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stevelacroix.crchuq.ca

Download - Laboratoire Steve Lacroix

THE JOURNAL OF COMPARATIVE NEUROLOGY 500:267–285 (2007)

Proinflammatory Cytokine Synthesis in

the Injured Mouse Spinal Cord:

Multiphasic Expression Pattern and

Identification of the Cell Types Involved

ISABELLE PINEAU AND STEVE LACROIX*

Department of Anatomy & Physiology, Laval University, Ste-Foy,

Québec, Canada G1V 4G2

ABSTRACT

We have studied the spatial and temporal distribution of six proinflammatory cytokines

and identified their cellular source in a clinically relevant model of spinal cord injury (SCI).

Our findings show that interleukin-1 (IL-1) and tumor necrosis factor (TNF) are rapidly

(5 and 15 minutes, respectively) and transiently expressed in mice following contusion. At

30–45 minutes post SCI, IL-1 and TNF-positive cells could already be seen over the entire

spinal cord segment analyzed. Multilabeling analyses revealed that microglia and astrocytes

were the two major sources of IL-1 and TNF at these times, suggesting a role for these

cytokines in gliosis. Results obtained from SCI mice previously transplanted with green

fluorescent protein (GFP)-expressing hematopoietic stem cells confirmed that neural cells

were responsible for the production of IL-1 and TNF for time points preceding 3 hours. From

3 hours up to 24 hours, IL-1, TNF, IL-6, and leukemia inhibitory factor (LIF) were strongly

upregulated within and immediately around the contused area. Colocalization studies revealed

that all populations of central nervous system resident cells, including neurons,

synthesized cytokines between 3 and 24 hours post SCI. However, work done with SCI-GFP

chimeric mice revealed that at least some infiltrating leukocytes were responsible for cytokine

production from 12 hours on. By 2 days post-SCI, mRNA signal for all the above

cytokines had nearly disappeared. Notably, we also observed another wave of expression for

IL-1 and TNF at 14 days. Overall, these results indicate that following SCI, all classes of

neural cells initially contribute to the organization of inflammation, whereas recruited

immune cells mostly contribute to its maintenance at later time points. J. Comp. Neurol. 500:

267–285, 2007. © 2006 Wiley-Liss, Inc.

Indexing terms: spinal cord injury; central nervous system; contusion; TNF; IL-1; IL-6; LIF; GM-

CSF; INF-; macrophage; microglia; impactor device; Wallerian degeneration

Although it is well established that immune cells and

their inflammatory mediators are induced following central

nervous system (CNS) injuries, whether these cells

and molecules are deleterious or beneficial to CNS tissue

is still a matter of great debate. Many groups have reported

that impeding inflammation, via the administration

of antiinflammatory drugs or by using macrophagedepletion

approaches, can prevent extensive tissue loss

and partially restore locomotor activity following spinal

cord injury (SCI) (Popovich et al., 1999; Lee et al., 2003;

Wells et al., 2003; Gris et al., 2004; Stirling et al., 2004;

Teng et al., 2004). Others have shown that treatment with

glucocorticoid or minocycline, a tetracycline derivative

with antiinflammatory effects, can also reduce the development

of the astroglial/fibroblastic scar after CNS injury

(Li and David, 1996; Teng et al., 2004). Nevertheless, the

Grant sponsor: The Canadian Institutes of Health Research (CIHR);

Grant number MOP-67139; Grant sponsor: the Rx&D Health Research

Foundation (Career Award to S.L.); Grant sponsor: the Canadian Foundation

for Innovation; Grant sponsor: Fonds de la Recherche en Santé du

Québec.

*Correspondence to: Steve Lacroix, Ph.D., CHUL Research Center and

Laval University, 2705, Laurier Blvd., Ste-Foy, Québec, Canada G1V 4G2.

E-mail: Steve.Lacroix@crchul.ulaval.ca

Received 27 March 2006; Revised 25 May 2006; Accepted 3 August 2006

DOI 10.1002/cne.21149

Published online in Wiley InterScience (www.interscience.wiley.com).

© 2006 WILEY-LISS, INC.


The Journal of Comparative Neurology. DOI 10.1002/cne

268 I. PINEAU AND S. LACROIX

use of antiinflammatory drugs to treat patients suffering

from SCI remains controversial, with several other studies,

including a few large randomized controlled trials,

reporting a lack of long-term effects of glucocorticoids on

functional recovery (George et al., 1995; Gerndt et al.,

1997; Nesathurai, 1998; Hurlbert, 2000). Work with nonsteroidal

antiinflammatory drugs in various models of

CNS insults has also yielded conflicting results with regard

to the potential of blocking inflammation to prevent

neuronal cell death (for review, see David and Lacroix,

2005).

Despite the evidence that immune cells could contribute

to the loss of neural tissue following injuries, other studies

have shown that when appropriately stimulated, macrophages,

microglia, and lymphocytes can promote axon

growth and regeneration, resulting in functional recovery

in animal models of SCI (Prewitt et al., 1997; Rabchevsky

and Streit, 1997; Rapalino et al., 1998). In addition,

primed autologous macrophages are being tested on SCI

patients in clinical trials (Schwartz and Yoles, 2005). Over

the years, many groups have reported that immune cells

can be a source of mitogenic, chemotactic, angiogenic, and

trophic factors supporting remyelination and neuronal

survival in various models of CNS insults and diseases

(for review, see Liberto et al., 2004). Another mechanism

by which immune cells may limit CNS toxicity and contribute

to creating an environment that is permissive for

regeneration and recovery is through elimination of foreign

materials and cellular debris (Vallières et al., 2006).

These results highlight the importance of clarifying the

role of the immune response in generating the pathological

changes at the site of CNS lesion, and its potential for

influencing regeneration and tissue repair.

SCI results in the almost-immediate activation of microglia

and astrocytes, followed by recruitment of bloodderived

immune cells to the primary site of lesion. Successively,

neutrophils, monocytes, and lymphocytes are

chemoattracted to the site of trauma and will occupy the

lesioned area for limited or prolonged periods, depending

on the cell type (Sroga et al., 2003). To organize an inflammatory

response that results in the phagocytosis of cellular

and axonal debris from sites of injury and from spinal

segments undergoing Wallerian degeneration (WD), immune

cells release and respond to a multitude of cytokines.

In rodents, analysis of these inflammatory mediators

in the context of CNS injury suggests that some of

these molecules could increase sensitivity to pain (Samad

et al., 2001; Miller, 2005). Others have shown that, at high

dosage, cytokines could also be toxic for neurons and glia

and cause demyelination and axon damage (Allan and

Rothwell, 2001).

Among all proinflammatory cytokines, tumor necrosis

factor (TNF) has received the most attention. It is a pleiotropic

cytokine with widespread effects on cellular responses

such as cell proliferation and differentiation, apoptosis,

and immunity and inflammation (Baud and

Karin, 2001). Of particular interest to the present study,

TNF also plays a crucial role in recruiting, but not activating,

macrophages following nerve injury (Liefner et al.,

2000). Interleukin-1 (IL-1) also appears to be a key

regulator of WD in the injured peripheral and central

nervous system (Shamash et al., 2002; Mi et al., 2004).

Like TNF, IL-1 can modulate the expression of cell adhesion

molecules (CAMs), which are required for the adhesion

of leukocytes to the cerebral endothelium and for

their migration to sites of injury. Other critical mediators

of immune and inflammatory responses in the injured

nervous system include the hematopoietic/neuropoietic cytokines

IL-6 and leukemia inhibitory factor (LIF). Work

done with LIF knockout mice has shown that it is essential

for the initial recruitment of neutrophils and macrophages

into crushed sciatic nerve (Sugiura et al., 2000).

Similarly, microglial and astroglial responses to cortical

injury were found to be compromised in LIF and IL-6

knockout mice compared with wild type (Penkowa et al.,

1999; Sugiura et al., 2000). Two other cytokines,

granulocyte/macrophage colony-stimulating factor (GM-

CSF) and interferon- (INF-), mediate many innate and

adaptive immune responses. GM-CSF stimulates hematopoiesis,

whereas INF- is one of the principal macrophageactivating

cytokines. Little is known about the role of

GM-CSF and INF- in SCI. Still, it has been suggested

that INF- could play a role in astrocyte proliferation

(Yong et al., 1991). However, recent studies have reported

that INF- is not expressed following SCI in mice (Ousman

and David, 2001; Perrin et al., 2005).

