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Cloning of m-ehk2 from the murine inner ear, an eph family receptor tyrosine kinase expressed in the developing and adult cochlea.

To identify receptor tyrosine kinases (RTKs) present in the murine inner ear, a degenerate polymerase chain reaction (PCR) methodology was employed to clone partial cDNAs encoding RTKs from embryonic day-17.5 mouse whole inner ear RNA. At least 20 distinct TKs were identified within the first 50 subcloned PCR products obtained by this analysis (Davis/Lee et al., 1996). One of the receptor RTKs identified encoded an eph-related kinase not previously described in the mouse. Analysis of full-length cDNAs revealed that this RTK is the mouse homolog of the rat ehk-2 gene product (Maisonpierre et al., 1993). Differences in the carboxyl terminal of the mouse and rat ehk2 RTKs suggest that differential splicing of this gene may occur resulting in transcripts encoding truncated and nontruncated forms of the ehk2 RTK. Multiple transcripts corresponding to this RTK were detected by Northern blot analysis only in the mouse brain. RT-PCR analysis revealed the presence of transcripts encoding this kinase in adult mouse brain, inner ear, testes, ovary, thymus, and spleen. Transcripts encoding this kinase were localized using in situ hybridization to the postembryonic day 1 cochlear ganglion neurons in the inner ear and to neurons in discrete regions of the nervous system. This is the first report of eph-related RTK in inner ear tissue that is present in both the developing and adult inner ear tissue. Because this is a member of a family of RTKs that is implicated in establishing the specificity of neuron-target cell interactions (Garrity and Zipursky, 1995), additional studies to determine if the ehk-2 gene product is involved in such processes in the murine cochlea are warranted.

Amino Acid Sequence↗

Embryonic inner ear cells reaggregate into specific patterns in vitro.

The sensory epithelia of the mammalian inner ear consist of a highly precise pattern of sensory hair cells and supporting cells. The mechanisms regulating this patterning are only beginning to be determined. The present study describes a method for culturing dissociated embryonic inner ear cells and the resulting patterning that occurs in these cultures. The results indicate that developing inner ear cells aggregate into precise patterns on a two-dimensional substrate, suggesting that intrinsic patterning mechanisms remain active in vitro. Using antibodies and scanning electron microscopy to detect hair cells and nonsensory cells, it was determined that only a subset of aggregates contained sensory hair cells. The hair cells were organized into specific patterns and surrounded by supporting cells, similar to the in vivo pattern. Additionally, hair cells increased their immunoreactivity and number of stereocilia over time, suggesting that hair cells continue to mature in vitro. Thus, the study reveals that the cells of the developing inner ear provide the necessary signals that direct sensory hair cells and supporting cells to reassociate into very precise patterns in vitro and that these patterns are reminiscent of the patterning that occurs in vivo.

Animals↗

Expression of myeloperoxidase in the inner ear of cisplatin-treated guinea pigs.

Cisplatin is known to cause inner ear damage (ototoxicity). The role of myeloperoxidase (MPO) in the inner ear of the guinea pigs after injections of cisplatin i.p. was examined immunohistochemically. Three days after the injection, the animals were sacrificed with intracardiac perfusion of fixative, and temporal bones were removed and processed for immunohistochemistry with the anti-MPO antibody. MPO could be detected after 3 days in the lateral wall, the organ of Corti, supporting cells of the sensory epithelium and dark cells. These results suggest that MPO and reactive oxygen species are involved in the inner ear dysfunction after the application of cisplatin.

Animals↗

Immunological damage to the inner ear: current and future therapeutic strategies.

There is considerable evidence to suggest that hearing and vestibular function can be influenced by immunity in the inner ear. While immunity can protect against infections of the labyrinth, immune response also has the capacity to damage the delicate tissues of the inner ear. Antigenic challenge of the inner ear of sensitized animals leads to rapid accumulation of leukocytes, antibody production, hearing loss and tissue damage. Moreover, a number of systemic autoimmune disorders include hearing loss and vertigo as part of their constellation of symptoms. It also appears that autoimmune damage can exist as an entity confined to the labyrinth. Immune disorders of the inner ear are of special interest since they are among the few forms of hearing loss that are currently amenable to medical treatment. In addition, recent developments in understanding the intracellular pathways that participate in damage to the inner ear provide new opportunities for pharmacotherapy of immune-mediated disorders of hearing and balance.

