Experimental Neurology 211 (2008) 259–270

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Experimental Neurology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex n r

The systemic inflammatory response after spinal cord injury damages

lungs and kidneys
Denis Gris 1, Eilis F. Hamilton, Lynne C. Weaver ⁎
The Spinal Cord Injury Laboratory, BioTherapeutics Research Group, Robarts Research Institute and Department of Physiology and Pharmacology and Program in Neuroscience,
University of Western Ontario, PO Box 5015, 100 Perth Drive, London, Ontario, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Spinal cord injury (SCI) triggers a well characterized, acute, local inflammation leading to secondary damage
Received 6 December 2007 at the lesion site. Another little recognized problem may be the activation of circulating inflammatory cells
Revised 25 January 2008 that potentially damage tissues outside the cord. We investigated this problem using severe clip-compression
Accepted 30 January 2008
SCI in rats. We studied systemic inflammation after SCI and its effects on lungs and kidneys, as dysfunction of
Available online 4 March 2008
these organs is a frequent, early complication after SCI. From 2–24 h after SCI, the number of circulating
neutrophils (especially immature cells) significantly increased by 3–10 fold. Flow cytometry experiments
Keywords:
revealed that SCI transiently activates these neutrophils, causing increased oxidative responses to
Inflammation
phorbolmyristic acid at 2 h after SCI; then, from 4–24 h, the neutrophils were less responsive. Neutrophil
Neutrophil
Macrophage longevity was increased (30–50% decrease in apoptosis) at 2–8 h after SCI. Immunohistochemical analyses
Oxidative burst demonstrated the invasion of neutrophils into lungs and kidneys (2 h–7 d after SCI) and more phagocytic
Apoptosis macrophages in lungs (12 h, 3 d after SCI). Myeloperoxidase and matrix metalloproteinase-9 activity in lung
Myeloperoxidase and kidney homogenates increased (12 h-7 d after SCI). Expression of COX-2 increased and lipid peroxidation
Proteinase also occurred within this time. Control experiments inducing local cord damage by excitotoxic quisqualate
Lipid peroxidation injection verified that SCI per se is sufficient to trigger systemic inflammation and organ damage. In summary,
Organ damage
SCI mobilizes and activates neutrophils that then migrate into visceral organs, a phenomenon occurring in
Treatment of spinal cord injury
parallel with their well-known entry into the cord injury site. The systemic inflammatory response to SCI
should be targeted in the development of new therapeutic strategies to treat SCI.
© 2008 Elsevier Inc. All rights reserved.

Introduction
Traumatic spinal cord injury (SCI) triggers an acute inflammatory
response that leads to secondary damage at the cord lesion site (Blight,
1992; Fleming et al., 2006; Popovich et al., 1997; Saville et al., 2004; Taoka
et al.,1997). Many of the intraspinal events causing this secondary damage
have been documented (Bao and Liu, 2004) but, as the inflammatory cell
activation begins within the circulation, this systemic response may also
damage tissues outside the CNS. In turn, these systemic responses may
impact on the progression of, or recovery from, spinal cord damage.
Historically, the leading cause of death after SCI was kidney failure, closely
followed by respiratory failure (Catz et al., 2002; Devivo et al., 1999;
O'Connor, 2005). With advances of contemporary care, kidney dysfunc-
tion no longer constitutes the greatest danger to the cord-injured patient,
and instead, respiratory failure has become the leading cause of mortality
(Catz et al., 2002; Devivo et al., 1999; O'Connor 2005; Pickett et al., 2006).
⁎ Corresponding author. Fax: +1 519 663 3789.
E-mail address: lcweaver@robarts.ca (L.C. Weaver).
1
Current address: University of North Carolina-Chapel Hill School of Medicine
Lineberger Comprehensive Cancer Center CB#7295 450 West Drive University of North
Carolina at Chapel Hill Chapel Hill NC 27599, USA.
Life-threatening lung dysfunction has been considered the most difficult
condition to treat (Bhatia et al., 2005; Kyono and Coates, 2002). Indeed,
clinical studies show that organ failure contributes significantly to the
morbidity and mortality of SCI patients (Acosta et al., 1998). The
mechanism of multiple organ dysfunction after severe general trauma,
such as that caused by multiple bone fractures or extensive burns, is
associated with induction of systemic inflammation mediated, in part, by
neutrophils that become more responsive (or primed) to stimulation
(Bhatia et al., 2005; Ogura et al., 1999; Tanaka et al., 1991). The organ
responses to SCI may resemble those to severe general trauma.
Recent studies show that, in addition to the local inflammatory
response in the spinal cord, SCI causes an influx of leukocytes into the
liver, due to a triggering of hepatic chemokine expression within 2 h of
the injury (Campbell et al., 2003; Campbell et al., 2005b; Perry et al.,
2003; Wilcockson et al., 2002). The presence of inflammatory cells in the
liver can increase the production of acute-phase proteins such as
complement protein C3 and C-reactive protein as well as the CXC and
CC chemokines, CINC1 and CCL-2, respectively; all of these proteins are
released into the circulation (Campbell et al., 2005b; Wilcockson et al.,
2002). Such systemic inflammation likely contributes to organ dysfunc-
tion after SCI, as it does after brain injury and other traumatic insults
(Acosta et al., 1998; Baskaran et al., 2000; Bhatia et al., 2005; Ott et al.,