Although much progress has been achieved in the field

of neuroimmunology, the exact role of immune cells and

molecules during the course of neuropathological conditions

is still uncertain. It is possible that under certain

conditions, the recruitment and activity of macrophages,

microglia, and astrocytes would need to be suppressed to

avoid extended damage, whereas in other conditions, restricting

the inflammatory response may impair regenerative

processes. Characterization of the signals that mediate

immune cell recruitment and activity could have a

major impact on the development of new strategies aimed

at manipulating the immune responses to treat CNS injuries.

In this study, we present the complete distribution

and temporal mRNA expression pattern of some of the key

cytokines (IL-1, TNF, IL-6, LIF, GM-CSF, and INF-) at

the site of SCI and in distal segments undergoing WD.

Furthermore, we identified the phenotype of the cells expressing

these cytokines in the injured spinal cord.

MATERIALS AND METHODS

Animals

A total of 234 adult female C57BL/6 mice (8–12 weeks

old) weighing 18–20 g (Charles River) were used in this

study. Mice had free access to food and water. All surgical

procedures were approved by the Laval University Animal

Care Committee and followed CCAC guidelines. The experimental

protocol included three groups of animals: naive

(n 10), sham-operated (n 79), and SCI (n 145)

mice. The distribution of these mice among the different

experiments performed in the present study is reported in

Table 1.

Experimental spinal cord injury

C57BL/6 female mice were deeply anesthetized with

isoflurane and underwent a laminectomy at vertebral

level T9–10, which corresponds to spinal segment T10–

11. Briefly, the vertebral column was stabilized and a

contusion of 70 kdyn was performed by using the Infinite

Horizon (IH) SCI device (Precision Systems & Instrumentation,

Lexington, KY). For the sham-operated mice, the

exposed spinal cord was left untouched. Overlying muscular

layers were then sutured and cutaneous layers sta-


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CYTOKINE mRNA EXPRESSION FOLLOWING SCI

269

Group

Noninjured

TABLE 1. Overview of the Number of Animals Included in Different Experiments

Time after spinal cord injury

Minutes Hours Days

5 15 30 45 1 3 6 12 24 2 4 7 14 28 35

In situ

1. Naive 7

2. Sham 2 6 7 7 6 7 7 7 6 6 6

3. SCI 2 2 3 2 10 10 10 10 10 11 10 10 10 10

Double labeling

1. Naive

2. Sham 2 2 2 2 2

3. SCI 4 4 4 4 4 4

Chimeric mice

1. Naive 3

2. Sham 1 1

3. SCI 1 2 2 1 1 1 1 1 1

Abbreviation: SCI, spinal cord injury.

pled. Postoperatively, animals received manual bladder

evacuation twice daily to prevent urinary tract infections.

SCI and sham-operated mice were sacrificed by perfusion

at 5, 15, 30, and 45 minutes and 1, 3, 6, 12, and 24 hours

and 2, 4, 7, 14, 28, and 35 days post contusion.

Tissue processing, histology and 3D spinal

cord reconstruction

Mice were overdosed with a mixture of ketamine and

xylazine and transcardially perfused with 40 ml of cold

0.9% saline solution followed by 50 ml of 4% paraformaldehyde

(PFA), pH 9.5, in borax buffer. After perfusion

with the fixative, spinal cords were dissected out, postfixed

for 2 days, and placed overnight in a 4% PFA-borax/

10% sucrose solution. Spinal cords were then blocked into

4-mm segments. For each animal, a total of three spinal

cord segments were cut by using a cryostat (model

CM3050S; Leica Microsystems, Richmond Hill, ON, Canada).

The first segment was centered at the site of the

trauma and for that reason included 2 mm on each side of

the lesion. The rostral and caudal 4-mm segments were

located 2–6 mm distal to the injury site, on each side of the

lesion. On the day of the sectioning, spinal cord segments

were placed side by side, the rostral end of each segment

facing up, into plastic molds and covered with Tissue-Tek

embedding medium (OCT compound; Canemco, St-

Laurent, QC, Canada). The entire mold was then quickly

frozen over dry ice and left in place until tissue processing.

Then 30-m-thick cryostat coronal sections were collected

directly onto slides that have a permanent positive

charged surface (Surgipath Canada, Winnipeg, Manitoba,

Canada) and separated into seven different series of adjacent

sections. Slides were stored at 20°C until histochemical

staining, in situ hybridization, or immunofluorescence

was performed.

One series of adjacent sections was stained with luxol

fast blue (LFB) and then counterstained with cresyl violet

(CV). LFB/CV-stained sections were used to identify white

matter sparing (myelin sparing, visualized by LFB) and

gray matter sparing. (The CV counterstaining allows one

to distinguish cellular debris from intact tissue at high

magnification; Popovich et al., 1999.) For LFB staining,

sections were dehydrated and then incubated in a 0.1%

LFB solution at 37°C overnight. The next day, slides were

cooled at 4°C, incubated in a 0.05% lithium carbonate

solution, and differentiated in 70% ethanol. Finally, slides

were counterstained with 0.1% CV, dehydrated through

graded concentrations of ethanol (80, 95, and 100%), defatted

in Hemo-D, and coverslipped with DPX mounting

medium (Electron Microscopy Sciences, Hatfield, PA).

These sections were used to identify the lesion epicenter

and for the three-dimensional (3D) reconstruction of the

lesion.

3D spinal cord reconstructions were performed by using

the BioQuant Nova Prime computerized image analysis

system (Bioquant Topographer XP plug-in; Nashville,

TN). Briefly, the outline of 1 out of 14 (IL-1, IL-6, and

LIF) and 1 out of 28 (TNF) coronal sections within a

predetermined spinal cord segment, including the lesion

epicenter and sections located up to 2 mm (for IL-1, IL-6,

and LIF) or 5 mm (for TNF) distal to the center of the

lesion in both directions (i.e., rostral and caudal), were

traced manually at low magnification, as well as the outline

of necrotic and damaged tissue at higher magnifications.

Areas where normal spinal cord architecture was

absent and areas containing cellular debris were considered

as areas of tissue damage. The Topographer (Atlas

Shop and Atlas Modeler) offered with the Bioquant program

was then used to reconstruct the injured spinal cord.

Topographic mapping of cells expressing cytokines in 3Dreconstructed

injured spinal cords was also performed.

In situ hybridization

In situ hybridization was carried out to detect the mR-

NAs coding for the following cytokines: IL-1, TNF, IL-6,

LIF, GM-CSF, and INF-. Full-length cDNAs for the

mouse IL-1, TNF, IL-6, LIF, and INF- cloned into expression

vectors pBluescript II SK (IL-6, TNF), pCRII-

TOPO (IL-1), pcDNA (LIF), and pGEMEX (INF-) were

kindly provided by Dr. Serge Rivest (Laval University,

QC, Canada). The full-length cDNA coding for the mouse

GM-CSF was obtained from Open Biosystems (Huntsville,

AL). For the latter cDNA, sequence corresponding to nucleotides

12–384 from IMAGE: 1428783 was amplified by

using the following primers: GM-CSF (forward, 5-gcatcgaattccaagaagctaacatgtgtgcagac-3;

reverse, 5-ggtcactcgaggctatactgccttccaagtcgtgctg-3).

The sequence chosen

for probe synthesis was selected to match only the intended

gene, as verified by BLAST analysis in Genbank.

Polymerase chain reaction (PCR) product was subcloned

into pBluescript II KS at the EcoR1-XhoI restriction

sites and then sequenced to confirm gene identity. After

linearization of the plasmids, radiolabeled cRNA probes

were synthesized by using the Riboprobe Combination


The Journal of Comparative Neurology. DOI 10.1002/cne

270 I. PINEAU AND S. LACROIX

System SP6/T7 (Promega) and T3 RNA polymerase (Promega).

In situ hybridization was performed according to a

previously described method (Lacroix et al., 1998, 2002;

Lacroix and Rivest, 1998). RNA probes used for in situ

hybridization were labeled with [- 35 S] UTP (NEN, Boston,

MA). All sections were prehybridized, hybridized, and

posthybridized in parallel to equalize background intensity.

