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ARTICLE |
Different Regulation of Connexin26 and ZO-1 in Cochleas of Developing Rats and of Guinea Pigs with Endolymphatic Hydrops
Toshihiro Suzukia,b, Masahito Oyamadaa, and Tetsuro Takamatsuaa Departments of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan
b Otolaryngology, Kyoto Prefectural University of Medicine, Kyoto, Japan
Correspondence to: Masahito Oyamada, Dept. of Pathology and Cell Regulation, Kyoto Prefectural Univ. of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail: oyamada@basic.kpu-m.ac.jp
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Using confocal microscopy and morphometry, we analyzed the expression of connexin26 (Cx26) and ZO-1 in rat cochlea during the postnatal period to elucidate spatiotemporal changes in gap junctions and tight junctions during auditory development. We also studied changes in these junctions in experimental endolymphatic hydrops in the guinea pig. In the adult rat cochlear lateral wall, Cx26 was detected in fibrocytes in the spiral ligament and in the basal cell layer of the stria vascularis, whereas ZO-1 was detected in the apical surfaces of marginal cells and in the basal cell layer. During postnatal development, Cx26 expression increased mainly in the spiral ligament, whereas ZO-1 expression increased in the basal cell layer. The morphometry of Cx26 showed a sigmoid time course with a rapid increase on postnatal day (PND) 14, whereas that of ZO-1 showed a marked increase on PND 7. In experimental endolymphatic hydrops, the expression of Cx26 significantly decreased, whereas there were no obvious changes in the expression of ZO-1. These results indicate that gap junctions and tight junctions in the cochlea increase in a different spatiotemporal manner during the development of auditory function and that gap junctions and tight junctions in the cochlea are differentially regulated in experimental endolymphatic hydrops. (J Histochem Cytochem 49:573–586, 2001)
Key Words: gap junctions, tight junctions, connexin, ZO-1, cochlea, stria vascularis, spiral ligament, endocochlear potential, endolymphatic hydrops
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Introduction |
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The mechanoelectrical transduction mechanisms in the cochlea depend on an electrical potential of about 80 millivolts that exists between the endolymph and the perilymph, with positivity inside the scala media and negativity outside, which is called the endocochlear potential. The maintenance of endocochlear potential needs not only barriers between endolymph and perilymph but also ionic flows, including K1 between endolymph and perilymph through the stria vascularis and spiral ligament. Several models for the generation of the endocochlear potential focusing on ionic transport mainly in the stria vascularis have been proposed, and among them the "two-cell model" is currently considered to be the best model (
The tight junction is another intercellular junction that plays two distinct roles in selective permeability barrier functions. One is as a barrier that seals neighboring cells together in a sheet to prevent leakage of molecules between them, and the other is to prevent diffusion of membrane proteins, including ion channels and glycolipids, between the apical and basolateral regions of the plasma membrane. Several proteins associated with tight junctions have been identified. These include not only the integral proteins claudin and occludin but also the peripheral proteins ZO-1, ZO-2, and symplekin (
Gap junctions and tight junctions were detected in the cochlea by electron microscopy (
In this study we performed confocal and morphometric analyses on the expression and localization of Cx26 and ZO-1 in the cochlea during the postnatal period and in an experimental model of endolymphatic hydrops to elucidate spatiotemporal changes in cochlear gap junctions and tight junctions not only during normal development of auditory function but also in the pathological condition. For the developmental study, we used a rat species in which the endocochlear potential has been shown to dramatically increase during the postnatal period, especially from 11–14 days after birth (
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Materials and Methods |
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Animals
Postnatal Development.
A total of 35 Wistar rats free of middle ear infection were used for assessment of developmental changes in gap junctions and tight junctions in the cochlea. Five rats were sacrificed at each of the following postnatal days (PNDs): 1, 4, 7, 11, 14, 21, and 70, under deep pentobarbital anesthesia. The animals were perfused transcardially with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, 4C). Temporal bones were removed bilaterally and the cochleas were dissected from them and placed in the same fixative at 4C for 3 hr. The cochleas were then decalcified in 0.12 M EDTA at room temperature (RT) for a week. After washing in PBS, pH 7.4, the cochleas were rinsed as follows: (a) 10% sucrose in PBS for 3 hr; (b) 15% sucrose in PBS for 3 hr; (c) 20% sucrose in PBS for 3 hr. The specimens were infused with Tissue-Tek OCT compound (Miles; Elkhart, IN), frozen in disposable vinyl specimen molds (Tissue-Tek Cryomold; Miles) by partial immersion in liquid nitrogen, so that the axis of the cochlea was parallel to the bottom of the mold, and stored at -80C. For comparison between immunohistochemistry of ZO-1 and that of occludin, adult rat cochlear lateral walls were frozen without fixation and decalcification.
