Brain Research 822 Ž1999.

43–51

Research report

Sequential changes in anti-GAL-1 staining of the rat organ of Corti following

amikacin exposure
´ Bartolome´ a , Philippe Vago b,c , Pablo Gil-Loyzaga a, ) , Gullienne Humbert b ,
M. Visitacion
Raymonde Joubert-Caron d , Remy ´ Pujol b , Marc Lenoir b
a
Centro CultiÕos Celulares (CAI-UCM) and Departamento de Cirugıa ´ II (ORL), Facultad Medicina, UniÕersidad Complutense, Madrid, Spain
b
INSERM U. 254, UniÕersite´ Montpellier I, Faculte´ de Medecine,
´ Montpellier, France
c
´ ´
Histologie-Embryologie-Cytogenetique, UniÕersite´ Montpellier I, Faculte´ de Medecine,
´ Montpellier, France
d
´
Laboratoire de Biochimie et Technologie des Proteines, UniÕersite´ Paris Nord, Bobigny, France
Accepted 15 December 1998

Abstract

Hair cell loss and a non-functional epithelial reorganization appeared in the organ of Corti after acoustic or toxic damage. Moreover,
in the drug damaged organ of Corti, transient atypical cells were recently described with characteristics of both immature hair cells
andror non-sensory epithelial cells. The phenotype of these atypical cells has been now investigated by using the galectine 1 ŽGAL-1.
antibody. In the normal organ of Corti, this antibody recognizes all the epithelial cells except the sensory hair cells and their supporting
cells. At PD 21, transient atypical cells were not stained by GAL-1 antibody, suggesting that they were originated from hair cells or their
supporting cells. Later, the organ of Corti was substituted by an epithelial scare, GAL-1 stained. This study also emphasizes the particular
resistance of the cochlear apex to degeneration after antibiotic intoxication. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Transdifferentiation; b-galactoside; Lectin; Ototoxicity; Cochlea; Rat

1. Introduction These atypical cells, occupying the region of the missing

In the mammalian organ of Corti, the inner ŽIHCs. and
the outer ŽOHCs. hair cells are very sensitive to acoustic
trauma or to drug toxicity. Whereas lost hair cells are
replaced by new hair cells in the damaged basilar papilla
of birds w1,7,8,35x, the loss of IHCs and OHCs is consid-
ered to be irreversible in adult mammals. In these condi-
tions, the supporting cells exhibited an important spatial
reorganization in vivo w5,10,17,23,28,29,34x and in vitro
w36x.
Amikacin, a well-known ototoxic aminoglycoside an-
tibiotic, produced extensive hair cell losses w21x in particu-
lar during the period of cochlear enhanced sensitivity to
ototoxicity, i.e., from postnatal day 9 to 16 w27x. Recently,
the presence of atypical cells has been observed in the
cochleae of young rats treated with amikacin w9,18,19,37x.
)
Corresponding author. Universidad Complutense, Apartado de
Correos 60.075, 28080 Madrid, Spain. Fax: q34-91-3941-383; E-mail:
loyzaga@eucmax.sim.ucm.es
OHCs, shared morphological and phenotypical character-
istics with both immature hair cells and non-sensory ep-
ithelial cells: their apical pole was covered with a tuft of
actin rich microvilli and their cell body contained cytoker-
atins w9x. As proliferative cells were absent from the dam-
aged region, it was hypothesized that atypical cells arise
through direct transformation of some of the non-sensory
epithelial cells which reorganize during and after hair cell
degeneration w9,37x. The presence of these atypical cells in
the organ of Corti after amikacin administration could
suggest an attempt at hair cell transdifferentiation w9,18,-
19,37x.
In the present study, we were interested in Ži. providing
new information on the sequential changes occurring in the
organ of Corti after amikacin administration, and ii. clari-
fying some aspects of phenotype of the transient atypical
cells.
The use of specific cell markers was required to differ-
entiate sensory and non-sensory cell derivatives w13–
16,32,33x. In this way, we have investigated the immuno-
histochemical expression of galectine-1 ŽGAL-1., a b-

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 0 7 0 - 7
44 M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51

galactoside-binding endogenous lectin w4,6x, in the organ specifically expressed in non-sensory cells but the support-
of Corti of amikacin treated rats. In the developing and ing cells, i.e., border, phalangeal, pillar, and Deiters’ cells
adult normal organ of Corti, this lectin was shown to be w11,31x. A total absence of this lectin was observed in the