Hybridized sections were then exposed at 4°C to X-ray

films (Kodak) for a period of 24 hours, defatted in Hemo-D,

and dipped in NTB nuclear emulsion (Kodak; diluted 1:1

in distilled water). Slides were exposed for 10 days (TNF),

14 days (IL-1, IL-6, and LIF), or 19 days (GM-CSF and

INF-) at 4°C, developed in a D19 developer (Kodak), and

fixed in a rapid fixer. Sections were then dehydrated,

cleared in Hemo-D, counterstained in 0.25% thionin, and

coverslipped with DPX mounting medium. The presence

of each transcript was detected by the agglomeration of

silver grains within cell cytoplasm. Digital microscopic

images were collected with a high-resolution Retiga QI-

CAM fast color 1394 camera (1,392 1,040 pixels; QImaging,

Burnaby, BC, Canada) installed on a Nikon Eclipse

80i microscope. These images were then imported into

Adobe Photoshop (v. 7.0) and adjusted to optimize brightness,

contrast, and sharpness. Off-tissue backgrounds

were darkened for clarity. To generate the final figures,

individual images were exported as TIF files into Adobe

Illustrator (v. 12.0.1) and assembled into plates.

Quantification of in situ hybridization signal

For SCI mice, the lesion epicenter was determined as

being the section containing the most tissue damage. For

the animals that received laminectomy only, because of

the absence of spinal cord lesion, the section having the

highest cell count in the 4-mm segment analyzed was

considered as the center of the lesion. Cells expressing

mRNAs coding for specific cytokines were then counted

from evenly spaced coronal sections (420-m intervals for

IL-1, IL-6, and LIF and 840-m intervals for TNF) located

on both sides of the lesion epicenter up to a rostrocaudal

distance of 2.1 mm for IL-1, IL-6, and LIF and 5

mm for TNF. Because of the impossibility of distinguishing

single cells in meningeal structures, meningeal layers

were excluded from the quantitative analyses. Cells were

quantified at 10 magnification. Results were expressed

as total number of cells expressing mRNA signal per cross

section at each level analyzed and then compared among

all subjects of this study to generate mean standard

error of the mean (SEM). Quantification was performed by

a single observer.

Combination of in situ hybridization with

multiple immunofluorescence labeling

To identify the cell populations expressing a number of

cytokine mRNAs, in situ hybridization was combined with

multiple immunofluorescence labeling. Prehybridization,

hybridization, and posthybridization steps were performed

as previously described with the sole exception

that dehydration steps were omitted. Instead, tissue sections

were rinsed in KPBS and then immediately processed

for multiple immunofluorescence labeling. The following

antibodies were used to identify cell types: the

ionized calcium-binding adaptor molecule 1 (Iba1) polyclonal

antibody, or the galactose-specific lectin-3

(Galectin-3) monoclonal antibody, or the CD11b monoclonal

antibody for macrophages/microglia; the carbonic anhydrase

II (CAII) polyclonal antibody for oligodendrocytes;

glial fibrillary acid protein (GFAP) polyclonal

antibody for astrocytes; and the neuron-specific nuclear

protein (NeuN) monoclonal antibody for neurons. A more

complete description of the primary antibodies used in

this study is given in Table 2.

Briefly, one series of spinal cord sections was processed

for multiple immunofluorescence labeling by using cover

well incubation chambers and the following protocol: 1)

blocking in KPBS 0.25% Triton-X 5% normal serum

for 1 hour, 2) incubation for 2 hours in primary antibody at

room temperature, 3) incubation for 2.5 hours in secondary

antibodies conjugated with either the fluorophore Alexa

488 (dilution 1:200; Invitrogen Canada Inc., Burlington,

ON, Canada) or the fluorophore Rhodamine Red-X

(dilution 1:200; Jackson ImmunoResearch, West Grove,

PA), and 4) counterstaining in DAPI (dilution 1:5,000;

Invitrogen Canada Inc.) for 20 minutes. Sections were

dried under vacuum and exposed at 4°C to X-ray film for

24 hours. The following day, slides were dipped in NTB

nuclear emulsion and exposed in the dark at 4°C. The

dehydration and defatting steps that normally precede

dipping into nuclear emulsion were omitted to avoid fading

of the fluorescence. Thereafter, slides were developed

and coverslipped as before. The presence of each mRNA

was detected by the agglomeration of silver grains in the

cell bodies. Immunofluorescence labeling was visualized

with a fluorescent microscope by using an ultraviolet (UV)

excitation filter for DAPI and absorption spectra filters of

bandpasses 515–555 and 590 for Alexa-488 and Rhodamine

Red-X, respectively. Digital microscopic images were

collected with a high-resolution Retiga QICAM fast color

1394 camera installed on a Nikon Eclipse 80i microscope.

Images were edited by using Photoshop and Illustrator

(Adobe Systems) software, as described before.

Immunoperoxidase staining and

immunoblotting

Immunoperoxidase staining and immunoblot analyses

were performed to determine the specificity of the primary

antibodies used in the present study. Immunoperoxidase

staining was performed on tissue sections directly

mounted on slides by using CoverWell incubation chambers

(Invitrogen Canada Inc.) and our previously published

protocol (Lacroix et al., 2002). Spinal cord sections

obtained from both naive and SCI mice were used for

immunolabeling. Dilutions used for primary antibodies

are given in Table 2.

Immunoblotting was performed on cell lysate extracts

obtained from the spinal cord of naive (n 2) and SCI (n

1; sacrificed at 48 hours post SCI to detect Galectin-3)

mice as well. Briefly, spinal cords were dissected out and

frozen immediately on dry ice. Spinal cord tissues were

homogenized in a protein extract buffer composed of 8 M

urea, 0.5% sodium dodecyl sulfate (SDS), 2%

-mercaptoethanol, and Protease Inhibitor Cocktail for

use with mammalian cell and tissue extracts. (All reagents

were purchased from Sigma, Mississauga, ON,

Canada.) Homogenates were incubated on ice for 1 hour

and then cleared by centrifugation at 13,000 rpm for 20

minutes at 4°C. Protein concentration was determined by

using the bicinchoninic acid (BCA) kit (Sigma).

Proteins from spinal cord lysates were separated on 12%

polyacrylamide gels by using the Mini-PROTEAN 3 sys-


The Journal of Comparative Neurology. DOI 10.1002/cne

CYTOKINE mRNA EXPRESSION FOLLOWING SCI

271

TABLE 2. Summary of the Primary Antibodies Used in the Present Study

Antibody Specificity Immunogen/clone Dilution Source Catalog/lot # References

1:750 Wako 019-19741/HNH3687 Imai et al., 1996;

Vallières and Sawchenko, 2003

Synthetic peptide of C-terminus iba1,

N-PTGPPAKKAISELP-C

17-kDa EF hand protein expressed

on macrophages and microglia

Polyclonal

–Ionized calcium-binding

adaptor molecule 1

(iba1)

GFAP present in astrocytes GFAP isolated from cow spinal cord 1:750 Dako Z 0334/5193 Vallières et al., 2006

–Glial fibrillary acid

protein (GFAP)

1:2000 Invitrogen A-6455/93E1-1 Lacroix et al., 2002

GFP Purified from jellyfish Aequorea

victoria

–Green fluorescent

protein (GFP)

CAII found in oligodendrocytes Purified protein from rat hemolysates 1:2,000 Dr. Said Ghandour N/A Ghandour et al., 1980;

Jalabi et al., 2005

–Carbonic anhydrase II

(CAII)

1:500 ATCC TIB-166/211921 Vallières et al., 2006

Spleen cells fused with NS-1

myeloma cells/clone M3/38

32-kDa Mac-2 glycoprotein on

macrophages/activated microglia

Monoclonal

–Galactose-specific lectin-

3 (Gal-3)

1:250 Chemicon MAB377/24120257 Vallières and Sawchenko, 2003

NeuN protein found in neurons Purified cell nuclei from mouse brain/

clone A60

–Neuron-specific nuclear

protein (NeuN)

1:500 BD Pharmingen 557397/M075043 Sroga et al., 2003

C57BL/10 mouse splenic T cells and

concanavalin A-activated

splenocytes/clone M1/70

Murine M chain of Mac-1 found

on microglia/macrophages

–CD11b (integrin M

chain, Mac-1 chain)

CD45 molecule on leukocytes Mouse thymus or spleen/clone 30-F11 1:500 BD Pharmingen 553082/M074786 Vallières and Sawchenko, 2003

–CD45 (leukocyte

common antigen, Ly-5)

tem (Bio-Rad, Mississauga, ON, Canada) and electrotransferred

to nitrocellulose Hybond-ECL membranes

(GE Healthcare, Baie d’Urfé, QC, Canada). After

blocking for 1 hour in 5% nonfat dried milk in TBS/0.1%

Tween-20, blots were incubated overnight with primary

antibodies at the following dilutions: anti-Iba-1 (dilution

1:500), anti-GFAP (dilution 1:3,750), anti-CAII (dilution

1:10,000), anti-Galectin-3 (dilution 1:1,500), anti-NeuN

(dilution 1:500). (For a complete description of these primary

antibodies, please refer to Table 2.) The next day,

membranes were washed and incubated with horseradish

peroxidase (HRP)-conjugated secondary antibodies (antirabbit,

anti-mouse, or anti-rat IgG antibodies; dilution

1:5,000; Jackson ImmunoResearch). Immunodetection

was performed by using enhanced chemiluminescence

(ECL) reagents (GE Healthcare).

