Spatiotemporal expression of inducible nitric oxide synthase and ...

Spatiotemporal expression of inducible nitric oxide
synthase and cyclooxygenase 2 in the spinal cord
during early stage sciatic nerve crush injury
Abstract
BACKGROUND: Previous studies have shown that inducible nitric oxide synthase (iNOS) and
cyclooxygenase 2 (COX-2) participate in inflammatory immune responses and neuropathic pain
following peripheral nerve injury. However, few reports have addressed time-dependent expression
of iNOS and COX-2 following peripheral nerve injury.
OBJECTIVE: To investigate spatiotemporal expression of iNOS and COX-2 during early stage
sciatic nerve crush injury.
DESIGN, TIME AND SETTING: The randomized, controlled, animal study was performed at the
Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South
University, China from September 2006 to September 2007.
MATERIALS: Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal
antibody (Transduction Laboratory, USA), as well as biotinylated rabbit anti-mouse IgG and
biotinylated rabbit anti-goat IgG (Santa Cruz Biotechnology, USA) were used in the present study.
METHODS: A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three
groups. In the model group (n = 32), crush injury to the right sciatic nerve was established using an
artery clamp. The model group was further assigned to four subgroups according to survival time (6,
12, 24, and 72 hours), respectively (n = 8). Sham surgery (n = 8) and normal control (n = 8) groups
were also established.
MAIN OUTCOME MEASURES: iNOS and COX-2 expression was detected in the L4-6 spinal cord
with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and
posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the
anterior horn of spinal cord, were measured.
RESULTS: iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn
of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury (P < 0.05)
and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly
increased in the anterior horn of spinal cord on the damaged side (P < 0.05).
CONCLUSION: iNOS and COX-2 expression increased in the spinal cord during early stage sciatic
nerve crush, which suggested that iNOS and COX-2 participates in occurrence and development of
inflammatory immune responses following peripheral nerve injury.
Key Words: inducible nitric oxide synthase; cyclooxygenase 2; sciatic nerve; spinal cord; peripheral
nerve injury; neural regeneration
INTRODUCTION
Inflammatory reactions are key elements in degeneration
and regeneration following peripheral nerve injury [1–4] .
The enzyme inducible nitric oxide synthase (iNOS)
catalyzes formation of nitric oxide, and free radicals
generated during this catalysis induce toxic injury. A
previous study [5] confirmed increased iNOS activity in
rats at 12 hours following severe cerebral trauma, which
peaks at 1–3 days following damage and lasts for 1 week.
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iNOS activity decreases to normal levels by 2 weeks
following damage. Simultaneously, the iNOS-specific
inhibitor aminoguanidine significantly relieves
neurological functional impairment following brain
damage, but the NOS substrate L-arginine aggravates
nerve damage. In addition, little change takes place in
endothelial nitric oxide synthase mRNA expression,
whereas iNOS mRNA expression is significantly
increased and peaks at 24 hours following spinal cord
injury [6] . In ischemia/reperfusion sciatic nerve injury
models, iNOS expression also increases, and early iNOS
inhibition reduces ischemia/reperfusion injury [7] .
1

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme
involved in conversion of arachidonic acid to prostanoids,
is a key inflammatory factor, whereas prostanoids is an
important inflammatory mediator that exhibits a crucial
effect on production and modulation of nerve pain [8] .
Neuronal COX-2 expression increases at 4 hours and
peaks at 24–48 hours following acute spinal cord injury [8] .
At day 1, COX-2, and not COX-1, protein expression
significantly increases, but decreases to normal levels in
the spinal cord and thalamus at 72 hours following sciatic
nerve injury [9] . Following L 5 spinal nerve injury in the rat
sciatic nerve, COX-2 protein expression increases at 24
hours [10] . Nitric oxide produced by iNOS could activate
COX-2 and increase production of reactive oxygen
species [11] . However, spatiotemporal expression of
inflammatory factors iNOS and COX-2 during early stage
sciatic nerve crush in the spinal cord remains unclear.
