Experimental studies of ischemia of the cochlea. Part 1: Method]

For the etiology of certain inner ear diseases e.g. sudden hearing loss, an impaired cochlear blood flow is discussed. The model of the ferromagnetic thrombosis is an atraumatic method to produce a selective ischemia of the inner ear. The measurement of the inner ear potentials EP, MP and CAP can help to elucidate which inner ear structures are primarily impaired by local ischemia. During the action of a magnetic field to the right cochlea an intravenous injection of small iron particles leads to a thrombosis of the cochlear veins. EP, MP and CAP were measured in the thrombosed ear and MP and CAP in the undisturbed contralateral ear. The methods of microsurgery on the middle and inner ear, the electrophysiological measurement techniques of the inner ear potentials and the method of producing a ferromagnetic thrombosis are described in detail.     

Experimental studies on ischemia of the cochlea. 2. Pathobiochemistry]

Biochemical analyses of organic substances (protein, glucose, amino acids) as well as inorganic substances (potassium and sodium) in inner ear fluids were performed for the first time under local ischemic conditions. The perilymph of the thrombosed right ear was collected from various animals after different durations of ischemia. The normal left cochlea served as control. The concentrations of potassium and sodium and the total protein content remain unchanged in the perilymph of the guinea pig during the first two hours of ischemia. The decrease in the glucose concentration in the perilymph shortly after the thrombosis is a new finding. It points to the possible existence of a reservoir of energy carriers in the fluid of the inner ear that become available to cochlear tissues during ischemia. The increase in the amino acid concentration during ischemia suggests metabolic processes in the cochlea. The biochemical analysis of the perilymph during ischemia of the cochlea provides evidence of two possible mechanisms that secure the survival of the organ of Corti. The glucose reserves of the perilymph are utilized by the cochlear tissues and the concentration of certain amino acids secure the supply.     

Vulnerability of the gerbil cochlea to sound exposure during reversible ischemia.

Cochlear ischemia induces a sensorineural hearing loss, in part through a fast functional impairment of outer hair cellls. Assuming that the cochlea is rendered fragile during ischemia and reperfusion and that stimulation itself can jeopardize its functional recovery, we used a model of reversible selective cochlear ischemia in Mongolian gerbils to establish what type of sound exposure can be deleterious during and immediately after reversible ischemia. Several groups of gerbils were used, with different ischemia durations and levels of sound exposure. Control groups were only exposed to tones at 80 and 90 dB SPL during 30 min, while other groups underwent complete and fully reversible blockage of the labyrinthine artery, during 5.5 or 8 min, and were exposed to 60 or 80 dB SPL tones during 30 min. The amount of ischemia and reperfusion was measured by means of laser Doppler velocimetry, whereas outer hair cells' function was continuously monitored through distortion-product otoacoustic emissions (DPOAEs). The losses of DPOAE levels after 8 min transient ischemia and 60 dB SPL exposure were as large as those induced by 80 dB SPL exposures combined with 5.5 min ischemia, or 90 dB SPL exposures without ischemia, with a maximum loss around 25-30 dB, half an octave above the stimulus frequency. These results give evidence for an extremely high cochlear vulnerability to low-level sound exposure when associated with reversible ischemia. This vulnerability may have important clinical consequences in patients with cochlear circulatory disturbances.     

Noise and the RNA synthesis of the cochlea. Autoradiographic studies.

In cochlear basal turns of 22 guinea pigs with autoradiography, grain density was measured after injection of 3H-cytidine. Following high initial nuclear labelling, a low incorporation in cytoplasm was observed, even after 24 hours. Synthesis of ribosomal RNA in the organ of Corti seems to be slow. Low grain densities in the whole cochlea presuppose a blood-lymph barrier to cytidine. After white noise (85 dB SPL, 12 hours) were seen a more rapid incorporation into nuclei and after 24 hours an increased labelling of cytoplasm of ganglion cells, fibrocytes and inner hair cells, less of outer hair cells. This might be induced by increased blood flow, concentration of the precursor in the lymphs, cell permeability of alterations of the cellular nucleotide pool. But a real stimulation of cochlear RNA synthesis seems to be the most likely. On comparing different turns, a peak in grain density was found over nuclei of the organ of Corti after 1 hour in the 2nd turn. After 24 hours, cytoplasmic labelling showed a decrease only in outer hair cells towards apical turns.     

Incorporation of D-[3H]-glucosamine and L-[3H]-fucose into the developing rat cochlea.

The uptake of two tritiated carbohydrates, D-[3H]-glucosamine and L-[3H]-fucose, to the developing rat cochlea was examined using light and electron microscopic radioautography. Both carbohydrates, administered to in vitro developing rat cochleas, shared a similar ultrastructural labeling pattern on the microvilli and apical cell region and on the tectorial membrane (TM) fibrils. On embryonic day 18, the radiolabeling appeared on the apical surface of the undifferentiated epithelium that will develop into both spiral limbus and K?lliker's organ (KO), while on postnatal day (PD) 1, it was only located on the apical surface of the KO. When D-[3H]-glucosamine was administered in vivo to newborn rats, the radiolabeling was observed in the TM covering the KO at PD 3. Lastly, D-[3H]-glucosamine administered in vivo to PD 7 rats, appeared at PD 9 in the TM region lying just above the organ of Corti. The present findings support the previously suggested leading role of the spiral limbus and KO in the secretion of the TM during cochlear development. The secretion of carbohydrates, and probably of other matrix components, starts on the spiral limbus and KO region and progressively extends to the organ of Corti.     

