Hair cell loss as a function of age in the normal cochlea of the guinea pig

The surface specimen technique was used to study both spiral organs of 28 normal guinea pigs of four age groups: less than 24 hours, 6 weeks, 3 months and 1 year. Damaged hair cells were recorded for the whole of each spiral organ on cochleograms. The mean percentage number of outer hair cells damaged per age group was found to increase as a power function of age. In the animals aged less than 24 hours the mean percentage of damaged outer hair cells was 0.45%; in the 6-week animals, 1.85%; in the 3-month animals, 3.19%; and in the 1-year animals, 6.82%. At all ages outer hair cell loss was maximal in the third row, and towards the apex of the cochlea. Inner hair cell loss was very slight, with a maximum of 9 damaged inner hair cells per cochlea.     

Burns of the hand and upper limb--a review

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer Dil. In the vestibular system, trkC+/- heterozygosity aggravates the trkB-/- mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthal's canal of the cochlea. trkC-/- mutation alone or in combination with trkB+/- heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB-/- mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB-/- mutant mouse combined with trkC+/- heterozygosity. In addition, the trkB-/- mutation combined with trkC+/- heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC-/- mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type: trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.     

Correspondence between middle frequency auditory loss in vivo and outer hair cell shortening in vitro

The aromatic hydrocarbon, toluene, has been reported to disrupt auditory system function both in occupational epidemiological and in laboratory animal investigations. This agent, along with several other organic solvents, impairs hearing preferentially at middle frequencies - a finding that distinguishes these agents from the traditional high frequency impairment observed with ototoxic drugs such as aminoglycoside antibiotics and cisplatin. Prior investigations performed in vivo have identified the outer hair cell as a probable target for toluene exposure. The purpose of this investigation was to determine directly whether outer hair cells isolated from the guinea pig cochlea show morphological alterations consistent with the toxic response seen in physiological studies with toluene exposure. The effect of toluene superfusion on outer hair cell shortening was assessed for cells harvested from different locations within the cochlea. Control studies included assessment of cell shortening among outer hair cells exposed to trimethyltin and cells exposed to benzene. Trimethyltin disrupts high frequency hearing preferentially and benzene does not produce hearing loss in vivo. Toluene at a concentration of 100 microM produced a marked shortening of outer hair cells although the effect was significantly greater among cells isolated from the apical half of the cochlea than from the basal half of the cochlea. By contrast, trimethyltin at the same concentration produced a preferential shortening among outer hair cells from the base of the cochlea. Benzene (100 microM) did not disrupt outer hair cell length of cells harvested from the apex. The results indicate that intrinsic features of outer hair cells contribute significantly to the site of ototoxic impairment observed in vivo for toluene.     

Nitroprusside suppresses cochlear potentials and outer hair cell responses

Biochemical and pharmacological evidence supports a role for nitric oxide (NO) in the cochlea. In the present experiments, we tested sodium nitroprusside (SNP), an NO donor, applied by intracochlear perfusions on sound-evoked responses of the cochlea (CM, cochlear microphonic; SP, summating potential; EP, endocochlear potential; CAP, compound action potential) and in vitro on outer hair cell (OHC) voltage-induced length changes and current responses. In vivo application of SNP in increasing concentrations (10, 33, 100, 330 and 1000 microM) reduced all sound-evoked responses starting at about 300 microM. The responses continued to decline after a postdrug wash. At 1 mM SNP decreased EP slowly (approximately 80 min) whereas at 10 mM it reduced EP more rapidly (approximately 20 min). Ferricyanide (1 mM) and S-nitroso-N-acetylpenicillamine (SNAP; 1 mM) had no effect on sound-evoked cochlear potentials. Ferricyanide (1 mM and 10 mM) and ferrocyanide (10 mM) had no effect on EP. In vitro, SNP (10 mM) significantly reduced both OHC voltage-induced length changes and whole-cell outward currents. Results suggest that SNP, possibly acting by released NO, influences cochlear function through effects at the stria vascularis and at the OHCs.     

