www.elsevier.com/locate/heares
Hearing Research 200 (2005) 51–62
Inner ear histopathology in ‘‘nervous Pointer dogs’’ withsevere hearing loss
Angelique G. Coppens a,*, Shana Gilbert-Gregory b, Sheldon A. Steinberg b,Claus Heizmann c, Luc Poncelet a
a Department of Anatomy and Embryology, Laboratory of Veterinary Anatomy, Faculty of Medicine, Free University of Brussels,
808 Lennik Street, B-1070 Brussels, Belgiumb Section of Neurology/Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, USA
c Department of Paediatrics, Division of Clinical Chemistry, University of Zurich, Switzerland
Received 23 April 2004; accepted 12 August 2004
Available online 13 October 2004
Abstract
Ten puppy dogs (82, 131 or 148 days-old) from a Pointer cross-colony, exhibiting a juvenile severe hearing loss transmitted as an
autosomal recessive trait, were used for histopathological characterization of the inner ear lesion. Immunostaining with calbindin,
Na,K-ATPase, cytokeratins, S100, S100A1 and S100A6 antisera were helpful in identifying the different cell types in the degenerated
cochleae.
Lesions, restricted to the Corti�s organ and spiral ganglion, were bilateral but sometimes slightly asymmetrical. Mild to severe
lesions of the Corti�s organ were unevenly distributed among the different parts of the middle and basal cochlear turns while the
apical turn remained unaffected at 148 days.
In 82 day-old puppies (n = 2), severe lesions of the Corti�s organ, meaning that it was replaced by a layer of unidentifiable cells,
involved the lower middle and upper basal turns junction area, extending in the upper basal turn. Mild lesions of the Corti�s organ,with both hair and supporting cells abnormalities, involved the lower middle turn and extended from the rest of upper basal turn
into the lower basal turn. The outer hair cells (ohc) were more affected than the inner hair cell (ihc). The lesions extended towards the
basal end of the cochlea in the 131 (n = 5) and 148 (n = 3) day-old puppies. Additionally, the number of spiral ganglion neurons was
reduced in the 131 and 148 day-old puppies; it is earlier than observed in most other canine hereditary deafness. These lesions were
interpreted as a degeneration of the neuroepithelial type. This possible animal model might provide information about progressive
juvenile hereditary deafness and neuronal retrograde degeneration investigations in human.
� 2004 Elsevier B.V. All rights reserved.
Keywords: Dog; Hearing loss; Hereditary; Neuroepithelial degeneration
0378-5955/$ - see front matter � 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.heares.2004.08.019
Abbreviations: BAER, brainstem auditory evoked potentials; BSA,
bovine serum albumin; NHS, normal horse serum; PD, postnatal days;
SPL, sound pressure level; TBS, Tris buffer saline* Corresponding author. Tel.: +32 2 555 63 76/20; fax: +32 2 555 63
78.
E-mail address: [email protected] (A.G. Coppens).
1. Introduction
Deafness has been reported in 53 dog breeds and
hereditary factors are suspected in dog breeds with high
prevalence of congenital deafness (Strain, 1996). Innerears pathological changes have been described in 19
dog breeds only and deafness is usually associated
with a cochleosaccular (the most frequently described)
or neuroepithelial type of degeneration (Hiraide and
52 A.G. Coppens et al. / Hearing Research 200 (2005) 51–62
Paparella, 1988; Strain, 1996; Coppens et al., 2000b,
2001a, 2003b).
In cochleosaccular type of degeneration, lesions are
characterized by atrophy of the stria vascularis, collapse
of the cochlear duct, degeneration of the Corti�s organ,abnormal tectorial membrane, and collapse of the sac-culi (Steel and Bock, 1983; Strain, 1996). The degenera-
tion of the spiral ganglion is delayed by months to years
(Mair, 1976; Strain, 1996; Niparko and Finger, 1997).
Cochleosaccular degeneration has been commonly de-
scribed in Dalmatian in which the inherited nature of
deafness has been well documented although the mode
of inheritance remains controversial (Greibrockk,
1994; Famula et al., 1996, 2000, 2001; Muhle et al.,2002).
Neuroepithelial type of degeneration has been de-
scribed in a family of Dobermann and in Shropshire ter-
rier dogs (Igarashi et al., 1972; Wilkes and Palmer,
1992). Lesions are characterized by degeneration of
Corti�s organ with normal stria vascularis, normal tecto-
rial membrane, no collapse of the cochlear duct and nor-
mal sacculi (Steel and Bock, 1983; Wilkes and Palmer,1992; Strain, 1996). The degeneration of the spiral gan-
glion is largely delayed too (Igarashi et al., 1972; Steel
and Bock, 1983; Wilkes and Palmer, 1992). An addi-
tional type of neuroepithelial degeneration, character-
ized by Corti�s organ degeneration and early spiral
ganglion neurons loss, has been described in a congeni-
tally deaf Rottweiler puppy (Coppens et al., 2001a).
