Hearing Research 129 (1999) 35^49

Ultrastructure and immunohistochemical identication of the

extracellular matrix of the chinchilla cochlea
Vladimir Tsuprun, Peter Santi *
Department of Otolaryngology, University of Minnesota, Rm. 121, Lions Research Bldg., 2001 Sixth St. SE, Minneapolis, MN 55455, USA
Received 11 August 1998; received in revised form 17 November 1998; accepted 27 November 1998

Abstract
The molecular composition and three-dimensional organization of the extracellular matrix (ECM) was studied by
immunofluorescent microscopy, transmission and scanning electron microscopy in three connective tissue structures of the cochlea:
the spiral limbus, basilar membrane and spiral ligament. Type II collagen, fibronectin, tenascin, chondroitin sulfate proteoglycans,
Kv and L1 integrins were immunolocalized in the ECM of these connective tissue structures. Electron micrographs showed a
continuum of cross-striated collagen fibrils having a similar diameter and axial periodicity that spread from the spiral limbus via the
basilar membrane and into the spiral ligament. Some of collagen fibrils were aggregated laterally into bundles. Bundle images, and
their digital Fourier transformations, showed a major 67-nm axial D-repeat characteristic for collagen fibrils. Transmission electron
microscopy showed numerous proteoglycans associated with the collagen fibrils. The spiral limbus, basilar membrane and spiral
ligament demonstrated regional differences in molecular composition and structural organization of their ECM. The glycoproteins
fibronectin, tenascin and KV integrin were immunolocalized mainly in the basilar membrane. Collagen fibrils of the spiral limbus and
spiral ligament did not appear to be strongly oriented. However, most of the collagen fibrils in the basilar membrane were arranged
into radially directed bundles. Collagen fibrils in the basilar membrane were also surrounded by a homogeneous matrix, which was
immunoreactive to fibronectin and tenascin antibodies. A more complete understanding of the composition and structural
organization of the ECM in these connective tissue structures in the cochlea provides a foundation upon which micromechanical
models of cochlear function can be constructed. z 1999 Elsevier Science B.V. All rights reserved.
Key words: Collagen; Extracellular matrix; Spiral limbus; Basilar membrane; Spiral ligament; Immunohistochemistry;
Transmission electron microscopy; Scanning electron microscopy

1. Introduction
The spiral limbus, basilar membrane and spiral ligament are connective tissue structures that support the
tectorial membrane, organ of Corti, and stria vascularis, respectively. The basilar membrane extends from
the inner osseous spiral lamina and spiral limbus to
the spiral ligament and forms the base of the organ of
Corti. The basilar membrane is composed primarily of
radially directed brous bundles embedded in a homo-

* Corresponding author. Tel.: +1 (612) 626-9881;

Fax: +1 (612) 626-9871; E-mail: santip@tc.umn.edu

geneous ground substance (Iurato, 1962 ; Angelborg

and Engstrom, 1974 ; Cabezudo, 1978). On an anatomical level, the basilar membrane can be divided into
two zones, called the pars arcuata and pars pectinata.
The pars arcuata contains compactly arranged, woven
brils, whereas in the pars pectinata the brils are
tightly packed into parallel bundles, which have been
called the `auditory strings' (Iurato, 1962 ; Katori et al.,
1993 ; Mikuni et al., 1994, 1995). Sound waves induce
vibrations of the basilar membrane together with the
tectorial membrane, and these vibrations stimulate the
sensory hair cells in the organ of Corti (von Bekesy,
1960).
The stiness and mass of basilar membrane, which
are important factors for cochlea micromechanics, are

0378-5955 / 99 / $ ^ see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 2 1 9 - 6

