Novel In Vivo Imaging Analysis of an Inner Ear Drug Delivery System in Mice: Comparison of Inner Ear Drug Concentrations over Time after Transtympanic and Systemic Injections

Objective

Systemic steroid injections are used to treat idiopathic sudden-onset sensorineural hearing loss (ISSHL) and some inner ear disorders. Recent studies show that transtympanic (TT) steroid injections are effective for treating ISSHL. As in vivo monitoring of drug delivery dynamics for inner ear is lacking, its time course and dispersion of drugs is unknown. Here, we used a new in vivo imaging system to monitor drug delivery in live mice and to compare drug concentrations over time after TT and systemic injections.

Methods

Luciferin delivered into the inner ears of GFAP-Luc transgenic mice reacted with luciferase in GFAP-expressing cells in the cochlear spiral ganglion, resulting in photon bioluminescence. We used the Xenogen IVIS® imaging system to measure how long photons continued to be emitted in the inner ear after TT or systemic injections of luciferin, and then compared the associated drug dynamics.

Results

The response to TT and IP injections differed significantly. Photons were detected five minutes after TT injection, peaking at ∼20 minutes. By contrast, photons were first detected 30 minutes after i.p. injection. TT and i.p. drug delivery time differed considerably. With TT injections, photons were detected earlier than with IP injections. Photon bioluminescence also disappeared sooner. Delivery time varied with TT injections.

Conclusions

We speculate that the drug might enter the Eustachian tube from the middle ear. We conclude that inner-ear drug concentration can be maintained longer if the two injection routes are combined. As the size of luciferin differs from that of therapeutics like dexamethasone, combining drugs with luciferin may advance our understanding of in vivo drug delivery dynamics in the inner ear.     

In Vitro and In Vivo Evaluation of a Hydrogel Reservoir as a Continuous Drug Delivery System for Inner Ear Treatment

Fibrous tissue growth and loss of residual hearing after cochlear implantation can be reduced by application of the glucocorticoid dexamethasone-21-phosphate-disodium-salt (DEX). To date, sustained delivery of this agent to the cochlea using a number of pharmaceutical technologies has not been entirely successful. In this study we examine a novel way of continuous local drug application into the inner ear using a refillable hydrogel functionalized silicone reservoir. A PEG-based hydrogel made of reactive NCO-sP(EO-stat-PO) prepolymers was evaluated as a drug conveying and delivery system in vitro and in vivo. Encapsulating the free form hydrogel into a silicone tube with a small opening for the drug diffusion resulted in delayed drug release but unaffected diffusion of DEX through the gel compared to the free form hydrogel. Additionally, controlled DEX release over several weeks could be demonstrated using the hydrogel filled reservoir. Using a guinea-pig cochlear trauma model the reservoir delivery of DEX significantly protected residual hearing and reduced fibrosis. As well as being used as a device in its own right or in combination with cochlear implants, the hydrogel-filled reservoir represents a new drug delivery system that feasibly could be replenished with therapeutic agents to provide sustained treatment of the inner ear.     

Design of a Robotized Magnetic Platform for Targeted Drug Delivery in the Cochlea

Inner ear disorders' treatment remains challenging due to anatomical barriers. Robotic assistance seems to be a promising approach to enhance inner ear treatments and, more particularly, lead to effective targeted drug delivery into the human cochlea. In this paper we present a combination of a micro-macro system that was designed and realized in order to efficiently control the navigation of magnetic nanoparticles in an open-loop scheme throughout the cochlea, considering that the magnetic particles cannot be located in real time.In order to respect the anatomical constraints, we established the characteristics that the new platform must present then proceeded to the design of the latter. The developed system is composed of a magnetic actuator that aims to guide nanoparticles into the cochlea. Mounted on a robotic manipulator, it ensures its positioning around the patient's head. The magnetic device integrates four parallelepiped-rectangle permanent magnets. Their arrangement in space, position and orientation, allows the creation of an area of convergence of magnetic forces where nanoparticles can be pushed/pulled to. To ensure the reachability of the desired orientations and positions, a 3 DOF robot based on a Remote Centre of Motion (RCM) mechanism was developed. It features three concurrent rotational joints that generate a spherical workspace around the head. The control of the latter is based on kinematic models.A prototype of this platform was realized to validate the actuation process. Both magnetic actuator and robotic manipulator were realized using an additive manufacturing approach. We also designed a virtual human head with a life-size cochlea inside. A laser was mounted on the end effector to track the positioning of the actuator. This permitted to experimentally prove the capacity of the robotic system to reach the desired positions and orientations in accordance with the medical needs.This promising robotic approach, makes it possible to overcome anatomical barriers and steer magnetic nanoparticles to a targeted location in the inner ear and, more precisely, inside the cochlea.     

