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Role of adaptor protein MyD88 in TLRmediated preconditioning and neuroprotection

after acute excitotoxicity
Article in Brain Behavior and Immunity February 2015
Impact Factor: 5.89 DOI: 10.1016/j.bbi.2015.02.019 Source: PubMed

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Brain, Behavior, and Immunity 46 (2015) 221231

Contents lists available at ScienceDirect

Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Role of adaptor protein MyD88 in TLR-mediated preconditioning

and neuroprotection after acute excitotoxicity
Antoine Larochelle, Marc-Andr Bellavance, Serge Rivest
Neuroscience Laboratory, CHU de Qubec Research Center and Department of Molecular Medicine, Faculty of Medicine, Laval University, 2705 Laurier Blvd., Qubec G1V 4G2, Canada

a r t i c l e

i n f o

Article history:
Received 10 November 2014
Received in revised form 3 February 2015
Accepted 20 February 2015
Available online 27 February 2015
Keywords:
Excitotoxicity
Toll-like receptors
MyD88
Microglia
Preconditioning
Neuroprotection

a b s t r a c t
Excitotoxic cell death is a crucial mechanism through which neurodegeneration occurs in numerous
pathologies of the central nervous system (CNS), such as Alzheimers disease, stroke and spinal cord
injury. Toll-like receptors (TLRs) are strongly expressed on microglial cells and are key regulators of
the innate immune response to neuronal damage. However, it is still unclear whether their stimulation
is protective or harmful in excitotoxic contexts. In this study, we demonstrate that systemic administration of lipopolysaccharide (LPS) or Pam3CSK4 24 h prior to an intrastriatal injection of kainic acid (KA)
signicantly protected cortical neurons in the acute phase of injury. Protection could not be detected with
the TLR3 ligand poly-IC. Histological analyses revealed that microglia of LPS and Pam3CSK4 pre-conditioned group were primed to react to injury and exhibited a stronger expression of Tnf and Tlr2 mRNA.
We also found that mice decient for MyD88, a critical adaptor protein for most TLR, were more vulnerable than WT mice to KA-induced excitotoxicity at early (12 h and 24 h) and late (10 days) time points.
Finally, bone-marrow chimeric mice revealed that MyD88 signaling in CNS resident cells, but not in cells
of hematopoietic origin, mediates the protective effect. This study unravels the potential of TLR2 and
TLR4 agonists to induce a protective state of preconditioning against KA-mediated excitotoxicity and
further highlights the benecial role of cerebral MyD88 signaling in this context.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Excitotoxicity is a pathological process through which neurons
are damaged or killed by dysregulated Ca2+ signaling following
excessive stimulation by excitatory neurotransmitters. Excitotoxic
processes are believed to underlie neurodegeneration and thereby
contribute to permanent disabilities observed in many chronic neurodegenerative diseases including Alzheimers disease, Parkinsons
disease and Amyotrophic lateral sclerosis. These pathologic
phenomena were also reported to provoke collateral (secondary)
neuronal death following stroke and spinal cord injuries
(Mattson, 2003).
Shortly after excitotoxic lesions, microglia, the main immune
cell and sentinel of the CNS, initiate an important inammatory
response. This response is triggered by the binding of danger signals released by necrotic neurons to various pattern recognition
receptors (PRRs) located at their surface (Zhang and Zhu, 2011).
Activated microglia are observed within the injured brain regions
in various neurological diseases. They produce inammatory factors that mobilize other immune cells, support neuronal survival
Corresponding author. Tel.: +1 418 654 2296; fax: +1 418 654 2761.
E-mail address: Serge.Rivest@crchudequebec.ulaval.ca (S. Rivest).
http://dx.doi.org/10.1016/j.bbi.2015.02.019
0889-1591/ 2015 Elsevier Inc. All rights reserved.

through synthesis of neurotrophic factors and establish an

environment supportive for regeneration by clearing debris and
other neurotoxic compounds (Neumann et al., 2009). Numerous
studies support the neuroprotective roles of microglia after brain
injury (Hao et al., 2007; Lalancette-Hbert et al., 2007; Shaked
et al., 2005). However, excessive or chronic activation of microglial
cells can induce collateral damage because of sustained release of
pro-inammatory cytokines, nitric oxide (NO) and reactive oxygen
species (Block et al., 2007; Chao et al., 1992).
Toll-like receptors (TLRs) are among the main PRRs responsible
for microglial recognition of danger signals. TLRs are innate
immune receptors that recognize both pathogen-associated
molecular patterns (PAMPs) and damage-associated molecular
patterns (DAMPs) (Takeuchi and Akira, 2010). TLRs use two main
signaling pathways depending either on Myeloid differentiation
factor-88 (MyD88) or Toll/interleukin-1 receptor (TIR)-domaincontaining adapter protein inducing interferon-b (TRIF/TICAM1)
(Kawai et al., 1999; Yamamoto et al., 2003). The MyD88-dependent
pathway is used by all TLRs (except TLR3), while the TRIFdependent pathway is used only by TLR3 and TLR4. The MyD88dependent pathway culminates in the activation of transcription
factors NF-jB and activator protein 1 (AP-1), which both lead to
the secretion of pro-inammatory cytokines such as IL-1b, TNF-a