Expression of the major mammalian stress protein in the rat cochlea following transient ischemia.

The transient expression of stress proteins in cells exposed to adverse environmental conditions is thought to provide a survival advantage. In the present study, the expression of the major mammalian stress protein, Hsp72, was characterized in the rat peripheral auditory system following varying periods of unilateral cochlear ischemia and reperfusion. Western blot analysis of whole cochlear homogenates revealed Hsp72 expression after as little as 5 minutes of cochlear ischemia. Cochlear Hsp72 immunoreactivity following 10 minutes of ischemia was observed within 2 hours, remained evident through 6 hours, and decreased toward control levels by 12 hours. Preliminary immunocytochemical localization data revealed Hsp72 expression in the outer hair cells. The characterization of Hsp72 expression following transient ischemia represents the first step toward understanding the role that stress proteins have in the protection of the mammalian auditory system.     

Flexible fiberoptic endoscopy of the cochlea: human temporal bone studies

New bone formation within the cochlea has been found more frequently than expected during cochlear implantation. Although implantation of partially ossified cochleas with long multichannel electrodes is possible, in practicality it may be difficult to determine whether electrode insertion is feasible prior to attempting it. In spite of use of high resolution computerized tomography, the presence, nature, and extent of new bone formation in the scala tympani is often difficult to ascertain. Flexible fiberoptic otologic endoscopes, 0.7 mm and 1.0 mm in diameter, were developed for the purpose of visualizing the fluid-containing scalae of the cochlea. In order to determine the efficacy of the device in visualizing the interior of the cochlea, as well as determining possible damage caused by its use, ten formalin fixed human temporal bones were studied. The flexible fiberoptic otologic endoscope was found to be capable of passage through the scala tympani of the cochlea in the inferior segment and of identifying normal and abnormal structures. Passage beyond this point resulted in damage to the spiral ligament. We conclude that human trials of use of the endoscope during cochlear implantation on previously deafened individuals is feasible.     

Electrophysiology of the cochlea.

With careful techniques it is possible to demonstrate three electric potential regions of the inner ear: an intracellular negative potential, a high positive potential in the endolymph that appears bounded at the scala media side of the tectorial membrane rather than at the reticular lamina, and the extracellular spaces, which contain a fluid at near zero potential. The tectorial membrane is also found to be at zero potential. Further research may yet uncover more details of the resting potential distribution as we become expert at placing microelectrodes and learn better ways of correlating potential with position. Surely the study of these resting potentials would contribute to our understanding of the mechanisms of fluctuant hearing loss because these first order potentials are intimately related to the state of health of the inner ear.     

心钠素对耳蜗缺血再灌注损伤的影响

目的观察心钠素对豚鼠耳蜗缺血再灌注损伤的影响。方法将豚鼠分为4组：实验组（A1、B1）及对照组（A2、B2）。采用造血栓后溶栓的方法制备耳蜗缺血再灌注模型。实验组A1在建模前10min静脉注射心钠素，实验组B1在再灌注即刻静脉注射心钠素，对照组（A2、B2）在相应时间静脉注射等量生理盐水。实验过程中采用激光多普勒血流量仪监测耳蜗血流量（cochlear blood flow，CoBF）并测定豚鼠听性脑干反应（auditory brainstem response，ABR）值。结果缺血前实验组A1的CoBF较对照组A2高，再灌注后至实验结束，2组CoBF值未见明显差别。实验组B1和对照组B2用药前的CoBF无明显差别，再灌注后对照组B2恢复到实验开始时的70％左右，而实验组B1恢复到实验开始时相同水平。缺血前4组听阈差异无统计学意义。缺血30min时，实验组A1的听阈较对照组A2低（t=7．761，P〈0．05）。实验组B1和对照组B2听阈差异无统计学意义。再灌注30min和60min时，实验组A1与对照组A2差异无统计学意义。实验组B1比对照组B2显著降低（t值分别为9．846和19．242，P值均〈0．05）。结论缺血再灌注后即刻应用心钠素，可以减轻耳蜗的缺血再灌注损伤，可以在增加耳蜗血流的同时降低听反应阈。为临床内耳微循环障碍疾病提供一种新的药物治疗方法。     

Acoustically induced hearing loss: Intracellular studies in the guinea pig cochlea

Intracellular recordings were made from both inner and outer hair cells (IHC. OHC) in the basal coil of the guinea pig cochlea before, during and after the animal was exposed to loud, pure tones. Following multiple loud tones both types of sensory cells demonstrate a culmulative decrease in their voltage responses to a test tone. A loss in sensitivity of the compound action potential (CAP) of the eighth nerve co-incides with a decrease in both the amplitude of the IHC receptor potential and the positive summating potential (+SP) recorded at the round window. The largest decreases in sensitivity of IHCs are found at the characteristic or best frequency (CF) of each cell and this frequency selective loss of sensitivity results in a decrease in the tuning of the hair cell. During and following a loud tone the nonlinear properties of IHCs are also reduced.     

