Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 1
Original research article
published: 23 June 2010
Bartke, 2007; Llorens-Martin et al., 2009). Partial or total IGF-I
deficit is associated with severe alterations to the nervous system
and disease. In humans, different mutations in the IGF1 gene pro-
duce the functional loss of this protein, a genetic condition associ-
ated with the presence of severe intrauterine and postnatal growth
impairment, microcephaly, mental retardation and sensorineural
deafness, among other problems (Woods et al., 1997; Bonapace
et al., 2003; Walenkamp et al., 2005; Walenkamp and Wit, 2007;
Netchine et al., 2009). Indeed, sensorineural hearing loss is asso-
ciated with poor growth rates in infancy and adolescence (Welch
and Dawes, 2007), adult short stature (Barrenas et al., 2005) and
Turner’s syndrome (Barrenas et al., 2000).
Accordingly, mice lacking the Igf1 gene suffer severe growth
defects that are coupled to diminished survival and delayed nerve
myelination, among other alterations (D’Ercole et al., 2002; Zeger
et al., 2007). We have shown that cochlear gene expression is affected
in the Igf1−/− null mouse, this organ displaying morphological
Neural development and the activity of the nervous system are
regulated by a complex network of local and systemic factors.
Insulin-like growth factors are fundamental modulators of nervous
system structure, proliferation, growth, differentiation and meta-
bolic demands during fetal and postnatal development (LeRoith,
2008). In mammals, the insulin gene family is comprised of three
factors, insulin and the insulin-like growth factors I and II, which
are recognized by three receptors (Varela-Nieto et al., 2007). These
factors and receptors are expressed in neural cells during devel-
opment in specific spatiotemporal patterns. Insulin-like growth
factor-I (IGF-I) expression peaks in the nervous system during late
embryonic and neonatal periods. While its expression is reduced in
the adult but it is maintained in areas of high plasticity such as the
olfactory bulb and the hippocampus (Aleman and Torres-Aleman,
2009; Aberg, 2010). In the adult brain, IGF-I is essential to maintain
normal brain physiology and to promote neurogenesis (Sun and
A comparative study of age-related hearing loss in wild type
and insulin-like growth factor I deficient mice
Raquel Riquelme1†, Rafael Cediel1,2,3†, Julio Contreras1,2,4†, Lourdes Rodriguez-de la Rosa1,2, Silvia Murillo-
Cuesta1,2, Catalina Hernandez-Sanchez5,6, Jose M. Zubeldia2,7, Sebastian Cerdan1 and Isabel Varela-Nieto1,2*
1 Instituto de Investigaciones Biomedicas “ Alberto Sols” , CSIC-UAM, Madrid, Spain
2 Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain
3 Departamento de Medicina y Cirugia, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
4 Departamento de Anatomia, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
5 Centro de Investigaciones Biologicas, CSIC, Madrid, Spain
6 Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabolicas, Madrid, Spain
7 Servicio de Alergia, Hospital General Universitario Gregorio Marañon, Madrid, Spain
Insulin-like growth factor-I (IGF-I) belongs to the family of insulin-related peptides that fulfils a
key role during the late development of the nervous system. Human IGF1 mutations cause
profound deafness, poor growth and mental retardation. Accordingly, Igf1−/− null mice are dwarfs
that have low survival rates, cochlear alterations and severe sensorineural deafness. Presbycusis
(age-related hearing loss) is a common disorder associated with aging that causes social and
cognitive problems. Aging is also associated with a decrease in circulating IGF-I levels and this
reduction has been related to cognitive and brain alterations, although there is no information as
yet regarding the relationship between presbycusis and IGF-I biodisponibility. Here we present a
longitudinal study of wild type Igf1+/+ and null Igf1−/− mice from 2 to 12 months of age comparing the
temporal progression of several parameters: hearing, brain morphology, cochlear cytoarchitecture,
insulin-related factors and IGF gene expression and IGF-I serum levels. Complementary invasive
and non-invasive techniques were used, including auditory brainstem-evoked response (ABR)
recordings and in vivo MRI brain imaging. Igf1−/− null mice presented profound deafness at all the
ages studied, without any obvious worsening of hearing parameters with aging. Igf1+/+ wild type
mice suffered significant age-related hearing loss, their auditory thresholds and peak I latencies
augmenting as they aged, in parallel with a decrease in the circulating levels of IGF-I. Accordingly,
there was an age-related spiral ganglion degeneration in wild type mice that was not evident
in the Igf1 null mice. However, the Igf1−/− null mice in turn developed a prematurely aged stria
vascularis reminiscent of the diabetic strial phenotype. Our data indicate that IGF-I is required
for the correct development and maintenance of hearing, supporting the idea that IGF-I-based
therapies could contribute to prevent or ameliorate age-related hearing loss.
Keywords: aging, auditory brainstem responses, deafness, Igf1−/− null mouse, insulin-like factors, in vivo brain imaging,
presbycusis, sensorineural deafness
Miguel A. Merchán, Universidad de
Matthew C. Holley, The University of
Julie A. Chowen, Hospital Infantil
Universitario Niño Jesús, Spain
Isabel Varela-Nieto, Instituto de
Investigaciones Biomédicas “ Alberto
Sols” , CSIC-UAM, Arturo Duperier 4,
28029 Madrid, Spain.
†Raquel Riquelme, Rafael Cediel and
Julio Contreras have contributed
equally to this work.
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 2
Riquelme et al. IGF-I and age-related hearing loss
alterations (Camarero et al., 2001, 2002; Sanchez-Calderon et al.,
2010) associated with bilateral hearing loss at the age of 1 month
in these mice (Cediel et al., 2006).
In humans, IGF-I levels are highest during puberty and they
decline with age (Leifke et al., 2000; Gomez, 2007). Aging is associ-
ated with a reduction in certain cognitive functions, such as hearing
loss, and the onset of diseases like atherosclerosis and Alzheimer’s
disease (Dik et al., 2003; Watanabe et al., 2005). Similarly, changes
in circulating IGF-I levels are also correlated with cognitive per-
formance in older humans, as well as with the onset of age-related
neurodegenerative diseases. For example, serum IGF-I levels are
diminished in Alzheimer’s disease and in other degenerative brain
disorders, whereas there is an up-regulation after brain injury, sug-
gesting that IGF-I is involved in neuroprotection (Torres-Aleman,
2008). Since recombinant human IGF-I therapy has been approved
in humans to treat poor linear growth, several groups have con-
templated the use of rhIGF-I in animal models of brain insults to
explore its utility as a protective or neural repair agent (Bright et al.,
2009; Torres-Aleman, 2010).
