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Hearing impairment, cochlear morphology, and peroxynitrite (ONOO − ) formation in adult and aging NOS II knockout mice
Abstract
Conclusion:
Nitric oxide synthase (NOS) II induction is a protective mechanism against age-related degeneration of the cochlea.
Objectives:
An induction of NOS II has been described in different inner ear pathologies. The objective was to examine the role of NOS II in age-related degeneration of the cochlea.
Methods:
The hearing ability in adult and aging NOS II knockout mice (KO) and their wildtype (WT) littermates was explored via auditory brainstem response (ABR) measurements. Inner ear morphological differences were studied with scanning electron microscopy (SEM). Immunohistochemistry was used to examine the induction of NOS II in the inner ear of aging WT mice. Expression of nitrotyrosin, a marker protein for the reactive oxygen species peroxynitrite, was compared between KO and WT mice using immunohistochemistry.
Results:
Adult KO mice exhibited a mild hearing impairment. WT mice showed an induction of NOS II after 6 months of age. Age-related hearing deterioration was accelerated in KO mice, which was accompanied by increased nitrotyrosin formation and outer hair cell loss.
... However, overproduction of free radicals disturbs the normal redox state of cells, which leads to oxidative stress and toxic effects. Of these reactive species, peroxynitrite (ONOO − ) has generated considerable interest in biological fields because of the identification that toxicity of nitric oxide, a key cellular signaling molecule, is mainly attributable to the production of ONOO − rather than itself [1][2][3]. ONOO − , the product of a reaction between nitric oxide and superoxide, is a potent and versatile oxidant implicated in a number of pathophysiological processes [4,5]. It has been documented Abstract Peroxynitrite (ONOO − ) is a potent and versatile oxidant implicated in a number of pathophysiological processes. ...
... This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO − could result in a base-to-apex gradient injury of 1 3 that ONOO − is able to diffuse freely across phospholipid membrane bilayers to react with a wide variety of molecular targets. However, responses evoked by exposure of cells to ONOO − depend on the environment and cell types [2,6]. ...
... In this work, we found that ONOO − -induced HC loss varied along the length of cochlea, which was started from the basal turn of cochlea and progressed to the apex eventually (Figs. 1, 2a-d). This appearance of a base-to-apex gradient injury of cochlear HCs indicates that the extent of ONOO − -elicited cytotoxicity is closely related to the anatomical position, which is an typical lesion pattern of cochlea under the most situations of oxidative stress [2]. Other studies have also reported the similar HC damage pattern in aminoglycosides-induced ototoxicity models [6,7]. ...
Peroxynitrite (ONOO⁻) is a potent and versatile oxidant implicated in a number of pathophysiological processes. The present study was designed to investigate the effect of ONOO⁻ on the cultured cochlear hair cells (HCs) of C57BL/6 mice in vitro as well as the possible mechanism underlying the action of such an oxidative stress. The in vitro primary cultured cochlear HCs were subjected to different concentrations of ONOO⁻, then, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy (TEM), the apoptosis was determined by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay, the mRNA expressions of Caspase-3, Caspase-8, Caspase-9, Apaf1, Bcl-2, and Bax were analyzed by RT-PCR, and the protein expressions of Caspase-3 and AIF were assessed by immunofluorescence. This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO⁻ could result in a base-to-apex gradient injury of HCs in a concentration-dependent manner. Furthermore, ONOO⁻ led to much more losses of outer hair cells than inner hair cells mainly through the induction of apoptosis of HCs as evidenced by TEM and TUNEL assays. The mRNA expressions of Caspase-8, Caspase-9, Apaf1, and Bax were increased and, meanwhile, the mRNA expression of Bcl-2 was decreased in response to ONOO⁻ treatment. Of interesting, the expression of Caspase-3 had no significant change, whereas, the expression alteration of AIF was observed. These results suggested that ONOO⁻ can effectively damage the survival of cochlear HCs via triggering the apoptotic pathway. The findings from this work suggest that ONOO⁻-induced apoptosis is mediated, at least in part, via a Caspase-independent pathway in cochlear HCs.
