Fixation-induced shrinkage of Reissner's membrane and its potential influence on the assessment of endolymph volume

doi:10.1016/S0378-5955(97)00153-6

Hearing Research

Volume 114, Issues 1–2, December 1997, Pages 62-68

https://doi.org/10.1016/S0378-5955(97)00153-6 Get rights and content

Abstract

The quantification of endolymph volume by histological techniques or by magnetic resonance (MR) microscopy requires the inner ear to be first treated with chemical fixatives. If the fixative induces soft-tissue shrinkage, it would tend to return a distended Reissner's membrane towards a straight position, since this membrane is anchored to bone at its medial and lateral edges. The goal of this study was to determine the degree of Reissner's membrane shrinkage induced by different fixation protocols to establish methods which minimize tissue shrinkage. Fragments of fresh Reissner's membrane were dissected from isolated cochleae in an artificial perilymph. Specimens were viewed with an inverted microscope during infusion of fixatives, and changes recorded on video tape. Size changes of the specimen were quantified, usually over a 20 min period. Heidenhain-Susa, a fixative which is widely used in histological studies of hydropic cochleae, caused substantial shrinkage of Reissner's membrane, decreasing the length of specimens by an average of 15.1%. Other fixation procedures induced far less shrinkage. The use of 3X/0 glutaraldehyde in Hanks' balanced salt solution produced a mean length decrease of only 0.3%. The inclusion in the fixation medium of 4.5`/0 mercuric chloride, corresponding to the concentration which is present in Heidenhain-Susa and which acts to increase the contrast of Reissner's membrane in MR microscopy, contributes significantly to specimen shrinkage. We can conclude that the degree of endolymphatic hydrops may be underestimated in specimens fixed with media containing high levels of mercuric chloride.