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aected by the structural organization of macromolecules of the extracellular matrix (ECM). Collagens are
major constituents of ECMs. Type II collagen has been
detected biochemically and immunohistochemically in
the mammalian tectorial membrane, in the spiral limbus, basilar membrane and spiral ligament (Richardson
et al., 1987; Thalmann et al., 1987, Thalmann, 1993 ;
Yoo and Tomoda, 1988; Ishibe et al., 1989; Ishibe and
Yoo, 1990 ; Slepecky et al., 1992; Ishii et al., 1992 ;
Kaname et al., 1994; Khetapal et al., 1994). Lesser
amounts of type IX and XI collagens were also reported for these connective tissue structures (Richardson et al., 1987; Slepecky et al., 1992; Thalmann, 1993).
Expression and localization of type IIA1 collagen
mRNA were studied in human fetal cochlea (Khetapal
et al., 1994). Defects in genes of type II collagen (Byers,
1989 ; Kuivaniemi et al., 1991) or proposed type II collagen autoimmunity (Joliat et al., 1992) were reported
to contribute to certain hearing disorders. However,
pathological changes in the ultrastructure of collagen
and other ECM molecules in the cochlea have not yet
been described. In addition to type II collagen, some
other ECM molecules were revealed in the cochlea.
They include keratan and/or chondroitin sulfate cartilage-specic proteoglycans (PGs) in the tectorial membrane, spiral limbus and basilar membrane (Ishii et al.,
1992 ; Thalmann et al., 1993; Munyer and Schulte,
1994 ; Swartz and Santi, 1997a). Two glycoproteins, bronectin (Santi et al., 1989; Woolf et al., 1992, 1996 ;
Keithley et al., 1993) and tenascin (Swartz and Santi,
1997b), have been immunolocalized within the ground
substance of the basilar membrane. Laminin, entactin,
type IV collagen and heparan sulfate PG were found in
large amounts within cochlear basement membrane
(Ishii et al., 1992 ; Cosgrove et al., 1996; Cosgrove
and Rogers, 1997 ; Santi et al., 1997). The ultrastructure
of the ECM was studied in the tectorial membrane
(Hasko and Richardson, 1988; Slepecky et al., 1992 ;
Tsuprun and Santi, 1996, 1997), spiral limbus (Ishii et
al., 1992), basilar membrane (Katori et al., 1993 ; Mikuni et al., 1994, 1995) and spiral ligament (Kaname et
al., 1994). All these connective tissue structures contain
a considerable amount of type II collagen, although
some type IX and XI collagens were also demonstrated
by immunoelectron microscopy in the tectorial membrane (Slepecky et al., 1992). Thalmann et al. (1987)
showed that about 40% of the protein in the tectorial
membrane from the guinea pig consists of type II
collagen. A similar level of this type of collagen is
present in the basilar membrane (Thalmann, 1993).
Abundant collagen brils were observed by electron
microscopy in the tectorial membrane (Hasko and Richardson, 1988; Slepecky et al., 1992; Tsuprun and
Santi, 1996, 1997), spiral limbus (Ishii et al., 1992)
and spiral ligament (Kaname et al., 1994). However,
collagen brils were not identied at the ultrastructural

level in the basilar membrane (Iurato, 1962 ; Mikuni et

al., 1994, 1995).
The binding of cells to the ECM is mediated by cell
surface receptors. The primary class of these receptors
is a family of transmembrane receptors known as integrins. Integrins are glycoproteins, consisting of K/L heterodimers. They bind to collagens, bronectin, tenascin,
laminin, entactin and other ECM molecules. Integrins
are also involved in the ECM assembly. The K and
L subunits, in various combinations, form a great variety of integrin types. The L1 and KV subunits appear to
be particularly versatile. They can combine with many
other K and L subunits in heterodimers, providing the
ability to recognize various ECM molecules (see for
review: Ruoslahti, 1991 ; Hynes, 1992 ; Brakebusch et
al., 1997). Integrins appear to play an important role in
ECM organization and cell/matrix interactions of
highly complex connective tissues of the inner ear ; however, there is no information about their distribution in
the cochlea.
The present study was performed in order to localize
some ECM molecules and to determine the structural
organization of the matrix in the spiral limbus, basilar
membrane and spiral ligament. On the basis of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) investigations, it was previously
suggested that the thin brils of the basilar membrane
appeared to be elastin-associated and elastin-independent microbrils (Mikuni et al., 1994, 1995). In the
present work, we will show ultrastructural and immunohistochemical evidence that the thin brils of the spiral limbus, basilar membrane and spiral ligament are
similar and appear to be composed of type II collagen.
Interaction of collagen brils and other ECM macromolecules (proteoglycans, bronectin, tenascin, basement membrane proteins) and their receptors may provide unique structural organization of the cochlear
connective tissues and contribute signicantly in their
mechanical characteristics.
2. Materials and methods
2.1. Tissue preparation
Samples of cochlear tissue from our tissue bank were
obtained from normal chinchillas that were approximately 1 year old. For harvesting of cochlear tissues
the animals were deeply anesthetized with ketamine hydrochloride (90 mg/kg) and sodium pentobarbital
(20 mg/kg). After the chinchillas reached a surgical level
of anesthesia, whole cochleas were removed from the
temporal bones and xed by perilymphatic perfusion
via round and oval windows, as previously described
(Santi et al., 1990). The care and use of the animals
for this study were reviewed and approved by the Uni-

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versity of Minnesota's Institutional Animal Care Committee.

