototoxic drugs (curcumin, CUR and tanshinone IIA, TSA) and perfluorohexane (PFH). Fe3O4 provides magnetic actuation, while PFH ensures acoustic propulsion. Upon ultrasound stimulation, the vaporization of PFH enables a microshotgun-like behavior, propelling the drugs through barriers and driving them into the inner ear. Notably, the proportion of drugs entering the inner ear can be precisely controlled by varying the feeding ratios. Furthermore, in vivo studies demonstrate that the drug-loaded microrobot exhibits superior protective effects and excellent biosafety toward cisplatin (CDDP)-induced hearing loss. Overall, the microrobot-based strategy provides a promising direction for on-demand multidrug delivery for ear diseases.

The sensory cells of the inner ear, called hair cells, do not regenerate spontaneously and therefore, hair cell loss and subsequent hearing loss are permanent in humans. Conversely, functional hair cell regeneration can be observed in non-mammalian vertebrate species like birds and fish. Also, during postnatal development in mice, limited regenerative capacity and the potential to isolate stem cells were reported. Together, these findings spurred the interest of current research aiming to investigate the endogenous regenerative potential in mammals. In this review, we summarize current in vitro based approaches and briefly introduce different in vivo model organisms utilized to study hair cell regeneration. Furthermore, we present an overview of the findings that were made synergistically using both, the in vitro and in vivo based tools.

Auditory neuropathy is a cause of hearing loss that has been studied in a number of animal models. Signal transmission from hair cells to spiral ganglion neurons plays an important role in normal hearing. CYLD is a microtubule-binding protein, and deubiquitinase involved in the regulation of various cellular processes. In this study, we used Cyld knockout (KO) mice and nerve cell lines to examine whether CYLD is associated with auditory neuropathy.

Hearing of Cyld KO mice was studied using the TDT RZ6 auditory physiology workstation. The expression and localization of CYLD in mouse cochlea and cell lines were examined by RT-PCR, immunoblotting, and immunofluorescence. CYLD expression was knocked down in SH-SY5Y cells by shRNAs and in PC12 and N2A cells by siRNAs. Nerve growth factor and retinoic acid were used to induce neurite outgrowth, and the occurrence and length of neurites were statistically analyzed between knockdown and control groups.

Cyld KO mice had mild hearing impairment. Moreover, CYLD was widely expressed in mouse cochlear tissues and different nerve cell lines. Knocking down CYLD significantly reduced the length and proportion of neurites growing from nerve cells.

The abnormal hearing of Cyld KO mice might be caused by a decrease in the length and number of neurites growing from auditory nerve cells in the cochlea, suggesting that CYLD is a key protein affecting hearing.

Disabling hearing loss is the most common sensorineural disability worldwide. It affects around 466 million people and its incidence is expected to rise to around 900 million people by 2050, according to World Health Organization estimates. Most cases of hearing impairment are due to the degeneration of hair cells (HCs) in the cochlea, mechano-receptors that transduce incoming sound information into electrical signals that are sent to the brain. Damage to these cells is mainly caused by exposure to aminoglycoside antibiotics and to some anti-cancer drugs such as cisplatin, loud sounds, age, infections and genetic mutations. Hearing deficits may also result from damage to the spiral ganglion neurons that innervate cochlear HCs. Differently from what is observed in avian and non-mammalian species, there is no regeneration of missing sensory cell types in the adult mammalian cochlea, what makes hearing loss an irreversible process. This review summarizes the research that has been conducted with the aim of developing cell-based strategies that lead to sensory cell replacement in the adult cochlea and, ultimately, to hearing restoration. Two main lines of research are discussed, one directed toward the transplantation of exogenous replacement cells into the damaged tissue, and another that aims at reactivating the regenerative potential of putative progenitor cells in the adult inner ear. Results from some of the studies that have been conducted are presented and the advantages and drawbacks of the various approaches discussed.