Fig. 1. Scanning Ža, and insert. and transmission Žb. electron microscopy in amikacin treated rats ŽPD 21.. Ža. Surface view of the organ of Corti in the
apical region of the cochlear spiral. All the hair cells are missing. Note the presence of small tufts of microvilli Žarrowheads. in the area of preexisting
OHCs ŽO.. ŽP s pillar cells; ISS s inner spiral sulcus; OSS s outer spiral sulcus.. Scale bar s 50 mm. Insert: tufts of microvilli at higher magnification.
Scale bar s 0.5 mm. Žb. Transverse section of the damaged organ of Corti in the lower apical region of the cochlea. The tunnel of Corti ŽtC. is largely open
and the pillar cells Žp. look well-preserved. Both the OHCs and the IHCs are absent. Deiters’ cells ŽD. expansions have invaded the region of pre-existing
OHCs. Epithelial cells of the OSS occupy a large area of the epithelium and partly cover the Deiters’ cells. Note: the two epithelial cells Ž1 and 2. in the
area of the missing IHC. Scale bar s 20 mm.
 M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51 45

Corti’s organ sensory cells during development and in the
adulthood w11,31x.
Amikacin was injected to rats from postnatal day 9 to
16 days, i.e., during the period of cochlear enhanced-sensi-
tivity to ototoxicity w21,27x. The cochleae were processed
for scanning ŽSEM., transmission ŽTEM. electron mi-
croscopy, and for immunostaining. They were examined at
21, 28, 45 and 70 days, when the cochlea is considered to
be both anatomically w20,22x and functionally w26x adult-
like.
2. Material and methods
The care and use of animals in this study were in strict
accordance with the animal welfare guidelines of the Dec-
laration of Helsinki.
A total number of 28 Wistar rats were used in this
study. Twenty-four animals received a daily subcutaneous
injection of 500 mgrkg of amikacin ŽBristol. for 8 consec-
utive days between postnatal day ŽPD. 9 and PD 16, the
day of birth being PD 0. Twenty-one of these treated rats

Fig. 2. Surface preparations of the organ of Corti in the apical region of the cochlea Žconfocal microscopy.. Ža,b. Non-treated rat ŽPD 21.. Ža. Rhodamine
phalloidin. Note the strong labelling at the level of the cuticular plates and stereocilia of OHCs ŽO. and IHCs ŽI. and at the level of the junctions between
the Deiters’ cells and the OHCs ŽISS s inner spiral sulcus; OSS s outer spiral sulcus.. The pillars of Corti Žp. are also well labelled. Žb. Anti-GAL-1
immunoreactivity. Only the cells located in the OSS are labelled. Žc,d. Amikacin treated rat ŽPD 21.. Žc. Rhodamine phalloidin. The cuticular plates and
stereocilia of the hair cells are absent. In the region of the pre-existing OHCs, some atypical cells Žarrowheads. can be identified: they showed a central dot
of labelling corresponding to the actin present within the tuff of microvilli. Žd. Anti-GAL-1 immunoreactivity. The labelling is restricted to the cells in the
OSS. The atypical cells are not stained. Scale bars s 10 mm.
46 M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
 M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51 47