Outer hair cell activity is not required for the generation of the forward masking curve

Forward masking of the auditory brainstem response (ABR) was achieved by increasing the time interval from 0 to 12 ms between the masker offset and the probe onset. The forward masking response demonstrated a near linear function with an approximate 3.0-dB increase in masking threshold for every millisecond interval increase in the control guinea pig. The slope of the masking curve at selected frequencies together with the quantification of hair cell loss through the analysis of cochlear surface morphology was studied before and after chemical insult. The intracochlear infusion of sodium salicylate caused an approximately 45-dB threshold shift of the ABR whereas the slope of the forward masking curve was not significantly different from the control values at the tested frequencies (1, 4, and 8 kHz). Systemic kanamycin administration (400 mg/kg body weight for 9 consecutive days) caused a permanent ABR threshold shift of 43-63 dB at 1, 4, and 8 kHz. The slope of the forward masking curve was not significantly different at 1 kHz despite significant outer hair cell loss. The slope of the forward masking curve at 4 and 8 kHz showed significant reductions at the time intervals between 0 and 4 ms. Analysis of the kanamycin-treated cochleae revealed not only significant outer hair cell loss throughout the cochlea but significant inner hair cell and inner pillar cell loss in the basal end of the cochlea. The results suggest that the outer hair cells are not needed for maintaining a normal forward masking curve, whereas the slope of the forward masking curve is sensitive to alterations induced to either the inner hair cells or the inner pillar cells.     

Ultrastructural, physiological, and molecular defects in the inner ear of a gene-knockout mouse model for autosomal Alport syndrome

It is well documented that damage to the chick cochlea caused by acoustic overstimulation or ototoxic drugs is reversible. Second-order auditory neurons in nucleus magnocellularis (NM) are sensitive to changes in input from the cochlea. However, few experiments studying changes in NM during cochlear hair cell loss and regeneration have been reported. Chicks were given a single systemic dose of gentamicin, which results in maximal hair cell loss in the base of the cochlea after 5 days. Many new hair cells are present by 9 days. These new hair cells are mature but not completely recovered in organization by 70 days. We counted neurons in Nissl-stained sections of the brainstem within specific tonotopic regions of NM, comparing absolute cell number between gentamicin- and saline-treated animals at both short and long survival times. Our data suggest that neuronal number in rostral NM parallels hair cell number in the base of the cochlea. That is, after a single dose of gentamicin, we see a loss of both cochlear hair cells and NM neurons early, followed by a recovery of both cochlear hair cells and NM neurons later. These results suggest that neurons, like cochlear hair cells, can recover following gentamicin-induced damage.     

Postnatal depression and social supports in Vietnamese, Arabic and Anglo-Celtic mothers

Nitric oxide synthase III (NOS III) was identified in the guinea pig cochlea on an ultrastructural level using a post-embedding immunolabeling procedure. Ultrathin sections of London Resin (LR) White-embedded specimens were incubated with various concentrations of a commercially available antibody to NOS III and the immunoreactivity visualized by a gold-labeled secondary antibody. Analysis of ultrathin sections of the organ of Corti in the second turn of the cochlea showed that NOS III could be localized in the endothelial cells of the blood vessels under the basilar membrane, which was comparable to its location in similar cells types in various biological systems. Besides this, NOS III was also found in the cytoplasm and in the nuclei of inner and outer hair cells. Immunoreactivity was not distributed homogeneously within receptor cells. Numerous gold particles could be identified at the border of the cuticular plates, in the middle parts of the stereocilia and in the cytoplasm. Gold-labeled anti-NOS III antibodies in these sites were seen mostly on the cytoplasmic side of the submembranous cisterns in the vicinity of mitochondria and in the central parts of the hair cells, whereas the cisterns were nearly free from any immunoreactivity. NOS III was also detected in the efferent and afferent nerve endings that were located at the basal and basolateral side of the outer hair cells. Some immunoreactivity was visible in different nerve fibers of the inner and outer spiral tunnels. Besides this, gold-labeled antibodies were also present in the cuticular plate of inner and outer pillar cells, in the cytoskeletal elements located in the apical parts of Deiters cells, forming the lamina reticularis, and in the cytoskeletal-containing region of the cytoplasm of those Deiters cells located at the basal side of the outer hair cells. The role of the NOS III immunoreactivity identified in the organ of Corti was consistent with respect to hair cell and tissue modulation.     

G protein Gi2 alpha in the cochlea: cloning and selective occurrence in receptor cells

A cDNA library was made from the mouse cochlea and screened with a G protein-cDNA like molecule obtained from cochlear tissue by polymerase chain reaction. The nucleotide sequence of a clone, named cochlear Gi2 alpha, had 99.2% identity to mouse macrophage Gi2 alpha. Using an antibody which is selective for Gi2 alpha, expression of the cochlear Gi2 alpha was localized in outer and inner hair cells of the organ of Corti. Possible functional roles of this G protein in hair cells are discussed.     

Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?

In the adult mammalian cochlea, post-injury hair cell losses are considered to be irreversible. Recent studies in cochlear explants of embryonic rodents show that the organ of Corti can replace lost hair cells after injury. We have investigated this topic in vivo during the period of cochlear development. Rat pups were treated with a daily subcutaneous injection of 500 mg/kg amikacin for eight consecutive days between postnatal day 9 (PND 9) and PND 16. During this period the organ of Corti is not fully mature, but hair cells are hyper-sensitive to aminoglycoside antibiotics. Scanning and transmission electron microscopy was used to evaluate morphological changes in the organs of Corti during the treatment and at different post-treatment periods, up until PND 90. A massive loss in outer and inner hair cells was observed at least as early as PND 14. A prominent feature in the apical part of cochleas at PND 21 and 35 was the transient presence of small atypical cells in the region of pre-existing outer hair cells. These atypical cells had tufts of microvilli reminiscent of nascent stereociliary bundles. A second striking observation was the replacement of degenerating inner hair cells by pear-shaped supporting cells throughout the cochlea. These cells were covered with long microvilli, and their basal pole was contacted by both afferent and efferent fibers, as in the early stages of inner hair cell maturation. At PND 55 and 90, these features were not clearly observed due to further cytological changes in the organ of Corti. It is possible that an attempt at hair cell neodifferentiation could occur in vivo after an amikacin treatment in the rat during the period of cochlear hyper-sensitivity to antibiotic.     

Pathologic mechanoelectric transduction of outer hair cells as the cause of recruitment

It is believed that the sound-induced travelling wave in the mammalian cochlea is enhanced and sharpened by a positive feedback mechanism. This causes the passive linear basilar membrane growth function to become non-linear. The present paper shows that nonlinear basilar membrane vibration is due to the nonlinear growth function of the receptor potential of outer hair cells, which can be described by a 2nd-order Boltzmann function. Since intensity coding in the inner ear depends on an interaction of nonlinear basilar membrane motion and nerve fibers with three different types of synaptic threshold and growth function, the process is directly dependent on an intact mechanoelectrical transduction of outer hair cells. According to the proposed model, a loss in efficiency of outer hair cell mechanoelectrical transduction must lead to both a reduction in gain (i.e., hearing loss) and a linearizing of the response. As a result, once above threshold, the changes of stereociliary displacement, basilar membrane displacement and neural firing rate per unit change of sound intensity must be larger than for the healthy cochlea with its compressive nonlinearity.     

Evidence for opening of hair-cell transducer channels after tip-link loss

The mechanosensitive transducer channels of hair cells have long been proposed to be gated directly by tension in the tip links. These are thin, elastic extracellular elements connecting the tips of adjacent stereocilia located on the apical surface of the cell. If this hypothesis is true, the channels should close after destruction of tip links. The hypothesis was tested pharmacologically using receptor currents obtained in response to mechanical stimulation of the stereociliary bundle of outer hair cells isolated from the adult guinea pig cochlea. Application of elastase (20 U/ml) or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid (BAPTA; 5 mM), both of which are known to disrupt tip links in other hair-cell preparations, led to the expected irreversible loss of receptor currents. However, the cells then displayed a maintained inward current, implying that channels were left permanently open. This current was similar in magnitude to the receptor current before treatment and was reduced reversibly by known blockers of mechanosensitive channels, namely, dihydrostreptomycin (100 microM), amiloride (300 microM), and gadolinium ions (1 mM). These observations suggest that the maintained current flows through the mechanosensitive channels. Electron microscopical analysis of isolated hair cells, exposed to the same concentrations of elastase or BAPTA as in the electrophysiological experiments, demonstrated an almost total loss of tip links in hair bundles that showed no evidence of other mechanical damage. It is concluded that although the tip links are required for mechanoelectrical transduction, the channels are not gated directly by the tip links.     

Pudendal nerve somatosensory evoked potentials in probable multiple sclerosis

One of the most frequent causes of sensorineural hearing loss in childhood is damage to outer hair cells of the cochlea. The presence of otoacoustic emissions, generated by outer hair cells, provides evidence for normal hearing. This finding, however, may give rise to false reassurance, because even severe hearing loss, localized behind the cochlea, can be associated with normal otoacoustic emissions. The coexistence of otoacoustic emissions and hearing loss calls for the prompt exclusion of neurological disease.     