The genetic component of hearing loss is very diverse and
involves hundreds of different genes (Dror and Avraham, 2009).
Age-related hearing loss (ARHL – presbycusis) is the most com-
mon cause of adult auditory deficiency. It is usually a sensorineural
hearing disorder in which hair cells and auditory nerve cells in the
inner ear are damaged or lost. The genetic and molecular bases of
susceptibility to ARHL are largely unknown and only a few genes
that influence late onset or progressive hearing loss have been dis-
covered (van Wijk et al., 2003; Zhu et al., 2003; Van Eyken et al.,
2007; Dror and Avraham, 2009). However, many of these genes
are likely to be linked to the aging and neurodegeneration related
to susceptibility to oxidative stress, excitotoxicity and cell death
IGF-I signaling regulates lifespan, apoptosis, sensitivity to
oxidative stress and the programmed response to DNA damage
across species (Christensen et al., 2006; Niedernhofer et al., 2006;
Sanchez-Calderon et al., 2007; Narasimhan et al., 2009). Despite
the importance of IGF-I in both aging and deafness, the influence
of an IGF-I deficit in age-related hearing loss has not yet been
studied. Here, we have carried out a longitudinal study of auditory
function, cochlear and brain morphology, and IGF-I levels in wild
type and Igf1−/− null mice to study the relationship between IGF-I
deficit and hearing performance during aging. Our findings sup-
port the idea that IGF-I levels may predict premature age-related
MaterIals and Methods
Mouse handlIng and genotypIng
Heterozygous mice with a targeted disruption of the Igf1 gene
were bred and maintained on a hybrid MF1 and 129/sv mouse
genetic background to increase nullizygous Igf1 mutant sur-
vival (Liu et al., 1993). Null mouse mortality before adulthood
is high, although between 20 and 30% survived. Both wild type
and null mice were studied at the time points indicated to fol-
low their progression from young adults (1 month) to aged mice
(1 year). Mouse genotypes were identified using the REDExtract-
N-AmpTMTissue PCR Kit (XNAT, Sigma) according to the manu-
facturer’s instructions. The PCR was conducted with the following
thermal cycle program: 1 cycle of 94°C for 10 min; 30 cycles of
94°C for 1 min, 59°C for 1 min, 72°C for 1 min; and a final
elongation step at 72°C for 10 min. The wild type Igf1 allele was
detected using the 5′-GTCTAACACCAGCCCATTCTGATT-3′ and
5′-GACTCGATTTCACCCACTCGATCG-3′ primers, which pro-
duced a 250-bp amplicon. The neomycin cassette was detected
using primers 5′-GCTTGGGTGGAGAGGCTATCC-3′ and
5′-CCAGCTCTTCAGCAATATCACGGG-3′, producing a 658-bp
band. All primers were used simultaneously with no evidence of
interference. All animal handling procedures were carried out in
accordance with the European Council Directive (86/609/ECC) and
with the approval of the Bioethics Committee of the CSIC.
audItory BraInsteM recordIng (aBr)
Mice were anesthetized by i.p. administration of ketamine (Ketolar
© 50, Parke Davis Labs, 100 mg/kg) and xylazine (Rompum © 2%,
Bayer Labs, 4 mg/kg), and they were maintained at 37°C throughout
the testing period to avoid hypothermia. Both female and male mice
were used; no sex-associated parameters were identified in this study.
ABR was carried out in a sound-attenuating chamber in an electri-
cally shielded room. Stimulus presentation, ABR amplification and
data acquisition were performed with TDT System 3™ equipment
and SigGeRPTM™ software (Tucker Davis Technologies, Alachua
FL 32615), as described previously (Cediel et al., 2006). Briefly,
an ES-1 electrostatic speaker was placed 10 cm from the animal’s
head and its hearing of different sound stimuli was tested. Tone-
burst stimuli (4, 8, 16 and 32 kHz), with a 1-ms rise/fall time and
a 5-ms plateau, or click stimuli (1–16 kHz) were applied. Stimuli
were presented at decreasing intensities in steps from 90 dB SPL to
10 dB SPL until no waveform was obtained. The stimulus intensity
was then increased in 5 dB SPL steps and two more responses were
recorded in order to determine the auditory threshold. Auditory
profiles were recorded using stainless steel needle electrodes (Spes
Medica S.r.l., 20090 Milan, Italy) placed as follows: vertex-positive,
mastoid area-negative and hip-ground. Responses were collected
and amplified by a factor of 1 × 106, and the system was calibrated as
recommended by the manufacturer with an ACO Pacific™ ¼ inch
microphone connected to a specific TDT calibration module. We
analyzed the following parameters obtained from waves registered
during the ABR tests: auditory thresholds, latencies of peaks I–V
and interpeak latencies.