2.2. Transmission electron microscopy
For tannic acid treatment, cochleas were xed for 1 h
in a solution containing 2% tannic acid, 2% paraformaldehyde, 1.5% glutaraldehyde, 4 mM calcium chloride in
0.075 M sodium cacodylate buer at pH 5.3 for 1 h,
and rinsed in 0.13 M cacodylate buer at pH 6.8 for
45 min. The tissues were postxed in 1% osmium tetroxide for 1 h and rinsed in 0.13 M cacodylate buer
at pH 6.8 for 45 min.
To increase the contrast of negatively charged glycosaminoglycan residues of PGs the treatment of cationic
dye cuprolinic blue at a ``high critical electrolyte concentration'' (Scott, 1980, 1985) was used. Cochleas were
xed for 1 h in solution containing 0.2% cuprolinic
blue, 2.5% glutaraldehyde and 0.2^0.3 M magnesium
chloride buer at pH 5.6, as previously described (van
Kuppevelt et al., 1987; Santi et al., 1990). After xation, the cochleas were rinsed in the same solution without cuprolinic blue for 10 min and immersed for 2 h in
buer containing 2.5% glutaraldehyde, 0.5% sodium
tungstate, and 0.2^0.3 M magnesium chloride. After
rinsing in the same solution without sodium tungstate,
cochleas were immersed in 2.5% glutaraldehyde containing 0.2% cuprolinic blue, 0.5% sodium sulfate, and
corresponding concentration of magnesium chloride for
2 h.
For ruthenium red/alcian blue treatment, cochleas
were xed in a solution of 0.2% ruthenium red and
0.2% alcian blue, 2.5% glutaraldehyde in 0.1 M sodium
cacodylate buer (pH 7.2) for 4 h, rinsed in 0.1 M
cacodylate buer and stored in 0.2% ruthenium red/alcian blue in 0.1 M cacodylate buer overnight. The
tissues were postxed in 1% osmium tetroxide, 0.2%
ruthenium red/alcian blue in 0.05 M cacodylate buer
for 4 h and rinsed in 0.1 M cacodylate buer for
45 min.
The cochleas were dehydrated, embedded, and sectioned on an LKB Nova ultramicrotome, as previously
described (Santi, 1986). The sections were not counterstained with uranyl acetate or lead citrate. The specimens were examined with JEOL 1010 electron microscope at 60 kV and magnications of 5000^15 000U.
The photographs of the electron microscope images
were digitized with Linotype-Hell atbed scanner interfaced to Macintosh computer. The periodicity of the
structures was studied using Fourier transformation of
the images (256U256 pixels) within NIH Image (ver.
1.60) software. The periodicity of the structure on specimen level was calculated from the position of diraction peaks (using Fourier transformation of the image
with known periodicity for calibration) and the magnication of the print.

37

2.3. Scanning electron microscopy

Chinchilla cochleas were xed in 2.5% glutaraldehyde, 0.5% cytylpyridinium chloride, 2 mM calcium
chloride in 0.15 M cacodylate buer, pH 7.3. Then
they were dehydrated, critical point dried, coated with
approximately 10 nm of platinum, and examined on
Hitachi S-900 eld emission scanning electron microscope at 1.5^5 kV.
2.4. Immunouorescence microscopy
Indirect immunouorescence microscopy was performed on frozen xed tissue sections. Cochleas were
xed in 100% acetone for 18 h. Tissues were cryoprotected by immersion in increasing concentration of
5^20% sucrose, and frozen in a 2:1 mixture of 20%
sucrose/OCT (Barthel and Raymond, 1990). Tissue
blocks were cryosectioned at 5 Wm. Cryosections,
mounted on gelatin-coated slides, were dehydrated
with 10 mM phosphate buered saline (PBS), pH 7.4,
blocked with 1% non-fat dry milk in PBS, and incubated with about 1:50 dilution of primary antibodies
in 1% bovine serum albumin (BSA) for 45 min. After
repeated washes with PBS, the sections were incubated
with FITC-conjugated, secondary antibodies diluted
1:100 in 1% BSA for 45 min. Finally they were rinsed
in PBS and examined with a Vanox ANBT3 (Olympus,
Lake Success, NY) light microscope.
2.5. Antibodies
Goat anti-type II collagen, polyclonal (Southern Biotechnology Association, Birmingham, AL) ; anti-proteoglycan v Di 6S monoclonal (clone 3-B-3/C1, Chemical
Credential, Lisle, IL), which recognizes 4-sulfated (4S)
and 6-sulfated (6S) chondroitin sulfate; rabbit anti-human decorin, raised against synthetic peptides (LF-30),
was a generous gift from Dr. Larry Fisher (Fisher et al.,
1987); mouse anti-human bronectin, monoclonal
(clone FN-3E2, Sigma, St. Louis, MO); mouse antihuman, monoclonal (clone BC-24, Sigma, St. Louis,
MO) or polyclonal rabbit, anti-human tenascin (Chemicon, Temecula, CA) ; rabbit anti-integrin L1 and KV
subunits, polyclonal (Chemicon, Temecula, CA) were
used as primary antibodies.
3. Results
3.1. Immunohistochemistry of the ECM and integrin
receptors
The ECM of the spiral limbus, basilar membrane
and spiral ligament contains abundant brillar elements. To identify these brillar elements we used anti-

