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Behavioural Brain Research 372 (2019) 112069
Contents lists available at ScienceDirect
Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Hearing loss as a risk factor for cognitive impairment and loss of synapses in
the hippocampus
Munyoung Changa,1, Haeng Jun Kimb,1, Inhee Mook-Jungb,c, Seung-ha Ohc,d,
T
⁎
a
Department of Otorhinolaryngology-Head and Neck Surgery, Chung-Ang University College of Medicine, 102 Heukseok-ro, Dongjak-gu, Seoul, 06973, Republic of Korea
Department of Biomedical Sciences, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea
c
Department of Biochemistry, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea
d
Department of Otolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Hearing loss
Alzheimer’s disease
Dementia
Amyloid-β
Hippocampus
Although epidemiological studies have identified an association between hearing loss and cognitive impairment,
there is a lack of biological evidence detailing the mechanisms underlying this association. The present study
investigated the effects of hearing loss on cognitive impairment using an at-risk model. In this animal model,
amyloid-β (Aβ) was administered to the brain to such an extent that it did not cause cognitive impairments but
made the brain vulnerable to risk factors. This study included four experimental groups based on hearing level
and Aβ administration. Behavioral tests were conducted to evaluate cognitive function, and synaptic protein
levels were measured in the hippocampus and prefrontal cortex. The group with hearing loss and Aβ administration showed significantly greater deficits on cognitive tests associated with the hippocampus than the other
three groups (only Aβ administration, only hearing loss, and without hearing loss or Aβ administration). The
hearing loss and Aβ administration group also had significantly lower levels of synaptic proteins in the hippocampus than the other groups. The present results suggest that hearing loss may act as a risk factor for cognitive
impairment in Alzheimer’s disease. Additionally, the present findings indicate hearing loss may cause hippocampal synapses to be more vulnerable to Aβ-induced damage.
1. Introduction
In 2016, approximately 43.8 million people suffered from dementia
worldwide. Furthermore, the worldwide death rate associated with
dementia was 2.4 million people, which made it the fifth leading cause
of death [1]. The leading cause of dementia is Alzheimer’s disease (AD)
[2] and, therefore, there is an urgent need for the development of
treatments for AD. Although much research has been conducted in this
area, the currently available treatments for AD have yet to achieve
significant clinical efficacy in that they can partially stabilize the
symptoms of this disease but not correct it [3].
It is also important to identify risk factors for AD, as this information
will allow us to develop methods preventing AD development or
slowing disease progression. Age, family history, and heredity are the
most important risk factors of AD [4] and can be used to predict its
occurrence. However, these factors cannot be modified and, thus,
cannot contribute to the prevention of AD. Recent epidemiological
evidence suggests that there is an association between hearing loss and
cognitive impairment [5–8] and other studies have shown that hearing
loss may be a potentially modifiable risk factor of AD [9]. Approximately one-third of elderly people 65 years of age and older have
hearing loss, which can be ameliorated by hearing aids and cochlear
implants. Therefore, if hearing loss is a risk factor of cognitive impairment and its mechanisms can be identified, then the treatment of
hearing loss can contribute to the prevention of AD. However, the
causal relationship between hearing loss and AD remains controversial.
For example, it has been suggested that the association between hearing
loss and AD exists due to difficulties in cognitive function tests that
patients with hearing loss experience due to poor verbal communication. Furthermore, the biological mechanisms that underlie this association have yet to be elucidated.
Thus, the present study employed animal models to investigate
Abbreviations: Aβ, amyloid-β; AD, Alzheimer’s disease; OPT, object-in-place task; OLT, object location task; NOR, novel object recognition task; ABR, auditory
brainstem response; NH-SA, normal hearing-subthreshold amyloid-β; deaf-SA, deaf-subthreshold amyloid-β; NH-NA, normal hearing-non amyloid-β; deaf-NA, deafnon amyloid-β
⁎
Corresponding author.
