Journal of Otology & Rhinology

Okada et al., J Otol Rhinol 2015, S1:1
http://dx.doi.org/10.4172/2324-8785.S1-009
Journal of Otology &
Rhinology
Research Article
A SCITECHNOL JOURNAL
Postnatal Development and
Maturation of the Vestibular Organ
in Dominant-Negative Connexin
26 Transgenic Mouse
Hiroko Okada, Kazusaku Kamiya, Takashi Iizuka and Katsuhisa
Ikeda*
Department of Otorhinolaryngology, Juntendo University Faculty of Medicine,
Tokyo 113-8421, Japan
*Corresponding
113-8421,
author: Katsuhisa Ikeda, MD, 2-1-1 Hongo, Bunkyo-ku, Tokyo
Japan, Tel: +81-3-5802-1094; Fax: +81-3-5689-0547; E-mail:
ike@juntendo.ac.jp
Rec date: Nov 17, 2014 Acc date: Feb 23, 2015 Pub date: March 06, 2015
Abstract
observed in the developing supporting cells of the Gjb2 R75W
transgenic mouse model include:
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•
•
•
The absence of the tunnel of Corti, Nuel’s space, or spaces
surrounding the OHCs.
Reduced numbers of microtubules in the pillar cells.
Shortening of height of the organ of Corti.
Increase of the cross-sectional area of the cells of the organ of
Corti.
Immunohistochemical studies have revealed that Cx26 exists not
only in the cochlea but also in the vestibular organs [6]. K+ cycling
involving gap junction protein Cx26 in the vestibular labyrinth, which
is similar to that in the cochlea, is thought to play a fundamental role
in the endolymph homeostasis and sensory transduction [7]. The
incidence of vestibular dysfunction patients with congenital deafness
related to GJB2 mutation was statistically higher than in both patients
with congenital deafness unrelated to GJB2 mutation and healthy
controls [8], suggesting that GJB2 mutation play a critical role in the
vestibular function.
Backgrounds: Immunohistochemical studies have revealed
that connexin 26 (Cx26) exists not only in the cochlea but also
in the vestibular organs. K+ cycling involving gap junction
protein Cx26 in the vestibular labyrinth, which is similar to that
in the cochlea, is thought to play a fundamental role in
endolymph homeostasis and sensory transduction.
In this study, we analyzed morphological and functional
development of the vestibular organ in R75W transgenic mice between
0 days after birth (P0) and P140, which was compared with that of
littermate control mice.
Methods: We analyzed the morphological and functional
development of the vestibular organ in Cx26 transgenic mice
between 0 days after birth (P0) and P140, which was
compared with that of littermate control mice (non-Tg).
All mice used for this study were obtained from a breeding colony
of R75W transgenic mice [4] and maintained at the Institute for
Animal Reproduction (Ibaraki, Japan). R75W transgenic mice were
maintained on a mixed C57BL/6 background and intercrossed to
generate R75W transgenic animals. The animals were genotyped using
DNA obtained from tail clips and amplified with the Tissue PCR Kit
(Sigma, Saint Louis, MO, USA). The animals were deeply anesthetized
with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine
(10 mg/kg) in all experiments. All experiment protocols were
approved by the Institutional Animal Care and Use Committee at
Juntendo University School of Medicine, and were conducted in
accordance with the US National Institutes of Health Guidelines for
the Care and Use of Laboratory Animals.
Results: The gross structure of the inner ear in non-transgenic
and transgenic mice revealed no hydrops, no defects, no
degeneration in either the cochlea or the vestibule throughout
the postnatal period. Light microscopic observations in the
sensory epithelium revealed normally developed and matured
utricula macula, saccular macula, and ampulla in the
transgenic mice, which were very similar to those of the nontransgenic mouse.
Conclusions: The present study clearly demonstrated that
postnatal development and maturation in the vestibular organ
were morphologically and functionally completed in Cx26
transgenic mice.
Keywords: Connexin 26; Postnatal Development; Vestibular Organ
Introduction
Heredity deafness affects about 1 in 2,000 children and mutations in
the connexin 26 gene (GJB2) are the most common genetic cause of
congenital bilateral non-syndromic sensorineural hearing loss [1]. In
the organ of Corti, most gap junctions are assembled from connexin
(Cx) protein subunits, predominantly connexin 26 (Cx26, Gjb2 gene)
and co-localized Cx30 [2]. Mouse models have confirmed that Cx26
encoded by Gjb2 is essential for cochlear function [3,4]. A dominantnegative Gjb2 R75W transgenic mouse model shows incomplete
development of the cochlear supporting cells, resulting in profound
deafness from birth [5]. Characteristic ultrastructural changes
Materials and Methods
The animals were deeply anesthetized and perfused intracardially
with 0.01 M phosphate-buffered saline (PBS; pH 7.2), followed by 4%
paraformaldehyde (PFA; pH 7.4) in 0.1 M phosphate buffer (PB; pH
7.4). The mice were decapitated and their vestibules were dissected out
under a microscope and placed in the same fixative at room
temperature for overnight. Vestibular specimens were then placed in
0.12 M ethylenediaminetetraacetic acid (EDTA; pH 7.0) in PBS for
decalcification for a week, dehydrated and embedded in paraffin.