... Oxidative stress is the disturbance in the balance between the generation of free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), and antioxidant defenses, and can cause oxidative damage to diverse cellular components, such as membranes, proteins, and DNA. Among RNS molecules, peroxynitrite, a product of superoxide anion and nitric oxide, is one of the most prominent one (Liu et al., 2011(Liu et al., , 2012Labbe et al., 2016;Cao et al., 2017;Ramdial et al., 2017). Peroxynitrite can oxidize a wide variety of biomolecules, which, in turn, modulates cell signal transduction pathways, interferes with mitochondrial function, impairs DNA, and finally mediates necrosis and apoptosis in different cell types (Korkmaz et al., 2009). ...
Glutathione peroxidase 1 (GPX1) is a crucial antioxidant enzyme that prevented the harmful accumulation of intra-cellular hydrogen peroxide. GPX1 might contribute in limiting cochlear damages associated with aging or acoustic overexposure, but the function of GPX1 in the inner ear remains unclear. The present study was designed to investigate the effect of GPX1 on cochlear spiral ganglion neurons (SGNs) against oxidative stress induced by peroxynitrite, a versatile oxidant generated by the reaction of superoxide anion and nitric oxide. Here, we first found that the expression of GPX1 in cultured SGNs was downregulated after peroxynitrite exposure. Then, the GPX1 mimic ebselen and the gpx1 knockout (gpx1–/–) mice were used to investigate the role of GPX1 in SGNs treated with peroxynitrite. The pretreatment with ebselen significantly increased the survived SGN numbers, inhibited the apoptosis, and enhanced the expression of 4-HNE in the cultured SGNs of peroxynitrite + ebselen group compared with the peroxynitrite-only group. On the contrary, remarkably less survived SGNs, more apoptotic SGNs, and the higher expression level of 4-HNE were detected in the peroxynitrite + gpx1–/– group compared with the peroxynitrite-only group. Furthermore, rescue experiments with antioxidant N-acetylcysteine (NAC) showed that the expression of 4-HNE and the apoptosis in SGNs were significantly decreased, while the number of surviving SGNs was increased in peroxynitrite + NAC group compared the peroxynitrite-only group and in peroxynitrite + gpx1–/– + NAC group vs. peroxynitrite + gpx1–/– group. Finally, mechanistic studies showed that the activation of nuclear factor-kappa B (NF-κB) was involved in the SGNs damage caused by peroxynitrite and that GPX1 protected SGNs against peroxynitrite-induced damage, at least in part, via blocking the NF-κB pathway activation. Collectively, our findings suggest that GPX1 might serve as a new target for the prevention of nitrogen radical-induced SGNs damage and hearing loss.
... First, there is a negative correlation between the degree of ARHL and cochlear cGMP concentrations in CBA/J mice (310). Second, NOSII knockout mice have a mild hearing impairment with loss of OHCs, with an acceleration of ARHL in NOSII-deficient mice (159). Third, NO has the ability to regulate apoptosis of inflammatory cells (133). ...
Although hearing loss is the most common sensory deficit in western societies, there are no successful pharmacological treatments for this disorder. Recent experiments have demonstrated that manipulation of intracellular cyclic guanosine monophosphate (cGMP) concentrations can have both beneficial and harmful effects on hearing. In this review, we will examine the role of cGMP as a key second messenger involved in many aspects of cochlear function, and discuss the known functions of downstream effectors of cGMP in sound processing. The nitric oxide-stimulated soluble guanylyl cyclase system (sGC) and the two natriuretic peptide-stimulated particulate GCs (pGCs) will be more extensively covered because they have been studied most thoroughly. The cochlear GC systems are attractive targets for medical interventions that improve hearing while simultaneously representing an under investigated source of sensorineural hearing loss.
Peroxynitrite (ONOO−), one of the reactive oxygen/nitrogen species (ROS/RNS) found in vivo, plays crucial roles in many physiological and pathological processes. The ability to selectively and sensitively determine ONOO− in vivo is important for the understanding of its biological roles. Thus, by utilizing the excellent chemical stability and photostability, high luminescence efficiency, and long luminescence lifetime of iridium complex, we developed a novel near-infrared (NIR) phosphorescent iridium(III) complex (FNO2) probe to detect ONOO− within seconds. The probe FNO2 had better selectivity towards ONOO− over other interfering-biomolecules, including O2− and ClO−. Moreover, it possessed a long luminescence lifetime, that was able to successfully eliminate the interference from background fluorescence in vitro (simulated by Rhodamine B) in time-resolved emission spectra. Finally, in addition to its low cytotoxicity, the probe FNO2 showed emission wavelengths in the NIR region and was able to specifically sense ONOO− induced in living cells and inflamed mouse models.
