THE JOURNAL OF COMPARATIVE NEUROLOGY 3303521-532 (1993)
Reorganization of the Chick Basilar
Papilla After Acoustic Trauma
Kresge Hearing Research Institute, The University of Michigan, Ann Arbor,
The auditory epithelium in birds and mammals consists of a postmitotic population of hair
cells and supporting cells. Unlike mammals, birds can regenerate their auditory epithelia dter
trauma. Recent evidence indicates that supporting cells undergo mitosis after acoustic trauma,
suggesting that supporting cells may transdifferentiate into hair cells. The goals of this study
were to 1) characterize the responses of hair cells and supporting cells to acoustic trauma, and
2) determine whether hair cell loss is a prerequisite for generation of new hair cells. Chicks were
exposed to an octave-band noise and their inner ears assayed with fluorescence or scanning
electron microscopy, In one area of the basilar papilla, defined as the center of the lesion,
extensive hair cell degeneration occurred. Expanded supporting cells obliterated degenerating
hair cells and invaded spaces normally occupied by hair cells. Aggregates of DNA were found
within the basilar papilla, suggesting that hair cell death and disintegration may occur within
the epithelium. The epithelial sheet appeared structurally confluent at all times examined.
Supporting cells exhibited altered apical contour in distal regions of the basilar papilla, where
hair cell damage was mild or inconspicuous. Four days after noise exposure, newly generated
hair cells were found in the center of the lesion and in the distal areas, where no hair cell loss
could be detected. The results suggest that supporting cells may play an important role in
maintenance and repair of the traumatized basilar papilla and raise the possibility that production of
new hair cells is not dependent on hair cell loss in the immediate vicinity.
Key words: noise, repair, regeneration, mitosis
18 iw wiiey-~ks, Inc.
In chick, hair cells that are lost after acoustic or chemical
trauma are replaced by new, normal appearing hair cells
(Cotanche, '87; Corwin and Cotanche, '88; Ryals and Rubel,
'88). The mechanisms that initiate and control this regener-
ative process are largely unknown.
Mechanisms that control regeneration vary between
different sensory epithelial tissues. In the experimentally
damaged teleost fish eye, rod precursors can give rise to
more than one retinal cell type, suggesting that cues from
the surroundings influence the regenerative process (Ray-
mond, '91). Experiments with cultured aggregates of chick
retinal pigment epithelium have shown that transdifferen-
tiation of these cells is modulated by cell shape, the
composition of the extracellular matrix, and the presence of
bFGF (reviewed in Reh et al., '911. In the olfactory neuroep-
ithelium, degenerated neurons can be replaced by new
neurons that arise via mitotic divisions of basal cells
(Graziadei and Monti Graziadei, '85). The possibility that
extracellular matrix proteins and growth factors regulate
growth and differentiation in olfactory neuroepithelium
has also been investigated (Calof et al., '911, but the
mechanism of regulation has yet to be determined. In the
postembryonic Oscar (Astronotus ocellatus), a teleost fish,
"embryonic like" neuroepithelial cells serve as stem cells,
which can give rise to new hair cells and supporting cells
(Presson and Popper, '90). Nevertheless, it is not known
how proliferation and regeneration of new hair cells and
supporting cells are regulated. Thus, data at the cellular,
molecular, and genetic levels indicate that control mecha-
nisms that regulate epithelial regeneration are species and
3H thymidine studies in traumatized chick inner ears
showed that labeled hair cells and supporting cells were
present in the basilar papilla (BPI after several days of
recovery from acoustic overstimulation (Corwin and Co-
tanche, '88; Girod et al., '89). Thus, cell divisions appear to
play a role in repopulating the BP after trauma. Recent data
showing that supporting cells divide within 2 days after
trauma (Raphael. '92) have implicated supporting cells as a
source of newly generated hair cells. The signal(s1 regulat-
Acceptcd December 23, 1992.
Preliminary result,s were presented in part at the First Meeting on
Molecular Biology of Hearing and Deafness, San Diego, May 1992, and the
29th Workshop on Inner Ear Biology, Engelberg, Septemher 1992.
Q 1993 WILEY-LISS, INC.
ing supporting cell proliferation and differentiation in the
noise exposed BP are not known.
To explain the regulatory mechanism that controls gener-
ation and differentiation of new cells in the basilar papilla,
Corwin et al. ('91) suggested that presence of intercellular
contacts with hair cells inhibits supporting cells from
proliferating. According to this hypothesis, loss of two or
more hair cells would deprive supporting cells of hetero-
philic contacts, leading to removal of lateral inhibition and
a subsequent regenerative response.
