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Microanatomy of the mouse osseous cochlea: a scanning electron microscopic study.

This is the first scanning electron microscopic demonstration of the three-dimensional architecture and detailed surface structures of the entire osseous labyrinth of the cochlea. Mouse cochleae were observed after dissolving the soft tissues with KOH and NaOCl solutions. The precise shapes, surface structures, and orientations of the primary osseous spiral lamina and secondary osseous spiral lamina in the cochlea were observed along their entire course from the hook at the base to the helicotrema at the apex. The primary osseous spiral lamina showed three half turns after the hook; the lengths of the hook and each half turn and the slope angle of the spiral were obtained. The widths of the primary and secondary spiral laminae and the spiral fissure for the basilar membrane between the free edges of the two spiral laminae were measured along the course of the cochlear duct. The surface of the lateral wall under the stria vascularis was also viewed. Scanning electron microscopy can provide more precise microanatomical data than has been previously available for the osseous cochlea, giving a better understanding of hearing mechanisms with regard to the width, support, and movement of the basilar membrane and the functions of various components of the cochlea.

Animals↗

Glycocalyx heterogeneity in normal and hydropic cochleas of the guinea pig.

The endo- and perilymphatic glycocalyx of the cochlear epithelia were investigated ultrastructurally in normal and hydropic cochleas using the electron-dense markers cationized ferritin and colloidal thorium. In the normal cochleas all epithelial surfaces showed reactivity with both markers. Using cationized ferritin, no regional differences in reactivity could be demonstrated. With colloidal thorium, however, the apical membranes of the outer hair cells reacted more intensely than either the basolateral membranes or the membranes of the supporting cells of the organ of Corti. Cationized ferritin reactivity was not affected by digestion with either neuraminidase or hyaluronidase. In contrast, colloidal thorium reactivity of the endolymphatic surfaces was greatly reduced after neuraminidase digestion. Reactivity of the cell membranes lining the perilymphatic compartments of the cochlea was less affected by neuraminidase digestion, except for the basolateral membranes of the sensory and supporting cells of the organ of Corti, which demonstrated a greatly reduced reactivity. These findings indicate that the glycocalyx of the epithelial surfaces lining the endolymphatic compartment has a high content of sialic acid. The differences that are observed in normal cochleas in regard to colloidal thorium reactivity between the apical membranes of the outer hair cells and the membranes of the Deiters' cells, could not be demonstrated in hydropic cochleas. This probably contributes to the early functional changes in outer hair cells observed in experimental hydrops.

Animals↗

Cochlear implantation in children: labyrinthitis following pneumococcal otitis media in unimplanted and implanted cat cochleas.

Pneumococcal otitis media is frequent in young children and could lead to labyrinthitis post-implantation. To assess the risk, and methods of minimizing it by a graft to the round window around the electrode entry point, we have used a cat animal model of pneumococcal otitis media. Twenty-one kittens were used in the study. Thirty-two cochleas were implanted when the kittens were 2 months of age. Fourteen cochleas were implanted without using a graft (12 were available for study); 9 had a fascial graft, and 9 a Gelfoam graft (7 were available for study). The implanted kittens had their bullae inoculated with Streptococcus pneumoniae 2 months after implantation and were sacrificed 1 week later. There were also 9 unimplanted control ears which were inoculated when the animals were 4 months of age. Labyrinthitis occurred in 44% of unimplanted control, 50% of implanted ungrafted, and 6% of implanted grafted (fascia and Gelfoam) cochleas. There was no statistically significant difference between the unimplanted control and the implanted cochleas (p < 0.05). There was, however, a difference between the implanted-ungrafted and implanted grafted cochleas, but not between the use of fascia and Gelfoam to graft the round window entry point. As a result, the data indicates that cochlear implantation does not increase the risk of labyrinthitis following pneumococcal otitis media, but it is desirable to use fascia as a graft to the round window around the electrode entry point.

Animals↗

NG-methyl-L-arginine protects the guinea pig cochlea from the cytotoxic effects of pneumolysin.