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V. Tsuprun, P. Santi / Hearing Research 129 (1999) 35^49

Fig. 1. Immunohistochemical demonstration of various ECM components within the connective tissue structures of the cochlea in the chinchilla
with antibodies against (a) type II collagen (COL II), (b) 4S/6S chondroitin sulfate (CS), (c) decorin (DC), (d) bronectin (FN), (e) tenascin
(TN), (f) L1 integrin, (g) K1 integrin. Bar = 100 Wm (a^g). TM, the tectorial membrane; L, the spiral limbus; PA and PP, pars arcuata and pars
pectinata zones of the basilar membrane (BM); SL, the spiral ligament.

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39

Fig. 2. Low-power TEM view of radial section of the spiral limbus after tannic treatment showing numerous, thin brils in the matrix (M).
Bar = 1 Wm. TM, the tectorial membrane; IS, the inner sulcus cells.

bodies directed against dierent matrix proteins and

their receptors. Antibodies against type II collagen
were reactive to bers of the spiral limbus, basilar membrane and spiral ligament. Type II collagen was abundant in the spiral limbus, traveled in the thin layer of
the pars arcuata zone of the basilar membrane, then
split into two layers in the pars pectinata, and continued into the spiral ligament (Fig. 1a). Chondroitin sulfate PGs were also seen in the ECM using antibodies
against anti-proteoglycan v Di 6S (Fig. 1b), and were
co-localized with type II collagen in the spiral ligament,
basilar membrane and spiral ligament. The small chondroitin sulfate-containing PG, decorin, showed a distribution similar to v Di 6S PG (Fig. 1c).
Two glycoproteins, bronectin and tenascin, showed
strong immunoreactivity within the homogeneous ma-

trix of the basilar membrane. Intense immunoreactivity

of bronectin was observed both in pars arcuata and
pars pectinata zones (Fig. 1d). Within the pars pectinata, bronectin reactivity was present in two regions of
the homogeneous matrix which is divided by a medial
layer of brils. Fibronectin in the lower region of the
homogeneous matrix, especially near mesothelial cells,
reacted more strongly compared with the upper region.
The same pattern of immunoreactivity was observed for
tenascin (Fig. 1e). Similar distribution of bronectin
and tenascin indicates their co-localization in the basilar
membrane.
We have also studied the distribution of L1 and KV
integrins. L1 integrin immunoreactivity was observed at
the cell-basement membrane interface of the interdental
cells and the inner sulcus cells in the spiral limbus,

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Fig. 3. a, b: Low-power TEM view of radial section of the basilar membrane (BM) and spiral ligament (SL) after tannic acid treatment. The
BM shows tightly aggregated thin brils running in a radial direction. The brils are arranged in one layer (arrow) in pars arcuata (a) and
then divided into two layers (arrows) in pars pectinata (b, c) of the BM. Loosely arranged brils are also extended from BM into matrix of
the SL (c, d). Bars = 5 Wm (a^d). OP, outer pillar cells; M, mesothelial cells; D, Deiters' cells; B, Boettcher's cells; C, Claudius' cells; ES, external sulcus cells.