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

OBJECTIVE Shunt obstruction by cells and/or tissue is the most common cause of shunt failure. Ventricular catheter obstruction alone accounts for more than 50% of shunt failures in pediatric patients. The authors sought to systematically collect explanted ventricular catheters from the Seattle Children's Hospital with a focus on elucidating the cellular mechanisms underlying obstruction. METHODS In the operating room, explanted hardware was placed in 4% paraformaldehyde. Weekly, samples were transferred to buffer solution and stored at 4°C. After consent was obtained for their use, catheters were labeled using cell-specific markers for astrocytes (glial fibrillary acidic protein), microglia (ionized calcium-binding adapter molecule 1), and choroid plexus (transthyretin) in conjunction with a nuclear stain (Hoechst). Catheters were mounted in custom polycarbonate imaging chambers. Three-dimensional, multispectral, spinning-disk confocal microscopy was used to image catheter cerebrospinal fluid-intake holes (10× objective, 499.2-μm-thick z-stack, 2.4-μm step size, Olympus IX81 inverted microscope with motorized stage and charge-coupled device camera). Values are reported as the mean ± standard error of the mean and were compared using a 2-tailed Mann-Whitney U-test. Significance was defined at p < 0.05. RESULTS Thirty-six ventricular catheters have been imaged to date, resulting in the following observations: 1) Astrocytes and microglia are the dominant cell types bound directly to catheter surfaces; 2) cellular binding to catheters is ubiquitous even if no grossly visible tissue is apparent; and 3) immunohistochemical techniques are of limited utility when a catheter has been exposed to Bugbee wire electrocautery. Statistical analysis of 24 catheters was performed, after excluding 7 catheters exposed to Bugbee wire cautery, 3 that were poorly fixed, and 2 that demonstrated pronounced autofluorescence. This analysis revealed that catheters with a microglia-dominant cellular response tended to be implanted for shorter durations (24.7 ± 6.7 days) than those with an astrocyte-dominant response (1183 ± 642 days; p = 0.027). CONCLUSIONS Ventricular catheter occlusion remains a significant source of shunt morbidity in the pediatric population, and given their ability to intimately associate with catheter surfaces, astrocytes and microglia appear to be critical to this pathophysiology. Microglia tend to be the dominant cell type on catheters implanted for less than 2 months, while astrocytes tend to be the most prevalent cell type on catheters implanted for longer time courses and are noted to serve as an interface for the secondary attachment of ependymal cells and choroid plexus.

Hearing loss is currently an incurable degenerative disease characterized by a paucity of hair cells (HCs), which cannot be spontaneously replaced in mammals. Recent technological advancements in gene therapy and local drug delivery have shed new light for hearing loss. Atoh1, also known as Math1, Hath1, and Cath1, is a proneural basic helix-loop-helix (bHLH) transcription factor that is essential for HC differentiation. At various stages in development, Atoh1 activity is sufficient to drive HC differentiation in the cochlea. Thus, Atoh1 related gene therapy is the most promising option for HC induction. DAPT, an inhibitor of Notch signaling, enhances the expression of Atoh1 indirectly, which in turn promotes the induction of a HC fate. Here, we show that DAPT cooperates with Atoh1 to synergistically promote HC fate in ependymal cells in vitro and promote hair cell regeneration in the cultured basilar membrane (BM) which mimics the microenvironment in vivo. Taken together, our findings demonstrated that DAPT is sufficient to induce HC-like cells via enhancing of the expression of Atoh1 to inhibit the progression of HC apoptosis and to induce new HC formation.

Neuroblasts migrate as directed chains of cells during development and following brain damage. A fuller understanding of the mechanisms driving this will help define its developmental significance and in the refinement of strategies for brain repair using transplanted stem cells. Recently, we reported that in adult mouse there are ionic gradients within the extracellular spaces that create an electrical field (EF) within the rostral migratory stream (RMS), and that this acts as a guidance cue for neuroblast migration. Here, we demonstrate an endogenous EF in brain slices and show that mimicking this by applying an EF of physiological strength, switches on chain migration in mouse neurospheres and in the SH-SY5Y neuroblastoma cell line. Firstly, we detected a substantial endogenous EF of 31.8 ± 4.5 mV/mm using microelectrode recordings from explants of the subventricular zone (SVZ). Pharmacological inhibition of this EF, effectively blocked chain migration in 3D cultures of SVZ explants. To mimic this EF, we applied a physiological EF and found that this increased the expression of N-cadherin and β-catenin, both of which promote cell-cell adhesion. Intriguingly, we found that the EF up-regulated P2Y purinoceptor 1 (P2Y1) to contribute to chain migration of neuroblasts through regulating the expression of N-cadherin, β-catenin and the activation of PKC. Our results indicate that the naturally occurring EF in brain serves as a novel stimulant and directional guidance cue for neuronal chain migration, via up-regulation of P2Y1.