Sensorineural hearing loss is a major sequela of the bacterial meningitis associated in particular with Streptococcus pneumoniae. Recent studies have shown pneumolysin, a toxin elaborated by S. pneumoniae, to be cytotoxic to the guinea pig cochlea. The mechanisms of this cytotoxicity are, however, not fully understood. In the present study this deleterious action of pneumolysin has been shown to be blocked by pretreating the cochlea with NG-methyl-L-arginine, a known inhibitor of nitric oxide synthesis. Furthermore, pretreatment of the cochlea with MK-801, an NMDA receptor antagonist, was also found to confer marked protection from the action of pneumolysin. This latter finding is consistent with previous reports that excess stimulation of NMDA receptors within the cochlea, an event known to lead to excess nitric oxide release, have similar effects on the cochlea as pneumolysin perfusion. It would therefore appear that nitric oxide may represent a significant link in the chain of events leading to the deafness of bacterial meningitis.

Action Potentials↗

[Activating effect of ATP on NO/cGMP pathway in guinea pig cochlea].

The present investigation was to study the relationship between ATP and nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway. Forty healthy purebred albino guinea pigs with sensitive pryer's reflex were randomly divided into five groups. Their cochleae were dissected and perfused immediately with different solutions. For the control group, the cochleae (group 1) were perfused with artificial perilymph basal solution (APBS, containing 100 micromol/L dipyridamole, 100 micromol/L L-Arg and 1 mmol/L IBMX). Other groups were respectively perfused group 2 with 330 micromol/L ATP, group 3 with 100 micromol/L L-NNA+330 micromol/L ATP, group 4 with 10 micromol/L ODQ+330 micromol/L ATP and group 5 with 10 micromol/L A-23187. All these reagents were freshly dissolved in artificial perilymph basal solution (APBS). The cochlear tissue specimens were collected and the average cGMP content was measured with (125)I-cGMP RIA kit. The results showed that there was no significant difference in the average cochlear tissue weights among different groups. The concentration of cGMP in the cochlear tissue of the groups perfused with ATP (59.541+/-8.744 fmol/mg) and A-23187 (55.416+/-7.018 fmol/mg) was significantly higher than those of the control group (30.089+/-4.876 fmol/mg), the groups perfused with L-NNA+ATP (28.761+/-5.019 fmol/mg) and ODQ+ATP (34.209+/-13.658 fmol/mg). No significant difference was observed between the group perfused with ATP and the one with A-23187, as well as among the control group and the groups perfused respectively with L-NNA+ATP and ODQ+ATP. These results suggest that ATP elevated the concentration of cGMP in cochlear tissue while administration of nonselective nitric oxide synthase inhibitor L-NNA and soluble guanylate cyclase inhibitor ODQ could prevent the increase of cGMP concentration induced by ATP. It is indicated that ATP is involved in the activation of NO/cGMP pathway by elevating concentration in the cytoplasm of the cochlea. In turn, NO/cGMP pathway may exert a negative action on the effects of ATP. It is suggested that there is an ATP/Ca(2+)-NO/cGMP pathway in the guinea pig cochlea. ATP and NO/cGMP pathway jointly regulate the function of the cochlea.

Adenosine Triphosphate↗

Enhanced horseradish peroxidase uptake in the electrically stimulated cochlea of the guinea pig.

Stimulation with alternating current at the round window causes an accelerated uptake of extracellular HRP by endocytotic vesicles in inner hair cells at the base of the stimulated cochlea, near the site of the electrode, but does not noticeably affect uptake into outer hair cells. After unilateral electrical stimulation, efferent endings on outer hair cells at the cochlear base show increased vesicular and endosomal labelling in both the ipsilateral and contralateral ears. It is concluded that round-window electrical stimulation increases afferent synaptic activity in inner hair cells near the electrode site, and also increases synaptic activity in OHC efferent endings, not only of the ipsilateral cochlea but also of the corresponding region of the contralateral cochlea. Rapid diffuse cytoplasmic labelling of inner and outer hair cells also occurs sporadically in both stimulated and unstimulated cochleae. However, in efferent terminals, diffuse labelling is restricted to stimulated animals, and occurs bilaterally, in corresponding basal regions of the cochlea.

Animals↗

[Overexpression of thymosin beta4 in the cochlea of senescence-accelerated mouse].

