Post on 26-Apr-2023
Upregulation of Insulin-Like Growth Factor and Inter-leukin 1b Occurs in Neurons but not in Glial Cells inthe Cochlear Nucleus Following Cochlear Ablation
Ver�onica Fuentes-Santamar�ıa,* Juan Carlos Alvarado, Mar�ıa Cruz Gabald�on-Ull, and Jos�e Manuel Juiz
Institute for Research on Neurological Disorders (IDINE), Faculty of Medicine, University of Castilla-La Mancha, 02006,
Albacete, Spain
ABSTRACTOne of the main mechanisms used by neurons and glial
cells to promote repair following brain injury is to
upregulate activity-dependent molecules such as
insulin-like growth factor 1 (IGF-1) and interleukin-1b
(IL-1b). In the auditory system, IGF-1 is crucial for
restoring synaptic transmission following hearing loss;
however, whether IL-1b is also involved in this process
is unknown. In this study, we evaluated the expression
of IGF-1 and IL-1b within neurons and glial cells of the
ventral cochlear nucleus in adult rats at 1, 7, 15, and
30 days following bilateral cochlear ablation. After the
lesion, significant increases in both the overall mean
gray levels of IGF-1 immunostaining and the mean gray
levels within cells of the cochlear nucleus were
observed at 1, 7, and 15 days compared with control
animals. The expression and distribution of IL-1b in the
ventral cochlear nucleus of ablated animals was tempo-
rally and spatially correlated with IGF-1. We also
observed a lack of colocalization between IGF-1 and IL-
1b with either astrocytes or microglia at any of the
time points following ablation. These results suggest
that the upregulation of IGF-1 and IL-1b levels within
neurons—but not within glial cells—may reflect a plastic
mechanism involved in repairing synaptic homeostasis
of the overall cellular environment of the cochlear
nucleus following bilateral cochlear ablation. J. Comp.
Neurol. 521:3478-3499, 2013.
VC 2013 Wiley Periodicals, Inc.
INDEXING TERMS: astrocyte; microglia; growth factor; interleukin; auditory system
The disruption of glial–neuronal homeostasis follow-
ing brain injury triggers a microglial activation response
that promotes the synthesis and release of activity-
dependent molecules, which are critical for restoring
functional integrity and preventing further damage
(Bruce-Keller, 1999; Hanisch and Kettenmann, 2007;
Cullheim and Thams, 2007). One of these molecular
signals is insulin-like growth factor 1 (IGF-1), a growth-
promoting peptide that plays a pivotal role in regulating
neuronal function and synaptic plasticity during brain
development and maturation (Guthrie et al., 1995; Fer-
nandez et al., 1998). Previous studies carried out in
IGF-1–deficient mice have demonstrated that this factor
is required for the proper development and mainte-
nance of hearing, as loss of IGF-1 results in delayed
inner-ear maturation (Camarero et al., 2001, 2002). In
the adult brain, IGF-1 modulates synaptic activity by
regulating neuronal excitability and synaptogenesis,
which has led to the suggestion that increased IGF-1
levels may contribute to synaptic rearrangements within
auditory nuclei following cochlear damage (Suneja
et al., 2005; Alvarado et al., 2007; Fuentes-Santamaria
et al., 2007).
The cytokine interleukin 1b (IL-1b) is also involved in
the regulation of synaptic signaling between glia and
surrounding neurons in both healthy and diseased ani-
mals (Fogal and Hewett, 2008; Mishra et al., 2012). In
healthy adult brains, low levels of IL-1b are detectable
in neurons but not in glial cells (Breder et al., 1988;
Additional Supporting information may be found in the online versionof this article.
Grant sponsor: Program I3 of the Ministry of Science and Innova-tion; grant numbers: I320101590 (to VFS); I320101589 (to JCA);Grant sponsor: Government of Castilla-La Mancha; grant number:PE110901526233 (to JMJ); Grant sponsor: Ministry of Science andInnovation; grant number: BFU2009-13754-C02-01 (to JMJ).
*CORRESPONDENCE TO: Ver�onica Fuentes-Santamar�ıa, Ph. D. Facultadde Medicina, Universidad de Castilla-La Mancha, Campus de Albacete, C/Almansa, 14, 02006 Albacete, Spain. E-mail: Veronica.Fuentes@uclm.es
Received February 24, 2013; Revised April 30, 2013;Accepted for publication May 3, 2013.DOI 10.1002/cne.23362Published online May 16, 2013 in Wiley Online Library(wileyonlinelibrary.com)VC 2013 Wiley Periodicals, Inc.
3478 The Journal of Comparative Neurology | Research in Systems Neuroscience 521:3478–3499 (2013)
RESEARCH ARTICLE
Lechan et al., 1990), which supports the hypothesis
that under normal physiological conditions this cytokine
may modulate neuron–neuron or neuron–glia interac-
tions (Watt and Hobbs, 2000; Parish et al., 2002). In
addition to its function as an activity-dependent signal-
ing molecule, IL-1b also plays a crucial role in the regu-
lation of axonal sprouting by providing guidance and
trophic support to newly formed synaptic connections
(Parish et al., 2002) as well as by modulating neurite
growth and regeneration (Hendrix and Peters, 2007;
Boato et al., 2011). Increased expression of IL-1b is
usually associated with microglial activation (Giulian
et al., 1986; Hetier et al., 1988), which implies that this
proinflammatory cytokine—whose rapid production and
secretion are largely regulated by glial cells in response
to cellular damage—is a key mediator of the pathogene-
sis and progression of several degenerative neurological
disorders (Vezzani et al., 1999; Allan 2000; Patel et al.,
2001; Boutin et al., 2001; Ralay et al., 2006; Prow and
Irani, 2008).
This idea is further supported by studies carried out
in the auditory system of rodents, which have demon-
strated that following acoustic trauma there is an
inflammatory response within the inner ear that
requires the migration of mononuclear phagocytes to
the affected cochlear areas (Hirose et al., 2005). This
response also involves increased cytokine levels during
its early stages, demonstrated by the fact that when
cytokines are inhibited by blocking upstream signaling
pathways, inflammation of the cochlea is suppressed
and hearing impairment improved (Fujioka et al., 2006;
Wakabayashi et al., 2010).
Deprivation of cochlear afferent activity has been
reported to initiate a series of degenerative and regen-
erative synaptic processes, which may occur simultane-
ously within auditory nuclei with the ultimate function
of repairing synaptic stability (Morest et al., 1998;
Altschuler et al., 1999; Potashner et al., 1997; Mully
et al., 2002). Previous studies provide evidence that
these events are accompanied by persistent glial activa-
tion within the cochlear nucleus following cochlear abla-
tion (Rubel and MacDonald, 1992; Lurie and Rubel,
1994; De Waele et al., 1996; Campos-Torres et al.,
1999, 2005). Furthermore, they indicate that the occur-
rence of long-term apposition between microglial cells
and deafferented cochlear nucleus neurons may reflect
a mechanism for facilitating an exchange of cellular sig-
nals for restoring synaptic transmission and survival
(Fuentes-Santamaria et al., 2012). In this respect, it has
been proposed that synaptogenesis-promoting signals
released by glial cells are required to facilitate axonal
sprouting and synaptic recovery following brain damage
(Pfrieger and Barres, 1997; Cullheim and Thams, 2007).
An issue that remains unexplored, however, is the
nature of the glia-derived activation signals that modu-
late microglial responses within the cochlear nucleus
following injury. In this study, we expand upon the
aforementioned observations by investigating the time-
window during which dysfunctional neuron-glial signal-
ing—caused by bilateral cochlear ablation—may lead to
increased synthesis and release of the intercellular
mediators IGF-1 and IL-1b by neurons, activated astro-
cytes, and microglia within the cochlear nucleus.
MATERIALS AND METHODS
Results were obtained from 24 adult rats (16 experi-
mental and 8 age-matched unoperated control rats).
Following bilateral cochlear ablation, experimental ani-
mals were sacrificed after 1 (PA1d, n 5 4), 7 (PA7d, n
5 4), 15 (PA15d, n 5 4), or 30 days (PA30d, n 5 4).
All animal protocols used in this study were approved
by the Institutional Animal Care and Use Committee at
the University of Castilla-La Mancha. Experimental pro-
cedures were carried out in accordance with the guide-
lines of the European Communities Council (Directive
2010/63/EU) and current national legislation (R.D. 53/
2013; Law 32/2007) for the care and use of research
animals.
Auditory brainstem responses (ABRs)Experimental rats were recorded the day before the
procedure of bilateral cochlear ablation and at the end
of each survival time. ABR recordings were performed
as described previously (Alvarado et al., 2012). Animals
were anesthetized with isoflurane (4% for induction,
1.5–2% for maintenance with a 1 L/min O2 flow rate)
and placed in a sound-attenuating electrically shielded
booth (Eymasa/Incotron, Barcelona, Spain), which was
located inside a sound-attenuating room. Subdermal
needle electrodes (Rochester Electro-Medical, Tampa,
FL) were placed at the vertex (positive) and under the
right (negative) and left (ground) ears. The stimulation
and recording were performed with a Tucker-Davis
(TDT) BioSig System III (Tucker-Davis Technologies,
Alachua, FL). The stimuli were generated digitally by
using the SigGenRP software (Tucker-Davis Technolo-
gies) and the RX6 Piranha Multifunction Processor hard-
ware (Tucker-Davis Technologies) and consisted of
tones (5-ms rise/fall time with no plateau, with a cos2
envelope, at 20/sec) at different frequencies across 7
octaves from 0.5 to 32 kHz. They were delivered mon-
aurally (right ear) by using the EDC1 electrostatic
speaker driver (Tucker-Davis Technologies) and the EC-
1 electrostatic speaker (Tucker-Davis Technologies),
which was placed into the external auditory meatus of
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3479
the rat. Prior to the experiments, stimuli were calibrated
by using the SigCal software (Tucker-Davis Technolo-
gies) and the ER-10B1 low noise microphone system
(Etymotic Research, Elk, Groove, IL). The evoked poten-
tials were filtered (0.3–3.0 kHz), averaged (500
waveforms) and stored for later analyses on a
computer.
