Chinchilla Cochlea Research Articles

The caspase pathway in noise-induced apoptosis of the chinchilla cochlea.

We previously reported that intense noise exposure causes outer hair cell (OHC) death primarily through apoptosis. Here we investigated the intracellular signal pathways associated with apoptotic OHC death. Chinchillas were exposed to a 4 kHz narrowband noise at 110 dB SPL for 1 h. After the noise exposure, the cochleas were examined for the activity of each of three caspases, including caspase-3, -8, or -9 with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The cochleas were further examined for cytochrome c release from mitochondria by immunohistology and for DNA degradation by the TUNEL method. The results showed that the noise exposure triggered activation of caspase-3, an important mediator of apoptosis. The noise exposure also caused the activation of caspase-8 and caspase-9, each of which is associated with a distinct signaling pathway that leads to activation of caspase-3. Caspase activation occurred only in the apoptotic OHCs and not in the necrotic OHCs. These results indicate that multiple signaling pathways leading to caspase-3 activation take place simultaneously in the apoptotic OHCs. In addition to caspase activation, noise exposure caused the release of cytochrome c from mitochondria, resulting in a punctate fluorescence in the cytosol. In contrast to activation of caspases, the release of cytochrome c took place in both apoptotic and necrotic OHCs. Moreover, the release of cytochrome c in a subpopulation of OHCs took place early in the cell death process, prior to any outward signs of necrosis or apoptosis. These data suggest that in this subpopulation there exists a common step that is shared by cell death pathways before entering either necrosis or apoptosis. Lastly, use of the TUNEL assay in combination with PI labeling provides a more accurate discrimination between apoptosis and necrosis.

Immunolocalization of voltage-gated calcium channel alpha1 subunits in the chinchilla cochlea.

The immunohistochemical localization of alpha1A, alpha1B, alpha1C, alpha1D, and alpha1E voltage-gated calcium channel subunits was investigated in the chinchilla organ of Corti and spiral ganglia with the use of specific antipeptide antibodies. The inner and outer hair cells were immunoreactive for alpha1A and alpha1D subunit antibodies. Alpha1C immunoreactivity localized to the nerve terminals innervating inner hair cells and the basal pole of the outer hair cell. There was only non-specific staining to alpha1B and alpha1E. Supporting cells were non-immunoreactive. Spiral ganglia neurons were alpha1B, alpha1C, and alpha1D immunoreactive. A few spiral ganglia neurons were alpha1E immunoreactive. The importance of alpha1D, the pore-forming subunit of the L-type channel, in outer and inner hair cell function has been clearly demonstrated in electrophysiological, molecular biological, and knockout models. The presence of alpha1A, the pore-forming subunit of the P/Q type channels, has not previously been demonstrated in inner and outer hair cells, and its function in the cochlear hair cell is unknown.

Effects of nitric oxide on morphology of isolated cochlear outer hair cells: possible involvement in sensorineural hearing loss.

One of the inflammatory mediators of otitis media, nitric oxide, can damage cochlear outer hair cells. Free radicals, including nitric oxide, have been detected in middle ear effusion. Increasing evidence implicates free radicals in the pathogenesis of otitis media and possibly in the development of sensorineural hearing loss. Isolated outer hair cells from adult chinchilla cochlea were exposed to standard bathing solution (Control Group 1) or the nitric oxide-producing compounds, S-nitroso-N-acetyl, l-penicillamine (1-1.5 mg/ml, Experimental Group 1) or 3-morpholinosynonimine (1-1.5 mg/ml, Experimental Group 2). Since nitric oxide is readily converted to nitrite and nitrate in vivo, a second control group using sodium nitrite was used to separate potential effects of nitric oxide from nitrite (Control Group 2). All experiments were performed at an osmolality of 305 +/- 5 mOsm at room temperature, and with exposure time up to 90 minutes. The cells were observed using an inverted microscope, and the images were recorded and analyzed on the IMAGE Pro-Plus program. Outer hair cells exposed to either standard bathing solution or sodium nitrite (Control Groups 1 and 2) showed no significant change in cell shape or length. Cells exposed to S-nitroso-N-acetyl and l-penicillamine or 3-morpholinosynonimine exhibited ballooning and significant shortening in mean cell length (p < 0.01). This study demonstrates that exposure to nitric oxide causes irreversible morphologic changes in isolated outer hair cells, suggesting possible involvement of nitric oxide radical in the development of sensorineural hearing loss as a sequela of chronic otitis media.

