Expression pattern of adenylyl cyclase isoforms in the inner ear of the rat by RT-PCR and immunochemical localization of calcineurin in the organ of Corti.

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.e. in the stria vascularis and endolymphatic sac. In the organ of Corti and vestibulum the Ca2+-modulated isoforms types 1, 6 and 9 were expressed. The regulation of adenylyl cyclase 9, which is the major isoform expressed in the organ of Corti, proceeds via the Ca2+-activated protein phosphatase 2B (calcineurin, PPP3). PCR with specific primers for calcineurin demonstrated its abundant expression in the organ of Corti. Using a monoclonal antibody we localized calcineurin immunochemically to the cochlear nerve, the nerve fibers and the inner hair cells. In the cochlear and vestibular nerves a characteristic neuronal expression pattern of adenylyl cyclase isoforms was observed, i.e. adenylyl cyclases types 2, 3 and 8. The functional consequences of the adenylyl cyclase expression pattern in the inner ear are discussed in conjunction with its unique sensory performance.     

Characteristics and drug responses of cochlear and vestibular adenylate cyclase

Summary Adenylate cyclase activity of dissected cochlear and vestibular structures was assayed with the ATP-analog adenylyl imidodiphosphate as substrate. High activities (per mg protein) were found in stria vascularis and in vestibular preparations, lower activities in spiral ligament, VIIIth nerve, and organ of Corti. The enzyme from all structures was stimulated by fluoride, guanylyl imidodiphosphate or manganese(II) ions, and strongly inhibited by ethacrynate and lead ion. The anti-cancer drug cis-diammine-dichloro platinum significantly inhibited adenylate cyclase from stria vascularis.Presented at the 16th Workshop of Inner Ear Biology in Bern 1979     

Pathological findings in the human auditory system following long-standing gentamicin ototoxicity

Summary The clinical, audiovestibular and histopathological findings in a patient who suffered from a long-standing gentamicin-induced deafness are reported. In both temporal bones, the organ of Corti was completely absent, with only a few nerve fibres remaining in the apical part of the cochlea. Regenerative ingrowth of nerve fibers into the area of the degenerative organ of Corti was present apically in both ears. The stria vascularis exhibited considerable degeneration in all turns and loss of microvasculature was found in the basilar membrane. The spiral ganglion cells, the cochlear nerve and the central auditory pathways and nuclei appeared to be unaffected.     

Pathological findings in the human auditory system following long-standing gentamicin ototoxicity

The clinical, audiovestibular and histopathological findings in a patient who suffered from a long-standing gentamicin-induced deafness are reported. In both temporal bones, the organ of Corti was completely absent, with only a few nerve fibres remaining in the apical part of the cochlea. Regenerative ingrowth of nerve fibers into the area of the degenerative organ of Corti was present apically in both ears. The stria vascularis exhibited considerable degeneration in all turns and loss of microvasculature was found in the basilar membrane. The spiral ganglion cells, the cochlear nerve and the central auditory pathways and nuclei appeared to be unaffected.     

Autoradiographic demonstration of deoxyglucose uptake in guinea pig cochlea

The 2-deoxyglucose autoradiographic method was applied to whole-body cryosectioning to include the cochlea. The highest levels of 2-deoxyglucose uptake were observed in the vascular stria, spiral ligament and spiral prominence. The cochlear nerve showed the next highest level of uptake, while the organ of Corti and the spiral ganglion showed low levels. The functional significance of the results was briefly discussed.     

Electron microscopic temporal bone histopathology in experimental pneumococcal meningitis.

Bacterial meningitis is one of the most common causes of acquired profound sensorineural deafness in children. Measurement of hearing and examination of the cochlea is limited in patients suffering from acute meningitis. A rabbit model of pneumococcal meningitis was developed to identify the temporal bone histopathologic changes that occur in meningogenic labyrinthitis caused by Streptococcus pneumoniae. Light microscopy was previously performed on temporal bones from acutely meningitic rabbits with profound hearing loss as determined electrophysiologically. Extensive inflammation of the cochlea with endolymphatic hydrops was observed. The organ of Corti, however, showed preserved architecture in the majority of these animals. In order to further investigate these findings, a protocol was used to create meningitic rabbits with hearing loss ranging from early high-frequency loss to profound deafness. The temporal bones from 7 rabbits were examined by transmission electron microscopy. In cases of mild hearing loss, partial degeneration of the inner row of outer hair cells, as well as edema of efferent cochlear nerve endings and marginal cells of the stria vascularis, was seen. With increasing degrees of hearing loss, the remainder of the organ of Corti and intermediate cells of the stria showed ultrastructural abnormalities. Spiral ganglion cells and basal cells of the stria vascularis remained intact in all subjects. This study provides unique information regarding the histology and pathophysiology of meningogenic deafness. The clinical implications of these findings are discussed, with an emphasis on potentially reversible changes and therapeutic intervention.     