Surgical procedures andimmunohistochemistry
Bilateral cochlear ablations were performed on adult
rats as previously described (Fuentes-Santamaria et al.,
2012). Animals were anesthetized, and under aseptic
conditions, a retroauricular incision was made in the
skin to expose the bulla. Once exposed, the cochlea
was removed by using fine forceps, and the remaining
cochlear contents were aspirated by using a vacuum-
aspiration system. During the surgical procedure, a
heating pad was used to maintain normal body temper-
ature and improve the recovery from anesthesia. Fol-
lowing surgery, the animals were observed as they
regained consciousness, after which time they were
returned to their cages and maintained with free access
to food and water.
At appropriate postoperative time points, control and
experimental rats were anesthetized with an intraperito-
neal injection of ketamine (100 mg/kg) and xylazine (5
mg/kg). Next, the rats were transcardially perfused
with a 0.9% saline wash followed by a fixative solution
consisting of 4% paraformadehyde in 0.1 M phosphate
buffer (PB; pH 7.4). Brains were removed, and coronal
sections (40 lm thick) were obtained using a sliding
microtome. After blocking for 1 hour in a solution con-
taining 10% normal goat serum diluted in Tris-buffered
saline (TBS; pH 7.4) with 0.2% Triton X-100 (TBS-Tx
0.2%), free-floating sections were incubated overnight
at 4�C in the same buffer solution with primary antibod-
ies against either glial fibrillary acidic protein (GFAP;
1:2,000), IGF-1 (1:100), or IL-1b (1:100). The following
day, the sections were washed in TBS-Tx 0.2% solution
and incubated for 2 hours at room temperature with a
biotinylated anti-rabbit secondary antibody (1:200, Vec-
tor, Burlingame, CA). The biotin–avidin procedure was
used to link the antigen–antibody complex to horserad-
ish peroxidase (HRP; ABC Elite, Vector), which was then
visualized with diaminobenzidine (3,30-diaminobenzidine
tetrahydrochloride [DAB]) histochemistry. The exposure
time to DAB was similar for control and experimental
samples. Finally, sections were thoroughly washed in
TBS, mounted on gelatin-coated slides, air-dried, dehy-
drated in ethanol, cleared in xylenes, and mounted with
Cytoseal (Stephens Scientific, Wayne, NJ). Three sets of
control experiments were performed to test the speci-
ficity of the immunohistochemical detection system: 1)
replacement of the primary antibody with TBS–bovine
serum albumin [BSA]; 2) omission of secondary antibod-
ies; and 3) omission of the ABC reagent. No immuno-
staining was detected under these conditions.
Antibody characterizationThe antibodies used in this study are listed in Table
1. Glial and neuronal antibodies included 1) mouse anti-
GFAP ; 2) rabbit anti-GFAP; 3) mouse anti-CD11b; 4)
mouse anti-neuronal nuclei (NeuN); 5) rabbit anti-IGF-1;
and 6) rabbit anti-IL-1 b.
The polyclonal and monoclonal anti-GFAP antibodies
were raised against GFAP protein from cow and porcine
spinal cord, respectively (manufacturer’s technical infor-
mation; Table 1). Both antibodies recognize a single 50-
kDa band that corresponds to GFAP protein on western
blot analysis (Debus et al., 1983; Yamanaka et al.,
2010) and label astrocytes in the mature nervous sys-
tem. The staining pattern of these antibodies in the rat
cochlear nucleus is consistent with previous reports
(White et al., 2010).
The monoclonal anti-CD11b antibody was raised
against rat peritoneal macrophages and recognizes an
integrin expressed on the surface of macrophages and
microglial cells. This antibody immunoprecipitates poly-
peptide chains of 160 and 95 kDa (Robinson et al.,
1986; Lu et al., 2011) and has been extensively used
to label microglial cells in rat brain (Milligan et al.,
1991). The staining pattern described herein is in
agreement with previous reports (Maroso et al., 2011).
The monoclonal anti-NeuN antibody was produced by
using purified cell nuclei from mouse brain
TABLE 1.
List of Primary Antibodies
Primary antibody Immunogen Host Code/clone Dilution Manufacturer
GFAP Porcine spinal cord GFAP Mouse MAB360 1:2,000 Millipore, Billerica, MAGFAP Cow spinal cord GFAP Rabbit Z0334 1:2,000 Dako, Glostrup, DenmarkCD11b Rat peritoneal macrophages Mouse MCA275G 1:100 Serotec, Oxford, UKNeuN Purified cell nuclei from mouse brain Mouse MAB337 1:100 MilliporeIGF-1 Recombinant human IGF-1 Rabbit PAB-Ca 1:100 Gropep, Adelaide, AustraliaIL-1b Highly pure recombinant mouse IL-1b Rabbit AB9722 1:100 Abcam, Cambridge, MA
V. Fuentes-Santamar�ıa et al.
3480 The Journal of Comparative Neurology |Research in Systems Neuroscience
(manufacturer’s technical information; Table 1). This
antibody recognizes the neuron-specific protein NeuN,
which is present in most neuronal cell types of the cen-
tral and peripheral nervous system (Rasmussen et al.,
2007). According to the manufacturer and previous
studies (Mullen et al., 1992; Lind et al., 2005), it recog-
nizes four bands between 45 and 75 kDa on western
blot of rat brain that may represent multiple phospho-
rylation states of the protein. The staining pattern of
this antibody agrees with previous observations in the
cochlear nucleus (Fuentes-Santamaria et al., 2012).
The polyclonal anti-IL-1b antibody was raised against
IL-1b protein from mouse (manufacturer’s technical
information; Table 1). This antibody recognizes a single
17-kDa band that corresponds to IL-1b protein on west-
ern blot analysis of mouse brain (Sozen et al., 2009).
The staining pattern with this antibody in the cochlear
nucleus matches previous descriptions in other brain
regions (Strecker et al., 2011). The polyclonal anti-IGF-1
antibody was produced by using recombinant human
IGF-1 (manufacturer’s technical information; Table 1).
The specificity of IGF-1 in the auditory pathway was
determined by preabsorbing the antibody with a 30-fold
excess of the human LongTMR3IGF-1 competitive con-
trol that completely abolished the IGF-1 immunostaining
in the sections (Alvarado et al., 2007). The staining pat-
tern of this antibody is agreement with previous studies
in the rat cochlear nucleus (Fuentes-Santamaria et al.,
2007). In addition, the specificity of this antibody has
been previously described by other authors (Hill et al.,
1999; Degger et al., 2000).
Double labelingSections were rinsed four times in TBS-Tx 0.2% and
incubated overnight with one of the following mixtures
of primary antibodies: 1) GFAP and NeuN; 2) IGF-1 and
GFAP; or 3) IL-1b and GFAP. Following four 15-minute
rinses in TBS-Tx 0.2%, the sections were incubated with
fluorescently labeled secondary antibodies for 2 hours
at room temperature (1:100, anti-mouse antibodies con-
jugated to Alexa 594 and anti-rabbit antibodies conju-
gated to Alexa 488; Molecular Probes, Eugene, OR) and
after several rinses in TBS, the sections were mounted,
coverslipped, and maintained overnight at 4�C.
Densitometric analysis of immunostainedsections
Subdivisions of the cochlear nucleus were defined as
described in previous studies (for review, see Cant and
Benson, 2003). Immunostained sections were examined
by using brightfield illumination on a Nikon Eclipse pho-
tomicroscope, and images were captured by using a
DXM 1200C digital camera attached to the microscope.
Color images of selected fields were digitized, and the
red channel was used to obtain images containing gray-
scale pixel intensities ranging from 0 (white) to 255
(black). Analysis of the immunostaining was performed
by using the public-domain image analysis software
Scion Image for Windows (version beta 4.0.2; devel-
oped by Scion), as previously described (Alvarado et al.,
2004; Fuentes-Santamaria et al., 2012).
Analysis of the GFAP immunostaining was performed
on six equally spaced coronal sections, located 160 lm
apart, along the rostrocaudal axes of the anteroventral
cochlear nucleus (AVCN) and the posteroventral coch-
lear nucleus (PVCN). For each section, three fields
(25.57 3 104 lm2; dorsal, middle, and ventral) were
sampled by using a 203 objective, equaling a total of
36 fields for each nucleus of each animal. To compare
across different samples, the images were normalized
by using an algorithm based on a principle of signal-to-
noise ratio (Alvarado et al., 2004), and the threshold
was set as 2 standard deviations above the mean gray
level of the field (Alvarado et al., 2007; Fuentes-
Santamaria et al., 2007). All immunostained profiles
(i.e., astrocytic cell bodies and processes) exceeding
this threshold were identified as “labeled.” To quantify
these labeled profiles, for each field, two quantitative
indexes were determined: 1) the mean gray level of the
nucleus—which was used as an indirect indicator of
GFAP levels within the ventral cochlear nucleus; and 2)
the immunostained area—which was calculated as the
summed area of cell bodies and processes labeled
above the threshold in each field and provides informa-
tion about the area occupied by the immunostained
astrocytes in the ventral cochlear nucleus.