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Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI
2015, Hearing Research
Citation Excerpt :
Endolymphatic hydrops in experimental models has to a large extent been demonstrated using histological analysis which requires several manipulations; fixation, decalcification, embedding, and sectioning. One concern with histological methods is thus that the degree of hydrops may be influenced by the fixation and processing [Brunschwig and Salt, 1997]. MRI studies on guinea pigs support however the induction of EH induced surgically or induced by other treatments [Zou et al., 2003a,b].
From histopathological specimens, endolymphatic hydrops has been demonstrated in association with inner ear disorders. Recent studies have observed findings suggestive of hydrops using MRI in humans. Previous studies suggest that vasopressin may play a critical role in endolymph homeostasis and may be involved in the development of Ménière's disease. In this study we evaluate the effect of vasopressin administration in vivo in longitudinal studies using two mouse strains. High resolution MRI at 9.4 T in combination with intraperitoneally delivered Gadolinium contrast, was performed before and after chronic subcutaneous administration of vasopressin via mini-osmotic pumps in the same mouse. A development of endolymphatic hydrops over time could be demonstrated in C57BL6 mice (5 mice, 2 and 4 weeks of administration) as well as in CBA/J mice (4 mice, 2 weeks of administration; 6 mice, 3 and 4 weeks of administration). In most C57BL6 mice hydrops developed first after more than 2 weeks while CBA/J mice had an earlier response. These results may suggest an in vivo model for studying endolymphatic hydrops and corroborates the future use of MRI as a tool in the diagnosis and treatment of inner ear diseases, such as Ménière's disease. MRI may also be developed as a critical tool in evaluating inner ear homeostasis in genetically modified mice, to augment the understanding of human disease.
This article is part of a Special Issue entitled <IEB Kyoto>.
Endolymphatic Hydrops: Pathophysiology and Experimental Models
2010, Otolaryngologic Clinics of North America
Citation Excerpt :
One concern with all these methods is whether the degree of hydrops was influenced by the fixation and processing methods. It was shown that dissected, fresh Reissner membrane segments in vitro underwent substantial shrinkage during fixation with some protocols, suggesting that the degree of hydrops could be underestimated based on measurements from fixed tissues.79 Recently, there have been major advances in MR imaging that have allowed endolymphatic hydrops to be visualized and quantified in the live ears of animals and humans.80–82
Differential passage of gadolinium through the mouse inner ear barriers evaluated with 4.7 T MRI
2010, Hearing Research
Magnetic resonance imaging (MRI), supplemented by contrast agents, is a powerful tool that can be used to visualise the structures of the inner ear in vivo and assess some aspects of physiology, such as the permeability of agents through membranes. The mouse is an excellent animal species for investigating human diseases, including hearing loss but detailed MRI studies with contrast have not been reported. In this work, we aimed to demonstrate the limits of MR imaging resolution of the fine inner ear structures in the mouse and to explore the permeability of the intracochlear barriers to gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) administered by intravenous injection (IV) or intratympanic (IT) routes. Twenty-three female FVB mice were imaged with a 4.7-T MR scanner using both 2D and high resolution 3D sequences. Inner ear region of interest (ROI) signal intensities and perilymph volumes were evaluated. Finer structures were studied using 3D acquisition and reconstruction techniques and comparisons were made to similarly oriented histological sections that were examined by light microscopy. Gd-DOTA enhancement occurred in the perilymphatic compartment and highlighted the contiguous inner ear structures, but enhancement did not appear within the endolymph. The dynamic uptake of Gd-DOTA in the perilymphatic compartments reached an initial plateau 80 min after IV administration and continued to slightly increase to a maximum level by 100 min. The perilymph volume demonstrated by Gd-DOTA uptake was statistically significantly larger in the IV group (1.72 mm³) than in the IT group (1.28 mm³) (p < 0.05).
Imaging the living inner ear using intravital confocal microscopy
2007, NeuroImage
Confocal laser scanning microscopy permits detailed visualization of structures deep within thick fluorescently labeled specimen. This makes it possible to investigate living cells inside intact tissue without prior chemical sample fixation and sectioning. Isolated guinea pig temporal bones have previously been used for confocal experiments in vitro, but tissue deterioration limits their use to a few hours after the death of the animal. In order to preserve the cochlea in an optimal functional and physiological condition, we have developed an in vivo model based on a confocal microscopy approach. Using a ventral surgical approach, the inner ear is exposed in deeply anaesthetized, tracheotomized, living guinea pigs. To label the inner ear structures, scala tympani is perfused via an opening in the basal turn, delivering tissue culture medium with fluorescent vital dyes (RH 795 and calcein AM). An apical opening is made in the bony shell of cochlea to enable visualization using a custom-built objective lens. Intravital confocal microscopy, with preserved blood and nerve supply, may offer an important tool for studying auditory physiology and the pathology of hearing loss. After acoustic overstimulation, shortening and swelling of the sensory hair cells were observed.
The rat cochlea in the absence of circulating adrenal hormones: An electrophysiological and morphological study
2000, Hearing Research
Circulating adrenal hormones affect strial function. Removal of endogenous levels of adrenal steroids by bilateral adrenalectomy (ADX) in rats causes a decrease of Na⁺/K⁺-ATPase activity in the cochlear lateral wall [Rarey et al., 1989. Arch. Otolaryngol. Head Neck Surg. 115, 817–821] and a decrease of the volume of the marginal cells in the stria vascularis [Lohuis et al., 1990. Acta Otolaryngol. (Stockh.) 110, 348–356]. To study further the effect of absence of circulating adrenocorticosteroids on cochlear function, 18 male Long Evans rats underwent either an ADX or a SHAM operation. Electrocochleography was performed 1 week after surgery for tone bursts in a frequency range of 1–16 kHz. Thereafter, the cochleas were harvested and examined histologically. No significant changes in the amplitude growth curves of the summating potential (SP), the compound action potential (CAP) and the cochlear microphonics (CM) were detected after ADX. However, visually, there appeared to be a decrease of endolymphatic volume (tentatively called imdrops). Reissner’s membrane (RM) extended less into scala vestibuli in ADX animals than in SHAM-operated animals. The ratio between the length of RM and the straight distance between the medial and lateral attachment points of RM were used as an objective measure to quantify this effect in each sub-apical half turn of the cochlea. The decrease in length of RM was statistically significant. Thus, circulating adrenal hormones appear to be necessary for normal cochlear fluid homeostasis. Absence of one or more of these hormones leads to shrinkage of the scala media (imdrops). However, the absence of adrenal hormones does not affect the gross cochlear potentials. Apparently, the cochlea is capable of compensating for the absence of circulating adrenal hormones to sustain the conditions necessary for proper cochlear transduction.
Signs of endolymphatic hydrops after perilymphatic perfusion of the guinea pig cochlea with cholera toxin; a pharmacological model of acute endolymphatic hydrops
1999, Hearing Research
There are indications that endolymph homeostasis is controlled by intracellular cAMP levels in cells surrounding the scala media. Cholera toxin is a potent stimulator of adenylate cyclase, i.e. it increases cAMP levels. We hypothesized that perilymphatic perfusion of cholera toxin might increase endolymph volume by stimulating adenylate cyclase activity, providing us with a pharmacological model of acute endolymphatic hydrops (EH). Guinea pig cochleas were perfused with artificial perilymph (15 min), with or without cholera toxin (10 μg/ml). The endocochlear potential (EP) was measured during and after perfusion. The summating potential (SP), evoked by 2, 4 and 8 kHz tone bursts, was measured via an apically placed electrode 0, 1, 2, 3 and 4 h after perfusion. Thereafter, the cochleas were fixed to enable measurement of the length of Reissner’s membrane, reflecting EH. After perfusion the EP increased significantly over time in the cholera toxin group as compared to the controls. Also, the SP increased gradually at all frequencies in the cholera toxin group. Comparison within animals showed that the increase in SP became significant after 2 h at 4 kHz, after 3 h at 2 kHz and after 4 h at 8 kHz. In the control group the SP did not change significantly. The compound action potential (CAP) amplitude decreased monotonically over time at all frequencies in both the cholera toxin group and the control group, but it decreased faster in the cholera toxin group. Also, the cochlear microphonics amplitude decreased over time at all frequencies in both groups, but the decrease was significant only in the cholera toxin group after 3 h at 2 and 4 kHz. Quantification of the length of Reissner’s membrane showed a small but insignificant enlargement in the cholera toxin treated animals compared to controls. These results are in accord with our view that EH is accompanied by an increase in SP and a decrease in CAP. Our results partially confirm previous results of Feldman and Brusilow (Proc. Natl. Acad. Sci. USA (1973) 73, 1761–1764). New aspects in relation to that study are the significantly increased EP and SP. In the classical EH model, based on obstruction of the absorptive function of the endolymphatic sac, increased SPs are accompanied by decreased EPs. In this cholera toxin model of EH, it is unlikely that the endolymphatic sac is involved. Apparently, EH can be based on mechanisms located in the cochlea itself as opposed to mechanisms located in the endolymphatic sac.

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Research paperFixation-induced shrinkage of Reissner's membrane and its potential influence on the assessment of endolymph volume

Abstract

Hear. Res.

Am. J. Otolaryngol.

Hear. Res.

Hear. Res.

Hear. Res.

Research paper
Fixation-induced shrinkage of Reissner's membrane and its potential influence on the assessment of endolymph volume