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A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

and IL-6. On the other hand, the TRIF-dependent pathway activates

both NF-jB and IRF-3, thereby triggering the production of NF-jB
inammatory cytokines as well as type-1 interferons. Studies using
TLR2 or TLR4-decient mice suggest a deleterious role of these
receptors following KA-induced excitotoxicity (Hong et al., 2010;
Maroso et al., 2010) and cerebral ischemia (Hua et al., 2007;
Lehnardt et al., 2007). However, TLR signaling also promotes
regenerative processes needed for long-term recovery after stroke
or injury to the spinal cord (Bohacek et al., 2012; Kigerl et al.,
2007). Recently, Stirling et al. showed that the TLR2 agonist
Pam2CSK4 promotes an alternative microglial response that was
neuroprotective following laser-induced spinal cord injury
(Stirling et al., 2013). Interestingly, systemic injection of specic
TLR agonists prior to ischemic injury was reported to induce a state
of preconditioning that decreased both infarct size and cellular
death (Marsh et al., 2009; Rosenzweig et al., 2004).
The impact of TLRs preconditioning has been studied extensively in animal models of stroke (Mallard, 2012), but critically
remains ill-dened in other relevant pathological contexts.
Furthermore, not much attention was accorded to microglia during
TLR-preconditioning while recent studies suggest that these cells
are crucial effectors of lipopolysaccharide (LPS)-preconditioning
(Chen et al., 2012; Mirrione et al., 2010). In this study, we therefore
sought to determine if TLR2, TLR3 or TLR4 agonists would yield
benecial or detrimental outcomes to a subsequent excitotoxic
challenge induced by intra-cerebral injection of kainic acid (KA)
and how they would respectively inuence microglial response
to injury. KA is a strong agonist of kainate and AMPA receptors,
and has been commonly used to evoke excitotoxic lesions in mice
for decades (Wang et al., 2005). To this end, the TLR2 agonist
Pam3CSK4, the TLR3 agonist Poly-IC or the TLR4 agonist LPS was
injected 24 h prior to injection of KA in the striatum. We then
evaluated the role of MyD88 and TRIF signaling in response to
excitotoxic cell death. This study demonstrates that pretreatment
with Pam3CSK4 or LPS signicantly prime microglial cells and protects cortical neurons in the early phase of KA mediated excitotoxic
neuronal death. It further highlights the role of cerebral MyD88 in
conferring long-term protection against acute excitotoxicity.
2. Materials and methods
2.1. Animals
Adult and age-matched (difference < 1 month) mice were used.
Wild-type (WT) C57BL/6 mice were purchased from Jackson
Laboratories (Bar Harbor, ME). GFP+/ mice were initially obtained
from Charles River Laboratories. MyD88 / mice were kindly provided by Dr. S. Akira (Osaka University, Osaka, Japan) and TRIF /
mice were a generous gift from Dr. T.J. Lin (Dalhousie University,
Halifax, Canada). All mice were kept on a C57BL/6 background.
Mice were acclimated to standard laboratory conditions (12 h
lightdark cycle) with ad libitum access to mouse chow and water.
All protocols were conducted according to the Canadian Council on
Animal Care guidelines, as administered by the Laval University
Animal Welfare Committee.
2.2. Pam3CSK4, Poly-IC and lipopolysaccharide (LPS) preconditioning
Prior to stereotaxic surgery (24 h before), mice were injected
intra-peritoneally (i.p.) with sterile 0.9% saline (vehicle) or one of
the following TLR agonists: Pam3CSK4 (5 mg/kg body weight,
TLR2-TLR1 ligand, InvivoGen, San Diego, CA), Poly-IC LMW
(10 mg/kg body weight, TLR3 ligand, InvivoGen) and LPS from
Escherichia coli 055:B5 (1 mg/kg body weight, TLR4 ligand,
Sigma, Saint-Louis, MO). Doses were validated in-house to ensure

that they induce a signicant inammatory response (data not

shown).
2.3. Kainic acid and vehicle stereotaxic injection
Mice were anaesthetized at a surgical level using isourane and
the top of the head was shaved and sterilized before skin was cut to
reveal the skull. The site of injection was reached using a stereotaxic instrument (David Kopf Instruments, Tujunga, CA). The coordinates from bregma were 0.0 mm anteriorposterior, 2.0 mm
lateral and 3.2 mm dorsoventral in order to reach the right striatum. 1 ll of vehicle (sterile 0.9% saline) containing 150 ng of kainic
acid was injected over 2 min. A 10 ll Hamilton syringe mounted on
a UltraMicroPump II connected to a microprocessor controller
Micro4 (World Precision Instruments) was used to control the rate
and time of injection. Insertion of the needle into the mouse brain
induced a small localized injury referred to as a stab injury. A 2 min
rest was allowed after the injection and the syringe was raised progressively over a 2 min period. Mice were then rehydrated by 2
subcutaneous injections of 0.5 ml of saline and housed singly in
new cages until sacrice.
2.4. Generation of bone-marrow chimeric mice
Bone-marrow (BM) chimeric mice were generated as previously
described (Lampron et al., 2012). Briey, WT and MyD88 / were
myeloablated with a total dose of 80 mg/kg of busulfan (two injections per day during 4 days) followed by two daily injections of
100 mg/kg of cyclophosphamide. After a day of rest, mice were
then transplanted with 1  107 freshly collected and puried BM
cells from either GFP+/ WT mice or MyD88 / mice. Six weeks
later, blood samples (120 ll) were collected from the facial vein
and myeloid cells were isolated to determine the level of chimerism in CD45+ cells by ow cytometry analysis as previously
detailed (Michaud et al., 2013). Briey, 65 ll of blood was incubated on ice 20 min with CD16/CD32 (Mouse BD Fc block, BD bioscience) and further stained for 40 min with the following antimouse antibodies at their predetermined optimal concentration:
CD45-V500, CD11b-A700, CD115-APC, Ly6G-PE, Ly6C-V450.
Chimerism was assessed only in mice that received GFP+/ WT
BM cells, and was thus assumed to be equivalent for mice receiving
MyD88 / BM cells as they were treated identically. Experimental
protocols were initiated two weeks later.
2.5. Tissue collection
Mice were deeply anaesthetized with an intraperitoneal injection of ketamine hydrochloride (91 mg/ml) and xylazine (9.1 mg/
ml) and then perfused transcardially with cold 0.9% saline and
4% PFA (pH 9.0) in 0.1 M borax buffer (5 min each). Brains were
rapidly removed from the skull and postxed in the same PFA solution for 3 days. Then, they were placed overnight in a 4% PFA (pH
9.0) solution containing 20% sucrose and were positioned on a
microtome (ReichertJung, Cambridge Instruments, Deereld, IL),
frozen with dry ice and sliced in 25 lm coronal sections. Brain
slices were kept in a cold cryoprotective solution (0.05 M sodium
phosphate buffer, pH 7.3, 30% ethylene glycol, 20% glycerol) at
20 C until analysis.
2.6. Fluorojade B staining
Brain slices, separated by 300 lm, from the start of the prefrontal cortex to the end of the cerebral cortex were mounted on
Colorfrost/Plus microscope slides (Fisher Scientic). Slides were
then dried overnight and xed with 4% PFA vapors at 37 C for
2.5 h. Dehydration and rehydration was performed through graded

