Electron microscopic study on inner ear barotrauma: with emphasis on the cochlear damage

In order to investigate inner ear barotrauma, guinea pigs were subjected to rapid decompression between 2 absolute pressure (ATA) and 1 ATA in a chamber. After pressure loading and observation for absence of Preyer's reflex, they were sacrificed immediately, 1 day, 1 week and 1 month later, respectively. Then, morphological changes of the organ of Corti and stria vascularis were studied under TEM and SEM. The immediate features noted were fracture of stereocilia with minimal intracellular changes. One day later, there was marked degeneration of outer hair cells and expansion of supporting cells. The damage to stereocilia clearly preceded morphological alterations within hair cell bodies and cannot be interpreted as arising secondary to hair cell degeneration. Most of outer hair cells eventually disappeared and were replaced by supporting cells. Inner hair cells degenerated slowly; 1 month later, some of them remained almost intact, despite disappearance of stereocilia. The continuity of reticular lamina was maintained not only immediately but also through the period of hair cell degeneration, thus preventing any leakage of endolymph into the organ of Corti. There was reversible dendritic swelling of inner hair cells immediately following the trauma. No changes of stria vascularis were observed over passage of time. The mechanism of hair cell damage due to inner ear barotrauma is presumed to be a deformity of the organ of Corti caused by pressure discrepancy between perilymph and endolymph resulting in an injury to stereocilia.     

Volumetric and dimensional analysis of the guinea pig inner ear

The objective of this study was to provide accurate volumetric data on the fluid spaces and soft tissue in the guinea pig inner ear by measuring all histologic serial sections by means of Metamorph Imaging Software at 400x to 1,000x magnification. The total endolymph volume of the inner ear was 4.691 mm3, of which 1.501 mm3 was in the cochlea, 3.090 mm3 in the vestibular labyrinth, and 0.100 mm3 in the endolymphatic duct and sac. The total perilymph volume was 15.938 mm3, of which 8.867 mm3 was in the cochlea and 7.071 mm3 in the vestibular labyrinth. The volume of the organ of Corti per millimeter length increased toward the apex, but the volumes of the stria vascularis, spiral ligament, and spiral limbus decreased. The volume of the macula utriculi was larger than that of the macula sacculi. The measurement of the luminal surface area of the stria vascularis was 3.944 mm2, and that of the vestibular dark cells was 5.772 mm2.     

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear

CONCLUSION: The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. OBJECTIVE: To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. MATERIAL AND METHODS: Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. RESULTS: Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.     

Detection of nitric oxide in the guinea pig inner ear, using a combination of aldehyde fixative and 4,5-diaminofluorescein diacetate

Localization of nitric oxide (NO) production sites in the inner ear of the guinea pig was investigated using a combination of glutaraldehyde fixative and a new fluorescence NO indicator. 4,5-diaminofluorescein diacetate (DAF-2DA). The cochlea and vestibular end organs were examined to locate NO production sites. The fluorescence persisted after glutaraldehyde fixation and embedding with water-soluble resin. NO production in the cochlea was observed in the outer and inner hair cells, nerve endings, nerve fibers and supporting cells of the organ of Corti, stria vascularis, spiral ligament, ganglion cells, etc. In the vestibular end organs, both type I and type II sensory cells, nerve fibers, blood vessels and dark cells displayed fluorescence. This localization was exactly identical to that of NO synthase. Thus, detection of intracellular NO production by using a combination of glutaraldehyde fixation and DAF-2DA is useful for examining the function of NO in cells, both in situ and in vivo.     

