Long-term interaction between microglial cells and cochlear nucleus neurons after bilateral cochlear...

Long-Term Interaction Between Microglial Cells andCochlear Nucleus Neurons After Bilateral CochlearAblation

Ver�onica Fuentes-Santamarı́a,* Juan Carlos Alvarado, and Jos�e Manuel Juiz

Facultad de Medicina e Instituto de Investigaci�on en Discapacidades Neurol�ogicas (IDINE), Universidad de Castilla—La Mancha,

02006 Albacete, Spain

ABSTRACTThe removal of afferent activity has been reported to

modify neuronal activity in the cochlear nucleus of adult

rats. After cell damage, microglial cells are rapidly acti-

vated, initiating a series of cellular responses that influ-

ences neuronal function and survival. To investigate

how this glial response occurs and how it might influ-

ence injured neurons, bilateral cochlear ablations were

performed on adult rats to examine the short-term (16

and 24 hours and 4 and 7 days) and long-term (15, 30,

and 100 days) changes in the distribution and morphol-

ogy of microglial cells (immunostained with the ionized

calcium-binding adaptor molecule 1; Iba-1) and the

interaction of microglial cells with deafferented neurons

in the ventral cochlear nucleus. A significant increase in

the mean cross-sectional area and Iba-1 immunostain-

ing of microglial cells in the cochlear nucleus was

observed at all survival times after the ablation com-

pared with control animals. These increases were

concomitant with an increase in the area of Iba-1 im-

munostaining at 24 hours and 4, 7, and 15 days posta-

blation. Additionally, microglial cells were frequently

seen apposing the cell bodies and dendrites of auditory

neurons at 7, 15, and 30 days postablation. In sum-

mary, these results provide evidence for persistent glial

activation in the ventral cochlear nucleus and suggest

that long-term interaction occurs between microglial

cells and deafferented cochlear nucleus neurons follow-

ing bilateral cochlear ablation, which could facilitate the

remodeling of the affected neuronal circuits. J. Comp.

Neurol. 520:2974–2990, 2012.

VC 2012 Wiley Periodicals, Inc.

INDEXING TERMS: glia; deafferentation; neural plasticity; image analysis

Sensorineural hearing loss resulting from cochlear

ablation is a model commonly used to study the degener-

ative and reparative mechanisms that occur in the adult

central auditory system in response to damage to audi-

tory hair cells and their cochlear innervation (Bilak et al.,

1997; F€orster and Illing, 2000; Alvarado et al., 2007;

Fuentes-Santamaria et al., 2007). Studies performed with

different animal models have shown that, when the coch-

lea is damaged by a mechanical lesion or noise exposure,

the resulting loss of sensory cells is associated with a

gradual degeneration of the spiral ganglion neurons;

these neurons, when deprived of continuous neurotrophic

support (which is crucial to maintain appropriate neuronal

function), initiate a complex series of activity-dependent

changes in the auditory neurons found at subcortical and

cortical levels (for review see Syka, 2002). More specifi-

cally, in some neurons of the cochlear nucleus, these re-

gressive changes include cell atrophy (Moore, 1990; Tier-

ney et al., 1997), abnormalities in intracellular calcium

signaling pathways (Lohmann and Friauf, 1996; Caicedo

et al., 1997; Fuentes-Santamaria et al., 2005), and synap-

tic rearrangements (Potashner et al., 1997; Muly et al.,

2002), which may occur as early as 30 minutes postle-

sion (Sie and Rubel, 1992). These changes can result in

long-term degenerative processes that may develop into

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: Programa I3 del Ministerio de Ciencia e Innovaci�on;Grant number: I320101590 (to V.F.-S.); Grant number: I320101589 (toJ.C.A.); Grant sponsor: Gobierno de Castilla—La Mancha; Grant number:PE110901526233; Grant sponsor: Ministerio de Ciencia e Innovaci�on;Grant number: BFU2009-13754-C02-01.

*CORRESPONDENCE TO: Ver�onica Fuentes-Santamarı́a, PhD, Facultadde Medicina, Universidad de Castilla—La Mancha, Campus de Albacete,C/ Almansa 14, 02006 Albacete, Spain. E-mail: [email protected]

VC 2012 Wiley Periodicals, Inc.

Received November 22, 2011; Revised December 23, 2011; AcceptedFebruary 15, 2012

DOI 10.1002/cne.23088

Published online February 20, 2012 in Wiley Online Library(wileyonlinelibrary.com)

2974 The Journal of Comparative Neurology | Research in Systems Neuroscience 520:2974–2990 (2012)

RESEARCH ARTICLE

neurodegenerative disorders under chronic conditions

(Morest et al., 1998).

A growing body of evidence suggests that microglial

activation is a hallmark of several neurological disorders

that feature neuronal degeneration (for reviews see Mrak

and Griffin, 2005; Dheen et al., 2007). In response to neu-

ronal injury, microglial cells (the macrophage population

of the central nervous system) concentrate at the site of

injury, become rapidly activated, and initiate a series of

cellular responses, including morphological transforma-

tion and proliferation and the synthesis and release of

growth factors and cytokines, which may play a pivotal

role in promoting the remodeling of affected neuronal cir-

cuits (for reviews see Bruce-Keller, 1999; Hanisch and

Kettenmann, 2007; Cullheim and Thams, 2007). Although

these nonneuronal elements appear to exert primarily

neuroprotective effects (especially following localized

damage), when injury is sustained and irreversible they

become persistently activated and can have deleterious

effects on neuronal function and survival (Streit, 2000;

Nakajima and Kohsaka, 2001).