A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

concentrations of alcohol (50%, 70%, 100%, 70% and 50% EtOH, 1 or

3 min each). Next, slides were rinsed for 1 min in MilliQ water and
treated with 0.06% potassium permanganate solution for 10 min.
Slides were rinsed again before being submerged in a solution containing 0.004% FJB (Histochem, Jefferson, AR), 0.1% acetic acid and
0.0002%
4,6-diamidino-2-phenylindole
(DAPI;
Invitrogen,
Burlington, ON, Canada) for 20 min. Slides were thereafter rinsed
in distilled water (3  1 min) and dried overnight in the dark.
Slides were nally dipped in xylene (3  3 min), and coverslipped
with DPX mounting media (Electron Microscopy Sciences,
Washington, PA).
2.7. Fluorojade B quantication
Visualization of FJB+ cells was done using a C-80 Nikon microscope and a super-high-pressure mercury lamp (Nikon, Melville,
NY) tted with a Retiga EXi Fast digital camera (QImaging,
Burnaby, BC, Canada) connected to a Precision 660 workstation
(Dell Computer, Round Rock, TX). The zone of interest (i.e. the
dorsolateral cortex) was traced on a Wacom pen tablet
(Vancouver, WA) by using the StereoInvestigator software package
(version 6, Microbrighteld Bioscience, Williston, VT). The FJB+
cells were counted in the dorsolateral cortex, on a slice located
closest to bregma 1.5 mm of the anteriorposterior axis. For mice
sacriced 12 h or 24 h after KA injection, FJB+ neurons were quantied in the ipsilateral cortex. For mice sacriced 10 days post-surgery, the quantication was made in the cortex of both
hemispheres since KA-induced neurodegeneration was observed
bilaterally at this time point.
2.8. In situ hybridization (ISH)
Expression of multiple TLRs, inammatory and neurotrophic
genes were revealed on every 12th section of the entire rostro-caudal with a 35S-Labeled cRNA probes as described previously
(Laamme et al., 2003; Laamme and Rivest, 1999). See Table 1
for the list of cRNA probes used. After the post-hybridization treatment was done, gene expression was revealed by placing slides
covered by a X-ray lm (Biomax; Kodak) in a Autoradiography
Cassette (Fisher Scientic, Pittsburgh, PA) for 13 days, depending
on the expression levels of the gene.
The relative area and intensity of positive hybridization signals
on two separate brain slices ( 1.22 mm and 1.70 mm on the
anteriorposterior axis) were measured on Biomax X-ray lms

223

(Kodak, Rochester, NY) as reported previously (Turrin, 2006).

Briey, transmittance values (referred to as optical density OD)
were measured by using a Northern Light desktop illuminator
(Imaging Research, Saint-Catherines, ON, Canada) and a Sony
(Tokyo, Japan) camera video system attached to a MicroNikkor
55-mm Vivitar extension tube set for a Nikon lens and coupled
to a Dimension GX270 personal computer (Dell Computer, North
York, ON, Canada). The OD value for each pixel was calculated with
the ImageJ software (version 1.23; W. Rasband, National Institutes
of Health, Bethesda, MD) by using a known standard of intensity
and distance measurements from a logarithmic specter adapted
from BioImage Visage 110s (Millipore, Ann Arbor, MI). Each value
was corrected for background signal by substracting the OD value
measured at a brain area devoid of positive signal.
2.9. Immunohistochemistry (IHC)
Free-oating sections were washed with potassium phosphate
buffered saline (KPBS; 3  10 min) and then incubated at RT for
30 min in a permeabilization/blocking solution containing 4% goat
serum, 1% bovine serum albumin and 0.4% triton in KPBS. Sections
were then incubated overnight at 4 C in a solution containing 50%
KPBS and 50% permeabilization/blocking solution and one of the
following primary antibody: Rabbit anti-ionized calcium binding
adaptor molecule 1 (Iba1) polyclonal antibody (1:3000, EMD;
Millipore) or rat anti-CD68 monoclonal antibody (1:500, AbD
Serotec). The sections were rinsed at RT in KPBS (3  20 min) and
incubated 2 h with a biotinylated goat anti-rabbit IgG (1:1000,
for Iba1) (or a biotinylated goat anti-mouse IgG (1:1000, for
CD68)) diluted in 0.5  permeabilization/blocking solution.
Binding was visualized using the peroxidase-based Vectastain
ABC kit (Vector Laboratories). Sections were then rinsed 3 more
times in KPBS (3  10 min), mounted on Colorfrost/Plus microscope slides (Fisher Scientic) and allowed to dry under vacuum
overnight. Sections were subsequently dehydrated by successive
dips in (50, 70, 100 EtOH) before being bathed in xylene
(3  3 min) and coverslipped with DPX mounting media.
Immunoreactivity for Iba1 was quantied as the % of area
covered by positive signal. Pixel intensity threshold was set using
Image J and held constant for images obtained at equal objectives
and light intensities in a single session. CD68+ cells were counted
using a similar method to the one described for quantifying FJB+
neurons. Data for each mouse represents the density of CD68+ cells
in the deeper half of the ipsilateral cortex.
2.10. Behavioral analysis