Animals↗

CT and MR imaging of acquired abnormalities of the inner ear and cerebellopontine angle.

INTRODUCTION: Several entities of acquired lesions may affect the inner ear and cerebellopontine angle. The imaging of these lesions depends on the clinical history, and should be adapted to the lesion searched for and suspected by the otolaryngologist. In this paper, the modality of CT and MR imaging which is suited to delineate the acquired lesions of this region will be presented. MATERIALS AND METHODS: CT and/or MR imaging of the inner ear and cerebellopontine angle was performed in all cases in which an acquired lesion of this region was suspected by the otolaryngologist. CT was performed in the axial and coronal plane with the use of a high-resolution bone-window-level-setting. MRI was performed in the axial plane using high-resolution 3D T2-weighted fast spin echo sequences and 3D T1-weighted gradient echo sequences before and after the i.v. application of gadopentate dimeglumine. The obtained images were evaluated for the depiction of the acquired lesions. RESULTS: CT best depicted osseous lesions such as traumatic affections or lesions leading to ossification of the inner ear. Tumorous lesions were delineated in those cases in which they yielded to bony changes. Inflammatory or tumorous lesions not yielding to bony changes or intralabyrintine calicifications were not depicted. MRI delineated very well all lesions leading to soft tissue changes, and moderately depicted traumatic changes yielding to less severe fractures affecting the investigated region. CONCLUSION: CT and MR imaging are suited differently to delineate the acquired lesions of the inner ear and cerebellopontine angle. CT is excellently suited to depict osseous lesions, while MRI is excellently suited to delineate lesions affecting the soft tissue structures. These two imaging modalities should be used depending on the clinical question, and are supposed to be complementary methods.

Cerebellar Diseases↗

Analysis of immunocompetent cells following inner ear immunostimulation.

Immunohistochemical methods were used to investigate the development of inner ear immunocompetent cells in healthy, BALB/c mice over a 3-week period as a result of a secondary inner ear response against keyhole limpet hemocyanin. Antibodies against murine macrophages and granulocytes (anti-Mac-1), T-cells (anti-Lyt-1, anti-Lyt-2), and immunoglobulins (anti-IgM, anti-IgG, anti-IgA) were used. Mac-1 positive (Mac-1+) cells were observed at 6 hours post-challenge in the endolymphatic sac and cochlea and rapidly increased in both sites. Lyt-1+ cells gradually increased in the endolymphatic sac after challenge, peaking at 2 and 3 weeks post-challenge. In the cochlea, Lyt-1+ cells were detected at 1 day post-challenge and then increased. Lyt-2+ cells were detectable in the endolymphatic sac and the cochlea by 3 weeks post-challenge. The predominant immunoglobulin-bearing cell in the endolymphatic sac was IgG, followed by IgM, with IgA seen late in the response. We conclude that the inner ear has the capacity to mount an immune response through the accumulation of the needed immunocompetent cells for antigen processing, antibody production, and modulation of the response through T-helper and suppressor cell activity.

Animals↗

[Spontaneous cerebrospinal fluid fistula in association with congenital inner ear anomaly: a case report].

A case of inner ear anomaly (Mondini dysplasia) associated with recurrent meningitis found in a 10 year-old boy is reported. The patient had suffered from bacterial meningitis and CSF rhinorrhea five times in 7 years. The origin of CSF leakage had not been detected by bifrontal craniotomy or trans-sphenoidal operation. In May 1985, the dysplasia of the temporal bone and the inner ear had been pointed out by an otologist. And he underwent operations a few times for closing the CSF fistula at the floor of the internal auditory meatus by translabyrinthine approaches, but in vain. On the 7th of November 1985, under the control of intracranial pressure by continuous ventricular drainage, re-operation was performed. Packing the internal auditory meatus with several pieces of muscle and gelatine sponge stopped the CSF leakage and the recurrence of meningitis.

Cerebrospinal Fluid Otorrhea↗

Changes in aquaporin expression in the inner ear of the rat after i.p. injection of steroids.