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41

Fig. 4. SEM views of the fractured tissue of the basilar membrane (a^d) and spiral ligament (e) exposing brillar structures. a: Fibrous bundles
(arrows) in the pars pectinata zone of the basilar membrane (BM) viewed from scala media side (bar = 10 Wm). b: Each bundle consists of parallel collagen brils (bar = 1 Wm). The D-periodic cross-striation of the brils is shown in the inset (bar = 100 nm). c,d: Fibrous bundles viewed
from scala tympani side near the pars arcuata zone of the basilar membrane (bars = 1 Wm). M, mesothelial cell. e: Network of collagen brils
in the spiral ligament, which form numerous bundles of dierent size and diameter (bar = 1 Wm). An axial striation of the brils is shown in inset (bar = 100 nm).

across the basilar membrane and beneath the external

sulcus cells (Fig. 1f). This integrin was also observed on
the scala tympani side of the basilar membrane adjacent
to mesothelial cells. Antibodies against KV integrin
showed reactivity mainly at the cell membrane of Claudius' and Boettcher's cells in the areas of contact with
the basement membrane of basilar membrane and
around the mesothelial cells (Fig. 1g).
3.2. Distribution of the brils
Radial sections of the spiral limbus after treatment
with tannic acid show poorly oriented, straight brils,
which had a tendency to aggregate (Fig. 2). The brils
continued into the basilar membrane (Fig. 3a,b) where
they ran in a radial direction. In the basilar membrane

the brils were tightly aggregated and arranged in one

layer in pars arcuata (Fig. 3a) and two layers in pars
pectinata (Fig. 3b,c). Loosely oriented brils spread
from the basilar membrane into the matrix of the spiral
ligament (Fig. 3c) toward the stria vascularis (Fig. 3d).
Thus, spiral limbus, basilar membrane and spiral ligament appear to form a contiguous network of brils.
The distribution of brils coincides with the reactivity
of antibodies against type II collagen (Fig. 1a) indicating that these brils contain type II collagen.
Fractured, connective tissue structures occasionally
expose bundles of thin brils of the basilar membrane.
SEM images of parallel bundles in the pars pectinata
zone of the basilar membrane viewed from the scala
media and the scala tympani sides are shown in Figs.
4a,b and 4c,d, respectively. The bundles are 0.5^2 Wm in

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V. Tsuprun, P. Santi / Hearing Research 129 (1999) 35^49

diameter and consist of thin brils running in the radial

direction. Toward the pars arcuata zone the brils were
less straight and appeared woven within the bundles
(Fig. 4c). At higher magnication, SEM images demonstrate that brils are aligned parallel in their bundles
and show cross-striation, with a repeat about 67 nm
and characteristic of the periodicity for collagen brils
(Fig. 4b). In contrast to the basilar membrane, collagen
brils in the spiral ligament are diusely organized (Fig.
4e). However, they also have a tendency to aggregate
laterally and to form numerous bundles of dierent size
and diameter, displaying axial D-periodic striations
(Fig. 4e, inset).
3.3. Spiral limbus
In order to better clarify the molecular and supramolecular organization of the thin brils and other
ECM molecules, we performed a TEM study with histological procedures using tannic acid, cuprolinic blue
and ruthenium red/alcian blue xatives. A fragment of
the spiral limbus attached to the tectorial membrane
after tannic acid treatment (Fig. 5a) shows interdental
cells, which are surrounded by the basement membrane,
and the ECM. The lamina densa of the basement membrane forms a layer approximately 30 nm thick. The
ECM contains individual thin brils and small bundles
of brils embedded in the ground substance.
The cationic dye cuprolinic blue ``at a high critical
electrolyte concentration'' selectively binds to negatively
charged glycosaminoglycan chains of PGs (Scott, 1980,
1985). Micrographs of sections of the spiral limbus
treated with cuprolinic blue (Fig. 5b) showed abundant,
electron-dense, rod-shaped PGs attached to the lightly
stained collagen brils. The most prominent dierence
is the size of the PGs in the spiral limbus compared to
the PGs of the tectorial membrane. PGs in the ECM of
the spiral limbus were randomly associated with collagen brils and had a wide size distribution of their
projected length (10^150 nm) and width (10^30 nm).
In the tectorial membrane, PGs were less than 65 nm
in length and 12 nm in width, and were D-periodically
associated with collagen brils (Tsuprun and Santi,
1997). Most brils in the spiral limbus are individual,
however, some brils were assembled into bundles (Fig.
5c). The bundles consisted of parallel brils, with an
average spacing of 20^22 nm. Typical Fourier transformation from one of the bundle images shows diraction