0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2008.01.033
260 D. Gris et al. / Experimental Neurology 211 (2008) 259–270
1994). Moreover, such systemic inflammation potentially augments the
inflammation at the original injury site. However, acute effects of SCI on
organs other than the liver have not been investigated.
The cellular and biochemical features of secondary damage to the
injured spinal cord resemble features of the damage to organs caused
by the systemic inflammatory response to severe general trauma. In
both cases, neutrophils play a major role in the initiation and
propagation of the inflammatory response. The same key proteins
participate in the cascade of the tissue-damaging inflammation. For
example, the oxidative enzyme myeloperoxidase (MPO) is increased
in the injured spinal cord, and in the lung, kidney, and liver during
multiple organ failure after trauma (Botha et al., 1995; Jones et al.,
2005). Matrix metalloproteinase-9 (MMP-9) mediates damage to the
injured spinal cord (Yong et al., 2007) and to the injured lung in
respiratory diseases (Chakrabarti and Patel, 2005); cyclooxygenase-2
(COX-2) inflicts secondary damage on the injured spinal cord (Resnick
et al., 1998) and also is a key player in inflammatory conditions outside
the CNS (O'Brien et al., 2005). Accordingly, the players in possible
organ dysfunction after SCI might be predicted by studies of
peripheral inflammatory conditions and of multi-organ failure caused
by severe general trauma.
The postulate of our work is that a spinal cord injury triggers a
systemic inflammatory response that generates injury not only in the
spinal cord but also in the lung and kidney. We propose a model in which
the systemic inflammatory response is initiated by pro-inflammatory
mediators known to be released into the circulation after the SCI
(Campbell et al., 2005b; Wang et al., 1997). These activate circulating
neutrophils as well as endothelial cells throughout the vasculature,
providing the opportunity for the neutrophils to engage in adhesion
molecule-mediated diapedesis through small vessel walls into unin-
jured organs such as the lungs and kidneys as well as the injured spinal
cord. This influx of neutrophils leads to organ damage and dysfunction
that would negatively impact on recovery from the traumatic SCI. This
investigation addresses several aspects of this model: namely, neutro-
phil influx into the lung and kidney after SCI, oxidative activity within
these organs and structural damage to the organs.
Materials and methods
Spinal cord injury
All animal procedures were done following the Canadian Guide to
Care and Use of Experimental Animals. Sixty-two male Wistar rats
(Harlan Bioproducts, Indianapolis, Indiana) were premedicated and
anaesthetized with halothane as described previously (Weaver et al.,
2001). The T4 spinal cord segment was exposed by a dorsal
laminectomy and injured severely, without disrupting the dura, by
60 s of 50 g clip compression (Weaver et al., 2001). This method causes
hemorrhage, tissue necrosis and infiltration of inflammatory cells that
extend at least one segment rostral and caudal to the segment of injury.
At least 80% of the gray and white matters are damaged at the center of
the injury. Post-operative care including antibiotics, analgesics and
urinary bladder evacuation was provided as described previously
(Weaver et al., 2001). Sham-injured rats had a skin incision over the T3
vertebra, dissection of muscle over this vertebra and a partial
laminectomy that did not actually expose the T4 spinal cord segment.
In an additional group of rats, the spinal cord was injured chemically by
injection of an excitotoxic dose of the excitatory amino acid quisqualic
acid, thereby generating the SCI (Plunkett et al., 2001) with minimal
trauma to surrounding tissue. A small skin incision was made between
the dorsal processes of the 3rd and 4th thoracic vertebra. A 28 G needle
was passed between these two vertebrae into the spinal cord and 5 μl of
125 μM quisqualic acid was injected directly into the cord, approxi-
mately 2 mm below the dorsal surface. This method causes an
excitotoxic injury to the majority of the grey matter at the segment of
injection as the volume of injection was large for the spinal cord
(Plunkett et al., 2001). This injury caused less complete paralysis than
the compression SCI.
Flow cytometry
Venous blood samples were collected from the tail vein into
heparinized syringes before injury and 2, 4, 8, 12, and 24 h after SCI. At
each time period of study, a blood sample was taken from every animal.
Each 100 μl blood sample was diluted to a volume of 1.0 ml with Roswell
Park Memorial Institute (RPMI) 1640 media only (Gibco, Invitrogen,
Carlsbad, CA) or RPMI containing dihydrorhodamine (DHR)123 only
(Molecular Probes, Invitrogen, Carlsbad, CA), or DHR + phorbolmyristic
acid (PMA Sigma Chemical Company, St. Louis, MO). PMA stimulates
oxidative burst in leukocytes (Wymann et al.,1989). Final concentrations
were as follows: DHR123 41.5 ng/ml and PMA 100 ng/ml. The samples
were prepared in duplicate and incubated at 37 °C or on ice for 30 min.
The data samples were acquired using FACS Caliber cell analyzer and Cell
Quest software (BD Biosciences, San Jose, CA). The analyzer was
calibrated before every use using calibration beads (FACS Calibrite 3,
BD Biosciences) and FACS Comp software (BD Biosciences). The data
were analyzed using FloJo for PC (Tree Star, Ashland, OR). Oxidative burst
was measured by the intensity of oxidized DHR123 that becomes the
fluorescent rhodamine123 (R123) in the stained neutrophils, which
were selected using side scatter (SSC) versus forward scatter (FSC) plots.
The R123 fluorescence of monocytes and lymphocytes was negligible.
To assay leukocytes for apoptosis, erythrocytes in 100 μl of blood
were lysed in buffer containing 154 mM Ammonium Chloride, 10 mM
Potassium Bicarbonate, and 86 mM EDTA (all from Sigma, Saint Lois MO,
USA) for 5 min, centrifuged at 500 G, and the remaining cells washed
twice in PBS (5 ml). Apoptosis of the cells was then assessed by staining
with Annexin V and 7-Amino-Actinomycin D (7AAD) following the
protocol of the Annexin V-PE Apoptosis Detection Kit I (BD Pharmingen,
Franklin Lakes, NJ). The following controls were used: unstained cells,
cells stained with Annexin V only, and cells stained with 7AAD only.
Annexin V identifies cells during the initial stages of apoptosis, whereas
the 7AAD stain labels cells that are dead or in the late stages of
apoptosis. The percentage of apoptotic neutrophils was defined as the
percentage of cells that were Annexin V-positive and 7AAD-negative.
Counts of neutrophils in venous blood
For differential analysis of the leukocytes, the total number of
leukocytes in venous blood obtained from the tail vein was counted
using a hemocytometer. In addition, blood smears were made on glass
slides and stained with the Protocol Hema-3 Stain Set (Fisher
Diagnostics, Middletown, VA). Using microscopy, the percent of
neutrophils within one hundred cells of the leukocyte population was
determined. The neutrophils were identified by their characteristic
segmented nucleus. Immature neutrophils, known as band cells, had
horseshoe-shaped nuclei and mature cells had nuclei with several lobes.
Tissue sampling for histological analysis
At 12 h,1 d, 3 d or 7 d after SCI, rats were anesthetized with ketamine
and xylazine (110 and 0.5 mg/kg, respectively, i.p.) and perfused
transcardially with saline followed by 4% formaldehyde. Lungs were
perfused separately via the pulmonary artery. Pieces of left kidney
(including cortex and medulla) and left lung were post-fixed for 12 h
and then embedded in paraffin, sectioned at 5 μm thickness into four
serial sets of sections and mounted onto positively charged slides. One