To confirm the relationship between maturation of endocochlear potential, and development of gap junctions and tight junctions, neonatal and adult guinea pigs, in which species endocochlear potential is mature at birth (
Experimental Endolymphatic Hydrops
Three albino guinea pigs weighing approximately 300 g were used. Experimental endolymphatic hydrops was induced by endolymphatic duct and sac obliteration as described by
At 3 months after the operation, brainstem-evoked responses were recorded. Under pentobarbital anesthesia, the head was stabilized by a clamp and a sound source was placed in the external auditory meatus. All recordings were made in an electrically shielded soundproof chamber. Evoked responses were recorded between a needle electrode placed into the skull at the vertex and in the pinna of the test ear. A needle electrode in the contralateral pinna served as a ground. Thresholds were measured for clicks and tone pips. The tone pips were presented with a two-cycle rise and fall time, with no plateau, at 1, 2, 4, and 8 kHz. At each frequency the threshold was separately measured for the unoperated left and then the operated right ear. Stimulus intensities were decreased in 5 dB steps until no responses could be detected visually, and the threshold was defined as the minimal intensity at which a response was evoked. After recording, the animals were sacrificed in the anesthetized condition. Cochlea specimens from not only the experimental ears on the right side but also the left control ears were prepared and stored at -80C in a way similar to that used for the rats, except for 3-week decalcification in 0.12 M EDTA at RT. All animal experiments described in this study adhered to the standards detailed in "Principles of Laboratory Animal Care" (NIH publication no. 85 23, revised 1985), and also to the "Rules and Regulations of Animal Research, Kyoto Prefectural University of Medicine."
Immunohistochemistry
Serial 10-µm frozen midmodiolar sections were cut with a cryostat microtome (Bright 5030 microtome), placed on silane-coated slides (Matsunami; Osaka, Japan), air-dried, and fixed in absolute ethanol at -20C for 15 min. After incubation in PBS with 5% skim milk for 15 min at RT to block nonspecific labeling, primary antibodies, such as a rabbit polyclonal antibody against a Cx26-specific peptide (amino acid residues 101–119;
Confocal Laser Scanning Microscopy
The sections were observed with a confocal laser scanning microscope (Olympus FLUOVIEW system; Olympus, Tokyo, Japan) equipped with an oil immersion objective (Plan Apo x60, NA = 1.4; Olympus). An argon–krypton laser produced excitation bands at 488 nm for FITC, 568 nm for Texas Red, and monochromatic light for differential interference contrast (DIC) images. Fluorescence images were collected with emission filters for 510–550 nm for FITC and 585–610 nm for Texas Red. Simultaneous images (800 x 600 pixels, 12 bits each) of the FITC label and either DIC images or Texas Red label were acquired and stored. Digital images were transferred to a Macintosh computer (Apple Computer) equipped with the Photoshop Program (Adobe System; Tucson, AZ) and printed on an ink-jet printer (PM-750C; Epson, Tokyo, Japan)
Morphometric Analysis
For morphometric analysis, all aspects of tissue processing, immunolabeling, and imaging were strictly standardized. For example, only the sections through the axis of the cochlea were utilized, and to exclude the possibility that variations in immunolabeling at different dates affected the morphometric data, one section each for PND 1, 4, 7, 11, 14, 21, and 70 was included in a single immunolabeling run and all of the sections were analyzed at the same time. Digital images were collected using the x60 objective lens and a zoom setting of 2 under the same conditions, i.e., laser power = 35 mW, pinhole size = 100 µm, ND filter = 20%, photomultiplier voltage = 600 V, and scanning speed = 8.7 sec/frame. As a result, all digital images showed that the spectrum of label intensities was within the 4096-level scale. Three section planes at 1-µm steps along the z-axis were obtained for the same x–y axis.
