# Inhibitory Effect on Cerebral Inflammatory Response  following Traumatic Brain Injury in Rats:  A Potential Neuroprotective Mechanism of N-Acetylcysteine

Authors: Gang Chen; Jixin Shi; Zhigang Hu; Chunhua Hang
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Although N-acetylcysteine (NAC) has been shown to be neuroprotective for traumatic brain injury (TBI), the mechanisms for this beneficial effect are still poorly understood. Cerebral inflammation plays an important role in the pathogenesis of secondary brain injury after TBI. However, it has not been investigated whether NAC modulates TBI-induced cerebral inflammatory response. In this work, we investigated the effect of NAC administration on cortical expressions of nuclear factor kappa B (NF-κB) and inflammatory proteins such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) after TBI. As a result, we found that NF-κB, proinflammatory cytokines, and ICAM-1 were increased in all injured animals. In animals given NAC post-TBI, NF-κB, IL-1β, TNF-α, and ICAM-1 were decreased in comparison to vehicle-treated animals. Measures of IL-6 showed no change after NAC treatment. NAC administration reduced brain edema, BBB permeability, and apoptotic index in the injured brain. The results suggest that post-TBI NAC administration may attenuate inflammatory response in the injured rat brain, and this may be one mechanism by which NAC ameliorates secondary brain damage following TBI.


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## Body

## 1. Introduction

Although traumatic  brain injury (TBI) represents a significant public health
problem in the world, there are currently no treatments that improve clinical
outcome measures [1, 2]. Recently, several reports from experimental studies
have demonstrated that N-acetylcysteine (NAC) played neuroprotective roles in
TBI including restoration of
TBI-induced mitochondrial dysfunction, increasing the reduced antioxidant
enzyme, and protecting neurons [3–5]. Most of these previous studies focused on the modulation of NAC on
oxidative stress in the brain following TBI, but ignored the influence of NAC
on cerebral inflammation, which also played crucial roles in the mechanisms of
secondary brain damage after TBI [6]. Furthermore, no study was
found in the literature to investigate the effects of NAC administration on brain edema, blood-brain barrier (BBB) permeability, and
cortical apoptosis following TBI.Several clinical and experimental studies have demonstrated that
secondary brain injury can be magnified by numerous immune mediators, which are
frequently upregulated in response to TBI [7–9]. Increased levels of these
molecules within the injured brain, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6
(IL-6), and intercellular adhesion
molecule-1 (ICAM-1), are believed to contribute to the cerebral damage, cell
death, and BBB dysfunction [10]. A pivotal player in the regulation of these
molecules is the nuclear factor kappa B (NF-κB) family (cRel, RelA/p65, RelB,
p50 and p52) of transcription factors [11, 12]. In our previous studies, we
have demonstrated that cortical contusion trauma could induce a concomitant and
persistent upregulation of NF-κB, TNF-α, IL-6, and ICAM-1 in the injured rat brain, which might play a central
role in the injury-induced inflammatory response of brain [13, 14].NAC is a compound that increases the pool of glutathione. The latter is an important
cellular antioxidant. It is a reactive oxygen species (ROS) scavenger and can
restore the reduced cellular glutathione [15]. It is also cytoprotective since
it inhibits the activation of NF-κB and TNF-α production by lipopolysaccharide [16, 17]. Xiong etal.
have reported that NAC could greatly restore brain glutathione levels and
mitochondrial glutathione levels after TBI [3]. Also, in a rat model of
experimental stroke, administration of NAC after ischemia onset protected the
brain from free radical injury, apoptosis, and inflammation, with a wide
treatment window [18]. Nevertheless, it is still unknown up till now whether NAC can affect the production of inflammatory
agents in the brain after TBI.The aim of the current study was to determine whether NAC could attenuate
the TBI-induced brain inflammation in the pericontusional area. We hypothesized that the effect
of NAC on modulating cerebral inflammatory response might be a mechanism by which NAC reduced
cerebral edema, protected BBB, and repressed cortical apoptosis after TBI.


## 2. Materials and Methods

### 2.1. Animals

Male Wistar rats (250–300 g) were
purchased from Animal Center of Chinese Academy of Sciences, Shanghai, China. The rats were
housed in temperature and humidity controlled animal quarters
with a 12-hour light/dark cycle. All procedures were approved by the
Institutional Animal Care Committee and were in accordance with the guidelines
of the National Institutes of Health on the care and use of animals.


