Inner ear histopathology in “nervous Pointer dogs” with severe hearing loss

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

mailto:[email protected]

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.


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·).


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·.


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·.


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


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·.



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-


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’’.


References

Borg, E., Lofqvist, L., 1982. A lower audiometric limit for auditory

brainstem response (ABR). Scand. Audiol. 11, 277–278.

Coppens, A.G., Resibois, A., Poncelet, L., 2000a. Immunolocalization

of calbindin and calretinin in the dog cochlea during postnatal

development. Hear. Res. 145, 101–110.

Coppens, A.G., Resibois, A., Poncelet, L., 2000b. Bilateral deafness in

a Maltese Terrier and a great Pyrenean puppy: Inner ear

morphology. J. Comp. Path. 122, 223–228.

Coppens, A.G., Kiss, R., Heizmann, CW., Deltenre, P., Poncelet, L.,

2001a. An original inner ear neuroepithelial degeneration in a deaf

Rottweiler puppy. Hear. Res. 161, 65–71.

Coppens, A.G., Kiss, R., Heizmann, CW., Schafer, B., Poncelet, L.,

2001b. Immunolocalization of the calcium binding S100A1,

S100A5 and S100A6 proteins in the dog cochlea during postnatal

development. Develop. Brain Res. 126, 191–199.

Coppens, A.G., Salmon, I., Heizmann, C.W., Kiss, R., Poncelet, L.,

2003a. Postnatal maturation of the dog stria vascularis. An

immunohistochemical study. Anat. Rec. 270A, 82–92.

Coppens, A.G., Steinberg, SA., Poncelet, L., 2003b. Inner ear

morphology in a bilaterally deaf Dogo Argentino pup. J. Comp.

Path. 128, 67–70.

Famula, T., Oberbauer, A., Sousa, C., 1996. A threshold model

analysis of deafness in Dalmatians. Mam. Genome 7, 650–653.

Famula, T., Oberbauer, A., Sousa, C., 2000. Complex segregation

analysis of deafness in Dalmatians. Am. J. Vet. Rec. 61, 550–553.

Famula, T., Oberbauer, A., Williams, D.C., 2001. Gender effects in

hearing loss in Dalmatians. Prev. Vet. Med. 48, 15–24.

Feghali, J.G., Lefebvre, P., Staecker, H., Kopke, R., Frenz, D.A.,

1998. Mammalian auditory hair cell regeneration/repair and

protection: a review and future directions. ENT-Ear, Nose Throat

J. 77, 280–285.

Greibrockk, T., 1994. Hereditary deafness in the dalmatian: relation-

ship to eye and coat color. J. Am. Anim. Assoc. 30, 170–176.

Hardisty, R.E., Fleming, J., Steel, J.P., 1998. The molecular genetics of

inherited deafness – current knowledge and recent advances. J.

Laryngol. Otol. 112, 432–437.

Harvey, S.J., Mount, R., Sado, Y., Naito, I., Ninomiya, Y., Harrison,

R., Jefferson, B., Jacobs, R., Thorner, P.S., 2001. The inner ear of

dogs with X-linked nephritis provides clues to the pathogenesis of

hearing loss in X-linked Alport syndrome. Am. J. Pathol. 159,

1097–1114.

Hiraide, F., Paparella, M.M., 1988. Histopathology of the temporal

bones of deaf dogs. Auris nasus Larynx (Tokyo) 15, 97–104.

Igarashi, M., Alford, B.R., Cohn, A.M., Saito, R., Watanabe, T.,

1972. Inner ear abnormalities in dogs. Ann. Otol. Rhinol.

Laryngol. 81, 249–255.

Ilg, E.C., Schafer, B.W., Heizmann, C.W., 1996. Expression pattern of

S100 calcium-binding proteins in human tumors. Int. J. Cancer 68,

325–332.

Klein, E., Steinberg, S.A., Weiss, S.R.B., Matthews, D.M., Uhde,

T.W., 1988. The relationship between genetic deafness and fear-

related behaviors in nervous pointer dogs. Physiol. Behav. 43, 307–

312.

Lalwani, A.K., Linthicum, F.H., Wilcox, E.R., Moore, J.K., Walters,

F.C., San Augustin, T.B., Mislinski, J., Miller, M.R., Sinninger, Y.,

Attaie, A., Luxford, W.M., 1997. A five-generation family with

late-onset progresive hereditary hearing impairment due to coch-

leosaccular degeneration. Audiol. Neurootol. 2, 139–154.