Histopathological findings in clinical gentamicin ototoxicity

The temporal bone histopathological findings in a case of gentamicin sulfate-induced hearing loss and vertigo in an anephric patient undergoing hemodialysis are presented. A study of the sensory neuroepithelium of the cristae and maculae disclosed the presence of vacuoles with clubbing of the sensory cells. In the cochlea, loss of the innermost row of outer hair cells in the basal turn was the most prominent feature. These findings are discussed in light of reports of similar morphological changes in laboratory studies of gentamicin ototoxicity.     

Histopathologic observations of the aging gerbil cochlea

Age-related histopathologic changes were examined in cochleas from 17 gerbils born and kept in a quiet environment until near the end of their life expectancy. Hearing loss varied greatly as did the loss of outer hair cells (OHC). Inner hair cells (IHC) were seldom missing even in cochleas with severe hearing losses. Flask- and spherical-shaped OHCs were frequently seen in the apical turn. Stereocilia were usually present and orderly on OHCs, but the tallest row of stereocilia on IHCs was often disarrayed and sometimes missing. Alterations in supporting cells were sometimes present in regions of extensive OHC loss. Although pillar cells were seldom missing, the nuclei of outer pillar cells were commonly displaced from their normal basal position. The density of radial fibers appeared similar to that in young gerbils except in the apical turn of one old ear where a marked loss of radial fibers occurred without an attendant loss of IHCs. All of the quiet-aged cochleas showed a characteristic clustering of epithelial cells lining the scala media surface of Reissner's membrane. This structural rearrangement was not accompanied by a significant decrease in the total number of cells forming Reissner's membrane and did not appear to be associated with hearing loss. The findings confirm and extend earlier work showing that several different types of cells are susceptible to histopathologic changes in old ears. The extent of histopathologic changes varied widely as did the degree of hearing loss in animals with a restricted genetic background and maintained under carefully controlled environmental conditions. It was not possible, based on these initial findings, to relate specific structural to specific functional changes in the aging cochlea. Further light and electron microscopic analysis of other regions from these aged cochleas may provide more conclusive data.     

Ototoxicity of carboplatin: comparing animal and clinical models at the Hospital for Sick Children

Carboplatin was introduced to the paediatric population as an alternative chemotherapeutic agent in the management of various malignant neoplasms, including sarcomas of the head and neck, in the hope that it would have fewer side effects than pre-existing agents. While many investigators have considered the ototoxicity of this drug only incidentally, others have presumed it to be of negligible importance. A recent animal model of its use has demonstrated damage to the inner hair cells of the cochlea, particularly at the basal turn, with a corresponding high incidence of hearing loss. Similarly, 11 of 22 patients who received this agent at the Hospital for Sick Children over the past 2 years demonstrated a sensorineural hearing loss in the 4 to 12 kHz range. This complication occurred as early as after the first dose and was generally progressively worse with subsequent doses. Consequently, we recommend careful audiologic monitoring of children receiving this agent.     

Alterations of insulin response to different beta cell secretagogues and pancreatic vascular resistance induced by N omega-nitro-L-arginine methyl ester

1. We studied a possible interplay of pancreatic NO synthase activity on insulin secretion induced by different beta cell secretagogues and also on pancreatic vascular bed resistance. 2. This study was performed in the isolated perfused pancreas of the rat. Blockage of NO synthase was achieved with Nw-nitro-L-arginine methyl ester (L-NAME); The specificity of the antagonist was checked by using its D-enantiomer as well as by substitutive treatments with sodium nitroprusside (SNP) as a NO donor in studies of glucose-induced insulin secretion. 3. Arginine (5 mM) induced a monophasic response which was, in the presence of L-NAME at equimolar concentration, very strongly potentiated and converted into a 13 times higher biphasic one. D-NAME (5 mM) was only able to induce a 3 times higher response, but provoked a similar vasoconstrictor effect. 4. The small biphasic insulin secretion induced by L-leucine (5 mM) was also strongly enhanced, by 8 times, in the presence of L-NAME (5 mM) vs 2 times in the presence of D-NAME (5 mM). 5. beta cell responses to KCl (5 mM) and tolbutamide (0.185 mM) were only slight increased by L-NAME (5 mM) to values not far from the sum of the effects of L-NAME and of the two drugs alone. D-NAME (5 mM) was totally ineffective on the actions of both secretagogues. 6. L-NAME, infused 15 min before and during a rise in glucose concentration from 5 to 11 mM, was able in the low millimolar range (0.1-0.5 mM) to blunt the classical biphasic pattern of beta cell response to glucose and, at 5 mM, to convert it into a significantly greater monophasic one. In contrast, D-NAME (5 mM) was unable to induce similar effects. 7. SNP alone at 3 microM was ineffective but at 30 microM substantially reduced to second phase of insulin response to glucose; however, at both concentrations the NO donor partly reversed alterations in insulin secretion caused by L-NAME (5 mM) and restored a biphasic response.     