Within the auditory system, the unilateral removal of

afferent activity results in a reactive microglial and astro-

cytic response in the cochlear nucleus, the magnitude

and intensity of which decreases over time, depending on

a combination of factors, such as the species, the age at

which deafferentation occurred, survival time, and type of

deprivation (Lurie and Rubel, 1994; De Waele et al.,

1996; Campos-Torres et al., 1999; Insausti et al., 1999;

Lurie and Durham, 2000; Zhao and Lurie, 2004). The

importance of such glial activation in modulating cell sur-

vival is highlighted by the fact that blockage of certain

cytokines released by microglial cells under normal condi-

tions (Fujioka et al., 2006) eliminates the inflammatory

response that occurs in the cochlea after noise exposure

and decreases hearing impairment in mice (Wakabayashi

et al., 2010). Despite the increasing evidence suggesting

the involvement of glial-related mechanisms in restoring

neuronal function after injury, none of these studies has

addressed how microglial cells become activated in the

cochlear nucleus following bilateral sensorineural hearing

loss. In the present study, we performed bilateral coch-

lear ablations in adult rats to examine short-term (16 and

24 hours and 4 and 7 days) and long-term (15, 30, and

100 days) changes in the distribution and morphology of

microglial cells immunostained with the ionized calcium-

binding adaptor molecule 1 (Iba-1).

MATERIALS AND METHODS

Animal subjectsAll animal protocols were approved by the Institutional

Animal Care and Use Committee at the University of Cas-

tilla—La Mancha. These protocols were in accordance

with the guidelines of the European Communities Council

(directive 2010/63/EU) and current national legislation

(R.D. 1201/2005) for the care and use of research ani-

mals. Data were obtained from 35 adult rats (28 experi-

mental and seven age-matched unoperated control rats).

After bilateral cochlear ablation, the experimental animals

survived for 16 hours (PA16h; n ¼ 4), 24 hours (PA24h; n

¼ 4), 4 days (PA4d; n ¼ 4), 7 days (PA7d; n ¼ 4), 15 days

(PA15d; n ¼ 4), 30 days (PA30d; n ¼ 4), or 100 days

(PA100d; n ¼ 4).

Auditory brainstem responsesThe auditory brainstem response (ABR) recordings

were performed on the day before the bilateral cochlear

ablation and at the end of each survival time. Rats were

anesthetized with isoflurane (4% for induction, 1.5–2% for

maintenance with a 1 L/minute O2 flow rate) and placed

in a sound-attenuating, electrically shielded booth

(Eymasa/Incotron S.L., Barcelona, Spain), which was

located inside of a sound-attenuating room. Subdermal

needle electrodes (Rochester Electro-Medical, Tampa,

FL) were placed at the vertex (positive) and behind 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 using the Sig-

GenRP software (Tucker-Davis Technologies) and the RX6

Piranha Multifunction Processor hardware (Tucker-Davis

Technologies) and consisted of tones (5 msec rise/fall

time with no plateau, with a cos2 envelope, at 20/sec-

ond) at different frequencies across seven octaves from

0.5 to 32 KHz. They were delivered monaurally (right ear)

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 the rat. Prior to the experi-

ments, stimuli were calibrated with SigCal software

(Tucker-Davis Technologies) and the ER-10Bþ low-noise

microphone system (Etymotic Research, Elk Groove, IL).

The evoked potentials were filtered (0.3–3.0 kHz), aver-

aged (500 waveforms), and stored for later analyses on a

computer.

Procedure of bilateral cochlear ablationExperimental animals were anesthetized with isoflur-

ane as described above (4% for induction, 1.5–2% for

maintenance). Under aseptic conditions and as previously

described (Fuentes-Santamaria et al., 2003; Alvarado

et al., 2004), the skin behind the ears was shaved, and a

retroauricular incision was made to identify the external

auditory canal, which was followed to the tympanic mem-

brane. Once the tympanic membrane and the auditory

Glial activation in the cochlear nucleus after deafferentation

The Journal of Comparative Neurology | Research in Systems Neuroscience 2975

ossicles had been removed, the bulla was exposed, the

cochleus was removed with a fine forceps, and any

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 for recovery from anesthesia. Once awake, ani-

mals were returned to their cages and maintained with

free access to food and water for the survival period.

After the appropriate survival time, the animals were

perfused (see below), and the extent of the cochlear abla-

tions was microscopically assessed.

Characterization of antibodiesThe monoclonal and polyclonal antibodies used in this

study were obtained from commercial sources and are

listed in Table 1.

Iba-1Rabbit polyclonal anti-Iba-1 was raised against a syn-

thetic peptide corresponding to the C-terminal fragment

of rat Iba-1 (manufacturer’s technical information; see

also Table 1). This antibody was purified by using antigen-

affinity chromatography from rabbit antisera and recog-

nizes a single 17-kD band that corresponds to Iba protein

on Western blot analysis of rat brain (Imai et al., 1996). It

specifically labels ramified and activated microglia and

macrophages but not neurons and astrocytes by immuno-

histochemistry in both primary mixed brain cell cultures

and fixed rat brain sections (Ito et al., 1998). In this study,

the staining pattern of this antibody was in accordance

with previous observations for the auditory system

(Okano et al., 2008) as well as for the hippocampus (Su

et al., 2010), spinal cord (Benton et al., 2008; Yamanaka

et al., 2011) and retina (Bosco et al., 2011). This antibody

is included in the Journal of Comparative Neurology

primary antibody database (ID No. AB 839504).

NeuNThe mouse monoclonal antineuronal nuclei antibody

(NeuN) was produced by using purified cell nuclei from

mouse brain (manufacturer’s technical information; see

also Table 1). This antibody recognizes the neuron-spe-

cific protein NeuN, which is present in most neuronal cell

types of the central and peripheral nervous systems (Ras-

mussen et al., 2007; Ravizza et al., 2008). It does not

stain glial cells (Todd et al., 1998). According to the man-

ufacturer and previous studies (Mullen et al., 1992; Lind

et al., 2005), it recognizes four bands between 45 and 75

kDa on Western blots of rat brain, which may represent

multiple phosphorylation states of the protein. The stain-

ing pattern of this antibody agrees with previous observa-

tions from our region of interest, the adult cochlear nu-

cleus (Zheng et al., 2011), and also from the auditory

cortex (Hackett and de la Mothe, 2009). This antibody is

also included in the Journal of Comparative Neurology pri-

mary antibody database (ID No. AB-2298770).

Iba-1 immunohistochemistryAfter the appropriate postoperative survival time, con-

trol and experimental rats were anesthetized with an

intraperitoneal injection of ketamine (100 mg/kg) and

xylazine (5 mg/kg) and perfused transcardially with a

0.9% saline wash, followed by a fixative solution of 4% par-

aformadehyde in 0.1 M phosphate buffer (PB; pH 7.3).