Table 1
Plasmids and enzymes used for the synthesis of the cRNA probes.
Plasmid

Vector

Length
(bp)

Enzymes
used for
antisense
probe

Primer

Tlr2

PCR Blunt-II
Topo

2278

Sp6

TNF-a

PCR II Topo

1384

Sp6

MyD88

PCR Blunt-II

890

Sp6

TRIF

PCR II-Topo

912

Sp6

Upper: GGC-TCT-TCT-GGATCT-TGG-TGG-CC Lower:

GGG-CCA-CTC-CAG-GTAGGT-CTT-GG
Upper: CCA-GAA-CTC-CAGGCG-GTG-CCT-ATG-T Lower:
TAC-ACC-CCA-TCG-GCT-GGCACC-ACT-A
Upper: ATG-TCT-GCG-GGAGAC-CCC-CGC-GT Lower:
TCA-GGG-CAG-GGA-CAAAGC-CTT-GC
Upper: CTT-CAC-GGT-GCTGCA-CGA-GCC-T Lower: GTTGCA-AGG-AGG-CAG-AGATGT-C

Nesting behavior was assessed daily post-surgery using a

slightly modied version of Deacons scale (Deacon, 2006). A score
was assessed from 0 to 5 (0 = untouched nestlet, 1 = nestlet partially torn, 2 = nestlet half torn 3 = nestlet shredded, but no
identiable nest site, 4 = identiable but at nest 5 = near perfect
nest covering the mice). In addition, the neurological performance
of mice was analyzed 5 and 8 days post-surgery using a slightly
modied version of the Garcia scale (Garcia et al., 1995). The mice
were therefore subjected to 5 different tests: spontaneous activity
(score between 03), symmetry in the movement of four limbs
7(03), forepaw outstretching (03), climbing (13) and body proprioception (13). The max score is 15 and the minimum score is 2.
Every mouse was tested for both tests before stereological injection to ensure that no differences were present between groups.
2.11. Statistical analysis
Data are expressed as mean standard error of the mean (SEM).
Statistical analyses were performed using unpaired t-test with or

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A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

without Welchs correction and 1 or 2-way ANOVA followed by

Bonferroni post hoc test. All analyses were performed using
Graph Pad Prism v6.0 software and alpha level was set to 0.05.
3. Results
3.1. Systemic preconditioning with Pam3CSK4 or LPS protects cortical
neurons against KA-induced excitotoxic neuronal death
Seizures started approximately 1 h after the intra-striatal injection of KA, whereas excitotoxic neuronal death, as revealed by FJB,
peaked at 24 h (Gosselin et al., 2013). To investigate the impact of
TLR signaling on neuronal survival, Pam3CSK4 (5 mg/kg), Poly-IC
(10 mg/kg) or LPS (1 mg/kg) were administered i.p. 24 h before
the excitotoxic challenge and brains were collected 24 h after the
stereotaxic surgery (Fig. 1a and b). Quantication of FJB+ neurons
in the dorsolateral cortex of the ipsilateral hemisphere (Fig. 1c
and d) revealed that mice treated with Pam3CSK4 or LPS exhibited
signicantly less neuronal death than saline treated mice
(p = 0.0028 and p = 0.0038, respectively; Fig. 1d and e). A trend
toward neuroprotection was also observed in animals pre-treated
with Poly-IC, but this was not statistically signicant from the saline-treated group (p = 0.095). Overall, these data suggest that preconditioning with the TLR2 ligand Pam3CSK4 or the TLR4 ligand
LPS 24 h before an intra-striatal injection of KA increases survival
of cortical neurons in the acute phase (i.e. 24 h later).
3.2. Systemic preconditioning with Pam3CSK4 or LPS enhances
microglial activation following KA administration
An experiment using pilocarpine-induced seizures in mice
established that microglia are needed to induce systemic LPS preconditioning (Mirrione et al., 2010). To determine whether microglial cells from mice that received a preconditioning regimen
exhibited a different state of activation than those of saline