The cochlea of the spontaneously diabetic mouse. II. Electron microscopic observations of non-obese diabetic mice

We used transmission electron microscopy to examine the cochlea of non-obese diabetic mice as animal models for human type I or non-insulin-dependent diabetes mellitus. Pathological changes were observed in the organ of Corti of the basal turn and in the stria vascularis of each turn. Major findings in the stria vascularis were protrusion or condensation of marginal cells, swelling of intermediate cells, and widening of the intercellular spaces. Principal findings in the organ of Corti involved degenerative changes of the outer and inner hair cells and replacement of hair cells by supporting cells. No prominent pathological changes were observed in the capillaries. The possible mechanism of diabetic involvement in cochlear pathology is discussed.     

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear

Conclusion. The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. Objective. To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. Material and methods. Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. Results. Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.     

Inner ear pathology in the Palmerston North autoimmune strain mouse.

The Palmerston North autoimmune strain mouse is a model for spontaneous systemic lupus erythematosus. Inner ear structure and function were examined during the onset and progression of systemic autoimmune disease to identify potentially correlated auditory system pathology. The onset of systemic disease occurred at 4 to 5 months of age and was characterized by elevated serum immune complexes, cryoglobulins, and antinuclear antibodies. Coincident with the onset of autoimmune disease was degeneration of the apical turn stria vascularis and outer hair cells. These cochlear changes progressed basalward. At 10 months of age, auditory brainstem response thresholds were elevated and the stria vascularis area was measurably smaller throughout the cochlea. Immunohistochemical staining showed immunoglobulin G deposits within the organ of Corti, the vas spirale of the basilar membrane, the scala tympani, and marrow cavities of the bony otic capsule. These results suggest that cochlear pathology may be immune mediated in this mouse, which would make the strain suitable for the study of the mechanisms relating inner ear abnormalities and autoimmune disease.     

Immunohistochemical localization of the epithelial sodium channel in the rat inner ear

Endolymph in membranous labyrinth is a K+-rich and Na+-poor fluid, and perilymph is conversely Na+-rich and K+-poor. Electrolyte transport between endolymph and perilymph is important for regulation of volume and osmotic pressure of the labyrinth. The epithelial sodium channel (ENaC) is a good candidate protein for Na+ transport in the tight epithelia, which has been well demonstrated in other tissues such as kidney, colon and lung. The purpose of the present study was to investigate the cellular localization of ENaC subunits in the rat inner ear immunohistochemically with the specific polyclonal rabbit antibodies against the rat alpha-, beta- and gamma-ENaC. All three subunits of ENaC were extensively labeled in the cochlea including the stria vascularis, spiral ligament, organ of Corti, spiral limbus, Reissner's membrane and spiral ganglion, and in the vestibule including the sensory epithelia and stroma cells of the macula utriculi, macula sacculi and ampullary crest. In conclusion, our results suggest that functional ENaC in the labyrinth may work in concert with other Na+ and K+ transport molecules to regulate endolymph and to maintain homeostasis in the inner ear.     

Expression of vascular endothelial growth factor and its receptors in the cochlea of various experimental animals

CONCLUSION: The results of this study indicate that vascular endothelial growth factor (VEGF) may be an important regulator of the vascular network of the inner ear and suggest that the VEGF signalling pathway may play a role in pathophysiologic conditions. OBJECTIVE: In order to clarify the role of vascular growth factor in the modulation of the vascular network of the cochlea, we studied the expression of VEGF and its receptors-fms-like tyrosine kinase (Flt-1) and foetal liver kinase (Flk-1)-in the inner ear of 3-month-old rodents of different species: C57BL/6J mice, Wistar albino rats and Hartley albino guinea pigs. MATERIAL AND METHODS: Qualitative immunohistochemical studies were performed by using specific antibodies to VEGF and its receptors on paraffin sections of the cochlea. The expression levels of VEGF and its receptors were quantified by means of Western blot analysis of cochlea protein extracts. RESULTS: We demonstrated that VEGF and its receptors are expressed in the cochlea and described their distribution in the inner ear. In particular, VEGF and Flt-1 are present at the level of the modiolus, spiral ganglion, spiral ligament, basilar membrane, supporting cells, outer and inner hair cells and stria vascularis. Flk-1 was less strongly expressed in the cochlea and was not detected in the organ of Corti.     