The goals of this study were 1) to characterize the process
of hair cell degeneration and the relationships between hair
cell degeneration and the response of supporting cells, and
2) to test the hypothesis that changes in cell-cell contacts
that result from the loss of hair cells and their replacement
by expanded supporting cells are necessary to induce prolif-
eration of supporting cells. To that end, the responses of
supporting cells and hair cells to acoustic trauma were
documented by fluorescence or scanning electron micros-
copy on wholemounts of the BP.
MATERIALS AND METHODS
Animals and noise exposure
One week old white Leghorn chick hatchlings were placed
for 4 hours in a mesh-wire cage beneath a horn speaker that
delivered octave-band noise with a center frequency of 1.5
kHz and intensity of 120 dB SPL. The signal was generated
by a random noise generator (General Radio Company,
model 1381), filtered, and amplified (MC 2105, McIntosh
Power Amplifier). Intensity was calibrated with a Precision
Sound Level Meter (type 2203, Bruel & Kjaer). Chicks were
killed at 0 (n = lo), 6 (n = 5), 24 (n = 41, 48.(n = 8), or 96
(n = 6) hours after noise exposure. Seven chicks were used
as untreated controls.
Histo- and immunocytochemistry
Chicks were anesthetized and perfused intracardially
with 3% paraformaldehyde in 0.15 M phosphate buffer at
pH 7.35. Temporal bones were rapidly removed and BP
harvested. Tissues were prepared as wholemounts, perme-
abilized with 0.1% Triton X-100 in phosphate buffered
saline (PBS) for 5 minutes and stained with one or more of
the following probes: cingulin-specific antibodies were used
to label tight-junctions, phalloidin to stain F-actin, and
bisbenzimide trihydrochloride (Hoechst) to label DNA.
Nonspecific immunoreactivity was blocked in normal goat
serum (5% in PBS) for 30 minutes. Samples were then
incubated in rabbit anti-cingulin antibody (1:600 dilution;
Citi et al., '88, '89) for 60 minutes, rinsed thoroughly, and
immersed in a PBS solution containing Hoechst (2 mg/ml,
Sigma): rhodamine phalloidin (1: 100; Molecular Probes,
OR), and FITC-conjugated goat anti rabbit secondary anti-
body (1:lOO; Cappel, Durham, NC) for 30 minutes. After
rinsing, papillae were mounted in 60% glycerol in sodium
carbonate buffer (pH 8.5) with p-phenylenediamine as an
anti-bleach agent. Controls for specificity of labeling in-
cluded omission of primary antibodies and labeling of
several nonauditory tissues.
Preparations were photographed with a Leitz Orthoplan
microscope equipped for epifluorescence with x 50 and
~ 1 0 0 oil objectives, or with a Bio-Rad laser confocal
microscope with an xl00 oil objective. Photography was
performed with Kodak T-max 400 film exposed at 1,600
Scanning electron microscopy (SEM)
Animals were fixed as described above for histochemistry
and then postfixed in 1% aqueous osmium tetroxide for 30
minutes. Wholemounts were processed using routine meth-
ods (Raphael, '92). Tissues were analyzed and photo-
graphed with an AMRAY lOOOB scanning electron micro-
scope operated at 10 kV.
The use of wholemounts for histochemical analysis en-
abled visualization of the BP in its entirety (Fig. 1A). Using
wholemounts with double- and triple-labeling proved to be
an excellent method for assessing the tissue organization,
distribution of junctional complexes, and location and
integrity of nuclei. In control tissues, F-actin (microfila-
ments) was present in the stereocilia and the cuticular plate
(terminal web) of every hair cell (Fig. lB), as previously
described (Baphael, '91). Hair cells were normally orga-
nized in rows with similar spaces between each other.
Co-localization of actin (Fig. 1B) and Hoechst (Fig. 1C) in
control tissues made it possible to locate the nucleus in
relation to the actin cytoskeleton of each cell. Normal hair
cells exhibited round nuclei organized in rows, visible at a
focal plane immediately beneath the luminal surface. Sup-
porting cell nuclei also labeled by Hoechst stain were
located at a deeper focal plane closer to the basement
membrane (not shown). Thus, nuclei of cells in the BP had
a laminar distribution, with hair cell nuclei immediately
beneath the reticular lamina (RL) and supporting cell
nuclei beneath them, closer to the basal lamina, as previ-
ously reported (Raphael, '92). Cingulin (Fig. lD), a protein
specific to tight-junctions (Citi et al., '88, '89), appeared as a
narrow line at the focal plane of the RL. Thus, cingulin-
specific label was found in homophilic (supporting cell-
supporting cell) and heterophilic (support cell-hair cell)
junctions at the RL. Because the apical surface area of
supporting cells was very slender, cingulin label often
appeared as a single line. Nevertheless, it should be noted
that cingulin was present all around the apical contour of
supporting cells. Therefore, the pattern of cingulin distribu-
tion was a good marker to delineate the polygonal apical
contours of hair cells and the narrow apical contour of
supporting cells (Figs. ID, 4A). In the polygon that sur-
rounds each hair cell, contact points between adjacent
supporting cells may be at the sharp corners, as depicted in
Figure 4A', or, alternatively, along the sides of the polygon.