OBJECTIVE: To develop gene expression profiles of young and old senescence-accelerated mouse (SAM) cochlea and identify genes responsible for aging-related hearing loss. METHODS: Gene micro-array slides containing 1101 mouse genes were hybridized to cDNA micro-arrays (Atlas Glass Array Mouse 1.0) that were synthesized using total RNAs from the cochlea of 2 mounts and 12 mounts mouse. Hybridization signals were visualized with cyanine-3 fluorescent reporter molecules, and the fluorescence intensities of the images were analyzed. Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to validate the micro-array results. Immunofluorescence was used to identify the located region of the protein encoding by the candidate gene in the cochlea. RESULTS: Expression of a majority of the 1101 genes represented on the micro-array slides was not altered during aging; nonetheless, changes in the expression of 3 genes were detected between young and old mouse cochlea. RT-PCR results confirmed the changes in expression of thymosin beta4 of 3 genes examined. Through the using of immunofluorescence, it was shown that thymosin beta4 was primarily located in the tectorial membrane and the supporting cells of outer hair cell. CONCLUSIONS: Using commercially available slide micro-arrays, the results show that aging of the mouse cochlea is associated with changes in patterns of gene expression. This analysis suggests that thymosin beta4 may play a role in aging-related hearing loss. These studies lay the foundation for future studies defining the genetic basis of aging-related hearing loss.

Aging↗

[Effects of acute hypoxia on expression of heat shock protein 70 in cochlea of guinea pigs].

OBJECTIVE: To investigate the changes of the expression of heat shock protein 70 (HSP70) in the cochlea of guinea pigs under acute hypoxia. METHOD: Different degrees of acute hypoxia were imposed on guinea pigs and slices of the cochlea were examined by immunohistochemistry to detect HSP70. RESULT: In normal situation, HSP70 is expressed in guinea pig's cochlea, especially in spiral ganglion and Corti's organ, under acute hypoxia, this expression was strengthened, and the degree of this strengthening showed no correlation with degree of hypoxia. CONCLUSION: These results suggest that acute hypoxia can strengthen the expression of HSP70 in guinea pig's cochlea, and the degree of hypoxia has no effects on the degree of this strengthening. The expression of HSP70 in the cochlea may be a kind of protection to the structure and the function of the inner ear.

Animals↗

Cell cycle progression in gentamicin-damaged avian cochleas.

Hair cells, the sensory receptors of the auditory, vestibular, and lateral-line organs, may be damaged by a number of agents including aminoglycoside antibiotics and severe overstimulation. In the avian cochlea, lost hair cells can be replaced by regeneration. These new hair cells appear to be derived from a support cell precursor which is stimulated to divide by events associated with hair cell loss. Little is known about the timing and sequencing of events leading to new hair cell production. In this study cell cycle-associated events in the avian cochlea were analyzed at early and late time intervals following a single high dose of gentamicin. This single dose protocol has been shown to consistently result in extensive morphological damage and hair cell loss in the proximal region of the cochlea while sparing a morphologically undamaged distal cochlear region. This allowed for the differential analysis of the underlying support cell populations with respect to local hair cell loss. Three cell cycle associated markers were used to evaluate which cells entered and progressed through the cell cycle: statin, a G0 associated nuclear marker; proliferating cell nuclear antigen (PCNA), a G1, S and G2 associated marker; and 5-bromodeoxyuridine (BrdU), an S phase associated marker. Using these markers we found evidence for reversible changes in cell cycle status throughout the cochlea, while progression through S phase and mitosis was restricted to the region of the cochlea which sustained hair cell loss.

Aging↗

[The histological study of the cochlea of the human temporal bone with the diagnosis of fetal distress].

Hypoacusia is a common audiologic sequela of hypoxia associated with fetal suffering. Nonetheless, the structural features of the cochlear lesions associated with the disorder have not been studied much. We analyzed histopathologically the cochleas of 21 newborns diagnosed as fetal suffering. Each cochlea was dissected from the temporal bone, decalcified in EDTA, included in methacrylate, sliced into serial 10 microns-thick sections, and stained with hematoxylin and eosin. The cochleas of eight newborns were eliminated because of intense autolysis. Of the 13 newborns whose cochlea were considered suitable for histopathologic study, seven had hemorrhage of the vestibular ramp and one had a ruptured Reissner's membrane. The other cochlea was were within the limits of normality. The histologic technique is discussed and the critical factor judged to be the post mortem period prior to sample fixation.

