Conditional immortalization of hair cells from the inner ear

The aim of this work was to culture conditionally immortalized cells that possess the potential to differentiate into mechanosensory hair cells. Utricular epithelia at embryonic stage E16 were cultured from the vestibular system of the H2kbtsA58 transgenic mouse (Immortomouse) that carries a conditionally expressed immortalizing gene derived from the simian virus 40. Immunolabelling showed that the immortalizing transgene product, the T antigen (Tag), was expressed in utricular cells under permissive conditions and that it was inactivated under non-permissive conditions. Several morphologically distinct cell types proliferated when Tag was expressed, including those that resembled fibroblasts, nerve cells and epithelial cells. Mixed cultures of cells from the utricle, passaged up to 50 times every 3-4 days over a period of 5 months, were subsequently allowed to differentiate for 10 days by transferring them to non-permissive conditions. Monoclonal antibody markers were used to locate expression of hair cell specific antigens. One antibody that normally labels stereociliary bundles from postnatal stage P4-6 labelled cellular projections from a population of spheroid cells that were distributed across the culture surface. A second antibody that normally labels stereociliary bundles did not label the same structures. We conclude that utricular hair cell progenitors can be derived from the H2kbtsA58 transgenic mouse but that under the experimental conditions used they do not follow the normal pattern of differentiation.     

Potassium currents in presynaptic hair cells of Hermissenda

Serum samples from eight endogamous Indian tribal populations of Madhya Pradesh (Dhurwa, Halba, Bhatra, Muria, Maria) and Orissa (Deshia Khond, Binjhal, Kisan) with a total of n = 731 unrelated individuals were typed for G1M (1,2,3,17), G3M (5,10,11,13,14,15,16,21, 26), and KM (1). In seven of these populations five different GM haplotypes were found: GM* 1,17;21,26; GM* 1,17;10,11,13,15,16; GM* 1,2, 17;21,26; GM* 1,3;5,10,11,13,14,26; and GM* 3;5,10,11,13,14,26. In the Kisan sample the haplotype GM* 1,2,17;21,26 is absent. The intergroup variability in the distribution of these haplotypes is considerable and statistically highly significant. The reasons for that can be attributed to the ethnohistory and to the genetic isolation of these eight endogamous tribal populations. The GM haplotype distribution pattern of all these groups is quite different from that of the non-tribal populations of India, whereas it is in good agreement with that of the so far tested other tribal populations from India. This can be explained by different origin and history of the Indian tribal and non-tribal populations. In the KM system, too, remarkable variability is seen in the distribution of phenotype and allele frequencies among the eight tribal populations under study.     

Macrophage and microglia-like cells in the avian inner ear

Recent studies suggest that macrophages may influence early stages of the process of hair cell regeneration in lateral line neuromasts; numbers of macrophages were observed to increase prior to increases in hair cell progenitor proliferation, and macrophages have the potential to secrete mitogenic growth factors. We examined whether increases in the number of leukocytes present in the in vivo avian inner ear precede the proliferation of hair cell precursors following aminoglycoside insult. Bromodeoxyuridine (BrdU) immunohistochemistry was used to identify proliferating cells in chicken auditory and vestibular sensory receptor epithelia. LT40, an antibody to the avian homologue of common leukocyte antigen CD45, was used to label leukocytes within the receptor epithelia. Macrophages and, surprisingly, microglia-like cells are present in normal auditory and vestibular sensory epithelia. After hair cell loss caused by treatment with aminoglycosides, numbers of macrophage and microglia-like cells increase in the sensory epithelium. The increase in macrophage and microglia-like cell numbers precedes a significant increase in sensory epithelial cell proliferation. The results suggest that macrophage and microglia-like cells may play a role in releasing early signals for cell cycle progression in damaged inner ear sensory epithelium.     

The efferents interconnecting auditory inner hair cells

The work describes the system of efferent terminals that interconnect inner hair cells through a chain of direct somatic synapses organized in repetitive patterns. The efferent boutons were discovered in the apical turns of 12-day-old (hearing) mice. Clusters or short rows of vesiculated boutons are located between adjoining hair cells at the lower half of the receptors, close to their modiolar side. The individual endings, about 1.2 microns in diameter, adjoin inner hair cells and form one synapse per hair cell. On the hair cell side, the synaptic contact is apposed by a classical postsynaptic cisterna. Within a cluster of endings, some synapse simultaneously with either or both neighbouring inner hair cells. The efferent boutons also connect synaptically with each other and with other--different in type--vesiculated and nonvesiculated endings. These endings seem to derive from the climbing collaterals of the inner spiral bundle, and we believe them to be GABAergic.     