Progression of Outer Hair Cell Death in the Chinchilla Cochlea following Traumatic Noise Exposure

Progression of Outer Hair Cell Death in the Chinchilla Cochlea following Traumatic Noise Exposure

A three-dimensional nonlinear active cochlear model analyzed by the WKB-numeric method

A physiologically based nonlinear active cochlear model is presented. The model includes the three-dimensional viscous fluid effects, an orthotropic cochlear partition with dimensional and material property variation along its length, and a nonlinear active feed-forward mechanism of the organ of Corti. A hybrid asymptotic and numerical method combined with Fourier series expansions is used to provide a fast and efficient iterative procedure for modeling and simulation of the nonlinear responses in the active cochlea. The simulation results for the chinchilla cochlea compare very well with experimental measurements, capturing several nonlinear features observed in basilar membrane responses. These include compression of response with stimulus level, two-tone suppressions, and generation of harmonic distortion and distortion products.

Structure of outer hair cell stereocilia side and attachment links in the chinchilla cochlea.

The structure and symmetry of chinchilla outer hair cell (OHC) stereocilia side and attachment links were investigated by transmission electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. The side links run laterally between and across the rows of the stereocilia and connect the stereocilia together within the bundle. Attachment links form a crown-like array around the tips of only the tallest OHC stereocilia and attach these stereocilia to the Type B fibrils of the tectorial membrane. Computer averaging of the side links from tannic acid-treated tissues showed a central dense region of the link between adjacent stereocilia and a smaller dense portion at the plasma membrane end of the link. Computer averaging of Cuprolinic blue-treated tissues showed low electron density of the central region of the link, and the plasma membrane ends of the link were electron dense. After tannic acid treatment, the attachment links showed a diffused radial distribution around the tips of the tallest OHC stereocilia. After Cuprolinic blue treatment, the attachment links appeared as electron-dense, membrane-bound granular structures arranged with radial symmetry. The central regions of the side links are reactive to tannic acid. These regions appear to contain neutral and basic residues of proteins and participate in side-by-side association of the side links in regular aggregates. Cuprolinic blue-reactive regions of the side and attachment links appear to contain acidic sulfated residues of glycoproteins or proteoglycans, which may be involved in the attachment of these links to the stereocilium membrane.

Involvement of apoptosis in progression of cochlear lesion following exposure to intense noise

It has been known for some time that noise-induced outer hair cell (OHC) death in the cochlea continues well after the termination of a noise exposure. However, the underlying mechanisms leading to the expansion of a cochlear lesion are not fully understood. Here we report involvement of the apoptotic pathway in the progression of OHC death in the chinchilla cochlea following exposure to a 4 kHz narrow band noise at 110 dB SPL for 1 h. Morphological examination of OHC nuclei revealed nuclear condensation and fragmentation, typical morphological features of apoptosis. OHC apoptosis developed asymmetrically toward the apical and basal parts of the cochleas following the noise exposure. Two days after the noise exposure, there was still active OHC pathology with condensed and fragmented nuclei in the basal part of the cochleas. Detection of caspase-3 activation, an intracellular marker for apoptosis, showed a spatial agreement between the apoptotic nuclei and activated caspase-3. These results clearly implicate the apoptotic pathway in the post-exposure progression of OHC demise.