set was stained with hematoxylin and eosin using a standard protocol.
Immunocytochemical analysis of tissue sections
For immunohistochemical analysis, tissue sections were processed
using an antigen retrieval protocol consisting of incubation of tissue
 D. Gris et al. / Experimental Neurology 211 (2008) 259–270
sections in citric acid buffer, pH 6, at 95 °C for 20 min, followed by
blocking with 10% goat serum (Cedarlane, Hornby, ON Canada) for at
least 1 h and then by incubation overnight with one of the following
primary antibodies diluted in 5% goat serum: rabbit anti-rat neutrophil
antibody diluted 1:20,000 (gift of Dr. Daniel Anthony, Oxford
University, Oxford, UK) or rabbit anti-human MPO antibody 1:10,000
(Dako Cytomation, Glostrup, Denmark). Sections were next incubated
overnight with biotinylated anti-rabbit antibody made in donkey
(1:1000 dilution) (Jackson Laboratories, West Grove, PA). The
immunoreactivity was visualized using diaminobenzidine [(DAB)
Sigma Chemical Company, as above] as a chromogen. The sections
were counterstained with hematoxylin. The slides were viewed using
an Olympus microscope (BX50, Olympus America Inc., Center Valley,
PA) and photomicrographs were acquired using a digital camera
(Retiga, Quantitative Imaging Corporation, Burnaby, BC, Canada) and
Image Pro software (Media Cybernetics, Silver Spring, MD).
Photomicrographs of lung and kidney tissue sections were
examined for the presence of neutrophils. These organs were
taken from four to six animals per time period of analysis and two
to four sections per animal were analyzed. The tissue sections were
taken from the same region of each organ, namely, an entire cross
section of kidney and approximately 1.0 cm2 of the middle lobe of
the right lung. The analysis of these sections included qualitative
descriptions of the gross morphological changes and extravasation of
neutrophils into the organs after SCI. After thorough scanning,
photomicrographs of representative fields were taken at low (2 or
10×) and high (100×) power magnification. Quantification of the
numbers of neutrophils that had been stained with the anti-
neutrophil antibody was done by counting all cells in ten randomly
selected 25× fields of view of one lung and one kidney section from
each animal.
Tissue sampling for homogenate assays
Animals were transcardially perfused with 400 ml of ice-cold
saline. Again, the lungs were perfused separately, via the pulmonary
artery with 200 ml of ice-cold saline. Tissue was dissected and pieces
of left kidney and left lung were stored at −80 °C. Tissue samples were
weighed and homogenized using a polytron at 14,000 rpm in RIPA
buffer [20 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.5% sodium
deoxycholate, 1% Triton X-100, 0.1% SDS] for immunoblotting or
using K-PBS buffer for MPO and malondialdehyde (MDA) assays, and
zymography. The samples were then centrifuged at 14,000 rpm in an
Eppendorf 5810R centrifuge with the F45-30-11 rotor (Eppendorf,
Hamburg, Germany) for 40 min and the supernatant stored at −80 °C.
The quantification of the protein was performed using a DC protein
Assay (Biorad, Hercules, CA, USA) and BioRad protocol that was
adapted for micro plates.
MPO assay
Homogenate samples (10 μl) were incubated in a 96-well plate in
a developing buffer that consisted of 100 μl of K-PBS and 100 μl of o-
dianosinisidine (12.5 mg/10 ml distilled water and 9 μl of H2O2). The
reaction was stopped by addition of 100 μl of 1% NaH3 in each well.
The plate was scanned using a 96-well plate reader (Multiskan
Ascent, Thermo Fisher Scientific, Waltham, MA) at a wavelength of
450 nm. For every plate, one standard curve in triplicate was
performed using MPO from human leukocytes (Sigma Chemical
Company, St. Louis, MO).
Thiobarbituric acid reactive substances (TBARS) assay for lipid
peroxidation
Tissue homogenate (25 μl) was added to 475 μl of thiobarbituric
acid (TBA) buffer (25 μl 8.1% sodium dodecyl sulfate (SDS), 200 μl 20%
261
acetic acid adjusted to pH 3.5 with NaOH, 200 μl 0.8% TBA in 50% acetic
acid adjusted to pH 3.5 and 50 μl water). The mixture was incubated at
95 °C for 1 h. The mixture was allowed to cool to room temperature
and 250 μl of separation buffer (n-butanol:pyridine, 15:1) was added.
Then samples were centrifuged for 10 min at 4000 rpm (centrifuge
and rotor as above) and 200 μl of the top (organic) layer was placed
into the wells of 96-well plates. Plates were scanned in a 96-well plate
reader using a 550 nm filter (Multiskan Ascent, Thermo Fisher
Scientific, Waltham, MA). For every plate, one standard curve in
triplicate was performed using malondialdehyde standards (Sigma
Chemical Company, St. Louis, MO).
Immunoblotting
Homogenized tissue samples (40 μg of protein) were loaded into
10% polyacrylamide gel and separated using 100 V for 1.5 h. Then the
proteins were transferred to polyvinylidene difluoride membranes
(Millipore, Billerica, MA). The membranes were first blocked in 10%
non-fat powdered milk and then incubated overnight with rabbit anti-
human COX-2 (diluted 1:20,000, Cayman Chemical, Ann Arbour, MI)
or mouse anti-rat ED-1 (1:20,000, Serotec, Raleigh, NC) antibodies.
Membranes were next incubated with the following secondary
antibodies made in donkey (diluted 1:10,000): HRP-conjugated anti-
rabbit antibody for COX-2 staining or HRP-conjugated anti-mouse
antibody for ED-1 staining (Jackson ImmunoResearch Laboratories
Inc., West Grove, PA). Finally, they were incubated in ECL Plus Western
blotting detection reagents (Amersham, NJ) according to manufac-
turer's specifications. Immunoreactive bands were scanned by an
imaging densitometer (BioRad GF-700, Hercules, CA) and results were
quantified using Multi-Analyst software (BioRad). All values of protein
expression were normalized as a percent of β-actin expression. To
verify equal protein loading of the gels, after scanning the ED-1 or
COX-2 bands, the membranes were stripped and re-probed with
1:10,000 mouse anti-β-actin antibody (Sigma, Saint Lois, MO).
Zymography protocol for MMP-9
Polyacrylamide gels were prepared according to BioRad manual
specifications for the mini-protein 3 system. The resolving gel was 8%
polyacrilamide containing 1% gelatin and the stacking gel was 4%
polyacrilamide. The tissue homogenate samples were re-suspended in
2X Tris Glycine SDS buffer and incubated for 10 min at room
temperature. The samples were then loaded with 40 μg of protein
per lane. The electrophoresis was conducted at 100 V for 1.5 h or until
bromphenol blue from the loading dye reached the bottom of the gel.
After electrophoresis, gels were incubated twice for 15 min in
renaturing buffer (Triton X 25% in distilled water). After renaturation,
gels were incubated in developing buffer (Tris–HCl 50 mM, NaCl 0.2 M,
CaCl2 5 mM, and Brij 35 0.02%) for 15 min at room temperature with
gentle agitation and then overnight at 37 °C. All reagents were
supplied by Sigma Chemical Company (St. Louis, MO). Latter gels were
stained in 1% Coomassie blue, in 20% methanol and 10% acetic acid, for
1 h. Then gels were washed several times in destaining buffer until
good differentiation was achieved. The quantification was performed
using inverse densitometry with a GelDoc system and software by
BioRad (BioRad, Hercules, CA).
Statistical Analyses
Mean values are expressed ±standard error of the mean (SE).
Group data were analyzed by 1-way or 2-way analysis of variance
(ANOVA), using repeated measures design when appropriate (Sokol
and Rohlf, 1981), followed by Dunnett's test for 2-tailed comparisons
to control or the Student Neumann Keuls test for comparisons
between groups. When only two groups were compared, a paired
Student's t test was used. Normalized data were square root
262 D. Gris et al. / Experimental Neurology 211 (2008) 259–270