pre-treated group, we performed an immunostaining for Iba1, a

protein that is expressed by resting microglia and its level
increased in activated microglia (Ito et al., 2001). Both Pam3CSK4
and LPS pre-treated mice displayed higher immunoreactivity for
Iba1 than control mice (saline) 24 h following intra-cerebral injection of the vehicle (sterile saline) (p < 0.001 for Pam3CSK4 and
p = 0.0143 for LPS). Following the intra-cerebral injection of KA,
Iba1 immunoreactivity was signicantly more widespread in the
ipsilateral cortex of Pam3CSK4 and LPS pre-treated animals than
in those administered with saline (Fig. 2a and b). No signicant differences were observed for Iba1 immunoreactivity between PolyIC and saline pre-treated groups 24 h after either vehicle or KA
injection. The density of activated microglia was also assessed in
the deeper half (layers 56) of the ipsilateral cortex by CD68
immunostaining. CD68 is a lysosomal protein that is not (or very
weakly) expressed in resting microglia, thus it represents a good
marker for microglial activation (Fig. 2c). No differences were
observed between TLR preconditioned and saline pre-treated
groups (Fig. 2d). Although differences were not signicant, more
CD68+ cells were observed in groups that displayed higher levels
of FJB+ neurons/area (saline and Poly-IC).
Microglia activation can also be monitored reliably by analyzing
Tlr2 mRNA expression in the brain (Lalancette-Hbert et al., 2009;
Rivest, 2009). Interestingly, the stab injury (injury caused by the
insertion of the syringes needle into the brain parenchyma)
induced a very strong expression of Tlr2 mRNA transcripts, even
in cortical regions remote from the injection site in the animals
of the vehicle-injected groups that were pre-treated with
Pam3CSK4 or LPS (Fig. 2e, left column). Tlr2 gene expression levels
were also higher in the brain of Pam3CSK4- or LPS-preconditioned
mice in response to KA injection (Fig. 2e, right column). Poly-IC did
not inuence Tlr2 expression neither in the vehicle nor in the KA
group (data not shown). These results correlate with Iba1
immunoreactivity, but not with CD68 density. Cerebral Tnf-a
expression is needed to induce TLR9-mediated preconditioning in

Fig. 1. Preconditioning with Pam3CSK4 or LPS protects cortical neurons against intra-striatal injection of kainic acid (KA). (a) Schematic illustration of the preconditioning
experiment. Intra-peritoneal injection of Pam3CSK4, Poly-IC or LPS was performed 24 h prior to an intra-striatal injection of KA. Mice were then sacriced 24 h after KA
injection and their brains were collected for analysis. (b) 150 ng of KA diluted in 1 ll of 0.9% saline solution was injected in the right striatum. Intra-striatal injection of KA
induces massive neuronal death in the cortex, thalamus and hippocampus. (c) Fluorojade B positive neurons were quantied in the cortex of the ipsilateral hemisphere. (d)
Representative photomicrograph of FJB staining showing absence of neuronal death in all the vehicle injected group (n = 4 for each group) and cortical neurodegeneration, as
depicted by FJB staining, in saline, Pam3CSK4, Poly-IC and LPS pre-treated group 24 h after KA injection (n = 1113 mice per group). (e) Stereological quantication of FJB+
cells/area represented as relative % of the saline pre-treated group mean value; data are mean SEM. p < 0.01 compared by one-way ANOVA and Bonferroni post hoc test.
Results were obtained from two independent experiments. Scale bar in d = 1 mm, scale-bar close-up: 250 lm.

A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

225

Fig. 2. Microglial cells reactivity following KA injection in Pam3CSK4- or LPS-pre-treated mice. (a) Representative photomicrographs of Iba1 immunostaining following
vehicle or KA injection. Panel at the bottom left corner represents higher magnication of the deeper half of the cortex (b) Graph showing immunopositive area % for Iba1 in
the ipsilateral hemisphere, n = 4 mice for vehicle groups and 79 mice for KA injected groups. (c) Photomicrographs showing no CD68+ cells in the cortex of vehicle-injected
mice and a clear increase in the number of CD68+ cells in the cortex 24 h following KA injection. (d) Stereological quantication of CD68+ cells in the deeper half of the cortex
in mice preconditioned with saline or TLR agonists, n = 79. Representative pictures showing Tlr2 (X-ray lm photomicrographs), left column represents the control group
injected with vehicle (stab injury) and the right column represents animals injected with KA (e) and Tnf-a (dark-eld photomicrograph) (f) mRNA expression using in situ
hybridization. p < 0.01; p < 0.05 compared by two-way b or one-way ANOVA d and Bonferroni post hoc test. Scale bar in a and c = 1 mm, close-up = 250 lm and f = 500 lm.

stroke (Packard et al., 2012b). We observed a strong expression of

Tnf-a mRNA transcripts in the brain parenchyma of Pam3CSK4 and
LPS preconditioned mice 24 h after KA injection (Fig. 2f). Tnf-a
mRNA transcripts were barely detected in saline treated KA group
and were undetectable in the vehicle groups. Together, these data
indicate that Pam3CSK4 or LPS preconditioning enhances microglia
responsiveness to both stab and excitotoxic injuries.
3.3. Signaling through MyD88, but not TRIF, increases survival of
cortical neurons following KA-induced neurodegeneration
TRIF/IRF3 signaling is critical to induce neuroprotection in the
ischemic LPS-preconditioning animal model (Vartanian et al.,

2011). We rst determined expression of the gene encoding either

MyD88 and/or TRIF following KA-induced brain injury. A strong
upregulation of Myd88 mRNA levels was observed 24 h and 3 days
after KA, whereas Trif mRNA remained unchanged (Fig. 3a). We
also tested LPS preconditioning in MyD88 / mice and found that
LPS was no longer efcient to protect cortical neurons in these
mice after KA injection (Fig. 3b). The role of MyD88 and TRIF in
KA-induced cortical neuronal death was also assessed without
preconditioning. Interestingly, quantication of FJB+ neurons 24 h
after KA injection revealed that MyD88 / mice were more
susceptible to KA-induced neuronal death than WT mice
(p = 0.0299) (Fig. 3c). No differences were observed in the number
of FJB+ neurons in the brain of TRIF / and WT mice (p = 0.602)