Beside information about dog deafness, detaileddescriptions of inner ear lesions in this species may also
be useful as dogs have been proposed as a suitable ani-
mal model to study human inner ear diseases and
cochlear implant evaluation (Niparko et al., 1993;
Lalwani et al., 1997; Niparko and Finger, 1997; Har-
vey et al., 2001; Sockalingam et al., 2002; Rak et al.,
2002). Progress in gene localization is now under way
in the canine species and Myo XVA gene, known tobe involved in human deafness, has been recently evi-
denced in this species, opening prospects for its use
as an animal model in heritable deafness studies (Rak
et al., 2002).
A high incidence of juvenile bilateral deafness has
been described in a colony of Pointer dogs selectively
bred for excessive nervous behaviour (Klein et al.,
1988; Steinberg et al., 1994). Although 74% of nervousdogs have a bilateral hearing loss (no brainstem audito-
ry-evoked response (BAER) could be detected), this
study has pointed out that hearing defect does not con-
tribute to the nervous behaviour, as deafness is not al-
ways a shared defect among nervous dogs. Excepted
for the hearing defect and the stereotypical behaviour,
no other pathological change or vestibular defect has
been observed in this colony (Klein et al., 1988; Stein-berg et al., 1994). Brainstem auditory evoked response
testing have revealed that hearing deficit may appear
as soon as 21 days of age, progressively leading to pro-
found deafness during the first half of the second month
of life (Steinberg et al., 1994). Breeding experiments
have indicated that deafness in this canine colony is an
autosomal recessive trait that is fully penetrant (Stein-
berg et al., 1994).Precise morphological descriptions are needed to
classify hearing losses and the purpose of the present re-
port is to detail the histopathological changes found in
this readily available possible animal model.
Immunohistochemistry using calbindin, cytokeratins,
Na,K-ATPase, S100, S100A1 and S100A6 antibodies
were also undertaken in this study as they make it pos-
sible to recognize specific cell types or structure in thedegenerated cochleae (Coppens et al., 2000a, 2001a,b,
2003a).
2. Materials and methods
2.1. Animals
Inner ears were collected from 10 Beagle-Pointer
cross-puppies (males and females) from three different
litters (Table 1). Pointer dogs came from a family of
dogs selectively bred for excessive nervous behaviour,
but showing also a high prevalence of deafness among
the behavioural nervous dogs. This deafness is an auto-
somal recessive trait that is fully penetrant (Steinberg
et al., 1994). No clinical vestibular involvement was de-tected in any dogs in this family. Subsequently, a colony
was established by outcross and F1 backcross breeding
to hearing beagle and keeshond Pointer dogs. The 10
puppies in this study are derived from that colony.
The dogs were cared for according to the principles of
the NIH Guide for the Care and Use of Laboratory Ani-
mals (NHI Grant RR02512).
For hearing by BAER tests, the puppies were sedatedwith morphine (1.5 mg/kg) and acepromazine maleate
(0.5–1.2 mg/kg) intramuscularly. They also received
atropine (0.02 mg/kg) by the same route. Averaged
brainstem auditory evoked potentials were recorded
with an electrodiagnostic apparatus (Dantec Cantata,
Medtronic, Skovlunde, Denmark). Rarefaction click
stimuli, (100 ms in duration, 10 Hz repetition rate) were
delivered to the tested ear through an insert phone, theuntested ear receiving a masking noise, 40 dB below
the click level (Steinberg et al., 1994). Both ears were
successively tested. Thresholds were looked for by
decreasing the click stimuli from 132 dB SPL (sound
pressure level, maximum output of the stimulator) by
3 dB steps and threshold was defined as the highest stim-
ulus where no BAEP could be recorded (gain setting 2
lv/div). The dogs were considered as suffering severehearing loss when the threshold was 102 dB SPL or
above, i.e. 70 dB above average threshold in normal
Table 1
Puppies used in the study and histopathological study outline
Litter 1 Litter 2 Litter 3
Puppies # 350 352 390 391 392 396 399 400 401 403
Sex F M F F F F M M M M
Age of profound hearing loss Before 38 day-
old (estimated*)
Before 55 day-old,
(estimated*)
Between 30 to 37 day-old (puppies were weekly
tested)
Age of euthanasia 82 day-old 148 day-old 131 day-old
Interval* 44 days
(estimated*)
111 days (estimated*) 94 days
Inner ear fixation Formol 10%
during weeks
Formol 10% during weeks Paraformaldehyde 4% for 48 h
Investigated inner ears R, L R, L R, L L L R, L R, L L R, L R, L
Calbindin + + + + 0 + + 0 + +
S100 + + FI + 0 + + 0 + +
S100A1 + + FI 0 0 + + 0 + +
Cytokeratins FI FI FI + 0 FI + 0 + +
Na,K-ATPase FI FI + 0 0 + + 0 + +
S100A6 + + + 0 0 + 0 0 + +
F: female; M: male; Age of profound hearing loss: age at which the severe hearing loss was confirmed by BAER assessment; estimated *: for puppies
of the litter 1 and 2, the estimated age of severe hearing loss (38 PD estimation) was below the BAEP confirmed detection date; Interval*: estimated
(litters 1 and 2) and observed (litter 3) interval between severe hearing loss detection and euthanasia. Investigated inner ears: inner ears histologically
investigated; R: right inner ear; L: left inner ear; calbindin: anti-calbindin immunolabelling; cytokeratins: anti-cytokeratins immunolabelling; S100:
anti-S100 immunolabelling; S100A1: anti-S100A1 immunolabelling; S100A6: anti S100A6 immunolabelling; Na,K-ATPase: anti-Na,K-ATPase
immunolabelling; +: successful immunolabelling; FI: failed immunolabelling for technical reasons; 0: immunolabelling test not performed.
