Keywords
congenital hypothyroidism - cochlea - hearing loss
Introduction
The diagnosis of hearing loss during childhood can cause several disturbances in the
quality of life, which may include changes in the social, educational, emotional,
and language behaviors.[1] Among the many causes of hearing loss, congenital hypothyroidism stands out as a
factor of great importance.[2]
[3] Early diagnosis of the low maternal level of thyroid hormones (THs) allows their
replacement and may avoid fetal and subsequent adult damages. Importantly, it is a
disease that can be prevented and treated.[4]
Experimental studies have shown the repercussion of hypothyroidism during gestation
in the organ of hearing when the hormonal replacement is not done.[2]
[5] Particularly, abnormalities in the organ of Corti have been reported as some of
the main consequences of the lack of THs.[2]
[3] Congenital hypothyroidism may also be experimentally induced when methimazole, an
inhibitor of TH production, is added to the drinking water of pregnant and lactating
rats.[3]
[6]
Thyroid hormone receptors are also of paramount importance to the organ of hearing
development during gestation, particularly to the development of the middle and inner
ear (ear ossicles[7]) and the organ of Corti.[2]
[6] In hair cell-specific mutation of TRα1 or deletion of TRβ using the Cre-loxP system,
hearing procedures such as the measurement of distortion product otoacoustic emission
(DPOE) showed impairment of the outer hair cell and auditory brainstem responses (ABRs).[8]
Despite the essential previous studies, there are still many aspects that need to
be clarified to provide a better understanding of the impact of prenatal THs on the
offspring's auditory system development. In this study, we evaluate the effect of
prenatal and postnatal hypothyroidism on the auditory function of adult offspring
in rats.
Material and Methods
Ethical Approval
All animals and audiological procedures were approved by the Brazilian Ethics Committee
on Animal Research (Protocol # 21/2017- CEPA/UFS ), following the Brazilian Guide
for the Care and Use of Animals in Teaching and Scientific Research Activities (Diretriz Brasileira para o Cuidado e a Utilização de Animais em Atividades de Ensino
ou de Pesquisa Científica - DBCA), the National Institute of Health Guide for the Care and Use of Laboratory Animals
(NIH Publication no. 8023, revised 1978), and the Declaration of Helsinki. All efforts
were made to minimize animal suffering and to reduce the number of animals used.
Animals and the Induction of Gestational Hypothyroidism
All animals were obtained from the animal care facility of Universidade Federal de
Sergipe and maintained in a controlled light/dark cycle (12/12 h) with a room temperature
of 23 ± 2° C. Animals had free access to standard chow (Presence – Nestlé Purina Petcare,
St. Louis, MO, USA) and drinking water.
Female Wistar rats (∼ 200 g) had their estrus cycle monitored daily by vaginal smears.
Once the proestrus phase was detected, adult males (∼ 300 g) were put in female cages
for overnight mating. The presence of spermatozoa on vaginal smears on the following
morning confirmed gestational day (GD) 0. To induce hypothyroidism, dams were given
0.02% methyl mercaptoimidazole (MMI – Sigma-Aldrich, Saint Louis, MO, USA) in drinking
water from GD 9 up to the postnatal day (PND) 15, during lactation, as described by
Knniper et al.[6]
The offspring from MMI-treated dams (OMTD) were compared to the corresponding control
offspring (offspring from water-treated dams; OWTD). The newborns were sexed on postnatal
day (PND) 3, and the litter size was adjusted to 8 pups (4 females and 4 males). Pups
were weaned on PND 21, and males and females were separated at weaning. From PND 30,
all data obtained were analyzed only in males (OWTD and OMTD).[9] For this study, offspring were arbitrarily classified as either preadult (PND 30)
or adult (PND 60–120).