peaks located on three layer lines (Fig. 5c). The distances of the layer lines (Fig. 5c) from an equatorial line
are in back proportion to the periodicities of 67 nm
(rst line), 33.5 nm (second line), 22.3 nm (third line)
on specimen level (see Section 2) and correspond to
axial D-repeat of collagen brils, about 67 nm.
In thin sections of tissues from the spiral limbus,
basilar membrane and spiral ligament, after ruthenium
red/alcian blue treatment, brils were positively stained
and clearly showed D-periodic collagen patterns (Fig.
5d). Electron-dense PG particles, collapsed after xation, were observed along collagen brils. This is in
agreement with immunouorescent analysis, which
showed co-localization of type II collagen (Fig. 1a)
and chondroitin sulfate PGs (Fig. 1b).
3.4. Basilar membrane
TEM of radial sections of the pars arcuata and pars
pectinata zones of the basilar membrane after tannic
acid treatment show, respectively, one and two brous
layers, surrounded by homogeneous ground substance
(Fig. 3a^c). The location of the brous layers coincides
with type II collagen immunoreactivity in the basilar
membrane (Fig. 1a). The brous layer of pars arcuata
and the upper layer of pars pectinata form a continuous
layer, directly associated with the basilar membrane at
the undersurface of Deiters', Claudius' or Boettcher's
cells (Fig. 3a^c). At higher magnications, cross-striated, thin brils, with a spacing of 20^25 nm between
them, can be seen in the brous layers of the basilar
membrane (Fig. 6a). Typical Fourier transformation
from the layer image shows diraction peaks located
on three layer lines (Fig. 6a, inset). The rst meridianal
peak corresponds to a periodicity of about 67 nm, the
same as D-repeat of collagen brils in the spiral limbus
(Fig. 5c). TEM images of the layers (Fig. 6b) are similar
to their SEM images (Fig. 4b, inset) and also show 67
nm, periodic cross-striation.
Fig. 6c shows the cross-section of the pars pectinata
zone of the basilar membrane perpendicular to the axes
of brils. Individual parallel bundles of thin brils are
clearly visible in a lower, scala tympani layer. These
bundles apparently correspond to those observed by
SEM (Fig. 4a^d). In a high-power view, collagen brils
appear as dark-spot projections. Fibrils are compactly
arranged with a center-to-center space of 20^25 nm.
Micrographs of radial sections of the basilar mem-

C
Fig. 5. TEM views of the spiral limbus. a: Micrograph of the spiral limbus after tannic acid treatment showing interdental cells (ID), the tectorilal membrane (TM), extracellular matrix (ECM) and basement membrane (open arrows) (bar = 50 nm). b: Cuprolinic blue treatment reveals
abundant, electron-dense PGs (small arrows) in the spiral limbus and small PGs (arrowheads) in the TM (bar = 50 nm). c: Typical bundle of
parallel brils from the matrix area (bar = 100 nm) and digital Fourier transform from its image showing diraction peaks corresponding to
67 nm, axial D-repeat of the collagen bris. d: Ruthenium red/alcian blue treatment shows striated D-band pattern of the collagen brils associated with PG (arrows) particles (bar = 100 nm).

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Fig. 6. TEM views of the basilar membrane after tannic acid treatment showing two layers of brous bundles (arrows), ground substance
(GS), basilar membrane (arrowheads), mesothelial cells (M), Claudius' cell (C) and Boettcher's cells (B). a: Radial section of the par pectinata
zone (bar = 1 Wm). Inset shows fragment of the bundle of parallel brils (bar = 100 nm) and digital Fourier transformation from its image corresponding to 67 nm periodicity of the brils. b: High-power image of brillar bundle showing cross-striation characteristic for collagen brils
with a repeat about 67 nm (bar = 500 nm). c: Cross-section of pars pectinata zone. Individual parallel bundles are clearly visible in a lower
layer (bar = 5 Wm). Inset shows high-power view of collagen brils (dark spots) in the bundle (bar = 100 nm).
6

brane treated with cuprolinic blue (Fig. 7) show a bundle of collagen brils associated with randomly oriented, rod-shaped PGs of similar projected length and
width as in the spiral limbus (see Fig. 5b). PGs were not
observed in the ground substance of the basilar membrane. Electron-dense PG particles, apparently, correspond to 4S/6S chondroitin sulfate or decorin, which
were co-localized with type II collagen in basilar membrane by immunouorescent microscopy (Fig. 1a^c).
Fibrous, nodular structures, less than 12 nm in diameter and morphologically dierent from collagen brils,
were visible within ground substance. The brous structures are not straight and are approximately parallel to
collagen brils. The diraction patterns from the images of local areas of the ground substance (Fig. 7,
inset) indicate that these structures are unidirectional
and arranged with an averaged space of about 35 nm.