The brains were removed from the cranium, and frozen

sections 40 lm thick were cut on a sliding microtome in

a coronal plane. Free-floating sections were processed in

three alternating series; the first series of sections was

processed for Iba-1 immunostaining, and the other two

series were used for double-labeling studies (see below)

and NeuN immunohistochemistry, respectively. After

blocking for 1 hour in a solution containing 10% normal

horse serum diluted in Tris-buffered saline (TBS; pH 7.4)

with 0.2% Triton X-100 (TBS-Tx 0.2%), sections were incu-

bated overnight at 4�C in the same buffer solution with

rabbit anti-Iba-1 primary antibody. On the next day, sec-

tions were washed in TBS-Tx 0.2% solution and incubated

for 2 hours at room temperature in biotinylated anti-rab-

bit secondary antibody (1:200; Vector Laboratories, Bur-

lingame, CA). Then, after several rinses in TBS-Tx (0.2%),

sections were incubated with the avidin-biotin complex

for 1 hour, rinsed in TBS, and treated with diaminobenzi-

dine (DAB) histochemistry. The exposure time to DAB was

similar across control and experimental cases. Finally,

the sections were washed thoroughly, mounted on gela-

tin-coated slides, air dried, dehydrated in ethanol, cleared

in xylene, and coverslipped with Cytoseal (Stephens Sci-

entific, Wayne, NJ). Three sets of control experiments

TABLE 1.

Antibodies Used for Immunohistochemistry

Primary antibody Immunogen Host Code/clone Dilution Manufacturer

Iba-1 C-terminus of Iba10

(N0-PTGPPAKKAISELP-C0)Rabbit 019-19741, lot No. CDQ5232 1:2,000 Wako Pure Chemical Industries,

Neuss, GermanyNeuN Purified cell nuclei

from mouse brainMouse MAB337, lot No. LV1825845 1:200 Millipore, Billerica, MA

Fuentes-Santamarı́a et al.

2976 The Journal of Comparative Neurology |Research in Systems Neuroscience

were performed to test the specificity of the immunohis-

tochemical detection system: 1) omission of the primary

antibody by replacement with TBS-BSA, 2) omission of

secondary antibodies, and 3) omission of ABC reagent.

No immunostaining was detected under these conditions.

Double labeling for glial and neuronalmarkers

Sections were rinsed four times in TBS-Tx (0.2%) and

blocked for 1 hour in the same buffer solution containing

10% normal goat serum. Next, sections were incubated

overnight in a solution of primary antibodies containing

Iba and NeuN. After four 15-minute rinses in TBS-Tx

(0.2%), sections were incubated in a cocktail of fluores-

cently labeled secondary antibodies for 2 hours at room

temperature (1:200, anti-mouse conjugated to Alexa 594

for NeuN and anti-rabbit conjugated to Alexa 488 for Iba-

1; Molecular Probes, Eugene, OR), and after several

rinses in TBS they were mounted, counterstained with

DAPI, coverslipped, and maintained overnight at 4�C.

Measurements of the cross-sectional area ofIba-immunostained cells

The cross-sectional area of Iba-immunostained cells in

both control and ablated animals was measured using the

public-domain image analysis software Scion Image for

Windows (Scion, Frederick, MD; beta 4.0.2). With a �60

objective, three fields (25.16 � 103 lm2; dorsal, middle,

and ventral) were sampled randomly in every third section

throughout the rostrocaudal extent of the anteroventral

cochlear nucleus (AVCN) and the posteroventral cochlear

nucleus (PVCN). Only cells with a well-defined cell body

were measured and included in the analysis.

Evaluation of Iba-1 immunostainingDigital image capture

Iba-1-immunostained sections from control and experi-

mental animals were examined with brightfield illumina-

tion using a Nikon Eclipse photomicroscope with a �40

objective, and images were captured with a DXM 1200C

digital camera attached to the microscope. Color images

of each field were digitized, and the resultant eight-bit

image contained a gray scale of pixel intensities that

ranged from 0 (white) to 255 (black).

Densitometric analysisThe densitometric procedure for the evaluation of the

immunostaining was performed by using Scion Image for

Windows (beta 4.0.2; developed by Scion Corp.), as previ-

ously described (Fuentes-Santamaria et al., 2003, 2005,

2007; Alvarado et al., 2004, 2005, 2007). Cochlear nu-

cleus subdivisions were defined in accordance with previ-

ous terminology (for review see Cant and Benson, 2003).

The analysis of Iba-1 immunostaining was performed in

six equally spaced coronal sections, 120 lm apart,

extending throughout the rostrocaudal dimension of the

AVCN and PVCN. In each section, three fields (55.25 �103 lm2; dorsal, middle, and ventral) were sampled using

a �40 objective, for a total of 36 fields for each nucleus

in each animal. To perform an appropriate comparison of

Iba-1 immunostaining among cases, a macro was

designed to process and analyze the captured images

(Alvarado et al., 2004). Images were normalized by using

an algorithm based on the signal-to-noise ratio that nor-

malizes each pixel, adjusting the gray-scale range of the

image (Herborn et al., 2002; Alvarado et al., 2004). After

normalization, the threshold level was set as 2 standard

deviations above the mean gray level of the field, and im-

munostained cells exceeding this threshold were identi-

fied as labeled. For each field, two quantitative indexes

were measured in the cochlear nucleus. 1) The mean

gray of Iba-1 immunostaining was used as an indirect in-

dicator of the levels of Iba-1 within cells. Modifications in

antigen concentration (Iba-1) are reflected in changes in

the intensity of the immunostaining, so this index pro-

vides a general estimation of the effect of bilateral coch-

lear ablation on the immunostaining of glial cells. 2) The

immunostained area of Iba-1 immunostaining was calcu-

lated as the summed area of all profiles (cells and proc-

esses) labeled as being above the threshold in each field

and gives us an estimate of the area occupied by micro-

glial cells at each survival time in comparison with control

rats.

Preparation of figures and statisticalanalysis

Adobe Photoshop 5.5 and Deneba Canvas 6.0 were

used to adjust size, brightness, and contrast of images

for publication. All the data were expressed as mean 6

standard error. Comparisons among groups were ana-

lyzed statistically by using the one-factor analysis of var-

iance and the Duncan’s post hoc analysis to evaluate the

effect of the survival time after the cochlear ablation over

the microglial immunostaining in the cochlear nucleus.