The early postnatal development of the hamster cochlea]

The Early Postnatal Development of the Hamster Cochlea.Inborn deafness can be diagnosed very early. Deafness in early childhood can be caused by: genetic defects (30 % - 40 %), embryopathies, embryonic noxae (60 %) and pre-, peri- and postnatal noxae. Which structures of the cochlea are disturbed, is unknown in most cases. Systematic studies are necessary with directed experimental impairments of the cochlea in the early development to elucidate these mechanisms. An animal model has to be established in which directed impairments can be carried out in single individuals. The cochlea of the hamster is at birth in a very early state of development. In the apical turn the epithelial layer of the cochlear duct is undifferentiated, whereas in the basal turn the papilla basilaris (K?lliker's organ), the lateral wall and Reissner's membrane are visible. Between the base of the cochlea (1st coil lateral) and the apical part (3rd coil lateral) the difference in the development of the hamster cochlea is exactly four days. This is evident for the 1st and the 5th day after birth. The cochlea development of the hamster is very rapid. The development during one day represents one week of development in the human cochlea. The difference in development in one hamster cochlea from the apex to the base at a certain day representing four days of development is comparable with the development time of four weeks in human. The different developmental stages which are present in the hamster cochlea of a certain day represent the development of the human cochlea of a whole month. The hamster cochlea at the 1st day after birth covers the 2nd embryonic month of the human (4th to 8th week) and at the 5th day the 3rd embryonic month (8th to 12th week). The hamster cochlea at the 1st and the 5th day after birth is especially suitable to study experimental disturbances in the early stages of the development of the cochlear duct when the tectorial membrane, the spiral limbus, the stria vascularis, Reissners membrane and the external spiral Sulcus with the root cells are differentiating. The biggest advantage is that the noxae (hypoxia, teratogenic substances, ototoxic antibiotics and intense noise) can be applied to single individuals which offers better control than the treatment of the mother.     

Susceptibility of the hair cells of the newborn rat cochlea to hypoxia and ischemia

Hypoxia and ischemia are thought to be important pathogenetic factors in bringing about hearing loss. In order to study the effect of these determinants on the loss of inner and outer hair cells (IHCs/OHCs), we used an in vitro hypoxia and ischemia model of the newborn rat cochlea. The specimens of the organ of Corti were exposed either to hypoxia (10-20 mm Hg) or to normoxic glucose deprivation or to both (ischemia) in artificial perilymph for different exposure periods. The number of IHCs and OHCs was counted and the hair cell loss was compared to controls. Normoxic aglycemia did not cause significant hair cell loss as compared to controls. Hypoxia and ischemia led to hair cell loss in a dose-dependent manner, with the loss in the ischemia groups found to be markedly higher than that in the hypoxia groups. Hypoxia resulted in a mean loss of 8% OHC and of 14% IHC after an 8-h exposure. Ischemia increased the loss to 19% OHC and 39% IHC after the same exposure period of 8 h. Our findings suggest that IHCs are more susceptible to hypoxia/ischemia than OHCs.     

3D-finite element model of the human cochlea including fluid-structure couplings.

The propagation of acoustic waves in the inner ear in vivo could not be quantified completely yet. This is in particular true in conjunction with the micromechanical structures of the organ of Corti, though these data are important for the explanation and discussion of clinical measurements like otoacoustic emissions and auditory brainstem responses. To access these problems a three-dimensional mechanical model of the cochlea including the fluid-structure couplings is developed and evaluated numerically by finite elements. Although the complex cochlear partition is covered by passive mechanical elements, the results fit early experiments (1928), which studied the wave propagation in the cochlea with fresh human cadavers [G. von Békésy: Experiments in Hearing. New York, McGraw-Hill, 1960]. Additionally it is now easy to calculate the mechanical input impedance of the cochlea. These results agree with recent experiments [S.N. Merchant et al.: Hear Res 1996;97:30-45].     

Sexual dimorphism and development of the human cochlea. Computer 3-D measurement.

The length of the cochlear turns was measured in 9 pairs of temporal bones from age-matched male and female individuals (1 day to 76 years old), using a computer-aided three-dimensional reconstruction and measurement method. The mean cochlear length was significantly longer (Two-way analysis of variance, F = 31.87, d.f. = 1, p less than 0.01) in males (37.1 +/- 1.6 mm) than in females (32.3 +/- 1.8 mm), whereas it did not vary with postnatal age in either sex. Sexually dimorphic cochlear length may pose a new issue in auditory physiology in man. The lack of postnatal elongation also indicates that length of the cochlea becomes close to its maximum during fetal life.