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats

2115 cases of chest neoplasms were registered during the first six months of 1995 in Pulmonary Outpatient Departments in Poland. In 865 (40.8%) patients squamous cell cancer was diagnosed, in 344 (16.2%)--small cell lung cancer, in 174 (8.2%)--adenocarcinoma, in 107 (5%) other types of lung cancer and in 5.6% of cases--lung cancer with undefined histology. Predominated subjects aged from 60 to 69 years (43.5%). Lung cancer under 50 years of age was observed significantly more often among women (25.8%) than among men (13.5%). Moreover adenocarcinoma was much more frequent among women than men and significantly more cases of lung cancer were recorded in nonsmoking women. Diagnosis was established during the first 4 weeks in 38.9% of patients but 23.6% of patients have waited for it more than 39 weeks. Surgical treatment was advised in 16.9% of subjects, radiotherapy in 14.3% and 26.4% of patients underwent chemotherapy. Treatment analysis was presented according histological type of lung cancer. Occupation, performance status, clinical stage of the disease, prevalence of cancer in patients families were also analysed.     

Induction of hair growth in ear wounds by cultured dermal papilla cells

In the adult hair follicle the dermal papilla plays a crucial role in the dermal-epidermal interactions that control hair production and events of the growth cycle. It has previously been shown that cultured cells from rat vibrissa follicle dermal papillae can stimulate hair growth when implanted into amputated follicles. This study investigated the effects of implanting low-passage cultured papilla cells into small incisional wounds in the rat ear pinna. The groups of fibers that emerged from wound sites were much larger than local hairs, and often had vibrissa-type characteristics. Later-passage papilla cells or cultured skin fibroblasts failed to elicit the same response. Histology revealed that big follicles were formed when papilla cells were trapped between the cut edges of the epidermis. Abnormally large follicles were seen at wound sites many months post-operatively. Independent of epidermal influence, cultured papilla cells in the wound dermis formed rounded papilla-like aggregates that also persisted until biopsy. A previously described method of wrapping papilla cells in glabrous epidermis was less successful in percentage terms but resulted in the production of one very large vibrissa-type follicle and fiber. These results further illustrate that the inductive powers and developmental information retained by cultured dermal papilla cells parallel the properties of their embryonic precursors; the findings may have implications for human hair growth.     

Laser applications in inner ear surgery

Argon and carbon dioxide lasers can cause heat shrinkage of the membranous labyrinth without injury to hearing. The argon laser can penetrate the membranous labyrinth and cause destruction of targeted vestibula special sensory epithelium also without injury to hearing. Consequently, these lasers have been used successfully in two treatment techniques for benign paroxysmal positional vertigo, which are discussed in detail. The early techniques for laser assisted resection of the pars superior portion of the inner ear with hearing preservation are discussed.     