E-mail address: shaoh@snu.ac.kr (S.-h. Oh).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bbr.2019.112069
Received 1 May 2019; Received in revised form 9 June 2019; Accepted 1 July 2019
Available online 02 July 2019
0166-4328/ © 2019 Published by Elsevier B.V.
Behavioural Brain Research 372 (2019) 112069
M. Chang, et al.
weeks after surgery and the Y-maze test was performed every 2 weeks
in all rats starting at 7 weeks after the surgery. The results of the first
stage were used to determine the timepoints at which hearing loss induced a significant effect on cognitive impairment.
In the second stage, 26 rats were randomly divided into four experimental groups: a normal hearing-non Aβ group (NH-NA; n = 6)
that underwent a sham surgery but not infusion of subthreshold Aβ, a
normal hearing-subthreshold Aβ group (NH-SA; n = 6) that underwent
a sham surgery and the infusion of subthreshold Aβ, a deaf-non Aβ
group (deaf-NA; n = 7) that underwent bilateral cochlear ablation but
not infusion of subthreshold Aβ, and a deaf-subthreshold Aβ group
(deaf-SA; n = 7) that underwent bilateral cochlear ablation and the
infusion of subthreshold Aβ. The infusion of subthreshold Aβ for two
weeks began 9 weeks after surgery and cognitive tests including the Ymaze test, object-in-place task (OPT), object location task (OLT), and
novel object recognition task (NOR), were performed to all rats 11
weeks after surgery. After the cognitive function tests, tissue samples
were harvested from the hippocampus and prefrontal cortex. One animal in the deaf-SA group exhibited postural asymmetry when picked
up after the bilateral cochlear ablation and was excluded from the experiment. During the breeding period, one animal in the NH-SA group
and one animal in the deaf-SA group died. Ultimately, the NH-NA, NHSA, deaf-NA, and deaf-SA groups consisted of 6, 5, 7, and 5 animals,
respectively. We performed an additional experiment using another
nine rats to assess whether the animals preferred familiar or novel
objects in the NOR.
whether hearing loss would be a risk factor for AD and to assess the
mechanisms by which hearing loss may act as a risk factor. Because
several empirical cases and other evidence indicates that hearing loss
alone does not lead to cognitive impairment [10], a subthreshold
amyloid-β (Aβ) model of AD [11] was used in the present study. In this
model, Aβ is administered to the brain to such an extent that it does not
cause cognitive impairments but makes the brain vulnerable to risk
factors so that it might be possible to verify whether hearing loss would
be a risk factor for cognitive impairment.
2. Methods
2.1. Experimental design
This study was approved by the Institutional Animal Care and Use
Committee of Chung-Ang University (2016-00086) and Seoul National
University Hospital (16-0133-C1A0) and all experiments were conducted in accordance with relevant guidelines and regulations. Sevenweek-old male Wistar rats (200–250 g) were used and all animals were
adapted to laboratory conditions for 1 week prior to the start of the
experiment and housed in a temperature- and humidity-controlled
room with a 12 -h light:dark cycle with food and water available ad
libitum. Auditory brainstem response (ABR) recordings and surgical
procedures were performed under anesthesia induced by the intraperitoneal administration of ketamine hydrochloride (100 mg/kg;
Ketamine®, Yuhan Co.; Seoul, Korea) mixed with xylazine (10 mg/kg;
Rompun®, Bayer-Korea; Seoul, Korea).
The present study consisted of two stages: determining the time
course of cognitive decline following hearing loss and then evaluating
changes in cognitive function and synaptic protein levels after induction of the hearing loss (Fig. 1). In the first stage, 10 rats were randomly
divided into two groups: a pilot-normal hearing-subthreshold Aβ group
(pilot-NH-SA; n = 5) that underwent a sham surgery and the infusion of
subthreshold Aβ and a pilot-deaf-subthreshold Aβ group (pilot-deaf-SA;
n = 5) that underwent bilateral cochlear ablation and infusion of subthreshold Aβ. The infusion of subthreshold Aβ for 2 weeks began 3
2.2. ABR recordings
ABR recordings were conducted in all rats before surgery and 1
week, 6 weeks, and 11 weeks after surgery to measure hearing levels.