Multicomponent stimulus interactions observed in basilar-membrane vibration in the basal region of the chinchilla cochlea

Multicomponent stimuli consisting of two to seven tones were used to study suppression of basilar-membrane vibration at the 3-4-mm region of the chinchilla cochlea with a characteristic frequency between 6.5 and 8.5 kHz. Three-component stimuli were amplitude-modulated sinusoids (AM) with modulation depth varied between 0.25 and 2 and modulation frequency varied between 100 and 2000 Hz. For five-component stimuli of equal amplitude, frequency separation between adjacent components was the same as that used for AM stimuli. An additional manipulation was to position either the first, third, or fifth component at the characteristic frequency (CF). This allowed the study of the basilar-membrane response to off-CF stimuli. CF suppression was as high as 35 dB for two-tone combinations, while for equal-amplitude stimulus components CF suppression never exceeded 20 dB. This latter case occurred for both two-tone stimuli where the suppressor was below CF and for multitone stimuli with the third component=CF. Suppression was least for the AM stimuli, including when the three AM components were equal. Maximum suppression was both level- and frequency dependent, and occurred for component frequency separations of 500 to 600 Hz. Suppression decreased for multicomponent stimuli with component frequency spacing greater than 600 Hz. Mutual suppression occurred whenever stimulus components were within the compressive region of the basilar membrane.

Representation of harmonic complex stimuli in the ventral cochlear nucleus of the chinchilla.

The representation of Schroeder-phase harmonic complex sounds in the ventral cochlear nucleus (VCN) of the anesthetized chinchilla was studied. Stimuli consisted of a series of harmonically related sinusoids, multiples of a fundamental frequency (f0), summed in either negative (-SCHR) or positive (+SCHR) Schroeder phase. Psychoacoustic experiments performed in humans by other investigators have revealed that masking effects of -SCHR stimuli are larger than those found using +SCHR stimuli as maskers. In our laboratory, basilar membrane measurements at the base of the chinchilla cochlea show that responses to -SCHR stimuli are less "peaked," or modulated, than responses to +SCHR stimuli. We also found that suppression of a characteristic-frequency (CF) tone by -SCHR stimuli is larger than that evoked by +SCHR stimuli. Rate-intensity functions display higher firing rates in responses to -SCHR stimuli than in those produced by +SCHR stimuli. Firing rates evoked by either -SCHR or +SCHR stimuli saturate at lower values than those obtained in responses to CF tones. Rate and synchrony suppressions by -SCHR stimuli were larger than those evoked by +SCHR stimuli. Auditory nerve fiber responses to Schroeder complex stimuli share most of the properties of VCN responses, indicating little additional processing by the VCN.

F-actin cleavage in apoptotic outer hair cells in chinchilla cochleas exposed to intense noise

Apoptosis is an active cell death pathway involved in a variety of pathological conditions, including noise-induced outer hair cell (OHC) death. During this process, the cytoskeletal proteins have been found to be either damaged and/or enzymatically disassembled in several cell types, leading to formation of apoptotic manifestations. This study was designed to examine the cleavage of filamentous actin (F-actin), an important cytoskeletal protein, in the cochlear OHCs after noise exposure. Chinchillas were exposed to a 4 kHz narrow band noise at 106 dB SPL for 1 h and cochleas were either collected immediately or 3 h after the noise exposure. The organs of Corti were double-stained using FITC-labeled phalloidin for F-actin and propidium iodide for OHC nuclei. The effect of noise on F-actin and nuclei was examined by confocal microscopy. The result showed that the fluorescence associated with F-actin was decreased in the OHCs possessing condensed nuclei, but remained unchanged in the OHCs with swollen nuclei. The change in F-actin labeling occurred coordinately with the changes in nuclear morphology of apoptotic cells and was prevented by administration of caspase-3 inhibitor (Z-DEVD-FMK). The results of this study indicate that F-actin cleavage is an important early cellular event in apoptotic development in OHCs following exposure to traumatic noise.