Cochlear IgG in the C3H/lpr autoimmune strain mouse.

The inner ear of the C3H/lpr autoimmune strain mouse was evaluated to identify potential mechanisms by which systemic autoimmune disease interferes with auditory function. The inner ears were immunohistochemically stained for IgG at ages before (2 months) and after (6-10 months) autoimmune disease onset and compared to age-matched nonautoimmune C3H/HeJ controls. Immunoreactivity for IgG was not seen in the 2 month C3H/lpr autoimmune mice or in either age group of the C3H/HeJ controls. On the other hand, all older C3H/lpr mice showed reaction product in the vessels of the cochlea, particularly the stria vascularis and bony capsule. Less frequent sites of staining were the geniculate ganglion, marrow cavities of the bony capsule, tensor tympani muscle, and on one occasion, a hair cell of the organ of Corti. These findings indicate that IgG is widespread within the cochlea and its vessels during systemic autoimmune disease and not directed against any specific sensorineural structure. This suggests a generalized or indirect mechanism whereby such systemic disease affects the inner ear.     

Insulin stimulates protein synthesis and phospholipid signaling systems but does not regulate glucose uptake in the inner ear

High-affinity insulin receptors exist in the organ of Corti (Kd = 1.1 +/- 0.5 nM) and in the lateral wall (stria vascularis and ligamentum spirale; Kd = 1.1 +/- 0.4 nM) of the inner ear of the guinea pig as determined by the binding of radiolabeled porcine or bovine insulin in vitro. Carrier-mediated transport of glucose (defined as the cytochalasin B-sensitive part of total uptake) was measured in vitro with 2-deoxy-D-glucose as the substrate. Its Km was 188 microM in the lateral wall (r = 0.99 and 0.94, respectively). Neither the Km nor the rates of transport (0.20 +/- 0.10 pmol/micrograms protein/hr in the organ of Corti, and 0.56 +/- 0.34 pmol/micrograms protein/hr in the lateral was) were affected by insulin. In contrast, 0.1 mM ouabain decreased deoxyglucose uptake in the organ or Corti by 37% and in the lateral wall tissues by 45% indicating the presence of an active, Na(+)-dependent transporter in these tissues. Insulin influenced both protein and lipid metabolism in the inner ear. Proteins and lipids were labeled in situ by perfusion of the perilymphatic space of the cochlea with [3]-leucine or [32P]-orthophosphate and [3H]-glycerol, respectively. Thirty nM insulin stimulated the incorporation of [3H]-leucine into protein of the organ of Corti from 39 to 56 pmol/mg protein but was ineffective in the tissues of the lateral wall. In the organ of Corti, [32P]-orthophosphate was incorporated into the phosphoinositides and phosphatidate, and 30 nM insulin increased this incorporation by 101 to 149%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular biology of hearing

The inner ear is our most sensitive sensory organ and can be subdivided into 3 functional units: organ of Corti, stria vascularis and spiral ganglion. The appropriate stimulus for the organ of hearing is sound which travels through the external auditory canal to the middle ear where it is transmitted to the inner ear. The inner ear habors the hair cells, the sensory cells of hearing. The inner hair cells are capable of mechanotransduction, the transformation of mechanical force into an electrical signal, which is the basic principle of hearing. The stria vascularis generates the endocochlear potential and maintains the ionic homeostasis of the endolymph. The dendrites of the spiral ganglion form synaptic contacts with the hair cells. The spiral ganglion is composed of neurons that transmit the electrical signals from the cochlea to the central nervous system. In the past years there was significant progress in research on the molecular basis of hearing. More and more genes and proteins which are related to hearing can be identified and characterized. The increasing knowledge on these genes contributes not only to a better understanding of the mechanism of hearing but also to a deeper understanding of the molecular basis of hereditary hearing loss. This basic research is a prerequisite for the development of molecular diagnostics and novel therapies for hearing loss.     

