A new immunohistochemical method for the detection of gentamicin in inner ear fluid compartments

A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immunohistochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri-and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.     

Chemical composition in various compartments of inner ear fluid.

Sodium, potassium, chloride, glucose and total protein were determined in samples of scala vestibuli perilymph, scala tympani perilymph, CSF, cochlear endolymph and utricular endolymph from normal cats. Small but significant differences were evident in the concentrations of sodium and potassium between the scala vestibuli and scala tympani perilymph. It was also apparent that each compartment of endolymph has different values for sodium and potassium concentrations. Compared with the endolymph, the perilymph was found to contain a higher concentration of glucose and total protein. These findings are discussed from the view point of biological significance.     

Species differences in inner ear fluids

Summary Inner ear fluids of guinea pigs and cats were analyzed for sodium, potassium, chloride, glucose, and total protein to determine species differences in chemical compositions. In the scala vestibuli perilymph and scala tympani perilymph, sodium, potassium, and choride levels in the guinea pig were lower than in the cat. The protein levels in the scala vestibuli perilymph and scala tympani perilymph of the guinea pig were lower than those of the cat. The glucose levels in the guinea pig were higher in the scala vestibuli perilymph and scala tympani perilymph, as compared to findings in the cat. Regarding the utricular endolymph, there were significant differences between guinea pigs and cats in sodium and potassium concentrations; the concentration in the former being higher in sodium and lower in potassium. These findings are pertinent for the phylogenetic studies on inner ear fluid biochemistry.This work was supported by Research Grant No. ROI-NS1026801 from the National Institute of Neurological Diseases and Strokes, USA     

Transmission of cerebrospinal fluid pressure changes to the inner ear and its effect on cochlear microphonics

Summary Alterations in inner ear fluid pressure and cochlear microphonics (CM) associated with increased cerebrospinal fluid (CSF) pressure were studied in the guinea pig. Hydrostatic pressure in the endolymph and perilymph of the cochlea were measured by use of a servo-controlled micropipet system. Endolymphatic and perilymphatic pressure increased in a linear manner with little or no time lag following pressure increases of 0–50 mm Hg. There was no pressure gradient between the endolymph and perilymph. CM in response to 500 Hz and 4000 Hz tone bursts was recorded before, during and after CSF pressurization. The input-output function of CM showed suppression of 2–8 dB in both maximum output and sensitivity unless the increase in CSF pressure exceeded a level which reduced the cochlear blood flow.     

内耳畸形伴脑脊液耳漏的高分辨率CT特征

目的:探讨先天性内耳畸形伴脑脊液耳漏的高分辨率CT(HRCT) 特征.方法:回顾性分析11例先天性内耳畸形伴脑脊液耳漏患者的HRCT表现.结果:共11例患者,耳蜗未发育4例,共腔畸形2例,耳蜗未未分隔型3例,耳蜗不全分隔型2例.内耳道底骨质缺损8例.所有患者患侧乳突气化和发育程度与对侧一致,前庭窗处都有软组织影.结论:内耳畸形伴脑脊液耳漏患者常存在内耳道底骨质缺损.前庭窗是脑脊液由内耳进入中耳腔的好发部位.HRCT能为先天性内耳畸形伴脑脊液耳漏患者的诊断及治疗提供可靠依据.     

Paracellular transport properties of inner ear barriers do not account for cisplatin toxicity in the rat

The interindividual variability for the ototoxic effect of the antineoplastic drug cisplatin has still to be explained. To examine if the variability can be related to differences in drug kinetics, the effect of cisplatin on the paracellular transport properties of the inner ear barriers was studied in vivo in cisplatin treated Long-Evans rats. The concentration of [³H]mannitol was followed in plasma, scala vestibuli perilymph, and endolymph after an intravenous infusion of the tracer. Cisplatin had no effect on paracellular transport of the inner ear barriers 3 days after administration of 8 mg/kg cisplatin. However, an interindividual variability for the transport of [³H]mannitol across the blood-perilymph barrier was evident, indicating a variability for the passive transport of solutes to the inner ear.     