were used for histochemical investigations: three of them
were used for surface preparations and eighteen rats for
paraffin sections at either PD 21 Ž n s 9., 28 Ž n s 3., 45
Ž n s 3. and 70 Ž n s 3.. Three treated rats were sacrificed
at PD 21 for morphological investigations using SEM and
TEM. Four non-treated ŽPD 21. rats served as control
animals.
2.1. Electron microscopy
The cochleae, from PD 21, were perilymphatically per-
fused with a fixative solution containing 2.5% glutaralde-
hyde in 0.1 M phosphate buffer saline at pH 7.4, 0.1 M
ŽPBS.. Three cochleae were prepared for SEM, and one
cochlea for TEM. For SEM, the bony capsule was re-
moved, later the stria vascularis and the tectorial mem-
brane were dissected away. Samples were dehydrated in
graded series of ethanol from 50 to 100%, then critical
point-dried in CO 2 . They were coated with 10 nm gold-
palladium and observed under a Hitachi S4000 micro-
scope.
For TEM, the cochleae were postfixed in 1% aqueous
osmium tetroxide, dehydrated in ethanol Ž50% to 100%.
and embedded in Spurr resin. Five series of about 10
transverse ultrathin sections each, were performed in the
apical region of each cochlea. Sections, mounted either on
formvar coated or 200 mesh grids and counterstained with
uranyl acetate and lead citrate, were examined using a
Hitachi 7100 microscope.
2.2. Histochemistry
2.2.1. Surface preparations
The left cochleae of three treated and two non-treated
rats Žall sacrificed at PD 21. were fixed with paraformal-
dehyde 4% in PBS, washed twice then, preincubated for
30 min in a 30% goat serum solution in PBS. Subse-
quently, the apical coils were dissected away and prepared
as surface preparations. Then, these surface preparations
were processed for a double stained with anti-GAL-1 and
actin. They were incubated for 2 h in a 1r2000 dilution of
rabbit anti-GAL-1 monoclonal antibody then, after wash-
ing, the samples were incubated for 2 h in a solution
containing fluorescein conjugated avidin ŽVector. Ž1r100
dilution.. For actin distribution the surface preparations
were incubated 2 h in a 1r30 dilution of rhodamine
phalloidin ŽMolecular Probes. in PBS. Finally, the speci-
mens were washed twice and mounted between slide and a
glass coverslip.
Surface preparations were observed under a MRC 1200
laser scanning confocal microscope ŽBioRad.. Optical sec-
tions of 1 mm thickness were recorder and noise was
reduced by Kalman averaging of 5 to 10 consecutive
scans. The FITC Ž520 nm. and rhodamine Ž620 nm. fluo-
rescences of 3 to 15 optical sections spaced from 1 to 5
mm were superimposed by computer to represent a single
plane.
2.2.2. Paraffin sections
The left cochleae of the 18 treated rats and the two
non-treated rats were fixed by perilymphatic perfusion in
2% acetic acid in 70% ethanol, postfixed in the same
solution for 72 h, dehydrated and embedded in paraffin.
Samples were then serially sectioned at 7 mm thickness
from the modiolar plane. The sections were rinsed three
times, for 5 min each, in PBS. Preincubation was carried
out for 30 min in a PBS solution and 30% goat serum. The
sections were then incubated overnight, at 48C, in a 1r2000
solution of anti-GAL-1 monoclonal antibody w11,31x. After
three washes Ž5 min each. in PBS, the sections were
incubated for 1 h in biotinylated goat anti-rabbit IgG
ŽVectastain, Vector, Laboratories. 1r200 in PBS. Anti-
gen–antibody immunoreaction was revealed by using the
avidin–biotin-peroxidase method ŽVectastain, Vector. as
previously described w3,12,24x. Negative controls were ob-
tained with the same procedure described above but omit-
ting the primary antibody.
3. Results
3.1. Scanning electron microscopy
Throughout the three cochleae investigated of PD 21
treated rats, the surface of the organ of Corti was devoid of
both IHC and OHC stereociliary bundles. In the apical
coil, small tufts of densely packed microvilli were seen in
the area where OHCs are normally present ŽFig. 1a.. At

Fig. 3. Paraffin sections of the organ of Corti Žlight microscopy.. Control animals ŽC.; Treated rats with amikacin ŽAK.; basal, medial and apical coil ŽBC,
MC, AC.. Ža. Non-treated rat, PD 21, medial coil. A strong positive immunoreaction is present in cells of the ISS Žasterisk. and of the OSS Žstar.. The
three OHCs ŽO. and the IHC ŽI. are not labelled, nor the pillar cells Žp. and the Deiters’ cells ŽD.. The tunnel of Corti ŽtC. is largely open. Žb–g. Treated
rats ŽAK.. Žb. PD 21, medial cochlear coil. The hair cells are missing. The region of pre-existing OHCs is covered by immunoreactive cells Žprobably the
Hensen’s cells. and unlabelled or weakly labelled Žprobably the Deiters’ cells and their apical expansions.. The pillar cells are unlabelled or are weakly
labelled. The cells in the region of the IHCs are not stained at all. Žc. PD 28, apical coil. The pattern of immunoreaction is essentially the same as in Žb.,
except that some cells are positive in the region of the Deiters’ cells. Žd,e. PD 45. Žd. Basal coil. All the cells of the epithelium are strongly labelled. Note
the absence of the tunnel of Corti. Že. Medial coil of the same cochlea as that shown in Žd.. The tunnel of Corti is still open but the region of the external
pillar and a part of the region of the Deiters’ cells appear immunoreactive. Žf–h. PD 70. Žf,g. Respectively the basal and the medial coil of the same
cochlea. The whole epithelium is composed of a single layer of strongly immunoreactive cells. Žh. Apical coil of the cochlea shown in Žf. and Žg.. The
pattern is essentially the same as that seen at PD 21 Žb. and PD 28 Žc.. Scale bars s 50 mm.
48 M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51