Statistical significance was set at P < 0.05.

RESULTS

Effects of bilateral cochlear ablationon hearing

To confirm that the bilateral cochlear ablations were

complete, ABR recordings were performed in experimen-

tal animals before (preablation ABR) and after (postabla-

tion ABR) ablation for each of the time points described

above. To eliminate the inclusion of animals already



suffering from hearing disorders, basal patterns for the

ABR recordings were established for each experimental

group. Preablation recordings showed a distinctive wave

pattern characterized by positive peaks generated after

a stimulus (left column in Fig. 1). The postablation ABRs

showed a complete elimination of the waves evoked af-

ter stimulus onset at all of the frequencies tested, indi-

cating that the auditory brainstem nuclei were not

receiving inputs from the cochlea (right column in Fig.

1). Figure 1 shows an example of an ABR after a short

(16 hours; Fig. 1A) or long (100 days; Fig. 1B) period fol-

lowing bilateral cochlear ablation. In both cases, the

normal preablation ABR was replaced by a nearly flat

postablation ABR.

Figure 1. ABR recordings depicting the effects of bilateral cochlear ablation in adult rats. In the experimental animals, ABR recordings

were performed before (ABR preablation, left column) and after (ABR postablation, right column) each survival time following bilateral

cochlear ablation. Note that at short-term (16 hours; A) and long-term (100 days; B) survival times following bilateral cochlear ablation,

the ABRs postablation showed a complete loss of response waves after stimulus onset at all of the frequencies tested, indicating that the

auditory brainstem nuclei did not receive any electrical stimulation arising from the cochlea. The dashed lines indicate stimulus onset.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



Distribution and quantification ofIba-immunostaining in the cochlear nucleusControl animals

In the control animals, analysis of coronal sections im-

munostained for Iba-1 showed that microglial cells were

distributed in both the AVCN (Fig. 2B) and the PVCN

(Fig. 3B). These cells had small, oval cell bodies (Fig. 4A,B,

Tables 2, 3) with processes that were highly ramified,

reflecting the typical staining pattern of resting microglia

(i.e., multipolar or, less frequently, bipolar morphology;

Fig. 5A,I). The distribution and morphology of these cells

were consistent with previously reported observations of

microglia in auditory nuclei (Campos-Torres et al., 1999).

Densitometric analysis of the immunostaining is shown in

Figure 6C,D and Tables 2 and 3.

Ablated animalsAfter bilateral cochlear ablation, the distribution and

morphology of Iba-1-immunostained cells observed in the

cochlear nucleus were different compared with control

Figure 2. Digital images depicting Iba-1 immunostaining in the AVCN in control (B), PA16h (C), PA24h (D), PA4d (E), PA7d (F), PA15d (G),

PA30d (H), and PA100d (I) rats. Iba-1 immunostaining was observed to increase by 16 hours postlesion (arrows in C); peaked at about 24

hours (arrows in D); and remained elevated at 4 (arrows in E), 7 (arrows in F), and 15 (arrows in G) days postlesion. Iba-1 levels began to

decline gradually by 30 and 100 days postlesion, without reaching normal levels (arrows in H,I). In A, the drawing indicates the location of

the AVCN, and the square boxes indicate the approximate locations of the fields represented in B–I. stt, Spinal trigeminal tract. Scale bar

¼ 500 lm in A; 50 lm in I (applies to B–I). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



animals (Figs. 2, 3). To characterize and quantify the

changes in microglial activation following bilateral coch-

lear ablation, three quantitative indices of Iba-1 immuno-

staining were evaluated in both the experimental and the

control animals: 1) the cross-sectional area of Iba-1-im-

munostained cells, as a measure of hypertrophic changes

occurring during microglial activation; 2) the mean gray

level of Iba-1 immunostaining, as an indirect indicator of

the levels of Iba-1 within cells; and 3) the Iba-1-immuno-

stained area, as an estimate of the area occupied by

microglial cell bodies and their processes in the cochlear

nucleus (for technical details see Materials and Methods).

Postablation 16 hoursA transition from resting-state to active-state microglia

was observed at 16 hours following bilateral cochlear

ablation. Microglial cells in the AVCN (Fig. 2C) and PVCN

(Fig. 3C) had larger cell bodies and thicker processes in

comparison with control rats (compare Figure 2C with B

in the AVCN and Figure 3C with B in the PVCN; see also

Figure 3. Digital images depicting Iba-1 immunostaining in the PVCN in control (B), PA16h (C), PA24h (D), PA4d (E), PA7d (F), PA15d (G),

PA30d (H), and PA100d (I) rats. Similar to the increase observed in the AVCN, an increase in Iba-1 immunostaining was first noticeable in

PA16h animals (arrows in C), although the highest levels were observed at about 24 hours postlesion (arrows in D). The immunostaining

remained elevated at 4 (arrows in E), 7 (arrows in F), and 15 (arrows in G) days postlesion, and staining began to decline gradually at lon-

ger survival times, compared with control animals (arrows in H,I). The boxed area in A indicates the approximate location of the fields rep-

resented in B–I. stt, Spinal trigeminal tract; icp, inferior cerebellar peduncle. Scale bar ¼ 500 lm in A; 50 lm in I (applies to B–I). [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



Figs. 4, 5B,J). Statistical analysis revealed that there was

a significant increase in the microglial cross-sectional

area in both the AVCN and the PVCN compared with the

control animals (Fig. 4, Tables 1, 2). This test also indi-

cated that the mean gray levels of Iba-1 immunostaining

(Tables 1, 2) and the extent of the immunostained areas

also increased significantly in both nuclei compared with

the control animals (Fig. 6, Tables 1, 2).