The influence of Er. YAG laser application in fenestration to the inner ear

OBJECTIVE: Erbium (Er.) YAG laser may be usable for middle ear surgery because of its ability to ablate bony tissue. We investigated the inner ear damage caused by the fenestration to the inner ear with Er. YAG laser. DESIGN: We investigated the influence of Er. YAG laser on the inner ear using electrophysiological technique. RESULTS: Several cases had a decrease in endocochlear potential (EP) and cochlear microphonics (CM) after the fenestration to the inner ear. CONCLUSIONS: Er. YAG laser is safe if it is used for the small and superficial fenestration to the stapes footplate. However, a few extra pulses after fenestration are dangerous.     

The preparation of acetic acid for use in otic drops and its effect on endocochlear potential and pH in inner ear fluid

The ototoxicity of an otic drop preparation containing 2% acetic acid and 3% propylene glycol (VoSol, Denver Chemical Co., Humacao, PR) was investigated according to measurements of endocochlear potential (EP) and inner ear fluid pH. The application of this preparation to the round window membrane for 30 minutes caused a depression in EP from 80.5 +/- 2.5 mV (mean +/- SD; n = 6) to 11.7 +/- 7.7 mV, and lowered inner ear fluid pH from 7.55 +/- 0.09 to 5.06 +/- 0.19 (n = 6) in perilymph and from 7.52 +/- 0.07 to 5.88 +/- 0.63 (n = 6) in endolymph. Two percent acetic acid produced similar changes after 30 minutes: EP was reduced from 83.0 +/- 2.2 mV to 34.0 +/- 2.9 mV and endolymphatic pH from 7.49 +/- 0.04 to 6.83 +/- 0.21 (n = 4). However, the application of artificial perilymph of pH 4 titrated with HCl induced no significant changes in either EP or endolymphatic pH. We suggest that the mechanisms of ototoxicity in the otic drop preparation are Na+ and K+-ATPase inhibition, and that such inhibition is due to the intracellular acidification of strial cells resulting from the penetration of acetic acid across the cell membrane, and to the direct and synergistic actions of propylene glycol.     

Melanin in the inner ear

Summary Following several studies on the effects of kanamycin toxicity on the inner ears of guinea pigs, we have studied the importance of melanin in this phenomenon. Transmission electron microscopy showed that, under the influence of kanamycin, the intermediate strial cells developed a secretory aspect similar to that seen in skin melanocytes. This aspect as yet has never been described for the inner ear cells. A planimetric, morphometric method was also used to determine the strial cell melanin status in control animals. Additional findings in the study confirmed an increase in the number of melanosomes during kanamycin poisoning. Statistical data are discussed.Presented at the First European Congress of Oto-Rhino-Laryngology and Cervico-Facial Surgery, Paris, 26–29 September 1988     

Cerebrospinal fluid otorrhea in a patient with congenital inner ear dysplasia

A case of a 10-month-old boy with a cerebrospinal fluid (CSF) fistula in his right ear is reported. In the same ear, the patient also showed congenital inner ear dysplasia. The CSF fistula was obstructed surgically. Surgical exploration showed a fistula superior to the oval window and a normally shaped stapes. The stapes was removed and the fistula was closed by obliteration of the vestibulum with the temporal fascia and fat tissue. The location of the fistula was very rare; to our knowledge, this is the first reported case of CSF fistula superior to the oval window. The relationship between perilymphatic fistula and the microfissure revealed by temporal bone study is discussed.     