Hearing Analysis Procedures
The pups were anesthetized with an intraperitoneal (i.p) mixture of ketamine (90 mg/kg)
and xylazine (10 mg/kg), and the hearing of offspring was measured on PNDs 30, 60,
90, and 120. All of them were evaluated by baseline otoscopic examination (Welch Allyn
Pocket Junior, model 22840, SP, Brazil). Rats with a clean external ear canal and
normal eardrum were included in the study to have reliable results. After anesthesia,
the animals were placed in a small acoustic measurement box, and environmental noise
was kept under 50 decibel sound pressure level (dB SPL). For all hearing procedures,
we inserted the earphone into the rat's external ear canal with a neonate probe after
the head of the animal was brought to a horizontal position.
Tympanometry Procedure
Tympanometry was performed with a portable tympanometer (Kamplex/Interacoustics, MT
10, Assens, Denmark) with a probe frequency of 226 hertz (Hz). This test measures
middle ear pressure using electroacoustic and manometric measurements. The results
were recorded by the air pressure of the ear canal, which corresponds to the peak
of the tympanogram. It reveals the measurement of compliance in milliliters (mL) and
the pressure in daPa. Moreover, compliance provides an index of the tympanic membrane's
mobility. Very high, low, or absent mobility indicates a dysfunctional middle ear.
Distortion Product Otoacoustic Emission (DPOAE)
The DPOAE measurements were performed using a standard commercial device (Otoread
– Interacoustics, Assens, Denmark). Distortion product otoacoustic emission was recorded
as distortion product diagrams (DPgrams). The intensity of the primary tones was constant,
and the DPOAE data were recorded at different frequencies, ranging from 3 to 12 kHz
(3, 4, 6, 8, 10, and 12 kHz). For the DPOAE, the intensity of the primary stimuli
was set equivalent to its level (L1= 65 dB SPL and L2 = 55 dB SPL). The frequencies
(f1 and f2) were adjusted to f2/f1 = 1.21. The noise floor level was measured at a
frequency of 50 Hz above the DPOAE frequency, using similar averaging techniques.
The evaluation of the DPOAE results was based on the amplitude (DPOAE level), which
should be ≥ 3 Db SPL. The average time of the test was 60 seconds for each animal.
Auditory Brainstem Response (ABR)
Auditory brainstem response measurements were performed using a standard commercial
ABR meter (EP25–Interacoustics, Assens, Denmark). The ABR stimulus was initially set
to 90 decibels sound pressure (dB SPL), 100 microseconds (µs) click (2–4 kHz) presented
at a rate of 26.8% with bandpass filtering (0.015 and 3 kHz). Replicate responses
(each representing the averaged responses to 1,024 clicks) were recorded. The responses
were differential voltage recordings from subdermal needle electrodes placed in the
scalp at the vertex (noninverting electrode), ipsilateral mastoid (inverting electrode),
and leg (ground electrode).
The presentation level was attenuated in 20 dB SPL steps from 90 dB SPL up to 30 dB
SPL. The lowest stimulus level that elicited a repeatable wave was considered as the
threshold. Finally, there is a similarity of electrophysiological and behavioral thresholds
in rat experiments.[10] The hearing loss classification was: threshold ≤ 20 dB SPL (normal); between 30
and 40 dB SPL (soft); between 45 and 70 dB SPL (moderate); between 80 and 90 dB SPL
(severe); and higher than 90 dB SPL (profound).[11]
Data Analysis
The IBM SPSS, Statistics for windows, version 20.0 (IBM Corp., Armonk, NY, USA) was
used for data analysis. Results were expressed as values of marginal mean and mean
standard error. Mixed analysis of variance (ANOVA) procedures were used for all main
and simple effects tests. When the analysis of variance indicated a significant difference,
a posthoc significant difference test (Tukey HSD) was performed. Differences were
considered significant at p ≤ 0.05.
The dependent variable for tympanometry was compliance; for DPOAE, it was amplitude
(DPOAE level); and for ABR, it was latency. Random factors included: litter and individual
animals, while fixed factors were: treatment during gestation, age, and both ears
(right and left).