Postablation 24 hoursBy 24 hours postablation, the majority of the microglial

cells had adopted a phenotype characterized by irregu-

larly shaped, hypertrophic cell bodies, with very few short

processes, compared with PA16h and control animals

(arrows in Figs. 2D, 3D; see also Fig. 4). Some of these

cells were characterized by rounded cell bodies and a

loss of processes, which was reminiscent of macrophage

cells (arrows in Fig. 5C,K). Post hoc analysis indicated

that the cross-sectional areas of the Iba-1-immuno-

stained cells were significantly increased compared with

PA16h and control rats (Fig. 4, Tables 1, 2). Also, at this

survival time, microglial cell immunostaining was much

darker, as confirmed by an increase in the mean gray lev-

els of Iba-1 immunostaining in comparison with PA16h

and control animals (Fig. 6A,B, Tables 1, 2). Similarly, the

quantification of the extent of the Iba-1-immunostained

areas revealed a significant increase in comparison with

PA16h and control animals, likely resulting from an

increase in microglial cell density (Fig. 6C,D, Tables 1, 2).

Postablation 4 daysAt 4 days postablation, the microglial cells had devel-

oped a morphology characterized by cell bodies that

Figure 4. Bar graphs showing the mean cross-sectional areas of

the Iba-1-immunostained cells in the ventral cochlear nucleus in

the control rats. There were significant increases in microglial cell

areas in both the AVCN (A) and the PVCN (B) at all of the survival

times studied compared with the control animals. The error bars

indicate the standard errors of the mean.

TABLE 2.

Iba-1 Immunostaining in the AVCN in Control and

Ablated Animals1

AVCN

Survival time CSA (lm2) MGL ISA (lm2)

Control (1) 34.9 6 0.8 124.3 6 1.9 2,592.2 6 51.916h (2) 49.9 6 1.2 133.1 6 2.4 2,812.3 6 44.824h (3) 74.9 6 1.9 173.1 6 2.4 3,968.2 6 32.44d (4) 56.6 6 1.6 178.2 6 3.3 3,240.9 6 55.67d (5) 53.3 6 1.2 177.1 6 2.1 3,164.7 6 48.615d (6) 54.0 6 1.4 182.7 6 2.9 3,166.1 6 55.930d (7) 51.5 6 1.3 177.7 6 3.4 2,946.5 6 103.7100d (8) 48.2 6 1.1 160.4 6 4.6 2,832.9 6 70.1

Statisticalcomparison

Significance levels

2 vs. 1 *** * **3 vs. 1 *** *** ***3 vs. 2 *** *** ***4 vs. 1 *** *** ***4 vs. 2 *** *** ***4 vs. 3 *** NS ***5 vs. 1 *** *** ***5 vs. 2 NS *** ***5 vs. 3 *** NS ***5 vs. 4 NS NS NS6 vs. 1 *** *** ***6 vs. 2 NS *** ***6 vs. 3 *** NS ***6 vs. 4 NS NS NS7 vs. 1 *** *** ***7 vs. 2 NS *** NS7 vs. 3 *** NS ***7 vs. 4 * NS ***7 vs. 5 NS NS *7 vs. 6 NS NS *8 vs. 1 *** *** **8 vs. 2 NS *** NS8 vs. 3 *** *** ***8 vs. 4 *** *** ***8 vs. 5 * *** ***8 vs. 6 ** *** ***8 vs. 7 NS *** NS

1Values are means 6 standard errors. CSA, cross-sectional area of

Iba-1-immunostained cells; MGL, mean gray level of Iba-1 immuno-

staining; ISA, immunostained area of Iba-1.

*P < 0.05.

**P < 0.01.

***P < 0.001.

NS, not significant.



were hypertrophic and from which emanated stout short

processes (Figs. 2–5). The Iba-1-immunostained cells

were often observed surrounding blood vessels (asterisk

in Fig. 5E). Quantitative analysis revealed a significant

decrease in the cross-sectional area of the cells com-

pared with PA24h rats and a significant increase com-

pared with PA16h and control animals (Fig. 4, Tables 1,

2). Analysis of the mean gray levels of Iba-1 immunostain-

ing indicated that there were significant increases in com-

parison with PA16h and control animals, but no changes

were observed compared with PA24h animals (Fig. 6A,B,

Tables 1, 2). However, the immunostained areas were sig-

nificantly decreased compared with PA24h animals and

were increased compared with PA16h and control rats

(Fig. 6C,D, Tables 1, 2).

Postablation 7 and 15 daysIn PA7d and PA15d rats, most of the microglial cells

still had relatively large cell bodies with thick, short proc-

esses (Figs. 2F,G, 3F,G). Statistical analysis demon-

strated that the cross-sectional area of these cells was

significantly decreased in PA7d and PA15d animals com-

pared with PA24h rats (Fig. 4, Tables 1, 2). Conversely,

there was an increase in size compared with control ani-

mals (Fig. 4, Tables 1, 2). At 7 and 15 days postablation,

the microglial cells were still darkly immunostained, as

indicated by significant increases in the mean gray levels,

in comparison with PA16h and control animals (Fig. 6A,B,

Tables 1, 2). With respect to the immunostained areas in

the AVCN at 7 and 15 days postlesion, significant

decreases were observed compared with PA24h rats, and

significant increases were observed compared with

PA16h and control animals (Fig. 6C,D, Tables 1, 2). In the

PVCN, there were also significant decreases in the immu-

nostained areas compared with PA24h animals and signif-

icant increases compared with control rats (Tables 1, 2).

Although microglial clusters were observed at all of the

survival times evaluated, they were particularly evident at

7 and 15 days postablation (Fig. 5N).

Postablation 30 daysIn PA30 animals, microglial cells with longer processes

showed statistically significant decreases in cross-sec-

tional area in the AVCN in comparison with PA4d and

PA24h cells but showed significant increases in cross-

sectional area compared with control animals (Fig. 4,

Tables 1, 2). In the PVCN, the microglial cross-sectional

area was significantly decreased compared with PA7d,

PA4d, and PA24h animals and significantly increased

compared with PA16h and control rats (Fig. 4, Tables 1,

2). At both survival times, the mean gray levels of Iba im-

munostaining were significantly higher in comparison

with PA16h and control animals (Fig. 6A,B, Tables 1, 2).

With respect to the extents of the immunostained areas

at 30 days postlesion, significant decreases were

observed compared with PA15d, PA7d, PA4d, and PA24h

animals; this was especially evident in the AVCN, in which

the cross-sectional area was greater than that of control

animals (Fig. 6C,D, Tables 1, 2).