Serial sections (6 µm) were stained with hematoxyline and eosin (H-E)
staining.
We assessed vestibular function by head tilt and swimming test. In
the swimming test, animals were placed in the center of a container
filled with comfortably warm water.
Results & Discussion
The inner ear was dissected at P0 (n=3), P7 (n=2), P14 (n=14), P49
(n=2), and P140 (n=2). The gross structure of the inner ear in nontransgenic and transgenic mice revealed no hydrops, no defects, no
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Citation:
Okada H, Kamiya K, Iizuka T, Ikeda K (2015) Postnatal Development and Maturation of the Vestibular Organ in Dominant-Negative Connexin 26
Transgenic Mouse. J Otol Rhinol S1:1.
doi:http://dx.doi.org/10.4172/2324-8785.S1-009
degeneration in either the cochlea or the vestibule throughout the
postnatal period (Figure 1).
Figure 2: Light microscopic observations in the sensory epithelium
of utricular macula. The cross-sections in the transgenic mice were
entirely similar to those of the non-transgenic (non-Tg) mouse.
Figure 1: Gross structure of the inner ear in non-transgenic(nonTg) andR75W transgenic mice. No hydrops, no defect, no
degeneration in either the cochlea or the vestibule throughout the
postnatal period were observed in non-transgenic and R75W
transgenic mice.
Light microscopic observations were performed in the sensory
epithelium of the utricula macula (Figure 2), saccular macula (Figure
3), and ampulla (Figure 4). The maculae of the saccule, utricule, and
ampullary crista consisted of a layer of tightly packed hair cells resting
on the top of a layer of supporting cells with irregularly shaped nuclei.
The hair cells were columnar and on their free ends bore stiff hairs.
The supporting cell nuclei were arranged in multiple layers at P0. At
P14, the sensory epithelium has attained a mature appearance. The
supporting cell nuclei were arranged in a monolayer and the thickness
of the sensory epithelium was reduced. The thickness was further
reduced at P49 and P140. The cross-sections of the maculae in the
transgenic mice were very similar to those of the non-transgenic mice.
Volume S1 • Issue 1 • S1-009
Figure 3: Light microscopic observations in the sensory epithelium
of saccular macula. The cross-sections in the transgenic mice were
completely similar to those of the non-transgenic (non-Tg) mouse.
• Page 2 of 4 •
Citation:
Okada H, Kamiya K, Iizuka T, Ikeda K (2015) Postnatal Development and Maturation of the Vestibular Organ in Dominant-Negative Connexin 26
Transgenic Mouse. J Otol Rhinol S1:1.
doi:http://dx.doi.org/10.4172/2324-8785.S1-009
the sensory epithelium of the vestibular organ is composed of a simple
structure, namely a layer of hair cells resting on a layer of supporting
cells. Thus, the vestibular supporting cells are unlikely to contribute to
the transduction mediated by vestibular hair cells.
There were two previous reports regarding vestibular function in
congenital deafness with GJB2 mutations [8,9]. Todt et al. [9] reported
that 5 of the 7 patients with GJB2 mutations showed no vestibular
evoked myogenic potential responses bilaterally and that only one case
had a unilateral pathological response in the caloric test, suggesting the
presence of severe saccular dysfunction. Our previous study [8]
demonstrated that among 7 patients with GJB2-related recessive
deafness, 5 showed abnormal responses in either or both test,
vestibular evoked myogenic potential responses and caloric test, and
that the incidence was apparently and significantly higher than in
patients with congenital deafness without GJB2 mutation.The
difference in the phenotype between human and mouse may be
explained by dominant and recessive forms and species differences.
Another possibility is the gap junction function in the vestibule is
compensated by Cx30. Cx26 and Cx30 coordinated expression is
necessary for the cochlear function [10]. However, Cx30 homomeric
channels without Cx26 may preserve the normal morphology and
function in the vestibular system. Further research is required.
Conclusions
We assessed the vestibular function and morphological
development of the vestibular organ in the control mice and the
transgenic mice. No morphological and functional differences of the
vestibular organ were observed at P0-P140.
References
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Figure 4: Light microscopic observations in the sensory epithelium
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No balance disorders such as head tilt or swimming abnormality
were observed in the R75W transgenic mice.
The present study clearly demonstrated that postnatal development
and maturation in the vestibular organ were morphologically and
functionally completed in Gjb2 R75W transgenic mice, which is
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Citation:
Okada H, Kamiya K, Iizuka T, Ikeda K (2015) Postnatal Development and Maturation of the Vestibular Organ in Dominant-Negative Connexin 26
Transgenic Mouse. J Otol Rhinol S1:1.
doi:http://dx.doi.org/10.4172/2324-8785.S1-009
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