Actin and cingulin were co-localized at the apical intercel-
lular contacts of' cells in the BP (Fig. 1E and D, respective-
ly). The pattern of cingulin and actin labels revealed similar
cellular organization of the BP in the proximal and distal
areas. The organization of the normal sensory mosaic of the
avian BP, shown with two fluorescent markers (Figs.
lD,E), is consistent with previous reports based on scan-
ning electron microscopy (Hirokawa, '78; Tanaka and
Smith, '78; Chandler, '84).
Structural alterations that accompany hair cell degenera-
tion were consistently observed in noise exposed chicks in a
region located 1.2 mm from the proximal end of the BP.
This region of damage is referred to as the "center of the
lesion" throughout this work (see Fig. 1A for location of
this region). Supporting cells in the center of the lesion
exhibited an expanded apical area, and occupied a signifi-
cant portion of the total surface of the RL, as previously
described (Cotanche, '87; Cotanche and Dopyera, '90; Marsh
REORGANIZATION IN TRAUMATIZED BASILAR PAPILLA
Gerschenson and Rotello, '92; Raff, '92). It is likely that
constriction of the apical actin belt in hair cells is an active
process, similar to constriction of the contractile ring that
separates two daughter cells from each other during cytoki-
nesis (Schroeder, '73). If true, the apical constriction in
damaged hair cells may constitute the first step in a cascade
of cellular activities leading to cell death. Thus, I raise the
possibility that hair cells are actively involved in their own
demise. A future goal will be to determine if degenerating
hair cells express any number of genes that encode apoptosis-
associated gene products.
Signals for supporting cell division and hair
A major finding in the present work is that new hair cells
may appear in distal areas of the BP, where hair cell
damage is minimal and hair cell loss could not be detected.
This finding raises the possibility that hair cell loss is not
necessary for production of new hair cells. It should be
noted that in this work, the lack of hair cell loss has been
determined by a subjective assessment based on the pres-
ence of orderly, uninterrupted rows of normal looking hair
cells. This subjective assessment cannot conclusively rule
out that single cells are eliminated unnoticed. Nevertheless,
it is rather unlikely that two or more neighboring hair cells
would be missing without being detected. Due to the
cellular organization at the RL, to completely deprive a
supporting cell of heterophilic contacts with hair cells, at
least two hair cells must be lost (see Corwin et al., '91, for
review). Thus, the data indicate that removal of lateral
inhibition is not necessary for producing new hair cells in
the traumatized BP. These findings corroborate previous
evidence that supporting cells that divide often maintain
contacts with preexisting hair cells (Raphael, '92).
Ionic leaks due to holes in the RL are also thought to
constitute a signal for regeneration after hair cell loss in the
BP (Corwin et al., '91). However, in this work the RL and
the network of tight junctions were structurally uninter-
rupted during hair cell degeneration and regeneration,
suggesting that changes in ionic composition do not play a
role in the signalling mechanism of regeneration. Neverthe-
less, a direct measurement of the functional integrity of the
RL as an ionic barrier should be performed before endolym-
phatic leak is unequivocally ruled out as a signal for
In conclusion, the present work demonstrated that after
intense noise exposure, damaged hair cells often display
constricted apical surface area and altered apical contour.
Dying hair cells may disintegrate within the epithelium
while the RL remains structurally undisturbed. Structural
changes in response to noise occur in supporting cells in the
distal BP, where hair cell damage is minimal and hair cell
loss is undetected. New hair cells are observed in the BP 4
days after acoustic trauma, in regions with or without
noticeable hair cell loss. Regenerated hair cells are appropri-
ately oriented in the BP as soon as they can be detected with
the present methods.
These findings emphasize the important role of support-
ing cells in the course of inner ear trauma. Based on the
present results, it is proposed that degenerating hair cells
may undergo active cell death. It is further speculated that
hair cell degeneration, supporting cell expansion, and hair
cell regeneration are mediated by tensile forces at the RL
and changes in cell shape. Experiments are now underway
to examine these hypotheses, and to determine whether
production of new hair cells in the center of the lesion is
regulated by the same mechanisms as in distal regions of
I offer many thanks to Peter Finger, Michael Lee, and Dr.
Yu Wang, who provided excellent technical support. I am
extremely grateful to Drs. Stephen Easter, Donna Martin,
Pamela Raymond, and Kathryn Tosney for their very
helpful comments on the manuscript. I thank Dr. Sandra
Citi (Cornell University Medical School, New York) for
kindly donating anti-cingulin antibodies. This work was
supported by a grant from the Deafness Research Founda-
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