急性次声波暴露后豚鼠位听功能及内耳超微结构的变化

目的观察次声波对豚鼠位听功能和内耳超微结构的影响。方法将豚鼠置于频率8Hz、声压级135dBSPL的次声声场中连续暴露90min。应用正弦摆动试验(sinusoidalpendulartest,SPT)、听性脑干反应（auditorybrainstemresponse,ABR）和畸变产物耳声发射(distortionproductionotoacousticemission,DPOAE)评价次声波暴露前后豚鼠前庭功能和听功能的变化,扫描电镜观察豚鼠内耳各结构表面超微形态的变化。结果次声波暴露后不同时间正弦摆动诱发的豚鼠前庭性眼震的最大慢相速度(slow-phasevelocity,SPV)和频率较次声暴露前轻微降低,但无显著性意义(P>0.05)。次声波暴露后各组动物ABR阈值较正常时略有升高,亦无统计学差异(P>0.05),各组动物ABR各波潜伏期和波间期与次声暴露前比较差异均无显著性(P>0.05);DPOAE的幅度值在各个频率段均有明显的降低(P<0.01)。扫描电镜下见各实验组动物内耳半规管壶腹嵴两囊斑及Corti器感觉毛细胞纤毛缺失、散乱、倒伏及融合,表皮板等结构均有不同程度的损伤。结论次声波对豚鼠前庭末梢感受器兴奋性可能有一过性的轻微抑制作用,但SPT无有意义改变。次声波可引起豚鼠内耳毛细胞超微结构的损伤,可导致豚鼠耳蜗外毛细胞功能明显减退,这种功能减退尚不足以引起听力的明显改变。     

Effects of inner ear immune responses on auditory function in guinea pigs]

Following direct challenge with KLH antigen primary or secondary (after systemic immunization) to the ES (endolymphatic sac) in guinea pigs, ECoG (electrocochleograms) were recorded from the round window induced by click and tone pips. The recordings were carried out on the 2nd, 7th and 21st days after local antigen challenge. There were no abnormal findings in the ECoG of the primary antigen challenged animals. On the other hand, prolongation of latencies, decrease in amplitudes of APs (compound action potentials) and increases in SP/AP ratios were observed in the 2nd day recordings of the secondary antigen challenged animals. However, all parameters of ECoG in the 7th day recordings were normal. Decreases in AP amplitudes were again found in the 21st day recordings. The ECoG findings with click and tone pip stimulation showed similar results. From these findings, in conjunction with morphological observations, it is speculated that these ECoG findings are caused by immuno-injury to the ES and cochlea, as well as the resultant endolymphatic hydrops which develops acutely and gradually subsides after secondary challenge.     

Free radicals in the guinea pig inner ear following gentamicin exposure

The purpose of this study was to investigate the occurrence of free radicals, nitric oxide (NO), superoxide (O-2) and peroxynitrite, in the inner ear of the guinea pig following intratympanic injection with 5 mg of gentamicin (GM). Forty-eight hours after GM injection, varying degrees of degeneration of the inner ear were observed. Immunohistochemical study revealed immunoreactivity to NO synthase II (which generates NO) and to xanthine oxidase (which generates O-2) in both the vestibular organ and the organ of Corti. Immunohistochemical investigation, using a specific antinitrotyrosine antibody, also showed intense staining, suggesting formation of peroxynitrite in the inner ear through the reaction of NO with O-2. Scanning electron-microscopic study showed that the ototoxic effects could be blocked with N-nitro-L-arginine methylester, a competitive inhibitor of NO synthase, with superoxide dismutase, an O-2 scavenger, and with ebselen, a scavenger of peroxynitrite. On the basis of these findings, it can be concluded that NO together with O-2, which form more reactive peroxynitrite, play an important role in GM ototoxicity in the guinea pig.     

Effect of acute inner ear pressure changes on low-level distortion product otoacoustic emissions in the guinea pig

Objective—To determine a relation between acute inner ear pressure changes and cochlear function as measured by low-level 2f₁–f₂ distortion product otoacoustic emissions (DPOAEs).