MagnetIc resonance IMagIng (MrI) and VoluMe estIMates
MRI of the brains from wild type Igf1+/+ and Igf1−/− null mice was
performed with a 7-T Bruker PharmaScan™ 7T. T1 or T2 weighted
images were acquired using the spin-echo sequence with values of
TR = 300 ms/TE = 10 ms or TR = 2500 ms/TE = 60 ms, respec-
tively. One day prior to the MRI experiments, animals were sub-
cutaneously injected with a 30-mM solution of MnCl2 at a dose of
0.4 mmol/kg body weight. Manganese is a positive MRI contrast
agent that is taken up in brain regions with higher neuronal activ-
ity (McDaniel et al., 2001; Watanabe et al., 2004; Uchino et al.,
2007). T1- and T2-weighted images were obtained every 1.1 mm
and volume estimates were obtained according to the principle of
Cavalieri (Gundersen and Jensen, 1987; Camarero et al., 2001). The
Cavalieri principle of systematic sampling in combination with
point counting is considered a reliable and efficient method to
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 3
Riquelme et al. IGF-I and age-related hearing loss
4–8%) (IDS Ltd., Boldon, UK) according to the manufacturer’s
recommendations. Briefly, mouse serum samples were incubated
with a reagent to inactivate binding proteins, and then diluted for
the assay. These diluted samples were then incubated for 2 h at
room temperature with a biotinylated polyclonal rabbit anti-rat
IGF-I antiserum, in polystyrene microtiter wells coated with puri-
fied monoclonal anti-Rat IGF-I antibodies. The wells were washed
and horseradish peroxidase-labeled avidin was added. After further
washing, tetramethyl-benzidine was added to develop the color
reaction and the absorbance of the developed reaction was read
at dual wavelength (A450–A650 nm). ELISA data are expressed
in ng/ml. Statistical comparisons of IGF-I sera levels between the
different age groups were performed with a Mann-Whitney rank
sum test using GraphPad InStat 3.06 (Software Inc., San Diego,
CA, USA). The results were considered to be statistically significant
when P < 0.05.
Unless indicated otherwise, statistical analysis was performed with
one-way ANOVA followed by Bonferroni adjustment as post hoc
method, using SPSS™ 12 software. The results were considered
significant at P < 0.05 and the data are presented as mean ± s.e.m.
Where indicated the Pearson product moment correlation coeffi-
cient (r) was used as a measure of the linear dependence between
two variables, r values are between +1 and −1 and 0 indicates
no correlation. Specific records are represented with Origin™
teMporal pattern of age-related hearIng loss In Igf1−/− null
and Igf1+/+ wIld type MIce
Hearing was tested in mice using ABR evoked by transient click
and tone-burst stimuli in 1, 3, 6, 9 and 12-month-old Igf1−/− null
and Igf1+/+ wild type littermates. The ABR curves obtained for the
mice of either genotype showed the characteristic five peaks during
the 10-ms post-stimulus period at all the ages studied (Figure 1).
The ABR thresholds in response to click stimuli were 40 and 35 dB
SPL in 1- and 3-month-old wild type mice, respectively, and they
increased steadily to a mean value of 75 dB SPL in 1-year-old mice
(Figures 1 and 2A). A different pattern of aging was reflected by
the auditory click-ABR thresholds in the Igf1−/− null mice. Young,
1- to 3-month-old Igf1−/− mice were profoundly deaf and had a
mean auditory threshold of 70 dB SPL, which remained con-
stant as they aged since 1-year-old mice had a threshold of 75 dB
SPL (Figure 2A). Hence, while the Igf1+/+ mice had significantly
lower auditory thresholds than the Igf1−/− mice at younger ages
(1–6 months), as the mice aged their ABR thresholds converged
to reach a similar value at 1 year of age (Figure 2A).
Pure tone audiograms were obtained in the same mice at the
frequencies of 4, 8, 16 and 32 kHz using tone-burst ABR stimuli.
At all ages tested, the general shape of the audiogram was similar in
both mouse genotypes, and while the ABR peaks I to IV were clearly
identified, peak V could only be distinguished in some recordings.
The lowest threshold was obtained at 16 kHz, the best frequency
for hearing in both the Igf1+/+ and Igf1−/− mice at the ages studied
(Figure 2B). At all four frequencies tested, an age-related increase
in the thresholds was evident in Igf1+/+ mice, whilst the audiograms
estimate volumes in MRI (Jelsing et al., 2005). The points hitting
the surface area of the cerebellum, brainstem, olfactory bulb and
total brain were counted for each MRI section.
Mice were anesthetized by i.p. injection of 0.12 mg/g ketamine
hydrochloride and perfused transcardially with 4% paraformal-
dehyde in 0.1 M phosphate-buffered saline (PBS) [pH 7.4]. The
animal’s brain was removed and weighed after separating the
medulla at the beginning of the spinal cord. Temporal bones were
isolated, post-fixed for 1 day, decalcified in 0.3 M EDTA [pH 6.5]
for 10 days and finally embedded in paraffin or gelatin. Paraffin
sections (10 μm) or cryosections (20 μm) of temporal bones from
Igf1+/+ and Igf1−/− mice were processed for Nissl staining, hematoxy-
lin/eosin or other techniques to study their cytoarchitecture. Serial
sections were collected to detect the Na+K+-ATPase β2 isoform (rab-
bit anti-NaK-ATPase, Upstate Biotechnology, dilution 1:200) and
Kir4.1 (rabbit anti-KCNJ10, Chemicon International, dilution
1:200), and phalloidin histochemistry was also performed (Alexa-
488 conjugated phalloidin, Molecular Probes, Invitrogen, Carlsbad,
CA, USA, dilution 1:100: Murillo-Cuesta et al., 2009, 2010a, 2010b;
Sanchez-Calderon et al., 2010). For immunohistochemistry, paraf-
fin sections were processed by the avidin-biotin-peroxidase (ABC)
method using 3,3-DAB as chromogen.
rna IsolatIon and quantItatIVe rt-pcr
Total RNA was isolated using the Trizol reagent (Invitrogen)
from the brainstem (BS), olfactory bulb (OB), cerebellum (CBL)
and the remainder of the telencephalon-diencephalon (TD) of
at least three to five postnatal day P90 mice per genotype. The
purity of the RNA was assessed with an Agilent Bioanalyzer 2100
(Agilent Technologies). The reverse transcriptase reaction (RT)
was typically performed on 5 μg RNA with the Superscript III Kit
and random primers (Invitrogen). Quantitative PCR (RT-PCRq)
was performed with the ABI Prism 7900HT Sequence Detection
System (Applied Biosystems, Weiterstadt, Germany) using the
TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied
Biosystems) and probes from the Universal Probe Library (UPL:
Roche Applied Science). The primer sequences used and the
respective UPL probes were: Igf1 Universal ProbeLibrary probe:
#67 (left primer CCGAGGGGCTTTTACTTCA and right primer
CACAATGCCTGTCTGAGGTG); Igf2 Universal ProbeLibrary
probe: #40, (left primer CGCTTCAGTTTGTCTGTTCG and right
primer GCAGCACTCTTCCACGATG; Igf1r Universal ProbeLibrary
probe: #55 (left primer CCTGAAGAACCTTCGTCTCATC
and right primer TGGTTGTCTAGGACATAGAAGGAGT);
and 18 rRNA Universal ProbeLibrary probe: #70 (left
primer TGCGAGTACTCAACACCAACA and right primer:
TTCCTCAACACCACATGAGC). Assays were carried out accord-
ing to the manufacturer’s instructions and using the expression
levels of eukaryotic 18S rRNA as a reference. The data are presented
in arbitrary units.
