Original Research—Otology and Neurotology
The Astroglial Reaction along the Mouse
Cochlear Nerve following Inner Ear
Damage
Otolaryngology–
Head and Neck Surgery
2014, Vol 150(1) 121–125
Ó American Academy of
Otolaryngology—Head and Neck
Surgery Foundation 2013
Reprints and permission:
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DOI: 10.1177/0194599813512097
http://otojournal.org
Zhengqing Hu, MD, PhD1*, Baofu Zhang, MS1*,
Xuemei Luo, MS, MD1,2*, Lei Zhang, MD1, Jue Wang, MD1,
Dennis Bojrab II, MD1, and Hui Jiang, MS, MD1,3
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Abstract
Objective. Determine how the astroglial cells of the peripheral and central nervous system transitional zone (PCTZ)
react to sensorineural hearing loss using a mouse cochlear
nerve model.
Study Design. Prospective, basic science.
Setting. Research laboratory.
Subjects and Methods. Neomycin was injected into the
mouse inner ear to cause chemically induced hearing loss.
Auditory brainstem responses (ABRs) were used to determine hearing threshold shifts after neomycin treatment.
Immunofluorescence was used to detect the expression of
proteins specific for hair cells, spiral ganglion neurons, astrocytes, and the myelin components of both oligodendrocytes
and Schwann cells.
Results. ABR threshold shifts and immunofluorescence results
supported that hair cells and spiral ganglion neurons were
damaged in neomycin-treated mice. Immunofluorescence
showed the peripheral and central nervous system (PNS and
CNS) transitional zone of the cochlear nerve at the interface
of the myelin components of the PNS and CNS. In the control mice, the expression of glial fibrillary acidic protein
(GFAP) was observed proximally to the PCTZ closer to the
CNS, which is their normal location. However, in neomycintreated animals the expression of GFAP was detected distally
to the PCTZ and was found close to the spiral lamina level in
the basal cochlear turn, suggesting that GFAP-expressing
astrocytes migrated across the PCTZ and reached the PNS.
Conclusion. The GFAP positive astrocyte processes extended
across the PCTZ along the mouse cochlear nerve following
chemically induced sensorineural hearing loss.
Keywords
astrocyte, astroglial reaction, cochlear nerve, hearing loss,
inner ear damage, transition zone
Received July 22, 2013; revised September 16, 2013; accepted October
18, 2013.
Introduction
In vertebrates the peripheral and central nervous systems
(PNS and CNS) connect at specific zones where motor
axons exit and sensory axons enter the CNS. These zones
are made up of cell boundaries and delineate territories with
different glial components. The main glial components in
the CNS are astrocytes and oligodendrocytes, while the corresponding cells in the PNS are Schwann cells. The boundary between the PNS and CNS is characterized by a tight
junction between the Schwann cells and oligodendrocytes.1
This area is called the PNS and CNS transition zone
(PCTZ).2
The peripheral and central glial cells close to the PCTZ
usually respond to damage to the nervous system. It has
been reported that Schwann cells can cross this zone in both
rodent models with demyelinating spinal cord lesions and in
humans with neurologic conditions such as spinal cord
injury or multiple sclerosis.3-5 Astrocytes can also respond
to insults of the nervous system. This is called the astroglial
reaction and causes an inhibitory microenvironment that
impedes regrowth of the axons.2,6-8 The extreme case of the
astroglial reaction is scar formation that inhibits axon regeneration. Nonetheless, accumulating evidence suggests that
the astroglial reactions are finely gradated changes that
range from subtle alterations in gene expression to scar
1
Department of Otolaryngology-HNS, Wayne State University School of
Medicine, Detroit, Michigan, USA
2
Department of Otolaryngology, Fudan University Zhongshan Hospital,
Shanghai, China
3
Department of Otolaryngology, Fudan University Jinshan Hospital,
Shanghai, China
*
These authors contributed equally to this article.
Corresponding Author:
Zhengqing Hu, MD, PhD, Department of Otolaryngology-HNS, Wayne
State University School of Medicine, 550 E Canfield St, 258 Lande, Detroit,
MI 48201, USA.