Fine structure histopathology of labyrinthitis ossificans in the gerbil model

Labyrinthitis ossificans (LO) is the pathological deposition of new bone within the lumen of the cochlea and labyrinth. This process occurs most commonly as a result of infection or inflammation affecting the otic capsule. Trauma and vascular compromise can also lead to neo-ossification within the otic capsule. The mechanism that regulates this process remains unestablished. This study details the end-stage histopathology in high-resolution plastic thin sections. Twenty Mongolian gerbils were infected by intrathecal injection of Streptococcus pneumoniae type 3 followed by subcutaneous penicillin G procaine (8 days) and were painlessly sacrificed 3 months later. The cochleas were serially divided and sectioned for light and electron microscopy. Sixteen of 20 animals (27 of 40 cochleas) demonstrated LO. Cochlear damage was most extensive in the vestibule and basal turn and decreased toward the apex, which often appeared normal. The histopathologic findings consisted of 1) new bone, calcospherites, osteoid, and fibrosis without dense connective tissue or osteoblasts extending from the endosteal wall into the lumen of the vestibule and scala tympani; 2) areas of dense connective tissue and osteoid enclosed by epithelial cells conjoined with the organ of Corti, stria vascularis, spiral ligament, and vestibular (Reissner's) membrane; and 3) partial to complete loss of the organ of Corti, spiral ligament cell bodies, stria vascularis, and spiral ganglion cells. Osteoblastic activity was not demonstrated in end-stage ossification in LO in the gerbil model. Neo-ossification appears to occur by calcospherite deposition along collagen-like fibrils within osteoid. The destruction of the organ of Corti, spiral ganglion cells, stria vascularis, and cells of Reissner's membrane and the spiral ligament occurs even in the absence of ossification of the cochlear duct.     

The cochlea of the spontaneously diabetic mouse

Summary We used transmission electron microscopy to examine the cochleae 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.     

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.     

Acoustic stimulation alters deoxyglucose uptake in the mouse cochlea and inferior colliculus

Deoxyglucose uptake and activities of hexokinase and glucose-6-phosphatase in auditory structures (organ of Corti, stria vascularis and spiral ligament, modiolar section of VIIIth nerve, inferior colliculus) and non-auditory tissues (heart, kidney, liver) of the mouse were analyzed. [³H]Deoxyglucose was given as a pulse into the tail vein and uptake was quantitated by microdissection of tissues and scintillation counting. Radioactivity in cochlear tissues was maximal after 45–60 min and declined with a half-life of 30–60 min. Deoxyglucose 6-phosphate represented ca. 60% of total radioactivity (heart, inferior colliculus. > 80%). The ratio of hexokinase to glucose-6-phosphatase activity was considerably lower in the auditory periphery than in brain. The rank order was inferior colliculus > VIIIth nerve ≈ heart > stria vascularis and spiral ligament > kidney > organ of Corti ≈ liver.Exposure to broadband noise increased glucose utilization in all auditory structures. Uptake was maximally (2- to 3-fold) stimulated at moderate noise intensity (55–85 dBA). In addition, the auditory system showed two salient features: at high intensities (100 and 115 dBA) deoxyglucose uptake decreased from the maximum; and the non-sensory tissues of the cochlea (stria vascularis and spiral ligament) responded to sound parallel to the sensory structures at all levels of stimulus intensity. There were no effects of acoustic stimulation on serum glucose levels, serum kinetics of deoxyglucose. or deoxyglucose uptake into other body tissues.     

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

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.     

Maintenance of cochlear function with artificial oxygen carriers

By means of vascular perfusion via the anterior inferior cerebellar artery with a blood substitute containing the perfluorochemical FC 47 as oxygen carrier, it is possible to maintain normal or near normal levels of the cochlear microphonics and the endolymphatic potential of the guinea pig for periods of 90 min. or longer. Following 60 min. of perfusion with artificial blood, the levels of ATP and 5′AMP in the stria vascularis and the organ of Corti are comparable to those of nonperfused control animals maintained at optimal metabolic conditions. Following the same period of perfusion, the appearance of the organ of Corti is normal, but small vacuoles, presumably deposits of FC 47, are visible in the marginal cells of the stria vascularis. Preliminary experiments concerning the survival time and the revival time of the cochlear potentials, as well as the response to furosemide, ouabain, and mersalyl are presented to illustrate the value of this method in elucidating various biochemical and pharmacological problems of the cochlea.     