For the densitometric analyses of IGF-1 and IL-1b
immunostaining, the field sampling, normalization, and
thresholding procedures were performed as described
above for GFAP. As changes in the overall levels of IGF-
1 and IL-1b immunostaining within the cochlear nucleus
could be due to changes either in the immunostained
neuropil or in the immunostained neurons, two quantita-
tive indices were measured for each field: 1) the mean
gray level of the nucleus and 2) the mean gray level
within cells, which were used as indirect indicators of
IGF-1 and IL-1b levels within the nucleus and within
neurons, respectively.
Preparation of figures and statisticalanalysis
Photoshop (Adobe v5.5) and Canvas (Deneba v6.0)
were used to adjust size, brightness, and contrast of publi-
cation images. All the data were expressed as means 6
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3481
standard errors. Comparisons among groups were ana-
lyzed statistically by using the one-factor analysis of var-
iance and Duncan’s post hoc analysis to evaluate the
effect of the survival time after the cochlear ablation over
the immunostaining in the cochlear nucleus. Statistical sig-
nificance was set at a level of P < 0.05.
RESULTS
ABR recordingsTo evaluate the effects of bilateral cochlear ablation
on hearing, preablation and postablation ABR record-
ings were performed on experimental rats for each of
the time points described in Materials and Methods.
Similarly to what was observed in control animals, prea-
blation recordings showed a distinctive wave pattern
characterized by positive peaks following stimulus (Fig.
1A). Conversely, postablation recordings showed no
waves at any of the tested frequencies (Fig. 1B), indi-
cating that following loss of cochlear integrity, no
responses were evoked from the auditory brainstem
nuclei after stimulus presentation. Representative exam-
ples of pre- and postablation ABRs at PA7d after bilat-
eral cochlear ablation are shown in Figure 1.
Upregulation of GFAP immunostainingfollowing bilateral cochlear ablationControl and ablated animalsIn control animals, GFAP immunostaining was restricted
to a few thin, short-branched processes that were
scattered throughout all regions of the AVCN (Fig. 2)
and PVCN (Fig. 3). Following bilateral cochlear ablation,
the pattern of GFAP immunostaining was different from
that observed in control animals. At all time points
studied, an increase in GFAP immunostaining was
observed in both the AVCN and PVCN. To quantify
these changes, two quantitative indices of GFAP immu-
nostaining were determined for both the experimental
and control animals: 1) the mean gray level within the
nucleus and 2) the immunostained area (see Materials
and Methods). At PA1d, GFAP-immunostained astrocytic
processes were darker and had thicker primary proc-
esses compared with control animals (compare Fig. 2B
with 2C and Fig. 3B with 3C). These qualitative observa-
tions were consistent with significant increases in both
the mean gray level of GFAP immunostaining (Figs. 2,
3G, Table 2) and immunostained area (Figs. 2H, 3H,
Table 2) within the ventral cochlear nucleus compared
with control animals. The astrocytic response reached a
maximum in PA7d and PA15d animals, in which astro-
cytic processes were strongly immunostained, highly
ramified, and formed an interconnecting network (Figs.
2D,E, 3D,E, Table 2). At these time points, the mean
gray levels of GFAP immunostaining within both the
AVCN and PVCN were significantly higher than levels
observed in PA1d or control animals (Figs. 2G, 3G,
Table 2). With respect to the immunostained areas
within the AVCN at PA7d and PA15d, significant
increases were observed in comparison with PA1d and
Figure 1. ABR recordings depicting the effects of bilateral cochlear ablation in adult rats. Preablation (A) and postablation (B) ABR record-
ings were performed in experimental animals to evaluate how a lack of cochlear integrity affects normal wave patterns, which are charac-
terized by positive peaks generated following stimulus. At all time points studied, postablation ABRs showed a complete loss of response
waves following stimulus onset at all tested frequencies, indicating that the auditory brainstem nuclei did not receive any electrical stimu-
lation arising from the cochlea. Arrows indicate stimulus onset. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
V. Fuentes-Santamar�ıa et al.
3482 The Journal of Comparative Neurology |Research in Systems Neuroscience
control animals (Table 2). In PA30d animals, the mor-
phology and staining of astrocytes were similar to that
observed in control animals (Figs. 2F, 3F, Table 2).
Quantification of the mean gray levels of GFAP immu-
nostaining in PA30d animals indicated that they were
decreased compared with PA15d and PA7d animals,
and no differences were observed compared with PA1d
or control rats (Figs. 2G, 3G, Table 2). Densitometric
analyses of the GFAP-immunostained areas within the
AVCN in PA30d animals revealed significant decreases
compared with PA15d and PA7d animals, but no
differences were observed in comparison with PA1d or
control rats (Figs. 2H, 3H, Table 2). However, in the
PVCN, immunostained areas were increased when com-
pared with control animals, but not when compared
with the other time points (Figs. 2H, 3H, Table 2).
Appositions between astrocytic cells andcochlear-nucleus neuronsDouble-labeling experiments for astrocytic and neuronal
markers showed that in PA1d animals the processes of
immunostained astrocytes lay in close proximity to and
Figure 2. Digital images depicting glial fibrillary acidic protein (GFAP) immunostaining in the AVCN in control (B), PA1d (C), PA7d (D),
PA15d (E) and PA30d (F) rats. GFAP immunostaining was increased at PA1d, peaked around PA7d, and gradually declined during PA15d
and PA30d. Bar graphs depict the mean gray levels of GFAP immunostaining (G) and the immunostained areas (H) in control and ablated
animals. In A, the drawing shows the location of the AVCN, and the square box indicates the approximate location of the fields repre-
sented in B–F. The error bars indicate the standard errors of the mean. Abbreviation: stt, spinal trigeminal tract. Scale bar 5 250 lm in
A; 50 lm in F (applies to B–F). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3483
partially surrounded the presumptive deafferented coch-
lear nucleus neurons within both the AVCN and PVCN,
indicating an initial and early interaction between these
two cell types (asterisks in Figs. 4C, 5C). The incidence
of astrocytic–neuronal appositions increased in PA7d
and PA15d animals, in which astrocytic processes
formed a dense network of fine processes that
completely surrounded the soma and dendrites of the
cochlear nucleus neurons (asterisks in Figs. 4D,E, 5D,E).
At later time points, the occurrence of these appositions
decreased in comparison with the other survival time
points and control animals (asterisks in Figs. 4F, 5F). An
example of multiple astrocytic appositions onto an audi-
tory neuron in a PA7d animal is shown in Figure 5G–I.
Effects of bilateral cochlear ablation on IGF-1 immunostainingControl animalsIGF-1 immunostaining was light within the neuropil, and
cells were moderately stained within both the AVCN
and PVCN (Figs. 6B, 7B). Immunostaining within
Figure 3. Digital images depicting glial fibrillary acidic protein (GFAP) immunostaining in the PVCN in control (B), PA1d (C), PA7d (D),
PA15d (E), and PA30d (F) rats. Similar to what was observed in the AVCN, an increase in GFAP immunostaining was first evident in PA1d
animals, although the highest levels were found around PA7d. Immunostaining gradually declined during PA15d and PA30d compared with
control animals. Bar graphs depict the mean gray levels of GFAP immunostaining (G) and the immunostained areas (H) in control and
ablated animals. In A, the drawing indicates the location of the PVCN, and the square box shows the approximate location of the fields
represented in B–F. The error bars indicate the standard errors of the mean. Scale bar 5 250 lm in A; 50 lm in F (applies to B–F). [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
V. Fuentes-Santamar�ıa et al.
3484 The Journal of Comparative Neurology |Research in Systems Neuroscience
individual auditory cells was mainly observed in the
cytoplasm and in subsets of the primary dendritic proc-
esses emerging from the soma (Figs. 6B, 7B).
Ablated animalsFollowing bilateral cochlear ablation, the patterns of
IGF-1 immunostaining observed in the AVCN and PVCN
showed temporal changes compared with control ani-
mals (Figs. 6,7). To quantify these changes, two quanti-
tative indices of IGF-1 immunostaining were determined
for both the experimental and control animals: 1) the
mean gray level of the nucleus and 2) the mean gray
level within cells (see Materials and Methods for
details).
At 1 day post ablation, IGF-1 immunostaining was
increased in both the neuropil and within cells of the
ventral cochlear nucleus compared with control animals
(arrows in Figs. 6B, 7B). Statistical analyses showed
increases in both the mean gray level of immunostain-
ing within the AVCN and PVCN and the mean gray level
within immunostained cells (Fig. 8, Table 3). In PA7d
animals, these increases remained significant compared
with control animals, although no differences were
observed in comparison with PA1d animals (Figs. 6D,
7D, 8, Table 3). In PA15d animals, IGF-1 immunostain-
ing remained elevated in both the AVCN and PVCN
(Figs. 6E, 7E, 8, Table 3). However, at later time points
(PA30d), the staining pattern of IGF-1 was similar to
that observed in control animals (Figs. 6F, 7F, 8), con-
sistent with the densitometric analyses of IGF-1 immu-
nostaining (Fig. 8, Table 3). It was also observed that
the IGF-1–immunostained cells had numerous close
appositions from astrocytic processes at all time points
studied, but these were particularly evident in PA7d
and PA15d animals (asterisks in Figs. 6G–K, 7G–K).