The effect of blood pressure on inner ear blood flow

Summary We investigated the effects of blood pressure on the blood flow in the inner ear as compared with other organs in normotensive rats (NR), spontaneously hypertensive rats (SHR) and renal hypertensive rats (RHR). The cardiac output and the regional blood flow were measured by a radioactive microsphere technique in conscious rats. The haemodynamics of the inner ear and other organs and the effects of a converting-enzyme inhibitor (SA-446) on the regional blood flow were compared among NR, SHR, and RHR, the blood pressure being 107, 152, and 185 mm Hg, respectively. With the exception of the heart, the organ blood flow was lower in SHR than in NR in all organs, including the inner ear. It was also lower in RHR than in SHR. The blood pressure dropped after i.v. injection of SA-446, while the regional blood flow increased. The effect was strongest in RHR, followed by SHR and finally NR.     

Immunolocalization of tenascin in the chinchilla inner ear

Tenascin was immunolocalized in the chinchilla cochlea and vestibular system to better understand the functional morphology of the inner ear. Inner ear tissues were fixed with acetone, decalcified and cryosectioned. Indirect immunofluorescence, using antibodies directed against human tenascin epitopes, were used to detect tenascin. As a positive control, tenascin immunoreactivity was found in kidney, cortical mesangial cells and the extracellular matrix of glomeruli and medullary tubule interstitial spaces, concurring with previously reported results. In the cochlea, tenascin immunoreactivity was present in osteocytes, the mesothelial cells underlying the basilar membrane (BM) and within the fibrous matrix of the BM. Greater reactivity was observed in the mesothelial cells than in the fibrous matrix of the BM. In the vestibular system, tenascin immunoreactivity formed a diffuse band directly beneath the basal lamina of the ampullary and otoconial organs. Tenascin immunoreactivity was also observed in cup-shaped regions between the type I vestibular hair cells and their surrounding VIII nerve calyces in the ampullary and otoconial organs. This is the first report of the anatomical distribution of tenascin in the adult, mammalian inner ear, other than our previously published abstract P.A. Santi and D. Swartz, Soc. Neurosci. Abstr. 23 (1997) 731.     

Wideband tympanometry findings in inner ear malformations

ObjectiveThe deficits in the cochlea which is at the one end of the ear sound transfer system, may effect middle ear functions. Wideband typanometry (WBT) is frequently used to evaluate these transfer functions which play a crucial role in setting the impedance matching between the external ear and the cochlea. To this end, the aim of this study was to investigate the ear transfer functions in inner ear malformations via WBT, and to question whether these functions change depending on the types of inner ear malformation.MethodsThis prospective case-control study was conducted in a university hospital. One hundered-fifty-seven ears (aged 3–37 years) under the groups of cochlear hypoplasia, incomplete partition I, incomplete partition II, cochlear aplasia and complete labyrinthine aplasia were evaluated. In the control group, 30 ears with normal hearing were enrolled and WBT was carried out. Tympanometric peak pressure, equivalent middle ear volume, static admittance, tympanogram width, resonance frequency, average wideband tympanometry and absorbance measurements were analyzed.ResultsThe inner ear malformation groups demonstrated statistically significant differences than the control group and from each other in terms of traditional tympanometric parameters and WBT test parameters (p < 0.05). The most remarkable difference was between the group of complete labyrinthine aplasia and the control group, most probably because of complete labyrinthine aplasia’s structural effects. However, on some parameters, incomplete partition II and the control group showed similarities. In absorbance measurements, there was significant difference between all patient groups and the control group, especially at high frequencies (p < 0.05). The largest difference was between the control group and the group of complete labyrinthine aplasia which has revealed the lowest absorbance values (p < 0.05). In averaged-wideband tympanogram analysis, all patient groups obtained a lower amplitude peak than the control group; complete labyrinthine aplasia group had the flattest peaked amplitude, while the incomplete partition II group had a near-normal curve.ConclusionThe results of the study revealed the distinctive effects of inner ear malformations in middle ear transfer functions. It is concluded that the absence of inner ear structures causes negative effects on energy absorbance and the other transfer functions of the middle ear. WBT may provide additional information on diagnosis of patients with inner ear malformations.     