Table 1
Neutrophils in the venous blood and in organs after SCI

A. Venous blood

Uninjured 2h 4h 8h 12 h 16 h 24 h
9.6 ± 1.0 29.3 ± 2.9⁎ 31.3 ± 3.0⁎ 25.5 ± 2.4⁎ 99.2 ± 3.1⁎ 53.2 ± 8.4⁎ 34.1 ± 3.1⁎

B. Organs

Uninjured 12 h 3d 7d
Lung 41 ± 17 (6) 1132 ± 108⁎ (4) 839 ± 47⁎ (4) 693 ± 183⁎ + (4)
Kidney 5 ± 2 (4) 59 ± 7⁎ (5) 27 ± 4⁎ (4) 19 ± 4⁎ + (5)

A. Mean numbers of neutrophils ± SE x 105/ml. ⁎Significantly different from uninjured, 1-way ANOVA, Dunnett's test, P b 0.05, n = 6 rats.
B. Mean numbers of neutrophils ± SE per ten 25X fields of view. ⁎Significantly different from uninjured, +Significantly different from 12 h, 1-way ANOVA, SNK tests, P b 0.05. Kidney vs
lung significantly different 12 h, 3 d, 7 d, 2-way ANOVA, SNK test, P b 0.01. Numbers of rats/group are indicated in parentheses.

transformed prior to analysis. Linear regression was used to assess the
relationship between MPO activity and lipid peroxidation (Sokol and
Rohlf, 1981). Statistical differences were considered significant at
P b 0.05.
Results
Activation of circulating neutrophils after SCI
The total white blood cell count increased significantly at 12 h after
SCI (169 ± 4.1 × 105/ml, n = 5) compared to that before injury (84
± 5.4 × 105/ml). A neutrophil response contributed to this change as,
after SCI, the number of circulating neutrophils significantly increased
at all times from 2 h to 24 h, reaching a peak 10-fold increase at 12 h
(Table 1A). Whereas neutrophils constituted 10–15% of the leukocyte
population in uninjured rats, this proportion increased to as much as
60% after SCI. The proportion of immature neutrophils (band cells) in
the blood before SCI was 17 ± 3.2% and this proportion was
significantly greater (52 ± 3.3%) when assessed at 24 h after SCI
(n = 8, correlated 2-tailed Student's t test, P = 0.0001).
Neutrophils have a continuous basal level of oxidative activity.
Basal oxidative activity of neutrophils from uninjured rats yielded a
R123 fluorescence of 17 ± 2 median fluorescence units (mfu, n = 7, see
example in Fig. 1A). This basal activity did not change significantly
with time after SCI (17 ± 1, 20 ± 3, 25 ± 5, 21 ± 3 and 15 ± 3 mfu at 2, 4, 8,
12 and 24 h after SCI, respectively, n = 7, P = 0.401). Stimulation of

Fig. 1. Activation of neutrophils in the circulation after SCI. A) Overlay of representative histograms of neutrophil oxidative activity in samples from an uninjured rat (U) and from this
rat 2 h and 4 h after SCI. Histograms beneath line were obtained after stimulation with phorbolmyristic acid (PMA). R123 fluorescence is expressed as log fluorescence units.
B) Fold increases in oxidative activity of neutrophils after PMA stimulation in uninjured rats (U) and at 2–24 h after SCI. Values are groups means ± SE. n = 11 (U, 2 h, 8 h, 12 h), n = 7 (4 h,
24 h). ⁎, significantly different from U, 1-way ANOVA, Dunnett's test, P b 0.05. C, D). Apoptosis of circulating neutrophils. Scatter plot showing the distribution of neutrophils from
uninjured (C) and 4 h after SCI (D) defined by their staining with annexin-V after they were identified by side scatter and forward scatter. Cells that were stained by 7AAD were dead.
X and Y-axes represent fluorescence intensity. Arrowheads point to population of neutrophils that were considered apoptotic. Numbers in the right lower quadrant define annexin-V+
and 7AAD− neutrophils; numbers in the left lower quadrant define annexin-V− and 7AAD−. E) Decrease in percentage of circulating neutrophils that were 7 AAD− but annexin-V+ after
SCI. Values are groups means ± SE. n = 8 (U, 2 h, 4 h, 8 h); n = 3 (12 h, 24 h). ⁎Significantly different from U, 1-way ANOVA, Dunnett's test, P b 0.05.
 D. Gris et al. / Experimental Neurology 211 (2008) 259–270 263

neutrophils with PMA (100 ng/ml) was used to assess their priming
(i.e, greater capacity for oxidative burst responses) after SCI. Examples
in Fig. 1A show that PMA-induced activation of neutrophils from an
uninjured rat increased R123 fluorescence to ∼ 450 mfu. At 2 h after
SCI, such stimulation caused greater activation, yielding R123
fluorescence of ∼1050 mfu. This greater activation was evidence of
priming. In contrast, by 4 h after SCI the PMA-induced activation
caused only ∼ 90 mfu of R123 fluorescence. Mean responses to PMA
were quantified by expressing a ratio of the activity (R123 fluores-
cence) produced by PMA stimulation to the basal activity at the same
time in the same rat (Fig. 1B). The PMA-induced oxidative burst was
significantly greater than in uninjured rats (a 35% increase) at 2 h after
SCI. However, by 4 h after SCI, the oxidative burst became significantly
less than that in uninjured rats (an 83% decrease from uninjured) and
remained reduced for as long as 24 h after SCI (Fig. 1B). Circulating
monocytes and lymphocytes had little or no basal oxidative activity
and this did not change significantly after SCI. PMA treatment and/or
time after SCI had no effect on their oxidative activity (data not
shown).
In uninjured rats, about 40% of neutrophils were 7AAD- and
annexin+, i.e., apoptotic (Figs. 1C, E). At 2–8 h after SCI, this percentage
decreased significantly (Figs. 1D, E). At 2 h, 4 h, and 8 h after SCI, the
percentage of neutrophils in the initial stages of apoptosis was
reduced significantly from 40 ± 4.3% (intact) to 29 ± 2.8%, 22 ± 2.7% and
18 ± 1.8%, respectively (Fig. 1E). This reduction in numbers of apoptotic
neutrophils was transient with basal values returning by 12–24 h after
SCI. The number of 7AAD positive (dead) neutrophils in the blood
samples was negligible before and after SCI (Figs. 1C, D). The
proportion of apoptotic monocytes and lymphocytes (approximately
1–2%) did not change after SCI (data not shown).
Extravasation of neutrophils into the lungs after SCI
The most striking difference between the appearance of the lungs
of uninjured control rats and the lungs of the rats after SCI was an
increased cellularity (Figs. 2A, B). The uninjured control lungs had
normal alveolar spaces with fine interalveolar septa surrounding
them. Neutrophils and erythrocytes were not found outside the