Material and Methods—During and after a change in inner ear pressure induced by injection or aspiration of perilymph, the 2f₁–f₂ DPOAE at 4.5 kHz generated by low-level primaries was recorded in the guinea pig.

Results—Large changes in overall inner ear pressure produced only small changes in the 2f₁–f₂ amplitude and phase. During injection of 0.5 μl of artificial perilymph into the scala tympani over a 10-s period, the mean inner ear pressure increased by ≈500 Pa, with an accompanying mean increase in the 2f₁–f₂ amplitude of 0.7 dB. During aspiration of 0.5 μl of perilymph over a 10-s period, the mean inner ear pressure decreased by ≈700 Pa, with an accompanying mean decrease in the 2f₁–f₂ amplitude of 0.9 dB. Changes in DPOAE amplitude followed inner ear pressure changes with a delay of 1–2 s. The magnitude and sign of the amplitude changes can (partly) be explained by a change in oval window stiffness. No explanation was found for the measured delay.

Conclusion—Clinically, these experiments can be of value in gaining insight into the pathophysiological mechanisms of pathological pressure changes as seen in Ménière's disease and perilymphatic fistulae.     

Effect of acute inner ear pressure changes on low-level distortion product otoacoustic emissions in the guinea pig

OBJECTIVE: To determine a relation between acute inner ear pressure changes and cochlear function as measured by low-level 2f(1)-f(2) distortion product otoacoustic emissions (DPOAEs). MATERIAL AND METHODS: During and after a change in inner ear pressure induced by injection or aspiration of perilymph, the 2f(1)-f(2) DPOAE at 4.5 kHz generated by low-level primaries was recorded in the guinea pig. RESULTS: Large changes in overall inner ear pressure produced only small changes in the 2f(1)-f(2) amplitude and phase. During injection of 0.5 microl of artificial perilymph into the scala tympani over a 10-s period, the mean inner ear pressure increased by approximately 500 Pa, with an accompanying mean increase in the 2f(1)-f(2) amplitude of 0.7 dB. During aspiration of 0.5 microl of perilymph over a 10-s period, the mean inner ear pressure decreased by approximately 700 Pa, with an accompanying mean decrease in the 2f(1)-f(2) amplitude of 0.9 dB. Changes in DPOAE amplitude followed inner ear pressure changes with a delay of 1-2 s. The magnitude and sign of the amplitude changes can (partly) be explained by a change in oval window stiffness. No explanation was found for the measured delay. CONCLUSION: Clinically, these experiments can be of value in gaining insight into the pathophysiological mechanisms of pathological pressure changes as seen in Meniere's disease and perilymphatic fistulae.     

Effect of inner ear immune response on vestibular function in guinea pigs]

We examined vestibular dysfunction and histological damage caused by direct antigen challenge to the endolymphatic sac in guinea pigs. We observed spontaneous nystagmus every eight hours and performed caloric testing every week following endolymphatic sac secondary KLH challenge. Spontaneous nystagmus was seen in 12 of 18 animals, and nystagmus in all directed toward the unchallenged ear (paralytic). The caloric response time courses were classified into two types, which were irreversible type and reversible type after endolymphatic sac KLH challenge. The immune injury of animals with irreversible type was thought to be stronger than that of these with reversible type. The spontaneous nystagmus of irreversible type animals was longer than that of reversible type animals. The temporary vestibular dysfunction was thought to be similar to that observed in Meniere's disease.     

