In Vitro Survival of Human Mesenchymal Stem Cells is Enhanced in Artificial Endolymph with Moderately High Concentrations of Potassium.

While mesenchymal stem cells (MSCs) are promising candidates for inner ear hair cell regeneration, to date, there have been no convincing reports indicating whether MSCs can survive in the cochlea for more than a few weeks, as the high levels of potassium (K+) in the endolymph (EL) are thought to be toxic to transplanted stem cells. For conditioning the EL for MSC transplantation, we conducted this in vitro study to examine the effects of artificial EL with altered K+ concentration levels, in the range of 5-153.8 mM, on proliferation, apoptosis, and morphological changes in MSCs derived from various human tissues. Our findings demonstrate that altering the K+ concentration in artificial EL could significantly influence the survival of MSCs in vitro. We discovered that K+ concentrations of 55-130 mM in artificial EL could enhance the survival of MSCs in vitro. However, MSCs exhibited reduced proliferation regardless of K+ concentration.

Semi-quantification of endolymphatic size on MR imaging after intravenous injection of single-dose gadodiamide: comparison between two types of processing strategies.

PURPOSE: Many inner ear disorders, including Ménière's disease, are believed to be based on endolymphatic hydrops. We evaluated a newly proposed method for semi-quantification of endolymphatic size in patients with suspected endolymphatic hydrops that uses 2 kinds of processed magnetic resonance (MR) images. METHODS: Twenty-four consecutive patients underwent heavily T2-weighted (hT2W) MR cisternography (MRC), hT2W 3-dimensional (3D) fluid-attenuated inversion recovery (FLAIR) with inversion time of 2250 ms (positive perilymph image, PPI), and hT2W-3D-IR with inversion time of 2050 ms (positive endolymph image, PEI) 4 hours after intravenous administration of single-dose gadolinium-based contrast material (IV-SD-GBCM). Two images were generated using 2 new methods to process PPI, PEI, and MRC. Three radiologists contoured the cochlea and vestibule on MRC, copied regions of interest (ROIs) onto the 2 kinds of generated images, and semi-quantitatively measured the size of the endolymph for the cochlea and vestibule by setting a threshold pixel value. RESULTS: Each observer noted a strong linear correlation between endolymphatic size of both the cochlea and vestibule of the 2 kinds of generated images. The Pearson correlation coefficients (r) were 0.783, 0.734, and 0.800 in the cochlea and 0.924, 0.930, and 0.933 in the vestibule (P<0.001, for all). In both the cochlea and vestibule, repeated-measures analysis of variance showed no statistically significant difference between observers. CONCLUSION: Use of the 2 kinds of generated images generated from MR images obtained 4 hours after IV-SD-GBCM might enable semi-quantification of endolymphatic size with little observer dependency.

Imaging of endolymphatic and perilymphatic fluid after intravenous administration of single-dose gadodiamide.

To visualize endolymph as bright signal after intravenous injection of single-dose gadodiamide, we shortened the inversion time of heavily T(2)-weighted 3-dimensional (3D) fluid-attenuated inversion recovery (FLAIR) to 2050 ms. In 14 patients with suspected Ménière's disease, we observed high signal of vestibular endolymph in all ears, including 6 ears without vestibular endolymphatic hydrops. We observed high signal of cochlear endolymph in 17 ears with cochlear endolymphatic hydrops but not 11 ears without cochlear endolymphatic hydrops.

Ca(2+) regulation of endocochlear potential in marginal cells.

We examined the effect of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca(2+) chelator or inhibitors of Ca(2+)-permeable channels or Ca(2+)-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca(2+) channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca(2+)-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the gamma subunit of epithelial Na(+) channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 muM amiloride or 10 muM phenamil. Taken together, these findings suggest that [Ca(2+)](c) regulated by endoplasmic Ca(2+)-pump and Ca(2+)-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na(+) across the basolateral membrane in marginal cells.

Transplantation of mouse embryonic stem cells into the cochlea of an auditory-neuropathy animal model: effects of timing after injury.

Application of ouabain to the round window membrane of the gerbil selectively induces the death of most spiral ganglion neurons and thus provides an excellent model for investigating the survival and differentiation of embryonic stem cells (ESCs) introduced into the inner ear. In this study, mouse ESCs were pretreated with a neural-induction protocol and transplanted into Rosenthal's canal (RC), perilymph, or endolymph of Mongolian gerbils either 1-3 days (early post-injury transplant group) or 7 days or longer (late post-injury transplant group) after ouabain injury. Overall, ESC survival in RC and perilymphatic spaces was significantly greater in the early post-injury microenvironment as compared to the later post-injury condition. Viable clusters of ESCs within RC and perilymphatic spaces appeared to be associated with neovascularization in the early post-injury group. A small number of ESCs transplanted within RC stained for mature neuronal or glial cell markers. ESCs introduced into perilymph survived in several locations, but most differentiated into glia-like cells. ESCs transplanted into endolymph survived poorly if at all. These experiments demonstrate that there is an optimal time window for engraftment and survival of ESCs that occurs in the early post-injury period.

Electrochemical potentials and potassium concentration profiles recorded from perilymph, endolymph and associated inner ear tissues in adrenalectomized rats.

This study evaluated the electrochemical potentials and potassium concentration (Ck+) profiles in the perilymph, endolymph, marginal cells, and spiral ligament of adrenalectomized rats in which endogenous corticosteroids had been removed. Electrochemical potentials recorded at the four cochlear sites were not affected by adrenalectomy (ADX). Ck+ was greater in the endolymph of the ADX animals as compared to control animals. Additionally, there was an increase of Ck+ in the marginal cells, perilymph, and spiral ligament tissues of the ADX animals as compared to control animals, although the observed increases were not statistically significant. In a previous study (Ma et al., 1995a), it was found that potassium levels in the blood plasma of ADX animals were higher than those identified in normal rats; thus, ADX may have a systemic effect on Ck+ that is detectable in both tissues and fluids within the cochlea. Even though Ck+ was elevated within the cochlea in the ADX model, the functional response of the inner ear, as assessed electrophysiologically, was not altered.