Proteoglycan arrays in the cochlear basement membrane

Indirect immunofluorescence and transmission electron microscopy were used to investigate the composition and assembly of proteoglycans in the basement membranes of the spiral limbus, basilar membrane, spiral ligament, Reissner’s membrane, myelinated nerve fibers, and blood capillaries of the spiral ligament and stria vascularis in the chinchilla cochlea. Four types of basement membrane components: laminin, entactin/nidogen, type IV collagen and heparan sulfate proteoglycans were immunolocalized in all basement membranes in association with heparan sulfate proteoglycans. β 1 and α 1 integrin subunits were also detected along these basement membranes. The concentration of the basement membrane-associated proteins and integrin subunits differed according to the adjacent cell type. Electron microscopy showed that all basement membranes, with exception of those of stria vascularis, consist of two layers: lamina lucida and lamina densa. In the stria vascularis only a homogeneous lamina densa was observed. Cuprolinic blue treatment revealed heterogeneity in the ultrastructure and arrangement of proteoglycans in the cochlear basement membranes. Proteoglycans of the subepithelial basement membrane in the spiral limbus and spiral ligament formed quasi-regular, linear arrays within the lamina lucida, or were located at both sides of the lamina densa in the basilar membrane and Reissner’s membrane. In the basement membranes of nerve fibers, and capillaries in the spiral ligament and stria vascularis, proteoglycans were scattered throughout these basement membranes, but showed different concentration and ultrastructural appearance, which may be related to different filtration and mechanical properties. In the basilar membrane, PGs were located above and below the lamina densa. An additional layer of PGs below the lamina densa may function as increased mechanical support of organ of Corti by its interaction with underlying fibrillar collagen layer. In the stria vascularis capillaries, PGs were stained considerably less with Cuprolinic blue and were scattered through the lamina densa of the basement membrane compared to capillaries of spiral ligament. This observation is compatible with a higher permeability of the strial capillaries.

Growth of masker level with signal level as a function of preceding stimulation

Some recent data suggest that stimulating the auditory system with sound may turn down active processing. The basic paradigm used to show this is to measure the level of a notched-noise, long-duration masker required to just mask a short-duration tone that occurs near masker onset. The preceding stimulation has been a precursor that was either a fixed-level broadband noise [Strickland, J. Acoust. Soc. Am. (in press)], or an extension of the notched-noise masker, which varied in level [Strickland, J. Acoust. Soc. Am. 107, 2915 (2000)]. The present experiment was done to examine the effects of a notched-noise precursor that had the same frequency characteristics as the masker, but which would be fixed in level. The signal was a 10-ms, 4-kHz tone. The notched-noise masker was 200 ms, and the precursor was 205 ms. For a given precursor level, the growth of masker level with signal level was determined. This data was used to calculate the effective input–output functions of the signal and the masker. These functions will be compared to those published physiological measures from the chinchilla cochlea. [Work supported by NIH (NIDCD).]

Effects of Common Topical Otic Preparations on the Morphology of Isolated Cochlear Outer Hair Cells

Otic drops are commonly used not only for otitis externa but also for otorrhea in the presence of tympanostomy tube or tympanic membrane perforation. Many studies have demonstrated the ototoxicity of common otic preparations such as Cortisporin® otic drops. Recent studies have suggested the use of fluoroquinolone antibiotic drops as an alternative owing to their excellent antimicrobial coverage and no ototoxic effect. The purpose of this study was to assess the relative ototoxicity of four common otic preparations by direct exposure to isolated cochlear outer hair cells (OHCs). OHCs from adult chinchilla cochlea were exposed to standard bathing solution (control), Cortisporin, Cipro HC®, Ciloxan®, and Floxin®. The cells were observed using an inverted microscope, and the images recorded in digital still-frame and video, and analyzed on the Image Pro-Plus® 3.0 program. As measured by time to cell death and change in morphology of OHCs, Cortisporin was most toxic to OHCs. Among the fluoroquinolone drops, Floxin was more toxic than Ciloxan or Cipro HC.

Basilar membrane responses to broadband stimuli.