Extracellular nucleotide signaling in the inner ear

Extracellular nucleotides, particularly adenosine 5'-triphosphate (ATP), act as signaling molecules in the inner ear. Roles as neurotransmitters, neuromodulators, and as autocrine or paracrine humoral factors are evident. The diversity of the signaling pathways for nucleotides, which include a variety of ATP-gated ion channels (assembled from different subtypes of P2X-receptor subunit) and also different subtypes of G protein-coupled nucleotide receptors (P2Y receptors) supports a major physiological role for ATP in the regulation of hearing and balance. Almost invariably both P2X and P2Y receptor expression is apparent in the complex tissue structures associated with the inner-ear labyrinth. However P2X-receptor expression, commonly associated with fast neurotransmission, is apparent not only with the cochlear and vestibular primary afferent neurons, but also appears to mediate humoral signaling via ATP-gated ion channel localization to the endolymphatic surface of the cochlear sensory epithelium (organ of Corti). This is the site of the sound-transduction process and recent data, including both electrophysiological, imaging, and immunocytochemistry, has shown that the ATP-gated ion channels are colocalized here with the mechano-electrical transduction channels of the cochlear hair cells. In contrast to this direct action of extracellular ATP on the sound-transduction process, an indirect effect is apparent via P2Y-receptor expression, prevalent on the marginal cells of the stria vascularis, a tissue that generates the standing ionic and electrical gradients across the cochlear partition. The site of generation of these gradients, including the dark-cell epithelium of the vestibular labyrinth, may be under autocrine or paracrine regulation mediated by P2Y receptors sensitive to both purines (ATP) and pyrimidines such as UTP. There is also emerging evidence that the nucleoside adenosine, formed as a breakdown product of ATP by the action of ectonucleotidases and acting via P1 receptors, is also physiologically significant in the inner ear. P1-receptor expression (including A1, A2, and A3 subtypes) appear to have roles associated with stress, acting alongside P2Y receptors to enhance cochlear blood flow and to protect against the action of free radicals and to modulate the activity of membrane conductances. Given the positioning of a diverse range of purinergic-signaling pathways within the inner ear, elevations of nucleotides and nucleosides are clearly positioned to affect hearing and balance. Recent data clearly supports endogenous ATP- and adenosine-mediated changes in sensory transduction via a regulation of the electrochemical gradients in the cochlea, alterations in the active and passive mechanical properties of the cells of the sensory epithelium, effects on primary afferent neurons, and control of the blood supply. The field now awaits conclusive evidence linking a physiologically-induced modulation of extracellular nucleotide and nucleoside levels to altered inner ear function.     

Are inner or outer hair cells the source of summating potentials recorded from the round window?

Previous studies indicated that antidromic stimulation of capsaicin-sensitive vagal afferent fibers activated, via peripheral release of tachykinins, nonadrenergic, noncholinergic parasympathetic ganglion neurons that mediate relaxations of guinea pig trachealis. On the basis of the effects of selective agonists and inhibition with a nonselective receptor antagonist (SR 48968), we speculated that tachykinin-mediated activation of neurokinin3 (NK3) receptors might be involved. Using the recently developed NK3-selective receptor antagonist SR 142801, we further assessed the role of NK3 receptors in these relaxant responses. Relaxations of the guinea pig trachea elicited by antidromic stimulation of capsaicin-sensitive vagal afferent nerves were markedly inhibited by 0.3 microM SR 142801 and were abolished by a combination of SR 142801 and either of the NK1-selective receptor antagonists SR 140333 and CP 99994 (0.3 microM each). The NK3 receptor antagonist had similar effects on the relaxant responses elicited by capsaicin and substance P, but it had no effect on relaxations of the trachealis elicited by electrical field stimulation of the postganglionic nerves that innervate the trachealis or by stimulation of the preganglionic parasympathetic vagal nerves that innervate the trachea. These results and the observation that the ganglion neurons that mediate these responses are densely innervated by substance P-containing nerve fibers lead us conclude that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, nonadrenergic, noncholinergic inhibitory neurons innervating guinea pig trachealis via activation of both NK3 and NK1 receptors.     

Potassium currents of pear-shaped hair cells in relation to their location in frog crista ampullaris

Voltage-dependent K+ currents in pear-shaped hair cells of the frog crista ampullaris were investigated in thin slice preparations using the whole-cell variant of the patch-clamp technique. Microscopy observation revealed that pear-shaped cells are located in intermediate and peripheral regions of the crista, whereas they are absent in the central region. Voltage-clamp recordings in cells from the peripheral regions revealed that the total outward K+ current could be separated pharmacologically into three distinct components: a A-type K+ current (IA); an inactivating calcium-activated K+ current (IK(Ca)) and a delayed rectifier K+ current (IK). IK and IK(Ca) exhibited similar magnitude and accounted for most of the membrane cell conductance. The same experimental protocol applied to cells from the intermediate regions showed the presence of a large and sustained IK(Ca) which represented 95% of the total outward current. In this region IA was absent. The present results demonstrated that pear-shaped hair cells located in two discrete regions of frog crista ampullaris exhibit a different complement of voltage-dependent conductances, suggesting that they can play a different role in processing the natural stimulus.     

