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.

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