high magnification ŽFig. 1a, detail., these tufts were simi-
lar to the immature stereociliary bundles of cochlear hair
cells. The number of tufts ranged from 28 to 86 tufts.
3.2. Transmission electron microscopy
Although the organ of Corti already lacked both OHCs
and IHCs, the general morphology of the epithelium was
retained ŽFig. 1b.. The tunnel of Corti was still open. The
Deiters’ cells had expanded in the Nuel’s spaces and in the
area of the missing OHCs. In addition, the Hensen’s cells
had partially invaded this area on the outer side. On the
IHC side, two ovoid shaped epithelial cells were present in
the area of the missing sensory cell.
3.3. Histochemistry
3.3.1. Surface preparations
In non-treated rats, the cuticular plates and the stereocil-
iary bundles of the hair cells, the head of the pillar cells,
and the adherent junctions between the OHCs and the
Deiters’ cells were strongly labelled with rhodamine phal-
loidin ŽFig. 2a.. These structures were not labelled with the
anti-GAL-1 antibody ŽFig. 2b.. In contrast, the Hensen’s
and the Claudius’ cells were strongly labelled with the
anti-GAL-1 monoclonal antibody, and their junctions were
weakly labelled with rhodamine phalloidin.
In treated rats from PD 21, the cuticular plates and the
stereocilia of the hair cells were absent. The adherent
junctions at the surface of the epithelium and the head of
the pillar cells were stained with rhodamine phalloidin
ŽFig. 2c.. In the region of the pre-existing OHCs, some
cells Ž10 in one sample and 22 in the other one. showed an
apical dot of rhodamine phalloidin ŽFig. 2c, arrows., which
probably corresponded to the tufts of microvilli observed
at the surface of the atypical cells under SEM ŽFig. 1a..
These cells were not immunoreactivity for anti-GAL-1
antibody ŽFig. 2d.. External to this region, a large band of
epithelial cells was strongly immunolabelled with anti-
GAL-1 antibody, they probably corresponded the Hensen’s
and Claudius’ cells.
3.3.2. Paraffin sections
In non-treated rats, the results were identical to those
previously reported in normal adult rats w11,31x. A strong
positive immunoreaction was seen on both sides of the
organ of Corti, in the epithelial cells of the inner spiral
sulcus ŽISS. and of the outer spiral sulcus ŽOSS.. In
contrast, a total absence of immunoreactivity was observed
in both types of sensory cells ŽIHCs and OHCs., their
specific supporting cells, border cells, phalangeal cells,
inner and outer pillar cells and Deiters’ cells ŽFig. 3a..
In treated rats from PD 21, the pattern of distribution of
anti-GAL-1 immunostaining was identical at all studied
cochlear coils. A strong immunoreaction was seen in the
epithelial cells of both the OSS and the ISS ŽFig. 3b.. In
the OSS, labelled cells extended on the outer part of the
pre-existing OHCs region, similar to that observed in
surface preparation ŽFig. 2d.. Unlabelled cells, partially
covered by labelled cells, were also seen in this region,
from the basilar membrane to the top of the organ of Corti.
In the ISS, all the epithelial cells were labelled except for
the cells located in the region of IHCs. The tunnel of Corti
appeared open ŽFig. 3b, star..
The pattern of distribution of anti-GAL-1 immuno-
staining was essentially the same between treated rats from