ABRs on the left side were recorded with subdermal needle electrodes
between the left mastoid and the nape of the neck with the right
mastoid as the return while ABRs on the right side were measured by
reversing the direction of the electrodes. ABRs were recorded with highfrequency transducers (HFT9911–20–0035) and software (ver. 2.33)
Fig. 1. Experimental flow of the first (a) and second (b) stage.
Aβ, amyloid-β; NH-SA, normal hearing-subthreshold amyloid-β; deaf-SA, deaf-subthreshold amyloid-β; NH-NA, normal hearing-non amyloid-β; deaf-NA, deaf-non
amyloid-β.
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M. Chang, et al.
2.6.2. OPT, OLT, and NOR
The OPT, OLT, and NOR were conducted by modifying a previously
reported method [16]. Beginning 4 days before the tests, the rats were
placed in an open field box (58 × 42 × 35 cm) without stimuli for
10–15 min daily. Each session consisted of familiarization and test
phases and either the type or location of the stimulus objects in the test
phase was different from that in the familiarization phase. In the familiarization phase, the rats explored stimulus objects in the open field
box for 5 min and were then returned to their home cage for a fixed
amount of time (5 min for OPT and OLT and 3 h for NOR). Then, the
rats were placed in the box again and allowed to explore the stimulus
objects during the test phase. The experiment was video recorded in a
room without the experimenter and the recorded video was analyzed
later. Exploratory behavior was defined as directing the nose toward an
object at a distance of less than 2 cm or touching the object with the
nose or paws. A discrimination ratio was calculated as follows: (exploration time with the changed object – exploration time with the
unchanged object) / (total exploration time with the changed and unchanged object). When exploration time was shorter than 15 s during
the familiarization phase or shorter than 10 s in the test phase, the data
were excluded from the analysis.
The test conditions are shown in Fig. S1. For the OPT familiarization
phase, four different stimulus objects were placed in the corners of the
box (10 cm from the wall). During the OPT test phase, the positions of
two of the objects (which were both on the left or right of the box) were
switched. For the OLT familiarization phase, two identical objects were
placed in the corners of the box. During the OLT test phase, one object
was repositioned to the corner adjacent to its original position; thus, the
two objects were diagonal to each other. For the NOR familiarization
phase, two identical objects were placed in the corners of the box.
During the NOR test phase, one object was changed to a novel object.
Before the NOR, we performed an additional experiment to assess object bias. After adaptation to the open field box, another nine rats explored the two objects (a familiar and a novel object) to be used in the
NOR test phase for 5 min. The durations of time spent exploring each
object were measured and compared.
from SmartEP (Intelligent Hearing Systems; Glenvar Heights, FL, USA)
and the responses were amplified (100,000×), band pass-filtered
(100–1500 Hz), and averaged over 512 stimulus repetitions. Tone pips
of 8, 16, and 32 kHz were used as sound stimuli (5-ms duration, cos
shaping, 21 Hz) and stimulus intensity was reduced in 5 dB SPL decrements. Two researchers, blind to the experimental conditions, determined the lowest stimulus intensity that evoked a recognizable response, and that was regarded as the threshold.
2.3. Cochlear ablation
Cochlear ablation was performed on both sides as previously described [12]. Briefly, after a retroauricular incision, the external auditory canal was opened and the tympanic membrane and ossicles, except
for the stapes, were removed. Then, a small hole was made on the bony
wall of the cochlea and the contents of the cochlea were ablated with a
dental pick. A small amount of soft tissue was packed into the small
hole on the bony wall of the cochlea. In the sham surgery, the same
operative procedure was performed before the point of opening the
external auditory canal.
2.4. Behavioral tests for vestibular deficits
To exclude the effects of vestibular function deterioration during
cochlear ablation, the behavioral test for vestibular deficits was performed the day and week after surgery as previously described [13].
Briefly, the behavioral scoring for vestibular deficits consisted of three
components: postural asymmetry, head roll tilt, and nystagmus (Table
S1). If any deficits were found in any of these three components, the
animal was excluded from the experiment.