deterMInatIon of seruM Igf-I leVels By elIsa
The concentration of circulating IGF-I was measured in serum
samples taken at the times indicated using a standard OCTEIA
Rat/Mouse IGF-I kit (sensitivity 63 ng/ml and variability
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 4
Riquelme et al. IGF-I and age-related hearing loss
from Igf1−/− null mice corresponded to profoundly deaf animals
from the youngest age tested. Like the click stimuli, the tone-burst
hearing thresholds were similar in 12-month-old Igf1+/+ and Igf1−/−
mice, both genotypes showing ABR thresholds corresponding to
profound deafness, although a small number of Igf1−/− and wild
type mice had hearing thresholds above the electrostatic speaker
driver limit (90 dB SPL). The ABR data were further analyzed to
calculate the percentage of mice in each of the five age groups tested
with thresholds above the normal hearing threshold value of 50 dB
SPL (Figure 2C). There was a progressive increase with age of the
proportion of Igf1+/+ mice with moderate to profound deafness,
and while at 6 months of age 50% of the mice tested had auditory
thresholds over 50 dB SPL, that figure reached 100% by 9 months
of age. By contrast, the entire population of Igf1−/− mice presented
hearing deficiencies at all the ages studied (Figure 2C).
Figure 1 | ABr wave recordings from representative 3- and 12-month-
old Igf1+/+ wild type and Igf1−/− null mice. The waves represent the ABR in
response to intensities of click stimuli decreasing from 90 dB SPL to 30 dB
SPL of a representative mouse for each condition. (A,B) ABR waves from
3- (A) and 12- (B) month-old Igf1+/+ mice. (C,D) ABR waves from 3- (C) and 12-
(D) month-old Igf1−/− mice. In (A) and (C) the intensity of the stimulus for each
response is indicated, and the range of intensities is the same in (B) and (D).
Figure 2 | A comparison of age-related hearing loss in Igf1+/+ and Igf1−/−
mice. (A) ABR thresholds in response to click stimulus in Igf1+/+ (open bars)
and Igf1−/− mice (closed bars) at different ages (1, 3, 6, 9 and 12 months). Wild
type mice show an age-related increase in ABR thresholds, whereas the null
mutant mice were deaf from the youngest age studied. (B) ABR responses to
a 16 kHz stimuli were obtained at the same ages and showed a similar trait.
(C) Percentage of individuals for each genotype showing ABR thresholds
above normal hearing (≥50 dB SPL) at each of the ages studied. Wild type and
null mice are represented as open and closed circles, respectively. Statistical
analysis was performed with ANOVA and post hoc Bonferroni and Dunnet
tests. Data are presented as the mean ± s.e.m; *indicates comparison
between genotypes ***P < 0.001, **P < 0.01 and *P < 0.05; #indicates
comparison between consecutive ages ###P < 0.001. The number (n) of mice
studied were 15, 13, 9, 8 and 7 wild type mice of 1, 3, 6, 9 and 12 month old,
respectively; and 10, 11, 10, 6 and 5 null mice of the same ages.
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 5
Riquelme et al. IGF-I and age-related hearing loss
Igf1−/− mice when compared with wild type mice (Figures 3A,B).
The Peak I latency differences between wild type and Igf1−/− mice
became progressively shorter as the mice aged, differences that were
significant (P < 0.05) at 3 and 6 months of age (Figure 3). The mean
latency of peak IV followed a similar trend. Interestingly, mice of
both genotypes showed a steeper increase in the peak I and IV
latencies between 3 and 6 months of age. The age-related increase
The absolute ABR peak latencies increased with age in both
Igf1+/+ and Igf1−/− null mice (Figure 3). For the sake of simplicity and
to reflect the ABR response in the peripheral auditory and central
nervous system, data were only plotted for peaks I and IV (Figures
3A,B), the latencies of which can be considered as a readout of
auditory nerve and auditory BS functions, respectively (Ponton
et al., 1996). At all ages tested, the peak latencies were delayed in
Figure 3 | Age-related changes in absolute and interpeak latencies in both
genotypes. (A,B) Absolute Peak I (A) and peak IV (B) latencies obtained at
80 dB SPL. Absolute latencies are plotted for Igf1+/+ (open circles) and Igf1−/−
mice (closed circles) aged from 3 to 12 month old. (C) Percentage delay in
latency with aging. Open and closed bars indicate wild type and null mice,
respectively. The bars represent the percentage latency increase from 3 to
12 months of age for peaks I (bars to the left) and IV (bars to the right). The
asterisk indicates that the 11% difference in peak I latency between 3- and
12-month-old wild type mice is significant (P < 0.05). Data are presented as the
mean ± s.e.m. (D,e) Interpeak latencies (IPL) I–II (D) and I–IV (e) obtained at
80 dB SPL for Igf1+/+ (open circles) and Igf1−/− mice (closed circles) aged from 3
to 12 months old. (F) Percentage IPL increases with aging. As in (C), for wild
type (open bars) and null mice (closed bars), the IPL I–II is shown by the bars on
the left and IPL I–IV in the bars on the right. The number (n) of mice studied was:
7 , 9, 4 and 5 wild type and 6, 6, 6 and 3 null mice of 3, 6, 9 and 12 month
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 6
Riquelme et al. IGF-I and age-related hearing loss
glion neurons that progressed with aging, an alteration that might
represent the anatomical basis for the elevation in the age-related
hearing threshold and the delay in the absolute peak I latency. The
morphology of the cochlea from Igf1−/− null mice did not change
with age and the altered postnatal traits already reported persisted,
in agreement with the functional ABR results. Indeed, there was
a progressive degeneration of the stria vascularis in Igf1−/− mice
from 3 months of age, but not in control wild type mice, which
was shorter, thicker and with aberrant vascularization including
dilated intra-strial capillaries when compared with that in the
control Igf1+/+ mice (Figures 6A–H). Kir4.1 (Figures 6I–K) and
Na-K-ATPase levels (compare Figures 6A,E with Figures 6B,F)
in 3- and 12-month-old Igf1−/− null mice reflected the important
alterations in the stria vascularis.
in latency did not affect peaks I and IV in the same way, nor were
the changes similar in Igf1+/+ and Igf1−/− mice. Wild type Igf1+/+ mice
experienced a larger age-related change in latency between 3 to
12 months of age, that was 11% for peak I (P < 0.05). By contrast,
the peak I latency did not increase significantly in Igf1−/− mice over
the same period. Peak IV presented smaller age-related changes in
both genotypes (Figure 3C).