A.G. Coppens et al. / Hearing Research 200 (2005) 51–62 53
age-matched dogs (Poncelet et al., 2000). The puppies oflitter 3 were assessed from 21 days of age, and during
five sessions at one-week intervals, permitting to date
the severe hearing loss. The puppies from litters 1 and
2 were tested irregularly, only to confirm the severe
hearing loss in all puppies. At varying ages, puppies in
each litter were euthanasied (Table 1).
2.2. Procedure
The ages for histological examination were (Table 1):
82 day-old (puppies 350 and 352 from litter 1), 148 day-
old (puppies 390, 391 and 392 from litter 2) or 131 day-
old (puppies 396, 399, 400, 401 and 403 from litter 3).
Euthanasia was performed with an intravenous over-
dose of pentobarbital solution. Temporal bones were
immediately removed after death. For puppies from lit-ters 1 and 2, bullae were removed, round windows were
opened with a 26-gauge needle and a flexible short 18-
gauge tube was inserted into the round window. A syr-
inge driven by a pump that delivered 0.45 ml/min was
used to perfuse the membranous labyrinths with 10%
formalin for 10 min. The temporal bones were postfixed
in 10% formol for weeks. Inner ears from an 82 day-old
bilaterally hearing puppy (litter 1), were also available tocheck tissue preservation with this histological
procedure.
For puppies from litter 3, bullae were opened and
temporal bones were immediately fixed in 4% buffered
paraformaldehyde in 0.1 M PBS for 48 h. This second
schedule was chosen as it enhanced antigenic preserva-
tion for immunohistologic investigation (Coppenset al., 2000a, 2001b, 2003a).
The right temporal bones were decalcified for 145–
225 days in 0.12 M ethylenediaminetetraacetate (EDTA)
(Tritriplex III, Merck-Belgolabo, Belgium) in 0.01 M
PBS, pH 7.3. The left temporal bones were decalcified
in 5% formic acid for 24–126 days. The decalcified sam-
ples were embedded in paraffin and serially cut (6–9 lmthick) parallel to the modiolus.
The left inner ear of each puppy and right inner ear of
selected puppies 350, 352, 390, 396, 399, 401, 403 were
histologically investigated (Table 1).
Twenty to 30 sections throughout the cochlea width
(both inner ears of puppies 352, 390, 401, and 403) were
examined in an effort to recreate most of the complete
cochlear duct. For descriptive purposes the duct is di-
vided into apical, upper middle, lower middle, upper ba-sal, lower basal turns and basal end of the cochlea (Fig.
1). For the other inner ears, five sections passing
through the modiolus were examined. Sections were de-
waxed, hydrated, and stained with either haematoxylin–
eosin or cresyl for classical histology.
Sections from the vestibule of puppies 350, 352 were
stained with cresyl.
2.3. Immunohistochemistry
Dewaxed sections were treated for 30 min with
methanol containing 1% peroxide solution to inhibit
endogenous peroxidase activity, and then washed in
0.05 M Tris buffered saline, pH 7.4 (TBS). The sections
Fig. 2. The upper basal turn in 131 day-old Pointer puppy with severe
hearing loss. The cochlear duct (CD) was normally shaped and the
Reissner�s membrane (RM) was in its usual position, the Corti�s organhad disappeared, the tectorial membrane (TM) was normal, the stria
vascularis (SV) was recognizable. Haematoxylin–eosin 80·.
Fig. 1. The dog cochlea was divided in: apical turn, upper middle turn,
lower middle turn, upper basal turn, lower basal turn and basal end of
the cochlea.