Fig. 2. Extravasation of neutrophils into lung. A, B) Photomicrographs of hematoxylin and eosin-stained, 5 mm sections of the lung from an uninjured rat (A) and from a rat 12 h after
SCI (B). Note hemorrhage and increased numbers of cells in the interstitial and alveolar spaces after SCI (green arrows). The cells had morphology typical of neutrophils and
macrophages. Inset in B shows a neutrophil with a segmented nucleus (arrowhead). ar, artery; b, bronchiole; av, alveolus. Calibration bar in B is 100 μm and applies to A and B.
Calibration bar in inset is 5 μm. C–E) Neutrophil immunoreactivity of a lung section from an uninjured rat (C), and from rats 12 h (D) and 3 d (E) after SCI. Insets show higher power
images to reveal morphology of cells. Arrowheads point to elongated neutrophils. Calibration bar in D is 100 μm and applies to A–C. Inset calibration bar in C is 10 μm and applies to
insets in A–C. F) Myeloperoxidase activity in lung homogenates. Arbitrary units of activity are expressed as means ± SE. Sh, 24 h after sham injury; Quis, 12 h after intraspinal
quisqualate injection. n = 6 (U, 24 h), 5 (Sh, 12 h, 3 d, 7 d), 4 (Quis). ⁎Significantly different from U, 1-way ANOVA, Dunnett's test, P b 0.05.
264 D. Gris et al. / Experimental Neurology 211 (2008) 259–270

vasculature in uninjured or sham-injured rats, but macrophages were
present in the peri-alveolar space in modest numbers (Figs. 2A, C and
see also Fig. 3A). In contrast the lungs of cord-injured rats had an
intense infiltration of neutrophils (with characteristic segmented
nuclei), thickened interstitium between alveoli and large numbers of
macrophages. The interstitial space in the lungs of the injured animals
contained numerous neutrophils that were also present in the alveoli
(Figs. 2B–E). Moreover, erythrocytes in the interstitium and within the
alveoli revealed intrapulmonary hemorrhage. The alveolar mem-
branes also were often disrupted, leaving large air sacs.
After SCI, many neutrophils in the interstitial and alveolar space
had an elongated shape consistent with migratory activity (Figs. 2D, E
insets). The influx of neutrophils occurred as early as 6 h after SCI,
lasting up to 7 d after SCI (Figs. 2D, E; Table 1B). Neutrophil numbers in
the lungs at 12 h, 3 d and 7 d after SCI were significantly greater than
in the lungs of uninjured control rats (Table 1B). The declining number
at 7 d after injury was significantly different from that at 12 h. The
density of cells immunoreactive to MPO in the lungs increased from
6 h to 7 d after SCI, following the pattern of anti-neutrophil staining
during the first week after SCI (data not shown). These MPO-
immunoreactive cells appeared to be neutrophils. The homogenates
from lung tissues of the uninjured rats had very little MPO activity but
this activity increased significantly (by more than 5 fold) at 12 h and
was still significantly greater at 7 d after SCI (Fig. 2F). Sham-injured
rats were examined at 24 h after injury. MPO activity in the lungs of
uninjured and sham-injured rats was very similar.
The effect of a direct neurotoxic (excitatory amino acid) injury to
the spinal cord, without trauma to the surrounding tissue, was
assessed to determine whether an injury that affected only the spinal
cord was sufficient to initiate a systemic inflammatory response. The
MPO assay was chosen for this assessment because, in our study, both
the lung and the kidney had consistent changes in MPO activity after
SCI. MPO activity was significantly increased by 4.3 fold in lung
homogenates from rats at 12 h after intraspinal injection of 5 μl of
125 μM quisqualic acid (Fig. 2F). This response to cord injury alone was
similar in magnitude to that caused by the compression SCI that also
damaged adjacent bone and muscle.
Increased presence of macrophages in the lungs after SCI
The lungs of uninjured control rats contained small cells
distributed within the interstitial spaces that expressed the macro-
phage marker ED-1 and were likely peri-alveolar macrophages (Fig.
3A). At 12 h and 3 d after SCI, the lungs contained large foamy,
apparently phagocytic, ED-1-expressing macrophages within the
interstitium and alveoli. Expression of ED-1 in the lung homogenates
also was greater than that in uninjured controls at 12 h and 3 d after
SCI (Fig. 3B). Sham injury had no influence on expression of ED-1 in
the lungs.
Extravasation of neutrophils into the kidney after SCI
Kidney sections stained by hematoxylin and eosin revealed a
large reduction of the tubular space after SCI and an increase in the
apparent cellularity of the parenchyma, both in the cortex and the
medulla (Figs. 4A–D). In addition, after SCI, Bowman's capsule of
some glomeruli appeared to be fused or collapsed (Fig. 3B), whereas
in others it was expanded. Staining with the anti-neutrophil
antibody revealed no neutrophils in the kidneys of the uninjured
rats (Fig. 4E). In contrast, cells with clear immunoreactivity to the
neutrophil antibody were apparent in the kidney sections from rats
at 12 h to 7 d after SCI (Figs. 4F, G, Table 1B). These cells were in the
interstitial space outside the tubular lumen and blood vessels.
Neutrophil numbers in the kidney sections at 12 h, 3 d and 7 d after

Fig. 3. Macrophages in the lung and kidney after SCI. A) Photomicrographs of 5 μm lung sections illustrating immunoreactivity for ED-1 in examples from an uninjured rat and from
rats 12 h and 3 d after SCI. Arrowheads point to phagocytic foamy macrophages. Calibration bar is 50 μm. B, C) Western blot analysis of ED-1 expression in homogenates of lung and
kidney. Format is as in Fig. 4. Lung: n = 8 (U),5 (Sh), 4 (12 h), 12 (24 h), 10 (3 d), 5 (7 d). Kidney: n = 4/group. ⁎Significantly different from U, 1-way ANOVA, Dunnett's test, P b 0.05.
 D. Gris et al. / Experimental Neurology 211 (2008) 259–270 265

Fig. 4. Extravasation of neutrophils into kidney. A–D) Photomicrographs of hematoxylin and eosin-stained, 5 mm sections of the kidney from an uninjured rat (A, C) and from a rat
12 h after SCI (B,D). A and B illustrate the renal cortex [note glomeruli (green g) and tubules (green t)]; C and D show the renal medulla (note tubules). Renal tubules have been
sectioned longitudinally in C and more often transversely in D. Calibration bar in D is 100 μm applies to A–D. Arrowhead in B points to collapsed Bowman's space. E–G)
Immunoreactivity for neutrophils in kidney sections from an uninjured rat (E) and from rats 12 h and 3 d after SCI (F, G). Insets show higher power images to reveal morphology of
cells. Arrowhead in inset G points to elongated neutrophil. Calibration bar in G is 100 mm and applies to E–G. Calibration bar in the inset in G is 10 mm and applies to insets in E–G. D)
Myeloperoxidase activity in kidney homogenates. Arbitrary units of activity are expressed as means ± SE. Sh, 24 h after sham injury; Quis, 12 h after intraspinal quisqualate injection.
n = 5 (all SCI groups), n = 4 (Quis). ⁎Significantly different from U, 1-way ANOVA, Dunnett's test, P b 0.05.