The interpeak latencies (IPL) reflected the transmission of audi-
tory information along the auditory pathway. The interval between
peaks I and II is assumed to represent the conduction time in the
auditory nerve axons and therefore, this value provides informa-
tion about the peripheral auditory system. On the other hand, the
value between peaks I and IV reflect axonal transmission along
the BS, offering information on the central auditory system. These
IPL were longer in Igf1−/− mice than in Igf1+/+ mice at all the ages
tested (Figures 3D,E), although in neither genotype were significant
changes in the IPL evident at any age (Figure 3F).
To further compare the temporal profile of hearing, the relation-
ship between age and auditory thresholds was assessed for both
mouse genotypes using product moment Pearson correlation
coefficients. A clear correlation was observed between both these
parameters in the Igf1+/+ animals (Figure 4A) but not in the Igf1−/−
mice (Figure 4B). The correlation was less clear between aging and
the increase in peak I latency as indicated by the product moment
Pearson correlation coefficient, which was not statistically signifi-
cant in Igf1+/+ wild type and Igf1−/− null mice (Figures 4C,D) Since
both the absolute latency for peak I and the auditory thresholds
increased with age, the peak latency increase could be caused by
the increase in the auditory threshold and not by aging. Product
moment Pearson correlation coefficients were obtained between
data pairs of auditory thresholds and absolute peak I latencies
values for both genotypes at all the ages studied (Figures 4E,F).
Accordingly, a significant correlation between auditory thresh-
olds and cochlear nerve time conductions were observed in Igf1+/+
(Figure 4E) but not in Igf1−/− mice (Figure 4F).
age-related reductIon In the densIty of spIral ganglIon
neurons In Igf1+/+ wIld type MIce and degeneratIon of the
strIa VascularIs In the Igf1−/− null MIce
Important cochlear alterations were evident in 1-month-old Igf1−/−
mice that included reduced cochlear volume, neuronal loss, hypo-
myelinization, an immature tectorial membrane and a general
delay in cell differentiation with increased presence of embry-
onic cell markers (Camarero et al., 2001, 2002; Sanchez-Calderon
et al., 2010). Here, the morphology of the cochlea in 3-, 7- and
12-month-old Igf1+/+ and Igf1−/− mice was analyzed. Generally, the
cochlear cytoarchitecture was similar in both genotypes at the
ages studied, although the differences reported in cochlear size
were maintained between both mouse genotypes. Damage to the
cochleae was evident in individual mice, although in general most
mice had intact inner and outer hair cells, and the general coch-
lear structure was preserved until 1 year of age, the oldest mice
studied (Figures 5A,B; Figure S1 in Supplementary Material). By
contrast, there were fewer spiral ganglion neurons in 1-year old
than in the younger Igf1+/+ mice (Figures 5C,E,G), resulting in a
similar density of neurons in 1-year-old mice from both genotypes
(Figures 5C–H). Hence, Igf1+/+ mice suffer a loss of spiral gan-
Figure 4 | Correlations between auditory thresholds and peak response
latencies with aging and genotype. Product moment Pearson correlation
coefficients were calculated for the pairs of parameters indicated. A clear
correlation was evident between ABR thresholds and age in the Igf1+/+ mice
[open circles; (A) P < 0.05; n = 24; r = 0.69] but not in the Igf1−/− mice [closed
circles; (B) ns; n = 19; r = 0.05]. The absolute peak I latency was weakly
correlated with age in both Igf1+/+ [(C) P < 0.1; n = 24; r = 0.33] and Igf1−/− [(D)
P < 0.25; n = 19; r = 0.24] mice. The Pearson product moment correlation
coefficient between peak I absolute latencies and auditory thresholds at
different ages in Igf1+/+ mice was statistically significant (P < 0.05) [(e)
P < 0.05; n = 24; r = 0.61]. The same correlation in Igf1−/− mice was not
statistically significant. [(F) ns; n = 19; r = 0.09].
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Riquelme et al. IGF-I and age-related hearing loss
Sera from wild type and null mice were collected at several times
between 1 and 10 months of age to analyze the circulating IGF-I lev-
els using a specific ELISA assay. An age-related decline in IGF-I was
observed in Igf1+/+ mice (Figure 7B), from the initial IGF-I levels in
1-month-old mice (1004 ± 220 ng/ml) to those in 10-month-old
mice (517 ± 151 ng/ml, P < 0.05). These progressive age-related
age-related changes In Body weIght, cIrculatIng Igf-I leVels
and BraIn weIght
The body weight of Igf1+/+ and Igf1−/− null mice was measured
at 1, 3, 6 and 12 months of age and both genotypes experienced
an age-related increase in body weight. As expected, at all the
ages studied the Igf1−/− mice were lighter than the Igf1+/+ mice
(Figure 7A). The greatest increase in body weight occurred at
younger ages, between 1 and 3 months of age in both genotypes,
whereas from 3 to 9 months of age their body weight reached a
plateau and showed only minor increases. Indeed, there was a
tendency to lose weight between 9 and 12 months in the wild type
mice. Moreover, a significant correlation was identified between
body weight and age.