54 A.G. Coppens et al. / Hearing Research 200 (2005) 51–62
were preincubated in a humid chamber for 30 min at 37
�C with 1/20 normal horse serum (NHS) in TBS. The
sections were covered for 24 h at 4 �C with the follow-ing antibodies diluted in TBS containing 2% bovine ser-
um albumin (BSA) (Sigma; Belgium): polyclonal rabbit
anti calbindin 28Kd (Swant, Switzerland), 1/3500; poly-
clonal rabbit Na,K-ATPase (b2 subunit) antiserum
(U.B.I., USA), 1/4000; monoclonal mouse cytokeratins
(RCK102) antiserum (Monosan; USA) against Cks 5
and 8, 1/10; monoclonal mouse vimentin (V6630) anti-
serum (Sigma; Belgium), 1/1200; polyclonal rabbit S100antiserum (Dako, Netherland), 1/12,000; polyclonal
goat S100A6 (Ilg et al., 1996) antiserum, 1/5000; poly-
clonal goat S100A6 antiserum (Ilg et al., 1996), 1/
3000. Dewaxed sections were pre-treated for 3 min at
room temperature with proteinase K (Ready-to-use,
Dako, The Netherlands) before labelling with
cytokeratins.
The primary immune antiserum was revealed by theavidin-biotin procedure as follows: 30 min incubation
either in biotinylated anti-rabbit, anti-mouse, or anti-
goat gamma globulin (Vector Labs, USA) diluted to 1/
100 in TBS with BSA 2%, followed by 30 min incuba-
tion with avidin-biotin-peroxidase complex standard
kit reagent (Vector Labs, USA). The intermediate wash-
ings were done with TBS. The staining was visualized by
diaminobenzidine/H2O2 solution (liquid DAB substratepack from Biogenex, USA). Immunolabelled sections
were not counterstained. Control sections from two
puppies of one and three months old from our personal
collection were included. Control sections, with the pri-
mary antibody omitted, remained unlabeled.
The results of the immunolabelling tests for each
puppy are detailed in the Table 1. For each antibody,
5–10 sections were immunolabelled for each cochlea.
3. Results
Pathological changes were confined to the cochlear
membranous labyrinth involving the Corti�s organ and
the spiral ganglion. Vestibular membranous labyrinth,
including saccule, utricule and semi-circular canals, were
found intact with normal cristae ampullaris and maculaeand the Scarpa ganglion was normal.
For the anatomopathological description, the cochlea
is divided in: apical turn, upper middle turn, lower mid-
dle turn, upper basal turn, lower basal turn and basal
end of the cochlea (nearest the vestibular endorgans)
(Fig. 1).
From base to apex, the cochlear duct was normally
shaped and the Reissner�s membrane was in its usualposition (Fig. 2).
From base to apex, the stria vascularis showed a nor-
mal three cell layer morphology (Fig. 3(a)) and the
S100A6 and Na,K-ATPase expression showed a similar
pattern to that observed in the normal stria vascularis of
dogs as previously described (Coppens et al., 2003a).
The lateral cell membrane and the numerous basal mem-
brane infoldings of marginal cells, that deeply penetrateinto the stria vascularis, strongly expressed Na,K-AT-
Pase b2 isoform (Fig. 3(b) and (c)). The S100A6 immu-
nolabelling evidenced the presence of intermediate cells,
regularly scattered between the marginal and basal cells
layers (Fig. 3(d) and (e)).
Immunolabellings were also used to help in identify-
ing the different cell-types in the degenerated Corti�s
Fig. 3. The stria vascularis of normal Beagle dog (b and d) and 131 day-old Pointer dog with severe hearing loss (a, c and d). (a) Haematoxylin–eosin
staining in 131 day-old Pointer stria vascularis; the stria vascularis showed a normal three cell layer morphology with marginal cells (MC) lining the
endolymphatic space, basal cells (BC) resting on the underneath spiral ligament and intermediate cells (IC) scattered between marginal and basal cell
layers. 500·. (b and c) Na,K-ATPase immunolabelling in normal Beagle (b) and 131 day-old Pointer (c) stria vascularis; the lateral cell membrane
and the numerous basal cell membrane infoldings of marginal cells (arrows) deeply penetrated into the stria vascularis and strongly expressed Na,K-
ATPase b2 isoform. 500·. (d and e) S100A6 immunolabelling in normal Beagle (d) and 131 day-old Pointer (e) stria vascularis; the S100A6
immunolabelling evidenced the presence of intermediate cells, regularly scattered between the marginal and basal cell layers (500·).
A.G. Coppens et al. / Hearing Research 200 (2005) 51–62 55
organ. In normal Corti�s organ of dogs (Fig. 4(a)), cal-
bidin antiserum (Fig. 4(b)) specifically labels hair cells
with their cuticular plate and stereocilia. Cytokeratins
are found in the apical and basal pole of supporting
cells, in the cup of Deiters cells and in the rods of
Fig. 4. The Corti�s organ of normal Beagle. (a) Cresyl staining in Corti�s o
(OHC) hair cells with their cuticular plate and stereocilia (arrow) were immu
cells (CD) supporting the outer hair cells, the reticular membrane (RM) the B
the Hensen cells were immunolabelled, defining the Corti�s organ cytoarchitec
(P), the rods of Corti (RC), the Hensen�s cells (H), the Deiters cell body (D), p
reticular membrane were immunolabelled, the inner and outer hair cells wer
pillar cells, the rods of Corti, the Hensen�s cells, the Deiters cells, phalangeal
membrane (RM) were immunolabelled, the inner (IHC) and outer hair (OH
Corti, defining the Corti�s organ cytoarchitecture (Fig.