The aquaporins (AQPs) are a family of small transmembrane water transporters. It has recently been revealed that they play a role in regulating homeostasis in the inner ear fluids. Steroid therapy is usually administered to patients with inner ear disorders; however, the mechanism of steroid effects has not been clearly determined. To elucidate the points of action of steroids in the inner ear, we recently examined the distributions of AQP isoform mRNAs in the rat inner ear and identified AQP1-6 mRNAs in the rat cochlea and AQP1, 3, 4, 5 and 6 mRNAs in the rat endolymphatic sac by means of reverse transcriptase polymerase chain reaction (PCR). In this study, we investigated changes in expression of AQP mRNAs in the rat inner ear after i.p. injections of steroids using real-time quantitative PCR and found that AQP3 mRNA in the endolymphatic sac was significantly upregulated in both dose- and time-dependent manners. This result suggests that steroids may effect water homeostasis in the rat inner ear via AQPs.

Animals↗

A novel vestibular approach for gene transfer into the inner ear.

The aim of gene transfer to the cochlea and vestibular organ is to protect the inner ear from different disorders. Although various vectors for gene delivery have been used with some success, there remains a need for a reliable transfer of genes into the inner ear without damaging cochlear function. Here, we have tested a novel application method for gene transfer into the rat inner ear in vivo using herpes simplex virus type-1(HSV-1)-based amplicon vectors. Our goal was to find an entry route into the inner ear that leaves its function intact. Besides other non-invasive and invasive application techniques, we applied the viral vector via injection through a small opening of the utriculus. Using this method, efficient -galactosidase reporter gene expression was achieved in nearly all neurons in the vestibulum and cochlea, without functional hearing deficits. At the time point of maximal expression 5 days after injection, -galactosidase activity was also observed in axonal fibres and synaptic endings close to inner and outer hair cells. Our results thus describe an efficient and reliable protocol for short-term expression of potential therapeutic genes in the neuronal compartment of the inner ear.

Animals↗

[Delayed damage of ionizing radiation on the inner ear].

To investigate the feature and mechanism of delayed effect of ionizing radiation on the inner ear, the function and structure of the inner ear of the guinea pigs were observed during 8 month's period after exposure to fractional doses of gamma radiation (2 Gy per day) with a total dose of 60 Gy. Morphological study was performed using light microscopy, transmission and scanning electron microscopy while the function was measured with electrocochleography (ECochG) and electronystagmography (ENG). The results showed that the mean CAP response threshold was 35.67 +/- 6.78 dB before radiation, and 41.17 +/- 7.76 dB, 47.00 +/- 8.82 dB and 71.00 +/- 7.63 dB at 1st day, 3rd month and 8th months after completion of treatment, respectively. No significant change was demonstrated in the auditory responses at the 1st day of radiation (P > 0.05), but there was obvious hearing loss at 3rd and 8th month (P < 0.01). The nystagmus duration induced by ice water (ENG) was 40.00 +/- 5.44 seconds before radiation, 22.71 +/- 7.93 seconds at 8th month after radiation (P < 0.01). Histologic examination at 8th month after radiation revealed atrophy and degeneration of stria vascularis, a reduced number of capillaries, degeneration of endotheliocyte in vessels, outer hair cell and supporting cell of Corti's organ. The percentage of damaged hair cells in basal turn of the cochlea was significantly higher than those of the upper turn. Degeneration of vestibular hair cell was found. It is suggested that radiation from routine radiotherapy doses may result in a delayed impairment of the inner ear. The main mechanism of the radiation damage may be interference of oxygen supply and metabolism of the inner ear by radiation vascular lesion.

Animals↗

Trks and p75 genes are differentially expressed in the inner ear of human embryos. What may Trks and p75 null mutant mice suggest on human development?