Lefebvre, P.P., Malgrange, B., Lallemend, F., Staecker, H., Moonen,

G., Van De Water, T.R., 2002a. Mechanism of cell death in the

injured auditory system: otoprotective strategies. Audiol. Neuroo-

tol. 7, 165–170.

Lefebvre, P.P., Staecker, H., Van De Water, T.R., Moonen, G.,

Malgrange, B., 2002b. Pharmacologic treatment of inner ear: from

basic sciences to the patient. Acta Oto-Rhino-Laryngol. Belg. 56,

45–49.

Mair, I.W.S., 1976. Hereditary deafness in the Dalmatian dog. Arch.

Otorhinolaryngol. 212, 1–14.

Malgrange, B., Rigo, J.M., Van De Water, T.R., Staecker, H.,

Moonen, G., Lefebvre, P.P., 1999. Growth factor therapy to the

damaged inner ear: clinical prospects. Int. J. Pediatr. Otorhinolar-

yngol. 49, S19–S25.

Miura, M., Sando, I., Hirsch, B.E., Orita, Y., 2002. Analysis of spiral

ganglion cell populations in children with normal and pathological

ears. Ann. Otol. Rhinol. Laryngol. 111, 1059–1065.

Muhle, A.C., Jaggy, A., Stricker, C., Steffen, F., Dolf, G., Busato, A.,

Kornberg, M., Mariscoli, M., Srenk, P., Gailmlard, C., 2002.

Further contributions to the genetic aspect of congenital sensori-

neuronal deafness in Dalmatians. Vet. J. 163, 311–318.

Nadol, J.B., Young, Y.S., Glynn, R.J., 1989. Survival of spiral

ganglion cells in profound sensorineural hearing loss: implications

for cochlear implantation. Ann. Otol. Rhinol. Laryngol. 98, 411–

416.

Niparko, J.K., Finger, P.A., 1997. Cochlear nucleus cell size changes in

the Dalmatian: model of congenital deafness. Otolaryngol. Head.

Neck. Surg. 117, 229–235.

Niparko, J.K., Pfingst, B.E., Johansson, C., Kileny, P.R., Kemink,

J.L., Tjellstrom, A., 1993. Cochlear wall titanium implants for

auditory nerve stimulation. Ann. Otol. Rhinol. Laryngol. 102, 447–

454.

Poncelet, L., Meurris, S., Coppens, A.G., Deltenre, P., 2000. Matu-

ration of the auditory system in normal dogs as reflected by BAEP

wave V latency-intensity curve and rarefaction-condensation dif-

ferential potentials. Am. J. Vet. Res. 61, 1343–1348.

Rak, S., Drogemuller, C., Kuiper, H., Leeb, T., Quignon, P., Andre,

C., Distl, O., 2002. Cloning and chromosomal localization of

MYO15A to chromosome 5 of the dog (Canis familiaris). Chro-

mos. Res. 10, 407–410.

Sockalingam, R., Charles, B., Fillippich, L., Murdoch, B., 2002.

Cisplatin-induced ototoxicity and pharmacokinetics: preliminary

findings in a dog model. Ann. Otol. Rhinol. Laryngol. 111, 745–750.

Staecker, H., Kopke, R., Malgrange, B., Lefebvre, P.P., Van De

Water, T.R., 1996. NT-3 and/or BDNT therapy prevents loss of

auditory neurons following loss of hair cells. Neuroreport 7, 889–

894.

Steel, K.P., Bock, GR., 1983. Hereditary inner ear abnormalities in

animals: relationship with human abnormalities. Arch. Otolaryn-

gol. 109, 22–29.

Steinberg, S.A., Klein, E., Killens, R.L., Uhde, T.W., 1994. Inherited

deafness among nervous pointer dogs. J. Hered. 85, 56–59.

Strain, G.M., 1996. Aetiology, prevalence and diagnostic of deafness in

dogs and cats. Brit. Vet. J. 152, 17–36.

Wilkes, M.K., Palmer, A.C., 1992. Congenital deafness and vestibular

deficit in the Dobermann. J. Sm. Anim. Pract. 33, 218–224.

Inner ear histopathology in “nervous Pointer dogs” with severe hearing loss

Documents

Transcript of Inner ear histopathology in “nervous Pointer dogs” with severe hearing loss