4(c)). S100 and S100A1 are expressed in supporting
cells in the normal Corti�s organ of dogs including
border, pillar, Hensen�s and Deiters cells (Fig. 4(d)
and (e)).
rgan 250·. (b) Calbidin immunolabelling; the inner (IHC) and outer
nolabelled 300·. (c) Cytokeratins immunolabelling; the cup of Deiters
order cell (B), the rods of Corti (RC) limiting the tunnel of Corti (TC),
ture 250·. (d) S100 immunolabelling; the border cell (B), the pillar cells
halangeal process of Deiters cells (PP), the cup of Deiters cells and the
e unlabelled. X 250. (e) S100A1 immunolabelling; the border cell, the
process of Deiters cells, the cup of Deiters cells (CD) and the reticular
C) cells were unlabelled 250·.
56 A.G. Coppens et al. / Hearing Research 200 (2005) 51–62
In the Pointer cross-puppies, the Corti�s organ of the
apical turn remained morphologically normal at all
studied ages. Elsewhere, two types of degeneration of
the Corti�s organ could be found: mild (Fig. 5) or severe
(Fig. 6) lesions. From base to apex, the tectorial mem-
brane appeared normal, even where the Corti�s organwas degenerated (Fig. 2).
In the mild lesions, the general cytoarchitecture of the
Corti�s organ was recognizable (Fig. 5(a) and (b)). Cal-
bindin immunolabelling evidenced at least the inner hair
cell (ihc) and one to three outer hair cells (ohc) (Fig. 5(c)
and (d)). The most lateral ohc disappeared most often,
while the medial one was most often preserved (Fig.
5(b) inset). Remaining hair cells showed always anabnormal spherical shape with a centrally located nu-
cleus. Their cuticular plate and cilia remained sometimes
distinguishable.
Fig. 5. Corti�s organ showing mild lesions in Pointer dog with severe heari
organ was recognizable 250·. (b) Haematoxylin–eosin staining; only the two
adjacent section permitted to identify the inner hair cell (IHC) and only one o
centrally located nucleus 200·. (c and d) Calbindin immunolabellings; the in
remaining hair cells were shortened, showing an abnormal cylindrical shape
disorganized but the immunolabelligs permitted to define the border cells (B)
Deiter�s and Hensen�s cells, the phalangeal process (PP) and the cup of D
immunolabellings; the Corti�s organ was collapsed, the lumen of the tunnel
The border cell surrounding the ihc was S100 and
S100A1-immunomarked and could be identified (Fig.
5(e) and (f)). The two pillar cells were recognizable.
Immunolabellings permitted to define the thin and elon-
gated pillars constituting the rods of Corti and their
massive pillar head (Fig. 5(e)–(g)). Nevertheless, therods of Corti were distorted and they were usually col-
lapsed so that the lumen of the tunnel of Corti was par-
tially to totally occlude (Fig. 5(g)).
Deiter�s and Hensen�s cells were disorganized but
immunolabellings permitted to evidence the phalangeal
process and the cup of Deiter�s cells. Narrow Nuel�sspaces separated the remaining ohc from each other
and from the external pillar cell. The reticular mem-brane evidenced by the cytokeratins, S100 and S100A1
immunolabellings, anchored the apices of the remaining
ohc (Fig. 5(e)–(g)).
ng loss. (a) Cresyl staining; the general cytoarchitecture of the Corti�spillar cells were identified 300·. (b) Inset, calbindin immunolabeling in
uter hair cell (OHC), remaining hair cells showed spherical shape with a
ner hair cell (IHC) and one to three outer hair cells (OHC) remained,
300·. (e and f) S100A1 (e) and S100 (f) and, the supporting cells are
surrounding the inner hair cell, the pillar cells and the rods of Corti, the
eiter�s cells and the reticular membrane (RM) 300·. (g) Cytokeratinsof Corti was reduced between the collapsed rods of corti (RC) 250·.
Fig. 6. Corti�s organ showing severe lesions in Pointer dog with severe hearing loss. (a) Cresyl staining; the Corti�s organ was replaced by a layer of
unidentifiable cells 300·. (b) Calbindin immunolabelling, no hair cell was evidenced 300·. (c and d) Cytokeratins (c) and S100 (d) immunolabellings;
the Corti�s organ was replaced by a layer of cells that were cytokeratins and S100-immunlabelled 300·. (e) Calbindin immunolabelling; only rare
spherical inner hair cells close to the inner sulcus, exhibiting recognizable immunolabelled cilia (arrow), were sometimes evidenced 300·. (f and g)
S100A1 immunolabellings; only remnants of Border (B) or pillar (P) cells were sometimes presumably detected 300·.
A.G. Coppens et al. / Hearing Research 200 (2005) 51–62 57
In severe lesions, the Corti�s organ was replaced by a
layer of unidentifiable cells cytokeratins, S100 and
S100A1-immunlabelled but calbindin-negative (Fig.