All antibodies that were used as cellular markers

stained the appropriate pattern of cellular morphology

and distribution in our tissue sections (Fig. 1A–G). Immunoblotting

experiments were also performed on spinal

cord extracts to assess further the specificity of the primary

antibodies used in the present study. In agreement

with the manufacturer’s description, primary antibodies

reacted specifically in immunoblotting. Specifically, the

anti-NeuN recognized two bands in the 46-48 kDa molecular

weight range (Fig. 1H, lane 1). These two bands

represent the different phosphorylated isoforms of NeuN

(Lind et al., 2005; manufacturer’s technical information).

Anti-Galectin-3 and anti-CAII stain a single band of approximately

32 and 29 kDa, respectively, on Western blot

(Fig. 1H, lanes 2 and 3). These numbers are in agreement

with the known molecular weights of the two proteins.

(Refer to the supplier’s datasheet for Galectin-3 and to

Ghandour et al., 1992 for CAII.) The GFAP antiserum

produced a prominent band running at approximately 50

kDa and a weaker band at 45 kDa (Fig. 1H, lane 4),

which apparently is a proteolytic fragment of the protein

or an alternate transcript from the GFAP gene (Nielsen et

al., 2002). Unfortunately, we were unable to obtain detectable

staining above background levels for Iba1 by Western

blot, using total protein extracts prepared from noninjured

or injured spinal cords. However, by using the same

antibody, Imai and colleagues have previously detected a

single 17-kDa band corresponding to Iba1 in proteins extracted

from cultured microglia and hematopoietic cells

but not from neurons, astrocytes, and fibroblasts (Imai et

al., 1996; Ito et al., 1998). At least two studies have shown

that the anti-CD11b monoclonal antibody (clone M1/70)

immunoprecipitates polypeptide chains of 190 and 105

kDa found in macrophages (Springer et al., 1979;

Springer, 1981). Finally, the anti-CD45 antibody (clone

30F-11) has been shown to immunoprecipitate specifically

a protein of 175 kDa, which was identified as an isoform of

CD45, on the surface of thymocytes (Uemura et al., 1996).

Production of chimeric mice

To establish the origin of the inflammatory cells that

synthesize cytokines following SCI (i.e., hematogenous

versus resident CNS cells), expression of proinflammatory

molecules was evaluated in SCI mice previously irradiated

and transplanted with bone marrow cells expressing

the green fluorescent protein (GFP) marker. Briefly,

C57BL/6J female mice were lethally irradiated with one

dose of 12.5 Gray by using a cesium-137 source (Gammacell

Exactor, MDS Nordion, Vancouver, BC, Canada) to


Figure 1

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The Journal of Comparative Neurology. DOI 10.1002/cne

CYTOKINE mRNA EXPRESSION FOLLOWING SCI

destroy hematopoietic stem cells (HSCs). Six hours following

irradiation, the animals were injected via a tail vein

with 7.5 10 6 bone marrow cells freshly collected from

GFP mice (The Jackson Laboratory, Bar Harbor, ME).

Bone marrow cells were aseptically harvested by using the

method published by Vallières and Sawchenko (2003).

Irradiated mice transplanted with these cells were housed

in autoclaved cages and treated with antibiotics (2.5 ml of

Septra/200 ml of drinking water; GlaxoSmithKline, Mississauga,

ON, Canada) for 1 week before and 2 weeks after

irradiation. After a 6-month recuperation period from the

irradiation and transplantation procedures, bone marrow

recipient mice received a spinal cord contusion, as previously

described. The day of the sacrifice, mice were deeply

anesthetized with a mixture of ketamine and xylazine,

and a blood sample was collected from the heart of the

animal by using a 27-gauge needle. Blood samples were

kept in blood collection tubes (Vacutainer plus, BD Biosciences,

Mississauga, ON, Canada) until the degree of

chimerism was analyzed. This was immediately followed

by transcardiac perfusion of the animal by using 4% PFA

(pH 9.5), following the protocol described before.

Chimerism studies were performed to determine the

ratio of white blood cells expressing the GFP marker at 6

months post irradiation/transplantation. For this, the

blood was diluted with Dubelco’s phosphate-buffered saline

(DPBS) and gently deposited over a Ficoll gradient.

After centrifugation, leukocytes were collected, washed

twice with DPBS supplemented with 2% normal goat serum

(NGS; Invitrogen Canada), and then incubated for 15

minutes on ice with Cy-Chrome-labeled antibodies to the

CD45 antigen (a leukocyte marker) and phycoerythrinlabeled

antibodies to the CD-11b antigen (a macrophage

marker; see Table 2). Cells were washed twice with DPBS

supplemented with NGS and then transferred into

fluorescent-activated cell sorting (FACS) analysis tubes.

Ten thousand cells were analyzed by FACS.

To discriminate cytokines produced by blood-derived

leukocytes from those derived from neural cells, in situ

hybridization was combined with immunofluorescence to

detect the GFP marker. Spinal cord sections obtained

from SCI-GFP chimeric mice were incubated for 2 hours

with primary antibody (anti-GFP; see Table 2) at room

temperature and processed as before.

Fig. 1. Specificity of the primary antibodies used in the present

study. The specificity of the primary antibodies used in this study was

established by immunoperoxidase labeling on spinal cord tissue section

and by immunoblotting on spinal cord lysates prepared from both

naive and SCI mice. A–G: Low-power magnification photomicrographs

showing representative examples of Galectin-3 (A,B), Iba1 (C),

CAII (D), GFAP (E), NeuN (F), and CD11b (G) immunoperoxidase

staining (DAB) on spinal cord tissue sections obtained from naive

(A,C–F) and SCI (B,G) mice. B–G, insets: Cells positive for

Galectin-3, Iba1, CAII, GFAP, NeuN, and CD11b immunoreactivity

have the characteristic size and morphology of macrophages/activated

microglia, microglia, oligodendrocytes, astrocytes, neurons, and

macrophages/microglia, respectively. Note that Galectin-3-

immunoreactive macrophages/activated microglia were not observed

when the anti-Galectin-3 antibody was used to stain spinal cord tissue

sections taken from a naive mouse (A), whereas many Galectin-3-

positive cells were detected in sections from a SCI mouse (B). H: Immunoblot

analyses confirmed that primary antibodies used in this

study recognized proteins of appropriate molecular weight. Lane 1,

NeuN; lane 2, Galectin-3; lane 3, CAII; and lane 4, GFAP. Scale bar in

G 100 m in A–G; 60 m in inset C; 40 m in insets B and D–G.

RESULTS

Tissue displacement and actual force

applied following severe spinal cord

contusions in C57BL/6 mice

273

In this study, a total of 145 C57BL/6 mice were subjected

to SCI by using the IH impactor device. As reported

by Scheff and colleagues (2003), the IH software allows

one to record several biomechanical variables during injury,

the most important being displacement of the spinal

cord following impact and the actual force applied. Tissue

displacement is defined as the distance, in m, that the

impactor tip travels from the initial contact to the point at

which the force preselected by the user has been attained.

The predetermined force used in this study was set at 70

kdyn. This particular force has been defined by the manufacturer

and at least one other group (Ghasemlou et al.,

2005) as the standard to create severe SCI in mice by

using the IH impactor device. On average, for a preselected

force of 70 kdyn, the actual force of impact on

SCI-mice was 78.0 0.9 kdyn. The extent of spinal cord

tissue displacement in these animals ranged from approximately

400 to 900 m. The mean displacement value was

596 16 m.

Cytokine mRNAs are not constitutively

expressed in the spinal cord of naive mice

By using in situ hybridization, we found no trace of

mRNA signal for any of the proinflammatory cytokines

analyzed on spinal cord sections obtained from naive mice.