Asphyxia Neonatorum↗

Segmental enhancement of the cochlea on contrast-enhanced MR: correlation with the frequency of hearing loss and possible sign of perilymphatic fistula and autoimmune labyrinthitis.

PURPOSE: To relate the finding of selective enhancement of different turns of the cochlea to the frequency range of the hearing loss measured by audiogram. METHODS: Six patients aged 23 to 53 years, four men and two women, who presented with sudden hearing loss and had segmental enhancement of different turns of the cochlea on contrast-enhanced MR imaging were included in this retrospective study. The patients were imaged on a 1.5 T MR imaging system using pre- and postcontrast axial T1-weighted images and postcontrast coronal T1-weighted images through the temporal bone. RESULTS: The basal turn of the cochlea enhanced selectively in five of the six patients and the apical turn enhanced in the sixth patient. All patients had sensorineural hearing loss. Three of the patients with basal turn enhancement had predominantly high-frequency hearing loss, whereas the patient with apical turn enhancement had predominantly low-frequency hearing loss. The two other patients with basal turn enhancement had complete hearing loss. Three patients had presumed autoimmune labyrinthitis. The other three patients had strong clinical and surgical evidence of a perilymphatic fistula. CONCLUSION: Contrast-enhanced MR imaging may demonstrate selective enhancement of different turns of the cochlea. In certain patients, the areas of enhancement may correlate with specific hearing loss in the frequencies mapped by the enhancing segment. Enhancement of the cochlea may represent radiologic evidence of cochlear inflammation secondary to a perilymphatic fistula.

Adult↗

Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.

Gene therapy is currently being used to treat many disorders including cancer, viral infection and the degenerative and fatal diseases of the cardiovascular and the central nervous systems. However, the potential use of gene therapy for alleviation of hearing impairment has not been investigated despite the absence of effective therapy for most forms of inherited hearing disorders. The purpose of this study was to assess the feasibility of introducing genetic material directly into the peripheral auditory system using adeno-associated virus (AAV) as the transfection vector and Hartley guinea pigs as the animal model. Approximately 10(5) particles of AAV containing the bacterial beta-galactosidase (beta-gal) sequence with Ad 2 major late promoter were infused into the cochlea of the animal with the aid of an osmotic minipump. Animals were killed after 2 weeks. Two Hartley guinea pigs with intracochlear saline infusion and four unoperated (nonperfused) animals served as negative controls. Both, the infused and the contralateral, non-infused cochleae were harvested from each animal, decalcified, and embedded in paraffin. Sections, 8 microns in width, were cut from the embedded cochleae and assayed for beta-gal expression via immunohistochemistry. Animals perfused with AAV showed intense immunohistochemical reactivity in the spiral limbus, spiral ligament, spiral ganglion cells and the organ of Corti in the perfused cochlea and a much weaker staining but with similar pattern in the contralateral ear. Cochleae from saline-infused and unoperated animals were devoid of the DAB stain. This study demonstrates for the first time in vivo expression of a foreign gene within the mammalian inner ear resulting from its localized, AAV-mediated introduction. The ability to introduce and stably express exogenous genetic material in the peripheral auditory system will have both experimental and therapeutic benefits. These results lay the groundwork for future studies assessing the potential use of gene therapy for alleviation of hearing impairment.

Animals↗

Localization of nitric oxide synthase and NADPH-diaphorase in guinea pig and human cochleae.