Therefore, the present study immunohistochemistry to
analyze iNOS and COX-2 expression in the rat L 4-6 spinal
cord at 6, 12, 24, and 72 hours following sciatic nerve
crush injury to determine spatiotemporal expression
patterns of iNOS and COX-2 during early stage
peripheral nerve injury.
MATERIALS AND METHODS
Design
A randomized, controlled, animal study.
Time and setting
Experiments were performed at the Laboratory of
Applied Anatomy, Department of Human Anatomy and
Neurobiology, Central South University, China from
September 2006 to September 2007.
Materials
A total of 48 healthy, adult, Sprague Dawley rats,
weighing (200 ± 20) g and of both genders, were
supplied by the Department of Experimental Zoology,
Xiangya School of Medicine, Central South University,
China. All rats were housed at room temperature and
were provided free access to food and water.
Experimental protocols were conducted in accordance
with Guidance Suggestions for the Care and Use of
Laboratory Animals, formulated by the Ministry of
Science and Technology of the People’s Republic of
China [12] .
Reagents and equipment used in the study were as
follows:
Reagents and equipment Sources
Mouse anti-rat iNOS
monoclonal antibody, goat
anti-rat
antibody
COX-2 monoclonal
Biotinylated rabbit anti-mouse
IgG, biotinylated rabbit anti-goat
IgG
2
Guo LY, et al. / Neural Regeneration Research. 2010;5(0):0000
Transduction
Laboratory, USA
Santa Cruz,
Biotechnology, CA,
USA
Optical microscope Motic, Wetzlar,
Germany
Methods
Grouping and model establishment
A total of 48 Sprague Dawley rats were randomly
assigned to normal control (n = 8), sham surgery (n = 8),
and model (n = 32) groups. The model group was further
subdivided into four subgroups according to survival time
(6, 12, 24, and 72 hours), respectively (n = 8). Rat
survival time in the sham surgery and normal control
groups was 24 hours.
In accordance with results from previously published
studies [13-15] , sciatic nerve crush was established in the
model group. The rats were peritoneally anesthetized
with 2% sodium pentobarbital (40 mg/kg) and were fixed
in a prone position. A 3-cm longitudinal incision was
sterilely made at the right lower limb, and a smooth jaw
artery clamp (average width of 2 mm) was utilized to
crush the sciatic nerve, which was vertical to the nerve
trunks at two different directions for 60 seconds each.
The length of damage was approximately 2 mm, and the
left side served as the control. Successful model
establishment was determined by a remarkable trace at
the crush site, a continuous and transparent membrane,
complete paralysis of legs and feet, and the
disappearance of unfolding claw reflection [14,15] .
With the exception of sciatic nerve crush, remaining
interventions in the sham surgery group were similar to
the model group, including 24-hour survival.
Rats in the normal control group were not injured.
Sample collection and sections
The rats were anesthetized with 2% sodium
pentobarbital, followed by rapid perfusion with 50–100
mL saline and 300–400 mL 4%
paraformaldehyde/phosphate buffer for 30 minutes.
Subsequently, the L 4-6 spinal cord segment connected to
the sciatic nerve was immersed in 4%
paraformaldehyde/phosphate buffer for 2 hours, followed
by immersion in sucrose (20 and 30% prepared in 0.1
mol/L phosphate buffer, pH 7.4) at 4 °C until the samples
sank. Samples were serially sliced into 20-μm thick
cryostat sections. One in every four sections was
collected and placed in 0.01 mol/L phosphate-buffered
saline (PBS).