Concerning Cx26 expression in the cochlear lateral wall, to cover the total cross-sectional area of the stria vascularis and spiral ligament in the second turn of the cochlea, two to four 800 x 600-pixel images were taken by moving slides in the x–y axis, and 6–12 images were acquired for each slide. A single set of images that were taken at the same z-axis and covered the total cross-sectional area of the stria vascularis and spiral ligament was then selected. The digital images were transferred to a Macintosh computer, converted into 8-bit images (256 levels), and analyzed using the NIH Image program (Wayne Rasband, NIH). After the area of the spiral ligament was enclosed manually, a binary overlay was created automatically by a set threshold of 40 on the 255-point gray scale to eliminate the background cell outlines. Then the binary overlaid area in the area of interest was analyzed. Data were first expressed as mean ± SE of overlaid areas (pixel x pixel) per areas of spiral ligament (pixel x pixel) and then converted so that the mean of each datum on PND 70 and of the control ear was 100% for the developmental study and experimental endolymphatic hydrops, respectively. Data were collected from five cochleas of different rats for each PND and from three cochleas of different guinea pigs for experimental endolymphatic hydrops. A total of 41 cochleas were actually analyzed.
Regarding ZO-1 expression in the strial basal cell layer, to cover the total cross-sectional area of the stria vascularis in the second turn of the cochlea, two 800 x 600-pixel images were taken. The same procedure was performed for the transfer and conversion of image data as for Cx26. Morphometric data were first expressed as mean ± SE of ZO-1-positive areas (pixel x pixel) per length of boundary lines (pixel) between the stria vascularis and spiral ligament and then converted so that the mean of each datum on PND 70 and of the unoperated ear was 100% for the developmental study and experimental endolymphatic hydrops, respectively.
Statistical analyses were performed using the StatView program (Abacus Concepts). Data on the rat postnatal development were examined by ANOVA and the Tukey–Kramer test. Data on the guinea pig between PND 1 and PND 70 were compared by t-test. Data on experimental endolymphatic hydrops were examined by paired t-test. Significant differences were defined by p<0.05.
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Cx26 and ZO-1 in Adult Rat Cochlea
Double-labeling for Cx26 and filamentous actin using the anti-Cx26 antibody (
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In the lateral wall of the adult rat cochlea, immunolabeling for ZO-1 was found mainly in two regions, the apical surfaces of the marginal cells and the basal cell layer (Fig 1C and Fig 1D). In midmodiolar sections, ZO-1-positive spots were localized at the most apical areas of the marginal cells. In the surface preparation, in which the surfaces of the marginal cells were viewed from the inside of the endolymphatic space, hexagonal or hexagonal-like labeling for ZO-1 was observed (data not shown). At the basal cell layer, ZO-1-immunolabeled spots were tightly packed in a linear manner. In addition, small ZO-1-positive spots were found in the vessels and among fibrocytes. The localization of ZO-1 (Fig 1E) in the cochlear lateral wall was similar to that of occludin (Fig 1F), a tight junction-specific integral protein, when serially sliced sections were immunolabeled for ZO-1 and occludin.
To elucidate the localization of Cx26 and ZO-1 in the border zone of the stria vascularis and the spiral ligament in more detail, we performed double-labeling for Cx26 and ZO-1. Double-labeling for Cx26 and ZO-1 showed that, in the adult rat cochlea, they were closely associated at the lower surfaces of basal cells of the stria vascularis, and that Cx26-positive spots were localized at the side of the spiral ligament, whereas ZO-1-positive spots were distributed at the side of the stria vascularis (Fig 1G and Fig 1H).
Developmental Changes in Cx26 and ZO-1 in Rat Cochlea During the Postnatal Period
On PND 1, the morphology of the rat cochlear lateral wall was different from that of the adult, i.e., the boundary between the stria vascularis and spiral ligament was ill-defined because the proper alignment of the basal cells was absent. Cx26-immunolabeled spots were localized in the border zone between the stria vascularis and spiral ligament and the number was small (Fig 2A and Fig 2B). Immunolabeling for ZO-1 at the apical surface of the marginal cells was detected on PND 1, and the pattern did not change much during postnatal development (Fig 3). On the other hand, in the basal cell layer, only a few ZO-1-positive spots were found (Fig 3A and Fig 3B).
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On PND 4, cells at the border region between the stria vascularis and spiral ligament showed a tendency to align, but there was no distinct separation of basal cells and fibrocytes. The localization of Cx26-immunolabeled spots was still limited to the border zone between the stria vascularis and spiral ligament (Fig 2C and Fig 2D). In the basal cell layer, only a few ZO-1-positive spots were found (Fig 3C and Fig 3D).