The distributions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and nitric oxide synthase (NOS) in mammalian cochlea were studied at light and electron microscope levels by NADPH-d histochemistry and brain NOS (bNOS) immunohistochemistry. The cochleae from 15 albino guinea pigs were perilymphatically fixed with 2% periodate-lysine-paraformaldehyde, decalcified in 10% EDTA and processed for light and electron microscopy after NADPH-d or NOS staining in frozen and vibratome sections respectively. One human cochlea was available for light microscope examination of NADPH-d or bNOS stained sections. Light microscope results revealed that type I neurons and nerve fibers of the spiral ganglion cells were labeled by bNOS immunohistochemistry as well as NADPH-d histochemistry in both guinea pig and human cochleae. At subcellular level, NADPH-d reaction product was localized in the mitochondria of the neuronal cytoplasm and axoplasm and in the cytoplasm of the vascular endothelium. The immunoreaction products of bNOS were evenly distributed in the neuronal cytoplasm and axoplasm. Myelinated and unmyelinated fibers in the intraganglionic spiral bundle and the inner spiral and inner radial fibers below the inner hair cells were labeled for bNOS. The nerve endings below the outer hair cells were not stained. NOS immunoreaction product was also found in the outer hair cells, Schwann cells of myelinated nerve fibers, Deiter's cells, pillar cells and the tympanic lamina cells. No difference was found in the staining pattern of both NADPH-d and NOS reaction products between human and guinea pig cochleae at the light microscope level. The results suggest that NO plays an important role in the maintenance of auditory function in the mammal.

Animals↗

Rapid changes in protein synthesis and cell size in the cochlear nucleus following eighth nerve activity blockade or cochlea ablation.

Destruction of the cochlea causes secondary changes in the central auditory pathway through transynaptic regulation. These changes appear to be mediated by an activity-dependent process and can be detected in the avian auditory system as early as 30 minutes after deafferentation. We compared the early changes in cochlear nucleus neurons following deafferentation by cochlea ablation with those seen following activity deprivation by perilymphatic tetrodotoxin (TTX) exposure. Protein synthesis and size of large spherical cells in the anteroventral cochlear nucleus (AVCN) of 14-day-old gerbils were measured during the first 48 hours after the manipulations. Both cochlea ablation and TTX produced a reliable decrease in protein synthesis by AVCN neurons (30-40%) by 1 hour. The magnitude of change in tritiated leucine incorporation was similar at all survival times, in both experimental groups. In contrast to the rapid changes in protein synthesis, the decrease in cell size was first evident 18 hours after TTX exposure and 48 hours after cochlea ablation. There was no significant change in protein synthesis or cell size in control groups at any of the survival times. These findings are consistent with changes in the avian auditory system in response to deafferentation and TTX exposure. Cochlea ablation and TTX exposure induced similar transneuronal changes, supporting the hypotheses that activity of auditory afferents in young mammals plays a regulatory role in the metabolism and morphology of their target neurons in the central auditory pathway, and that early changes following destruction of the peripheral receptor are due to reduction of activity-dependent interactions of presynaptic and postsynaptic cells.

Analysis of Variance↗

Ontogenesis of rat cochlea. A quantitative study of the organ of Corti.

A systematic quantitative set of data concerning the organ of Corti in developing Sprague-Dawley rats at intervals from 18 days of gestation to 10 days after birth (DAB) is provided in this study. Using phalloidin staining, the total number of inner and outer hair cells, the whole length of cochlea, as well as the diameter of inner and outer hair cells and the intercellular space between inner hair cells were determined in order to analyze the quantitative change of inner and outer hair cells during development and to explore some roles of the factors regulating the growth of cochlea. The results show that: (1) The length of cochlea approached its adult size by 7DAB. (2) The growth of the extreme part of the apex was responsible for the delayed elongation of the cochlea. (3) Growth in the cochlear length mainly results from an increase of cell diameter tempered by a decrease of intercellular space. (4) The adult size of inner and outer hair cells was obtained by 7-14DAB. (5) The final number of inner and outer hair cells was reached at 3DAB and remained constant through adulthood. No significant hair cell overproduction and cell death were observed during ontogenesis of the cochlea. The negligible importance of overproduction and missing hair cells during hair cell differentiation suggest that there is a precise regulation phenomenon for producing the right spatial organization of the organ of Corti.

Actins↗

Expression patterns of ion transport enzymes in spiral ligament fibrocytes change in relation to strial atrophy in the aged gerbil cochlea.