iNOS and COX-2 immunohistochemistry
In accordance with the streptavidin-biotin-peroxidase
complex method (free-floating method) [13] , sections were
washed twice in 0.01 mol/L PBS (pH 7.4) for 5 minutes,
followed by 3% H 2O 2 for 20 minutes to block endogenous
peroxidase, three washes in 0.01 mol/L PBS for 10
minutes, and blocking with bovine serum albumin
containing 0.3% TritonX-100 for 1 hour. Sections were
incubated with mouse anti-rat iNOS monoclonal antibody
(1:200) or goat anti-rat COX-2 monoclonal antibody
(1:300) supplemented with 0.1% TritonX-100 at 4 °C
overnight, followed by three washes with 0.01 mol/L PBS
for 10 minutes. The sections were then incubated with
biotinylated rabbit anti-mouse IgG (1:200) or rabbit
anti-goat IgG (1:200) for 2 hours, rinsed three times with
0.01 mol/L PBS for 10 minutes, followed by incubation

with streptavidin-biotin-peroxidase complex (1:200) at
room temperature for 1.5 hours and three washes with
0.01 mol/L PBS for 10 minutes. The sections were
developed by 0.05% 3, 3’-diaminobenzidine + 0.03%
H 2O 2 in the dark at room temperature for 3–5 minutes.
The reaction time was controlled under a microscope
and terminated by a thorough wash with 0.01 mol/L PBS.
Slices were mounted, air-dried, dehydrated in gradient
ethanol, permeabilized with xylene, and mounted in
neutral resin. Negative controls were treated with 0.01
mol/L PBS, rather than primary antibody, and the
remaining procedures were identical to sections from the
other groups [16] .
Five sections were randomly selected from each animal
and were subjected to immunohistochemistry. Under a
Motic optical microscope, five fields (100×) from each
section were randomly selected to measure gray values
of iNOS- and COX-2-positive cells in the anterior and
posterior horns of the spinal cord using Motic3.2 image
analysis software (Motic, Wetzlar, Germany). The
number of iNOS- and COX-2-positive cells were
quantified under a Motic microscope (20×).
Main outcome measures
Gray values of iNOS- and COX-2-postive cells in the
spinal cord anterior and posterior horns were measured,
and the number of iNOS- and COX-2-positive cells in the
spinal cord anterior horn was quantified.
Statistical analysis
Data are expressed as mean ± standard deviation and
were analyzed using SPSS 13.0 software (SPSS,
Chicago, IL, USA). All data from each group were
analyzed utilizing one-way analysis of variance. Paired
comparison of intragroup data was analyzed using the
LSD-t test. Lesion and control sides from the same
sample were compared utilizing paired t-test. A value of
P < 0.05 was considered statistically significant.
RESULTS
Quantitative analysis of experimental animals
A total of 48 rats were included in the final analysis.
Changes in iNOS expression following spinal cord
injury
In normal control and sham surgery groups,
iNOS-positive cells were detected in the anterior and
posterior horns of the L 4-6 spinal cord segment.
Immunoreactive iNOS products were observed in
neuronal cytoplasm and processes, and no significant
difference was detected between left and right
hemispheres. In the model group, iNOS expression
gradually increased in the lesion side at 6, 12, 24, and 72
hours following spinal cord injury. iNOS immunoreactive
products were distributed in the anterior horn and
surrounded the central canal and neuronal cytoplasm
and processes of the spinal cord posterior horn. Gray
values of immunoreactive products were less in the
anterior and posterior horns of the lesioned hemisphere,
compared with the contralateral hemisphere and normal
Guo LY, et al. / Neural Regeneration Research. 2010;5(0):0000
control group at 6, 12, 24, and 72 hours (P < 0.05)
(Figure 1, Table 1). Quantification of iNOS-positive cells
in the spinal cord anterior horn is displayed in Table 2.
Spinal cord
anterior horn
Spinal cord
posterior horn
Figure 1 Inducible nitric oxide synthase (iNOS) expression in
the anterior and posterior horns of the rat L4-6 spinal cord
(immunohistochemistry). iNOS expression is less in the spinal
cord anterior and posterior horns of the normal control group
compared with the model group at 27 h following injury. Arrow
indicates iNOS-positive cells.