Colocalization of IGF-1 with neuronal and glialmarkersAs a previous study carried out in the rat cochlear
nucleus has reported persistent microglial activation fol-
lowing bilateral cochlear ablation (Fuentes-Santamaria
et al., 2012), double-labeling experiments with neuronal
and glial markers were performed in both control and
ablated animals to determine whether IGF-1 levels are
increased in glial cells following deafferentation. We
observed colocalization of IGF-1 with the neuronal
marker NeuN (Fig. 9A–C)—but not with the astroglial
marker GFAP (Fig. 9D–F) or the microglial marker
CD11b (Fig. 9G–I)—indicating that neurons within both
the AVCN and PVCN were the only cell types express-
ing IGF-1 during the time points studied. Despite the
lack of colocalization of IGF-1 with glial markers, appo-
sitions between glial cells and IGF-1–immunostained
TABLE 2.
GFAP Immunostaining in the AVCN and PVCN in Control and Ablated Animals1
AVCN PVCN
Survival times
Mean gray
level of the
nucleus
Immunostained
area (lm2)
Mean gray level of
the nucleus
Immunostained
area (lm2)
Control (1) 125.72 6 2.4 2229.50 6 20.24 129.17 6 4.0 2,181.26 6 72.11 d (2) 139.16 6 7.6 2352.26 6 52.4 142.77 6 2.5 2,476.27 6 51.67 d (3) 160.11 6 3.8 2683.47 6 55.9 173.10 6 4.9 2,624.35 6 47.315 d (4) 151.92 6 3.8 2428.41 6 35.5 165.10 6 5.4 2,523.24 6 49.830 d (5) 133.03 6 4.9 2385.52 6 49.1 141.79 6 3.4 2,456.42 6 61.9
Statistical comparison Significance levels Significance levels
2 vs. 1 2 2 2 33 vs. 1 4 4 4 43 vs. 2 3 4 4 NS4 vs. 1 4 4 4 34 vs. 2 2 NS 4 NS4 vs. 3 NS 4 NS NS5 vs. 1 NS 2 2 35 vs. 2 NS NS NS NS5 vs. 3 4 4 4 NS5 vs. 4 3 NS 4 NS
1Values are means 6 standard errors.2P < 0.05.3P < 0.01.4P < 0.001.
NS, not significant.
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3485
neurons were clearly evident in ablated animals in com-
parison with control animals (Fig. 9F,I).
Effects of bilateral cochlear ablation onIL-1b immunostainingControl animalsAnalyses of the expression and distribution of IL-1b
within the AVCN and PVCN of control animals revealed
that the levels of IL-1b immunostaining within the ven-
tral cochlear nucleus (Figs. 10B, 11B) were lower than
those of IGF-1, which were confirmed by quantifying
the overall mean gray level of immunostaining and
mean gray level within cells (Table 4).
Ablated animalsThe expression and distribution of IL-1b in ablated ani-
mals was temporally and spatially correlated with that
of IGF-1. At PA1d, there was a striking upregulation of
IL-1b levels compared with control animals, which was
maintained for up to 7 days following bilateral cochlear
ablation (Figs. 10,11). These qualitative observations
were consistent with increases in both the mean gray
level of immunostaining within the AVCN and PVCN and
the mean gray level within immunostained cells (Table
4). At PA15d, IL-1b immunostaining throughout the
AVCN and PVCN was decreased compared with PA7d
animals (Figs. 10,11; also see Table 4), and IL-1b
immunostaining had returned to control levels by
PA30d (Figs. 10,11; also see Table 4).
Colocalization of IL-1b with neuronal and glialmarkersTo determine whether IL-1b expression was increased
in glial cells following bilateral cochlear ablation,
double-labeling experiments with neuronal and glial
markers were performed in both control and ablated
animals (Fig. 12). The results were similar to those
observed for IGF-1, and they demonstrate that neurons
are the sole source of IL-1b within the ventral cochlear
nucleus in control animals and following bilateral coch-
lear ablation (Fig. 12A–C). A lack of colocalization of IL-
Figure 4. Interactions between astrocytes (GFAP, green) and cochlear nucleus neurons (NeuN, red) within the AVCN in control (A,B) and
ablated (C–F) rats. Following bilateral cochlear ablation, close appositions between astrocytic processes and neurons (asterisks in B–F)
were first observed at PA1d (C), increased in frequency during PA7d (D) and PA15d (E), and returned to control levels by PA30d (F). The
square box in A indicates the approximate location of the high-magnification images shown in B–F. A magenta–green copy is available as
Supplementary Figure 4. Scale bar 5 500 lm in A; 40 lm in F (applies to B–F). [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
V. Fuentes-Santamar�ıa et al.
3486 The Journal of Comparative Neurology |Research in Systems Neuroscience
1b with either GFAP (Fig. 12D–F) or CD11b (Fig. 12G–I)
was observed at all of the time points tested.
DISCUSSION
A recent study of the ventral cochlear nucleus in
adult rats has demonstrated that changes in neuron–
glia communications in response to bilateral cochlear
damage lead to microglial activation, which could be
critical for the remodeling of auditory circuitry (Fuentes-
Santamaria et al., 2012). Our findings demonstrate that
such activation is accompanied by a protracted upregu-
lation of GFAP immunostaining that peaks around PA7d
compared with control animals. In addition to increased
astrocytic activation, our findings also indicate that
activity-dependent molecules such as IGF-1 and IL-1b
may be involved in restoring basal synaptic transmis-
sion following bilateral cochlear ablation. Accordingly,
Figure 5. Interactions between astrocytes (GFAP, green) and cochlear nucleus neurons (NeuN, red) within the PVCN in control (A,B) and
ablated (C–I) rats. Similar to what was observed in the AVCN, astrocytic processes were observed apposing cochlear nucleus neurons (aster-
isks) by PA1d (C), although the frequency of these close appositions significantly increased during PA7d (D) and PA15d (E); levels were simi-
lar to control animals (B) by PA30d (F). Note that multiple astrocytes may appose the same cochlear nucleus neuron (arrows in I).
The square box in A indicates the approximate location of the high-magnification images shown in B–F. Astrocytic nuclei (DAPI, blue) in H
are indicated by numbers. Scale bar 5 500 lm in A; 40 lm in F (applies to B–F); 20 lm in G (applies to G–I). [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3487
there were increases both in the mean gray level and in
the area of IGF-1 and IL-1b immunostaining at 1, 7,
and 15 days post ablation within the ventral cochlear
nuclei of ablated animals. Upregulation of IGF-1 and IL-
1b levels was observed in neurons but not in either
astrocytes or microglia during any of the time points
studied following ablation. These findings suggest that
deafferented cochlear nucleus neurons do not require
additional IGF-1 and IL-1b synthesis by glial cells to re-
establish damaged synaptic connections following bilat-
eral cochlear ablation.
Deprivation of afferent activity following cochlear
ablation disturbs homeostasis and diminishes trophic
support to cochlear nucleus neurons (Winsky and
Figure 6. Digital images depicting IGF-1 immunostaining in the anteroventral cochlear nucleus (AVCN) in control (A,B) and ablated (C–F)
rats. Following cochlear ablation, IGF-1 immunostaining was increased in the neuropil and within cells of the AVCN at PA1d (arrows in B),
PA7d (arrows in D), and PA15d (arrows in E) compared with control animals (B). At the latest time point (PA30d), the staining pattern of
IGF-1 (F) was similar to that observed in control animals (B). At all of the time points studied (G–K), but particularly in PA7d (I) and PA15d
(J) animals, GFAP-immunostained astrocytic processes were observed to closely appose IGF-1–immunostained cells. These appositions are
indicated by asterisks in G–K. The square box in A indicates the approximate location of the high-magnification images shown in B–F.
Abbreviation: stt, spinal trigeminal tract. A magenta–green copy is available as Supplementary Figure 6. Scale bar 5 250 lm in A; 50 lm
in F (applies to B–F); 25 lm in K (applies to G–K). [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
V. Fuentes-Santamar�ıa et al.
3488 The Journal of Comparative Neurology |Research in Systems Neuroscience
Jacobowitz, 1995; Potashner et al., 1997; Fuentes-
Santamaria et al., 2005; Suneja et al., 2005; Wang
et al., 2011), resulting in a series of rapid cellular
events that range from changes in microglial phenotype
that occur within a few hours following injury, to close
associations between microglia and injured neurons
(Campos-Torres et al., 1999; Fuentes-Santamaria et al.,
2012). Hypertrophy of microglia and upregulation of
Iba-1 levels in response to bilateral cochlear ablation in
these cells are first evident in the cochlear nucleus at
16 hours post lesion, peak at approximately 1 day, and
persist for at least 100 days, as demonstrated by
increases both in the cross-sectional area of Iba-1–
immunostained neurons and in the mean gray levels of
Iba-1 immunostaining (Fuentes-Santamaria et al., 2012).