2.5. Infusion of subthreshold Aβ
The Aβ peptide solution was continuously administered into the
intracerebroventricular space (160 pmol/day) for 2 weeks using a brain
infusion cannula (Brain Infusion Kit 2, Alzet; Cupertino, CA, USA) that
was connected to a mini-osmotic pump (Osmotic Pump 2002, Alzet).
The infusion cannula was implanted into the right cerebral lateral
ventricle (AP: −0.3, L: 1.2, V: 4.5) according to the coordinates of
Paxinos and Watson (2006) [14]. The composition of the Aβ peptide
solution, which does not induce cognitive impairment, has been described previously [11]. Briefly, a Aβ1-42 peptide solution (AnaSpec
Inc.; San Jose, CA, USA) was dissolved in 35% acetonitrile/0.1% trifluoroacetic acid. The mini-osmotic pump was removed 2 weeks after
implantation, and the remaining volume of Aβ1-42 peptide solution
measured to confirm that the expected volume had been delivered; we
subtracted the residual from the initial volume.
2.7. Western blot analysis
After completion of the behavioral tests, all animals were euthanized and brain tissue samples were harvested from the hippocampus and prefrontal cortex based on the coordinates of Paxinos and
Watson (2006) [14]. For the Western blot analyses, tissues from the
hippocampus and prefrontal cortex of all groups were lysed in a
radioimmunoprecipitation assay buffer (RIPA) buffer (iNtRON Biotechnology; Seoul, Korea) containing a protease inhibitor cocktail
(Sigma; St. Louis, MO, USA), protein phosphatase inhibitor cocktail (AG
Scientific; San Diego, CA, USA), and phenyl-methylsulfonyl fluoride
(PMSF; Sigma). Then, the brain lysates were sonicated to ensure thorough lysis. The concentrations of the protein lysates were determined
with a BCA assay and an identical amount of protein from each sample
was electrophoretically separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in 4–12% Bis–Tris gels and
then transferred to polyvinylidene difluoride (PVDF) membranes. The
membranes were blocked in 5% non-fat dry milk in Tris-buffered saline
(TBS) and 0.1% Tween-20 (TBS-T) and then incubated with the following primary antibodies at 4℃ overnight: postsynaptic density protein 95 (PSD95; ab18258, Abcam; Cambridge, UK), synaptophysin
(mab268, Millipore; Burlington, MA, USA), Ca2+/calmodulin-dependent protein kinase II (CaMKII; ab52476, Abcam), phosphorylated
CAMKII (pCaMKII; 3361 s, Cell Signaling Technology; Danvers, MA,
USA), N-methyl D-aspartate receptor subtype 2B (NR2B; 06–600, Millipore), and α-tubulin (05–829, Millipore). Next, the membranes were
washed with TBS-T for 30 min and incubated with secondary IgG-HP
antibodies against each primary antibody for 1 h. Then, the membranes
were washed with TBS-T and incubated with an ECL chemiluminescent
2.6. Cognitive testing
2.6.1. Y-maze test
Cognitive function was assessed by recording spontaneous alternation behavior in a single session in the Y-maze; the protocol for this task
has been previously reported [15]. Briefly, each arm of the maze was
40 cm long, 30 cm high, and 15 cm wide and converged in a central
triangle area. None of the animals had ever experienced a Y-maze before. All arms were brushed with 10% ethanol prior to each session to
remove the possible effects of odor cues and the experimenter was not
in the room during testing. Each rat was placed on one arm tip of the Ymaze and then allowed to walk around the maze for 7 min without
restriction. Each session in the Y-maze was video recorded and analyzed
later. The rat was considered to have entered the arm when its hind
paws entered the arm and alternation was defined as successive entries
into three arms based on overlapping triplets. The alternation percentage was calculated as follows: actual alternations / possible alternations (total number of arm entries minus two).