On PND 7, the alignment of basal cells became clearer and the density of fibrocytes appeared to increase. The distribution of Cx26-immunolabeled spots barely changed and was restricted to the border region (Fig 2E and Fig 2F). Double-labeling for Cx26 and F-actin showed that Cx26 was not localized in the marginal cell layer but was expressed at the border region between the stria vascularis and spiral ligament, and that most Cx26-immunolabeled spots were closely associated with F-actin (Fig 4A). In contrast, a dramatic increase in the expression of ZO-1 occurred in the basal cell layer. ZO-1-positive spots were localized on the cell membrane between overlapping basal cells (Fig 3E and Fig 3F).
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On PND 11, the basal cells became flatter and aligned more regularly than on PND 7. Afterward, the general morphology of the lateral wall did not change so much. The localization of Cx26-immunolabeled spots extended a little way into the spiral ligament (Fig 2G and Fig 2H). Double-labeling for Cx26 and F-actin demonstrated that Cx26-immunolabeled spots were present not only at the upper and lower surfaces of the basal cells but also on the strial side of the spiral ligament (Fig 4B). ZO-1-positive spots were localized along the basal cell layer (Fig 3G and Fig 3H).
On PND 14, the distribution of Cx26-immunolabeled spots spread dramatically throughout the entire spiral ligament (Fig 2I and Fig 2J). Double labeling for Cx26 and F-actin showed that Cx26-immunolabeled spots were widely distributed in the spiral ligament, including the border with the basal cells in the stria vascularis (Fig 4C). ZO-1-positive spots in the basal cell layer were aligned in a continuous pattern (Fig 3I and Fig 3J).
On PND 21, the density of Cx26-immunolabeled spots moderately increased compared with that of PND 14. The expression pattern of Cx26 (Fig 2K and Fig 2L) appeared similar to that in the adult rat cochlea (PND 70). ZO-1-positive spots formed a linear structure that surrounded basal cells (Fig 3K and Fig 3L).
Morphometric analysis for Cx26 expression during postnatal development of the rat demonstrated that the Cx26-positive area per area of spiral ligament followed a sigmoid time course, i.e., there was a gradual increase from PND 1 to PND 11, a steep rise from PND 11 to PND 14, and a moderate increase from PND 14 to adulthood (Fig 5A). Morphometric analysis demonstrated that ZO-1 expression markedly increased on PND 7 and afterward the increase gradually slowed (Fig 5B).
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Cx26 and ZO-1 in the Cochlea of Neonatal and Adult Guinea Pigs
To confirm the relationship between maturation of endocochlear potential, and development of gap junctions and tight junctions, neonatal and adult guinea pigs, in which species endocochlear potential is mature at birth, were analyzed by Cx26 and ZO-1 immunolabeling. On PND 1, Cx26 was already expressed widely in the spiral ligament of the guinea pig (Fig 6A and Fig 6B). There was no apparent difference in the Cx26 expression in the guinea pig cochlea between PND 1 and PND 70 (Fig 6C and Fig 6D). The guinea pigs showed abundant expression of ZO-1 both at the apical surfaces of the marginal cells and in the basal cell layer from PND 1 (Fig 6E and Fig 6F). Morphometric data confirmed that on PND 1, the Cx26-positive area per area of spiral ligament reached the adult level in guinea pigs (no significant difference between PND 1 and PND 70) (Fig 6I), which coincided with the endocochlear potential of neonatal and adult guinea pigs reported by
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Changes in Cx26 and ZO-1 in the Cochlea of the Guinea Pig with Experimental Endolymphatic Hydrops
Fig 7K shows the brainstem response thresholds for the operated and unoperated ears at 3 months after right endolymphatic duct and sac obliteration. The operated ears had higher thresholds of responses to the stimulus at each frequency, i.e., 1, 2, 4, and 8 kHz, than the unoperated ears in the same animals. Light microscopic examination demonstrated distention of Reissner's membrane in all turns of the operated ears (Fig 7B), whereas the unoperated control ears on the left side had no hydrops (Fig 7A). No severe cell injury was noted in the operated ears, including the stria vascularis and spiral ligament, although mild atrophy of the stria vascularis at all turns and a decrease in the number of spiral ganglion cells at the apical turns (data not shown) were observed.
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In the hydropic ears, the number of Cx26-immunolabeled spots decreased in the spiral ligament and in the basal cell layer of the stria vascularis in each turn (Fig 7E and Fig 7F). Morphometric analysis for Cx26 expression in the cochlea (Fig 7L) demonstrated a significant difference between control and hydropic ears (p<0.05).