Table 1
Summary of results obtained by immunocytochemical distribution of anti-GAL-1 in different types of cells of the organ of Corti in the control and treated
rats
Rats Basal coil Medial coil Apical coil
Control rats IHCŽy., OHCŽy., DCŽy., Fig. 3a, IHCŽy., OHCŽy., IHCŽy.,OHCŽy., DCŽy.,
ECŽq., ACŽnp. DCŽy., ECŽq., ACŽnp. ECŽq., ACŽnp.
AK treated Ž21 PD. IHCŽy., OHCŽnp., DCŽy., Fig. 3b, IHCŽy., OHCŽnp., IHCŽy., OHCŽnp., DCŽy.,
ECŽq., ACŽy. DCŽy., ECŽq., ACŽy. ECŽq., ACŽy.
AK treated Ž28 PD. IHCŽy., OHCŽnp., DCŽy., IHCŽy., OHCŽnp., DCŽy., Fig. 3c, IHCŽy., OHCŽnp.,
ECŽq., ACŽnp. ECŽq., ACŽy. DCŽy., ECŽq., ACŽy.
AK treated Ž45 PD. Fig. 3d, IHCŽnp., OHCŽnp., Fig. 3e, IHCŽy., OHCŽnp., IHCŽy., OHCŽnp., DCŽy.,
DCŽnp., ECŽq., ACŽnp. DCŽnp., ECŽq., ACŽnp. ECŽq., ACŽy.
AK treated Ž70 PD. Fig. 3f, IHCŽnp., OHCŽnp., Fig. 3g, IHCŽnp., OHCŽnp., Fig. 3h, IHCŽy., OHCŽnp.,
DCŽnp., ECŽq., ACŽnp. DCŽnp., ECŽq., ACŽnp. DCŽy., ECŽq., ACŽy.