Hearing loss is the most common sensory deficit in developed societies. Hearing impairment in children, particularly of prelingual onset, has been shown to negatively affect educational achievement, future employment and earnings, and even life expectancy. Sensorineural hearing loss (SNHL), which refers to defects within the cochlea or auditory nerve itself, far outweighs conductive causes for permanent hearing loss in both children and adults. The causes of SNHL in children are heterogeneous, including both congenital and acquired causes. This article identifies potential mechanisms of intervention both at the level of the hair cell and the spiral ganglion neurons.

Ginkgo biloba extract (GBE) has been widely used for treatment of neural damage and disorders. Neural stem cells (NSCs) hold promise as a treatment of hearing loss caused by neural damage. However, the biological functions of GBE in modulating NSC behaviors in the cochlea are still largely elusive. In this study, we sought to explore the effects of GBE on the differentiation and performance of NSCs from mouse cochlea. Our data showed that GBE treatment promotes cell survival and NSC proliferation. In addition, GBE treatment also increases NSC differentiation to neurons and enhances the performance of mature neural networks evident by the increased frequency of calcium oscillation. Moreover, neurite outgrowth is also dramatically increased upon GBE treatment. Overall, our study demonstrates the positive regulatory role of GBE in NSC proliferation and differentiation into functional neurons in vitro, supporting the potential therapeutic use of GBE in hearing loss recovery.

Spiral ganglion neurons (SGNs) of the eighth nerve serve as the bridge between hair cells and the cochlear nucleus. Hair cells use Cav1.3 as the primary channel for Ca(2+) inflow to mediate transmitter release. In contrast, SGNs are equipped with multiple Ca(2+) channels to mediate Ca(2+)-dependent functions. We examined directly the role of Cav1.3 channels in SGNs using Cav1.3-deficient mice (Cav1.3(-/-)). We revealed a surprising finding that SGNs functionally express the cardiac-specific Cav1.2, as well as neuronal Cav1.3 channels. We show that evoked action potentials recorded from SGNs show a significant decrease in the frequency of firing in Cav1.3(-/-) mice compared with wild-type (Cav1.3(+/+)) littermates. Although Cav1.3 is the designated L-type channel in neurons, whole-cell currents recorded in isolated SGNs from Cav1.3(-/-) mice showed a surprising remnant current with sensitivity toward the dihydropyridine (DHP) agonist and antagonist, and a depolarization shift in the voltage-dependent activation compared with that in the Cav1.3(+/+) mice. Indeed, direct measurement of the elementary properties of Ca(2+) channels, in Cav1.3(+/+) neurons, confirmed the existence of two DHP-sensitive single-channel currents, with distinct open probabilities and conductances. We demonstrate that the DHP-sensitive current in Cav1.3(-/-) mice is derived from Cav1.2 channel activity, providing for the first time, to our knowledge, functional data for the expression of Cav1.2 currents in neurons. Finally, using shRNA gene knockdown methodology, and histological analyses of SGNs from Cav1.2(+/-) and Cav1.3(+/-) mice, we were able to establish the differential roles of Cav1.2 and Cav1.3 in SGNs.

Hearing loss is the most common sensory deficit in humans, with some estimates suggesting up to 300 million affected individuals worldwide. Both environmental and genetic factors contribute to hearing loss and can cause death of sensory cells and neurons. Because these cells do not regenerate, the damage tends to accumulate, leading to profound deafness. Several biological strategies to restore auditory function are currently under investigation. Owing to the success of cochlear implants, which offer partial recovery of auditory function for some profoundly deaf patients, potential biological therapies must extend hearing restoration to include greater auditory acuity and larger patient populations. Here, we review the latest gene, stem-cell, and molecular strategies for restoring auditory function in animal models and the prospects for translating these approaches into viable clinical therapies.