Figure 5 | Comparative study of cochlear morphology with aging in
Igf1+/+ wild type and Igf1−/− null mice. (A,B) Cochlear cross-section and close
up of the cochlear basal turn in a representative 3-month-old null mice. The
disposition of the cells in the organ of Corti is apparently normal. IHC: inner
hair cell; OHC: outer hair cells; BM: basilar membrane. (C–H) Comparison in
both genotypes of the spiral ganglion morphology and of the neuronal density
in mice at the ages studied. The neuronal density decreased in the aged spiral
ganglion, such that at the older ages studied the cochlear ganglia was
morphologically similar in both genotypes. Some specimens displayed heavy
hair cell loss at this age, which was correlated with the increase in hearing
thresholds (data not shown). Scale bar 50 μm [0.5 mm in (A)].
Figure 6 | Comparative study of the stria vascularis with the aging of
Igf1+/+ wild type and Igf1−/− null mice. Na-K-ATPase (A,B,e,F) and cresyl
violet staining (C,D,g,H) expression in the stria vascularis of 3- (A–D) and
12-month-old mice (e–H) showing the morphological differences associated
with the aging of each genotype. The stria vascularis in the Igf1−/− mice had an
abnormal morphology, it was shorter and thicker, and with evident dilation of
the vascular spaces (arrows). Kir4.1 (KCNJ10) expression (i–K) in the stria
vascularis of aged null mice was altered and there was a relative loss of
expression in the stria and sacular dilations. Four to six mice per condition
were analyzed. Scale bar 50 μm.
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Riquelme et al. IGF-I and age-related hearing loss
Figure 7 | A comparison of age-related changes in igF-i levels, and body
and brain weight in both genotypes. (A) Body weight of Igf1+/+ (open circles)
and Igf1−/− null (closed circles) mice was measured at 1, 3, 6 and 12 months of
age. Statistical analysis in (A) and (C) was performed with ANOVA and post hoc
Bonferroni and Dunnet tests. Data are presented as the mean ± s.e.m. Both
genotypes presented a significant difference at all the ages studied (P < 0.001)
and an age-related increase in body weight. (B) The serum IGF-I levels were
followed during aging in Igf1+/+ and Igf1−/− mice in parallel. An age-related decline
in serum IGF-I was observed in wild type mice (open circles) whereas no IGF-I
could be detected at any time in the null mice (closed circles). Data are given in
ng/ml as median ± s.e.m. At least six to eight mice were studied per group. (C)
Brain weight measurements did not change significantly as the mice aged,
although there was a significant difference between the two genotypes at the
ages studied (P < 0.05). (D) The brain/body weight ratio was calculated for each
age group of Igf1+/+ wild type (black bars) and Igf1−/− null mice (white bars). This
ratio did not vary with age in any genotype. The number (n) of mice studied in
(A), (C) and (D) were 19, 7 , 9, 8 and 5 wild type and 8, 6, 7 , 6 and 5 null mice of 1,
3, 6, 9 and 12 month old, respectively.
decline in serum IGF-I levels mirrored the changes in body weight.
By contrast, no detectable levels of IGF-I were measured at any time
in the Igf1−/− null mice. IGF-I levels decline data were compared
with those of increasing ABR thresholds (Figure 2A) in the different
groups of wild type mice studied between 1 and 10 months. This
comparison strongly suggest that there is a correlation between
both parameters (Pearson r = −0.33; n = 35; P < 0.05).
Unlike body weight, brain weight measurements did not notice-
ably change as the animals aged, neither in Igf1+/+ nor Igf1−/− null
mice (Figure 7C). The brain/body weight ratio was calculated for
each age group of wild type and null mice and the ratio was higher
for Igf1−/− null mice than for wild type mice (Figure 7D). At each
age tested, the Igf1+/+ mouse brain represented less than 2% of the
total body weight, whilst the Igf1−/− null mouse brain represented
roughly 4% of their total body weight. Aging did not seem to affect
these ratios, although the results indicated that there was an age-
related tendency towards a small reduction in the brain to body
age-related and genotype-dependent BraIn Morphology
In vivo serial MRI of the brain has provided valuable insights into
the changes associated with a range of neurodegenerative and aging
conditions (Liu et al., 2003; Paul et al., 2009). MRI is a non-invasive
procedure that allowed both mouse populations to be studied in
parallel with the hearing tests to further study the brain areas par-
ticipating in auditory processing (Figures 8A–C). Stereological
procedures were used to calculate areas and volumes from the MRI
images, and to analyze their progress over time (Figure 8B and
Table 1). The gross brain morphology was similar between wild
type and null mice, taking into account the difference in size, and
except for the OB (Figure S2 in Supplementary Material), all the
main brain regions that could be observed in the wild type mice
were also observed in the null mice (Figure 8C). Brain images were
obtained from 3-, 6-, 9- and 12-month-old wild type and null mice,
and the volumes of the whole brain and selected brain parts were
obtained. Since it was not possible to identify the auditory BS, the
whole BS was considered as an individual part, and this region
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 9
Riquelme et al. IGF-I and age-related hearing loss
more Igf1 transcripts than in the BS, and 10-fold more than in the
TD (excluding the OB) and CBL. Null mouse brain did not express
Igf1 as expected (data not shown). Since brain size is less strongly
affected by Igf1 deletion than the size of the rest of the body, we
examined the possible compensatory effect of up-regulating Igf1r
and/or Igf2 expression in the brain. Igf1r and Igf2 mRNA expres-
sion were analyzed by RT-qPCR in the four brain regions and they
were not significantly different in Igf1−/− null mice than in the wild
type mice (Figures 9A,B).
IGF-I is a member of the family of insulin-related peptides that
fulfils a prominent role in the development of the central nerv-
ous system and in adult neurogenesis. Circulating levels of IGF-I
decrease with age, this reduction being related to age-associated
cognitive and brain alterations. Congenital mutations in the gene
coding for IGF-I cause sensorineural deafness in mice and men,
although it is not known whether hearing loss in the elderly popula-
tion is related to the age-dependent reduction in IGF-I levels.