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A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

Fig. 3. MyD88, but not TRIF, is upregulated and involved in neuronal survival following KA-induced excitotoxicity. (a) Photomicrographs showing upregulation of MyD88
mRNA following KA-induced neuronal death in mice sacriced 24 h, 3 days and 7 days post-injection. Trif mRNA expression is shown at 24 h, but remained unchanged also at
3 days and 7 days. (b) MyD88 / mice were subjected to saline or LPS preconditioning (as in Fig. 1a) and cortical FJB+ neurons were quantied 24 h after KA injection (n = 3
and 5 mice). (c and d) Stereological quantication of FJB+ cells/mm2 in the cortex of MyD88 / (n = 10) and WT mice (n = 7) c and TRIF / (n = 6) and WT (n = 6) mice d
sacriced 24 h after KA injection. Data are presented as % relative to WT (or saline for LPS pre-conditioning) and are expressed as mean SEM (bars). p < 0.05, signicant
differences were established using unpaired t test b, c and d.

(Fig. 3d). These results suggest that MyD88, but not TRIF, signaling
promotes neuronal survival following excitotoxic insult.
3.4. MyD88 deciency exacerbates short- and long-term neuronal
injury but does not restrain microglial activation
To investigate the neuroprotective role of MyD88-dependent
signaling in the acute and chronic phases of excitotoxicity, KA-induced neuronal death was quantied in mice sacriced at 12 h
and 10 days post-injection. Interestingly, MyD88 / mice were signicantly more vulnerable to KA even at the early 12 h post-injection time point (p = 0.0125) (Fig. 4a and b). As expected,
signicantly more FJB+ neurons were detected in the cortex of
MyD88 / mice than in WT mice 10 days after KA treatment
(p = 0.0051) (Fig. 4a and c). Iba1 immunoreactivity was similar in
the brain of both mouse groups 12 h after KA injection (Fig. 4d
and e). In contrast, the area of Iba1 immunoreactivity was signicantly greater in MyD88 / than WT mice 10 days following the
injection of KA (p = 0.011; Fig. 4d and f). At this time point,
MyD88 / exhibited signicantly more amoeboid microglia and
greater density values of CD68+ cells that WT mice (p = 0.007;
Fig. 4g and 4 h). CD68-ir signal was too weak to be analyzed in

the brain of mice that were killed 12 h after KA injection. These

results provide evidence that MyD88 signaling plays a neuroprotective role in both early and late time points and that microglia
exhibit a highly activated state even in its absence following
excitotoxic neuronal death.
3.5. MyD88 signaling is neuroprotective in resident microglia
Using chimeric mice, our group recently reported that KA-induced neuronal death leads to a massive but transient engraftment
of monocyte-derived macrophages (MDMs) within the lesioned
sites of the brain (Bellavance et al., 2014). This study also unraveled a neuroprotective role for patrolling monocytes in this context. To determine whether the protective effect of MyD88 comes
from the action of peripheral cells or resident CNS cells, we generated bone-marrow chimeric mice. Three types of chimeric mice
were generated: (1) WT mice transplanted with GFP+/ BM cells
(GFP+/ ? WT) or (2) with MyD88 / BM cells (MyD88 / ? WT)
and (3) MyD88 / mice transplanted with GFP+/ BM cells
(GFP+/ ? MyD88 / ). The use of GFP+/ BM cells was motivated
by the possibility of measuring the level of chimerism in recipient
mice. Flow cytometry analysis of blood samples revealed an

A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

227

Fig. 4. MyD88 promotes both short-term and long-term survival of cortical neurons following KA injection. (a) FJB staining showing cortical neuronal death in WT and
MyD88 / mice sacriced 12 h and 10 days post-injection of KA. (b and c) Stereological quantication of FJB+ cells in the ipsilateral hemisphere of the dorsolateral cortex at b
12 h post-injection and in both hemisphere of the dorsolateral cortex c 10 days post-injection. (d) Representative photomicrographs of microglial cells as depicted by Iba1
immunostaining in WT and MyD88 / mice 12 h and 10 days after KA injection. Panel at the bottom left corner represents higher magnication of the deeper half of the
cortex. Graphs showing % of area immunopositive for Iba1 in the ipsilateral cortex of WT and MyD88 / mice (e) 12 h and (f) 10 days post-injection of KA. (g) CD68
immunostaining in the cortex of WT and MyD88 / mice 10 days after KA. (h) Stereological quantication of CD68+ cells in the deep layers of the ipsilateral cortex of WT and
MyD88 / mice 10 days post-injection of KA. Data are expressed as mean SEM, n = 78. p < 0.01; p < 0.05 compared by unpaired t test with Welchs correction in b or
without Welchs correction in c, e, f and h. Scale bar in a, d and g = 1 mm, and for close-up d and g = 250 lm.

average of 95.7% of chimerism for CD45+ cells (Fig. 5a). We used 2

different tests (nesting score and neuroscore) to determine which
group of mice displayed the most substantial behavioral decits
following KA injection. All mice from the three groups showed a

perfect score for both test before the KA challenge (data not
shown). Following KA injection, GFP ? MyD88 / mice had a signicantly lower nesting score than both GFP+/ ? WT (day 4 to
day 10) and Myd88 / ? WT (day 5 to day 9) mice (Fig. 5b).