Recent work has shown the expression of Neurotrophins low (p75) and high affinity (Trk's A, B, and C) receptors in the developing inner ear sensory neurons of chick and mouse. Likewise the biological significance of such receptor expression was demonstrated by using both Trks and Neurotrophins null mutant mice. The present study was conducted to determine the expression of Trks and p75 proteins in the human inner ear throughout development. Hence to assess the potential role of Neurotrophins in the development of auditory and vestibular specific innervation in man. In other words, we intend to address the issue whether or not what null mutant mice for Trks and p75 have revealed on inner ear development may be relevant for human embryos. Fifty-two inner ears and their cochleovestibular ganglions (CVG) from human embryos and fetuses, ranging from 5 to 24 weeks of pregnancy were analyzed. Both Western blot and immunocytochemistry on frozen sections were used as complementary procedures. Quantitative Western blot studies revealed that Trk-B and C immunoreactivity (IR) appeared by embryonic week 5 in CVG neurons, increased at high levels between embryonic weeks 7 and 12, and later on, in 15 week-old specimens and older began to decrease to minimal levels. Trk-A IR was detected at just moderate levels during 5 and 7 weeks reflecting the presence of NGF high affinity receptors only at these earlier developmental ages. The p75 IR was detected at high degrees in the early stage of the 5th week and at abundant levels in all studied inner ears from the 7th to the 24th pregnancy week. These Western blot observations were corroborated by immunocytochemistry on frozen sections, which also revealed a major distribution of both p75 and Trks on neuronal bodies while p75 appears localized on supporting cells. Our findings reveal a tight correlation between p75 and Trks expression throughout human development and specific inner ear developmental events, such as target-dependent neuronal cell death and afferent hair cells innervation. That kind of association of p75 and Trks temporal pattern with distinctive steps in inner ear developmental schedule, is a feature shared between human embryos and other mammals, such as mouse. Based on the present results and considering them together with the reported phenotype of p75 and Trks null mutant mice, we hypothesize that p75 and Trk receptors, as well as, their binding Neurotrophins may be essential in human inner ear development. Accordingly, they may be required molecules for sensory epitheliums innervation and target-dependent neuronal cell death, during embryogenesis and even early postnatal life, in man.

Animals↗

Histochemical localization of carbonic anhydrase in the inner ear.

Carbonic anhydrase was histochemically located in chinchilla inner ear tissues. A strong carbonic anhydrase reaction was observed in the spiral ligament cells, Boettcher's cells, the external sulcus cells, and the stria vascularis (intermediate and/or basal cells). The enzymatic reaction was also positive in the supporting cells of all vestibular sensory epithelia, as well as in the dark cells and transitional cells of the utricle and saccule. Some epithelial cells of the endolymphatic sac were also positive. It is speculated that this enzyme may be involved in: 1) ionic or fluid regulation of the endolymph, 2) removal of CO2 from the inner ear tissue near the sensory cells, and 3) otoconia formation and maintenance.

Animals↗

Pax2 expression patterns in the developing chick inner ear.

The fate specification of the developing vertebrate inner ear could be determined by complex regulatory genetic pathways involving the Pax2/5/8 genes. Pax2 expression has been reported in the otic placode and vesicle of all vertebrates that have been studied. Loss-of-function experiments suggest that the Pax2 gene plays a key role in the development of the cochlear duct and acoustic ganglion. Despite all these data, the role of Pax2 gene in the specification of the otic epithelium is still only poorly defined. In the present work, we report a detailed study of the spatial and temporal Pax2 expression patterns during the development of the chick inner ear. In the period analysed, Pax2 is expressed only in some presumptive sensory patches, but not all, even though all sensory patches show the scattered Pax2 expression pattern later on. We also show that Pax2 is also expressed in several non-sensory structures.

Animals↗

Effects of intratympanic injection of steroids on changes in rat inner ear aquaporin expression.

Although steroid treatment is generally administered for patients with inner ear disorders, including Meniere's disease, the mechanism via which steroids exert their effects remains to be clarified. The aquaporins (AQPs) are a family of small transmembrane water transporters, and it has recently been revealed that they play a role in regulating homeostasis in the inner ear fluids. In order to elucidate the action points of steroids in the inner ear, we firstly identified AQPI, 2, 3, 4, 5 and 6 mRNAs in the rat cochlea and AQP1, 3, 4, 5 and 6 in the rat endolymphatic sac by means of reverse transcription-polymerase chain reaction. Subsequently, we found that intratympanic injections of steroids upregulated AQPI mRNA of the rat cochlea in a dose-dependent manner. These results suggest that steroids may affect water homeostasis in the rat inner ear mainly via AQP1.

Animals↗

Adenylate cyclase activity in the fetal and the early postnatal inner ear of the mouse.

The adenylate cyclase activity was analyzed in fetal, early postnatal and adult inner ears of the CBA/CBA mouse and also in approximately one month old inner ears from Shaker -1 and Shaker -2 mice. A comparison was made with the maturation of potassium levels in endolymph as investigated with the X-ray energy dispersive technique. Adenylate cyclase activity in the developing normal inner ear shows two significant periods of increases: from the 16th to the 19th gestational day in both the cochlear and vestibular parts of the labyrinth, and from birth to day 6 after birth in the lateral wall tissues of the scala media. During the first period the anatomical boundaries of the secretory epithelia are developing. The postnatal rise in adenylate cyclase activity correlates with the morphological maturation of stria vascularis at the cellular and subcellular levels and the rise in potassium content of endolymph. The rise of enzyme activity in the cochlear during the maturation of endolymph supports a link between adenylate cyclase and the control of inner ear fluids. Adenylate cyclase activity in stria vascularis/spiral ligament of Shaker -1 and Shaker -2 mice were at normal levels and correlated better with the rather normal morphology of the tissues than the abnormal composition of endolymph in these mutants.