SCI were significantly greater than those in kidneys of uninjured the accumulation of neutrophils in the kidney was significantly
control rats (Table 1B). The smaller accumulation of neutrophils at smaller than that in the lungs at 12 h, 3 d, and 7 d after SCI (Table
7 d after injury was significantly different from that at 12 h. Overall, 1B). The distribution of cells immunoreactive for MPO in the kidney
266 D. Gris et al. / Experimental Neurology 211 (2008) 259–270

sections was similar to that of cells stained by the anti-neutrophil
antibody (data not shown). MPO activity in homogenates from
uninjured control kidneys was low but increased significantly by 30%
and 25% at 12 h and 24 h after SCI, respectively (Fig. 4H). Again, MPO
activity in kidneys of uninjured and sham-injured rats was very
similar. Selective cord injury by direct intraspinal injection of
quisqualic acid (as above) caused a significant, 66% increases in
MPO activity measured in the kidney homogenates (Fig. 4H),
demonstrating that the cord injury alone was sufficient to initiate
the systemic inflammatory response.
Expression of ED-1 in the kidneys was assessed to determine
possible changes in macrophages in this organ. ED-1 expression in the
kidneys did not change significantly after SCI (Fig. 3C). Sham injury
also had no influence on expression of ED-1 in the kidneys.
Oxidative and proteolytic damage in lungs and kidneys after SCI
An infiltration of neutrophils into tissue is often associated with an
increased presence of oxidative enzymes within the tissue and with
cellular damage evidenced by lipid peroxidation (Bao et al., 2004a;