Fibrocytes of the lateral wall function in conjunction with the stria vascularis (StV) to mediate cochlear ion homeostasis. Age-related changes in the expression patterns of ion transport enzymes in spiral ligament fibrocytes were investigated to ascertain their relation to metabolic presbyacusis in the gerbil. Immunoreactivity of fibrocytes for Na,K-ATPase (Na,K), carbonic anhydrase isozyme II (CA) and creatine kinase isozyme BB (CK) varied among and within cochleas from aged but not from young gerbils. The variable immunostaining was related to the extent and location of StV atrophy. Age-dependent degeneration and loss of Na,K in the StV occurred predominantly in the apex and lower base and hook of the cochlea, largely sparing more central regions. Immunostaining intensity for Na,K, CK, and CA in fibrocytes changed in relation to declines in strial marginal cell Na,K initially showing upregulation followed by downregulation. Spiral ligament fibrocytes in cochleas with more than two remaining normal turns often disclosed overexpression of CK in regions of strial atrophy. Conversely, CA in such cochleas was often increased in regions of normal StV adjacent to foci of atrophic StV. Senescent cochleas with two or fewer functional turns generally contained fibrocytes with diminished CK or CA immunoreactivity in regions of atrophic StV but in isolated instances exhibited fibrocytes with enhanced staining. Heightened staining for CK in type Ia fibrocytes underlying regions of complete or partial strial atrophy indicated an increased metabolic demand in fibrocytes in response to strial insufficiency. The findings provide further support for the role of spiral ligament fibrocytes in cochlear fluid and ion homeostasis.

Aging↗

Changes in the avian cochlea after single high-dose gentamicin.

PURPOSE: Define the time course of functional and anatomical damage and subsequent recovery (by regeneration) of hair cells in the chicken inner ear after a single high-dose of gentamicin. MATERIALS AND METHODS: Broiler chicks were given a single intraperitoneal dose (200 mg/kg) of gentamicin (n = 39) or saline (n = 39). Functional status was evaluated with auditory brainstem response (ABR) thresholds before injection and before sacrifice at 2, 5, 9, 16, 21, 28, and 70 days postinjection. The cochleae were then examined with scanning electron microscopy (SEM) to assess the extent of damage along the cochlea and absolute hair cell numbers in the basal 15% of the cochlea (high-frequency region). RESULTS: Considerable variability between animals was seen for both ABR and SEM changes. Damage was maximal at 5 days postinjection with an average ABR threshold shift of 12 dB (range -10 to 50 dB) and basal cochlear damage of 28% (range 12%-57%). Hair cell counts were significantly decreased in the basal 15% of the cochlea at 5 days. Hair cell regeneration resulted in rapid anatomical and functional recovery, but evidence of hair cell disorganization persisted at 70 days despite improved thresholds. CONCLUSION: A single high dose of gentamicin produces a significant but variable anatomical and functional insult in the chick cochlea. Hair cell regeneration results in rapid but incomplete recovery.

Analysis of Variance↗

[The Nucleus Double Array Cochlear Implant: a new concept in obliterated cochlea].

AIM: In order to increase the number of intracochlear electrodes to be inserted into a totally obliterated cochlea a special implant has been developed in collaboration with Cochlear Ltd. The implant features two separate electrode carriers containing 11 and 10 active electrodes, respectively, and a reference electrode on the receiver stimulator package. The potential stimulation modes include monopolar and bipolar stimulation as well as stimulation between both arrays. SURGICAL TECHNIQUE: A cochleostomy at the round window provides access to the scala tympani. Newly formed bone is removed as far as the anterior portion of the basal turn. Care is taken to identify and preserve the osseous border of the cochlea. A second cochleostomy is performed immediately caudal to the cochleariform process after removal of the incus. New tissue can be removed here in the same way. The two electrode carriers are then placed into the first and the second turn respectively. The remaining procedure corresponds to the procedure for cochlear implantation in cases in which the cochlea is not obliterated. PATIENTS: For the purpose of a clinical study n = 10 patients aged between 32-66 years with an obliterated cochlea were fitted with a double array cochlear implant. All patients showed signs of total obliteration of the basal turn either in preoperative imaging or during surgery. Intraoperative inspection revealed that the second turn was not obliterated in 4 of 10 patients. POSTOPERATIVE RESULTS: Postoperatively, a standard test battery was used to determine auditory performance over a period of time. All patients achieved significantly better speech understanding due to the additional apical electrode array. No complications occurred. CONCLUSION: In cases involving an obliterated cochlea, the number of intracochlear electrodes can be increased with the double array implant. As a result, the patients achieve significantly better auditory results.

Adult↗