Guinea Pig Endolymphatic Sac Research Articles

Is cochlear synapse loss an origin of low-frequency hearing loss associated with endolymphatic hydrops?

There is a strong association between endolymphatic hydrops and low-frequency hearing loss, but the origin of the hearing loss remains unknown. A reduction in the number of cochlear afferent synapses between inner hair cells and auditory nerve fibres may be the origin of the low-frequency hearing loss, but this hypothesis has not been directly tested in humans or animals. In humans, measurements of hearing loss and postmortem temporal-bone based measurements of endolymphatic hydrops are generally separated by large amounts of time. In animals, there has not been a good objective, physiologic, and minimally invasive measurement of low-frequency hearing. We overcame this obstacle with the combined use of a reliable surgical approach to ablate the endolymphatic sac in guinea pigs and create endolymphatic hydrops, the Auditory Nerve Overlapped Waveform to measure low-frequency hearing loss (≤ 1 kHz), and immunohistofluorescence-based confocal microscopy to count cochlear synapses. Results showed low- and mid-(1–4 kHz) frequency hearing loss at all postoperative days, 1, 4, and 30. There was no statistically significant loss of cochlear synapses, and there was no correlation between synapse loss and hearing function. We conclude that cochlear afferent synaptic loss is not the origin of the low-frequency hearing loss in the early days following endolymphatic sac ablation. Understanding what is, and is not, the origin of a hearing loss can help guide preventative and therapeutic development.

Localization of basement membrane components in the guinea pig endolymphatic sac

Localization of basement membrane components in the guinea pig endolymphatic sac

Expression of Aquaporins in the Cochlea and Endolymphatic Sac of Guinea Pig

It has been shown that the aquaporin (AQP) family forms membrane pores selectively permeable for water and some small solutes such as glycerol and urea, and thus plays important roles in regulating the fluid in many organs involved in fluid transport such as kidney, lung and brain. The aims of the present study were to investigate the cellular localization and its significance of aquaporins (AQPs) in various subregions of the cochlea and endolymphatic sac of guinea pig. The expression patterns of AQP1, 2, 3 and 4 were immunolabeled with the specific polyclonal rabbit antibodies against the rat AQP1, 2, 3 and 4. Our immunohistochemical examination showed that in the cochlea, AQP1, 3 and 4 were widely distributed in various locations including stria vascularis, spiral ligament, the organ of Corti and spiral ganglion in the similar patterns except that AQP3 in the stria vascularis was lightly weaker than AQP1 and AQP4. AQP2 was labeled only in Reissner’s membrane. In the endolymphatic sac, AQP1, AQP3 and AQP4 were strongly expressed in the epithelial cells and subepithelial cells similarly with the exception that AQP3 was lightly weaker than AQP1 and AQP4. No AQP2 immunoreactivity was detected in the endolymphatic sac. Theses results suggest that different members of the AQP family in the labyrinth may work in concert to regulate endolymph and to maintain homeostasis in the inner ear.

Quantitative anatomy of the guinea pig endolymphatic sac

The endolymphatic sac is believed to represent one of the primary loci for endolymph volume regulation in the inner ear. Quantitative analysis of physiologic measurements from the endolymphatic sac requires knowledge of the anatomy of the structure, specifically the luminal volume and the variation of cross-sectional area with distance along the sac. Recently techniques have become available to make these measurements. In the present study, fixed, isolated specimens of the guinea pig endolymphatic sac were imaged by high-resolution magnetic resonance microscopy (MRM) or by histological serial sections. Structures were reconstructed and quantified using image analysis software. In specimens imaged by MRM the endolymphatic sac volume, including tissue and lumen, was 359 nl for the intraosseous region and 106 nl for the extraosseous region, totaling 465 nl for the entire structure. The luminal volumes were 131 nl for the intraosseous region and 13 nl for the extraosseous region, totaling 144 nl. In histological specimens the volume, including tissue and lumen, was 414 nl for the intraosseous region and 121 nl for the extraosseous region, totaling 535 nl for the entire structure. The luminal volumes were 152 nl for the intraosseous region and 26 nl for the extraosseous region, totaling 179 nl. Differences in volume estimates obtained by the two methods were not statistically significant and variation was dominated by inter-specimen variation. Pooling the data, the total volume of the endolymphatic sac in the guinea pig including tissue and lumen was 506 nl (S.D. 100, n=17) and the volume of the lumen was 169 nl (S.D. 48, n=14).

Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the hydropic cochlea of guinea pigs – second report

Apoptosis was induced in the cochlea by the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac of guinea pigs and immunohistochemically examined. Keyhole limpet hemocyanin was injected into the right endolymphatic sac. The temporal bones were fixed via cardiac infusion of fixative and immunohistochemically stained for caspase-activated deoxyribonuclease or caspase 3. Endolymphatic hydrops became evident in the cochlea 1 day after the injection of keyhole limpet hemocyanin (n=6). The temporal bones in the control group did not show any caspase-activated deoxyribonuclease or caspase 3 immunoreactivity (n=6). Immunoreactivity for caspase 3 was detected in the supporting cells of the organ of Corti, the stria vascularis and the spiral ganglion cells. Caspase-activated deoxyribonuclease was also detected in the same areas. These findings suggest that apoptosis is involved in the pathogenesis of endolymphatic hydrops. This phenomenon could lead to cochlear dysfunction, as seen in endolymphatic hydrops.

Apoptosis in the hydropic cochlea of guinea pigs following immune reaction of the endolymphatic sac: immunohistochemical analysis.

Apoptotic change in the cochlea was studied by immunohistochemistry after the injection of keyhole limpet haemocyanin (KLH) into the right endolymphatic sac of guinea pigs. Apoptosis was examined with the specific antibody to single-stranded DNA (ssDNA). Endolymphatic hydrops became evident in the cochlea 1 day after the injection of KLH (n = 6). Increased ssDNA expression could be detected in the spiral ligament and the stria vascularis. The temporal bones in the control group did not show any ssDNA immunoreactivity (n = 6). Apoptosis is the process of the cell death. Our findings imply that apoptotic changes are involved in endolymphatic hydrops. These phenomena could lead to cochlear dysfunction as seen in endolymphatic hydrops.

Calcium-sensitive nonselective cation channel identified in the epithelial cells isolated from the endolymphatic sac of guinea pigs.

We identified a Ca2+-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 +/- 1.3 pS (n = 8) in our standard solution. The relative ionic permeability of the channel was in the order K+ = Na+ > Ca2+ >> Cl-. This channel was weakly voltage-dependent but was strongly activated by Ca2+ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca2+ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 microm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 microm), or ATP (1 mm), which increased intracellular Ca2+ concentration ([Ca2+]i), activated the channel at an estimated [Ca2+]i from 0.6 microm to 10 microm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca2+ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na+ under certain pathological conditions that will increase [Ca2+]i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells.

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the hydropic vestibule after injection of keyhole limpet hemocyanin into the endolymphatic sac of guinea pigs

This study was undertaken to examine the expression of inducible nitric oxide synthase (iNOS / NOS II) in the hydropic vestibule of guinea pigs. Animals were systemically sensitized with 500 microg of keyhole limpet hemocyanin. Two weeks after the first injection, keyhole limpet hemocyanin (100 microg/5 microl) was injected into the endolymphatic sac following the intradural approach, and the next day temporal bones were removed for the immunohistochemical examination. Endolymphatic hydrops was evidenced by the expansion of the Reissner's membrane in the cochlea after direct injection of keyhole limpet hemocyanin into the endolymphatic sac. Inducible nitric oxide synthase expression was increased in the sensory cells, supporting cells and vestibular ganglion cells, while temporal bones, where only phosphate buffered saline was injected, did not show any inducible nitric oxide synthase immunoreactivity. High levels of inducible nitric oxide synthase-catalyzed nitric oxide were detected prior to the development of the inner ear dysfunction. Our results suggest that the occurrence of inducible nitric oxide synthase immunoreactivity parallels the inner ear disturbance as seen in endolymphatic hydrops.