Experimental mumps labyrinthitis in monkeys (Macaca irus)--immunohistochemical and ultrastructural studies

Three monkeys (Macaca irus) were inoculated with mumps virus into unilateral cochleas and their inner ear were examined by immunofluorescent microscopy and transmission electronmicroscopy. The temporal bones were removed after survival period of 14 days when serological tests disclosed elevation of anti-mumps antibody titers. Immunofluorescent microscopy revealed that the viral antigen was positive in the stria vascularis. The ultrastructural study revealed that the pathologic changes in the cochleas were marked in the organ of Corti and stria vascularis. The outer hair cells were more susceptible to the infection than the inner hair cells. In the stria vascularis, both marginal and intermediate cells were affected. It was possible to find some of marginal cells in the basal turn shedding a large number of mature virions into the endolymph. These pathologic changes observed in the cochleas of the monkeys were similar to those previously revealed in the guinea pig cochleas and thus were considered as the specific features of acute mumps labyrinthitis.     

Detection of Nitric Oxide in the Guinea Pig Inner Ear,Using a Combination of Aldehyde Fixative and 4,5-Diaminofluorescein Diacetate

Localization of nitric oxide (NO) production sites in the inner ear of the guinea pig was investigated using a combination of glutaraldehyde fixative and a new fluorescence NO indicator, 4,5-diaminofluorescein diacetate (DAF-2DA). The cochlea and vestibular end organs were examined to locate NO production sites. The fluorescence persisted after glutaraldehyde fixation and embedding with water-soluble resin. NO production in the cochlea was observed in the outer and inner hair cells, nerve endings, nerve fibers and supporting cells of the organ of Corti, stria vascularis, spiral ligament, ganglion cells, etc. In the vestibular end organs, both type I and type II sensory cells, nerve fibers, blood vessels and dark cells displayed fluorescence. This localization was exactly identical to that of NO synthase. Thus, detection of intracellular NO production by using a combination of glutaraldehyde fixation and DAF-2DA is useful for examining the function of NO in cells, both in situ and in vivo.     

Evidence for an Akt-kinase/NO/cGMP pathway in the cochlea of guinea pigs

The protein kinase Akt (protein kinase B) can be activated by numerous growth factors via PI-3 kinase-generated phosphoinositides and is thought to have anti-apoptotic properties. Activated Akt/PKB boosts the activity of endothelial NO synthase (NOS III), which has been found in the key areas of the inner ear (e.g., hair cells and stria vascularis). In order to localize activated Akt/PKB (phospho-Akt) in the cochlea of guinea pigs, sections of ten temporal bones were observed immunohistochemically. The strongest immunoreactivity was found in and underneath inner hair cells (IHC). Within the organ of Corti, reactivity was found in supporting cells, while outer hair cells remained unstained. Spiral ganglion cells, the endothelium of the lateral wall and the vascular area of the modiolus showed moderate staining. The results give evidence that activated Akt/PKB influences the activity of the NO/cGMP pathway in the cochlea. Because of the antiapoptotic properties, activated Akt should now be examined under non-physiological conditions.     

Localization of Nitric Oxide Synthase Isoforms in the Human Cochlea

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.     

Localization of nitric oxide synthase isoforms in the human cochlea

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.     

Scanning electron microscopy of the normal human cochlea

Preservation of the fine structures of the human cochlea has been achieved by perfusing the cochlea with fixative shortly after death. Following the dissection of the temporal bone the surface of the organ of Corti and stria vascularis has been examined in the scanning electron microscope. The surfaces of the inner and outer hair cells can be seen and the stereocilia projecting from their surfaces closely examined. The number and length of the stereocilia of the outer hair cells changes linearly with distance along the cochlear duct. The surface of the stria vascularis is similar to that seen in other animals.