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A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

Fig. 5. MyD88 / signaling in CNS resident cells provides neuroprotection against KA-induced excitotoxicity. (a) Chimerism observed in myeloid cells 4 weeks after bonemarrow transplantation. (b) Nesting performance from day one to day 10 post-injection of KA in GFP ? WT, MyD88 / ? WT and GFP ? MyD88 / mice. (c) Assessment of
neuroscore 5 days and 8 days post KA injection, n = 48. (d) Representative photomicrographs of FJB+ cells in the cortex of WT mice transplanted with WT GFP+/ or MyD88 /
bone-marrow cells and MyD88 / mice transplanted with WT GFP+/ 10 days following intra-striatal injection of KA. (e) Graphs showing FJB+ cells/mm2 (% of GFP ? WT
group) in the three chimeric mice groups 10 days post-injection of KA. Data are expressed as mean SEM; n = 48. Signicant differences were established using Two-way or
One-way ANOVA with Bonferroni post hoc test. p < 0.01; p < 0.05. (a) Scale bar in d = 1 mm.

Their neurological scores were also tested and a small tendency

toward greater decits in GFP ? MyD88 / was observed both 5
and 8 days post-injection (Fig. 5c). Analysis of neuronal death
10 days after KA injection revealed a much greater density of FJB+
neurons in GFP ?MyD88 / mice than in GFP ? WT (p = 0.015)
(Fig. 5d and e). Together these results indicate that neuronal survival
during excitotoxicity critically relies on a MyD88-dependent mechanism in CNS resident cells rather than peripheral bone-marrow cells.

4. Discussion
Excitotoxic processes and dysregulation of calcium and glutamate signaling are implicated in a wide range of neurodegenerative diseases. In response to brain injury, microglial TLRs
recognize DAMPs released by dying neurons and participate in
initiating an inammatory response with the aim of promoting
brain repair. However, this inammatory response needs to be
controlled to avoid excessive production of neurotoxic molecules.
Here, we report, to the best of our knowledge, the rst evidence
of benecial TLR preconditioning in the early phase (24 h) after
KA-induced excitotoxic neuronal death. Furthermore, we also
establish that microglial cells are more reactive in Pam3CSK4 and
LPS preconditioned mice and suggest that TLR2/4-preconditioning
is dependent on MyD88 signaling. This study also highlights the
benecial role of MyD88 signaling in KA-induced degeneration of
cortical neurons and suggests that this benecial effect is mainly
mediated by CNS resident cells rather than cells of hematopoietic
origin.
The rst study to characterize TLR preconditioning in a model of
stroke was realized by Tasaki et al. in 1997 using LPS as the TLR
agonist (Tasaki et al., 1997). Since then, many studies have shown
the ability of other TLR agonists to induce a state of ischemic tolerance when injected as a preconditioning agent, including the TLR2
ligand Pam3CSK4 (Hua et al., 2008), TLR3 ligand Poly-IC (Packard
et al., 2012a), TLR7 ligand Gardiquimod (Leung et al., 2012) and
the TLR9 ligand CpG (Stevens et al., 2008). LPS preconditioning
has also been reported in traumatic brain injury and different seizure models (Dmowska et al., 2010; Longhi et al., 2011; Mirrione
et al., 2010). It is still not clear if TLR ligands can induce a state

of excitotoxic tolerance that is similar to the known state of

ischemic tolerance. The exact mechanisms of TLR preconditioning
have not been clearly established so far, but a study using chimeric
mice revealed that TNF-a expression in cerebral cells is needed
(Packard et al., 2012b). TNF-a has both deleterious (Blais and
Rivest, 2004; Zhu et al., 2010) and benecial roles in excitotoxicity
depending on timing, concentration and by which of its receptors it
signals (Bernardino et al., 2005; Cheng et al., 1994; Figiel, 2008).
Upregulation of CREB-binding protein, a protein involved in calcium homeostasis, in TNF-a preconditioned neurons also supports
a protective role for this cytokine (Saha et al., 2009). Microglia do
not respond to systemic injection with LPS when TNF-a is neutralized (Nadeau and Rivest, 2000). Pam3CSK4 or LPS injected systemically signals via their respective TLRs on endothelial cells,
perivascular macrophages and microglial cells located in the circumventricular organs and choroid plexus. This signaling was
shown to induce a progressive wave of TNF-a across the brain parenchyma, which leads to microglial cells priming. It is tempting to
suggest that this wave of TNF-a induces a state of tolerance/preconditioning in neurons and protects them against a subsequent
excitotoxic insult. In accordance with this hypothesis, Chen et al.
showed, in an elegant study, that hematogenous TLR4 is not
needed to induce microglial activation after intra-peritoneal injections of LPS (Chen et al., 2012). This study also suggests an innovative mechanism by which LPS-preconditioned microglia protect
neurons through reduction of inhibitory axosomatic synapses.
It is well known that microglia and macrophages are very plastic cells that can adopt different phenotypes with various characteristics. Our results regarding the Iba1 staining suggest
morphological changes of microglial cells in response to neuronal
injury when mice are preconditioned with Pam3CSK4 or LPS compared to control. These results contrast with the ndings of
Rosenzweig et al. (2004) who observed that microglial activation
was attenuated after stroke in LPS-preconditioned mice using
FACS with antibody directed against CD45 and CD11b. These differences might be explained by the different methods used to analyze microglial phenotype. However, the fact that the number of
CD68+ cells was not higher in preconditioned mice suggests that
while microglia are more reactive to neuronal injury, they might
not exhibit a pro-inammatory, highly phagocytic phenotype.