Expression of Inducible Nitric Oxide Synthase in the Cochlea following Immune Response in the Endolymphatic Sac of Guinea Pigs

Immunohistochemical study for inducible nitric oxide synthase (iNOS or NOS II) in the cochlea of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac. Morphological changes were observed in the cochlea of all animals after the injection of KLH. Increased iNOS expression was detected in the lateral wall, organ of Corti and ganglion cells. It is known that high levels of nitric oxide can lead to inner ear dysfunction. Our results suggest that iNOS may mediate the inner ear disturbance as seen in endolymphatic hydrops.

Effect of locally applied drugs on the pH of luminal fluid in the endolymphatic sac of guinea pig.

The aim of the present work was to assess the effect of various drugs applied locally on the pH of the luminal fluid (pH(lum)) in guinea pig endolymphatic sac. pH(lum) and transepithelial potential, when measured in vivo by means of double-barrelled pH-sensitive microelectrodes, were 7.06 +/- 0.08 and +6.1 +/- 0.34 mV (mean +/- SE; n = 84), respectively, which is consistent with a net acid secretion in the luminal fluid of the endolymphatic sac. Bafilomycin and acetazolamide increased and decreased, respectively, pH(lum). Amiloride, ethylisopropylamiloride, ouabain, and Schering 28080 had no effect on pH(lum). Results obtained with inhibitors of anionic transport systems were inconclusive; e.g., DIDS reduced pH(lum), whereas neither SITS nor triflocin had any effect. We conclude that bafilomycin-sensitive H(+)-ATPase activity accounts for the transepithelial acid gradient measured in the endolymphatic sac and that intracellular and membrane-bound carbonic anhydrase probably participates in regulating endolymphatic sac pH(lum). The relationship between acid pH, endolymph volume, and Ménière's disease remains to be further investigated.

Detection of single-stranded DNA in the hydropic vestibule after the direct injection of antigen into the endolymphatic sac of guinea pigs

Immunohistochemical study for single-stranded DNA (ssDNA) with vestibule of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the right endolymphatic sac. Endolymphatic hydrops became evident by expansion of the Reissner's membrane in the cochlea of all animals 1 day after the injection of KLH. Increased ssDNA expression was detected in the sensory epithelium and transitional area, while temporal bones in the control group did not show any ssDNA immunoreactivities. ssDNA is accompanied with the apoptotic change in the vestibule. Our results suggest that apoptotic changes could be involved in the hydropic vestibule and these phenomena lead inner ear disturbance as seen in endolymphatic hydrops.

アデノウィルスを用いた内リンパ嚢経由ｇｅｎｅ ｔｒａｎｓｆｅｒの試み

Gene transfer may be applied in a variety of organs, including the inner ear. For experimental purposes, adenoviral vectors have been inoculated into the scala tympani via cochleostomy or the round window membrane, which resulted in transgene expression restricted to the cochlear tissues in the perilymphatic spaces. In the current study, we injected an adenoviral vector expressing the bacterial lacZ reporter gene into the endolymphatic sac of guinea pigs, and observed the distribution of transgene expression. A large number of lacZ-positive cells were present in the endolymphatic duct and sac. LacZ was expressed in the saccule and utricle, as well as in the semicircular canals. In the cochlea, a greater amount of lacZ-positive cells was observed in basal turns compared to more apical turns; the lacZ-positive cells included Hensen cells and strial marginal cells. LacZ was not expressed in the hair cells in either the vestibule or the cochlea, suggesting that transgene expression to the sensory hair cells is difficult to achieve using an adenoviral vector. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation, and can expand the possibility of molecular research and therapy to more inner ear diseases.

Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.