Results
In the analysis of all procedures, the ears were all analyzed together, since there
was no significant difference between the right and left ears in the mixed ANOVA (p > 0.05).
The evidence that the protocol have ensured hypothyroidism was the OMTD low body mass
as shown in [Figure 1].
Fig. 1
Effect of experimental congenital hypothyroidism in offspring body weight at PND 30 (n = 18 OWTD/ n=16 OMTD), PND 60 (n = 18 OWTD/ n = 6O MTD), PND 90 (n = 16
OWTD/ n=5 OMTD), PND 120 (n = 15 OWTD/ n=5 OMTD). For analysis, mixed ANOVA followed
by the Tukey HSD post hoc test were used. Values are expressed as marginal mean ±
SEM. (*) p < 0.05 and (**) p < 0.01 vs. PND30. PND: Post-natal day, OWTD: Offspring from water-treated dams, OMTD:
offspring from MMI treated dams.
Tympanometry test results showed a decrease of compliance in OMTD advanced age of
analyses as presented in [Figure 2]. There was a significant main effect for time factor of OWTD and OMTD [F (3; 13.474) = 19.721,
p < 0.001]. Moreover, there was a significant main effect for experimental groups [F
(1; 4.397) = 10.368, p = 0.028]. The study also revealed a significant interaction between time factor and
experimental group [F (3; 12.723) = 6.239, p = 0.008]. The Tukey honestly significant difference (HSD) posthoc test revealed a
statistically significant difference at 30 and 60 days for OMTD (p < 0.05).
Fig. 2
Effect of experimental congenital hypothyroidism on compliance in offspring at PND 30 (n = 18 OWTD/ n = 16 OMTD), PND 60 (n = 18 OWTD/ n = 6OMTD), PND 90 )n = 16
OWTD/ n = 5 OMTD), PND 120 (n = 15 OWTD/ n = 5 OMTD), as measured by tympanometry.
For analysis, mixed ANOVA followed by the Tukey HSD post hoc test were used. Values
are expressed as marginal mean ± SEM. (*) p < 0.05 and (**) p < 0.01 vs. PND30. PND: Post-natal day, OWTD: Offspring from water-treated dams, OMTD:
offspring from MMI treated dams.
The DPOAE levels (amplitude) for OWTD and OMTD were similar for 2 kHZ, with values
below 3 dB SPL. The analyses of the OWTD group's amplitudes showed values higher than
those in the treated groups, with a significant main effect of experimental groups
[F (1; 4.519) = 7.762; p = 0.043] and time factor [F (3; 12.311) = 76.695; p = 0.006]. Also, a significant main effect was found for all frequencies [F (5; 24.435) = 162.027;
p = 0.001), and frequencies versus groups [F (5; 23.044) = 4.282; p = 0.007] ([Figure 3]). The Tukey's HSD posthoc test revealed a statistically significant difference for
frequencies higher than 6 kHz for OMTD (p > 0.005).
Fig. 3
Effect of experimental gestational hypothyroidism on amplitude and 2, 4,6,8,10,12
kHz frequencies in offspring (n = 18 OWTD/ n=6 OMTD) as measured with DPOAE test. For analysis, mixed ANOVA followed
by Tukey HSD post hoc test. Values are expressed as marginal mean ± SEM. (***) p < 0.001. PND: post-natal day, OWTD: offspring from water-treated dams, OMTD: offspring
from MMI treated dams, DPOAE: distortion product otoacoustic emission, kHz: kilohertz,
dBSPL: decibel sound pressure.
The 90 dB SPL stimulus were used to analyse ABR absolute latencies and there was no
effect of experimental gestational hypothyroidism on latency and absolute waves (peaks
I, II, III, IV, and V) in offspring. Therefore, the OWTD and OMTD groups had the same
ABR wave latencies. In contrast, there was an effect of experimental gestational hypothyroidism
on the incidence of hearing loss in offspring, once most of the rates in the OMTD
group had a hearing threshold up to 40 dB SPL, which is considered a mild-to-profound
hearing loss ([Figure 4]). Furthermore, ABR stimulus levels (dB SPL) with a significant main effect for the
experimental group were also found [F (1; 2.383) =18.564, p = 0.036].