Expression of a potassium current in inner hair cells during development of hearing in mice

Excitable cells use ion channels to tailor their biophysical properties to the functional demands made upon them. During development, these demands may alter considerably, often associated with a change in the cells' complement of ion channels. Here we present evidence for such a change in inner hair cells, the primary sensory receptors in the mammalian cochlea. In mice, responses to sound can first be recorded from the auditory nerve and observed behaviourally from 10-12 days after birth; these responses mature rapidly over the next 4 days. Before this time, mouse inner hair cells have slow voltage responses and fire spontaneous and evoked action potentials. During development of auditory responsiveness a large, fast potassium conductance is expressed, greatly speeding up the membrane time constant and preventing action potentials. This change in potassium channel expression turns the inner hair cell from a regenerative, spiking pacemaker into a high-frequency signal transducer.     

Evidence that inner hair cells are the major source of cochlear summating potentials

The role of the inner hair cells (IHCs) in generating the cochlear summating potentials (SP) was assessed by measuring SP, cochlear nerve action potentials (CAP), cochlear microphonics (CM) and 2f1-f2 distortion product otoacoustic emissions (DPOAEs) in 15 chinchillas with either acute chemical de-afferentation, accomplished by applying kainic acid to the round window, or surgical de-afferentation and basal IHC loss, which developed within two months after sectioning the auditory nerve. In the auditory nerve sectioned ears, type I ganglion cells disappeared whereas most, if not all, type II ganglion cells were still present. Histological analysis of surface preparations and sections through the modiolus verified the de-afferentation in both models and showed a large IHC loss at the base of the cochlea in the ears with the auditory nerve sectioned while other structures of the cochlea remained intact. Unoperated (left) ears of 9 animals served as controls. CM and DPOAEs were normal in all ears whereas the CAP was substantially depressed in de-afferented ears. Comparisons among the SP input-output functions suggest that (1) the IHCs are the major generator of SP recorded from the round window in chinchilla, in particular at low to moderate stimulus levels, (2) the SP recorded from the round window largely reflects the responses from hair cells at the base of the cochlea, and (3) kainic acid results in an increase of SP amplitude to high-level stimuli whereas the SP to low- to moderate-level stimuli remains in the normal range.     

Histologic demonstration of glycosaminoglycans in inner ear fluids

We have shown previously that rat pancreatic periacinar fibroblastoid cells (PFCs) can be cultured from isolated pancreatic acini. In the present study, immunocytochemical examination of the PFC extracellular matrix was performed using antibodies against prolyl hydroxylase alpha and beta subunits, types I, III, and IV collagen, fibronectin, and laminin. The PFC content of alpha-smooth muscle actin and platelet-derived growth factor (PDGF) receptor were studied by immunoblotting. We demonstrated that PFCs synthesized extracellular matrix and expressed alpha-smooth muscle actin and PDGF receptors. These results suggested that PFCs resemble myofibroblasts and may play a critical role in pancreatic fibrosis. Conversely, pancreatic-type phospholipase A2 (P-PLA2), one of the pancreatic digestive enzymes, has been shown to induce DNA synthesis of Swiss 3T3 fibroblasts. To determine whether this enzyme is involved in pancreatic fibrosis, we studied P-PLA2's proliferative and chemotactic effects on PFCs as well as its digestive activity. The proliferative and chemotactic effects were investigated using 3H-thymidine incorporation and a chemotactic assay, respectively. P-PLA2 had both proliferative and chemotactic effects. P-PLA2 is considered a growth factor for PFCs and is implicated in pancreatic fibrosis.     