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Behavioural Brain Research 372 (2019) 112069
M. Chang, et al.
reagent. Finally, peroxidase activity was detected with LAS 4000 (GE
Healthcare Life Science; Marlborough, MA, USA); the optical densities
were normalized with a standard protein.
2.8. Statistical analyses
IBM SPSS software version 21.0 (IBM; New York, NY, USA) was
used for all statistical analyses. ABR thresholds were analyzed with oneway analysis of variance (ANOVA) tests. Scores on the cognitive tests in
the first stage of the experiment were analyzed with repeated measures
ANOVA tests and scores at each timepoint were analyzed with unpaired
two-tailed Student’s t-test. Scores on the cognitive tests in the second
stage of the experiment were analyzed with one-way ANOVAs and
paired t-tests. The results of the Western blot analyses were analyzed
with one-way ANOVAs. All post hoc testing was performed using
Tukey’s tests.
3. Results
3.1. ABR recordings
Prior to surgery, the baseline ABR thresholds at 8, 16, and 32 kHz
ranged from 20 to 35 dB SPL in all animals; these values did not differ
significantly among the groups (p > 0.05). At 1 week, 6 weeks, and 11
weeks after surgery, the ABR thresholds at 8, 16, and 32 kHz ranged
from 20 to 35 dB SPL in the NH group but were higher than 80 dB SPL
in the deaf group (Fig. 2).
3.3. Time course of cognitive decline following hearing loss
Fig. 3. Cognitive test results. (a) Time course of cognitive decline following
hearing loss. Y-maze scores were significantly lower in the pilot-deaf-SA group
compared to the pilot-NH-SA group at 11 weeks after surgery. (b) In the Ymaze, OPT, and OLT tests, the deaf-SA group had significantly lower scores than
the other three groups in the second stage of the experiment. All data are
presented as a mean ± SEM. (a) Unpaired two-tailed Student’s t-test at each
timepoint. (b, c) One-way ANOVA followed by Tukey’s post-hoc test.
*P < 0.05, **P < 0.01, ***P < 0.001. SA, sub-amyloid-β; NH, normal hearing; OPT, object-in-place task; OLT, object location task; NA, non-amyloid-β. In the first stage of the experiment, the time course of cognitive decline following hearing loss was evaluated using the results of the Ymaze test (Fig. 3a). The influence of hearing loss was explored with a repeated measures ANOVA using the Y-maze scores across time as a repeated measure (7, 9, and 11 weeks after surgery) and the groups as fixed factors. Mauchly’s test of sphericity indicated that the assumption 3.2. Dose of Aβ1-42 peptide solution delivered The daily volumes of delivered Aβ1-42 peptide solution ranged from 12.0 to 11.9 μL, corresponding to 161.0 to 159.0 pmoL/day of the Aβ142 peptide, similar to the anticipated volumes. The daily doses did not differ significantly between the groups (p > 0.05).
Fig. 2. ABR thresholds before surgery and 1 week, 6 weeks, and 11 weeks after surgery. (a) pilot-NH-SA group. (b) pilot-deaf-SA group. (c) NH-NA group. (d) NH-SA
group. (e) deaf-NA group. (f) deaf-SA group. Error bars indicate standard deviation.
ABR, auditory brainstem response; SPL, sound pressure level; NH, normal hearing; SA, sub-amyloid-β; NA, non-amyloid-β.
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Behavioural Brain Research 372 (2019) 112069
M. Chang, et al.
hearing loss would act as a risk factor for AD and to identify the mechanisms underlying this association.
When planning the present experiments, it was important to consider that cognitive dysfunction will not be induced when only hearing
loss is present. The results of a follow-up study investigating cognitive
function in subjects who developed hearing loss in childhood reported
that long-term sensory impairment alone has a negligible effect on one’s
overall level of cognitive function [10]. Therefore, the present study
compared cognitive function in animals with hearing loss and normal
hearing using a model of subthreshold Aβ, which has been published
under the name of the at-risk model [11]. This model is intended to
represent individuals with a predisposition for Aβ buildup but normal
cognitive function. Thus, it is possible to investigate whether certain
factors may be risk factors of AD.