电钻噪声对豚鼠内耳功能和形态影响的实验研究

目的：观察耳科电钻噪声对豚鼠内耳功能和形态的影响,并探讨电钻噪声对内耳的损伤机制。方法：应用听性脑干反应（ＡＢＲ）记录技术评价电钻噪声在不同时间暴露前后豚鼠听功能的变化。电钻噪声对豚鼠Ｃｏｒｔｉ器超微结构及组织化学的影响,采用定量组织化学技术测定豚鼠耳蜗外毛细胞（ＯＨＣ）的琥珀酸脱氢酶（ＳＤＨ）显色的灰度值,比较电钻噪声暴露前、暴露后６小时、１天、７天和１５天时ＯＨＣ的ＳＤＨ的活性变化,并在光镜下观察耳蜗改变的情况。应用扫描电镜观察电钻噪声暴露后即刻（２ｈ内）１天、７天和１５天豚鼠Ｃｏｒｔｉ器的超微结构改变。将豚鼠耳后切口置于电钻噪声连续暴露３０分钟和６０分钟。结果：听功能检测结果显示电钻噪声暴露后各组动物ＡＢＲ阈值、潜伏期和波间期较正常时略有升高,但无明显差异（Ｐ＞０．０５）。组织化学观察显示：电钻噪声连续 ６０分钟暴露后各组动物耳蜗 ＯＨＣ的 ＳＤＨ显色灰度值与正常对照组和空白对照组无明显差异（Ｐ＞０．０５）。扫描电镜观察发现各组动物耳蜗受损区域分布于外毛细胞第三排,尤以底转区和第二转明显,主要的病理变化有ＯＨＣ纤毛轻度散乱、倒伏、融合及个别脱落。结论：说明耳科电钻就当前临床应用的强度,应用时间范围内来说,     

Distribution of beta-tubulin in guinea pig inner ear

Our previous research had suggested that beta-tubulin might be an autoantigen for autoimmune inner ear disease. In this study, the expression of beta-tubulin in inner ears of normal and tubulin-immunized guinea pigs was examined by immunohistochemical staining. Strong immunoreactivity to beta-tubulin monoclonal antibody was found in stria vascularis, neurons of the spiral ganglion, cochlear nerve fibers and spiral ligament. Diffuse staining was found in the stria vascularis and the neurons of the spiral ganglion, while dense network staining was found in the spiral ligament, the nerve fibers and the vestibular end organs. The semicircular canals, endolymphatic duct and sac were also positively stained. In inner ears of guinea pigs challenged with beta-tubulin, staining intensity was diminished in the stria vascularis, the spiral ligament, and the neurons of the spiral ganglion. The results suggest that beta-tubulin is distributed to most structures of guinea pig inner ear. A challenge to the inner ear by tubulin could change the beta-tubulin distribution and cause degeneration in the spiral ganglion. The results support the hypothesis that beta-tubulin might be an autoantigen for autoimmune inner ear disease.     

Effects of reserpinization on the electrolytes distribution in inner ear fluids of guinea pig

Reserpine (2 mg/kg and 10 mg/kg, i.p.), an adrenergic blocker, was administered to normal guinea pigs, and samples of serum, CSF, scala tympani perilymph, scala vestibuli perilymph and cochlear endolymph were collected. The concentrations of Na and K in these fluids were then assessed to observe electrolyte distributions. Both Na and K concentrations were reduced in serum. In CSF, the Na concentration was markedly depressed, while the K concentration remained unchanged. Scala tympani perilymph showed a pattern of electrolyte changes similar to that in CSF. In scala vestibuli perilymph, the Na concentration was unaltered, but the K concentration was lowered. Cochlear endolymph exhibited no change of the Na concentration but there was a marked decrease in the K concentration. These changes are thought to be attributable to the indirect blockage of energy producing systems in membrane transport in the inner ear by the reserpine-induced depletion of catecholamines.     