A. Larochelle et al. / Brain, Behavior, and Immunity 46 (2015) 221231

Exposure of monocytes/macrophages to an endotoxin (like LPS)

modies the response to a subsequent inammatory challenge
toward an anti-inammatory response, this phenomenon is known
as endotoxin tolerance. At rst, we tough that preconditioning
with Pam3CSK4 or LPS induces a state of endotoxin tolerance in
microglial cells which would then generate a more antiinammatory/neurotrophic response when stimulated by DAMPs
released by dying neurons. However, we observed no difference
in the expression of anti-inammatory genes (TGF-b, IL-10 or
SOCS-3) between preconditioned and control groups after
KA-treatment using in situ hybridization, which is probably due
to their very high expression after KA injection (Data not shown).
The role of microglia in the early phase after neuronal injury is a
matter of great debate. We previously reported that TNF-a, but not
IL-1b, knockout mice exhibit an impaired early microglial response
that resulted in higher subsequent damage caused by the injection
of the excitotoxin sodium nitroprusside (Turrin, 2006). It is hard to
draw a conclusion about whether microglia activation is protectiveor harmful against neurotoxicity because usually, greater neurodegeneration leads to greater microglial activation. Such
correlative results may indicate that microglial activation compromises neurons survival. Here, we report that Pam3CSK4 or LPS
preconditioning induces stronger microglial activation but less
neurodegeneration early after KA injection.
In this study we showed that TLR preconditioning during acute
excitotoxicity is MyD88-dependent. MyD88 mRNA is strongly upregulated in this model of neuronal excitotoxicity and MyD88 signaling is neuroprotective at 12 h, 24 h and 10 days post KA
administration. On the contrary, Trif mRNA was not upregulated
after KA and TRIF / and WT mice exhibited similar neurodegeneration 24 h after KA injection. These data provide evidence
that MyD88 is a critical molecule for microglia to properly react
to dying neurons. The residual and strong activation of microglial
cells in MyD88 / mice is not surprising because there are other
well characterized PRRs and pathways through which microglia
may respond to injury. For example, other DAMPs include signaling
of nucleic acid through purinergic receptors (Davalos et al., 2005;
Ulmann et al., 2013). High levels of FJB+ neurons and very large
amoeboid microglia expressing CD68 were found across the deep
cortex of MyD88 / mice 10 days following KA administration.
These results suggest that while microglial cells from MyD88 /
mice can accumulate at the site of injury, they might have a
decient phagocytic response that prevent repair mechanisms
and regenerative process. Interestingly, phagocytic defects were
observed in MyD88 / mice after sciatic nerve lesion (Boivin
et al., 2007).
Higher susceptibility to neuronal death was already reported in
MyD88-decient mice following NO-induced excitoxicity (Simard
and Rivest, 2007). Two different studies using mouse model of
stroke reported no differences between WT, MyD88 / and
TRIF / mice (Famakin et al., 2011; Yang et al., 2008), but
Downes et al. recently reported a protective role for MyD88 signaling in hematopoietic cells after middle cerebral artery occlusion
(Downes et al., 2013). Our results using the KA model of excitotoxicity differ from these results as we found that MyD88 signaling
in CNS resident cells mediates the protective effects. These diverging results might be explained by fundamental differences in neuronal death in response to KA and MCAO models, different time
points for analyses as well as by the different methods used to
induce myeloablation in chimeric mice (chemotherapy vs. irradiation). In a study using bone-marrow chimeric mice, Lambertsen
et al. reported that microglia, but not peripheral cells, protected
neurons against ischemia by synthesizing TNF-a (Lambertsen
et al., 2009).
TLR2- and TLR4-decient mice have been shown to exhibit
milder signs of neuronal death after KA injection (Hong et al.,

229

2010; Maroso et al., 2010). It is possible that partially blocking

TLRs-dependent inammatory responses is benecial, whereas
blocking them completely by gene deletion exacerbates neuronal
damage. It is also plausible that the enhanced vulnerability of
MyD88 / mice to KA-induced excitotoxicity is due to a defective
brain development, as it was evidenced that TLRs are critically
involved in this process (Okun et al., 2011; Pimentel-Coelho
et al., 2013). Moreover, a recent study has showed that
MyD88 / mice display behavioral decits in specic tests
(Drouin-Ouellet et al., 2012). Therefore, it will be interesting to
investigate the role of MyD88 signaling in a cell- and time-specic
manner by using conditional/inducible knockout mice to verify
this possibility.
In conclusion, this study shows that TLR2 and 4 preconditioning
protects cortical neurons against KA-mediated excitotoxicity and
that both receptors modify the reactivity of microglial cells in
response to neuronal death. We have also shown that expression
of MyD88 in CNS resident cells is neuroprotective in the acute
and late phase of KA-induced cell death. Further studies aimed at
characterizing the exact molecular mechanisms underlying
neuroprotection conferred by TLR preconditioning and MyD88 signaling will signicantly help to decipher how TLR signaling by
DAMPs impact on the outcome of many neurological disorders,
such as Alzheimers disease, multiple sclerosis, stroke and epilepsy.
Conict of interest
The authors declare that they have no conict of interest.
Acknowledgments
The Canadian Institutes in Health Research (CIHR) and the
Multiple Sclerosis Scientic Research Foundation of Canada supported this research. Marc-Andre Bellavance was supported by a
Fonds de recherche QubecSant (FRQS) doctoral scholarship.
The authors thank Marie-Michle Plante for assistance with the
surgeries.
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