### 2.2. Experiment Protocol

Rat model of cortical contusion trauma: following
IP anesthesia with urethane (1000 mg/kg), animal head was fixed in the
stereotactic frame. A right parietal craniotomy (diameter 5 mm) was drilled under aseptic conditions 1 mm posterior
and 2 mm lateral to the bregma. We used a modification of Feeney's
weight-drop model in which a freefalling weight onto the exposed intact cranial dura produced a standardized
parietal contusion by letting a steel rod weighing 40 g with a flat end diameter of 4 mm fall onto a piston resting on
the dura from a height of 25 cm [19].
The piston was allowed to compress the tissue a maximum of 5 mm. After operation procedures, the rats were then returned to
their cages and the room temperature kept at 23 ± 1°C. Heart rate, arterial blood
pressure, and rectal temperature were monitored, and the rectal temperature was
kept at 37 ± 0.5°C, by using physical cooling (ice bag) when required, throughout
experiments. Sham-operated rats were anesthetized and mounted in the stereotactic
apparatus, with right parietal craniotomy surgically prepared alone and without
brain injury.We established 3 experimental groups in a randomized fashion: (a) the sham
operation group (SHAM;n = 15), (b) the TBI group (TBI; n = 18), and (c) the NAC
treatment group (TBI-NAC; n = 18). Rats of TBI-NAC group received injections of
150 mg/kg NAC IP at 15 minutes and 1, 2, and 3 days after the surgery. Rats of
SHAM and TBI groups received equal volumes of 0.9% saline solution. The dose
was chosen according to Hicdonmez etal. since they observed beneficial effects
on preventing trauma-induced oxidative brain tissue damage following TBI after
treatment with NAC using the same protocol [4]. The animals were decapitated 3
days after injury for tissue assays. The surrounding brain tissue of the
injured cortex (see Figure 1) was dissected on ice as described in our previous
study [14], some of which were
put into 10% buffered formalin, the others were stored at liquid
nitrogen immediately until use.Figure 1
Schematic representation of the cortical contusion area induced by weight-dropping trauma and the studied
region surrounding the injured brain.


### 2.3. Nuclear Protein Extract and Electrophoretic Mobility Shift Assay (EMSA)

Nuclear protein was extracted and
quantified as described [14, 20]. EMSA was performed using a commercial kit (Gel Shift
Assay System; Promega, Madison, Wis, USA) following the methods in our laboratory. The NF-κB oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′)
was end-labeled with [γ-32P] ATP (Free Biotech, Beijing, China). EMSA
was performed according to our previous study [14, 20].


### 2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of inflammatory mediators
were quantified using specific ELISA kits for rats according to the
manufacturers’ instructions (TNF-α from Diaclone Research, Besançon, France; IL-1β, IL-6 from Biosource Europe SA, Nivelles, Belgium)
and our previous studies [14, 20]. Values were expressed as ng/g protein.


### 2.5. Immunohistochemical Study

Immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded sections. The rabbit-antirat
monoclonal antibody of ICAM-1 (diluted 1 : 100, Santa Cruz Biotechnology,
Calif, USA) was used. Immunohistochemical assay was performed
according to our previous study [14]. The positive cells were identified, counted, and analyzed
under the light microscope by an investigator blinded to the grouping. The number of
positive microvessels in each section was counted in 10 microscopic fields (at
× 100 magnifications) and averaged for the positively immunostained vessel
number of per visual field.


### 2.6. Brain Water Content

Brain edema was determined using the wet/dry method as
previously described where the
percentage of brain water = [(wet weight − dry weight)/wet weight]×
100% [21]. Briefly, brains were rapidly removed from the skull and the
bilateral brains were separated. Both were placed separately into preweighed
and labeled glass vials and weighed. The vials were then placed in an oven for
72 hours at 100°C and then reweighed to obtain dry weight content. The number
of animals used in each group for brain edema study was as follows: for SHAM (n = 5), for TBI (n = 6), and for
TBI-NAC (n = 6).