In addition to these changes, several studies carried
out in different species have reported reactive gliosis in
the cochlear nucleus following either unilateral cochlear
Figure 7. Digital images depicting IGF-1 immunostaining within the posteroventral cochlear nucleus (PVCN) in control (A,B) and ablated
(C–F) rats. By PA1d, there was already a significant increase in immunostaining both in the neuropil and within neurons (C) compared
with control animals (B). IGF-1 immunostaining remained increased during PA7d (D) and PA15d (E), but had returned to control levels by
PA30d (F). Note that IGF-1–immunostained cells were closely apposed by astrocytic processes during all time points (asterisks in G–K),
although the frequency of these events was greatest during PA7d and PA15d (I,J). The square box in A indicates the approximate location
of the high-magnification images shown in B–F. A magenta–green copy is available as Supplementary Figure 7. Scale bar 5 250 lm in A;
50 lm in F; 25 lm in G.
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3489
Figure 8. Bar graphs indicating the mean gray level of insulin-like growth factor 1 (IGF-1) immunostaining and the mean gray level of IGF-
1 immunostaining within cells in the anteroventral cochlear nucleus (AVCN; A,C) and the posteroventral cochlear nucleus (PVCN; B,D) in
control and experimental rats. Following ablation, significant increases in the overall mean gray levels of IGF-1 immunostaining and the
mean gray levels within cells were observed in the ventral cochlear nucleus at PA1d, PA7d, and PA15d compared with control animals.
The error bars indicate the standard errors of the mean.
TABLE 3.
IGF-1 Immunostaining in the AVCN and PVCN in Control and Ablated Animals1
AVCN PVCN
Survival times
Mean gray level
of the nucleus
Mean gray level
within cells
Mean gray level
of the nucleus
Mean gray level
within cells
Control (1) 94.29 6 2.6 166.78 6 4.4 84.65 6 2.8 160.44 6 4.61 d (2) 114.67 6 3.0 198.96 6 2.6 112.21 6 1.9 199.06 6 3.87 d (3) 110.80 6 1.7 196.80 6 3.6 102.09 6 2.8 184.89 6 2.215 d (4) 101.15 6 2.0 191.30 6 2.2 96.55 6 3.3 181.94 6 3.930 d (5) 98.23 6 2.9 178.47 6 3.1 82.99 6 4.6 161.69 6 5.6
Statistical comparison Significance levels Significance levels
2 vs. 1 4 4 4 43 vs. 1 4 4 4 33 vs. 2 NS NS 2 24 vs. 1 NS 4 2 34 vs. 2 3 NS 3 24 vs. 3 2 NS NS NS5 vs. 1 NS NS NS NS5 vs. 2 3 2 4 45 vs. 3 2 2 4 35 vs. 4 NS NS 3 3
1Values are means 6 standard errors.2P < 0.05.3P < 0.01.4P < 0.001.
NS, not significant.
V. Fuentes-Santamar�ıa et al.
3490 The Journal of Comparative Neurology |Research in Systems Neuroscience
ablation (rats: De Waele et al., 1996; Campos-Torres
et al., 2005; chickens: Lurie and Rubel, 1994; Lurie and
Durham, 2000; monkeys: Insausti et al., 1999) or
tetrodotoxin-mediated blockage of cochlear afferent
activity (Canady and Rubel, 1992). More specifically, in
adult rats, increases in the expression, protein levels,
and immunostaining of GFAP in the cochlear nucleus
begin 1 day post ablation and remain elevated until 45
days after unilateral labyrinthectomy (Campos-Torres
et al., 2005).
Consistent with these findings as well as with previ-
ous observations using noise overstimulation (Feng
et al., 2012), entorhinal perforant path lesions (Jensen
et al., 1994), kainic acid–induced lesions of the hippo-
campus (J�rgensen et al., 1993), and facial-nerve axot-
omy (Tetzlaff et al., 1988; Graeber and Kreutzberg,
1988), the present findings provide evidence for astro-
glial activation, which reaches maximal levels at
approximately 7 days post ablation, the period during
which degeneration and synaptogenesis occur within
the cochlear nucleus following cochlear deafferentation
(Benson et al., 1997; Mully et al., 2002; Hildebrandt
et al., 2011). As opposed to the early (16 hours) and
long-lasting (>100 days) activation of microglia, these
results indicate that astrocytic responses can be more
protracted (24 hours) and less persistent (<30 days).
Although it is unclear why there are differences in
the temporal pattern of glial activation, our results–in
combination with previous observations carried out in
other lesion systems (Wilms and B€ahr, 1995; Bacci
et al., 1999; Bechmann and Nitsch, 2000; Slezak et al.,
2006)–provide evidence that microglia and astrocytes
play different roles in mediating synaptic repair follow-
ing deafness. Consistent with this hypothesis, peri-
neuronal microglia within the cochlear nucleus have
been observed to appose cochlear nucleus neurons 1
Figure 9. A–I: Digital images depicting the colocalization of IGF-1 with neuronal and glial markers in PA7d animals. Note that IGF-1–
immunostained cells (asterisks in A,D,G) in both the AVCN and PVCN colocalized with the neuronal marker NeuN (arrows in B) but not
with either the astroglial marker GFAP (arrows in E) or the microglial marker CD11b (arrows in H). A magenta–green copy is available as
Supplementary Figure 9. Scale bar 5 20 lm in I (applies to a–I). [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3491
day post ablation (Fuentes-Santamaria et al., 2012),
suggesting that microglial cells may be involved in dis-
placing degenerating boutons from presumptive deaffer-
ented neurons by interposing fine processes. This
observation is also supported by studies carried out in
various injury models, and it has been speculated that
this phenomenon may reflect an attempt to eliminate
nonfunctional synapses to facilitate the sprouting of
new connections, which could then compete to occupy
vacated synaptic sites following cochlear removal
(Schiefer et al., 1999; Cullheim and Thams, 2007).
Conversely, recent reports have emphasized the fact
that astrocytic processes are often intermingled with
microglial processes and that presynaptic boutons con-
tact postsynaptic injured neurons, which has led to the
suggestion that astrocytes also contribute to these
Figure 10. Digital images depicting interleukin 1b (IL-1b) immunostaining within the AVCN in control (A,B) and ablated (C–F) rats. Follow-
ing ablation, IL-1b immunostaining was increased in the neuropil and within cells of the AVCN at PA1d (arrows in B), PA7d (arrows in D),
and PA15d (arrows in E) compared with control animals (B). In PA30d (F) rats, IL-1b staining patterns were similar to what was observed
in control animals (B). Following ablation, quantification of the mean gray levels of IL-1b immunostaining and the mean gray levels within
cells identified increases in staining at PA1d, PA7d, and PA15d compared with control animals (G,H). The square box in A indicates the
approximate location of the high-magnification images shown in B–F. Scale bar 5 250 lm in A; 50 lm in F (applies to B–F). [Color figure
can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
V. Fuentes-Santamar�ıa et al.
3492 The Journal of Comparative Neurology |Research in Systems Neuroscience
processes (Reisert et al., 1984). Consistent with this
latter idea, our findings show that astrocytic processes
form a dense network of fine processes that completely
ensheath the soma and dendrites of cochlear nucleus
neurons at approximately 7 days post ablation, at the
period during which microglia with short or no proc-
esses were most frequently observed apposing the
soma and dendrites of deafferented cochlear nucleus
neurons following bilateral cochlear ablation (Fuentes-
Santamaria et al., 2012). However, it is important to
note that, depending on the species, age, lesion type
and extension, different synaptic stripping responses
may be triggered by microglia, astrocytes or both (Jinno
and Yamada, 2011). Several studies have also eval-
uated the contribution of astroglia to functional synapse
formation in primary cocultures of cortical, retinal, and
spinal cord neurons with glial cells, and they concluded
that neurons are unable to form functional synaptic
Figure 11. Digital images depicting IL-1b immunostaining within the posteroventral cochlear nucleus (PVCN) in control (A,B) and ablated
(C–F) rats. Analysis of the immunostained sections revealed increases in IL-1b immunostaining in the neuropil and within cells at PA1d
(arrows in B), PA7d (arrows in D), and PA15d (arrows in E) compared with control animals (B). These increases were confirmed by using
densitometric analysis of the immunostaining, as shown in G and H. The square box in A indicates the approximate location of the high-
magnification images shown in B–F. Scale bar 5 250 lm in A; 50 lm in F (applies to B–F). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3493
connections in the absence of astrocyte-derived signals,
which suggests that astrocytes may influence synaptic
transmission and neuronal survival (Pfrieger and Barres,
1997; Ullian et al., 2001). These observations, taken
together with our findings concerning early microglial
and protracted astrocytic activation processes in the
ventral cochlear nucleus, are consistent with the idea
that microglial cells and astrocytes may act jointly to
promote synaptic repair following deafness.