Gene transfer has been performed in a variety of organs. In the mammalian inner ear, viral vectors have been used to introduce exogenous reporter genes via the scala tympani into the cochlea. While scala tympani inoculation is clinically feasible, it is not without risks. Moreover, transgene expression has so far been restricted to the cochlear tissues in the perilymphatic spaces that are contiguous with the scala tympani. To achieve gene transfer of vestibular organs and cells surrounding the endolymphatic space, and to extend the clinical utility of inner ear gene therapy, we developed a new surgical approach for vector inoculation. A replication-deficient adenoviral vector, Ad.RSVntlacZ, was injected into the guinea pig endolymphatic sac. A large number of blue (LacZ-positive) cells was observed in the endolymphatic sac and duct, the vestibule, and the ampulla. Blue cells were also detected in the cochlea, mainly in cells bordering the endolymphatic space: marginal cells in the stria vascularis and supporting cells in the organ of Corti. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation.

Evidence for apical K conductance and Na-K-2Cl cotransport in the endolymphatic sac of guinea pig

The transepithelial potential in the endolymphatic sac (ESP) was recorded up to 60 min after apical injection of ouabain, bumetanide, quinine, barium, tetraethylammonium, and 4-aminopyridine. After control injection, ESP decreased by 74% and completely recovered at 30 min. After ouabain, barium, or quinine injection, the ESP time course was similar to that in the control group. After bumetanide, tetraethylammonium, or 4-aminopyridine injection, complete recovery was only observed at 60 min. These results suggest that apical K + conductance and Na-K-2Cl cotransporter could be involved in the genesis of ESP.

In Vivo Study of the Electrochemical Composition of Luminal Fluid in the Guinea Pig Endolymphatic Sac

The aim of this study was to investigate the ionic composition (sodium, potassium) of the luminal fluid in the endolymphatic sac and to correlate it with the transepithelial potential. Experiments were performed in guinea pigs using either an intradural posterior fossa approach or a translabyrinthine approach. The endolymphatic sac transepithelial potential (ESP) was measured and the luminal fluid was sampled. The sodium, potassium and protein concentrations were determined. The results were: i) the luminal fluid in the endolymphatic sac differs in composition from perilymph, on the one hand, and from both cochlear and vestibular endolymph, on the other hand, indicating that the endolymphatic sac maintains chemical (sodium, potassium) and electrical (ESP) gradients; ii

Low-amiloride-affinity Na+ channel in the epithelial cells isolated from the endolymphatic sac of guinea-pigs.

By using the whole-cell patch-clamp technique, an amiloride-sensitive Na+-selective conductance was found in epithelial cells from the endolymphatic sac (ES) epithelia of guinea-pigs. In the current-clamp configuration, the average resting membrane potential was -41.7+/-8.4 mV (n = 22). Application of amiloride at a concentration of 20 microM elicited a decrease in cation conductance that was responsible for a membrane hyperpolarization by 17.9+/-6.0 mV (n = 22). Substitution of N-methyl d-glucamine chloride (NMDG-Cl) for external NaCl led to a more significant membrane hyperpolarization by 28.4+/-8.3 mV (n = 22). At holding potential of -70 mV, amiloride and ethylisopropylamiloride (EIPA) blocked the inward current in a concentration-dependent manner over the range of concentrations of between 0.1 microM and 50 microM, with an inhibitory constant (Ki) of 1.3+/-0.4 microM (n = 7) and 1.5+/-0.3 microM (n = 5), respectively. In the voltage-clamp configuration, substitution of NMDG-Cl for external NaCl significantly reduced the inward current (n = 9), indicating that the whole-cell conductance has a high permeability for Na+. Superfusion with 20 microM amiloride induced a significant reduction of the inward current, shifted the reversal potential from -39.4+/-8.8 mV to -60.4+/-10.5 mV (n = 12), and decreased the inward conductance from 5.0+/-1.3 nS to 3.7+/-1.5 nS (n = 12). The permeability ratio of Na+ over K+, calculated from the difference in reversal potential between the currents before and after application of amiloride, was approximately 5:1. Additionally, the conductance was not activated by application of forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo-cAMP (8-Br-cAMP). These findings suggest that a low-amiloride-affinity Na+ channel localized in the ES epithelial cells may be involved in uptake of Na+ in the ES.

Outward K+ current in epithelial cells isolated from intermediate portion of endolymphatic sac of guinea pigs.