6(a)–(d). Calbindin immunolabelling evidenced only
rare spherical ihc close to the inner sulcus exhibiting rec-
ognizable immunolabelled cilia (Fig. 6(e)). Only rem-nants of Border, Hensen�s or pillar cells were
sometimes presumably detected based on S100A1
immunohistochemical reaction (Fig. 6(f) and (g)). When
the Corti�s organ was totally collapsed, immunohisto-
chemistry only gave a clue on the identity of abnormally
shaped cells.
Pathological changes that involved the Corti�s organand spiral ganglion were bilateral, but unevenly distrib-uted among the different parts of the cochlear duct and
lesions were sometimes asymmetrical between both
inner ears from a same individual. However, an age-
dependant pattern could be recognized in this investiga-
tion (Table 2).
In 82 day-old puppies (n = 2), severe lesions of the
Corti�s organ involved the lower middle and upper ba-sal turns junction area, extending most in the upper ba-
sal turn (Fig. 7(b) and (c)). Mild lesions involved the
lower middle turn (Fig. 7(a)) and extended from the
rest of the upper basal turn into the lower basal turn
(Fig. 7(d)).
In the basal end of the cochlear duct, severe lesions
were found and the Corti�s organ was totally missing
without any recognisable structure coating the basilarmembrane.
Table 2
Lesions of the Corti�s organ was age-related
Apex Upper middle Lower middle Junction area Upper basal Lower basal Basal end
82 days Normal Normal Mild Severe Severe to mild Mild Severe
131 days Normal Normal Mild Severe Severe Mild (396, 401, 403)
Severe (399, 400)
Mild (396, 401, 403)
Severe (399, 400)
148 days Normal Mild Mild Severe Severe Severe Severe
58 A.G. Coppens et al. / Hearing Research 200 (2005) 51–62
A loss of neuronal dendrites in the spiral lamina clos-
est to the degenerated Corti�s organ (Fig. 7(c)), restrictedto the lower middle and upper basal turns junction area,
was observed in one puppy (352). The spiral ganglion in
both 82 day-old puppies was found normal from base to
apex (Fig. 7(e)).
In 131 day-old puppies (n = 5), the lower middle turn
remained unchanged with mild lesions (Fig. 8(a)). Severe
lesions had progressed from the lower middle and upper
basal turns junction area up into a part of the lower ba-sal turn (Fig. 8(b) and (c)). In the rest of the lower basal
turn and in the basal end of the cochlear duct, lesions of
the Corti�s organ were inconsistent depending on the
individuals: the Corti�s organ was found either mildly
Fig. 7. Histopathological findings in inner ears of 82 day-old Pointer puppi
found in the lower middle turn. Cresyl 250·. (b) (puppy 350) and (c) (puppy 3
junction area, a loss of neuronal dendrites (arrow) in the spiral lamina closest
300·. (d) Mild lesions of the Corti�s organ were found in the lower basal turn
turns junction area; the spiral ganglion neurons were found normal 300·.
(396, 401, 403) (Fig. 8(d) and (e)) or severely affected
(399,400).Severe dendrite loss was usually associated with se-
vere lesions as it was observed from the lower middle
and upper basal turns junction area (Fig. 8(b)) to the
lower basal turn and in the end of the basal turn in pup-
pies 399 and 400.
A reduction of 30–50% in neuron cell count was ob-
served throughout the basal turn (Fig. 8(f)). In puppy
399 and 400, the neuronal population was also dimin-ished in the basal end of the cochlea. In most of the
remaining neurons, cresyl staining did not evidence
Nissl�s substance and the chromatin was condensed.
The severity and the spatial location of the spiral gan-
es with severe hearing loss. (a) Mild lesions of the Corti�s organ were
52): severe lesions were found in the lower middle and upper basal turns
to the degenerated Corti�s organ was observed in the puppy 352. Cresyl
. Cresyl 250·. (e) Spiral ganglion of the lower middle and upper basal
Fig. 8. Histopathological findings in inner ears of 131 day-old Pointer puppies with severe hearing loss. (a) Mild lesions of the Corti�s organ were
found in the lower middle turn. Cresyl 250·. (b and c) Severe lesions were found in the lower middle and upper basal turns junction area, in the upper
basal turn, extending up into a part of the lower basal turn, severe dendrite loss (arrow) was usually associated with severe lesions. Cresyl 300·. (d)Mild lesions of the Corti�s organ were found in the end of the lower basal turn (puppy 403). Cresyl 250·. (e) S100A1 immunolabelling in the lower
basal turn (puppy 403), supporting cells were disorganized 300·. (e) Inset, adjacent section: calbindin immunolabelling, the hair cells were abnormal
and distorted 200·. (f) Spiral ganglion of the basal turn (403 puppy), a reduction in neuron cell count was observed Cresyl 300·.