Glial cells—in response to changes in chemical and
electrical signals from damaged neurons—are endowed
with the capacity to produce and secrete growth fac-
tors and cytokines that are critical for synaptic mainte-
nance and repair (Hanisch, 2002; Mrak and Griffin,
2005). Growth factors are activity-dependent molecu-
lar signals released by neurons and glial cells, and
they have been shown to influence survival and synap-
tic plasticity of spiral ganglion cells following deafness
(Altschuler et al., 1999; Wei et al., 2007). Consistent
with this idea, decreases in growth factor levels have
been associated with sensorineural hearing loss in
patients (Salvinelly et al., 2002), indicating that upreg-
ulation (Suneja et al., 2005) or exogenous application
of such factors (Altschuler et al., 1999) could contrib-
ute to mechanisms that repair cochlear damage. Dur-
ing development, IGF-1 is a crucial factor required for
the correct development and maintenance of hearing
(Camarero et al., 2001, 2002), whereas in the adult
brain, IGF-1 plays a regulatory role in the maintenance
of neuronal excitability and synaptic transmission effi-
cacy throughout life (Ishii et al., 1994; Kar et al.,
1997; Aberg et al., 2006; Shi et al., 2005). In adult
ferrets, increases in IGF-1 immunostaining in the coch-
lear nucleus following unilateral ablation have been
associated with a transient upregulation of synapto-
physin levels, supporting a role for IGF-1 as a neuro-
trophic modulator of synaptic plasticity following
damage (Fuentes-Santamar�ıa et al., 2007). The pres-
ent findings also demonstrate the upregulation of IGF-
1 levels in the cochlear nucleus at 1, 7, and 15 days
post ablation, which is consistent with previous find-
ings suggesting that this molecular signal may partici-
pate in reparative processes involving newly formed
synaptic connections following bilateral cochlear abla-
tion in adult rats.
Cytokines such as IL-1b are also intercellular media-
tors involved in the modulation of the neuronal–glial
response following injury (for review, see Hanisch,
2002). In the central nervous system, only low levels of
IL-1b are detectable within healthy neurons, which
have been linked to normal physiological activity,
whereas overexpression of IL-1b by neurons and glial
cells following neuronal injury has been proposed to
play both detrimental (Vezzani et al., 1999, 2002;
TABLE 4.
IL-1b Immunostaining in the AVCN and PVCN in Control and Ablated Animals1
AVCN PVCN
Survival times
Mean gray
level of the nucleus
Mean gray level
within cells
Mean gray level
of the nucleus
Mean gray level
within cells
Control (1) 82.75 6 2.9 148.11 6 7.9 88.32 6 4.5 150.44 6 4.21 d (2) 99.40 6 2.8 179.84 6 3.4 103.84 6 3.2 175.99 6 3.97 d (3) 106.15 6 4.1 180.06 6 3.9 107.11 6 2.4 173.52 6 3.615 d (4) 87.54 6 5.7 165.26 6 6.1 85.93 6 5.7 168.41 6 6.930 d (5) 80.59 6 5.3 151.86 6 4.2 82.76 6 4.5 153.19 6 5.2
Statistical comparison Significance levels Significance levels
2 vs. 1 2 3 2 33 vs. 1 3 3 3 33 vs. 2 NS NS NS NS4 vs. 1 NS NS NS 24 vs. 2 NS NS 3 NS4 vs. 3 2 NS 3 NS5 vs. 1 NS NS NS NS5 vs. 2 2 3 3 35 vs. 3 3 3 3 25 vs. 4 NS NS NS 2
1Values are means 6 standard errors.2P < 0.05.3P < 0.01.
*** P < 0.001.
NS, not significant.
V. Fuentes-Santamar�ıa et al.
3494 The Journal of Comparative Neurology |Research in Systems Neuroscience
Touzani et al., 2002) or neuroprotective (Saavedra
et al., 2007) roles. Following noise exposure that ini-
tiates an inflammatory response in the inner ear and
induces hearing loss, increases in the levels of IL-1b
and Il-6 are observed between 3 and 6 hours post
lesion in adult rats (Fujioka et al., 2006). These findings
have led to the suggestion that cytokines are neuromo-
dulators induced by neuronal activity that regulate brain
function, and when cytokine activity caused by inflam-
mation of the cochlea is eliminated, hearing impairment
is improved (J€uttler et al., 2002; Fujioka et al., 2006;
Wakabayashi et al., 2010). In addition to the well-
known actions of IL-1b as an inflammatory and activity-
dependent signal, several studies have highlighted its
role in the modulation of growth factor levels and the
regulation of axonal sprouting by providing guidance
and trophic support to newly formed synaptic connec-
tions (Parish et al., 2002). This idea is supported by
recent findings demonstrating that simultaneous coad-
ministration of IL-1b and NT-3 to organotypic brain and
spinal-cord cultures increases neurite density and
length, suggesting that cytokines and growth factors
Figure 12. A–I: Digital images depicting the colocalization of IL-1b with neuronal and glial markers in PA7d animals. In both the AVCN
and PVCN, IL-1b–immunostained cells (asterisks in A,D,G) colocalized with the neuronal marker NeuN (arrows in B) but not with either the
astroglial marker GFAP (arrows in E) or the microglial marker CD11b (arrows in H). A magenta–green copy is available as Supplementary
Figure 12. Scale bar 5 20 lm in C (applies to A–C) and I (applies to D–I). [Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3495
may act synergistically during the regulation of axonal
plasticity (Boato et al., 2011).
Consistent with the aforementioned studies, our
findings show parallel increases in IGF-1 and IL-1b lev-
els at 1, 7, and 15 days following bilateral cochlear
ablation. We observed this upregulation in neurons but
not in either astrocytes or microglia during the posta-
blation time points studied, suggesting that the deaf-
ferentation paradigm used in the present study does
not require additional IGF-1 and IL-1b production by
glial cells, in contrast with other lesion models such
as ischemia (Touzani et al., 1999) and traumatic brain
injury (Rothwell and Luheshi, 2000; Madathil et al.,
2010).
In conclusion, the present findings indicate that
impairment of synaptic transmission by removal of
cochlear inputs leads to a series of cellular events
within the cochlear nucleus that activate both neurons
and non-neuronal cells such as microglia and astro-
cytes. Particularly, our results indicate that there is an
increased synthesis and secretion of IGF-1 and IL-1b in
neurons that may activate their respective receptors in
microglia and astrocytes. Interplay between these dis-
tinct activated cell types may lead to the induction of
synaptic repair mechanisms.
CONFLICT OF INTEREST STATEMENT
The authors declare that there are no conflicts of
interest including any financial, personal, or other rela-
tionships with other people or organizations within
three years of beginning the submitted work that could
inappropriately influence, or be perceived to influence,
their work.
ROLE OF AUTHORS
All authors had full access to all the data in the
study and take responsibility for the integrity of the
data and the accuracy of the data analysis. Study con-
cept and design: VFS and JCA. Acquisition of data: VFS,
JCA, and MCGU. Analysis and interpretation of data:
VFS and JCA. Drafting of the manuscript: VFS and JCA.
Critical revision of the manuscript for important intellec-
tual content: VFS, JCA, and JMJ. Statistical analysis:
VFS and JCA. Obtaining funding: VFS, JCA, and JMJ.
LITERATURE CITED
Aberg ND, Brywe KG, Isgaard J. 2006. Aspects of growth hor-mone and insulin-like growth factor-I related to neuropro-tection, regeneration, and functional plasticity in theadult brain. Sci World J 6:53–80.
Allan SM. 2000. The role of pro- and antiinflammatory cyto-kines in neurodegeneration. Ann N Y Acad Sci 917:84–93.
Altschuler RA, Cho Y, Ylikosk J, Pirvola U, Magal E, Miller JM.1999. Rescue and regrowth of sensory nerves followingdeafferentation by neurotrophic factors. Ann N Y AcadSci 884:305–311.
Alvarado JC, Fuentes-Santamaria V, Brunso-Bechtold JR, Hen-kel CK. 2004. Alterations in calretinin immunostaining inthe ferret superior olivary complex after cochlear abla-tion. J Comp Neurol 470:63–79.
Alvarado JC, Fuentes-Santamaria V, Franklin SR, Brunso-Bech-told JK, Henkel CK. 2007. Synaptophysin and insulin-likegrowth factor-1 immunostaining in the central nucleus ofthe inferior colliculus in adult ferrets following unilateralcochlear removal: a densitometric analysis. Synapse 61:288–302.
Alvarado JC, Fuentes-Santamaria V, Jare~no-Flores T, Blanco JL,Juiz JM. 2012. Normal variations in the morphology ofauditory brainstem response (ABR) waveforms: a study inwistar rats. Neurosci Res 73:302–311.
Bacci A, Verderio C, Pravettoni E, Matteoli M. 1999. The roleof glial cells in synaptic function. Philos Trans R SocLond B Biol Sci 354:403–409.
Bechmann I, Nitsch R. 2000. Involvement of non-neuronalcells in entorhinal-hippocampal reorganization followinglesions. Ann N Y Acad Sci 911:192–206.
Benson CG, Gross JS, Suneja SJ, Potashner SJ. 1997. Synap-tophysin immunoreactivity in the cochlear nucleus afterunilateral cochlear or ossicular removal. Synapse 25:243–257.
Boato F, Hechler D, Rosenberger K, L€udecke D, Peters EM,Nitsch R, Hendrix S. 2011. Interleukin-1 beta andneurotrophin-3 synergistically promote neurite growth invitro. J Neuroinflammation 8:183.
Boutin H, LeFeuvre RA, Horai R, Asano M, Iwakura Y, RothwellNJ. 2001. Role of IL-1alpha and IL-1beta in ischemicbrain damage. J Neurosci 21:5528–3554.