Similarly, no positive signal was observed when tissue

sections were hybridized with control mouse sense riboprobes.

SCI triggers the rapid and transient

expression of proinflammatory cytokines

Interleukin-1 (IL-1). Unless indicated otherwise,

all observations made throughout the Results section apply

to nonchimeric (i.e., nonirradiated) SCI mice. IL-1

mRNA-positive () cells were detected as early as 5 minutes

following SCI (Fig. 2A). At this time point, IL-1

cells were confined to damaged areas only. From 15 minutes

up to 45 minutes post SCI, the number of IL-1 cells

rapidly increased, and their distribution started to spread

out to the entire spinal cord segment analyzed (Fig.

2B,D,E). This generalized, widespread expression of IL-1

mRNA was very dynamic, however, and had almost completely

disappeared at 1 hour post SCI. By using SCI mice

previously transplanted with GFP-expressing hematopoietic

stem cells, we confirmed that neural cells were mainly

responsible for the production of IL-1 at time points

preceding 1 hour. This was shown by the absence of GFPpositive

cells expressing the IL-1 mRNA transcript at 1

hour post SCI (Fig. 2C). On average, we estimated that the

percentage of white blood cells expressing the GFP

marker in our transplanted mice (n 6) was equal to

87.2 1.2% at 6 months post irradiation/transplantation.

At this time point post irradiation/transplantation, many

GFP-labeled cells were detected throughout the CNS of

noninjured chimeric mice. As reported by Vallières and

Sawchenko (2003), the vast majority of these donorderived

cells were closely associated with blood vessels. It

should also be mentioned that the number of cells express-


The Journal of Comparative Neurology. DOI 10.1002/cne

274 I. PINEAU AND S. LACROIX

Fig. 2. IL-1 mRNA expression in the injured mouse spinal cord;

time points up to 1 hour post spinal cord injury (SCI). A,B: Darkfield

photomicrographs showing IL-1 mRNA expression at the lesion epicenter

at 5 minutes (A) and at a distance of 2,520 m rostral to the

lesion at 45 minutes (B) following spinal cord contusion in C57BL/6

mice. C: Brightfield photomicrograph showing the absence of colocalization

between IL-1 mRNA and cells expressing the GFP

marker (brown cells) at 1 hour post SCI. This picture was obtained

from an SCI mouse previously transplanted with GFP-expressing

hematopoietic stem cells. D,E: Three-dimensional reconstructions of

the injured spinal cord showing damaged areas (D) and the spatial

distribution of IL-1 cells (E) at 45 minutes following SCI. F: Time

course of IL-1 mRNA expression at the lesion epicenter following

SCI. Values for the number of IL-1 cells are means SEM.

G: Distribution of cells expressing IL-1 mRNA at 12 hours post SCI.

Positive values on the x-axis represent distances rostral to the lesion,

whereas negative values are caudal to the lesion. Scale bar 50 m

in A; 100 m inB;25m inC.

ing cytokine mRNA and the distribution of these cells in

the spinal cord was comparable between sham-operated

chimeric mice (i.e., irradiated mice) and sham-operated

nonirradiated animals, indicating that CNS resident cells

that could have been activated as a result of the irradiation

were not constitutively producing proinflammatory

cytokines before the injury. The fact that GFP-positive

cells did not express IL-1 mRNA at 1 hour post SCI

further supports this finding.

At the lesion epicenter, the number of cells expressing

IL-1 increased progressively from 1 hour up to 12 hours,

when expression was maximal (Fig. 2F). At this time,

IL-1 cells were once again restricted to damaged areas

extending about 1.5 mm on either side of the lesion epicenter

(Figs. 2G, 3A,B). At 24 hours, the number of IL-1

cells had already decreased by 81% compared with the

12-hour time point. The number of IL-1 cells continued

to decrease at 2, 4, and 7 days post SCI before reaching

another peak of expression at 14 days (4.5-fold increase;

Figs. 2F, 3C,D). Signal was almost back to normal values

at 28 days. These results are in agreement with our previous

study in which we reported a second peak of IL-1

expression at day 14 post SCI by using RNase protection

assays (Perrin et al., 2005).

At 1 hour, IL-1 cells were mainly found around the

central canal at the level of the lesion epicenter. At 3 and

6 hours, positive cells were mostly located in the dorsal

columns and at the dorsal root entry zone (DREZ). At the

time when expression peaked at the lesion epicenter (i.e.,

12 hours), IL-1 cells were already more dispersed


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CYTOKINE mRNA EXPRESSION FOLLOWING SCI

275

Fig. 3. IL-1 mRNA expression in the injured mouse spinal cord,

time points from 3 hours up to 28 days post SCI. A–D: Threedimensional

reconstructions of the injured spinal cord showing damaged

areas (A,C) and the spatial distribution of IL-1 cells (B,D) at

12 hours (A,B) and 14 days (C,D) following SCI. E: Darkfield photomicrographs

showing IL-1 mRNA expression at lesion epicenter at 12

hours post SCI. F,G,J: Co-localization of IL-1 mRNA within astrocytes

at 12 hours (F,G, green) and an endothelial cell at 14 days (J)

post SCI. Nuclear counterstaining with DAPI is shown in blue.

H,I: Brightfield photomicrographs showing co-localization of IL-1

mRNA within GFP cells at 12 hours post SCI. Note the presence of

several double-labeled cells attached to blood vessels (H, brown) and

in clusters within the injured spinal cord (I, brown), suggesting that

infiltrating leukocytes also contribute to IL-1 production at 12 h.

Scale bar 100 minE;10m in G (applies to F,G), I (applies to H,I),

and J.


The Journal of Comparative Neurology. DOI 10.1002/cne

276 I. PINEAU AND S. LACROIX

across the entire cross-section (Fig. 3E). IL-1 cells were

observed within spared white matter tracts, around the

ventral median fissure, in the dorsal columns, at the

DREZ, and in meningeal layers. In addition, many IL-1

cells were found in specific areas of the spinal gray matter

such as the ventral horns and around the central canal

(Fig. 3E). By using multilabeling techniques on the same

tissue sections, microglia and astrocytes were identified as

the main cellular sources of IL-1 at this particular time

point. That microglial cells produce IL-1 has also been

reported in another in vivo model of CNS trauma, the

brain stab wound injury model (Herx et al., 2000). Thus,

only examples for double-labeled astrocytes (GFAP-ir/

IL-1 mRNA) were included in Figure 3 (Fig. 3F,G). In

addition to glial cells, co-localization studies performed on

tissue sections obtained from SCI-GFP chimeric mice

showed that some infiltrating leukocytes, primarily found

attached to the blood vessels or already in clusters within

the damaged spinal cord, were also contributing to IL-1

expression at 12 hours post SCI (Fig. 3H,I). However, we

cannot preclude the possibility that CNS resident GFPpositive

cells that have migrated into the spinal cord prior

to injury, most likely perivascular macrophages, could be

responsible, at least in part, for some of the expression of

IL-1 at this time point. From 2 days up to 7 days, the

signal had nearly disappeared. However, IL-1 mRNA

expression rose again at 14 days post SCI (Figs. 2F, 3C,D).

At this time point, at least some IL-1 cells were clearly

associated with the microvasculature (Fig. 3J). Although

IL-1 mRNA was not found to be constitutively expressed

in the spinal cord of naive mice, it was detected in the

spinal cord of sham-operated animals, as reported by

Wang et al. (1997) in laminectomized rats.

Tumor necrosis factor (TNF). Cells expressing TNF

mRNA were detected as early as 15 minutes post SCI.

TNF mRNA levels then increased gradually to reach, at 1

hour post SCI, its highest levels of expression for the

entire duration of the experimental protocol (i.e., 28 days

after SCI; Fig. 4A). Notably, for all time points between 15

minutes and 1 hour, most TNF cells were found in spinal

cord cross-sections located both rostral and caudal to the

center of the lesion (Fig. 4B–D). At 1 hour, cells expressing

TNF mRNA could be seen over the entire 10-mm segment

analyzed (5 mm on either side of the lesion epicenter).

That the signal was not confined within damaged areas

was further confirmed by comparing 3D reconstructions of

injured spinal cords with spatial distributions of TNF

cells at 1 hour (Fig. 4C,D).