Fig. 5. Oxidative activity in lung and kidney homogenates. A, B). Western blot analysis demonstrating expression of COX-2 in the lung and kidney. Typical immunoblots are shown at
the top of each panel. Similar protein loading of each gel is shown by the β-actin bands below each COX-2 band. Densitometric values for each band were expressed as arbitrary units
(AU) and mean values ± SE are plotted in the bar graphs below the immunoblots for uninjured rats (U), 24 h after sham injury (Sh) and at 12 h, 24 h, 3 d, and 7 d after SCI. Lung: n = 4 (U,
Sh, 12 h), 7 (24 h), 5 (3 d, 7 d). Kidney: n = 4 all groups. ⁎Significantly different from U, 1-way ANOVA, Dunnett's test, P b 0.05 (#, difference is a decrease). C, D) Analysis of lipid
peroxidation by T-bars assay for malondialdehyde (MDA) in lung and kidney. Lung: n = 5/group; Kidney: n = 4 (U), 5 (Sh, 24 h, 7 d), 6 (12 h, 3 d). ⁎Significantly different from U, 1-way
ANOVA, Dunnett's test, P b 0.05. E) MMP-9 activity in lung determined by zymography. Typical zymographs are shown at the top of the panel. These bands were quantified by
densitometry and mean values ± SE are plotted in the bar graphs below the zymographs in arbitrary units. N = 5 rats/group. ⁎Significantly different from U, 1-way ANOVA, Dunnett's
test, P b 0.05.
 D. Gris et al. / Experimental Neurology 211 (2008) 259–270
Bao et al., 2004b). At 12 h after SCI, expression of the oxidative enzyme
COX-2 was significantly greater in homogenates of lung (100%
increase) and kidney (20% increase) than in uninjured controls (Figs.
5 A, B). COX-2 expression in the lung was also increased at 3 d after SCI
(Fig. 5A). In contrast, this response was not long-lasting in the kidney
and COX-2 expression became significantly smaller than in controls at
3 d and 7 d after SCI (Fig. 5B). The sham injury had no effect on
expression of COX-2 in lungs or kidneys.
SCI led to lipid peroxidation in lung tissue, demonstrated by the
presence of malondialdehyde (MDA) in the lung homogenates during
the first week after SCI. Lung MDA was significantly higher (up to 5-
fold increases) at 12 h, 24 h, 3 d and 7 d after SCI than in lungs of
control uninjured rats (Fig. 5C). In the kidney, at 12 h after SCI, the
levels of MDA were significantly but more modestly increased (by 2
fold) when compared to uninjured controls (Fig. 5D). Sham injury did
not lead to increased MDA in the lungs or kidneys. Because changes in
lipid peroxidation and MPO activity were particularly robust in the
lungs, an additional analysis was done to assess the possible
correlation between these two outcomes of SCI. The amount of MDA
and the activity of MPO in these organ homogenates were significantly
correlated (r2 = 0.71 Y = 0.2205 + 0.0486⁎X). Chemical injury of the
spinal cord with intraspinal injection of quisqualic acid also lead to
increased MDA in the lung homogenates at 12 h after SCI, consistent
with the presence of lipid peroxidation (Fig. 5C). This increase was
similar to that caused by compression SCI. Effects on the kidney were
not tested.
Activity of the proteolytic enzyme MMP-9 was low in the lung
homogenates from control or sham-injured rats (Fig. 5E). MMP-9
activity in the lungs increased 3-fold at 12 h after SCI and remained
significantly higher than in uninjured controls at 24 h and 7 d after the
injury.
Discussion
Responses of circulating neutrophils to spinal cord injury
SCI changed the character of neutrophils in the circulation,
transiently priming them for greater oxidative burst activity and
also mobilizing them to generate greater numbers, likely from
marginal pool stores as well as from bone marrow. The mobilization
yielded a population of young cells in the circulation that had greater
longevity. The initial priming, i.e., increased oxidative burst, found at
2 h after SCI can be attributed to pro-inflammatory cytokines and
chemokines (Campbell et al., 2005b; Wang et al., 1997), as well as to
other stimulatory molecules such as platelet activating factor (Botha et
al., 1996) that appear in circulation soon after such traumatic injury.
These proteins may be released by the injured cells in the cord and
surrounding tissue as well as the liver, once it is stimulated by the
trauma. Basal oxidative activity of the circulating neutrophils also was
slightly (although not significantly) increased from 4–12 h after SCI.
Upon entry into the injured cord or visceral organs, these neutrophils
would be prepared to release oxidative and proteolytic enzymes,
inducing or extending damage. Priming renders neutrophils more
reactive to stimulation, leading to greater production of reactive
oxygen species and enzymes such as elastase (Kobayashi et al., 2003;
Ogura et al., 1999). The pro-inflammatory cytokines and chemokines
that lead to priming also increase the lifespan of neutrophils (Varga et
al., 2004) (Dancey et al., 1976; Kobayashi et al., 2003; Tanaka et al.,
1991), a response that may have contributed to the increased
longevity of circulating neutrophil as early as 2 h after SCI.
By 4 h after SCI the population of neutrophils in the circulation was
less responsive to PMA, showing that it was less primed than in
uninjured rats. This reduced response remained for the 24 h of our
study and may be attributed to several changes ongoing in the
circulating neutrophil population after SCI. First, the neutrophils with
the greatest capacity for activation, such as those present at 2 h after
267
injury, may have migrated into the injured spinal cord or into organs
such as the lungs and kidneys. Primed neutrophils undergo upregula-
tion of adhesion molecules, enhancing their ability to migrate (Sunil et
al., 2002; Walzog et al., 1999; Wittmann et al., 2004). Second, from 2–
24 h after SCI, many neutrophils in the circulation were young,
characterized by lower expression of apoptotic genes and by the
presence of band cells as long as 24 h after SCI. These newly recruited
young neutrophils appeared less capable of full oxidative burst. We
propose that, as they matured in this capacity, they migrated into
tissues, leaving behind the less responsive population.
Although activation of the immune system, i.e., the systemic
inflammatory response, is a well-known response to trauma (as
described in the Introduction), another perturbation of immune
function after SCI is immunosuppression. Immune impairment likely
plays a role in the septicemia, infections of organs such as the lungs,
gastrointestinal tract and kidneys and development of decubitous
ulcers that are significant issues after SCI (Cruse et al., 1996; Devivo et
al., 1999). Depending on the level of SCI, suppression of the function
of natural killer cells, T-cells, B cells and macrophages has been
documented in humans and animals within weeks of the injury
(Cruse et al., 1992; Cruse et al., 2000; Lucin et al., 2007). Moreover,
expression of lymphocyte and neutrophil adhesion molecules can be
decreased by 2 weeks after injury (Cruse et al., 2000). Injuries in the
cervical or high thoracic region that cause significant sympathetic
nervous system dysfunction cause greater immunosuppression. In
addition, when studied at times greater than 3 months after injury,
neutrophils of tetraplegic but not paraplegic patients demonstrated
less phagocytic capacity (Campagnolo et al., 1997). In that study
neutrophils had normal cell-killing ability, suggestive of normal
production of oxidative and proteolytic enzymes. Such changes in
immune function after SCI are important considerations in the overall
management of the SCI patient, but these changes usually occur later
than the systemic inflammatory response examined in our study, that
commences within hours of the injury. Our data show that, in the
first days after SCI, the innate immune system is fully functional.
Further support for this contention comes from preliminary findings
in our laboratory showing that acute leukocyte entry into the lungs is
a specific, integrin-mediated response and reaffirms that these
leukocytes have the capacity to cause damage. Intravenous treatment
of rats, for 72 h after a T4 compression SCI, with a monoclonal
antibody to the α4 subunit of the α4β1 integrin, blocks the entry of
neutrophils into the lungs and decreases the presence of free radicals,
myeloperoxidase and lipid peroxidation in this organ (F. Bao, and D. Gris
unpublished observations).
Infiltration of neutrophils into the lungs and kidneys
The infiltration of neutrophils into the lungs and kidneys after SCI
coincided in time with the infiltration of neutrophils into the injured
spinal cord in our previous study [that used the same injury model
(Saville et al., 2004)]. The peak of neutrophil invasion in both cases
occurred between 12–24 h after injury. The largest influx of
neutrophils after SCI was observed in the lungs, consistent with a
filtering function of the lung vasculature that can bar the passage of
activated neutrophils through the organ (Bhatia et al., 2005; Worthen
et al., 1989). Few neutrophils were found in the lungs of uninjured
control or sham-injured rats, in the absence of pro-inflammatory
stimulation. A neutrophil infiltration into the lungs occurs in many
systemic inflammatory disorders such as burns, multiple trauma, and
infections (Bhatia et al., 2005; Crimi et al., 2006; Kielar et al., 2002;
O'Brien et al., 2005; Sunil et al., 2002). Although the triggers of the
inflammation may be different in each of these conditions, they all are
characterized by sequestration of activated neutrophils in the lungs,
an event that is followed by bystander tissue damage. SCI appears to
follow this model. The extravasation of neutrophils is not limited to
the lung, as other richly-vascularized organs such as kidney, liver, and
268 D. Gris et al. / Experimental Neurology 211 (2008) 259–270
intestines also can be infiltrated by these cells and damaged (Campbell
et al., 2005a; Crimi et al., 2006). Likewise, in our study, the kidneys of
rats after SCI contained neutrophils, but those of uninjured or sham-
injured did not. Normally, the elasticity of the neutrophil and
endothelial cell membranes allows neutrophils to pass through the
kidney, even through the complex and convoluted capillary system of
glomeruli and vasa recta (Heinzelmann et al., 1999). However, in the
pro-inflammatory state after SCI, neutrophils accumulated through-
out the cortex and medulla of the kidney.
Why do neutrophils and monocytes migrate into the lungs and
kidneys after SCI? Why are the resident macrophages activated? Our
study cannot answer these questions but a body of work has
addressed the migration of these cells into the liver after brain or
SCI (Campbell et al., 2003; Campbell et al., 2005b; Wilcockson et al.,
2002). After CNS injury the liver produces acute-phase proteins and