Basilar membrane (BM) responses to two types of broadband stimuli-clicks and Schroeder-phase complexes--were recorded at several sites at the base of the chinchilla cochlea. Recording sites (characteristic frequency, CF, in the range of 5.5-18 kHz) span the 1-4-mm basal region of the basilar membrane. BM responses to clicks consisted of undamped oscillations with instantaneous frequency that increased over time until it reached a value around CF. The time constant of this glide is CF dependent. Throughout the entire region under study, BM vibration exceeded umbo motion by up to 60 dB. Nonlinear properties of BM responses to clicks resemble those found in the more studied 8-10-kHz region. Amplitude spectra of Schroeder-phase complex stimuli, which consist of a series of sinusoidal components summed in negative (-SCHR) and positive Schroeder phase (+SCHR), are flat. The envelope of BM responses to +SCHR stimuli contains valleys, or dips, that are wider than those found in responses to the -SCHR stimuli. Hence, BM responses to the former stimuli are "peakier" than responses to the latter. Differences in response waveforms are less obvious in linear cochleae. Suppression of a near-CF tone by -SCHR stimuli was larger than that evoked by +SCHR stimuli.

Helical structure of hair cell stereocilia tip links in the chinchilla cochlea.

Outer-hair-cell stereocilia tip-link structure in the chinchilla cochlea was studied by transmission electron microscopy using tannic acid and Ruthenium red/ Alcian blue histochemical procedures. Tannic acid and Ruthenium red/Alcian blue treatments showed the tip link as a compact strand of filaments 9-12 nm thick. Fourier analysis of tip-link images showed that the strand is a three-start helical bundle of fine, coiled filaments which had an axial period of 22.5+/-1.5 nm. Each of three coiled filaments in the strand showed globular structures, 4.3+/-0.3 nm in diameter. The globular structures may correspond to individual protein subunits or they may be repeating identical domains of one polypeptide. The three filaments of the helical array may provide a rigidity to the tip link during stereocilia deflections. Alternatively, changes in the subunit or domain structure of each filament may result in a lengthening or shortening of the tip-link strand.

Study of mechanical motions in the basal region of the chinchilla cochlea.

Measurements from the 1-4-mm basal region of the chinchilla cochlea indicate the basilar membrane in the hook region (12-18 kHz) vibrates essentially as it does more apically, in the 5-9-kHz region. That is, a compressive nonlinearity in the region of the characteristic frequency, amplitude-dependent phase changes, and a gain relative to stapes motion that can attain nearly 10,000 at low levels. The displacement at threshold for auditory-nerve fibers in this region (20 dB SPL) was approximately 2 nm. Measurements were made at several locations in individual animals in the longitudinal and radial directions. The results indicate that there is little variability in the phase of motion radially and no indication of higher-order modes of vibration. The data from the longitudinal studies indicate that there is a shift in the location of the maximum with increasing stimulus levels toward the base. The cochlear amplifier extends over a 2-3-mm region around the location of the characteristic frequency.

The phases of auditory-nerve fiber responses to tones: Dependence on stimulus frequency and intensity

As stimulus intensity increases, the phases of responses to tones of squirrel monkey auditory-nerve fibers [ANFs; Anderson et al., J. Acoust. Soc. Am. 49, 1131–1139 (1971)] and guinea pig inner hair cells [IHCs; Dallos, Hearing Res. 22, 185–198 (1986)] undergo lags and leads, respectively, at frequencies lower and higher than the characteristic frequency (CF) and remain relatively constant at frequencies near CF. A similar pattern holds for basal sites of the basilar membrane (BM) in several species. In contrast, tectorial-membrane (TM) vibrations at apical sites of the chinchilla cochlea exhibit phase shifts of opposite polarity [Rhode and Cooper, Aud. Neurosci. 3, 101–121 (1996)]. To explore this inconsistency between neural and mechanical behavior at apical cochlear regions, we investigated the phases of responses to tones of ANFs recorded in deeply anesthetized chinchillas. For stimulus intensities lower than 70 dB SPL, responses of most ANFs (regardless of CF) exhibited phase shifts similar to those observed in squirrel monkey ANFs, guinea pig IHCs, and basal BM sites in several species. Thus, the inconsistency between neural and TM mechanical data cannot be ascribed to species differences or a dependence of phase behavior on CF. [Work supported by NIH Grant DC-00419.]