which appeared to contain, and be primarily composed

of type II collagen in the spiral limbus (Ishii et al.,
1992) and spiral ligament (Kaname et al., 1994). The
striation across thin brils of basilar membrane was
previously reported using TEM study (Mikuni et al.,
1994), however, the authors did not nd regular D-periodicity of collagen and related them to elastin-associated microbrils. X-ray diraction analysis (Iurato,
1962) and SEM (Mikuni et al., 1995) also did not iden-

3.5. Spiral ligament

Immunouorescent microscopy shows the presence
of type II collagen in the spiral ligament, extending
from the basilar membrane (Fig. 1a). Numerous
cross-striated, irregularly oriented, thin collagen brils
and their bundles consisting of parallel brils were seen
in electron micrographs of the spiral ligament after tannic acid treatment (Fig. 8a). Fourier transformation of
the bundle images shows a 67 nm axial repeat corresponding to collagen D-bands (Fig. 8a, inset). This repeat was unchanged, compared with the spiral limbus
and basilar membrane. Cross-sections of the collagen
bundles (Fig. 8b) demonstrate crystalline-like arrays
of collagen brils having tetragonal symmetry with a
regular space of 20^22 nm between the brils.
In sections of the spiral ligament treated with cuprolinic blue, elongated PG particles were visible in association with collagen brils (Fig. 8c). Their appearance
and size were similar to those observed in the spiral
limbus (Fig. 5b) and basilar membrane (Fig. 7).
4. Discussion
Type II collagen, bronectin, tenascin, 4S/6S chondroitin sulfate PG, decorin, KV and L1 integrins were
immunolocalized in the spiral limbus, basilar membrane
and spiral ligament of chinchilla cochlea. These connective tissue structures contain small diameter brils,

Fig. 7. TEM view of radial section of the basilar membrane after

cuprolinic blue treatment showing the bundle of collagen brils associated with randomly oriented PGs (arrows) and brous, nodular
structures (arrowheads) within ground substance (GS). Bar = 200
nm. Inset demonstrates typical Fourier transformation from the image of local area of ground substance containing brous structures.
Diraction peak (arrow) corresponds to an averaged space between
brous structures of about 35 nm.

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V. Tsuprun, P. Santi / Hearing Research 129 (1999) 35^49

Fig. 8. TEM views of the spiral ligament. a: Micrograph of the spiral ligament after tannic acid treatment showing numerous cross-striated
bundles of collagen brils (bar = 50 nm). Inset shows fragment of the bundle of parallel brils (bar = 100 nm) and digital Fourier transformation from its image with diraction peaks corresponding to 67 nm periodicity of the brils. b: Cross-section of the bundles of collagen bril
(bar = 100 nm). c: Fragment of the bundle of collagen brils associated with randomly oriented PGs (arrows) after cuprolinic blue treatment
(bar = 100 nm).

tify collagen brils in the basilar membrane. Furthermore, type II collagen was not detected immunohistochemically in the basilar membrane of guinea pig (Tomoda et al., 1984). Ishibe et al. (1989) were able to
demonstrate type II collagen only in the small region
of the basilar membrane of the guinea pig embryo, but

not in newborn animals. In contrast to these publications, our immunohistochemical, TEM and SEM data,
and image analysis of micrographs provided evidence
that the spiral limbus, basilar membrane and spiral ligament contain a continuum of straight, cross-striated
type II collagen brils in the chinchilla cochlea. These

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brils are of similar diameter and spread from the spiral