Fig. 9. Histopathological findings in inner ears of 148 day-old Pointer puppies with severe hearing loss. (a) Severe lesions of the Corti�s organ in the
lower basal turn (puppy 391) 350·. (b) The basal turn (puppy 392); dendrites were almost totally missing (arrow) in the areas where the Corti�s organwas severely affected and 90% of neurons lacked 120·. (c) Spiral ganglion of the end of the cochlear duct (puppy 390) 350·.
A.G. Coppens et al. / Hearing Research 200 (2005) 51–62 59
60 A.G. Coppens et al. / Hearing Research 200 (2005) 51–62
glion cells and dendrites loss were asymmetrical and de-
pended on individuals.
In 148 day-old puppies (n = 3), severe lesions of the
Corti�s organ extended from the lower middle and upper
basal turns junction area to the basal end of the cochlea
(Fig. 9(a)). Mild lesions of the Corti�s organ involved theupper and lower middle turns.
Dendrites were almost totally missing in the areas
where the Corti�s organ was severely affected in puppies
390 and 392 (Fig. 9(b)).
There was a moderate (puppy 391) to severe (puppies
390 and 392) loss of cochlear neurons from the lower
middle and upper basal turns junction area to the end
of the cochlear duct: 70–90% of neurons lacked in thebasal turn and in the basal end of the cochlea in puppies
390 and 392 (Fig. 9(b) and (c)).
The severity and the extent of the Corti�s organ and
spiral ganglion cell loss varied somewhat among deaf
age-matched puppies (Table 2) and pathological
changes slightly varied between both cochleae of the
puppies 350, 390, 401.
All these puppies suffered bilateral severe hearing loss(threshold above 102 dB SPL) weeks before euthanasia
(Table 1), and it could be observed as soon as 37 days
of age. The hearing loss was progressive, but profound
hearing loss did not necessarily mean absence of poten-
tials at the maximum output of the auditory stimulator
(132 dB SPL) (Fig. 10).
Fig. 10. BAEP in response to 132 dB SPL rarefaction click recorded
from puppy 401 right ear. Records obtained at 24, 56 and 95 days of
age (from top to bottom). Arrows point to the assumed BAEP wave V.
4. Discussion
The present study investigated pathological changes
involving inner ears of 10 Beagle-Pointer cross-puppies,
aged from 82 to 148 day-old, with severe bilateral hear-
ing loss that was electrophysiologically confirmed.Pointers came from a colony of dogs selectively bred
for excessive nervous behaviour and showing high prev-
alence of juvenile deafness (Klein et al., 1988; Steinberg
et al., 1994).
In the present study, BAER assessments evidenced
that bilateral hearing loss appeared between one to
two months. A previous communication has reported
that puppies eventually becoming deaf had almost nor-mal bilateral BAER at 21 days of age and that the re-
sponse gradually decreased, leading to total deafness
by two months of age (Steinberg et al., 1994). The func-
tional evidence of almost normal hearing at least during
a short postnatal period suggests that the cochlea
reaches an advanced postnatal maturation stage before
progressive degeneration.
This study investigated inner ears collected from pup-pies aged of 82, 131 and 148 days, i.e. beyond the age at
which severe hearing loss was electrophysiologically
confirmed. It is well established that anatomical and
functional changes are not strictly related, morphologi-
cal changes being delayed.
This histopathological investigation pointed out that
pathological changes involved the Corti�s organ and spi-
ral ganglion and lesions were interpreted as a progres-sive and age-dependant bilateral neuroepithelial type
of degeneration. The immunolabellings brought addi-
tional evidence in identifying the different cell-types in
the degenerated structure. The Corti�s organ progres-
sively degenerated passing through mild and severe le-
sions. Our observation suggested that outer hair cells
disappeared first, the lateral outer hair cell disappearing
before the medial one, followed by inner hair cell lossand supporting cells degeneration until the Corti�s organwas replaced by a layer of unidentifiable cells resting on
the basilar membrane.
The Corti�s organ changes found in the Pointer
cross-puppies were similar to the neuroepithelial degen-
eration described by Wilkes and Palmer in a family of
Dobermanns, in both cases outer hair cells were first al-
tered (Wilkes and Palmer, 1992). But, Dobermann pup-pies differed from Pointer cross-puppies; indeed,
Dobermann deaf puppies also showed signs of vestibu-
lar deficit and no BAER could be recorded in any of
the puppies of three weeks of age or older (Wilkes
and Palmer, 1992).
On the other hand, the Corti�s organ changes found
in the Pointer cross-puppies differed from our previous
observation in a congenitally deaf Rottweiler puppy inwhich inner hair cells were more affected than outer hair
cells, suggesting different molecular causes to neuroepit-
A.G. Coppens et al. / Hearing Research 200 (2005) 51–62 61
helial degeneration in canine species (Coppens et al.,
2001a).