It is shown here that Igf1+/+ wild type MF1/129/sv mice suffer
age-related progressive elevation in hearing thresholds, which by
the age of 1 year old reaches that presented at birth by the Igf1−/− null
MF1/129/sv mouse. From 1 to 12 months of age, the click-ABR
thresholds of Igf1+/+ mice experienced a progressive elevation of
30 dB SPL, which was paralleled by the progressive decrease in
circulating IGF-I levels. The Igf1−/− null mouse has elevated auditory
thresholds, and morphological alterations in the auditory recep-
tor and spiral ganglion could account for this functional disor-
der (Camarero et al., 2001; Cediel et al., 2006; Sanchez-Calderon
et al., 2010).
Age-related hearing loss is characterized by a progressive dete-
rioration of hearing sensitivity with increasing age, and it has been
studied in mice of different strains (Zheng et al., 1999; Ouagazzal
et al., 2006; Niu et al., 2007). The mice used in the present study are
on a hybrid genetic background of MF1 and 129/sv mouse strains,
and they present mild hearing loss when compared to mice on
other genetic backgrounds. By contrast, the 1-month-old Igf1−/− null
mouse is already deaf and does not present significant threshold
variations as it ages.
In both Igf1+/+ and Igf1−/− null mice the absolute latencies for
waves I through IV show age-related increments, although the
increment was larger in the Igf1+/+ than in the Igf1−/− null mice,
especially the peak I latency. These data confirm that the increase
in peak I latency is a trait associated with hearing loss in the mouse,
as reported in other mammals such as man (Rosenhall et al., 1986;
Cooper et al., 1990; Martini et al., 1990; Ingham et al., 1998; Torre
and Fowler, 2000; Burkard and Sims, 2002). However, the effect of
aging on IPL is not yet clear. Many authors have reported that the
interpeak I–V latency is prolonged in the elderly (Rosenhall et al.,
1986; Cooper et al., 1990; Fraenkel et al., 2003), whilst others argue
that aging is not associated with a delay in the central conduction
time (Martini et al., 1990; Burkard and Sims, 2002). Neither the
Igf1−/− nor the wild type mouse show significant variations in IPL,
since IPL I–II shows a small increase and IPL I–IV a small com-
pensatory decrease, neither of which are significant. These data
suggest that aging has no effect on the central conduction time,
in contrast with the acute impact of aging on auditory receptor
was considered as a representative region for auditory information
processing. The CBL and the OB were included for comparison
(Figure 8C). The relative BS volumes were similar in Igf1+/+ and
Igf1−/− null mice, and these volumes did not changed with aging
in either genotype (Table 1). No other morphological alterations
were observed by MRI at the BS level. A different orientation of
the cochlea with respect to the brain was observed when com-
paring wild type and null mice (Figures S2A,B in Supplementary
Material). At all the ages studied, the OB was the brain structure
that showed the most important differences between wild type and
null mice, as well as an aberrant laminar structure in the Igf1−/−
null mouse (Figures S2C,D in Supplementary Material; Table 1).
Finally, there was a clear alteration of the ventricular system in the
null mouse brain (Figures S2E,F in Supplementary Material). No
evident age-related changes in brain morphology were observed
in either genotype (Table 1).
The data presented above suggest that different brain areas may
have distinct expression of IGF-I or that they may possess dis-
tinct compensatory mechanisms. Indeed, the Igf1 gene is widely
expressed in the mouse brain, albeit with regional variations in
the levels of expression. Here, we quantified the Igf1 mRNA lev-
els in four regions of the adult P90 mouse brain by quantitative
RT-PCR. The highest levels of Igf1 expression were in the OB
(Figure S3 in Supplementary Material) where there were 5-fold
Figure 8 | In vivo brain images of Igf1+/+ wild type and Igf1−/− null mice. (A)
Complete series of brain slices in the sagittal plane of a representative
3-month-old Igf1−/− null mouse. (B) Sagittal MRI T2 weighted image of a
representative 3-month-old wild type mouse showing the stereological grid that
was randomly overlaid on each slide to estimate the volume. The inset shows
the specific grid used to estimate the volume of specific brain areas, such as the
cerebellum. (C) Comparison of the selected brain areas of Igf1−/− and Igf1+/+
mice: cerebellum (1), brainstem (2), diencephalon-telencephalon (3) and
olfactory bulb (4). Note the reduced olfactory bulb in the deficient mice (yellow).
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 10
Riquelme et al. IGF-I and age-related hearing loss
function that is associated with a loss in spiral ganglion cell density.
Age-related modification of ABR parameters has been associated
with increased auditory thresholds, whereas a tendency toward
increased ABR latencies has only been reported when threshold
increases were considerable (Hunter and Willott, 1987). Our data
support these concepts and indeed, the correlation analysis shows
that the increase in ABR latency with age is strongly related to the
The auditory alterations detected by physiological procedures
have morphological substrates. We previously showed that the coch-
lea of 1-month-old Igf1−/− mice have morphological alterations that
correlate with their elevated auditory thresholds (Camarero et al.,
2001, 2002; Cediel et al., 2006). When compared to the wild type,
Igf1−/− null mice have smaller cochlea and cochlear ganglion, with a
significant decrease in the number and average size of cochlear gan-
glion neurons (Camarero et al., 2001, 2002). With aging, the altered
cochlear morphology in Igf1−/− mice persists yet neuronal loss does
not progress, indicating that the early phenotype of neuronal loss
was not premature neurodegeneration but rather a failure in the late
developmental program. A transcriptome study of the null mouse
cochlea confirmed that the differentiation of neural cells and struc-
tures is delayed at birth (Sanchez-Calderon et al., 2010). However, the
Igf1+/+ cochlear ganglion suffers age-related neuronal loss, reaching
levels that are similar to that of the neonatal Igf1−/− mice. These data
suggest that a subpopulation of auditory neurons depend on IGF-I
for their final differentiation and/or for their survival, although
most adult cochlear neurons can survive in the absence of this fac-
tor. Accordingly, cochlear neurons of Igf1+/+ mice die as they age with
a time-course that follows the decay in circulating IGF-I levels.
Cochlear degeneration has been associated with a down-regula-
tion of IGF-I levels and aging in other models of ARHL (Riva et al.,
2007). No hair cell loss was evident in either genotype at the ages
studied but interestingly, a lack of IGF-I caused long-term degen-
eration in the stria vascularis, the metabolic center of the cochlea.