Fig. 4
Effect of experimental gestational hypothyroidism on incidence of hearing loss in
offspring (n = 18 OWTD/ n=6 OMTD) as measured with ABR test. For analysis, mixed ANOVA was
used. Values are expressed as marginal mean ± SEM. PND: post-natal day, OWTD: offspring
from water-treated dams, OMTD: offspring from MMI treated dams, ABR: auditory brainstem
response, dB SPL: decibel sound pressure.
Discussion
The induction of hypothyroidism in the animals was confirmed by the low body mass
of the rats in the OMTD group, as classically defined in the literature.[12]
[13]
[14] Besides, previous studies from our laboratory had already shown a reduction in the
T3 and T4 serum concentrations in pregnant rats that received similar treatment from
GD 9 until the delivery day.[15]
The function of the middle ear was evaluated, analyzing the tympanic ossicular system.
Although we found significant differences between the values obtained for OMTD at
30 and 60 days postnatal regarding the age-matched control, all the values obtained
were greater than 0.3 mL, thus indicating values of compliance in a normal pattern.
These results converge with previous works indicating the integrity of the tympanic
ossicular system.[16]
[17]
In mice, Cordas et al.[7] showed that the lack of maternal THs is essential for the development of the tympanic
ossicular system. The activation of thyroid receptors TRα1 and TRß is required in
the gestational period, and its absence can delay ossicular maturation, leading to
middle ear dysfunction. These data appear to be inconsistent with our results. However,
the authors used mutant mice devoid of all known TH receptors that exhibited disorders
of the pituitary-thyroid axis, growth, and bone maturation. The complexity of these
animals that are unable to respond to any TH is undoubtedly different from those used
in our study, in which there was a reduction in T3/T4 levels in perfectly responsive
animals. These differences may have been the cause of the differences found in our
animals, which, despite lower compliance, did not present functional loss.
Analyzing the values of the untreated animals (OWTD), in [Figure 2], there is a relationship between compliance and time factor (aging) leading to reduced
compliance. Although we have not studied older ages, these values may continue to
decline and lead to dysfunction of the middle ear. This idea is supported by physiological
changes caused by age as an increase in the stiffness and hardness of the tympanic
membrane due to deterioration of the elastin and collagen fibers of the tympanic membrane.[17] The use of an inbred, albino rat strain, the F344, which develops a progressive
hearing loss (high frequencies evolving to lower frequencies as the animal ages[9]), did not result in changes in compliance measurement associated with aging, although
the data obtained suggested damage to the outer hair cells and a relative hearing
and DPOAE loss. The genetic background of this strain, however, can be an essential
factor in the differences in sensitivity of the auditory system to TH.
Distortion product otoacoustic emission evaluates the outer hair cells of the inner
ear, specifically their electromotility properties.[18] As expected, our data showed the absence of DPOAE response at 2 kHz frequency in
both experimental groups. Evidence shows that this result is due to two main aspects;
first, the biophysical aspects of the rat auditory system, known as micro ear,[19] and, second, the fact that frequencies lower than 3 kHz generate vertical bands
that outer hair cells are unable to respond to.[20] Furthermore, pieces of evidence have been reported showing that this frequency has
little or no importance to a rat's auditory system.[21]
We also found an absence of DPOAE response in OMTD. This finding suggests a cochlear
dysfunction with reduced or absent DPOAE amplitude response. These data corroborate
previous studies and indicate that the reduced or absent DPOAE amplitude response
is due to the decrease of outer hair cell motility. This is a cochlear dysfunction,
which can be associated with hearing loss. Several morphological damages were previously
reported in the hearing organ when hypothyroidism was induced from DG 10 until PND
14.[2] Abnormalities of the cochlear duct, tectorial membrane, and organ of Corti development
were found. The membrane was thicker without hair cells contact. The organ of Corti
had a half-turn around the modiolus and a significant delay in development. Hair cells
exhibited a reduced number of cells with a smaller size, and irregular appearance.