Pathological specimens of rat temporal bone--comparison between different fixative solution

We investigated the difference in temporal bone specimens fixated by three sorts of fixative solution (10% formalin fixative, Wittmaack's fixative and Heidenhein-SuSa fixative). 1. 10% Formalin's fixative solution We found many pinkish precipitates, which are stained by hematoxy-eosin, in the scala media of the cochlea. Same substance is found in the perilymphatic space and endolymphatic space in the vestibulum. The fusion of the cells is indicated in the tectorial membrane, inner sulucus cell, outer sulucus cell especially in the apical turn of the cochlea. 2. Wittmaack's fixative solution The detachment of inner and outer sulucus cells are found from basilar membrane at the basal turn of the cochlea. The tendency in convex and concave form of the Reissner's membrane is most remarkable in these three fixative solution. 3. Heidenhein-SuSa fixative solution In this solution, no precipitates, no fusion of cells are found. No detachment of the inner or outer sulucus cells is also found. This study indicates that Heidenhein-SuSa fixation is most excellent fixative method.     

The cytochochleogram in atoxyl-treated guinea pigs

The earliest atoxyl induced changes in the cochlea appeared in the upper and medial parts of the 4th coil, whence the changes spread progressively downwards towards the round window, the extent of the changes depending on the amount of atoxyl administered and the duration of the treatment. The inner hair cells were more resistant to the effects of atoxyl than the outer hair cells which were affected first. The sensory cells in the 2nd and 3rd rows appeared more sensitive than the outer hair cells in the first row.     

Anticholinergic effects of strychnine in the cochlea do not involve muscarinic receptors

Central control of cochlear function is mediated by the cholinergic (medial) efferent system and both muscarinic and nicotinic acetylcholine receptors are thought to be present on outer hair cells. All the physiological effects of acetylcholine in the cochlea are blocked by strychnine and we therefore investigated whether strychnine interacts with muscarinic receptors in the cochlea. The effects of strychnine on both (3H)-quinuclidinyl benzylate binding and atropine sensitive carbachol-induced (3H)-inositol phosphate formation were examined. Strychnine (1 to 50 microM) has no effect on either quinuclidinyl benzylate binding or carbachol (1 mM)-induced inositol phosphate synthesis. Moreover, strychnine does not change basal inositol phosphate metabolism. These data indicate that muscarinic receptors are not sensitive to strychnine at concentrations which are known to block the effects of acetylcholine on outer hair cells.     

Nitric oxide modulates cGMP levels in neurons of the inner and outer retina in opposite ways

In the mammalian retina, neuronal nitric oxide synthase (NOS) is mainly localized in subpopulations of amacrine cells. One function of nitric oxide (NO) is to stimulate soluble guanylate cyclases which in turn synthesize cGMP. We used an antibody specific for cGMP to demonstrate cGMP-like immunoreactivity (cG-IR) in bovine, rat, and rabbit retinae and investigated the effects on cGMP levels of both exogenously applied NO and of endogenously released NO. We found that cGMP levels in inner and outer retina were controlled in opposite ways. In the presence of the NO-donors SNP, SIN-1 or SNAP, cG-IR was prominent in neurons of the inner retina, mainly in cone bipolar cells, some amacrine and ganglion cells. Retinae incubated in IBMX showed weak cG-IR in bipolar cells. Glutamate increased cG-IR in the inner retina, presumably by stimulating endogenous NO release, whereas NOS inhibitors or GABA and glycine decreased cG-IR in bipolar cells by reducing NO release. In somata, inner segments and spherules of rod photoreceptors the situation was reversed. cG-IR was undetectable in the presence of NO-donors or glutamate, was moderate in IBMX-treated retinae, but increased strongly in the presence of NOS inhibitors or GABA/glycine. We conclude that NO is released endogenously in the retina. In the presence of NO, cGMP levels are increased in neurons of the inner retina, but are decreased in rods.