As mentioned before, this first anatomopathological
study permitted to evidence progressive spatial exten-
sion of Corti�s organ during the studied period. Patho-
logical changes in the Pointer cross-puppies wereunevenly distributed among the different parts of the
cochlear duct and results suggest that the Corti�s organdegenerated first in the lower middle and upper basal
turns junction area, the site where lesions were severe
in the 82 day-old puppies. With age, severe lesions of
the Corti�s organ progressed towards the basal end of
the cochlea. In the Dobermann family, the Corti�s or-
gan also began to degenerate in the middle turn, butlesions extended throughout the whole cochlear duct,
resulting in almost complete loss of Corti�s organ by
77 day-old (Wilkes and Palmer, 1992). Our observa-
tions about spatial extension cannot be compared to
the neuroepithelial type of degeneration previously
described in Shropshire terrier and Rottweiler dogs be-
cause of the absence anatomopathological observations
in age-matched deaf puppies (Igarashi et al., 1972;Coppens et al., 2001a).
In the present description, dendrite loss followed by
spiral ganglion degeneration appeared secondary to the
Corti�s organ pathological involvement. Indeed, the
spiral ganglion cell count was found normal in the 82
day-old puppies but spiral ganglion neuron loss was al-
ready observed in 131 day-old puppies and massive
neuronal loss was observed in at least two 148 day-old puppies. In the progressive sensorineuronal degen-
eration previously described in dogs as well as in other
species, the retrograde neuronal degeneration is usually
slow and a time lag of several months to years sepa-
rates Corti�s organ degeneration and spiral ganglion
neuron loss (Igarashi et al., 1972; Mair, 1976; Steel
and Bock, 1983; Nadol et al., 1989; Wilkes and Palmer,
1992; Niparko and Finger, 1997; Miura et al., 2002).Our observation differed from this description in that
the spiral ganglion neurons disappeared early after
the Corti�s organ degeneration in Pointer cross-pup-
pies. The delay and location of retrograde neuronal
degeneration are important parameters in the cochlear
implant frame. Indeed the neuronal population preser-
vation in sensorineuronal deafness is determinative pre-
dictor of the cochlear implant performance inprofoundly deaf children and adults (Nadol et al.,
1989; Niparko et al., 1993; Niparko and Finger,
1997; Miura et al., 2002).
Dramatic threshold increase to absent BAEP re-
sponse was observed in animals with some degree of
Corti�s organ preservation along a large stretch of the
cochlea and with an apical turn devoid of detectable
changes. Some remaining potential could however beevidenced in some dogs with the highest output of the
auditory stimulator, suggesting some functional rem-
nant. It should also be kept in mind that the highly sus-
ceptible machinery of the Corti�s organ might become
ineffective before detection of lesions through light micr-
oscopy. Electron microscopy might detect subcellular,
functionally relevant changes where the cells looked
unchanged through light microcopy observation.Moreover, electrophysiological recordings may miss
substantial functional integrity in the lowest frequency
range corresponding the cochlear apical turn. In man
with high frequency hearing loss, auditory brainstem re-
sponses to 2 kHz tone stimuli could be unrecordable de-
spite preservation of hearing of the lowest frequencies
(Borg and Lofqvist, 1982).
Though heritability has been often suspected or evenaccepted in congenital or juvenile deafness, predictable
inherited deafness has been reported only rarely in dogs.
In the Pointer colony, the genetic relationship between
the deafness and the nervous trait remains unresolved
but cross-breeding experiments have indicated that deaf-
ness has a genetic origin of its own, consistent with an
autosomal recessive trait that is fully penetrant (Stein-
berg et al., 1994). As the heritability of deafness isknown in this nervous Pointer dog colony, selective
breeding can produce puppies destined to become bilat-
erally deaf. Further studies should characterize the ear-
lier evolution of the lesions providing data about the
nature and maybe the molecular cause of this original
neuroepithelial type of degeneration in the dog species.
The deaf dogs described in this study might be a read-
ily available possible animal model for studying auto-somal recessive deafness in childhood that represent
85% of the non-syndromic cases (Lalwani et al., 1997;
Hardisty et al., 1998).
An important current direction in deafness therapeu-
tic research and otoprotection lies in the intracochlear
administration of neurotrophins, growth factors or stem
cells in the future (Staecker et al., 1996; Feghali et al.,
1998; Malgrange et al., 1999; Lefebvre et al., 2002a,b).The next step towards this goal will be the use of animal
models with neurosensory deafness. In this context, dogs
offer three advantages when compared to rodents: sen-
sorineuronal hereditary deafness similar to that de-
scribed in human, longevity of dogs that permits study
of long-term effects of treatments, and the inner ear size
of dogs that permits easier access to the inner ear via col-
eostomies. Use of minipump and cochlear implant forhuman might also be tested without major technical
adaptations.
Acknowledgements
This study was supported by the NHI grant
RR02512, the Aldina Scaife Gates Research Fund andby the FNRS grant 1.5025.02 from the ‘‘Fonds National
de la Recherche Scientifique’’.
62 A.G. Coppens et al. / Hearing Research 200 (2005) 51–62
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