Ion currents in epithelial cells isolated from the intermediate portion of endolymphatic sac (ES) in guinea pigs were investigated with the use of the whole cell patch-clamp technique. Depolarizing voltage steps from a holding potential of -60 mV induced a time- and voltage-dependent outward current, which is comparable to that of delayed rectifying K+ currents. The average resting membrane potential in the current-clamp mode was -54.8 +/- 11 mV (n = 45), which was similar to the value of zero current potential (-55.6 +/- 0.8 mV, n = 32) obtained from current-voltage (I-V) relationships of outward currents in voltage-clamp mode. The I-V relationship of the tail current exhibited a reversal potential (Erev) of -78.1 +/- 0.9 mV (n = 19) in standard external solution. The Erev of the outward current was linearly related to the logarithm of extracellular K+ concentrations. The slope was 48 mV per 10-fold change in extracellular K+ concentrations. The time constants of K+ current activation, inactivation, and K+ tail current deactivation were voltage dependent. The steady-state activation and inactivation of K+ current exhibited a sigmoidal relationship to voltage. The 50% maximal activation voltage and slope factor were -21 and 11 mV (n = 8), respectively. The 50% maximal inactivation voltage and slope factor were -45 and 13 mV (n = 7), respectively. The K+ current was blocked by externally applied 1 mM 4-aminopyridine (4-AP), 5 mM Ba2+ and 20 mM tetraethylammonium chloride (TEA). The sensitivity of the current to 4-AP and Ba2+ was higher than that to TEA. Elimination of external Ca2+ and increase of internal Ca2+ failed to significantly change the current, suggesting that the K+ current may be Ca2+ independent. The results show that epithelial cells in the intermediate portion of the ES possess a delayed-rectifier K+ current, which may be involved in membrane stability or in the ion balance between the cytosol and the extracellular environment.

Insulin receptors on the endolymphatic sac: an autoradiographic study.

Light microscopic autoradiographs were prepared of isolated guinea pig endolymphatic sac (ES) tissue incubated with iodine (I)-125-labeled insulin (insulin I 125). These demonstrated specific binding of the insulin I 125 to receptors in the ES. Receptor-mediated endocytosis of the insulin-receptor complex is postulated to occur, and the relevance of insulin receptors in the ES is discussed, with particular emphasis on the previously observed synthesis of hyaluronan from glucose by the ES.

Fluctuating hearing loss following immune reaction in the endolymphatic sac of guinea pigs.

This study has investigated immune injuries to the inner ear auditory system of guinea pigs. Following secondary antigen challenge to the endolymphatic sac, the mean hearing threshold significantly increased in the early phase from day 1 to day 3 and thereafter recovered. In the early phase, hearing threshold significantly increased simultaneously to the elevation of perilymph antibody levels. The size of hydrops was not the only factor that causes an increase in hearing loss as well as in AP/SP ratio. Scale-out hearing loss was seen in 2 animals with severe degeneration of the stria vascularis as well as the organ of Corti associated with the inflammatory cellular infiltration especially in the perilymphatic space, even in the absence of keyhole limpet hemocyanin antigen in the cochlea. On the other hand, control animals did not suffer hearing loss. These results suggest that an immune reaction in the endolymphatic sac is a possible pathogenic etiology of Ménière's disease or sudden deafness.

Free radicals in inner ear immune responses; immunohistochemical study of myeloperoxidase

Previously, we reported that immune reactions in the endolymphatic sac of guinea pigs caused spontaneous nystagmus, a suppressed caloric response, and fluctuating hearing loss. In order to evaluate immune injury to the sensory organs of the inner ear, we conducted an immunohistochemical investigation of the distribution and time course of myeloperoxidase (MPO) following immune reaction in the endolymphatic sac. MPO staining can be seen in the endolymphatic sac, outer hair cells, stria vascularis and lateral canal (ampulla), from several hours to 3 weeks after a secondary KLH challenge, and its time course parallels the time course of neutrophil infiltration of the endolymphatic sac. This result indicates that free radicals in the MPO-H2O2-Cl system are responsible for the tissue damage and dysfunction resulting from inner ear immune reactions.