Nitric oxide (NO) is a versatile molecule that plays key roles in the development and survival of mammalian species by endowing brain neuronal networks with the ability to make continual adjustments to function in response to moment‐to‐moment changes in physiological input. Here, we summarize the progress in the field and argue that NO‐synthetizing neurons and NO signalling in the brain provide a core hub for integrating sensory‐ and homeostatic‐related cues, control key bodily functions, and provide a potential target for new therapeutic opportunities against several neuroendocrine and behavioural abnormalities.
Deficiency of cystathionine β-synthase (CBS) activity is the most common cause of increased homocysteine (Hcy). However, until now the underlying mechanisms why CBS activity decreased still remain unresolved. The goal of this study was to explore the contribution of nitrative stress to deficiency of CBS activity, and further identify the possible nitration sites of CBS protein. Results showed that in elderly people, there was an increased nitrative stress level, which was relative to elevated Hcy level. In natural aging rats and diet-induced hyperhomocysteinemia (HHcy) rats, the levels of Hcy and nitrative stress were both elevated, and interestingly, pretreatment with peroxynitrite (ONOO(-)) scavenger FeTMPyP ameliorated the elevation of Hcy as well as nitrative stress. Further experiments showed the reduction of CBS bioactivity and elevation of CBS nitration in two rats models were both reversed by FeTMPyP pretreatment. In vitro, replacement of tyrosine (Tyr, Y) residue (Tyr(163), Tyr(223), Tyr(381), Tyr(518)) in CBS with alanine (Ala, A) abolished the Hcy-mediated CBS inactivation. These results highlighted that deficiency of CBS activity was correlated with the nitration of CBS at Tyr(163), Tyr(223), Tyr(381) and Tyr(518), which may play a mutual role in the progression of HHcy. This discovery may shed a novel light on the pathogenesis of HHcy and provide a possible gene therapy target to HHcy.
Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality.
The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.
Presbycusis is a complex of high frequency hearing loss and disproportionate loss of speech discrimination that is seen concomitantly with physical signs of aging. Among the most extensively characterized strains of mice that show an early hearing loss is the C57B16/J strain, a strain that shows early onset of high frequency hearing loss at age 6 months and complete hearing loss by 1 year of age. The histopathology of this strain consists of loss of hair cells and spiral ganglion neurons in the basal turn, with a progression of loss of hair cells and ganglion neurons towards the apical portion of the cochlea as the animal ages. The process of aging has been extensively studied and although details differ in various organisms the consensus today is that oxidative stress, i.e. free radical-mediated tissue damage, is one of the core mechanisms of aging. Aerobic metabolism results in the creation of hydrogen peroxide and reactive oxygen species. These are normally detoxified by a variety of enzymes and free r...
The exact distribution of nitric oxide-synthases (NOS) and the NO-target enzyme soluble guanylyl cyclase (sGC) in the cochlea and vestibular organ is an issue of current discussion. The existence of NOS-isoforms in the cochlea of the guinea pig has been described recently, while information about the vestibular system are still rare and non-satisfying. In order to gain more information, immunostaining was performed, using specific antibodies to NOS I–III and to sGC, on paraffin sections of complete temporal bones from mice. NOS III could be detected in cochlea and vestibular ganglion cells, in nerve fibres, in outer hair cells of the cochlear and in the sensory epithelium of the maculae. Also, the spiral ligament and the limbus epithelium was positive to NOS III. NOS I was found in the sensory epithelium of the maculae and cristae ampullares, outer and inner hair cells of the cochlea, in nerve fibres and in ganglion cells. In contrast to that NOS II could not be detected at all. Furthermore, a strong NOS I immunoreaction was displayed on the endosteum of the bone, while the periosteum was lacking of NOS. NOS detection was accompanied by immunoreactivity to sGC. The findings imply that NOS I and III-generated NO is involved in neurotransmission and other regulative processes in the vestibulocochlear system.
Nitric oxide synthase (NOS) activity and NADPH diaphorase staining has previously been reported in mammalian cochlea. Here we demonstrate immunoreactivity for neuronal-type NOS I and endothelial-type NOS III in the cochlea of the guinea pig. NOS I immunoreactivity was seen in inner and outer hair cells, and spiral ganglion cells. Staining for NOS I was also shown in basal and intermediate cells of the stria vascularis, spiral ligament cells, and the media of vessels near the modiolus. An antibody to NOS III stained primarily vascular endothelial cells. Some NOS III immunoreactivity was also detected in spiral ganglion cells. An antibody to the inducible-type NOS II did not stain any structure of the guinea pig cochlea, suggesting that this isoform is not expressed under physiological conditions. Nitric oxide produced by NOS I and/or NOS III may act as a neuromodulator in the organ of Corti and could also play a role as a regulator of cochlear blood flow.