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.     

Otx1 and Otx2 activities are required for the normal development of the mouse inner ear

The Otx1 and Otx2 genes are two murine orthologues of the Orthodenticle (Otd) gene in Drosophila. In the developing mouse embryo, both Otx genes are expressed in the rostral head region and in certain sense organs such as the inner ear. Previous studies have shown that mice lacking Otx1 display abnormal patterning of the brain, whereas embryos lacking Otx2 develop without heads. In this study, we examined, at different developmental stages, the inner ears of mice lacking both Otx1 and Otx2 genes. In wild-type inner ears, Otx1, but not Otx2, was expressed in the lateral canal and ampulla, as well as part of the utricle. Ventral to the mid-level of the presumptive utricle, Otx1 and Otx2 were co-expressed, in regions such as the saccule and cochlea. Paint-filled membranous labyrinths of Otx1-/- mutants showed an absence of the lateral semicircular canal, lateral ampulla, utriculosaccular duct and cochleosaccular duct, and a poorly defined hook (the proximal part) of the cochlea. Defects in the shape of the saccule and cochlea were variable in Otx1-/- mice and were much more severe in an Otx1-/-;Otx2(+/)- background. Histological and in situ hybridization experiments of both Otx1-/- and Otx1-/-;Otx2(+/)- mutants revealed that the lateral crista was absent. In addition, the maculae of the utricle and saccule were partially fused. In mutant mice in which both copies of the Otx1 gene were replaced with a human Otx2 cDNA (hOtx2(1)/ hOtx2(1)), most of the defects associated with Otx1-/- mutants were rescued. However, within the inner ear, hOtx2 expression failed to rescue the lateral canal and ampulla phenotypes, and only variable rescues were observed in regions where both Otx1 and Otx2 are normally expressed. These results suggest that both Otx genes play important and differing roles in the morphogenesis of the mouse inner ear and the development of its sensory organs.     

A mutation in the Lunatic fringe gene suppresses the effects of a Jagged2 mutation on inner hair cell development in the cochlea

Recent studies have demonstrated that the Notch signaling pathway regulates the differentiation of sensory hair cells in the vertebrate inner ear [1] [2] [3] [4] [5] [6] [7] [8] [9]. We have shown previously that in mice homozygous for a targeted null mutation of the Jagged2 (Jag2) gene, which encodes a Notch ligand, supernumerary hair cells differentiate in the cochlea of the inner ear [7]. Other components of the Notch pathway, including the Lunatic fringe (Lfng) gene, are also expressed during differentiation of the inner ear in mice [6] [7] [8] [9] [10]. In contrast to the Jag2 gene, which is expressed in hair cells, the Lfng gene is expressed in non-sensory supporting cells in the mouse cochlea [10]. Here we demonstrate that a mutation in the Lfng gene partially suppresses the effects of the Jag2 mutation on hair cell development. In mice homozygous for targeted mutations of both Jag2 and Lfng, the generation of supernumerary hair cells in the inner hair cell row is suppressed, while supernumerary hair cells in the outer hair cell rows are unaffected. We also demonstrate that supernumerary hair cells are generated in mice heterozygous for a Notch1 mutation. We suggest a model for the action of the Notch signaling pathway in regulating hair cell differentiation in the cochlear sensory epithelium.     

人脂肪来源间充质干细胞植入受损耳蜗后定向归巢到并分化为毛细胞样细胞

目的：通过将人脂肪来源间充质干细胞（human adipose-derived mesenchymal stem cells,hAD-MSCs）移植到受损小鼠,探讨hAD-MSCs替代缺失/受损毛细胞的可行性。方法：将hAD-MSCs经尾静脉移植入药物致聋后的小鼠体内,用免疫染色及RT-PCR等方法检测移植后hAD-MSCs在耳蜗内的归巢和分化。结果：移植的hAD-MSCs能够定向归巢到受损耳蜗内,并至少存活2周,未观察到对移植细胞的免疫排斥反应。有少量细胞定位于耳蜗感觉上皮并表达毛细胞特异性抗体myosin 7a。结论：hAD-MSCs移植入药物性致聋小鼠后,能够定向归巢到耳蜗内,并分化为内耳毛细胞样细胞,是一种针对内耳损伤及退变性疾病治疗的潜在细胞来源。     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