In the present study, four experimental groups based on hearing
level and the subthreshold administration of Aβ were formed and
cognitive tests known to be related to specific brain regions were conducted. Cognitive tests associated with the hippocampus, such as the Ymaze, OPT, and OLT [16], revealed significant decreases in cognitive
function in the deaf-SA group after hearing loss, as compared to the
other groups. However, there were no significant differences among the
groups in the NOR. The hippocampus may affect NOR results when the
time between the familiarization and test phases is extended [19,20].
However, others have reported that the hippocampus does not influence NOR results regardless of the time interval between the two phases
[16,21–24]. The discrepancies may be attributable to differences in the
methods used to eliminate hippocampal function and the experimental
conditions under which NOR was performed. A study that evaluated
NOR exactly as we did reported that the hippocampus did not affect the
results [16]. Therefore, in our experiment, the hippocampus may not
affect NOR results. Taken together, these results suggest that hearing
loss affected the hippocampus and may be a risk factor for cognitive
impairment.
Comparisons of synaptic protein levels in the hippocampus between
the NH-NA and NH-SA groups revealed no significant differences. These
results indicate that the subthreshold administration of Aβ did not affect synaptic protein levels in the hippocampus in normal hearing animals. The changes in synaptic protein levels in the hippocampus after
hearing loss mirrored the results of the cognitive testing: the deaf-SA
group exhibited a significant decrease in synaptic proteins compared to
the other three groups. These data indicate that cognitive impairment
may be accelerated by the synergistic effects of hearing loss and Aβ due
to synaptic loss. In the case of prefrontal cortical synaptic protein levels,
some proteins in the deaf-SA group exhibited a reduction but these
changes were not consistent and were not likely to be affected by
hearing loss.
The present study demonstrated that hearing loss might act as a risk
factor for cognitive impairment in AD patients and that hearing loss
may cause hippocampal synapses to be more vulnerable to brain pathology. This finding indicates that there are connections between the
central auditory pathway and the hippocampus, which has been proposed in previous studies. For example, there are changes in the hippocampus following sound exposure [25–29] and the use of anterograde tracers revealed that the hippocampus receives signals from the
auditory cortex via the entorhinal cortex [30]. Therefore, degeneration
in the central auditory pathway induced by hearing loss [31,32] may
cause the degeneration of hippocampal synapses or make these synapses more vulnerable to damage. This hypothesis is supported by
findings showing that focal cortical infarction of brain regions that are
remote but connected to the hippocampus induce neuronal loss in the
hippocampus [33]. Further studies are needed to obtain solid conclusions.
The present study has several limitations that should be noted. First,
the development of hearing loss and Aβ deposition in the animal
models used in this study differ from those in actual humans. In most
humans, hearing loss and Aβ deposition progress slowly and, therefore,
of sphericity for time had been violated (p = 0.453) and, therefore, the
results for time are reported using the Greenhouse-Geisser correction
(ε = 0.832). The Y-maze scores changed over time (p = 0.046) and
there was a significant interaction between time and group (p = 0.032);
thus, the main effects for group are reported at each timepoint. The Ymaze scores of the pilot-NH-SA and pilot-deaf-SA groups did not significantly differ at 7 or 9 weeks after surgery (p = 0.624 and p = 0.208,
respectively) but the Y-maze scores of the pilot-deaf-SA group were
significantly lower than those of the pilot-NH-SA group at 11 weeks
after surgery (p = 0.014).
3.4. Cognitive function and synaptic maker protein levels after hearing loss
The cognitive testing results in the second stage of the experiment
are displayed in Fig. 3b and Table S2 and S3. The time spent by the
animals in exploration exceeded 15 s during the familiarization phases
and 10 s during the test phases of the OPT, OLT, and NOR. No animal
was excluded from the analysis. The total time spent exploring objects
during the familiarization and test phases of the OPT, OLT, and NOR
did not differ among the groups (Table S2). During the familiarization
phases of the OPT and OLT, no significant differences in the time spent
exploring objects that were switched and those not switched during the
test phases were apparent (Table S3). This was also the case for the
additional experiment of the NOR (21.8 ± 3.3 and 21.6 ± 4.0 s respectively, p = 0.852, paired t-test).