Email: [email protected]
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Otolaryngology–Head and Neck Surgery 150(1)
formation. These changes can exert both beneficial and detrimental effects in a context-dependent manner.9,10
The cochlear nerve is critical for normal hearing function
because it carries auditory signals from the inner ear to the
cochlear nucleus located in the brainstem. Additionally,
the cochlear nerve presents an exceptionally long segment
of central nervous tissue extending peripherally into the
PNS. The PCTZ is located within the internal acoustic
meatus.11,12 Many problems can affect the cochlear nerve,
and some examples can be found in sensorineural hearing
loss, acoustic neuroma, and traumatic head injuries that
involve the cochlear nerve. It was reported that mechanical
stress to the cochlear nerve caused a substantial outgrowth
of astrocytic processes from the transitional zone into the
peripheral portion of the cochlear nerve, which led to an
invasion of dense gliotic tissue in the cochlear nerve.13 In
case of sensorineural hearing loss, insults to the auditory
system usually damage hair cells and spiral ganglion neurons.14,15 However, how the astroglial cells of the PCTZ
react to sensorineural hearing loss has not yet been
reported. In this study, we generated sensorineural hearing
loss using an aminoglycoside in order to determine how
the astroglial cells along the mouse cochlear nerve react to
sensorineural hearing loss.
mean of 1024 samples of 7.7 ms electrophysiological activity were recorded after stimulation. Stimuli were provided
at various intensities to determine the threshold, which was
defined as the lowest stimulus intensity that evoked at least
a 0.2 mV replicable waveform between the negative wave II
and positive wave III (N2-P3).16
Neomycin was used to generate an inner ear damage
model.14,15 A postauricular approach was used to expose the
bulla of the mice. A custom-made needle was used to open
the bulla and gain access to the cochlea of the mouse. The
round window was identified and a 30 gauge needle was
used to penetrate the round window membrane. One ml of 1
mM neomycin (Sigma, St Louis, Missouri) was slowly
injected into the scala tympani for at least 2 min using a
microsyringe (Hamilton, Reno, Nevada). In the control
group, 1 ml of artificial perilymph (in mM: NaCl,137; KCl,
2.8; CaCl2, 1.5; NaH2PO4, 8.0; MgCl2, 1.0; KH2PO4, 4.7;
glucose, 11.0; pH 7.4 )16,17 was gently injected into the
scala tympani. After the injection, a subcutaneous fascia
was used to cover the round window and the wound was
sutured. The animals were monitored for vital signs and
signs of head tilt, abnormal gait, and infection for 2 to 4
hours postoperatively and daily afterwards. The sutures
were removed approximately 7 days after the surgery.
Materials and Methods
Animals and Groups
Histology
The animal study was approved by local Institutional
Animal Care and Use Committee (IACUC; registration
number: A04-04-09; 2009-05-21). Adult Swiss Webster
mice (2-3 months old) were used in this study. The mice
were paired by gender, age, and weight. They were then
randomly assigned to the control (treated with artificial perilymph) or neomycin groups. Therefore, 5 replicates were
included in each group (n = 5). Both the control animals
and the neomycin-treated animals were followed for 3 and 6
weeks.
Auditory Function Measurement and Inner Ear
Injection
Ketamine (80-100 mg/kg/ip) and xylazine (5-10 mg/kg/ip)
were used to anesthetize the mice for both the auditory
brainstem response (ABR) measurement and the inner ear
injection. All animals received ABR evaluation prior to
inner ear injection to assure the baseline hearing. The hearing of the mice was followed 3 and 6 weeks after neomycin
or artificial perilymph treatment.
The Tucker-Davis system was used to determine the
ABR threshold in response to click stimulation.16 Responses
were recorded with subdermal recording needle electrodes
placed at the vertex. The reference electrode and the ground
electrode were placed at the middle of the skull ~1 cm anterior to bregma and in the left thigh, respectively. In the
soundproof cabinet, computer-generated alternating polarity
voltage pulses (160 ms duration, 50 pps) were delivered to a
transducer positioned at the opening of the ear canal. A
At the end of the follow-up periods, the mice were euthanized with an overdose of pentobarbital. The cochlea together
with the cochlear nerve and the cochlear nucleus were dissected out and fixed in 4% paraformaldehyde at 4ºC overnight. The specimens were treated with 0.1 M EDTA for 6 to
7 days. Following decalcification, samples were embedded in
cryosection gel (Fisher Scientific, Hampton, New Hampshire)
and cryosectioned on a cryostat (Leica, Wetzlar, Germany).
Serial mid-modiolar sections (10 mm thickness) were collected for immunofluorescence analyses.
Immunofluorescence
Multiple-labeling immunofluorescence was used in this
study. The most middle modiolar sections were treated with
5% donkey serum in PBS containing 0.2%Triton X-100 for
30 minutes. Anti-myosin VIIa (1:200; Developmental Studies
Hybridoma Bank) and anti-b tubulin type III (TUJ1, 1:1000;
Covance) antibodies were used to label hair cells and spiral
ganglion neurons, respectively. Anti-GFAP antibodies (glial
fibrillary acidic protein, 1:100), anti-MOG antibodies (myelin
oligodendrocyte glycoprotein, 1:100), and anti-MPZ antibodies (myelin protein-zero, 1:100; all from Covance,
Princeton, New Jersey) were used as the primary antibody
for astrocytes, oligodendrocytes, and Schwann cells, respectively. DyLight-488, 549, or 649 conjugated antibodies
(1:500; Jackson Immunoresearch, West Grove, Pennsylvania)
were applied as secondary antibodies. The samples were
mounted in Anti-fade mounting medium (Invitrogen,
Carlsbad, California). A confocal microscope (Leica) and an
epifluorescence microscope system equipped with appropriate
filters (Leica) were used for observation.