Changes in CMDP and DPOAE during acute increased inner ear pressure in the guinea pig

During and after an increase of inner ear pressure, induced by injection of artificial perilymph, the 2f ₁ − f ₂ and f ₂ − f ₁ distortion products (DPs) in cochlear microphonics (CM) and otoacoustic emissions (OAE) were recorded in the guinea pig. An inner pressure increase of ∼600 Pa gave only small changes in CMDP and DPOAE. Along with a decrease in f ₁ amplitude, a small decrease in amplitude of the 2f ₁ − f ₂ and a small increase in the f ₂ − f ₁ were measured in CM. This matches a shift from a symmetrical position of the operating point for hair cell transduction, leading to an increase in even-order distortion and a decrease in odd-order distortion. Similar, a decrease in 2f ₁ − f ₂ DPOAE was expected. This might be the case at the generation sites but this effect was then more than compensated for by a better middle ear transfer, accounting for the increase of 0.4 dB of the 2f ₁ − f ₂ DPOAE amplitude. In conclusion, changes of overall inner ear fluid pressure have minor effects on cochlear function. This is a relevant finding for further understanding of diseases with changed inner ear fluid volumes, as Ménière’s.     

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.     

Immunolocalization of beta-lipotropin in the inner ear of the guinea pig

The occurrence of beta-lipotropin (beta-LTH) immunoreactive material was investigated in the inner ear of newborn and juvenile guinea pigs by means of Sternberger's PAP technique. Unlike met5-enkephalin and endorphin, beta-LTH could not be found in the organ of Corti but was identified in the spiral ganglion and the neuroepithelium of the crista ampullaris.     

Sensory hair fusion and glycocalyx changes following gentamicin exposure in the guinea pig vestibular organs

This study demonstrates the mechanism of sensory hair fusion and its relationship to glycocalyx on the hair cells in the vestibular epithelia of gentamicin-treated guinea pigs, using a ruthenium red staining technique. 0.1 ml of a solution containing 50 mg gentamicin sulphate per 1 ml was injected once into the middle ear. After 7 days, various stages of sensory hair degeneration were observed. The glycocalyx was clearly visualized by ruthenium red. The gentamicin-induced hair damage started with a local decrease in or loss of glycocalyx from neighbouring sensory hairs, followed by formation of local attachment areas of membrane sensory hairs. These findings suggest that the glycocalyx of the sensory cell may play an important role in separating the cilia as well as maintaining the organization of the bundle. Therefore, the decrease in or loss of glycocalyx from the hair bundle of sensory cells affected by gentamicin, either directly or secondarily, can be considered as closely related to the fusion of the sensory hairs.     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

Summary We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathologcial changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, and by a Research Grant for Specific Diseases from the Ministry of Health and Welfare to the Acute Profound Deafness Research Committee of Japan     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathological changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.     

Glycine immunoreactivity in the brainstem auditory and vestibular nuclei of the guinea pig

An immunohistochemical study was performed on the brainstem of the guinea pig, using a specific antibody against glycine. Glycine-like immunoreactivity was observed in stellate and multipolar neurons in the cochlear nucleus, in the medial and lateral nuclei of the trapezoid body and in the ventromedial periolivary cell group. No immunoreactive neurons were found in the vestibular nuclei. Positive fibre tracts were observed mainly in dorsal acoustic stria and lateral lemniscus. The results are consistent with electrophysiological and anatomical data from the literature concerning the response pattern in the fusiform layer of the dorsal cochlear nucleus and the phenomenon of binaural inhibition in the superior olivary complex.     

Expression of heat shock protein 70 in immune response of the guinea pig inner ear]

OBJECTIVE: To explore whether the immune response of the inner ear could induce heat shock protein (hsp) 70 in guinea pig cochlea. METHODS: A model of autoimmune inner ear disease (AIED) was established by systemically immunizing the guinea pig with the homologous crude inner ear antigen (CIEAg). The immunized cochleae and normal control cochleae were examined for the expression of hsp70 with techniques of immunohistochemistry and in situ hybridization. RESULTS: In the control animals, the expression of the hsp70-like protein appeared only in the spiral ganglion, whereas in the cochleae with CIEAg immunization, strong expression of the hsp70-like protein and its mRNA appeared in the spiral ganglion as well as in the stria vascularis and the spiral ligament. The hearing thresholds were significantly increased in 10 out of 28 cochleae (35.7%) with CIEAg immunization. CONCLUSION: The results suggest that the immune response of the inner ear can induce the expression of hsp70 in the guinea pig cochlea.