In the Y-maze, OPT, and OLT tests, the deaf-SA group had significantly lower scores than the other three groups (p < 0.05, Fig. 3b). There were no significant differences among the other three groups on those three tests and no significant differences among all four groups in the NOR test. The present study also investigated molecular changes in the hippocampus and prefrontal cortex of all groups by quantifying synaptic protein levels with Western blot analyses. In the hippocampus, there were significant decreases in NR2B and PSD95, which are post-synaptic markers, and synaptophysin, which is a pre-synaptic marker, levels in the deaf-SA group (Figs. 4a–d and S2) but no significant changes in the other three groups. Additionally, there were no significant changes in the phosphorylation levels of CaMKII (Fig. 4a and e). In the prefrontal cortex, PSD95 levels significantly decreased in the deaf-SA group compared to the NH-NA and deaf-NA groups (Fig. 4f and i). Synaptophysin levels significantly decreased in the NH-SA and deaf-SA groups compared to the NH-NA and deaf-NA group showed decreasing trends (Fig. 4f and h). The phosphorylation levels of CaMKII decreased in all other groups compared to the NH-NA group (Fig. 4f and j). NR2B levels in the prefrontal cortex did not significantly differ among the groups (Fig. 4f and g). 4. Discussion Although several epidemiological studies have suggested that hearing loss is a risk factor for cognitive decline [6–8,17], the underlying mechanisms remain unclear. Three representative hypotheses have been presented; they involve the effects of hearing impairments on cognitive load and brain structure and decreased social engagement [18]. The cognitive load hypothesis suggests that auditory perceptual processing requires more cognitive resources when the auditory signal is degraded, which results in the degradation of other cognitive processes, such as working memory. Another hypothesis proposes that impaired auditory signals and reduced stimulation from an impaired cochlea cause changes in brain structure. This would make the brain more vulnerable to brain pathology-causing factors, such as Aβ accumulation, neurofibrillary tangles, and microvascular disease, and lead to an increased risk of dementia. The third hypothesis suggests that cognitive function is degraded by social isolation due to hearing loss. However, few studies have provided evidence supporting these hypotheses. Thus, the present study attempted to determine whether 5 Behavioural Brain Research 372 (2019) 112069 M. Chang, et al. Fig. 4. Synaptic marker proteins are altered by hearing loss and Aβ infusion in the rat brain. Pre- and post-synaptic marker protein levels in the hippocampus decreased following hearing loss and Aβ infusion. (a) Representative images and (b–e) quantificational graphs (n = 5–7). Some pre- and post-synaptic marker protein levels in the prefrontal cortex decreased following hearing loss and Aβ infusion. (f) Representative images and (g–j) quantificational graphs (n = 5–7). All data are presented as a mean ± SEM. One-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. NA, non-amyloid-β; SA, sub-amyloid-β; Aβ, amyloid-β. it will be necessary to develop a novel animal model in which hearing loss and Aβ deposition progress in a manner similar to that of humans. Second, the present study showed that there was a decrease in hippocampal synapses following hearing loss. However, the locations and roles of the degenerated synapses could not be identified and further research will be necessary to clarify these findings. (2019) 88–106. [2] C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones, Alzheimer’s disease, Lancet 377 (2011) 1019–1031. [3] J. Cao, J. Hou, J. Ping, D. 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Bussey, Double dissociation between the effects of peri-postrhinal cortex and hippocampal lesions on tests of object recognition and spatial memory: heterogeneity of function within the 7 This article discusses how hearing loss opens up an opportunity for risks on cognitive disfunction and synapses loss in the hippocampus. From this research, it is clear that the mechanism behind suggesting risk factor that are posed to the loss of hearing for cognitive remains unclear (Chang et al., 2019). This study draws the three representative hypotheses that involve hearing disfunctions effects on brain structure, cognitive load and the effects of the hearing impairments on decreased social engagement. Each hypothesis here represents a different meaning and implication in this study, as explained in the Article (Chang et al., 2019). One critical point to note from these hypotheses is social isolation degrades cognitive functions when is there is loss of hearing. According