At PND 12, the tectorial membrane was still immature and closely adhering to the internal
spiral sulcus. At PND 15, the distortion over the cochlear duct was visible, and the
tectorial membrane had no contact with hair cells that displayed longitudinal appearance.
Other studies also described the morphological changes of the auditory system in hypothyroidism
as tectorial membrane malformation, decreased endocochlear potential, and delay of
sensorial epithelium differentiation.[22]
[23]
[24]
[25]
Evidence has also shown that hair cells cannot respond to the DPOAE test when morphologically
and functionally altered by hypothyroidism during their development. Indeed, Knipper
et al.[6] demonstrated that outer hair cells are not capable of electrical and mechanical
transduction beyond endocochlear potential and emphasized the importance of THs to
the organ of Corti development. These findings indicate that a thyroid hypofunction
leads to cochlear damage.[6]
[8]
[12]
It is well documented that the longer the induction period of hypothyroidism, the
greater the impact on the auditory function will be.[2]
[6]
[26] However, the analyses of the auditory neural pathways up to the brainstem level
in the central nervous system by ABR revealed the integrity of this via, since no
interaction between the experimental groups and wave latencies, and no latency modification
were found. Our results, nevertheless, did not agree with those in which increased
latency values, mainly for wave I, have been reported when the anti-thyroid drug was
given until PND 10,[27] PND 14[6] and PND 30.[28] The differences found in this study may be due to the gestational induction period
of hypothyroidism, which does not extend to lactation.
A significant and similar decrease in ABR latency in both groups was found during
aging in this study. Studies of Blatchley et al.[29] had already shown the decrease in the amplitude of waves I and II at 8 kHz along
the maturational process. Possible changes in amplitude related to the development
might include synaptic deficiency from cochlear nuclei to the brainstem, as well as
a decrease in the genesis and conduction of electrical potential, which may be implicated
in the results we obtained.
In this study, we used wave II as a parameter to determine the electrical hearing
threshold.[3] Our data suggest mild-to-profound hearing loss, as consensually described in the
literature for congenital hypothyroidism: mild,[30] severe,[28]
[30]
[31] and profound.[22] Hearing loss in hypothyroidism can be explained by the malfunctioning of outer hair
cells, such as abnormality in cortical cytoarchitecture and maturational process,
impacting mechanical and electrical transduction, and endocochlear potential.[32]
[33] Moreover, hypothyroidism led to a TRß receptor impairment, which conducts to a delay
of potassium channel expression that, in turn, affects the endocochlear potential.
Abnormalities of afferent dendrites and the absence of efferent innervation of inner
hair cells have also been described.[34] In addition, outer hair cells malformation may harm inner hair cells. Receptors
α and ß support the mechanical and electrical properties of outer hair cells, so an
inadequate control of this system can compromise the function of inner hair cells.[31] Cochlear failure is also a factor of relevance in the auditory system in hypothyroidism.
As previously reported, OMTD had a preserved neural integrity of the auditory brainstem
with a higher electrophysiological threshold.[8]
[22]
[24] However, a more intense stimulus is necessary to evoke a response in ABR when sound
transduction in cochlea machinery is deficient.[18]
Conclusion
In conclusion, this study suggests that perigestational (gestational/postnatal) exposure
to hypothyroidism may reduce the function of the organ of Corti, followed by hearing
loss in adult rat offspring, although no changes in the middle ear and neural circuitry
up to the brainstem were seen in this study. In summary, the findings reveal lifelong
programming of hearing (dys)function evoked by a maternal thyroid disorder during
the offspring's intrauterine life.