先天性内耳畸形并中耳畸形患者多通道人工耳蜗植入术3例报告

目的：探讨先天性内耳畸形并中耳畸形患者行多通道人工耳蜗植入术的方法及效果。方法：1995年5月-2002年5月我院为3例罕见的先天性内耳畸形并中耳畸形患者经乳突进路行人工耳蜗植入术,分别植入27、28、32个电极,植入后3个月行声场测听。结果：3例患者均成功地行人工耳蜗植入,术中、术后无严重并发症发生,术后声场测听听阈达35-40dBHL。结论：罕见的先天性内耳畸形并中耳畸形患者也可行人工耳蜗植入术,术后效果满意。     

Inner ear fine structures of the hamster in frozen sections used for immunohistochemical assays for inner ear diseases

Summary Frozen sections of the inner ear of the hamster enable detailed investigations of the fine structures in immunofluorescence assays. At high magnification single mitochondria can be identified by their reactions with an antiserum containing antibodies against mitochondria. In the positive reaction with an antiserum against nuclei, the typical green fluorescence is restricted to the nuclei, which are mostly separated by the surrounding cytoplasm. The method of immunohistochemical assay using frozen sections from the non-decalcified inner ear is very time-consuming and cannot be recommended for the routine diagnosis of inner ear diseases, although it may be useful in research and for studying critical clinical cases. Offprint requests to: N. R. Wei     

Otoconin 90蛋白在豚鼠内耳的表达

目的观察Otoconin 90蛋白在内耳的表达情况,探讨其在耳石代谢中的意义。方法采用免疫组织化学SP方法 (streptavidin-perosidase,链霉菌抗生物素蛋白-过氧化物酶连结法)检测正常豚鼠内耳切片各主要部位Otoconin 90表达情况。结果 Otoconin 90蛋白不只在椭圆囊和球囊囊斑的感觉上皮有表达,在与其临近和相对位置的非感觉上皮,以及壶腹、半规管和耳蜗等多个部位均有丰富表达,在椭圆囊和球囊囊腔、半规管内等也有染色阳性物质出现。结论 Otoconin 90蛋白在内耳呈现出"多点"表达的特点,提示内耳多处产生Otoconin 90,可能最后转运至椭圆囊和球囊囊斑,在此与钙离子相互作用形成耳石。     

The presence of laminin in the fetal human inner ear

Summary The expression of laminin was analyzed in the human fetal inner ear using immunohistochemical methods. In the 11-week-old human fetus, the presence of laminin was found in the basement membrane of the immature cochlea, endolymphatic sac and vestibular end organs. The reaction of the basement membrane of the endolymphatic sac was strong in the 15-week-old human fetus. A laminin reaction was seen in the cochlea, Reissner's membrane, epithelial cells of the limbus spiralis, the basilar membrane and the stria vascularis. In particular, the capillaries and basement membrane of the stria vascularis were strongly positive. These results suggest that laminin may be an essential component in the development of the inner ear and may possibly be related to filtration of the endolymph.     

The fine structure of lipofuscin in the human inner ear

Summary Lipofuscin inclusions in the human membranous labyrinth were studied by electron microscopy. Lipofuscin is morphologically an irregularly shaped, membrane-bound inclusion consisting of an electron-dense structure. The most common component was a fine, granular, osmiophilic substance which was always associated with a homogenous, spherical structure resembling a lipid droplet. The combination of these two components was frequently observed in the human inner ear. Distended inclusions containing lipofuscin components were also observed within the supporting cells, saccular, utricular and ampullar wall, the epithelial cells of the transitional zone and in the dark cells. Lipofuscin is closely associated with lysosome and is known to accumulate in the tissue as a result of aging. The high lysosomal activity possibly may result in lipofuscin formation in the human inner ears. Also some other unknown metablic conditions may provide the deposits of lipofuscin.I wish to express my thanks to Professor Nobuo Ueda of the Biochemistry Department for his advice and to Mr. Dominic W. Hughes for English editing of this paper.