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Hu et al
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Table 1. The auditory brainstem response thresholds of the mice before and after the inner ear injection.
Control group
Neomycin group
Average
Standard deviation
Average
Standard deviation
Before treatment (dB SPL)
3 weeks after treatment (dB SPL)
6 weeks after treatment (dB SPL)
10a
4
13a
5
10b
4
64b
7
17b
5
72b
10
a
There is no significant difference between the control and neomycin groups before treatment (P . .05, paired t test).
There is significant difference between the control and neomycin groups at 3 and 6 weeks after treatment (P \.01; paired t test).
b
Figure 1. Auditory brainstem responses show hearing thresholds
in the control and neomycin-treated mice. Error bars indicate the
standard deviation.
Statistical Analysis
In this study, the mice were paired by gender, age, and
weight, and they were randomly assigned to the control
group or neomycin group. A paired t test was used to analyze the ABR threshold of both the control and neomycintreated mice at baseline and at 3 and 6 weeks after treatment. In this study, statistical significance was defined as
P \ .05.
Results
All animals survived the auditory brainstem response measurements and inner ear injections. Two animals had a transient head tilt that recovered in approximately 2 to 4 days
after the neomycin treatment. Before the inner ear injection,
there was no significant difference between the ABR thresholds of the control and neomycin groups (P . .05, paired t
test) (Table 1). In the control group the ABR threshold
shifts were less than 10 dB SPL at both 3 and 6 weeks after
treatment. On the other hand, the ABR thresholds in the
neomycin-treated animals were 64 6 7 dB SPL at 3 weeks
and 70 dB SPL at 6 weeks after treatment (Figure 1). At
3 and 6 weeks after neomycin injection, statistical analysis
showed significant difference between the ABR thresholds
of the control and neomycin groups (P \ .01, paired t test)
(Table 1).
Six weeks after injection with artificial perilymph, the
mid-modiolar cryosections were stained with anti-myosin
VIIa and anti-TUJ1 (neuron-specific class III beta-tubulin)
antibodies. The immunofluorescence indicated that hair
cells were labeled with anti-myosin VIIa antibodies (arrowhead in Figure 2A), while the spiral ganglion neurons were
stained with anti-TUJ1 antibodies (arrow in Figure 2A). At
both 3 and 6 weeks after neomycin treatment, the cryosections were labeled with anti-myosin VIIa antibodies.
Immunoreactivity was not seen with the epifluorescence
microscope, which indicated that the hair cells were damaged (arrowheads in Figure 2B and 2C). Additionally, the
number of spiral ganglion neurons after neomycin treatment
was reduced at 3 weeks (arrow in Figure 2B), and few
spiral ganglion neurons survived at 6 weeks (arrow in
Figure 2C). This was shown with the anti-TUJ1 antibody
immunostaining in cryosections.
Six weeks after artificial perilymph treatment, the PCTZ
of the control group was identified along the cochlear nerve
near the interface of the myelin components of the PNS
(MPZ) and CNS (MOG) (dotted lines in Figures 3A13A2). We found that the expression of astrocyte marker
GFAP was weakly detected centrally to the PCTZ (dotted
lines in Figure 3A2). In addition, the expression of GFAP
was fully overlapped with the mature oligodendrocyte
marker MOG and was interfaced with the PNS myelin
marker MPZ at the PCTZ (dotted lines in Figures 3A13A2).
At 3 and 6 weeks following neomycin treatment the
PCTZ was visualized through immunostaining of the PNS
myelin marker MPZ and the CNS myelin marker MOG
(Figures 3B1-3B2, 3C1-3C2). The GFAP labeling, however, was enhanced and shown in a disarrayed manner at 3
weeks following neomycin treatment (Figure 3B2).
Remarkably, GFAP-expressing processes were observed distally to the PCTZ and extended beyond the spiral lamina
level at the basal cochlear turn (arrows in Figure 3B2). At
6 weeks following neomycin treatment, the processes with
positive GFAP labeling were found distally to the PCTZ
and beyond the spiral lamina level at the basal cochlea
(arrows in Figure 3C2). This indicated that the CNS astroglial outgrowth extended peripherally to the PCTZ in chemically induced sensorineural hearing loss.
