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.     

豚鼠耳蜗和内淋巴囊上皮钠通道的表达

目的 研究豚鼠耳蜗和内淋巴囊组织中上皮钠通道(epithelial sodium channel,ENaC)α、β、γ亚基的表达及其意义.方法 用兔抗大鼠ENaC α、β和γ亚基的多克隆抗体,采用免疫组化SP法观察豚鼠耳蜗和内淋巴囊组织中α、β、γ-ENaC的表达模式.另用α-ENaC cDNA质粒合成探针,原位杂交法检测豚鼠耳蜗和内淋巴囊组织中α-ENaC mRNA的表达.结果 免疫组化显示,在豚鼠耳蜗中,α-ENaC蛋白强烈表达于螺旋缘,而螺旋韧带、Corti器、Reissner膜等处的表达较弱;β-ENaC蛋白在螺旋韧带、螺旋缘和螺旋神经节、Corti器、Reissner膜等处的表达均呈弱阳性;γ-ENaC蛋白则在螺旋韧带的上半部、螺旋缘和螺旋神经节等处的表达呈强阳性,Corti器、Reissner膜等处也有阳性表达.三个亚基在血管纹中均呈阴性表达.在内淋巴囊中,α、β和γ亚基均较明显地表达于上皮细胞和上皮下纤维组织.原位杂交显示,α-ENaC mRNA除了表达于螺旋缘、螺旋韧带下部外,还表达于血管纹,而在内淋巴囊的上皮细胞和上皮下纤维组织也均呈阳性表达.结论 ENaC的各个亚基以不同的模式分布于豚鼠耳蜗和内淋巴囊的各个区域,形成功能性通道参与内淋巴的调节,从而保持内耳内环境的稳定.     

Adenylate cyclase and G-proteins as a signal transfer system in the guinea pig inner ear

In many eukaryotic cells G-proteins play a key role in signal transduction through outer cell membranes. To study this pathway in the auditory organ of mammals we examined tissue preparations from the stria vascularis and the organ of Corti from the guinea pig inner ear. The activity of adenylate cyclase was measured by stimulation at the site of the enzyme, the hormone receptors and the modulating G-proteins. In the organ of Corti we found a low enzyme activity in all cochlear turns. The stria vascularis, however, showed a constant high concentration of beta 2-adrenergic receptors and of stimulating G-proteins in all cochlear turns. In contrast, the activity of the enzyme increased from the apical to the basal turn. Adenylate cyclase could be stimulated or inhibited in a concentration-dependent manner by drugs selectively effecting the G-proteins. Our results suggest a structure of the adenylate cyclase complex in the inner ear similar to other organs. Pathophysiological correlations to hearing loss associated with pseudohypoparathyroidism are discussed.     

Histopathology of the temporal bones of deaf dogs

Histopathological studies were done in 22 deaf dogs consisting of 10 Dalmatians, 5 English setters, 2 Great Danes, 2 foxhounds, 1 shepherd, 1 bulldog and 1 Australian sheep dog. Hypoplasia or aplasia of the sensory cells of the organ of Corti, stria vascularis and macula sacculi (pars inferior) was noted in all deaf dogs examined, indicating the pathology of inner ear malformation in these dogs to be of Scheibe's type. A solidified and calcified tectorial membrane was noted in 19 out of the 22 deaf dogs. A calcified sclerosis of the tectorial membrane is thought to be a characteristic finding of genetically inherited anomaly of the inner ear in deaf dogs. Distortion of the tectorial membrane, absence of the sensory cells in the organ of Corti, agenesis of the stria vascularis and abnormalities of the saccule described Scheibe's dysgenesis of the pars inferior as the pathological correlate for deafness in these dogs as assessed functionally.     