Postablation 100 daysIn PA100d animals, the staining and morphologic char-

acteristics of the microglial cells had returned nearly to

normal (Figs. 2I, 3I). The cross-sectional areas of the

TABLE 3.

Iba-1 Immunostaining in the PVCN in Control and

Ablated Animals1

AVCN

Survival time CSA (lm2) MGL ISA (lm2)

Control (1) 36.7 6 0.8 129.9 6 7.4 2,612.3 6 58.716h (2) 45.6 6 1.4 142.3 6 1.4 2,851.6 6 28.224h (3) 68.4 6 1.9 189.4 6 2.8 4,095.1 6 71.44d (4) 59.2 6 1.3 185.7 6 2.7 3,367.8 6 60.67d (5) 57.7 6 1.3 184.8 6 3.0 2,982.4 6 68.215d (6) 54.9 6 1.3 193.7 6 2.6 3,039.6 6 80.730d (7) 52.4 6 1.2 194.1 6 3.2 2,740.8 6 148.8100d (8) 45.3 6 1.0 193.2 6 2.2 2,635.6 6 74.6

Statisticalcomparison

Significance levels

2 vs. 1 *** ** *3 vs. 1 *** *** ***3 vs. 2 *** *** ***4 vs. 1 *** *** ***4 vs. 2 *** *** ***4 vs. 3 *** NS ***5 vs. 1 *** *** ***5 vs. 2 NS *** NS5 vs. 3 *** NS ***5 vs. 4 NS NS NS6 vs. 1 *** *** ***6 vs. 2 NS *** NS6 vs. 3 *** NS ***6 vs. 4 NS NS NS7 vs. 1 *** *** NS7 vs. 2 *** *** NS7 vs. 3 *** NS ***7 vs. 4 *** NS ***7 vs. 5 *** NS *7 vs. 6 NS NS ***8 vs. 1 *** *** NS8 vs. 2 NS *** NS8 vs. 3 *** NS ***8 vs. 4 *** NS ***8 vs. 5 *** NS *8 vs. 6 *** NS ***8 vs. 7 *** NS NS

1Values are means 6 standard errors. CSA, cross-sectional area of

Iba-1-immunostained cells; MGL, mean gray level of Iba-1 immuno-

staining; ISA, immunostained area of Iba-1.

**P < 0.05.

**P < 0.01.

***P < 0.001.

NS, not significant.



microglial cells were decreased in comparison with

PA15d, PA7d, PA4d, and PA24h and were increased in

comparison with control animals (Fig. 4, Tables 1, 2). In

the PVCN, the glial cells also had decreased cross-sec-

tional areas in comparison with PA30d (Table 2). Quantifi-

cation of the mean gray levels of Iba-1 immunostaining in

the AVCN indicated that they were decreased compared

with PA30d, PA15d, PA7d, PA4d, and PA24h animals and

increased compared with PA16h and control rats (Fig. 6A,

Table 1). However, in the PVCN, there was an increase in

the mean gray levels compared with PA16h and control

rats, but no differences were found compared with the

other survival times (Fig. 6B, Table 2). Densitometric anal-

ysis of the Iba-1-immunostained areas revealed

decreases compared with PA15d, PA7d, PA4d, and

PA24h animals (Fig. 6C,D, Tables 1, 2). In contrast,

Figure 5. High-magnification digital images depicting Iba-1-immunostained cells in the AVCN and PVCN in the control rats and after bilat-

eral cochlear ablation. In the control rats, the microglial cells presented a multipolar or, less frequently, a bipolar morphology with highly

ramified processes (A,I). By 16 hours postlesion, Iba-1-immunostained cells had larger cell bodies and thicker processes compared with

control animals (B,J). One day after ablation, the cells adopted a phenotype characterized by irregularly shaped hypertrophic cell bodies

with very few short processes or no processes (arrows in C,K). In PA4d (D,L) and PA7–15d (F,M,N) animals, the microglial cells remained

hypertrophic, and their short processes were more noticeable. Note that, in PA4d animals, the microglial cells were frequently seen sur-

rounding blood vessels (asterisk in E). The presence of microglial clusters was especially evident in PA7d and PA15d animals (N). In

PA30d (G,O) and PA100d (H,P) animals, Iba-1-immunostained cells had decreased cell area and ramified processes, although their mor-

phologic characteristics were still different compared with control animals (A,I). Scale bar ¼ 10 lm. [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]



densitometric analysis of the Iba-1-immunostained areas

revealed increases in the AVCN compared with control

animals (Fig. 6C,D, Table 1).

Long-term appositions between microglialcells and cochlear nucleus neurons afterbilateral cochlear ablation

To determine the spatial relationship between micro-

glial cells and deafferented cochlear nucleus neurons, we

performed double-labeling experiments for microglial and

neuronal markers in both control and experimental ani-

mals. In control (white arrows and yellow asterisks in

Figs. 7B, 8B) and PA16h (white arrows and yellow aster-

isks in Figs. 7C, 8C) rats, microglial processes in both the

AVCN and the PVCN were rarely seen apposing cochlear

nucleus neurons. However, by 24 hours postablation,

activated microglial cell bodies were observed in close

apposition with the cochlear nucleus neurons (white

arrows and yellow asterisks in Figs. 7D, 8D). Such cellular

appositions were particularly evident at later survival

times and were found in PA4d (Figs. 7E, 8E), PA7d (Figs.

7F, 8F), and PA15d (Figs. 7G, 8G) animals, in which the

microglial cells (perineuronal microglia) with short or no

processes were most frequently observed apposing the

soma and dendrites of deafferented cochlear nucleus

neurons in both the AVCN and the PVCN. It is important

to note that these microglial–neuronal appositions

decreased in frequency by day 30 (Figs. 7H, 8H) but per-

sisted for at least 100 days (Figs. 7I, 8I) following bilateral

cochlear ablation in comparison with control animals, at

which time the staining and morphological characteristics

of the microglial cells had not yet returned to normal.

However, at all of the time points studied, the microglia

cells were also found in the neuropil without being closely

associated with cochlear nucleus neurons (Figs. 7, 8).