Discussion
Hearing loss is a major disability and affects the daily lives
of millions of people. Hearing prostheses, such as hearing
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Otolaryngology–Head and Neck Surgery 150(1)
Figure 2. Hair cells and spiral ganglion neurons were damaged after neomycin treatment. Scale: 50 mm shown in panel C.
Figure 3. The astroglial reaction was found along the cochlear nerve following neomycin treatment. CN, cochlear nucleus; ST, scala tympani; SGN, spiral ganglion neurons. Scale: 50 mm.
aids, cochlear implants, auditory brainstem implants, and
auditory midbrain implants, have all been developed in clinical practice. However, there is virtually no biological
approach to replace damaged auditory cells. Recent advances
in stem cell technology provide new hope for the treatment
of neurodegenerative diseases, including the reconstruction of
neural processes from the inner ear to the brain.14,18,19 In this
stem cell–based replacement, it is fundamental to understand
the astroglial reaction at the PCTZ along the cochlear nerve
following insults to the inner ear, which is critical for the
regeneration of the neural processes from the inner ear (PNS)
to the central auditory system.
In this study, we found that hair cells and spiral ganglion
neurons were damaged following neomycin treatment,
which was indicated by the lack of myosin VIIa and TUJ1
immunostaining (Figure 2). The sensorineural hearing
loss induced by neomycin was further supported by functional ABR measurements, an observation that is consistent
with previous studies.14,15 Our immunofluorescence study
showed that the expression of GFAP was increased and
GFAP-positive processes extended peripherally to the
PCTZ and even reached the spiral lamina level at the basal
cochlea. This was in response to the degeneration of the
peripheral auditory system and was not seen in the control
group. The outgrowth of astrocytic processes from the
PCTZ into the peripheral portion of the cochlear nerve was
also observed when the cochlear nerve was damaged centrally to the PCTZ at the cerebellopontine angle.13 In the
central cochlear nerve damage model,13 hypertrophic astrocytic processes were abundant in the cochlear nucleus.
However, increased expression of GFAP did not extend
centrally into the cochlear nucleus in the inner ear damage
model in this study. The reason for this difference has not
been determined.
In this study, we found that the oligodendrocyte-derived
myelin, which was labeled by MOG, remained central to
the PCTZ. The peripheral portion of the myelin in the PNS,
which was labeled by anti-MPZ antibodies, was situated distally to the PCTZ along the cochlear nerve. In a previous
study using myelin mutants, Schwann cells were found to
invade along the neuraxis and cause myelination of the
CNS in spinal cord, brainstem, and cerebellum that
increased in amount and distribution with age.4 In addition,
in a human spinal cord injury study, axonal demyelination
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Hu et al
125
was observed and Schwann cells may invade the PCTZ and
contribute to the myelination of some spinal axons.5 The
reason for lack of Schwann cell invasion in this study has
not been identified. Future studies are required to detail the
Schwann cell response following injury to the auditory
system. On the other hand, the relatively normal expression
of peripheral and central myelin proteins may prove to have
a beneficial effect during auditory pathway regeneration
because it is reported that myelinating glial cells may have
a fibroblast growth factor receptor signaling-mediated supportive effect on auditory neurons.20
In summary, this study reveals the astroglial reaction in
response to neomycin-induced inner ear damage. The astrocytes, which were indicated by GFAP labeling, were
observed to invade the PCTZ along the cochlear nerve and
travel distally to the PCTZ. The expression pattern of the
myelin components of oligodendrocytes and Schwann cells
remained unchanged along the cochlear nerve. Extensive
gliosis or glial scar was not observed using immunofluorescence in this study. The findings in this study may contribute to the design of future cochlear nerve regeneration
models that are able to replace damaged auditory pathways
in hearing loss and other auditory disorders.
Author Contributions
Zhengqing Hu, design the experiment; collect, analyze, and interpret the data; write and revise the manuscript; final approval of the
manuscript; Baofu Zhang, design and conduct the experiment, collect and analyze the data, revise the manuscript, final approval of
the manuscript; Xuemei Luo, design the experiment, analyze and
interpret the data, revise the manuscript, final approval of the
manuscript; Lei Zhang, design the experiment, collect and analyze
the data, revise the manuscript, final approval of the manuscript;
Jue Wang, design the experiment, collect and analyze the data,
revise the manuscript, final approval of the manuscript; Dennis
Bojrab II, design the experiment, analyze the data, revise the
manuscript, final approval of the manuscript; Hui Jiang, design
the experiment, collect and analyze the data, revise the manuscript,
final approval of the manuscript.
Disclosures
Competing interests: None.
Sponsorships: None.
Funding source: Deafness Research Foundation: study design and
conduct, American Hearing Research Foundation: study design and
conduct, Carls Endowment Trust: data analysis and writing the
manuscript, NIDCD/NIH: collection, analysis, and interpretation of
the data and writing the manuscript.
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