limbus via basilar membrane into the spiral ligament.
Collagen brils were frequently observed to be laterally
aggregated into striated bundles. Striation pattern and
periodicity of the bundle images appears unchanged
throughout the spiral limbus, basilar membrane and
spiral ligament. Fourier transformations of the bundle
images revealed 67 nm axial repeat, corresponding to
the D-periodicity of striated bands of collagen brils.
Numerous, randomly oriented PGs, specically
stained for TEM with cuprolinic blue in the ``critical
electrolyte concentration'' method or with ruthenium
red/alcian blue, were observed in the spiral limbus, basilar membrane and spiral ligament in association with
collagen brils. An association of type I and type II
collagen brils with PGs was observed in many other
connective tissues (Scott, 1988). Immunouorescent microscopy detected 4S/6S chondroitin sulfate PGs and
decorin, small chondroitin sulfate-containing PG, which
were co-localized with type II collagen (Fig. 1a^c).
Chondroitin sulfate-containing PGs were previously immunolocalized in the basement membrane of gerbils
(Munyer and Schulte, 1994). Another connective tissue
structure, the tectorial membrane, also contained abundant type II collagen brils associated with PGs (Hasko
and Richardson, 1988; Slepecky et al., 1992; Tsuprun
and Santi, 1996, 1997). In contrast to the spiral limbus,
basilar membrane and spiral ligament, collagen brils in
the tectorial membrane are interconnected via small
PGs or type B brils. PGs of the tectorial membrane
are smaller in size, orthogonally oriented and attached
D-periodically to collagen brils (Tsuprun and Santi,
1996, 1997). No immunoreactivity of 4S/6S chondroitin
sulfate or decorin was found in the tectorial membrane
(Fig. 1a).
Type II collagen in combination with PGs should be
considered as an important component of ECM, common to the spiral limbus, basilar membrane and spiral
ligament and contributing to their structural integrity.
However, these connective tissue structures demonstrate
regional dierences in distribution and arrangement of
their collagen brils. The spiral limbus and spiral ligament contain mainly individual collagen brils without
strong orientation, which occasionally form small bundles of parallel brils. The basilar membrane is a complex, highly organized connective tissue. Collagen brils
are running in the radial direction and are distributed in
one layer in the pars arcuata zone of the basilar membrane or in two layers in pars pectinata. Collagen brils
in the lower tympanic layer of the par pectinata are
arranged into about 50 parallel bundles that are 0.5^2
Wm in diameter and immersed in the homogeneous
ground substance. A similar three-dimensional arrangement of the brils was observed previously in the basilar membrane of the rat (Iurato, 1962), guinea pig (Katori et al., 1993) and mouse (Mikuni et al., 1994, 1995).

47

Control of growth of collagen brils, their assembly

into bundles and association with other ECM molecules
in connective tissues appears to occur in extracellular
compartments of these tissues (Birk et al., 1991).
Immunohistochemical analysis in the present study
showed dierent molecular composition of the ECM
and integrin receptors in the cochlea partitions : the
spiral limbus, basilar membrane and spiral ligament.
The glycoproteins bronectin, tenascin, KV integrin
were immunolocalized mainly in the basilar membrane,
but not in the spiral limbus and spiral ligament (Fig.
1g). The L1 integrin showed higher immunoreactivity in
the basilar membrane, compared to the spiral limbus
and spiral ligament (Fig. 1f). An interaction of collagen
with dierent ECM macromolecules in the spiral limbus, basilar membrane and spiral ligament appears to
result in dierent distribution and three-dimensional
organization of their collagen brils.
Recent investigations of stiness (Olson and Mountain, 1991, 1994) and morphometric data (Schweitzer et
al., 1996) of the basilar membrane suggest the critical
role of brous layers in cochlear mechanics. These
layers appear to provide tensile strength during
sound-induced vibration of the basilar membrane. In
addition to the collagen bundles, the ground substance
may also contribute to the mass and stiness of the
basilar membrane. Two glycoproteins, bronectin and
tenascin, were co-localized immunohistochemically in
the ground substance of the basilar membrane surrounding the collagen brils. These glycoproteins appear to be produced by mesothelial cells (Santi et al.,
1989 ; Woolf et al., 1996; Swartz and Santi, 1997b),
which form the tympanic covering layer. Fibronectin
shows high immunoreactivity in the basilar membrane
and at the ultrastructural level is a major candidate for
the thin brous structures running parallel to collagen
brils within ground substance of the basilar membrane
(Fig. 7). These structures were visible after treatment
with cuprolinic blue, which can react specically with
sulfated residues of PGs and glycoproteins. Morphologically similar brous structures, attributed to bronectin, were observed in vivo and in vitro for dierent cell
and ECM types, and appeared to be organized from
high molecular weight multimers of bronectin molecules (Mosher et al., 1991 ; Dzamba and Peters, 1991;
Chen et al., 1997). The high structural organization of
collagen brils and other related ECM molecules in the
cochlear connective tissue structures may provide the
necessary stiness, tensile strength and compression resistance of the organ of Corti to transfer sound-induced
vibrations from the basilar membrane to the hair cells.
Acknowledgments
The authors are grateful to Dr. D. Swartz for his

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V. Tsuprun, P. Santi / Hearing Research 129 (1999) 35^49

help in the immunohistochemical analysis and Dr. L.

Fisher for his generous gift of the anti-decorin antibody. This research was supported by grants from the
NIDCD.

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