In the contused area at 15–45 minutes, the majority of

TNF cells were small and scattered throughout spared

white matter tracts (Fig. 4E). In spinal cord areas located

away from the lesion epicenter at 1 hour, cells expressing

TNF mRNA had a small diameter and were mostly found

within spinal cord white matter, the dorsal horns of the

gray matter, and the dorsal columns (Fig. 4F). As reported

by Bartholdi and Schwab (1997), TNF cells were also

observed near the central canal. In general, the distribution

pattern described above for the spinal cord areas

located away from the epicenter was more pronounced

caudal to the lesion site. In spinal cord sections away from

the epicenter but rostral, TNF mRNA signal was primarily

found within the spinal cord dorsal columns (Fig. 4G).

After 3 hours, the number of cells expressing the TNF

mRNA transcript at the lesion epicenter had decreased by

66% compared with the 1-hour time point (Fig. 4A). Interestingly,

the more generalized, widespread expression of

TNF had also disappeared at this time point. At 12 hours,

TNF cells were confined within damaged areas, as demonstrated

by topographic mapping of cells expressing TNF

in a 3D-reconstructed injured spinal cord (Fig. 5A,B). At

24 hours post SCI, TNF cells could only be detected in

very limited number (3 1 positive cells per cross-section

at the lesion epicenter) and were also restricted to damaged

areas. By 2 days, TNF signal had returned to the

levels detected in mice that had received laminectomy

only. A striking feature of this time-course study was the

second peak of TNF mRNA expression observed at the

lesion epicenter at 14 and 28 days post SCI (Fig. 4A).

Notably, the number of TNF cells increased by approximately

7.5- and 11-fold at 14 and 28 days, respectively,

compared with the number of positive cells detected at the

7-day time point. Remarkably, the number of TNF cells

seen at the lesion epicenter at 28 days was not statistically

different from the number of cells observed at 1 hour,

when expression was maximal. Topographic mapping of

TNF cells in a 3D-reconstructed injured spinal cord revealed

that cells expressing TNF were confined within

damaged areas at 28 days post SCI (Fig. 5C,D).

Multilabeling analyses combining in situ hybridization

with immunofluorescence revealed that the four

major classes of neural cells residing within the spinal

cord (i.e., microglia, astrocytes, oligodendrocytes, and

neurons) synthesize TNF during the first few hours

following SCI. More specifically, we found that TNF

mRNA was located within Iba1 microglia/

macrophages and Galectin-3 activated microglia/

macrophages at 3 hours following SCI (Fig. 5E–H). The

rapid upregulation (within 15 minutes) and downregulation

(within 24 hours) of TNF, combined with the fact

that recruited monocytes normally required approximately

2–3 days to become predominant in the injured

rodent spinal cord (Popovich and Hickey, 2001), suggests

that resident microglia rather than blood-borne

macrophages are contributing to TNF synthesis early

on following SCI. In addition to microglial cells, multilabeling

analyses revealed that GFAP astrocytes,

CAII oligodendrocytes, and NeuN neurons also expressed

TNF mRNA at 1 hour following SCI (Fig. 5I–O).

For the second peak of expression observed at days

14–28 post SCI, cells expressing TNF were mostly

found in clusters within damaged areas (Fig. 5C,D).

These TNF cells were typically found at the lesion site

in proximity to Galectin-3 activated microglia/

macrophages (Fig. 5P) and, to a lesser degree, within

the scar (Fig. 5Q). The high density of macrophages (as

visualized by Galectin-3 immunofluorescence) and scar

tissue (GFAP immunofluorescence) at 14 and 28 days

post injury made any co-localization analyses almost

impossible at these two time points. Taken together,

these results indicate that following SCI, TNF transcription

was rapidly induced within endogenous glial

and neuronal cells. Our results also show that this early

expression was rapidly downregulated, within 24 hours,

and upregulated again at 14 and 28 days post lesion.

Interleukin-6 (IL-6). Cells expressing IL-6 mRNA

were detected at all time points analyzed between 3 hours

and 4 days post SCI (Fig. 6A). The number of IL-6 cells

peaked at 12 hours post SCI. This number corresponds to

roughly 15% of the number of IL-1 cells found at the


The Journal of Comparative Neurology. DOI 10.1002/cne

CYTOKINE mRNA EXPRESSION FOLLOWING SCI

277

Fig. 4. TNF mRNA expression in the injured mouse spinal cord,

time points up to 1 hour post spinal cord injury (SCI). A: Time course

of TNF mRNA expression at the lesion epicenter following spinal cord

contusion. B: Distribution of cells expressing TNF mRNA at 1 hour

post SCI. C,D: Three-dimensional reconstructions of the injured spinal

cord showing damaged areas (C) and the spatial distribution of

TNF cells (D) at 1 hour following SCI. E–G: Darkfield photomicrographs

showing expression of TNF mRNA at the lesion epicenter at 15

minutes post SCI (E) and at distances of 2,100 m caudal and 2,730

m rostral to the lesion at 1 hour (F,G). Arrows point to cells expressing

TNF mRNA. Scale bar 100 m in E and G (applies to F,G).

lesion epicenter at the same time. At 12 hours, cells expressing

the IL-6 transcript were dispersed over a distance

covering approximately 2 mm on either side of the

lesion epicenter (Fig. 6B,D).

A close examination of the spatial distribution of the

IL-6 signal revealed that IL-6 cells were closely associated

with meningeal structures at first, although some

IL-6 cells were found within spared white matter

tracts at 3 hours (Fig. 6E). In contrast, most IL-6 cells

detected at 6 and 12 hours were found within spared

white matter tracts and the spinal gray matter (Fig.

6F). At 12 hours, the IL-6 mRNA signal in meningeal

layers had almost completely disappeared, with the exception

of a small segment located directly above the

dorsal columns (i.e., where the impactor tip hit the

cord). At the lesion site at 12 hours, IL-6 cells were

mostly seen at the DREZ and in ventral areas such as

around the ventral median fissure and the central canal

(Fig. 6F). From 24 hours up to 4 days, the average

number of cells expressing the IL-6 transcript progressively

decreased to become almost nonexistent at 7 days

up to 28 days. Mice that received laminectomy only

showed a faint positive signal restricted to meningeal

layers at 3 and 6 hours. Although some studies could not

detect the IL-6 mRNA transcript following dorsal overhemisection

in mice by using nonradioactive in situ

hybridization (Bartholdi and Schwab, 1997), others

have shown an early and transient expression of IL-6


Fig. 5. TNF mRNA expression in the injured mouse spinal cord, time

points after 1 hour post SCI. A–D: Three-dimensional reconstructions of

the injured spinal cord showing damaged areas (A,C) and the spatial

distribution of TNF cells (B,D) at 12 hours (A,B) and 28 days (C,D)

following SCI. E–Q: Co-localization of TNF mRNA within activated

microglia/macrophages (E,H,P,Q; red), microglia/macrophages (F,G,

green), neurons (I,J; red), oligodendrocytes (K,L; green), and astrocytes

(M–Q; green) at 1 h (E,F,H–O), 3 hours (G), and 14 days (P,Q) post SCI.

Nuclear counterstaining with DAPI is shown in blue. Scale bar in P 10

m in E–O; 100 m in P,Q.


The Journal of Comparative Neurology. DOI 10.1002/cne

CYTOKINE mRNA EXPRESSION FOLLOWING SCI

279

Fig. 6. IL-6 mRNA expression in the injured mouse spinal cord.

A: Time course of IL-6 mRNA expression at the lesion epicenter

following spinal cord contusion. B: Distribution of cells expressing

IL-6 mRNA at 12 hours post spinal cord injury (SCI). C,D: Threedimensional

reconstructions of the injured spinal cord showing

damaged areas (C) and the spatial distribution of IL-6 cells (D) at

12 hours following SCI. E,F: Darkfield photomicrographs showing

mRNA following SCI in rats by using reverse transcriptase

(RT)-PCR (Streit et al., 1998; Hayashi et al., 2000).

The fact that we detected IL-6 mRNA following SCI in

expression of IL-6 mRNA at the lesion epicenter at 3 hours (E) and

12 hours (F) following SCI. G–M: Co-localization of IL-6 mRNA

within astrocytes (G–J, green), activated microglia/macrophages

(K, red), and neurons (L,M, red) at 6 hours post SCI. Nuclear

counterstaining with DAPI is shown in blue. Scale bar 100 m in

F (applies to E,F); 10 m in M (applies to G–M).

mice not only supports the latter studies but also shows

that our in situ hybridization method using radioactive

probes is highly sensitive.