AK: Amikacin.
PD: Postnatal day.
IHC: Inner hair cells.
OHC: Outer hair cells.
DC: Deiters’ cells.
EC: Epithelial cells.
AC: Atypical cells.
np: Not present Žsee electron microscopy..
Žq.: Positive immunoreaction.
Žy.: Negative immunoreaction.
 M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
PD 28 and PD 21. However, a weak immunoreaction was
noticed in some cells located in the region of the missing
OHCs, in particular at the cochlear apex region ŽFig. 3c..
An important change with respect to previous finding
was observed in the basal coil of the cochleae from treated
rats from PD 45. The tunnel of Corti collapsed, suggesting
a receptorial degeneration, and all the remaining cells of
the organ of Corti formed an epithelium, which showed a
strong immunoreactivity ŽFig. 3d..
In the medial coil, only the epithelial cells of the IHCs
area and inner pillar and head of external pillar cells
showed an absence of immunoreactivity for anti-GAL-1
monoclonal antibody. The tunnel of Corti was still open
ŽFig. 3e, star.. In the apical coil, the distribution of im-
munolabelling was similar to the apical coil of the cochlea
observed in PD 28 treated rats.
At PD 70 treated rats, in the basal ŽFig. 3f. and medial
ŽFig. 3g. cochlear coils, the epithelium of the organ of
Corti appeared as a single layer of strongly immunoreac-
tive cells. At the apex ŽFig. 3h., the central region of the
organ of Corti still showed an open tunnel ŽFig. 3h, star.
and some immunoreactive cells in the area of the Deiters’
cells, as was observed in the previous stages ŽFig. 3c..
The immunohistochemical results obtained with anti-
GAL-1 monoclonal antibody in cochleae of control and
treated rats were summarized in Table 1.
4. Discussion
In the present experiment, animals were treated with
500 mgrkg of amikacin between 9 and 16 PD, i.e., during
the period of cochlear enhanced-sensitivity to antibiotic
w21,27x. In previous studies, such treatment was shown to
produce extensive damage to OHCs and IHCs as early as
PD 17. It also resulted in the transient presence, from PD
21 to PD 35, of atypical cells in the area of the missing
OHCs in the apical coil of the cochlea w9,18,19x.
The atypical cells were covered with round tufts of
microvilli, similar to nascent stereociliary bundles of im-
mature cochlear hair cells w20x. The number of atypical
cells per cochlea varied from a few cells to several hun-
dred w9x. The present results are consistent with these
previous findings. They show a complete loss of both
types of hair cells from PD 21, at least, to PD 70, the
atypical cells were recognized at PD 21, in the region of
the missing OHCs. The tufts of microvilli of the atypical
cells were observed using SEM and identified under confo-
cal microscopy using rhodamine phalloidin.
The organ of Corti damaged by drugs administration
w10,23,28x or noise exposed w29x, a scarring process rapidly
occurs which protects the epithelium from further injury.
These processes, involving the spatial reorganization of
non-sensory epithelial cells, has been well described in the
region of missing OHCs w9x. As illustrated by the present
ultrastructural findings, the Deiters’ cells remained and the
49
Hensen’s cells invaded the Nuel’s spaces and the area of
the pre-existing OHCs.
When the anti-GAL-1 was used, the unlabelled cells, in
amikacin ŽPD 21 and PD 28. treated rats, probably corre-
sponded to pillars and Deiters’ cells Žw11,31x, see also the
present results.. The labelled cells were probably Hensen’s
cells or another cell type of the outer spiral sulcus, such as
Claudius’ or Boettcher’s cells, which are now extended to
ward the place of the missing OHCs. In fact, all these
types of epithelial cells were also strongly labelled in the
normal cochlea Žw11,31x, and the present results in con-
trols.. Altogether, these results agree with the notion that
all the non-sensory epithelial cells participate to the scar-
ring process in the damaged organ of Corti w23x.
The IHCs are generally less sensitive to noxious influ-
ences than OHCs, less is known about the scarring process
occurring in the inner spiral sulcus. In previous investiga-
tions in the developing rat cochlea w31x, it has been hypoth-
esized that the b-galactoside lectin expressed at the level
of the GAL-1-immunoreactive cells blocks an excessive or
abnormal nerve fiber distribution to non-sensory area.
Thus, in amikacin treated cochleae, the progressive colo-
nization of the scarring epithelium by GAL-1 immunoreac-
tive cells could be a limiting factor to the maintenance of
nerve fibers. Indirectly, this could have negative influences
on the regenerative potential of the organ of Corti, at least
on the survival of the atypical cells. Further morphological
and molecular experiments are need to investigate the
status of innervation of the amikacin treated organ of
Corti.
From recent investigations in cochleae of rats treated
with amikacin antibiotic w18,19x, it has been hypothesized
that the missing IHCs are spatially replaced by their
supporting cells, i.e., the border cell on the internal side
and the phalangeal cell on the external side. At the final
stage of the scarring, after drug administration, acoustic
trauma or genetic disorders, the non-functional organ of
Corti is composed of a single layer of unidentified cuboid
epithelial cells Žamong other examples see Refs. w25,30x..
In the present study, this pattern was seen at PD 45 in the
basal coil of the cochlea and extended to the medial coils
at PD 70 according to the baso-apical kinetics of the drug
ototoxicity Žamong other examples see Ref. w2x and Table
1.. All the cells composing this epithelium were strongly
immunoreactive to the anti-GAL-1 antibody. These fea-
tures probably reveal a dramatic epithelial reorganization
involving either a loss of the hair cell supporting cells,
without excluding the possibility of a change in their
phenotype. At PD 70, some cells of the organ of Corti, at
its very apical region, still retain a recognizable shape and
showed both GAL-1 non-immunoreactive and immunore-
active cells. This emphasizes the resistance of the cochlear
apex to antibiotic injury.
An other interest of the current experiment, was to
clarify the phenotype of the transient atypical cells which
appear in the OHCs region soon after amikacin administra-
50 M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
tion. Such as hair cells and their supporting cells in the
normal cochlea w31x, the atypical cells in the treated
cochleae were not GAL-1 immunoreactive. This result
supports the assumptions that Ži. atypical cells represent an
attempt at hair cell transdifferentiation and Žii. arise from
the transdifferentiation of supporting cells, probably Deit-
ers’ cells w9,18,19,37x. However, this does not preclude
that some Hensen’s cells which migrate toward the area of
ototoxic damage and change their GAL-1 pattern of im-
munostaining participate to the production of the atypical
cells.
Further morphological and molecular experiments are
needed to clarify the phenotype and innervation of the
atypical cells which appear in the OHCs region after
amikacin administration.
Acknowledgements
´
We would like to thank Teresa Rodrıguez-Benito for
histological assistance, and J.L. Pasquier for photographic
work. We also thank M. Galliego for animal care. A part
of the morphological investigations were performed in
´
Centre Regional d’Imagerie Cellulaire de Montpellier. This
work has been supported by grants from FISss 95-1540,
French-Spanish Integrated Action ŽHF 94-231. and Human
Capital and Mobility ERBCHRXCT940659.
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