DISCUSSION

The process of microglial activation in the ventral coch-

lear nucleus of the adult rat was investigated after bilat-

eral cochlear ablation, which results in the loss of hair

cells, degeneration of spiral ganglion neurons, and regres-

sive changes in the auditory nucleus. The present study

demonstrated an increase in microglial activation in the

AVCN and PVCN in rats as a consequence of bilateral

Figure 6. Bar graphs showing the mean gray levels of Iba-1 immunostaining (A,B) and the immunostained areas (C,D) in control and

ablated animals. At all of the survival times studied, there were significant increases in the mean gray levels of Iba-1 immunostaining in

the cochlear nucleus compared with the control animals. Concomitant with these changes were increases in the extent of the Iba-1-immu-

nostained areas at 24 hours and 4, 7, and 15 days following ablation in the AVCN and PVCN compared with the control animals. At 30

and 100 days postlesion, the immunostained areas were greater in the AVCN but not in the PVCN compared with the control rats. The

error bars indicate the standard errors of the mean.



deprivation of presynaptic cochlear activity. Specifically,

our data showed significant increases in the mean cross-

sectional areas of Iba-1-immunostained cells and

increases in the mean gray levels of Iba-1 immunostaining

within the AVCN and PVCN at all of the survival times

studied after bilateral cochlear ablation compared with

control animals. Correlated with these increases were

increases in the extent of the Iba-1-immunostained areas

at 24 hours and 4, 7, and 15 days following ablation in

both nuclei compared with the control animals. The im-

munostained areas were still increased at later survival

times in the AVCN but not in the PVCN compared with

the control animals. Double-labeling studies for Iba-1 and

the neuronal marker NeuN showed that, as early as 24

Figure 7. Appositions between microglial cells and neurons in the AVCN after bilateral cochlear ablation. In the control (arrows and aster-

isks in B) and PA16h (arrows and asterisks in C) rats, microglial processes in both the AVCN and the PVCN were rarely seen apposing

cochlear nucleus neurons. The frequency of appositions between microglial cells and neurons increased in PA4d (arrows and asterisks in

E), PA7d (arrows and asterisks in F), and PA15d (arrows and asterisks in G) animals, where microglial cell bodies were frequently observed

apposing the soma and dendrites of deafferented cochlear nucleus neurons. Although the number of appositions between the microglial

cells and the cochlear nucleus neurons decreased at later survival times (arrows and asterisks in H,I), these appositions were still present

100 days following bilateral cochlear ablation. The drawing in A indicates the location of the AVCN, and the boxed areas (A0,A00) indicate

the approximate locations of the high-magnification images shown in B–I. The dashed lines and asterisks indicate cochlear nucleus neu-

rons apposing (arrows) microglial cells. A magenta-green copy of this figure is available online as Supporting Information. Scale bars ¼250 lm in A; 50 lm in C (applies to C–I).



hours postablation, microglial cells were observed appos-

ing the presynaptically deprived cochlear nucleus neu-

rons and that such appositions, which peaked between 7

and 30 days postlesion, were still present 100 days later.

These findings demonstrate that there is persistent glial

activation in the ventral cochlear nucleus and indicate

that long-term interactions between microglial cells and

deafferented cochlear nucleus exist following bilateral

cochlear ablation.

Several lines of evidence suggest that, in the healthy

adult brain, glial cells are directly involved in the mainte-

nance of normal functionality and neuroplasticity (for

review see Todd et al., 2006). In particular, these dynamic

glial cells, whose processes and arborizations are highly

mobile (Nimmerjahn et al., 2005), are actively involved in

the control of neuronal activity and in the regulation of

synaptic strength and synaptogenesis (Araque et al.,

1999; Fields and Stevens-Graham, 2002). In this respect,

microglial cells are constantly screening their own envi-

ronment, receiving continuous information from neurons

and delivering the appropriate signals required for normal

functioning of the brain. After neuronal injury, microglial

Figure 8. Appositions between microglial cells and neurons in the PVCN after bilateral cochlear ablation. Similar to those observed in the

AVCN, microglial–neuronal appositions were most frequently observed at 4 (arrows and asterisks in E), 7 (arrows and asterisks in F), and

15 (arrows and asterisks in G) days postablation, although they were still present in PA30d (H) and PA100d (I) animals. The drawing in A

indicates the location of the PVCN, and the boxed areas (A0,A00) indicate the approximate locations of the high-magnification images shown

in B–I. The dashed lines, asterisks, and arrows indicate points of apposition between the glial cells and the cochlear nucleus neurons. A

magenta-green copy of this figure is available online as Supporting Information. Scale bars ¼ 250 lm in A; 50 lm in I (applies to B–I).



sensing arms detect subtle disturbances in the brain envi-

ronment, rapidly change to an active phenotype, and

elicit a complex series of events, potentially lasting for

months, to prevent further impairment (for reviews see

Bruce-Keller, 1999; Hanisch and Kettenmann, 2007).

Particularly in the auditory system, removal of afferent

activity by means of unilateral cochlear ablation has been

shown to induce astroglial and microglial proliferation as

well as hypertrophy in the cochlear nucleus of rats (De

Waele et al., 1996; Campos-Torres et al., 1999), monkeys

(Insausti et al., 1999), and chickens (Lurie and Rubel,

1994; Lurie and Durham, 2000). In rats, this microglial

response was first evident at 24 hours postlesion, peaked

at about 7–15 days, and persisted for at least 42 days

(Campos-Torres et al., 1999). Consistent with these find-

ings, the present study demonstrated similar timing of

microglial activation, although our data indicate that

microglial activation occurs within the first 16 hours fol-

lowing ablation and persists for at least 100 days in the

cochlear nucleus. Nevertheless, it is important to empha-

size that the conclusions drawn by the authors cited

above with respect to the effect of unilateral cochlear

ablation on microglial activation in the cochlear nucleus

are based on qualitative interpretations of immunostain-

ing at each time point. In the present study, we have char-

acterized these responses in detail by quantifying the

cross-sectional areas of the Iba-1-immunostained cells as

a measure of cell hypertrophy following lesion. Consistent

with an increased microglial response after deafferenta-

tion and in agreement with previous studies (Campos-

Torres et al., 1999), our results demonstrate clear hyper-

trophy in microglial cells at all of the survival times stud-

ied, but in particular 24 hours postlesion, compared with

the control animals. These increases in the microglial

cross-sectional areas were concomitant with increases in

the mean gray levels of Iba-1 immunostaining, which

were used as an index of intracellular levels of the

antigen Iba1.