Cochlear implantation in children with inner ear malformations

We performed a case study and intervention study, with follow-up of 1 to 5 years, in 4 children with inner ear malformations who underwent implantation of a multichannel cochlear implant (Nucleus, Cochlear Corporation) at ages 3 to 12 years. Malformations included a common cavity deformity, 2 incomplete partitions, and 1 case of isolated bilateral vestibular aqueduct enlargement. One child had a single-channel implant placed at 3 years of age, and this was exchanged for a 22-channel implant at age 9. One child had her implant placed at age 4.5 years, but due to complications from a cerebrospinal fluid (CSF) leak had the initial implant removed and replaced at age 5 years during repair of the CSF leak. Intraoperative findings included a CSF leak at the time of surgery in 3 patients. One patient contracted bacterial meningitis 7 months postimplantation that was thought to be secondary to acute otitis media in the unoperated ear. Bilateral CSF leaks were noted in the middle ear by a lumbar puncture radionuclide and fluorescein dye study. Successful repair of the CSF leaks and reimplantation of the cochlear implant was carried out in this patient. Mapping and programming of the implant was found to be challenging in each of these patients. All patients demonstrated improved performance after implantation. Two patients demonstrated some open-set speech perception. One patient demonstrates improved use of temporal cues in a structured closed set. One patient has achieved no significant speech recognition at this time, but does have improved sound detection and awareness. Cochlear implantation in children with congenital inner ear abnormalities can be a successful method of rehabilitation. It should be recognized that the postoperative speech perception results may be highly variable among patients, and that intraoperative complications may occur.     

Removal and processing inner ear specimens for morphological research on guinea pig ear

Seventy (70) dissections of temporal bones of grown-up guinea pigs were done by the AA. in order to get a suitable approach to obtain inner ear studies, aiming at a morphological investigation.     

Molecular cloning of a zinc finger gene eZNF from a human inner ear cDNA library, and in situ expression pattern of its mouse homologue in mouse inner ear

A mathematical model of posttetanic processes launched by rhythmic stimulation of the excitatory and inhibitory inputs to the dendritic spine of a pyramidal neuron in hippocampal field CA3 was used to study conditions for modifying the efficiency of the inhibitory input. The level of dephosphorylation of GABAa and GABAb receptors, which determines the GABA sensitivity of these receptors, was shown to depend on the Ca(2+)-dependent ratio of active protein kinases and protein phosphatases; the level of dephosphorylation decreased monotonically as the intracellular Ca2+ increased. Posttetanic increases and decreases in the Ca2+ concentration, as compared with the level achieved during the previous stimulation, led to increases or decreases respectively in the number of dephosphorylated GABA receptors and to induction of long-term potentiation and depression, respectively, in the efficiency of inhibitory transmission. The extent of the modification effect depended on the ratio of the quantities of inhibitory and excitatory mediators in the synaptic cleft. At very low or very high GABA concentrations, modification of inhibitory transmission was insignificant.     

Bacterial flora in chronic inner ear infections in adults

The human genome carries multiple copies of sequences related to endogenous retroviral DNA. We report here the distribution of a new multicopy long terminal repeat (LTR) sequence that has been a part of an endogenous retrovirus-like sequence RTVL-H2. Twenty-four human chromosomes were either separated by flow sorting or by using rodent cells carrying a single human chromosome, and the DNA was subjected to Southern analyses using the RTVL-H2 DNA as a probe. The RTVL-H2 LTRs were distributed among all the human chromosomes, but the density and the profile differed from chromosome to chromosome. The same chromosome obtained from different individuals showed essentially the same chromosome-specific patterns. The distribution of the RTVL-H2 LTRs among different chromosomes did not correlate with the distribution of LTRs from another endogenous retroviral DNA, HERV-A, strongly suggesting that there is no preferred chromosome or a region thereof, for the integration. The possibility of rearrangement or amplification after integration is discussed.     

Effect of ibudilast on microcirculation thrombosis in rat inner ear

Arterial wall thickening may be quantitatively assessed by measuring the intima-media thickness (IMT) with high resolution ultrasound. Previous studies have shown a good inter/intraobserver variability of IMT measurements in the common carotid. In this study we evaluated the inter/intraobserver variability of IMT measurements in 10 randomly selected asymptomatic subjects (age 55.4 +/- 6). Two carotids and two femorals were studied in each subject. IMT for each patient was the average of five IMT measurements at the artery bifurcation. Three observers repeated the scanning and the measurements twice with no knowledge of the previous readings. The between observer coefficient of variation (CV) was 8.45%; the intraobserver CV (mean of carotids and femorals) varied from 4.4 to 5.1% for the three observers who measured IMT three times. The mean absolute difference between the first and the third measurement was 0.0738 mm. In conclusion IMT measurement variability is mostly due to differences between observers. The intraobserver variability is very small. IMT measurements at the carotid and femoral bifurcations have a low variability and are a good expression of atherosclerosis as they consider early lesions at the bifurcation level which may not be observed in the common carotid.