Whereas prehearing spiral ganglion neurons (SGNs) rely faithfully on outputs from spontaneously active developing hair cells, the electrical phenotypes of posthearing neurons are shaped by distinct rapid and graded receptor potentials from hair cells. To date, technical difficulties in isolation of fragile posthearing neurons from the rigid bony labyrinth of the inner ear have hindered analyses of the electrical phenotype of SGNs. Therefore, we have recently developed new strategies to isolate posthearing mouse SGNs for functional analyses. Here, we describe the coarse and fine properties of Ca(2+) currents, which sculpt the firing properties of posthearing SGNs. Murine SGNs express multiple Ca(2+) channel currents to enable diverse functions. We have demonstrated that suppression of Ca(2+) currents results in significant hyperpolarization of the resting membrane potential (rmp) of basal SGNs, suggesting that Ca(2+) influx primes rmp for excitation. In contrast, removal of external Ca(2+) has modest effects on rmp of apical SGNs. The blockade of Ca(2+) currents with a mixture of specific blockers attenuates spontaneously active SGNs. Paradoxically, different subtypes of Ca(2+) currents, such as R-type currents, may activate resting outward conductances since blockage of the current results in depolarization of rmp. In keeping with whole-cell current data, single-channel records revealed multiple diverse Ca(2+) channels in SGNs. Additionally, there were differential expressions of distinct Ca(2+) current densities in the apicobasal contour of the adult cochlea. This report provides invaluable insights into Ca(2+)-dependent processes in adult SGNs.

To provide an overview of the methodologies involved in the field of hair cell regeneration. First, the author provides a tutorial on the biotechnological foundations of this field to assist the reader in the comprehension and interpretation of the research involved in hair cell regeneration. Next, the author presents a review of stem cell and gene therapy and provides a critical appraisal of their application to hair cell regeneration. The methodologies used in these approaches are highlighted.

The author conducted a narrative review of the fields of cellular, molecular, and developmental biology, tissue engineering, and stem cell and gene therapy using the PubMed database.

The use of biotechnological approaches to the treatment of hearing loss--approaches such as stem cell and gene therapy-has led to new methods of regenerating cochlear hair cells in mammals.

Incredible strides have been made in assembling important pieces of the puzzle that comprise hair cell regeneration. However, mammalian hair cell regeneration using stem cell and gene therapy are years--if not decades--away from being clinically feasible. If the goals of the biological approaches are met, these therapies may represent future treatments for hearing loss.

Neuronal M-channels are low threshold, slowly activating and non-inactivating, voltage dependent K(+) channels that play a crucial role in controlling neuronal excitability. The native M-channel is composed of heteromeric or homomeric assemblies of subunits belonging to the Kv7/KCNQ family, with KCNQ2/3 heteromers being the most abundant form. KCNQ2 and KCNQ3 subunits have been found to be expressed in various neurons in the central and peripheral nervous system of rodents and humans. Previous evidence shows preferential localization of both subunits to axon initial segments, somata and nodes of Ranvier. In this work, we show the distribution and co-localization of KCNQ2 and KCNQ3 subunits throughout the hippocampal formation, via immunostaining experiments on unfixed rat brain slices and confocal microscopy. We find intense localization and colocalization to the axonal initial segment in several regions of the hippocampus, as well as staining for non-neuronal cells in the area of the lateral ventricle. We did not observe colocalization of KCNQ2 or KCNQ3 with the presynaptic protein, synaptophysin.