Immunohistochemical investigations of the guinea pig cochlea, using a specific antibody to the inducible isoform of NO synthase (iNOS/NOS II), have been performed 3 weeks after closure of the right endolymphatic duct (n=7). Endolymphatic hydrops, the morphological substrate of Meniere’s disease, became evident by distension of the Reissner’s membrane. iNOS expression could be noted in endothelium, spiral ganglion cells, in nerve fibers, in supporting cells of the organ of Corti and cells of the spiral ligament. Temporal bones of non-operated controls (n=6) as well as of sham-operated animals (n=3) did not show structures positive to iNOS. These findings imply that iNOS-generated NO could be involved in the pathophysiology of cochlear dysfunction in Meniere’s disease.
Tuning curves were obtained from 100 to 150 auditory-nerve fibers spanning the range of characteristic frequencies (CFs) in each of eight cases of permanent noise-induced and three cases of permanent kanamycin-induced threshold shift. In each ear, from one to six neurons were intracellularly labeled with horseradish peroxidase. Locating the labeled terminals in plastic-embedded surface preparations of the cochlea enabled us to accurately correlate particular tuning-curve abnormalities with the condition of the sensory cells generating them. The correlations between structural and functional changes suggest that a normal tuning-curve tip requires that the stereocilia on both the IHCs and OHCs (especially those from the first row) be normal. Selective damage to the OHCs is associated with elevation of the tips and hypersensitivity of the tuning-curve tails. This tuning-curve pattern also originates from cochlear regions at the basal border of hair cell lesions where the local hair cells (and their stereocilia) appear completely normal at the light-microscopic level. Total destruction of the OHCs in a region in which the IHCs appear normal (as can happen in cases of kanamycin poisoning) is associated with bowl-shaped tuning curves which appear to lack a tip. Combined damage to the IHCs and OHCs (as typically happens in cases of acoustic trauma) is invariably associated with elevation of both tips and tails on the tuning curve. A framework for the interpretation of the results is suggested in which the activity of the OHCs is transmitted via the tectorial membrane to the tall row of stereocilia on the IHCs.
A survey of the temporal bone collection at the Massachusetts Eye and Ear Infirmary reveals 21 cases that meet the criterion for the clinical diagnosis of presbycusis. It is evident that the previously advanced concept of four predominant pathologic types of presbycusis is valid, these being sensory, neural, strial, and cochlear conductive. An abrupt high-tone loss signals sensory presbycusis, a flat threshold pattern is indicative of strial presbycusis, and loss of word discrimination is characteristic of neural presbycusis. When the increments of threshold loss present a gradually decreasing linear distribution pattern on the audiometric scale and have no pathologic correlate, it is speculated that the hearing loss is caused by alterations in the physical characteristics of the cochlear duct, and the loss is identified as cochlear conductive presbycusis. It is clear that many individual cases do not separate into a specific type but have mixtures of these pathologic types and are termed mixed presbycusis. About 25% of all cases of presbycusis show none of the above characteristics and are classified as indeterminate presbycusis.
Extended high-frequency (EHF) hearing was studied in children with and without histories of chronic or recurrent otitis media (OM). The EHF thresholds were found to have good test-retest repeatability. Children with OM histories had poorer EHF hearing than children without OM histories. The EHF hearing in OM children appeared to be related to OM severity. Children with residual tympanometric abnormalities had poorer EHF hearing than OM children with normal middle ear function. The results suggest evidence for middle ear and inner ear components of EHF hearing losses in children with OM.
The age-related induction of inducible nitric synthase (iNOS) and apoptotic cell death in spiral ganglion cells (SGCs) of ddy strain mice were studied with immunohistochemical method and TdT-mediated dUTP-biotin nick end labeling (TUNEL), respectively. A large amount of iNOS was expressed in SGCs of 18- to 24-month-old mice, but not in those of the mice less than 12 months of age. Moreover, these mice were accompanied by a great rise in auditory brainstem response threshold as well as a great decrease in the number of SGCs that seemed to be due to preceding cell death of the cells. However, we were unable to find apoptotic TUNEL-positive cells in the spiral ganglions. This was assumed to be due to a very short clearance time of the dead cell bodies of less than one hour. As has been known, NO produced by iNOS can implicate in causes for either protection of cells from peroxidation and cell death. In the present study, therefore, the steep augmentation of iNOS in the SGCs of senescent mice implies that the iNOS initially induced to protect SGCs from the cytotoxicity of cellular peroxidation eventually contribute to the cell death of SGCs themselves.