Recovery of kainic acid excitotoxicity in chinchilla cochlea.

The present study examines the recovery of the inner hair cell (IHC)/auditory nerve synapse following cochlear excitotoxicity induced by kainic acid (KA). Three hours after KA treatment, there was massive swelling of type I afferent endings under the IHCs. Five to ten days later, the pattern of IHC innervation appeared to be normal. Distortion-product otoacoustic emissions were normal during the whole experiment. The amplitude of the auditory nerve compound action potential (CAP) was significantly reduced immediately after KA treatment and then recovered over a 30-day period. However, it only took five days for the evoked response from the inferior colliculus (IC) to recover from a substantial depression. In contrast to amplitudes, thresholds for the CAP and IC recovered at the same rate and returned to normal within 5 days after KA. Single auditory nerve fibers were also assessed at various times after the KA treatment. Ten days after KA, these fibers had almost normal thresholds, tuning, spontaneous, and driven discharge rates. The results indicate that (1) excitotoxically damaged cochlear afferent neurons can rapidly regenerate and establish viable synapses with the IHCs, and (2) the central auditory system recovers more rapidly than the periphery.

Ablation of inner hair cells by carboplatin alters cells in the medial K + flow route and disrupts tectorial membrane

The thesis that K + effluxing from inner hair cells (IHCs) cycles medially back to endolymph through inner sulcus and interdental cells (IDCs) was tested by comparing control chinchilla cochleas with those in which IHCs were selectively destroyed by carboplatin. By light microscopy inner sulcus cells appeared tall and nearly empty in control ears, but 4 months after the carboplatin treatment many showed vacuolization and shrinkage. Inner pillar cells also consistently developed abnormal vacuoles after carboplatin treatment. Control cochleas exhibited lateral columns and central clusters of IDCs which at their apex possessed expanded presumably hydrated phalanges. Four months after carboplatin, the IDC epithelium enclosed empty looking spaces and the apical phalangeal compartment collapsed into a thin, apparently dehydrated layer. This alteration was accompanied by changes in the tectorial membrane (TM) whereby the membrane’s limbal zone thickened progressively to form a tall hollow mound in advanced lesions. The clear spaces in the epithelium and collapse of the phalanges are thought to reflect diminished flow of ions and fluid through IDCs. The accumulation of limbal TM supports the premise that IDCs secrete macromolecules for TM turnover as well as ions and fluid for promoting lateral migration of its precursor constituents. Occurring after ablation of IHCs by carboplatin, the changes in inner pillar, inner sulcus and IDCs and limbal TM can be viewed as a secondary effect of the interrupted ion efflux from IHCs and as further evidence that this effluent follows a medial route.

Immunolocalization of tenascin in the chinchilla inner ear

Tenascin was immunolocalized in the chinchilla cochlea and vestibular system to better understand the functional morphology of the inner ear. Inner ear tissues were fixed with acetone, decalcified and cryosectioned. Indirect immunofluorescence, using antibodies directed against human tenascin epitopes, were used to detect tenascin. As a positive control, tenascin immunoreactivity was found in kidney, cortical mesangial cells and the extracellular matrix of glomeruli and medullary tubule interstitial spaces, concurring with previously reported results. In the cochlea, tenascin immunoreactivity was present in osteocytes, the mesothelial cells underlying the basilar membrane (BM) and within the fibrous matrix of the BM. Greater reactivity was observed in the mesothelial cells than in the fibrous matrix of the BM. In the vestibular system, tenascin immunoreactivity formed a diffuse band directly beneath the basal lamina of the ampullary and otoconial organs. Tenascin immunoreactivity was also observed in cup-shaped regions between the type I vestibular hair cells and their surrounding VIII nerve calyces in the ampullary and otoconial organs. This is the first report of the anatomical distribution of tenascin in the adult, mammalian inner ear, other than our previously published abstract P.A. Santi and D. Swartz, Soc. Neurosci. Abstr. 23 (1997) 731.