to the study in this article, a significant decrease arises in the cognitive function after hearing loss for the deaf SA group, but the same is not true for the other group. The study also suggests that loss of hearing affects the hippocampus risks it against cognitive dysfunctions. Synergistic impacts due to loss of hearing have a high capability of increasing or rather speeding up the cognitive impairment. Hearing loss might make the hippocampal to be more vulnerable to pathology of the brain. Hearing loss might also cause lots of risks onto cognitive disfunctions in AD patients. Loss of Hearing induces degeneration in the central auditory pathway. This might make the hippocampus synapses more vulnerable to damage. There are various limitations that are highlighted in the current study (Chang et al., 2019). The main novelty of this article is to find out if the loss of hearing produces high vulnerability to risk for synapses loss in the hippocampus as well as cognitive disfunction (Chang et al., 2019). It seeks to identify the various impact that accompany the loss of hearing on disfunctions of cognitive parts utilizing model called an at-risk model. It is a model that involves a case where a system is represented in a mathematical format, typically including probability distributions (Silva et al., 2020). These models use the most relevant historical data and expert identification from people mentioned in the topic in question to understand the risk probability event as well as its probable severity. The results of this novelty are because of the most appropriate methodology applied in this research. The methods include the use of experimental design, whereby all the experiments were conducted per the relevant guidelines and regulations. Other methods include recordings of the ABR done in all the study rats before surgery as well as one week, six weeks, including eleven weeks after surgery to get the measurements on the level of hearing(Chang et al., 2019). Other methods that led to great results of this novelty include cochlear ablation, behavioral test for infusion of subthreshold, western blot analysis, cognitive testing, vestibular deficits, and statistical analyses. This study also noted that according to several epidemiological studies, the loss of hearing possesses a larger possibility of risk for cognitive disfunction (Chang et al., 2019). It was also known that a prolonged sensory disfunction alone has an overall negligible impact on someone's cognitive function. The hippocampus may impact the outcomes of NOR when there is an extension between familiarization and test phases (Chang et al., 2019). Therefore, the results expand on previous knowledge by conducting the various hypothesis to guide the research and provide valid responses (Chang et al., 2019). The results also support the previous knowledge by comparing what was there before and what has been done in the present study and extending or filling the gap where necessary. The results are an addition to the current field because they validate whether the loss of hearing has various impacts on the cognitive impairment, including the synapses loss in the hippocampus. Therefore, this provides clarity on the information that was not initially available. It is because up to the time of this research, few people had an idea on whether loss of hearing can be a factor of risk for cognitive disfunction. The outstanding questions include the suggestion that is it possible to avoid hearing loss totally? Is there a mechanism that has been put to educate the public on the severe effects of hearing loss? This is so that they live a well-defined life to avoid getting themselves into such scenarios. Is this research good enough to prove that loss of hearing opens up opportunity for lots of risk towards cognitive impairment? Do people have an understanding of what cognitive impairment is, hippocampus? The missing information that needs to be discussed is the various ways to curb these effects and the risk factors. The research also misses information on the other effects of hearing loss (Presacco, 2019). References Presacco, A., Simon, J. Z., & Anderson, S. (2019). Speech-in-noise representation in the aging midbrain and cortex: Effects of hearing loss. PloS one, 14(3), e0213899. Chang, M., Kim, H. J., Mook-Jung, I., & Oh, S. H. (2019). Hearing loss as a risk factor for cognitive impairment and loss of synapses in the hippocampus. Behavioural brain research, 372, 112069. Silva, V., Amo-Oduro, D., Calderon, A., Costa, C., Dabbeek, J., Despotaki, V., ... & Pittore, M. (2020). Development of a global seismic risk model. Earthquake Spectra, 36(1_suppl), 372-394. Purchase answer to see full attachment

  
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