This phenomenon is reminiscent of the diabetic cochlear phenotype
also seen in the insulin receptor substrate 2 null mice (unpublished
observation). Degeneration of the stria vascularis is one of the most
prominent aspects in the pathophysiology of presbycusis in several
species (Schulte and Schmiedt, 1992; Spicer and Schulte, 2002; Spiess
et al., 2002). The variations in absolute latency with age show that
the increase in latency is age-related in both genotypes and thus,
the impact of IGF-I deficiency in the central auditory pathways was
further explored in a longitudinal in vivo brain MRI study. Several
important alterations were observed and since the gross morphology
of the null mouse brain was maintained, brain size was less affected by
the loss of IGF-I than body weight, and no differences were associated
Table 1 | Body and brain of Igf1−/− and Igf1+/+ mice with age.
One month Three months Seven months One year
WT KO WT KO WT KO WT KO
Body weight (g)
Brain weight (g)
27 .1 ± 3.2 6.7 ± 1.2 42.8 ± 5.8 10.4 ± 1.8 42.4 ± 5.5 8.8 ± 4.6 34.6 ± 4.5 10.8 ± 3.1
0.49 ± 0.04 0.29 ± 0.01 0.47 ± 0.02 0.33 ± 0.04 0.59 ± 0.06 0.38 ± 0.03 0.47 ± 0.03 0.33 ± 3.2
1.8 4.3 1.1 3.1 1.4 4.3 1.3 3
239% 287% 309% 223%
Three months Seven months One year
WT KO WT KO WT KO
522 ± 32
63.5 ± 2.9
91.6 ± 5.6
29.5 ± 2.5
322 ± 13
41.4 ± 2.3
58.8 ± 2.3
10.7 ± 0.9
503 ± 37
62.3 ± 8.6
91.1 ± 6.6
27 .2 ± 2.6
316 ± 35
42.7 ± 2.2
59.2 ± 5
12.6 ± 1.42
469 ± 24
59.2 ± 4.2
86.7 ± 3.5
27 ± 2.3
324 ± 29
44.2 ± 3.8
52.3 ± 4.4
13.4 ± 1.7
(A) Body and brain weights of Igf1−/− (KO) and Igf1+/+ (WT) mice with aging. Brains were weighed after cutting off the medulla at the beginning of spinal cord. The
values are represented as the mean ± s.e.m. of at least four to six animals from each condition. An statistically significant difference between both genotypes
(P < 0.001) was found at all the ages studied. Change (%) indicates the % difference of each parameter between both genotypes, 100% corresponds to the wild
(B) Volume of total brain and of different brain areas in Igf1−/− (KO) and Igf1+/+ (WT) mice were estimated from MRI images taken in vivo at different ages. The
volume estimates were obtained by stereological analysis according to the Cavalieri principle (Gundersen and Jensen, 1987). Volumes are expressed in mm3 as the
mean ± s.e.m. of at least five animals for each genotype and age.
Relative volume indicates the % of the total brain volume that each brain area represents, being 100% the brain volume of each genotype at each age studied. When
genotypes were compared, a reduction in the olfactory bulb of the Igf1−/− deficient mice but not in the other structures was observed.
Frontiers in Neuroanatomy www.frontiersin.org June 2010 | Volume 4 | Article 27 | 11
Riquelme et al. IGF-I and age-related hearing loss
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No changes in the expression of related factors (e.g., insulin and
IGF-II), or of the high affinity receptor IGF1R are evident, in the
brain or cochlea of the null mouse when compared with the wild
type mouse (see also Sanchez-Calderon et al., 2010), nor in other
contexts (Moerth et al., 2007). Other compensatory factors, such as
neurotrophins, may act during late development, leading to FoxM1
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cochlear size with respect to body size in the Igf1−/− null mouse.
In summary, the Igf1−/− null mouse is a model of chronic IGF-I
deficit that leads to cochlear neuronal loss and profound deafness
from the onset of hearing. With aging, chronic IGF-I deficit caused
premature degeneration of the stria vascularis that was not observed
in wild type mice. These data suggest that IGF-I is a candidate factor
to control cochlear metabolic demands and hence, adult hearing.
Conversely, wild type mice show age-related hearing loss that was
associated with progressive loss of neurons and that paralleled the
reduction in serum IGF-I levels. These data suggest that IGF-I is
an otic protector whose levels may predict ARHL.
This work was partially supported by grants to IVN from DIGNA
Biotech, the Ministerio de Ciencia e Innovacion (SAF2008-00470)
and from the Fundacion Mutua Madrileña to IVN and JMZ. Silvia
Murillo-Cuesta and Lourdes Rodriguez-de la Rosa hold contracts
The Supplementary Material for this article can be found online at
Figure 9 | Brain distribution and expression of Igf2 and Igf1r. Igf1r
(A) and Igf2 (B) mRNA levels were measured by quantitative RT-PCR of RNA
from the indicated mouse brain regions and normalized to the levels of the
18S ribosomal RNA 3–5 mice per genotype were used. The results represent
the mean ± s.d. Brainstem (BS), olfactory bulb (OB), cerebellum (CBL) and the
remainder of the telencephalon-diencephalon (TD). Open and closed bars
indicate Igf1+/+ and Igf1−/− mice, respectively.
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Riquelme et al. IGF-I and age-related hearing loss
Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential conflict
Received: 31 March 2010; paper pending
published: 20 April 2010; accepted: 01 June
2010; published online: 23 June 2010.
Citation: Riquelme R, Cediel R, Contreras
J, Rodriguez-de la Rosa L, Murillo-Cuesta
S, Hernandez-Sanchez C, Zubeldia JM,
Cerdan S and Varela-Nieto I (2010) A
comparative study of age-related hearing
loss in wild type and insulin-like growth
factor I deficient mice. Front. Neuroanat.
4:27. doi: 10.3389/fnana.2010.00027
Copyright © 2010 Riquelme, Cediel,
Contreras, Rodriguez-de la Rosa, Murillo-
Cuesta, Hernandez-Sanchez, Zubeldia,
Cerdan and Varela-Nieto. This is an
open-access article subject to an exclusive
license agreement between the authors and
the Frontiers Research Foundation, which
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