Adenylyl Cyclases↗

[Immunopathology of the inner ear].

Immunologically mediated tissue lesions or functional disturbances can result from immune responses either to foreign antigens or autoantigenic constituents. Before a disease or a functional disorder can be called an (auto)immune disease, it must be established that immunological processes are involved in its etiology and/or pathogenesis. After reviewing the available data on immunology and on research on immunobiology of the inner ear, as well as clinical observations on disorders of the inner ear of possibly immunological origin, an attempt is made to determine whether (auto)immunopathology can be assumed in some patients with sensineural hearing loss. In patients with disorders presently recognized as autoimmune diseases, no hearing loss has been described so far. However, serum antibodies can be found via the indirect immunofluorescence method in some patients having functional disorders of the inner ear. These serum antibodies interact with normal human inner ear tissue. However, as long as immunopathological mechanisms have not been confirmed, these disorders should merely be called "diseases with autoimmune markers." It has been shown in animal experiments that the production of autoantibodies against inner ear components is associated with morphological and functional changes of the inner ear. The human endolymphatic sac releases secretory IgA into the lumen. Its epithelium contains (intraepithelial) lymphocytes, whereas lymphocytes, plasma cells (IgA, IgG) and macrophages occur in the perisaccular region. In this manner, the inner ear has its own immune system, which can possibly react independently of the systemic immune system.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Mercury deposition and its relationship to inner ear function in methylmercury-poisoned rats. A histological and immunohistochemical study.

Mercury deposition and its relationship to the inner ear function in methylmercury-poisoned rats were examined. Rats were poisoned with methylmercury chloride. After the appearance of signs of poisoning, the cerebellar and inner ear tissues were examined histochemically using an autoradiographic procedure. Mercury deposits were typically found in the cerebellum and appeared in parts of the vestibular nerves, cochlear nerves, spiral ganglion and stria vascularis. In the vestibule, a slight mercury deposition was observed in the acoustic maculae and in the cochlea; only one instance of mercury deposit in the organ of Corti was detected. The effect of mercury on the inner ear function was also assessed on the basis of immunohistochemical localization of substance P which is believed to be a neurotransmitter in the inner ear. However, no pathological changes were detected. These findings suggest that methylmercury deposition has little effect on the inner ear function.

Animals↗

Transcripts encoding three types of guanylyl-cyclase-coupled trans-membrane receptors in inner ear tissues of guinea pigs.

The distribution of membrane-bound guanylyl cyclase (GC) transcription in inner ear tissues of the guinea pig was addressed by a reverse transcription-PCR approach using consensus primers flanking a region of about 630 bp in the intracellular domains in the target sequences. Restriction mapping of such amplificates obtained from cochlear and vestibular specimens permitted us to demonstrate GC-A, GC-B, and GC-C expression by differentiating overall PCR signals. This assay indicated that GC-A was expressed in the cochlea and vestibular organ. PCR products resulting from transcripts of the GC-B gene were obtained at considerably lower abundance than amplificates typical of the GC-A gene. The consensus primer approach with subsequent restriction mapping provided the opportunity to examine at the same time expression of GC-C in the inner ear and revealed the occurrence of GC-C transcripts in both inner ear compartments under investigation. The distribution pattern found by analysing the intracellular domains of membrane-bound guanylyl cyclases was confirmed by demonstrating transcription of the corresponding extracellular receptor domains. In addition, single-strand conformation polymorphism analysis of cDNA amplificates comprising the catalytic domain of guanylyl cyclases also indicated the presence of GC-C expression in the inner ear tissues examined. The GC-C transcripts detected in inner ear tissues appeared to correlate with functional receptor expression, since the production of cyclic GMP catalyzed by cochlear and vestibular specimens was stimulated by 1 microM of heat-stable enterotoxin to 18 and 80% above basal levels, respectively. Thus, GC-C may be involved in the fluid regulation by typical ligands (e.g., the peptide hormone guanylin or the toxins causing travellers' diarrhea), not only in the intestine but also in the organs responsible for hearing and gravitational orientation.

Animals↗