Glutathione and glutathione-related enzymes protect against oxidative (free radical) cell injury. This study presents basic information on this antioxidant system in inner ear tissues and preliminary results of the influence of age, ototoxic drugs and noise. These conditions affect inner ear function, possibly through free radicals, and are therefore expected to affect cellular defense mechanisms. In 24-month old Fischer 344 rats, a standard model for aging, glutathione levels were significantly decreased in the auditory nerve by 86% as compared to 3-month old rats but remained unchanged in other cochlear tissues. In guinea pig, the common model for drug- and noise-induced trauma, glutathione levels in the cochlear sensory epithelium were about 8-fold higher (223 +/- 35 nmol glutathione/mg protein) than in the rat. Cochlear glutathione S-transferase and glutathione reductase activities were similar between the two species, whereas selenium-independent glutathione peroxidase was strikingly lower in guinea pig than in rat (9 +/- 3 nmol vs. 161 +/- 84 nmol glutathione converted/mg protein/min). Cisplatin treatment of guinea pigs (56 dB threshold shift at 18 kHz) significantly lowered cochlear glutathione levels by 65% and glutathione S-transferase activity by 44%. Gentamicin treatment (80 dB threshold shift at 18 kHz) and noise exposure (43 dB threshold shift at 18 kHz) did not affect glutathione at the tissue level. These results demonstrate species differences in cochlear glutathione and glutathione-related enzymes. The antioxidant system is sensitive towards environmental influences as seen for age and cisplatin. For gentamicin and noise trauma, whole tissue glutathione and enzyme levels do not correlate with functional damage. This indicates that glutathione homeostasis is largely maintained in the cochlea and that biochemical changes, if they occur under these conditions, may be limited to specific cells.
Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.
Since NO is believed to be involved in cochlear physiology, presence of the constitutive isoforms of nitric oxide synthase (NOS), and the target enzyme of NO, soluble guanylyl cyclase (sGC) in structures of the mammalian cochlea have been demonstrated. To date, no reports have been published regarding the detection of the inducible isoform (NOS II) in the cochlea. In order to show the capability of iNOS expression in cochlear tissue, a mixture of proinflammatory bacterial lipopolysaccharides (LPS) and tumor necrosis factor alpha (TNF-alpha) was injected into the tympanic cavity of guinea pigs, vs. saline-solution as control. Paraffin sections of LPS/TNF-alpha treated and saline-treated cochleae (6 h) were examined immunohistochemically with specific antibodies to neuronal, endothelial and inducible NOS and to sGC. Initiated expression of iNOS in the cochlea was observed in the wall of blood vessels of the spiral ligament (SL) and the modiolus, in supporting cells of the organ of Corti, in the limbus, in nerve fibers and in a part of the perikarya of the spiral ganglion after LPS/TNFalpha-treatment. iNOS was not detected in saline-treated control tissue. Expression of both constitutive NOS-isoforms (endothelial and neuronal NOS) and of sGC showed no significant differences in both experimental groups. Endothelial eNOS and neuronal bNOS were detected co-localized in ganglion cells, in nerve fibers, in cells of the SL and in supporting cells of the organ of Corti, but not in sensory cells. Strong labeling for bNOS became evident in the endosteum of the cochlea, while in the endothelium of blood vessels and in the epithelium of the limbus only eNOS could be labeled. sGC could be detected in SL, in supporting and sensory cells of the organ of Corti, in nerve fibers, ganglion cells, in the wall of blood vessels and in the limbus-epithelium. While small amounts of NO, generated by bNOS and eNOS, seem to support the cochlear blood flow and auditory function as well as neurotransmission, high amounts of iNOS-generated NO could have dysregulative and neurotoxic effects on the inner ear during bacterial and viral infections of the middle and inner ear.
The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.