The effect of noise on deoxyglucose uptake into inner ear tissues of the mouse

Summary The uptake of deoxyglucose into inner ear tissues was studied in the mouse. Sixty minutes after a single i.v. injection of 5 mCi 2-deoxy-D-[³H]glucose/kg body weight, stria vascularis/spiral ligament and organ of Corti as well as other body tissues were dissected and analyzed for radioactivity. Uptake into inner ear tissues was three to five times lower than into brain or heart. The ratio of deoxyglucose-6-phosphate to deoxyglucose was 6040 and the compounds were eliminated from the inner ear with a half life of approximately 60 min. Exposure to 100 dB of white noise during the radioactive pulse decreased uptake of deoxyglucose into both stria vascularis/spiral ligament and organ of Corti by 50%.This work was supported by grant NS 12881 from the National Institutes of Health and Program Project grant NS 05785 and training grant NS 07106     

Cisplatin ototoxicity in guinea pigs with special reference to toxic effects in the stria vascularis

The toxic effects of cisplatin (cis-diamminedichloroplatinum [II]) on the organ of Corti are well established. Few and conflicting data on this drug's effects on the stria vascularis exist. The present study presents animal experiments on the toxic effects of cisplatin in the stria vascularis and in the organ of Corti. Cisplatin-induced toxicity in albino and pigmented guinea pigs was evaluated morphologically and functionally, using light and transmission electron microscopy as well as auditory brainstem-evoked potentials on the organ of Corti and the stria vascularis. The results showed variability in hearing thresholds, ranging from no change to hearing loss of 30 dB, and prominent damage in the organ of Corti and in the stria vascularis. The toxic effects to both the organ of Corti and the stria vascularis should be considered when cisplatin is used in chemotherapy.     

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.     

Results of an ultrastructural study comparing stria vascularis with organ of Corti in guinea pigs treated with kanamycin

Ultrastructural study of ototoxicity is well documented with two points of interest: organ of Corti for aminoglycosides and stria vascularis for loop diuretics. As a previous study suggested initial lesions of stria vascularis, an attempt of comparison and of chronological study was made between the organ of Corti and stria vascularis lesions by kanamycin intoxication. The method was devised by J. M. ARAN, with electrophysiological control. We failed to find in the stria vascularis a radial or longitudinal pattern of lesions. We could not discern a chronological injury between the organ of Corti and stria vascularis because both were damaged even in the less deafened animals. Nevertheless, two facts were clarified: hair cell lesions are lysosomial as for the kidney lesions, while stria vascularis lesions are mitochondrial, melanine granulations play a part in drug metabolism (increased number, secretory aspect) and deserve further study.     

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.     

Application of argon laser to the inner ear

The effects of argon laser irradiation on the inner ear were studied morphologically. The stria vascularis showed focal coagulation, vesicle formation, perforation and atrophy after irradiation through the otic capsule. These changes vary according to the power and duration of irradiation. Atrophy of the stria vascularis can be produced without injuring the organ of Corti, by suitable application of the laser beam. The round window membrane transmits the argon laser. Tissues in the hook region and a part of the lower basal turn demonstrated changes of varying degree. Loss of the nerve fibres was observed in the osseous spiral lamina, which was fractured and dislocated. Irradiation through the stapedectomized oval window destroyed the macula sacculi without rupturing the saccular wall.     

Electrical coupling differs in the in vitro and in vivo organ of Corti

Electrical communication between the supporting cells of the guinea pig organ of Corti was studied. For in vitro experiments, the inner ear was rapidly removed and placed in a heated perfusion chamber. Medium 199 was used. The bony cochlea and the lateral wall (spiral ligament and stria vascularis) were removed to expose the top two coils of the organ of Corti. In vivo experiments were performed upon anesthetized animals whose cochleas were exposed surgically. A tiny fenestra was made in the bony cochlea which permitted the passage of electrodes through the lateral wall and into the organ of Corti of the third turn. Coupling was assessed by impaling neighboring cells with 3 M KCl electrodes, and noting the spread of intracellularly injected current. Coupling ratios in the in vitro preparation were consistently greater than those obtained in vivo (0.58 +/- 0.17 vs. 0.104 +/- 0.064). Differences exist between the in vitro and in vivo preparations which might account for these results. In vivo the supporting cells are bathed in two different media, endolymph apically, and perilymph basally. Consequently, on their apical side the supporting cells are exposed to fluid high in K+, low in Ca2+ and at a potential of 80 mV, the endolymphatic potential. In vitro the cells are bathed on all sides in fluid similar to perilymph. Intermixing the fluids in an in vivo preparation, by tearing away the stria vascularis and Reissner's membrane, increases the magnitude of the coupling ratio (0.455 +/- 0.209). Thus the unique microenvironment of the inner ear maintains lower coupling ratios, and smaller space constants for the supporting cells.     