Iba-1 is an EF hand calcium-binding protein that is

expressed specifically in cells from monocytic lineages

(Ito et al., 1998). Several studies have demonstrated that

the expression levels of Iba-1 within microglial cells are

increased during microglial activation following neuronal

damage. In particular, after facial nerve axotomy (Ito

et al., 1998) and ischemia (Ito et al., 2001), this protein is

highly up-regulated in the facial nucleus of the rat, which

suggests that Iba-1 may participate in calcium-related

mechanisms following injury. Consistent with this, in vivo

two-photon imaging studies monitoring intracellular Ca2þ

levels in cortical microglia have demonstrated that micro-

glial cells typically respond with large, generalized Ca2þ

transients that modify microglial activity as a result of

neuronal injury (Eichhoff et al., 2011). Therefore, changes

in the levels of Iba-1, similar to changes in other calcium-

binding proteins, might be related to changes in synaptic

activity. Deprivation of afferent activity produces an

imbalance in the normal levels of calcium-binding pro-

teins in auditory neurons, because unilateral cochlear

ablation at the time of hearing onset in ferrets results in a

significant up-regulation in calretinin immunostaining in

the cochlear nucleus (Fuentes-Santamaria et al., 2005),

superior olivary complex (Alvarado et al., 2004), and infe-

rior colliculus (Fuentes-Santamaria et al., 2003).

These observations, together with the observed

increases in the mean gray levels of Iba-1 immunostaining

within microglial cells in the cochlear nucleus reported in

the present study, are consistent with activity-dependent

changes resulting from deafferentation, and they suggest

that Iba-1 may play a crucial role in the regulation of

microglial activation following bilateral cochlear ablation.

Consistent with this possibility, recent reports suggest

that Iba-1, apart from its calcium-buffering capacity, also

has an actin cross-linking function, which could allow it to

function in the reorganization of the actin cytoskeleton

that accompanies the microglial reaction following injury

(Sasaki et al., 2001). Iba-1 has also been proposed to

facilitate microglial migration, proliferation, and phagocy-

tosis following injury (Ohsawa et al., 2000, 2004). Taken

together, these results strongly suggest that up-regula-

tion of Iba-1 within microglial cells may play a pivotal role

in the mechanisms and signaling pathways underlying

microglial activation after injury.

Our results also demonstrated increases in the extent

of the Iba-1-immunostained areas at 24 hours and 4, 7,

and 15 days postablation in the AVCN and PVCN com-

pared with the control animals. These increases in the im-

munostained area peaked at 24 hours when Iba-1-immu-

nostained cells increased in cell area, rather than density

(which was observed at 4, 7, and 15 d), compared with

the control animals. These results provide evidence that

the process of microglial activation in the cochlear

nucleus occurs as early as 16 hours postlesion, when the

intracellular levels of Iba-1 are first up-regulated, and

reaches an initial peak at 24 hours. Several lines of evi-

dence suggest that the activation of microglial cells can

be observed as early as 20 minutes following transient

cerebral ischemia in the rat hippocampus (Morioka et al.,

1991; Jørgensen et al., 1993; Abrah�am and L�az�ar, 2000);

however, it is unlikely that such an initial activation could

be caused by a degenerative process. It is more likely

that this early activation is caused by an initial shift in

microglial morphology resulting from imbalances in ion

homeostasis or a lack of neurotrophic support, leading to

modified neuronal activity (Jørgensen et al., 1993). Con-

sistent with this hypothesis, our double-labeling studies

demonstrated that, by 24 hours postlesion, activated



microglial cells are first seen in close apposition to coch-

lear nucleus neurons. Although the exact role of this inter-

action is unknown, it might reflect an initial attempt by

glial cells to respond quickly to the signals released by

injured neurons to prevent further neuronal damage and

to reestablish homeostasis following bilateral cochlear

ablation.

Degeneration of synaptic endings after cochlear abla-

tion has been shown to occur in the cochlear nucleus as

early as 7 days postlesion (Benson et al., 1997), and

these effects have been shown to last for at least 8

months following acoustic overstimulation in adult guinea

pigs (Bilak et al., 1997; Kim et al., 2004). Evidence sug-

gests that this degenerative process occurs simultane-

ously with the growth of new synaptic endings, suggest-

ing a synaptic reorganization after cochlear

deafferentation (Muly et al., 2002; Hildebrandt et al.,

2011). It has been suggested that activated microglia

contribute to synaptic recovery and remodeling following

brain damage (for review see Bruce-Keller, 1999). Along

these lines, it has been proposed that microglial cells par-

ticipate in a synaptic stripping process in which damaged

synaptic boutons are detached from postsynaptic neu-

rons by the intercalation of microglial processes (Cull-

heim and Thams, 2007). Although it is unclear why micro-

glial cells strip off synapses from the cell surface of

injured neurons, it has been hypothesized that this pro-

cess may facilitate axonal sprouting and synaptic recov-

ery. Our findings showed that, by 7 days postablation, the

time point at which degeneration and synaptogenesis

occur, there was an increase in the number of appositions

between microglial cells and the presumptive injured au-

ditory neurons and that such interactions were still pres-

ent 100 days after bilateral cochlear ablation. These

results indicate that direct microglial apposition with

cochlear nucleus neurons may reflect a two-way mecha-

nism to facilitate the exchange of cellular signals to

restore neuronal function following brain damage.

In summary, our results provide additional information

concerning the microglial activation that takes place in

the cochlear nucleus after bilateral deprivation of afferent

activity. Our findings are consistent with early (by 16

hours) and long-lasting (over a period of 100 days) micro-

glial activation that may act to minimize damage and pro-

mote neuronal repair. In addition, these results suggest

that interactions between microglial cells and cochlear

nucleus neurons are critical to facilitate the remodeling of

the affected neuronal circuits involved in auditory

processing.

ACKNOWLEDGMENTS

The authors thank Tania Jare~no, Mari Cruz Gabald�on,

and Jos�e Julio Cabanes for excellent technical assistance

and also Jos�e Ram�on Marı́n for valuable confocal micros-

copy support.

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