The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the cochlea of the guinea pig could elicit formation of inducible nitric oxide synthase (iNOS). Immunohistochemical study revealed that immunoreactivity to iNOS was seen below outer hair cells representing nerve fibers and synaptic nerve endings. iNOS-staining could also be observed in phalangeal dendrites of Deiter's cells pointing to the cuticular membrane, Hensen's cells and on stria vascularis 48 h after inoculation with LPS. Immunohistochemical investigation with a specific anti-nitrotyrosine antibody also revealed intense immunoreactivity identical to that of iNOS, suggesting formation of peroxynitrite in the organ of Corti by the reaction of NO with O(2)(-). On the basis of these findings, it can be concluded that NO together with O(2)(-), which form the more reactive peroxynitrite, are the most important pathogenic agents in LPS-induced damage of cochlea in the guinea pig.
An anatomically based frequency-place map was created for the mouse using C57BL/CBA F1 hybrids by matching noise-induced lesions in the organ of Corti with permanent hearing losses as determined by auditory brainstem response (ABR) thresholds. Twenty-six mice developed 'notched' ABR threshold shifts after exposure to an octave band of noise with a center frequency of 2 kHz at 120 dB SPL for 24 h, 4 kHz at 110 dB SPL for 4 h or 8 kHz at 100 dB SPL for 1 or 2 h. ABR thresholds were determined at several intervals post-exposure until thresholds stabilized (14-27 days). Once thresholds had stabilized, the mice were killed and their cochleas were prepared for phase-contrast microscopic examination as plastic-embedded flat preparations. Hair cell loss, stereocilia damage, and myelinated nerve fiber degeneration as a function of percentage distance from the cochlear apex were determined. Frequency-position matches could be made for 22 of the 26 mice by correlating areas of hair cell loss/stereocilia damage with permanent changes in ABR thresholds. These frequency-position data were fitted with the equation: % Distance from apex=56.6 log (f(Hz))-179.1; r(2)=0.810. This frequency-place function agrees well with Ehret's (1975) theoretical function based on critical bands and masked auditory thresholds.
Low NO concentrations synthesized by constitutively expressed NO synthases act on several signaling pathways activating transcription factors (TF), such as NF-kappaB or AP-1, and thereby influence gene expression. In contrast, during inflammatory reactions the inducible NO synthase produces NO for prolonged periods of time. The resulting nitrosative stress directly affects redox-sensitive TF like NF-kappaB, AP-1, Oct-1, c-Myb, or zinc finger-containing TF, but also additional mechanisms have been identified. Nitrosative stress in some cases induces expression of TF (AP-1, p53), indirectly modulates activity or stability of TF (HIF-1, p53) or their inhibitors (NF-kappaB), or modulates accessibility of promoters via increased DNA methylation or histone deacetylation. Depending on the promoter the result is induced, increased, decreased or even totally inhibited expression of various target genes. In unstimulated cells nitrosative stress increases NF-kappaB- or AP-1-dependent transcription, while in activated cells nitrosative stress rather abolishes NF-kappaB- or AP-1-dependent transcription. Sometimes the oxygen concentration also is of prime importance, since under normoxic conditions nitrosative stress activates HIF-1-dependent transcription, while under hypoxic conditions nitrosative stress leads to inhibition of HIF-1-dependent transcription. This review summarizes what is known about effects of physiological NO levels as well as of nitrosative stress on transcription.
Free radical-derived reactive oxygen species (ROS) are constantly generated in most living tissue and can potentially damage DNA, proteins and lipids. "Oxidative stress" occurs if ROS reach abnormally high concentrations. Harman was the first to propose that the damaging effects of ROS may play a key role in the mechanism of aging. Genetic studies of such distantly related species as C. elegans, Drosophila melanogaster, and mice support this hypothesis. However, ROS are not only a cause of structural damage, but also physiologically important mediators in biological signaling processes. Abnormally high levels of ROS may therefore lead to dysregulation of redox-sensitive signaling pathways. The redox-sensitive targets in these pathways are often signaling proteins with redox-sensitive cysteine residues which are oxidized to sulfenic acid moieties and mixed disulfides, thereby altering the signaling function of the protein. Because the formation of these mixed disulfides can also occur through a prooxidative shift in the intracellular thiol/disulfide redox status (REDST), the respective signaling pathways respond not only to ROS but also to changes in REDST. Information about the concentration of ROS in living tissue is scarce, but aging-related changes in REDST are well documented. Several studies with cell cultures or experimental animals have shown that the oxidative shift in the intracellular glutathione REDST is typically associated with cellular dysfunction. Complementary studies in humans have shown that oxidative changes in the plasma (i.e., extracellular) REDST are correlated with aging-related pathophysiological processes. The available evidence suggests that these changes play a key role in various conditions which limit the human life span. Several attempts have been made to ameliorate the consequences of aging by thiol-containing antioxidants, but this approach requires a detailed knowledge of the effects of thiol-containing antioxidants on cysteine homeostasis, REDST, and redox-sensitive signaling pathways of the host.