chemokines that promote this intra-hepatic invasion and tissue
damage. Furthermore, release of such proteins into the circulation
causes a systemic inflammatory response that leads to further
activation of circulating leukocytes that then can enter the original
injury, exacerbating it (Perry, 2004). The precise reason why the liver
has an immediate response to CNS injury remains unknown, but the
phenomenon is well established. Liver injury is also known to impact
the lung, precipitating lung pathology via an organ-to-organ commu-
nication that likely involves complex cytokine networks (Neff et al.,
2003). Therefore the influx of inflammatory cells into the lungs after
SCI may be related to the responses of the liver. We have confirmed
the influx of neutrophils into the liver after SCI, accompanied by an
increased hepatic expression of alkaline phosphatase, a marker of liver
damage (Gris, 2007). The lungs also may be a source of pro-
inflammatory cytokines, after stimulation by the initial entry of
neutrophils. Moreover, the pulmonary vessels probably upregulate
vascular adhesion molecules (see below). These interesting possibi-
lities remain to be investigated. Finally, the lungs, liver and kidney are
not likely to be the only organs involved in the systemic inflammatory
response and targets such as the gastrointestinal tract need to be
explored.
The extravasation of neutrophils into organs that are spared from
an initial trauma has general and specific components. The upregula-
tion of adhesion molecules and increase rigidity of the membrane
makes neutrophils more ‘sticky’. In this form neutrophils, given an
opportunity, are able to extravasate using diapedesis, which is tightly
regulated mechanism, dependent on the interaction of the adhesion
molecules and expression of appropriate chemokine receptors. The
vast capillary bed of the lung, liver and kidney presents such an
opportunity. Studies have shown that the upregulation of adhesion
molecules by endothelial cells throughout the circulation, during the
systemic inflammatory response, promotes leukocyte extravasation.
For example, ischemic reperfusion injury to the limb leads to a distant
response, i.e., lung damage by mechanisms that include upregulation
of pulmonary vascular intercellular adhesion molecule-1 (Seekamp et
al., 1993). Moreover, vascular permeability in the lungs increases,
leading to intrapulmonary hemorrhage. Therefore the inflammatory
infiltrate and hemorrhage in the lungs that we observed after SCI is
not unlike responses to other types of injury or responses of other
organs. We postulate that the systemic inflammatory response attacks
the lung and, to a lesser degree, the kidney, by activation of circulating
inflammatory cells, making them more aggressive in extravasation,
and by rendering the vascular endothelium more adhesive and/or
more permeable.
In the first few days after SCI, neutrophils in the lungs and kidneys
had an elongated shape suggesting a process of active migration
(Wymann et al., 1989). This migrating behavior implies the presence
of a chemotaxic gradient. The lungs and kidneys can produce such a
gradient in response to a variety of stimuli. For example, in a rat model
of lung inflammation, infiltration of the neutrophils into the lung
corresponded with an increased concentration of pro-inflammatory
chemokines in the bronchi-alveolar lavage (Yan et al., 2006). In the
kidneys, the epithelial cells of the proximal convoluted tubules come
into contact with pro-inflammatory messengers from the filtered
plasma, triggering their production of chemokines and cytokines, that,
in turn, may attract neutrophils (Daha and van Kooten, 2000; Gould et
al., 2002).
Lung and kidney damage after spinal cord injury
The histological evidence of neutrophil influx into the organs
prompted a search for oxidative and proteolytic enzymes known to be
produced by neutrophils. Indeed, the presence of neutrophils was
accompanied by an increase in these enzymes. For example, MPO
activity was increased in the lungs and kidneys. Increased MPO in
tissue can be considered a marker of neutrophil sequestration
(Reynolds et al., 2003; Tjoa et al., 2003) and, in our study, MPO
immunoreactivity in tissue sections correlated with increased density
of neutrophils identified by an anti-neutrophil antibody, and with
increases in the MPO activity in tissue homogenates. As MPO
expression generates oxidative activity, the presence of MPO implies
oxidative damage (Bao et al., 2004b).
The origin of MPO expression can be queried because MPO is also
produced by macrophages (Leskovar et al., 2000; Vega et al., 1999).
Alveolar macrophages in the lung constantly undergo some level of
metabolic activity, which explains the easily detected levels of basal
MPO activity in the lungs of uninjured rats (Grattendick et al., 2002;
Vega et al., 1999). In contrast, the kidney does not have an extensive
population of tissue macrophages, and accordingly the basal MPO
activity in the kidneys was much lower than in the lungs. The
sustained increase in MPO activity in the lung at 3 d and 7 d after SCI
may be due, in part, to the activation of the resident macrophages by
neutrophils or to the entry of activated hematogenous monocytes/
macrophages into the organ. In support of this idea, ED-1 immunor-
eactivity in the lung sections and expression detected by western
blotting demonstrated an increased macrophage presence at 12 h and
3 d after SCI. The first peak of ED-1 immunoreactivity most probably
can be attributed to resident macrophages since infiltration of the
monocytes/macrophages from circulation usually does not commence
until 24 h after the insult. Therefore, 12 h does not present sufficient
time for peripheral source of ED-1 to develop, while expression of ED-
1 at 3 d is probably driven by both: resident and infiltrating
macrophages. The later wave of MPO activity at 3 d and 7 d after SCI
was not observed in the kidney, likely because this organ does not
have many resident macrophages (that would be assumed to make up
the majority of the tissue macrophage population) (Sean and
Cockwell, 2005).
The expression pattern of COX-2 in the lungs also supports the idea
that early damage was caused by neutrophils whereas later effects
may be attributed to macrophages. In the lung, the expression of COX-
2 was bimodal: the first wave was probably due to the invasion of
neutrophils, while the second wave was likely caused by the activation
of macrophages (Radi and Ostroski, 2007). COX-2 in the kidney
increased only at 12 h after injury, consistent with the smaller, more
short-lived inflammatory response within this organ. Lung MMP-9
activity was robust immediately after SCI, and then remained
increased for at least 7 d. The later expression of lung MMP-9 cannot
be attributed to macrophages because lung MMP-9 is expressed
mostly in neutrophils (Chakrabarti and Patel, 2005; Nagase and
Woessner, 1999).
The lipid peroxidation within the lung and kidney correlated
temporally with the arrival of neutrophils and appeared to be
particularly related to MPO activity, as shown by the strong
correlation between MDA and MPO. Although MPO activity in the
kidney was not striking, lipid peroxidation in this organ increased and
decreased with the influx and clearance of neutrophils, respectively,
suggesting some relationship between neutrophils and this damage.
 D. Gris et al. / Experimental Neurology 211 (2008) 259–270
We questioned whether the organ inflammation, the activation of
oxidative and proteolytic enzymes and the ensuing damage could be
attributed to specific injury of the spinal cord or, instead, were simply
the net consequence of the overall general tissue trauma. Sham injury
that damaged only soft tissue and bone did not initiate any of these
organ responses, suggesting that the response to SCI is different from
trauma to tissues outside the spinal cord (and likely outside the CNS).
These findings are consistent with our study of human spinal cord
injury in which cord-injured patients have more robust systemic
inflammatory responses than patients with significant trauma that
includes bone fractures but no spinal cord injury (F. Bao, unpublished
observations). Moreover, the response to localized injection of
quisqualic acid into the cord also supports the idea that SCI initiates
a systemic inflammatory response with particular efficacy. This
discrete injury caused oxidative responses and tissue damage similar
to that caused by compression SCI, again suggesting that injury to the
spinal cord, rather than generalized tissue damage, initiated the
systemic inflammatory response and ensuing organ damage. The
chemical injury to the cord clearly initiated the same cascade of events
produced by compression injury. As presented above, we speculate
that this cascade consists of production and release of pro-inflamma-
tory cytokines and other stimulatory proteins by injured cells in the
spinal cord with amplification of the pro-inflammatory response by a
hepatic response to the injury. Trauma to the spinal cord per se,
however small, induces the systemic inflammatory response.
Conclusion and significance
The results of our study demonstrate that the developing secondary
injury at the lesion site after SCI occurs on the background of a systemic
inflammatory response. Indeed, the systemic inflammatory response
after SCI may play a key role in the development of secondary injury to
the cord. This phenomenon has been demonstrated in other disease
conditions: systemic inflammation superimposed upon existing CNS
pathology can trigger or augment the CNS disease. For example, multiple
sclerosis is often precipitated by an immune system-activating viral
infection (Sun et al., 2004). Activation of microglia by systemic infection
can provoke Alzheimer's and other neurodegenerative diseases (Perry
et al., 2003). Considering that secondary damage, in many aspects,
depends on the inflammatory processes that are triggered after SCI, the
systemic inflammatory response may serve as an important link in the
chain of destructive events after SCI. This response increases energy
demands and affects function of the organs that are critical for recovery
after SCI. Therefore, this response should be viewed with concern when
devising strategies for anti-inflammatory neuroprotection after SCI, as
organ protection should be addressed as well.
Acknowledgments
This research was supported by a grant from the Canadian
Institutes of Health Research (CIHR). D. Gris held a studentship from
the CIHR during the tenure of this research. We thank Dr. Canio Polosa
for his critical evaluation of the manuscript. We appreciate the
generous donation of anti-neutrophil antibody by Dr. Daniel Anthony,
Oxford University.
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