Breder CD, Dinarello CA, Saper CB. 1988. Interleukin-1 immu-noreactive innervation of the human hypothalamus. Sci-ence 240:321–324.
Bruce-Keller AJ. 1999. Microglial-neuronal interactions in syn-aptic damage and recovery. J Neurosci Res 58:191–201.
Camarero G, Avendano C, Fernandez-Moreno C, Villar A, Con-treras J, de Pablo F, Pichel JG, Varela-Nieto I. 2001.Delayed inner ear maturation and neuronal loss in post-natal Igf-1-deficient mice. J Neurosci 21:7630–7641.
Camarero G, Villar MA, Contreras J, Fern�andez-Moreno C,Pichel JG, Avenda~no C, Varela-Nieto I. 2002. Cochlearabnormalities in insulin-like growth factor-1 mousemutants. Hear Res 170:2–11.
Campos Torres A, Vidal PP, De Waele C. 1999. Evidence for amicroglial reaction within the vestibular and cochlearnuclei following inner ear lesion in the rat. Neuroscience92:1475–1490.
Campos-Torres A, Touret M, Vidal PP, Barnum S, De Waele C.2005. The differential response of astrocytes within thevestibular and cochlear nuclei following unilateral labyrin-thectomy or vestibular afferent activity blockade bytranstympanic tetrodotoxin injection in the rat. Neuro-science 130:853–865.
Canady KS, Rubel EW. 1992. Rapid and reversible astrocyticreaction to afferent activity blockade in chick cochlearnucleus. J Neurosci 12:1001–1009.
Cant NB, Benson CG. 2003. Parallel auditory pathways: pro-jection patterns of the different neuronal populations inthe dorsal and ventral cochlear nuclei. Brain Res Bull 60:457–474.
Cullheim S, Thams S. 2007. The microglial networks of thebrain and their role in neuronal network plasticity afterlesion. Brain Res Rev 55:89–96.
V. Fuentes-Santamar�ıa et al.
3496 The Journal of Comparative Neurology |Research in Systems Neuroscience
Debus E, Weber K, Osborn M. 1983. Monoclonal antibodiesspecific for glial fibrillary acidic (GFA) protein and foreach of the neurofilament triplet polypeptides. Differen-tiation 25:193–203.
Degger B, Upton Z, Soole K, Collet C, Richardson N. 2000.Comparison of recombinant barramundi and humaninsulin-like growth factor (IGF)-I in juvenile barramundi(Lates calcarifer): in vivo metabolic effects, associationwith circulating IGF-binding proteins, and tissue localisa-tion. Gen Comp Endocrinol 117:395–403.
De Waele C, Campos-Torres A, Josset P, Vidal PP. 1996. Evi-dence for reactive astrocytes in rat vestibular and coch-lear nuclei following unilateral inner ear lesion. Eur JNeurosci 8:2006–2018.
Feng J, Bendiske J, Morest DK. 2012. Degeneration in theventral cochlear nucleus after severe noise damage inmice. J Neurosci Res 90:831–841.
Fernandez AM, de la Vega AG, Torres-Aleman I. 1998. Insulin-like growth factor I restores motor coordination in a ratmodel of cerebellar ataxia. Proc Natl Acad Sci U S A 95:1253–1258.
Fogal B, Hewett SJ. 2008. Interleukin-1beta: a bridge betweeninflammation and excitotoxicity? J Neurochem 106:1–23.
Fuentes-Santamaria V, Alvarado JC, Brunso-Bechtold JK, Hen-kel CK. 2003. Upregulation of calretinin immunostainingin the ferret inferior colliculus after cochlear ablation. JComp Neurol 460:585–596.
Fuentes-Santamaria V, Alvarado JC, Taylor AR, Brunso-Bech-told JK, Henkel CK. 2005. Quantitative changes in calre-tinin immunostaining in the cochlear nuclei afterunilateral cochlear removal in young ferrets. J CompNeurol 483:458–475.
Fuentes-Santamaria V, Alvarado JC, Henkel CK, Brunso-Bech-told JK. 2007. Cochlear ablation in adult ferrets resultsin changes in insulin-like growth factor-1 and synapto-physin immunostaining in the cochlear nucleus. Neuro-science 148:1033–1047.
Fuentes-Santamaria V, Alvarado JC, Juiz JM. 2012. Long-terminteraction between microglial cells and cochlear nucleusneurons after bilateral cochlear ablation. J Comp Neurol520:2974–2990.
Fujioka M, Kanzaki S, Okano HJ, Masud M, Ogawa K, OkanoH. 2006. Proinflammatory cytokines expression in noise-induced damaged cochlea. J Neurosci Res 83:575–583.
Giulian D, Baker TJ, Shih LC, Lachman LB. 1986. Interleukin 1of the central nervous system is produced by ameboidmicroglia. J Exp Med 164:594–604.
Graeber MB, Kreutzberg GW. 1988. Delayed astrocyte reac-tion following facial nerve axotomy. J Neurocytol 17:209–220.
Guthrie KM, Nguyen T, Gall CM. 1995. Insulin-like growthfactor-1 mRNA is increased in deafferented hippocam-pus: spatiotemporal correspondence of a trophic eventwith axon sprouting. J Comp Neurol 352:147–160
Hanisch UK. 2002. Microglia as a source and target of cyto-kines. Glia 40:140–155.
Hanisch UK, Kettenmann H. 2007. Microglia: active sensorand versatile effector cells in the normal and pathologicbrain. Nat Neurosci 10:1387–1394.
Hendrix S, Peters EM. 2007. Neuronal plasticity and neurore-generation in the skin: the role of inflammation. J Neuro-immunol 184:113–126.
Hetier E, Ayala J, Denefle P, Bousseau A, Rouget P, Mallat M,Prochiantz A. 1988. Brain macrophages synthesizeinterleukin-1 and interleukin-1 mRNAs in vitro. J NeurosciRes 21:391–397.
Hildebrandt H, Hoffmann NA, Illing RB. 2011. Synaptic reor-ganization in the adult rat’s ventral cochlear nucleus
following its total sensory deafferentation. PLoS One 6:e23686.
Hill DJ, Hogg J, Petrik J, Arany E, Han VK. 1999. Cellular distri-bution and ontogeny of insulin-like growth factors (IGFs)and IGF binding protein messenger RNAs and peptidesin developing rat pancreas. J Endocrinol 160:305–317.
Hirose K, Discolo CM, Keasler JR, Ransohoff R. 2005. Mono-nuclear phagocytes migrate into the murine cochlea afteracoustic trauma. J Comp Neurol 489:180–194.
Insausti AM, Cruz-Orive LM, J�auregui I, Manrique M, InsaustiR. 1999. Stereological assessment of the glial reactionto chronic deafferentation of the cochlear nuclei in themacaque monkey (Macaca fascicularis). J Comp Neurol414:485–494.
Ishii DN, Glazner GW, Pu SF. 1994. Role of insulin-like growthfactors in peripheral nerve regeneration. Pharmacol Ther62:125–144.
Jensen MB, Gonz�alez B, Castellano B, Zimmer J. 1994. Micro-glial and astroglial reactions to anterograde axonaldegeneration: a histochemical and immunocytochemicalstudy of the adult rat fascia dentata after entorhinal per-forant path lesions. Exp Brain Res 98:245–260.
Jinno S, Yamada J. 2011. Using comparative anatomy in theaxotomy model to identify distinct roles for microglia andastrocytes in synaptic stripping. Neuron Glia Biol 7:55–66.
J�rgensen MB, Finsen BR, Jensen MB, Castellano B, DiemerNH, Zimmer J. 1993. Microglial and astroglial reactionsto ischemic and kainic acid-induced lesions of the adultrat hippocampus. Exp Neurol 120:70–88.
J€uttler E, Tarabin V, Schwaninger M. 2002. Interleukin-6 (IL-6):a possible neuromodulator induced by neuronal activity.Neuroscientist 8:268–275.
Kar S, Seto D, Dore S, Hanisch U, Quirion R. 1997. Insulin-like growth factors-I and -II differentially regulate endoge-nous acetylcholine release from the rat hippocampal for-mation. Proc Natl Acad Sci U S A 94:14054–14059.
Lechan RM, Toni R, Clark BD, Cannon JG, Shaw AR, DinarelloCA, Reichlin S. 1990. Immunoreactive interleukin-1 betalocalization in the rat forebrain. Brain Res 514:135–140.
Lind D, Franken S, Kappler J, Jankowski J, Schilling K. 2005.Characterization of the neuronal marker NeuN as a mul-tiply phosphorylated antigen with discrete subcellularlocalization. J Neurosci Res 79:295–302.
Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF, Shan Q.2011. Ursolic acid improves high fat diet-induced cogni-tive impairments by blocking endoplasmic reticulumstress and IjB kinase b/nuclear factor-jB-mediatedinflammatory pathways in mice. Brain Behav Immun 25:1658–1667.
Lurie DI, Durham D. 2000. Neuronal death, not axonal degen-eration, results in significant gliosis within the cochlearnucleus of adult chickens. Hear Res 149:178–188.
Lurie DI, Rubel EW. 1994. Astrocyte proliferation in the chickauditory brainstem following cochlea removal. J CompNeurol 46:276–288.
Madathil SK, Evans HN, Saatman KE. 2010. Temporal andregional changes in IGF-1/IGF-1R signaling in the mousebrain after traumatic brain injury. J Neurotrauma 27:95–107.