In contrast to Cx26, ZO-1 immunolabeling showed no drastic changes in the lateral wall of the hydropic ears, i.e., the expression of ZO-1 did not decrease or increase either on the apical surface of the marginal cells or in the strial basal cell layer (Fig 7I and Fig 7J). Morphometric analysis for the expression of ZO-1 in the basal cell layer of the cochlea (Fig 7M) showed no significant difference between the control and hydropic ears.
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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This study shows that gap junctions and tight junctions in the cochlear lateral wall increase in different spatiotemporal manners during postnatal development of rat auditory function. We found that Cx26 expression increased mainly in fibrocytes in the spiral ligament, whereas ZO-1 expression increased in the basal cell layer of the stria vascularis but did not change at the apical surfaces of the marginal cells of the stria vascularis. Our morphometric analysis demonstrated that Cx26 expression was rapidly augmented on PND 14, whereas ZO-1 expression was augmented on PND 7.
Our results also indicated that gap junctions and tight junctions in the cochlear lateral wall were differentially regulated during experimental endolymphatic hydrops of the guinea pig. The evidence showed that Cx26 expression significantly decreased in the operated ears compared with the unoperated ears in the same animals, whereas there were no obvious differences in ZO-1 expression between them.
Our present work has confirmed the observations of recent studies on rats (
First, we found a sigmoidal increase in the Cx26 expression in the cochlear lateral wall by confocal microscopy and morphometry. It was reported that postnatal development of endocochlear potential also shows a sigmoidal time course, as indicated in Fig 5C (quoted from
Second, new observations were made by comparison between rats and guinea pigs, also indicating a close relationship of Cx26 expression with endocochlear potential development. This is because in the guinea pig, in which species endocochlear potential is mature at birth (
As far as we know, this is the first report on ZO-1 in the cochlea. ZO-1 has been reported to localize not only in tight junctions but also in adherens junctions, depending on the cell type (
Our developmental study using rats and guinea pigs showed that ZO-1 expression in the basal cell layer of the stria vascularis was closely related to hearing development, suggesting involvement of tight junctions in auditory function. The evidence demonstrated that in rats, in which species endocochlear potential develops dramatically during the postnatal period, especially from days 11 to 14, ZO-1 expression markedly increased on PND 7. After this, the increase gradually slowed. In contrast, in the guinea pig, in which species endocochlear potential is mature at birth, ZO-1 was already expressed strongly in basal cells on PND 1 and there was no apparent difference in ZO-1 expression between PND 1 and PND 70. Our morphometric data on ZO-1 in rats are consistent with those on the increase in tight junction strands on lateral membranes of basal cells in gerbils obtained by freeze-fracture analysis (
The different spatiotemporal development of gap junctions and tight junctions in the rat cochlear lateral wall during postnatal development of auditory function suggests that maturation of these two intercellular junctions is independently regulated. However, we cannot exclude the possibility that during development these two junctions interact with each other, especially in the basal cells of the stria vascularis. This is because both junctions exist next to each other in some parts of basal cells and because a close relationship between connexins and tight junction-related proteins, including ZO-1 (
Different regulation of gap junctions and tight junctions in the cochlear lateral wall has also been demonstrated by the present data on experimental endolymphatic hydrops in the guinea pig. Cx26 expression significantly decreased in the operated ears, whereas there were no obvious changes in ZO-1 expression between the operated and control ears. These results indicate that hearing loss in this model is associated with dysfunction of gap junctions rather than of tight junctions. One possible explanation for the difference may be that fibrocytes in the spiral ligament are more seriously affected than other cells by experimental endolymphatic hydrops, and consequently the expression of Cx26 in these fibrocytes is reduced. This explanation is supported by the results reported by
In conclusion, using confocal microscopy and morphometry, we have shown that gap junctions and tight junctions in the cochlear lateral wall increase in different spatiotemporal manners during postnatal development of rat auditory function, and that gap junctions and tight junctions in the cochlear lateral wall are differentially regulated during hearing loss induced by experimental endolymphatic hydrops in the guinea pig. Because mutations in multiple connexin genes other than Cx26 have been found in human hereditary deafness (
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Acknowledgments |
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We gratefully acknowledge the generous gift of the polyclonal anti-connexin26 antibody from Dr Y. Shibata (Kyushu University School of Medicine; Fukuoka, Japan). We thank Mr Kim Barrymore for advice on English usage.
Received for publication September 18, 2000; accepted January 17, 2001.
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