Endocytosis and transepithelial transport of endolymph in the endolymphatic sac.

The fate of cationized ferritin (CF) injected into the endolymphatic space of the endolymphatic sac was observed by transmission electron microscopy. At 10 min after the injection, CF particles bound to the apical plasma membrane of epithelial cells of the sac and were then endocytosed with coated pits. However, they never passed through the junctional complexes between the epithelial cells. At 30 min after the injection, the CF particles were transferred to endosomes and lysosomes by small vesicles of 100-150 nm in diameter. CF particles were also found in small vesicles close to Golgi cisternae and in multivesicular bodies. Acid phosphatase positive lysosomes were found close to endosomes containing CF particles. In addition, a small fraction of the small vesicles containing CF particles became inserted into the basolateral plasma membrane. At 60 min after the injection, many CF particles were found in acid phosphatase positive secondary lysosomes. These observations suggest that endocytosis of endolymph is actively performed by the epithelial cells of the sac, and transepithelial vesicular transport across the epithelial cells occurs.

Ultracytochemical localization of Na+, K(+)-ATPase activity in the tegmentum vasculosum of the developing chick cochlea.

We studied the morphological changes and the precise localization of Na+, K(+)-ATPase activity in the developing chick tegmentum vasculosum (TV) with a cytochemical method utilizing K(+)-p-nitrophenylphosphatase (K(+)-NPPase). At stage 37, epithelial folds (lamellae) were relatively well developed, and the light and the dark cells were clearly identified. At stage 41 to 42, Na+, K(+)-ATPase activity appeared in the folded cytoplasmic processes of the dark cells which interdigitated with those of the light cells. The present results indicate that the TV is involved in the production of the cochlear endolymph prenatally (at least up to stage 41), and that the dark cell plays a central role in the maintenance of the ionic composition of the endolymph.

Scanning electron microscopic observations on the distended Reissner's and saccular membranes in the guinea pig.

The surface ultrastructures of membranous labyrinths in normal and hydropic guinea pig inner ears were studied. In normal specimens differences between Reissner's and saccular membranes were observed. The endolymphatic epithelial cells of the saccule were smaller in size and their microvilli varied more in size and distribution. Mesothelial cells of the saccule were often spherical in shape and bulged toward the vestibule. In hydropic guinea pig inner ears, distension of Reissner's and saccular membranes occurred by irregular enlargement of epithelial cells. Microvilli were reduced in size and number. On the severely distended Reissner's membrane, outpouchings, infoldings and holes were observed. Transmission electron microscopic examination revealed no significant difference between the tight junctions of epithelial cells in normal or hydropic Reissner's membranes. Mesothelial cells of Reissner's membrane were more severely degenerated than those of the saccule. Cochlear sensory cells were often degenerated in the apical turn where mesothelial cells were lacking, but saccular sensory cells showed very little change in their surface structures.

[The pathogenesis of the acute symptoms of Menière's disease (author's transl)]. / Zur Pathogenese der akuten Symptome des Morbus Menière.

The pathogenesis of vertigo attacks and hearing fluctuations in Menière's disease is discussed on the basis of new electronmicroscopic observations on the perilymph endolymph barrier. The zonulae occludentes (z.o.) of the epithelial cells lining the endolymphatic space are the main sealing elements which control the passive ion transport. They consist of parallel running intramembranous fibrils (cell membrane fusion lines). Recent investigations demonstrated conditions which partially destruct some of such fibrils (e.g. increased osmotic pressure, endocrine disorders). This also may be true in Menière's attacks: tight z.o. are transformed to leaky z.o. such that a high transepithelial ion exchange developes. This would result in reduced generation of the receptor potentials as well as in decreased activities of the first order afferent neurons as it was supposed earlier by other authors. This mechanism may be combined with a decompensation of active ion transport processes due to microcirculation disturbances. Finally it will be pointed out that true ruptures of the endolymphatic walls cannot be the usual mode of reversible lesions in Menières disease but a diffuse leakage of the perilymph-endolymph barrier.

[Animal experiment histological-histochemical studies on the development of hearing disorders related to hypothyroidism]. / Tierexperimentelle histologisch-histochemische Untersuchingen zur Formalgenese der hypothyreotisch bedingten Schwerhörigkeit

It has been known for a long time that hearing deficits may coexist in patients with thyroid disease, but without definite morphologic evidence present to correlate gland dysfunction with hearing disturbances. To clarify this relationship between thyroid dysfunction and hearing disturbances, the guinea pig was employed as an experimental model. 70 animals were thyroidectomized, and maintained in a hypothyroid state for varying periods of time. The animals were then sacrificed, and various histochemical studies then performed. These studies included analysis for glycosidase (beta-galactosidase, beta-glucuronidase, n-acetyl-beta-glucosamide), non-specific esterases, sulfatases, sulghydryl groups as well as mucous substances within the cochlea and saccus endolymphaticus of the experimental animals. Results indicated that hyaluronidase-sensitive mucous substances were increased in the scala of the inner ear. As a consequence of increased deposition of acid mucopolysaccharides, the relationship of potassium to sodium in endolymph and perilymph was found markedly altered. Marked swelling of the chambers of the inner ear was noted, and believed to represent hydropic induction by acid mucopolysaccharide-with consequent alteration of electrolyte relationships ("Electrochemical Theory").