Osseous inner ear structures and hearing in early marsupials and placentals

Based on the internal anatomy of petrosal bones as shown in radiographs and scanning electron microscopy, the inner ear structures of Late Cretaceous marsupials and placentals (about 65 Myr ago) from the Bug Creek Anthills locality of Montana, USA, are described. The inner ears of Late Cretaceous marsupials and placentals are similar to each other in having the following tribosphenic therian synapomorphies: a fully coiled cochlea, primary and secondary osseous spiral laminae, the perilymphatic recess merging with the scala tympani of the cochlea, an aqueductus cochleae, a true fenestra cochleae, a radial pattern of the cochlear nerve and an elongate basilar membrane extending to the region between the fenestra vestibuli and fenestra cochleae. The inner ear structures of living therians differ from those of their Late Cretaceous relatives mainly in having a greater number of spiral turns of the cochlea and a longer basilar membrane. Functionally, a coiled cochlea not only permits the development of an elongate basilar membrane within a restricted space in the skull but also allows a centralized nerve system to innervate the elongate basilar membrane. Qualitative and quantitative analyses show that, with a typical therian inner ear, Late Cretaceous marsupials and placentals were probably capable of high-frequency hearing.     

Hes1 is a negative regulator of inner ear hair cell differentiation

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, we have focused on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. We report here that targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions which are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells.     

ERK2-dependent activation of c-Jun is required for nontypeable Haemophilus influenzae-induced CXCL2 upregulation in inner ear fibrocytes

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.     

Genetic background modifies inner ear and eye phenotypes of jag1 heterozygous mice

Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene (Jag1del1) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed Jag1del1 heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in Jag1del1 heterozygous mice from this same cross.     

Deafness and stria vascularis defects in S1P2 receptor-null mice

The S1P(2) receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P(2) receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P(2) receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P(2) receptor-null mice showed markedly disturbed structures. JTE013, an S1P(2) receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P(2) receptor-null mice.     

The WFS1 gene,responsible for low frequency sensorineural hearing loss and Wolfram syndrome,is expressed in a variety of inner ear cells

Heterozygous mutations in the WFS1 gene are responsible for autosomal dominant low frequency hearing loss at the DFNA6/14 locus, while homozygous or compound heterozygous mutations underlie Wolfram syndrome. In this study we examine expression of wolframin, the WFS1-gene product, in mouse inner ear at different developmental stages using immunohistochemistry and in situ hybridization. Both techniques showed compatible results and indicated a clear expression in different cell types of the inner ear. Although there were observable developmental differences, no differences in staining pattern or gradients of expression were observed between the basal and apical parts of the cochlea. Double immunostaining with an endoplasmic reticulum marker confirmed that wolframin localizes to this organelle. A remarkable similarity was observed between cells expressing wolframin and the presence of canalicular reticulum, a specialized form of endoplasmic reticulum. The canalicular reticulum is believed to be involved in the transcellular movements of ions, an important process in the physiology of the inner ear. Although there is nothing currently known about the function of wolframin, our results suggest that it may play a role in inner ear ion homeostasis as maintained by the canalicular reticulum.     

Expression and localization of Tmie in adult rat cochlea

Loss-of function mutations in transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). However, the functional roles of TMIE in the cochlea remain unclear. A primary step toward the understanding of the role of TMIE in hearing and its dysfunction is the documentation of its cellular and sub-cellular location within the cochlea, the auditory organ. In this study, we located and determined the cellular expression of Tmie within the rat cochlea using a polyclonal anti-Tmie antibody. The anti-Tmie antibody identified a specific band of 17 kDa in a variety of rat tissues by using Western blot analyses. The expression products of Tmie were also detected in the spiral limbus, spiral ligament, organ of Corti, and stria vascularis by immunohistochemistry analysis and RT-PCR. Our results point out the presence and localization of Tmie products in the cochlea of rat. Knowledge of spatial distribution of Tmie will provide important insight into the mechanisms that lead to deafness due to mutations in the TMIE gene.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.