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.     

庆大霉素对豚鼠血管纹Na，K-ATP酶α亚单位异构体表达的影响

目的 观察正常豚鼠血管纹Ｎａ,Ｋ－ＡＴＰ酶α亚单位异构体的表达及庆大霉素对其表达的影响。方法 用鼠抗大鼠Ｎａ,Ｋ－ＡＴＰ酶α亚单位异构体特异性单克隆抗体,采用免疫组化ＳＰ法观察正常豚鼠血管纹中Ｎａ,Ｋ－ＡＴＰ酶α１异构体的表达及庆大霉素作用后表达的变化。结果 正常豚鼠血管纹Ｎａ,Ｋ－ＡＴＰ酶α１异构体表达呈阳性,而α２和α３异构体表达呈阴性。庆大霉素作用后α１异构体表达显著减弱。结论 庆大霉素显著     

Ototoxicity of Vasocidin drops applied to the chinchilla middle ear

Some widely used ototopical preparations are potentially toxic to the middle and inner ear. Vasocidin Ophthalmic Solution (sulfacetamide sodium and prednisolone sodium phosphate) has been advocated as an alternative agent that may have fewer toxic side effects in the treatment of otorrhea. Vasocidin was introduced into the bullae of nine chinchillas to investigate the effects on the middle and inner ear. The organ of Corti and stria vascularis were found to be entirely normal in 17 of the 18 temporal bones studied. Changes observed in the middle ears at one week included inflammation, hemorrhage, and effusion. Examination of specimens at four weeks revealed resolution of most of the inflammatory changes. The results of this experimental study indicate that Vasocidin causes reversible middle ear inflammation with little or no toxic effect on inner ear structures.     

An experimental study of inner ear pathology due to NaCl on the round window

An animal experiment was performed to evaluate structural and ultrastructural changes to the inner ear as a result of placing 3-4 crystals of reagent grade sodium chloride (NaCl) on the round window membrane. Chinchillas were sacrificed at 8 and 24 hours after treatment and the cochlear and vestibular tissues were examined by light microscopy and scanning electron microscopy. Inner ear pathology consisted of destruction of both sensory and supporting cells in the basal turn of the organ of Corti, atrophy of the stria vascularis and alterations to the otoconia and the maculae and ampullae of the vestibular system. This study demonstrates that NaCl in the middle ear does not provide a model for Meniere's disease as previously suggested by Arslan. It may, however, be utilized in the destructive treatment of selected inner ear disorders.     

Inner ear changes in the ferret model for Reye's syndrome

The acute effects of influenza B, aspirin, and hyperammonemia on the inner ear were examined using the ferret model for Reye's syndrome. Histopathologic examination revealed varying degrees of vacuolization in both the sensory and nonsensory endolymphatic tissues of the cochlear and vestibular membranous labyrinth. The secretory epithelial cells of the stria vascularis and the vestibular dark cells appeared to be more severely affected, as demonstrated by the presence of an extensive number of intracytoplasmic vacuoles. The cells of the perilymphatic tissues appeared to be unaltered. Although all neuroepithelial regions of the inner ear appeared to be altered, an unexpected observation was that only the inner hair cells of the organ of Corti were pathologically affected. These results suggest that metabolic and virus-induced derangements may alter inner ear microhomeostasis in patients with Reye's syndrome and may potentially result in loss of hearing in such patients.     

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.     

The Effect of Anaphylatoxin Component on Inner Ear Damage

It has already been reported that anaphylatoxin (AT) is closely related to inner ear lesions. In this study the pathology of inner ear damage caused by AT was investigated in Hartley strain and C4 deficient guinea pigs (C4D-GP). Although the biological activity of C3a and C5a is strong, it is of short duration, and the resulting ear damage is considered to be reversible. Because of the cellular degeneration caused by this slight damage, the damage owing to continuously activated C4a is thought to be irreversible, that is, atrophy in the stria vascularis, degeneration and sloughing of the cochlear neurons, and stretching of Reissner's membrane, and C4a is therefore considered to be requisite for the manifestation of inner ear damage.