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.     

Localization of glucocorticoid receptors and glucocorticoid receptor mRNAs in the rat cochlea

The distribution of glucocorticoid (GR) receptor messenger RNAs (mRNAs) and GR receptors was studied by in situ hybridization histochemistry and immunocytochemistry, respectively. In situ hybridization histochemistry was performed with a biotin-labeled riboprobe complementary to rat GR receptor mRNA. GR receptor mRNAs were demonstrated in spiral ligament cells, spiral limbus cells, and spiral ganglion cells. GR receptor mRNAs were demonstrated neither in cells of the stria vascularis nor in cells of the organ of Corti region. With the use of a monoclonal and a polyclonal antibody, GR receptors were observed in the spiral ligament cells, stria vascularis cells, spiral limbus cells, and spiral ganglion cells by immunocytochemistry. Binding of anti-GR-receptor antibodies to a lesser extent was observed in the organ of Corti region; however, cellular distribution of the GR receptors could not be resolved with the applied techniques. These results suggest that the GR receptor is expressed differently in the heterogeneous cochlear tissues.     

Viral labyrinthitis: early pathology in the human

The histologic findings in the temporal bones of three patients who died from viral encephalopathy are presented. Pathology was restricted to the scala media, vestibular labyrinth, and internal auditory canal and was considered to be expressions of viral labyrinthitis. The changes were different degrees of degeneration of the organ of Corti, early encapsulation of the tectorial membrane, degeneration of the stria vascularis, and round cell infiltration of the modiolus and contents of the internal auditory canal. A new finding in the organ of Corti and early stages of cystic degeneration of the stria vascularis are documented. In all cases, the saccule was degenerated with sloughing of the otolithic membrane and vestibular labyrinth was involved in varying degrees.     

Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea

Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti. We used an AAV-2 vector delivered hammerhead ribozyme, AAV–CBA–Rz, which specifically recognizes and cleaves wild type mouse clarin-1 mRNA. Cochleae of CD-1 mice were injected either with 1 μl of the AAV–CBA–Rz, or control AAV vectors containing the green fluorescent protein (GFP) marker gene (AAV–CBA–GFP). Additional controls were performed with saline only. At one-week and one-month post-injection, the animals were sacrificed and the cochleae were studied by histology and fluorescence imaging.

Mice injected with AAV–CBA–GFP displayed GFP reporter expression of varying fluorescence intensity throughout the length of the cochlea in the outer and inner hair cells and stria vascularis, and to a lesser extent, in vestibular epithelial cells. GFP expression was not detectable in the spiral ganglion. The pro-apoptotic effect of AAV–CBA-delivered anti-clarin-1 ribozymes was evaluated by TUNEL-staining. We observed in the AAV–CBA–Rz, AAV–CBA–GFP and saline control groups apoptotic nuclei in the outer and inner hair cells and in the stria vascularis one week after the microinjection. The vestibular epithelium was also observed to contain apoptotic cells. No TUNEL-positive spiral ganglion neurons were detected. After one-month post-injection, the AAV–CBA–Rz-injected group had significantly more apoptotic outer and inner hair cells and cells of the stria vascularis than the AAV–CBA–GFP group.

In this study, we demonstrate that AAV–CBA mediated clarin-1 ribozyme may induce apoptosis of the cochlear hair cells and cells of the stria vascularis. Surprisingly, we did not observe apoptosis in spiral ganglion cells, which should also be susceptible to clarin-1 mRNA cleavage. This result may be due to the injection technique, the promoter used, or tropism of the AAV serotype 2 viral vector. These results suggest the role of apoptosis in the progression of USH3A hearing loss warrants further evaluation.     

Ultrastructural and physiological defects in the cochlea of the Mpv17 mouse strain. A comparison between young and old adult animals

Ultrastructural investigations were performed in young (approximately 2 months) and old (7 months) Mpv17-negative and wild-type mice. The onset, the severity and the pattern of the degeneration significantly differed between both mice strains. In the wild-type mouse strain the degenerative changes of the cochlear structures were similar to the aging pattern described for other species. In contrast, the Mpv17 mutants showed degenerative changes of the cochlear structures already at the age of 2 months. The degenerative changes were patchy arranged throughout the entire length of the cochlea and involved the organ of Corti as well as the stria vascularis epithelia with alterations of the basement membrane of the capillaries. The severe sensorineural hearing loss and degenerative changes of the cochlear structures indicate that cochlear structures, especially the outer hair cells and the intermediate cells of the stria vascularis, are vulnerable to the missing Mpv17 gene product.     

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.