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280 I. PINEAU AND S. LACROIX

Fig. 7. LIF mRNA expression in the injured mouse spinal cord.

A: Time course of LIF mRNA expression at the lesion epicenter

following spinal cord contusion. B: Distribution of cells expressing LIF

mRNA at 6 hours post spinal cord injury (SCI). C–E: Darkfield photomicrographs

showing expression of LIF mRNA at the lesion epicenter

(C) and at distances of 1,260 m (D) and 5,250 m (E) caudal to the

lesion at 6 hours following SCI. F–H: Co-localization of LIF mRNA

within GFAP-ir astrocytes (green) at 6 hours post SCI. Nuclear counterstaining

with DAPI is shown in blue. Scale bar 100 m inE

(applies to C–E); 10 m in H (applies to F–H).

Like IL-1 and TNF, IL-6 mRNA was found within most

classes of neural cells residing in the spinal cord. Indeed,

multilabeling analyses showed that GFAP-immunoreactive

(ir) astrocytes, Galectin-3-ir activated microglia/macrophages,

and NeuN-ir neurons all express the IL-6 transcript during

the first few hours following SCI (Fig. 6G–M).

Leukemia inhibitory factor (LIF). Results show the

presence of LIF mRNA signal from 3 hours up to 4 days

following SCI, with the number of LIF cells peaking at 6

hours (Fig. 7A). LIF cells were most abundant in sections

immediately adjacent to the lesion site over a rostral-tocaudal

distance of approximately 4 mm (2 mm on either


The Journal of Comparative Neurology. DOI 10.1002/cne

CYTOKINE mRNA EXPRESSION FOLLOWING SCI

side of the lesion epicenter; Fig. 7B). Although LIF cells

were mostly associated with meningeal structures at 3–12

hours, some positive cells were also detected within the

spinal gray matter at 6–24 hours (Fig. 7C–E). In more

distal regions on either side of the lesion epicenter (i.e.,

distances greater than 2 mm from the lesion site), positive

hybridization signal was still present but was generally

associated with meninges located above the spinal cord

dorsal columns (Fig. 7E). Multilabeling analyses revealed

that GFAP-ir astrocytes were the major cellular source of

LIF mRNA (Fig. 7F–H). Mice that underwent laminectomy

only showed very weak positive signal. In these

mice, LIF cells were mainly found to be associated with

meningeal structures.

Granulocyte/macrophage colony-stimulating factor

(GM-CSF) and interferon- (INF-). GM-CSF and

INF- mRNA expression was not detected at any time

point analyzed following SCI (from 1 hour up to 28 days

post SCI). These results are in agreement with our previous

findings obtained in BALB/C mice following dorsal

hemisection of the spinal cord and by using RNase protection

assays as a detection method (Perrin et al., 2005).

DISCUSSION

In this study, we report the detailed analysis of the

spatiotemporal distribution of six proinflammatory cytokines

in the injured mouse spinal cord. Importantly, we

also define the cellular sources of these inflammatory mediators.

Our findings reveal that IL-1 and TNF were

produced almost immediately following SCI (5 minutes),

placing them at the beginning of the cytokine cascade.

This was followed by the expression of IL-6 and LIF,

which also were rapidly and transiently synthesized following

SCI. Co-localization studies revealed that glia,

neurons, and endothelial cells were mainly responsible for

the initial cytokine responses, which occurred between 5

minutes and 1 hour and between 3 and 24 hours post SCI.

Results obtained from SCI mice previously transplanted

with GFP hematopoietic stem cells confirmed that, during

the first hour post injury, cytokines were solely synthesized

by CNS resident cells. However, neural cells and

immune cells recruited from the periphery both contribute

to cytokine production at 3 hours up to 24 hours post SCI.

Our time-course study also revealed another wave of

IL-1 and TNF expression at 14–28 days post SCI. This

delayed cytokine response coincided with the entry of lymphocytes

and the formation of the glial scar. Taken together,

these findings indicate that, through the synthesis

of proinflammatory cytokines, most classes of cells that

reside within the CNS contribute to the initiation of the

inflammatory response following SCI. It is likely that the

initial cytokine responses occurring locally within the injured

CNS are responsible for induction of gliosis and the

recruitment of immune cells from the periphery and their

entry into injured tissues. We speculate that cytokines

released later on are responsible for the perpetuation of

inflammation and glial scarring. The results presented in

this study should help us to understand the regulatory

processes of CNS inflammation and the roles that immune

responses play in regeneration and degeneration following

SCI.

IL-1 and TNF

281

In peripheral tissues, IL-1 and TNF are early mediators

of innate immunity and inflammation. Although the

receptors for IL-1 and TNF are structurally different,

both cytokines share signaling molecules, like NF-kB and

AP-1 transcription factors (Nguyen et al., 2002). As a

result, both cytokines share many biological effects. Here,

we showed that IL-1 and TNF mRNAs are expressed

almost immediately following SCI (within 5–15 minutes).

Rapid upregulation of IL-1 and TNF mRNA and protein

levels has been reported in a variety of experimental models

of SCI but never so early following injury (Bartholdi

and Schwab, 1997; Wang et al., 1997; Streit et al., 1998;

Lee et al., 2000; Song et al., 2001; Pan et al., 2002; Bareyre

and Schwab, 2003; Yune et al., 2003; Perrin et al., 2005).

The present study is also the first in vivo demonstration

that the initial synthesis of IL-1 and TNF following SCI

is produced by CNS resident cells such as glia, neurons,

and endothelial cells. This issue was unresolved until now

mainly because of 1) the lack of cytokine-specific antibodies

that work for immunohistochemistry on fixed tissue

sections, and 2) the fact that cytokines are rapidly cleaved

and released from the cell membrane to become soluble,

biologically active proteins, which complicates immunohistochemical

detection of the cells producing these factors.

This explains why we have decided to examine cytokine

mRNA instead of protein.

An important feature of the early cytokine response

occurring during the first hour post SCI is the generalized

expression of IL-1 and TNF in microglia and astrocytes

over the entire spinal cord segment analyzed (i.e., 10 mm).

This result suggests that these cytokines may exert additional

actions beside their role in organizing the inflammatory

response at the site of injury. One possible consequence

of the rapid and widespread expression of IL-1

and TNF could be the modulation of astrogliosis. It is

noteworthy that astrocytes located around the lesion site

and in areas distal from the traumatic injury rapidly

produce increasing amounts of the intermediate filament

GFAP mRNA (within 6 hours) and protein (within 12

hours) (Condorelli et al., 1990; Yong, 1996), undergo hypertrophy,

and extend their processes. Also of relevance in

light of our results are earlier studies reporting that the

initial astrocytic reaction may extend up to 10 mm from

the site of SCI in rats (Eng et al., 1987; Reier and Houle,

1988). It should be emphasized that our results show a

very similar distribution for IL-1 and TNF following SCI

(over 10 mm) and that their expression preceded the increase

in GFAP levels. Supporting a role for these two

cytokines in the early evolution of astrogliosis is another

study demonstrating that IL-1 and TNF can stimulate

extensive astrogliosis when microinjected at the site of

cortical stab wound injury in neonatal mice, in which the

astroglial response is normally minimal following CNS

injury (Balasingam et al., 1994). In addition, astrogliosis

was found to be attenuated in the brain of adult IL-1 and

IL-1R1 knockout mice following injury (Herx and Yong,

2001; Basu et al., 2002).

Another possible consequence of the rapid and widespread

expression of IL-1 and TNF could be the stimulation

of microglia. Following CNS injury, the first signs of

microgliosis are normally observed within the first 24

hours (Raivich et al., 1999), a time course that once again

follows the temporal pattern of expression of IL-1 and


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282 I. PINEAU AND S. LACROIX

TNF after SCI. That IL-1 and TNF may regulate microgliosis

in the injured mouse spinal cord is supported by the

following results: 1) treatment of microglial cultures with

IL-1 or TNF increases the expression of antigenic markers

that are known to be upregulated following microglial

activation, such as the CR3 receptor (Yu et al., 1998); 2)

injection of IL-1 or TNF intracerebroventricularly or directly

into the mouse brain rapidly stimulates widespread

activation of microglia (Nadeau and Rivest, 2000; Proescholdt

et al., 2002); and 3) microglial cell activation is

abrogated in type 1 IL-1 receptor (IL-1R1) and TNF receptor

knockout mice compared with wild type following

brain injury (Bruce et al., 1996; Basu et al., 2002). Once

microglia are activated, it is likely that the presence of

dead cells and degenerating myelin will determine

whether they return to a resting state or phagocytose

cellular debris, as occurs during WD. A role for IL-1 and

TNF in WD following SCI is supported by at least two in

vivo studies (Ousman and David, 2001; Perrin et al.,

2005). Based on these studies and our findings, we speculate

that the expression of a select group of proinflammatory

mediators for a restricted period could explain the

lack of recruitment of peripheral macrophages into degenerating

white matter tracts. Indeed, by using bone marrow

chimeric rats, Popovich and Hickey (2001) demonstrated

that virtually all macrophages detected in white

matter undergoing WD following midthoracic spinal cord

contusion are derived from microglia. Considering that

activated microglia exhibit poor myelin removal in vivo

compared with hematogenous macrophages (Reichert and

Rotshenker, 1996), the distribution of these two cell types

following injury could explain why WD is slow in the CNS.