Hearing loss affects hundreds of millions of people worldwide by dampening or cutting off their auditory connection to the world. Current treatments for sensorineural hearing loss (SNHL) with cochlear implants are not perfect, leaving regenerative medicine as the logical avenue to a perfect cure. Multiple routes to regeneration of damaged hair cells have been proposed and are actively pursued. Each route not only requires a keen understanding of the molecular basis of ear development but also faces the practical limitations of stem cell regulation in the delicate inner ear where topology of cell distribution is essential. Improvements in our molecular understanding of the minimal essential genes necessary for hair cell formation and recent advances in stem cell manipulation, such as seen with inducible pluripotent stem cells (iPSCs) and epidermal neural crest stem cells (EPI-NCSCs), have opened new possibilities to advance research in translational stem cell therapies for individuals with hearing loss. Despite this, more detailed network maps of gene expression are needed, including an appreciation for the roles of microRNAs (miRs), key regulators of transcriptional gene networks. To harness the true potential of stem cells for hair cell regeneration, basic science and clinical medicine must work together to expedite the transition from bench to bedside by elucidating the full mechanisms of inner ear hair cell development, including a focus on the role of miRs, and adapting this knowledge safely and efficiently to stem cell technologies.

Because of its high prevalence and social impact, hearing impairment is a major public health problem. Whatever the cause--heredity, acoustic trauma, aminoglycoside antibiotics, noise exposure or aging--the hearing impairment is often caused by an irreversible loss of sensory hair cells. So far, hearing aids and cochlear implants are the only possibility to "treat" profound deafness. With the advent of regenerative medicine, extensive studies aimed to repair, regenerate or replace lost hair cells have been initiated. Recently, Stefan Heller and colleagues described a guidance protocol to induce mouse embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) to differentiate into mechanosensitive hair cells. The resulting hair cells hold promise as a tool for hair cell molecular physiology and physiopathology, drug discovery, and possibly also hair cell replacement. The next challenges, alternative strategies, their limitations and prospects are also discussed.

Delivery of medications to the inner ear has been an area of considerable growth in both the research and clinical realms during the past several decades. Systemic delivery of medication destined for treatment of the inner ear is the foundation on which newer delivery techniques have been developed. Because of systemic side effects, investigators and clinicians have begun developing and using techniques to deliver therapeutic agents locally. Alongside the now commonplace use of intratympanic gentamicin for Meniere's disease and the emerging use of intratympanic steroids for sudden sensorineural hearing loss, novel technologies, such as hydrogels and nanoparticles, are being explored. At the horizon of inner ear drug-delivery techniques, intracochlear devices that leverage recent advances in microsystems technology are being developed to apply medications directly into the inner ear. Potential uses for such devices include neurotrophic factor and steroid delivery with cochlear implantation, RNA interference technologies, and stem-cell therapy. The historical, current, and future delivery techniques and uses of drug delivery for treatment of inner ear disease serve as the basis for this review.

Sensory hair cells of the inner ear are responsible for translating auditory or vestibular stimuli into electrical energy that can be perceived by the nervous system. Although hair cells are exquisitely mechanically sensitive, they can be easily damaged by excessive stimulation by ototoxic drugs and by the effects of aging. In mammals, auditory hair cells are never replaced, such that cumulative damage to the ear causes progressive and permanent deafness. In contrast, non-mammalian vertebrates are capable of replacing lost hair cells, which has led to efforts to understand the molecular and cellular basis of regenerative responses in different vertebrate species. In this review, we describe recent progress in understanding the limits to hair cell regeneration in mammals and discuss the obstacles that currently exist for therapeutic approaches to hair cell replacement.

This review will focus on 'self-repair' of the mammalian inner ear sensory epithelium, including recruiting the in-situ proliferation and differentiation of endogenous cells at the damaged site and the autologous transplantation

Self-repair refers to a favorable structural and functional outcome of damaged inner ear sensory epithelium. Our advanced ability of manipulating the fate of inner ear sensory cells makes in-situ proliferation a possible candidate of hearing restoration. A practical alternative of the unavoidable immune rejection is to introduce autologous cells. Ependymal cells, induced pluripotent stem cells, and olfactory neuroepithelial cells have been recognized as promising sources, which will spur ongoing efforts to evaluate these new cell sources for cell replacement therapy.

Further exploration of the innate advantages of in-situ proliferation and use of novel cell sources for autologous transplantation may serve as rehearsals for clinical trials in the near future.