Several strains of mice hear well initially but show progressive sensorineural hearing loss. Affected cochlear cell types include all those known to be affected in human age-related hearing loss (ARHL), or presbycusis. Thus these mice have been offered as models of human ARHL. At present, however, few mouse ARHL models are sufficiently well described to serve as the basis for specific hypotheses about human ARHL. We examined 1-month-old and 15-month-old 129S6/SvEv (129S6) mice and compared them with BALB/cJ and CBA/J mice. Age-related elevation of compound action potential thresholds was interpreted in the light of endocochlear potentials and changes in hair cells, afferent neurons, fibrocytes in spiral limbus and ligament, and supporting cells within the organ of Corti. Aging in 129S6 mice was associated with high-frequency hearing loss. Four components of age-related cochlear degeneration emerged from quantitative analyses, including 1) basal loss of outer hair cells; 2) basal loss of type IV fibrocytes in the spiral ligament; 3) apical loss of fibrocytes in spiral limbus, and 4) anomalies of supporting cells in the cochlear base. Although neuronal loss was not consistently found, two mice showed loss of afferent dendrites and cell bodies in the cochlear apex without inner hair cell loss. Despite multifaceted degeneration, hearing loss in 129S6 mice appears to be best explained by degenerative changes in outer hair cells and in the organ of Corti, conforming to human sensory ARHL. Age-related changes in the apical spiral limbus may promote pathology of the medial organ of Corti and eventual loss of afferent neurons, with possible implications for human neural ARHL.
The aim of this study was to investigate the involvement of oxidative stress and apoptosis in an animal model of Meniere's disease. Endolymphatic hydrops (ELH) is generally accepted as the decisive histological characteristic of Meniere's disease. Closure of the endolymphatic duct (Kimura's method) was used to induce endolymphatic hydrops in guinea pigs. Sham-operated animals served as controls. After 4 weeks the animals operated showed a significant elevation of the hearing thresholds as measured by audiometric brainstem responses (ABR) pre- and postoperatively. Immediately after the second ABR measurement, the animals were sacrificed for further immunohistological examinations of the inner ear with specific antibodies to active caspase-3 (cas-3) as a marker for apoptosis and antibodies to 8-isoprostane (8-iso) and nitrotyrosine (NT) as indicators of oxidative stress. Compared with the sham-operated controls, hydropic cochleae showed strong immunostaining for both oxidative stress markers in spiral ganglion cells, in the blood-vessels and fibrocytes of the lateral wall, as well as in supporting cells of the organ of Corti. Activation of cas-3 in spiral ganglion cells and the lateral wall was found exclusively in hydropic cochleae. Our findings suggest that oxidative stress is involved in the development of endolymphatic hydrops and may lead to cellular damage which induces apoptosis by activation of cas-3. Apoptotic cell death might contribute to the sensorineural hearing loss found in later stages of Meniere's disease.
The mammalian inner ear loses its sensory cells with advancing age, accompanied by a functional decrease in balance and hearing. This study investigates oxidant stress in the cochlea of aging male CBA/J mice. Glutathione-conjugated proteins, markers of H2O2-mediated oxidation, began to increase at 12 months of age; 4-hydroxynonenal and 3-nitrotyrosine, products of hydroxyl radical and peroxynitrite action, respectively, were elevated by 18 months. Immunoreactivity to these markers was stronger in the supporting cells (Deiters and pillar cells) than the sensory cells and appeared later (23 months) in spiral ganglion cells and in the stria vascularis and spiral ligament. Conversely, antioxidant proteins (AIF) and enzymes (SOD2) decreased by 18 months in the organ of Corti (including the sensory cells) and spiral ganglion cells but not in the stria vascularis. These results suggest the presence of different reactive oxygen species and differential time courses of oxidative changes in individual tissues of the aging cochlea. An imbalance of redox status may be a component of age-related hearing loss.
Mechanisms and biological consequences of nitrosative stress
- K D Kroncke