Table 2 Quantification of inducible nitric oxide synthase-positive
cells in the spinal cord anterior horn (错误!不能通过编辑域代码
创建对象。±s, n = 8, cell/20-fold visual field)
Control
Group
Lesion hemisphere
hemisphere
Normal control 2.0±0.8 2.0±0.8
Sham surgery 2.0±0.9 2.1±0.8
6-h model 2.2±0.9 8.6±2.5 ab
12-h model 2.3±1.3 15.2±3.8 ab
24-h model 2.3±1.0 18.2±2.3 ab
72-h model 2.3±1.3 18.9±3.6 ab
a P < 0.05, vs. control hemisphere; b P < 0.05, vs. normal control
or sham surgery groups.
Normal control
group
Model group at
72 h
Changes in COX-2 expression following spinal cord
injury
In the normal control and sham surgery groups, COX-2
expression was low in the rat L 4-6 spinal cord, and
immunoreactive COX-2 products were distributed in the
cytoplasm and nuclear membrane. In the model group,
COX-2 expression increased within 24 hours following
injury and remained at a high level at 72 hours.
Immunoreactive COX-2 products were distributed in gray
matter layers, primarily in anterior horn motor neurons
and posterior horn cells. COX-2-positive cells were
rhombus, ellipsoid, or round in the spinal cord posterior
horn I–III layers, and the majority of cells exhibited small
bodies. Gray values in the spinal cord anterior and
posterior horns from the lesioned hemisphere were less
than from the contralateral hemisphere and normal
3

Spinal cord
anterior horn
Spinal cord
posterior
horn
control group following injury at various time points (P <
0.05) (Figure 2, Table 3). Quantification of
COX-2-positive cells in the spinal cord anterior horn is
displayed in Table 4.
Figure 2 COX-2 expression in the spinal cord
(immunohistochemistry). COX-2 expression is less in the spinal
cord anterior and posterior horns of the normal control group,
compared with the model group at 27 h following injury. Arrow
indicates COX-2-positive cells.
Table 4 Quantification of cyclooxygenase 2-positive cells in the
spinal cord anterior horn (错误!不能通过编辑域代码创建对象。
±s, n = 8, cell/20-fold visual field)
4
Normal control
group
Group
Control
hemisphere
Guo LY, et al. / Neural Regeneration Research. 2010;5(0):0000
Lesion
hemisphere
Normal control 1.2±0.8 1.2±0.8
Sham surgery 1.3±0.8 1.3±0.9
6-h model 2.3±1.1 9.6±2.1 ab
12-h model 2.3±1.1 14.0±2.5 ab
24-h model 2.5±1.1 16.7±3.1 ab
72-h model 2.6±1.2 6.8±2.8 ab
a P < 0.05, vs. control hemisphere; b P < 0.05, vs. normal control
or sham surgery groups.
DISCUSSION
Model group
at 72 h
Nitric oxide synthases belong to the family of eukaryotic
enzymes that catalyzes production of nitric oxide.
Previous results have demonstrated that iNOS
participates in apoptotic regulation [17] . Results from the
present study confirmed that iNOS expression increased
following nerve injury, which was consistent with
previously published results [5–7] . iNOS associates with
secondary inflammatory reactions following peripheral
nerve injuries, which suggests that appropriate inhibition
of iNOS activity during early stage peripheral nerve injury
could be beneficial for neuronal protection [18,19] .
COX has two types of isomers, i.e., COX-1 and COX-2
[20] . COX-2 is primarily expressed in the lumbar spinal
cord; it plays an important role in inflammatory reactions
and is one of the crucial factors that mediate cytotoxic
effects in inflammatory reactions [21] . COX-2 expressional
changes following sciatic nerve crush injury in the
present study were consistent with previously published
results [8–10] , demonstrating that COX-2 participated in
inflammatory reactions of spinal cord neurons during
early stage peripheral nerve injury. Therefore, a suitable
therapeutic time window for COX-2 inhibitor
administration following nerve injury could suppress
neuroinflammation and ultimately contribute to nerve
repair.