Maroso M, Balosso S, Ravizza T, Iori V, Wright CI, French J,Vezzani A. 2011. Interleukin-1b biosynthesis inhibitionreduces acute seizures and drug resistant chronic epilep-tic activity in mice. Neurotherapeutics 8:304–315.
Milligan CE, Cunningham TJ, Levitt P. 1991. Differential immu-nochemical markers reveal the normal distribution ofbrain macrophages and microglia in the developing ratbrain. J Comp Neurol 314:125–135.
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3497
Mishra A, Kim HJ, Shin AH, Thayer SA. 2012. Synapse lossinduced by interleukin-1b requires pre- and post-synaptic mechanisms. J Neuroimmune Pharmacol 7:571–578.
Morest DK, Kim J, Potashner SJBohne BA. 1998. Long-termdegeneration in the cochlear nerve and cochlear nucleusof the adult chinchilla following acoustic overstimulation.Microsc Res Tech 41:205–216.
Mrak REGriffin WS. 2005. Glia and their cytokines in pro-gression of neurodegeneration. Neurobiol Aging 26:349–354.
Mullen RJ, Buck CRSmith AM. 1992. NeuN, a neuronal spe-cific nuclear protein in vertebrates. Development 116:201–211.
Mully SM, Gross JS, Morest DK, Potashner SJ. 2002. Synapto-physin in the cochlear nucleus following acoustic trauma.Exp Neurol 177:202–221.
Parish CL, Finkelstein DI, Tripanichkul W, Satoskar AR, DragoJ, Horne MK. 2002. The role of interleukin-1, interleukin-6, and glia in inducing growth of neuronal terminalarbors in mice. J Neurosci 22:8034–8041.
Patel HC, Boutin H, Allan SM. 2003. Interleukin-1 in the brain:mechanisms of action in acute neurodegeneration. AnnN Y Acad Sci 992:39–47.
Pfrieger FW, Barres BA. 1997. Synaptic efficacy enhanced byglial cells in vitro. Science 277:1684–1687.
Potashner SJ, Suneja SK, Benson CG. 1997. Regulation of D-aspartate release and uptake in adult brain stem audi-tory nuclei after unilateral middle ossicle removal andcochlear ablation. Exp Neurol 148:222–235.
Prow NA, Irani DN. 2008. The inflammatory cytokine,interleukin-1 beta, mediates loss of astroglial glutamatetransport and drives excitotoxic motor neuron injury inthe spinal cord during acute viral encephalomyelitis. JNeurochem 105:1276–1286.
Ralay Ranaivo H, Craft JM, Hu W, Guo L, Wing, LK, Van EldikLJ, Watterson DM. 2006. Glia as a therapeutic target:selective suppression of human amyloid-beta-inducedupregulation of brain proinflammatory cytokine produc-tion attenuates neurodegeneration. J Neurosci 26:662–670.
Rasmussen S, Wang Y, Kivis€akk P, Bronson RT, Meyer M, Imi-tola J, Khoury SJ. 2007. Persistent activation of microgliais associated with neuronal dysfunction of callosal pro-jecting pathways and multiple sclerosis-like lesions inrelapsing–remitting experimental autoimmune encephalo-myelitis. Brain 130:2816–2829.
Reisert I, Wildemann G, Grab D, Pilgrim C. 1984. The glialreaction in the course of axon regeneration: a stereologi-cal study of the rat hypoglossal nucleus. J Comp Neurol229:121–128.
Robinson AP, White TM, Mason DW. 1986. Macrophage heter-ogeneity in the rat as delineated by two monoclonal anti-bodies MRC OX-41 and MRC OX-42, the latterrecognizing complement receptor type 3. Immunology57:239–247.
Rothwell NJ, Luheshi GN. 2000. Interleukin 1 in the brain:biology, pathology and therapeutic target. Trends Neuro-sci 23:618–625.
Rubel EW, MacDonald GH. 1992. Rapid growth of astrocyticprocesses in N. magnocellularis following cochlearemoval. J Comp Neurol 318:415–425.
Saavedra A, Baltazar G, Duarte EP. 2007. Interleukin-1betamediates GDNF up-regulation upon dopaminergic injuryin ventral midbrain cell cultures. Neurobiol Dis 25:92–104.
Salvinelli F, Casale M, Greco F, Trivelli M, Di Peco V,Amendola T, Antonelli A, Stampachiacchiere B, Aloe L.
2002. Nerve growth factor serum level is reduced inpatients with sensorineural hearing impairment: possi-ble clinical implications. J Biol Regul Homeost Agents16:176–180.
Schiefer J, Kampe K, Dodt HU, Zieglg€ansberger W, Kreutz-berg GW. 1999. Microglial motility in the rat facialnucleus following peripheral axotomy. J Neurocytol 28:439–453.
Shi L, Linville MC, Tucker EW, Sonntag WE, Brunso-BechtoldJK. 2005. Differential effects of aging and insulin-likegrowth factor-1 on synapses in CA1 of rat hippocampus.Cereb Cortex 15:571–577.
Slezak M, Pfrieger FW, Soltys Z. 2006. Synaptic plasticity,astrocytes and morphological homeostasis. J PhysiolParis 99:84–91.
Sozen T, Tsuchiyama R, Hasegawa Y, Suzuki H, Jadhav V,Nishizawa S, Zhang JH. 2009. Role of interleukin-1betain early brain injury after subarachnoid hemorrhage inmice. Stroke 40:2519–2525.
Strecker JK, Minnerup J, Gess B, Ringelstein EB, Sch€abitz WR,Schilling M. 2011. Monocyte chemoattractant protein-1-deficiency impairs the expression of IL-6, IL-1b and G-CSF after transient focal ischemia in mice. PLoS One 6:e25863.
Suneja SK, Yan L, Potashner SJ. 2005. Regulation of NT-3 andBDNF levels in guinea pig auditory brain stem nucleiafter unilateral cochlear ablation. J Neurosci Res 80:381–390.
Tetzlaff W, Graeber MB, Bisby MA, Kreutzberg GW. 1988.Increased glial fibrillary acidic protein synthesis in astro-cytes during retrograde reaction of the rat facial nucleus.Glia 1:90–95.
Touzani O, Boutin H, LeFeuvre R, Parker L, Miller A, Luhe-shi G, Rothwell N. 2002. Interleukin-1 influencesischemic brain damage in the mouse independentlyof the interleukin-1 type I receptor. J Neurosci 22:38–43.
Ullian EM, Sapperstein SK, Christopherson, KS, Barres BA.2001. Control of synapse number by glia. Science 291:657–661.
Vezzani A, Conti M, De Luigi A, Ravizza T, Moneta D, MerchesiF, De Simoni MG. 1999. Interleukin-1beta immunoreac-tivity and microglia are enhanced in the rat hippocampusby focal kainate application: functional evidence forenhancement of electrographic seizures. J Neurosci 19:5054–5065.
Vezzani A, Moneta D, Richichi C, Aliprandi M, Burrows SJ, Rav-izza T, Perego C, De Simoni MG. 2002. Functional role ofinflammatory cytokines and anti-inflammatory moleculesin seizures and epileptogenesis. Epilepsia 43(suppl 5):30–35.
Wakabayashi K, Fujioka M, Kanzaki S, Okano HJ, Shibata S,Yamashita D, Masuda M, Mihara M, Ohsugi Y, Ogawa K,Okano H. 2010. Blockade of interleukin-6 signaling sup-pressed cochlear inflammatory response and improvedhearing impairment in noise-damaged mice cochlea. Neu-rosci Res 66:345–352.
Wang Y, O’Donohue H, Manis P. 2011. Short-term plasticityand auditory processing in the ventral cochlear nucleusof normal and hearing-impaired animals. Hear Res 279:131–139.
Watt JA, Hobbs NK. 2000. Interleukin-1beta immunoreactivityin identified neurons of the rat magnocellular neurose-cretory system: evidence for activity-dependent release.J Neurosci Res 60:478–489.
Wei D, Jin Z, J€arlebark L, Scarfone E, Ulfendahl M. 2007. Sur-vival, synaptogenesis, and regeneration of adult mouse
V. Fuentes-Santamar�ıa et al.
3498 The Journal of Comparative Neurology |Research in Systems Neuroscience
spiral ganglion neurons in vitro. Dev Neurobiol 67:108–122.
White RE, McTigue DM, Jakeman LB. 2010. Regional heter-ogeneity in astrocyte responses following contusivespinal cord injury in mice. J Comp Neurol 518:1370–1390.
Wilms P, B€ahr M. 1995. Reactive changes in the adult ratsuperior colliculus after deafferentation. Restor NeurolNeurosci 9:21–34.
Winsky L, Jacobowitz DM. 1995. Effects of unilateral cochleaablation on the distribution of calretinin mRNA andimmunoreactivity in the guinea pig ventral cochlearnucleus. J Comp Neurol 354:564–582.
Yamanaka H, Kobayashi K, Okubo M, Fukuoka T, Noguchi K.2011. Increase of close homolog of cell adhesion mole-cule L1 in primary afferent by nerve injury and the con-tribution to neuropathic pain. J Comp Neurol 519:1597–1615.
Upregulation of IGF-1 and IL-1b in Cochlear Nucleus
The Journal of Comparative Neurology | Research in Systems Neuroscience 3499