Thus, restricted access of blood-derived macrophages to

injured white matter tracts and improper activation of

resident microglia might be attributable to the very transient

expression of a limited number of cytokines in these

areas.

In the rodent CNS, TNF receptors (TNF-R1 and TNF-

R2) and IL-1R1, the only IL-1R that possesses an intracellular

signaling domain, are expressed predominantly

by barrier-related cells, including cells associated with the

cerebral endothelium, meninges, and ependymal cell layer

bordering the ventricles (Ericsson et al., 1995; Nadeau

and Rivest, 1999). It is therefore likely that the early

release of IL-1 and TNF may also trigger a complex

series of signaling events in the target cells identified

above that could result in the recruitment of inflammatory

cells from the periphery into damaged tissues. One mechanism

by which IL-1 and TNF could attract and mediate

the entry of leukocytes is by stimulating endothelial cells

and macrophages associated with the cerebral endothelium

to secrete inflammatory mediators. For instance,

Thibeault et al. (2001) have previously found that IL-1

and TNF, but not IL-6, can rapidly trigger transcription of

the chemokine monocyte chemoattractant protein

(MCP)-1 throughout the CNS microvasculature when they

were injected into the bloodstream of mice. Studies have

also shown that activation of the receptors for IL-1 and

TNF leads to transcription of many other genes involved

in inflammation, including chemokines such as macrophage

inflammatory protein (MIP)-1 and -; cell adhesion

molecules (CAMs) such as intercellular CAM-1, vascular

CAM-1, and E-selectin; and cytokines such as IL-6

and LIF (for review, see Basu et al., 2004; John et al.,

2005; Lee and Brosnan, 1997). It should be emphasized

that these three classes of inflammatory mediators are

required for the recruitment of leukocytes toward sites of

injury, their adhesion to cerebral endothelium and migration

across the blood-brain barrier, and their activation,

respectively. Thus, our results suggest an important role

for IL-1 and TNF in the initial recruitment and activation

of leukocytes at sites of SCI.

IL-6 and LIF

Our results show that IL-6 and LIF are rapidly and

transiently synthesized following SCI. Although the exact

signals that stimulate IL-6 and LIF transcription are still

uncertain, it is likely that other cytokines, such as IL-1

and TNF, could be involved, as previously demonstrated

in astrocytes in culture (Norris et al., 1994). Supporting

this theory is our time-course study showing that IL-6 and

LIF expression followed IL-1 and TNF expression almost

perfectly. Co-localization studies revealed that most

classes of neural cells intrinsic to the spinal cord synthesized

IL-6, whereas LIF was predominantly produced by

astrocytes. Because of their critical role in the acute phase

of inflammation in peripheral tissues, IL-6 and LIF should

be considered as potential mediators of the initial phase of

the inflammatory response following CNS injury. Supporting

this hypothesis are studies showing that microglial

and astroglial reactivity is reduced and neutrophil

and macrophage infiltration is delayed in IL-6 and LIF

knockout mice compared with wild-type controls following

traumatic CNS injury (Klein et al., 1997; Penkowa et al.,

1999; Sugiura et al., 2000; Kerr and Patterson, 2004). In

addition to their involvement in the initial phase of the

inflammatory response, IL-6 and LIF could also directly

and/or indirectly influence neuronal survival and myelination

(Marz et al., 1999; Ishibashi et al., 2006). However,

chronic exposure of CNS tissue to IL-6 and LIF could lead

to neurodegeneration (Campbell, 1998; Lacroix et al.,

2002; Kerr and Patterson, 2004). Although the exact functions

of IL-6 and LIF following SCI are still not fully

understood, these studies strongly suggest that these two

hematopoietic/neuropoietic cytokines have potent effects

on inflammation and reactive gliosis.

Delayed IL-1 and TNF expression

As mentioned before, we observed another peak of IL-1

and TNF mRNA expression at 14–28 days. These results

are in agreement with our previous study in which we

reported early and delayed IL-1 responses by using a

different mouse model of SCI, the dorsal spinal cord hemisection

model (Perrin et al., 2005). It should be noted

that the delayed expression of IL-1 and TNF coincided

with the entry of T lymphocytes at sites of injury. Indeed,

it was recently reported that T cells start to infiltrate the

injured mouse spinal cord at day 14 post contusion, with

T-cell numbers increasing over the next few weeks (Sroga

et al., 2003). This increase in T-cell numbers between

weeks 2 and 6 post–contusion is in agreement with the

increase in TNF cell numbers observed in this study

between weeks 2 and 4. In peripheral tissues, macrophages

and lymphocytes are regarded as the major cellular

sources of IL-1 and TNF. Unfortunately, it was very

difficult to co-localize IL-1 and TNF mRNAs with any

particular cell type at the later time points because of the

high number of infiltrating cells and dense astroglial scar

found at the site of injury. Still, some Galectin-3-ir macrophages

and GFAP-ir astrocytes appeared to express the


The Journal of Comparative Neurology. DOI 10.1002/cne

CYTOKINE mRNA EXPRESSION FOLLOWING SCI

TNF transcript at 14 days post SCI. We also found clear

examples of endothelial cells producing the IL-1 mRNA

transcript at 14 days. Nevertheless, it remains unclear

whether the production of IL-1 and TNF by these cells

could serve to recruit T lymphocytes at sites of SCI. However,

other studies did reveal the presence of T lymphocyte

infiltrates into brains of mice overexpressing TNF under

the control of the GFAP promoter (Akassoglou et al.,

1997). Alternatively, it is possible that TNF and IL-1

secreted at 14–28 days could regulate astrogliosis (i.e.,

astrocyte reactivity, proliferation, and migration) in the

injured spinal cord. Interestingly, it has been shown that

in addition to the early and transient astroglial response

that takes place over a wide area in the injured CNS,

there is also a much later phase of astrocytic response that

begins at approximately 14 days post SCI. This later

phase of astrogliosis, known as glial scar, is long lasting

and limited to a narrow area surrounding the lesion site.

Glial scarring has been suggested to be a major cause of

axon regeneration failure following SCI (Fawcett and

Asher, 1999).

CONCLUSIONS

In summary, based on the dynamics of cytokine expression

and identity of the cellular source of these inflammatory

mediators following SCI, we propose that: 1) microglia

and astrocytes located throughout the injured spinal

cord rapidly produce IL-1 and TNF and become activated;

2) IL-1 expression at the site of injury rapidly

orchestrates a cascade of inflammatory signals including

IL-6 and LIF that lead to the recruitment and diapedesis

of leukocytes; 3) cytokine-activated microglia express receptors

that contribute to phagocytosis of myelin debris

during WD; and 4) IL-1 and TNF are released at 14–28

days post SCI to recruit T lymphocytes and regulate glial

scar formation. Such knowledge will be critical to develop

targeted immunotherapies aimed at neutralizing or stimulating

cytokine synthesis following CNS injury and diseases.

These approaches could potentially help to create

an environment that is more permissive for central regeneration

(e.g., by suppressing gliosis or accelerating WD)

and reduce tissue loss (e.g., by controlling the entry of

leukocytes at the site of injury) following CNS injury.

ACKNOWLEDGMENTS

We thank Nicolas Vallières and Nadia Fortin for their

technical assistance. We are grateful to Dr. Said Ghandour

(Université Louis Pasteur, Strasbourg, France) for

providing us with the carbonic anhydrase II polyclonal

antibody. We also thank Marc-André Laniel for his help

editing this manuscript.

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