Distribution and expressional changes of COX-2 and
iNOS were similar in the spinal cord following sciatic
nerve crush injury. Therefore, it was presumed that
COX-2 and iNOS play an important role in pain
conduction. A previous study [22] determined that iNOS is
activated in L 4-6 spinal cord regions in models of sciatic
nerve ligation, and iNOS transcription increases nitric
oxide levels. Propofol was utilized to block iNOS
transcription, which exhibited an anti-nociceptive effect.
In addition, soluble guanylate cyclase expression in the
N-methyl-D-aspartic acid receptor and nitric oxide
signaling pathway results in hyperalgesia in the spinal
cord during peripheral inflammation [23] . Interactions
between nitric oxide and diverse transmitters, such as
proinflammatory factors, excitatory amino acids,
prostaglandin, and substance P, increases pain signal
modulations in the spinal cord posterior horn and induces
pathological pain [24] . Once iNOS is synthesized, it
catalyzes production of nitric oxide [25] , which activates
COX-2 to produce prostaglandins [26,27] , enhance
sensitivity of nociceptors, and maintain prostaglandin
E 2-induced hyperalgesia [28] . Therefore, COX-2 mediates
inflammatory nerve pain by regulating the nitric oxide
pathway, inducing iNOS expression, and increasing nitric
oxide production [11] . However, the precise mechanisms
require further study.
In conclusion, iNOS and COX-2 expression exhibit an
increased tendency in the spinal cord during early stage
sciatic nerve crush injury. These results suggested that
iNOS and COX-2 participate in onset and development
of inflammatory immune responses and processes of
nerve pain following peripheral nerve injury.
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Table 1 Gray values of inducible nitric oxide synthase (iNOS) immunoreactive products in the spinal cord (错误!不能通过编辑域代码
创建对象。±s, n = 8, cell/100-fold visual field)
Group
Normal control
Spinal cord anterior horn Spinal cord posterior horn
Control
Lesion Control
Lesion
hemisphere hemisphere hemisphere hemisphere
204.19±5.13 206.55±5.67 200.10±5.23 198.79±4.89
Sham surgery 204.52±4.89 207.86±6.39 201.25±6.46 200.09±5.38
6-h model 200.63±4.67 189.29±4.18 ab 190.03±4.68 b 186.03±3.98 ab
12-h model 198.91±4.32 185.95±4.79 ab 180.52±5.40 b 173.60±4.39 ab
24-h model 196.47±5.12 170.07±4.11 ab 172.77±3.26 b 165.31±4.01 ab
72-h model 198.18±4.27 160.46±3.95 ab 168.17±4.11 b 160.61±3.93 ab
a P < 0.05, vs. control hemisphere; b P < 0.05, vs. normal control or sham surgery groups.
Table 3 Gray values of COX-2 immunoreactive products in the spinal cord ( x ±s, n = 8, cell/100-fold visual field)
Spinal cord anterior horn Spinal cord posterior horn
Group
Control Lesion Control Lesion
hemisphere hemisphere hemisphere hemisphere
5

Normal control
Sham surgery
6
Guo LY, et al. / Neural Regeneration Research. 2010;5(0):0000
208.37±3.23 207.86±3.89 205.17±5.23 205.38±3.97
208.25±3.56 208.65±4.02 205.26±5.54 206.21±4.02
6-h model 203.63±2.58 193.37±3.18 ab 203.38±4.14 193.21±5.28 ab
12-h model
202.18±5.40 190.50±4.19 ab 198.13±5.31 190.75±4.28 ab
24-h model 201.87±3.50 187.61±4.01 ab 195.46±3.87 180.25±4.71 ab
72-h model 202.15±4.00 186.66±3.75 ab 196.24±4.31 187.89±3.95 ab
a P < 0.05, vs. control hemisphere; b P < 0.05, vs. normal control or sham surgery groups.