### 2.7. Blood-Brain Barrier Permeability

Blood-brain barrier (BBB) permeability was determined by
Evans blue (EB) extravasation at 3 days after TBI. Briefly, 2% Evans blue was
injected IV at a dose of 2 mL/kg. Animals were then reanesthetized after 1 hour
with urethane (1000 mg/kg) and perfused using saline to remove intravascular EB dye. Animals were then
decapitated; the brains were removed and homogenized in phosphate buffered
saline. Trichloroacetic acid was then added to precipitate protein, and the
samples were cooled and centrifuged. The resulting supernatant was measured for
absorbance of EB at 610 nm using a spectrophotometer. The number of animals
used in each group for BBB permeability study was as follows: for SHAM (n = 5), for TBI (n = 6), and for TBI-NAC
(n = 6).


### 2.8. TUNEL Staining and Quantitation  of Apoptotic Cells

The formalin-fixed tissues were embedded in paraffin and
sectioned at 4μm thickness with a microtome. The sections were detected for
apoptotic cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL) method. TUNEL: in situ cell death detection kit POD (ISCDD, Boehringer Mannheim, Germany)
was used. The procedures were according to the protocol of the kit and our previous study
[14]. The positive cells
were identified, counted, and analyzed under the light microscope by an
investigator blinded to the grouping. The extent of brain damage was evaluated
by apoptotic index which was the average number of TUNEL-positive cells in
each section counted in 10 microscopic fields (at ×200 magnifications).


### 2.9. Statistical Analysis

All data were presented as mean ± SD. SPSS 12.0 was used for
statistical analysis of the data. All data were subjected to one-way ANOVA. Differences between experimental groups were determined by Fisher's LSD
posttest. Statistical  significance was inferred atP < .05.


## 2.1. Animals

Male Wistar rats (250–300 g) were
purchased from Animal Center of Chinese Academy of Sciences, Shanghai, China. The rats were
housed in temperature and humidity controlled animal quarters
with a 12-hour light/dark cycle. All procedures were approved by the
Institutional Animal Care Committee and were in accordance with the guidelines
of the National Institutes of Health on the care and use of animals.


## 2.2. Experiment Protocol

Rat model of cortical contusion trauma: following
IP anesthesia with urethane (1000 mg/kg), animal head was fixed in the
stereotactic frame. A right parietal craniotomy (diameter 5 mm) was drilled under aseptic conditions 1 mm posterior
and 2 mm lateral to the bregma. We used a modification of Feeney's
weight-drop model in which a freefalling weight onto the exposed intact cranial dura produced a standardized
parietal contusion by letting a steel rod weighing 40 g with a flat end diameter of 4 mm fall onto a piston resting on
the dura from a height of 25 cm [19].
The piston was allowed to compress the tissue a maximum of 5 mm. After operation procedures, the rats were then returned to
their cages and the room temperature kept at 23 ± 1°C. Heart rate, arterial blood
pressure, and rectal temperature were monitored, and the rectal temperature was
kept at 37 ± 0.5°C, by using physical cooling (ice bag) when required, throughout
experiments. Sham-operated rats were anesthetized and mounted in the stereotactic
apparatus, with right parietal craniotomy surgically prepared alone and without
brain injury.We established 3 experimental groups in a randomized fashion: (a) the sham
operation group (SHAM;n = 15), (b) the TBI group (TBI; n = 18), and (c) the NAC
treatment group (TBI-NAC; n = 18). Rats of TBI-NAC group received injections of
150 mg/kg NAC IP at 15 minutes and 1, 2, and 3 days after the surgery. Rats of
SHAM and TBI groups received equal volumes of 0.9% saline solution. The dose
was chosen according to Hicdonmez etal. since they observed beneficial effects
on preventing trauma-induced oxidative brain tissue damage following TBI after
treatment with NAC using the same protocol [4]. The animals were decapitated 3
days after injury for tissue assays. The surrounding brain tissue of the
injured cortex (see Figure 1) was dissected on ice as described in our previous
study [14], some of which were
put into 10% buffered formalin, the others were stored at liquid
nitrogen immediately until use.Figure 1
Schematic representation of the cortical contusion area induced by weight-dropping trauma and the studied
region surrounding the injured brain.


## 2.3. Nuclear Protein Extract and Electrophoretic Mobility Shift Assay (EMSA)

Nuclear protein was extracted and
quantified as described [14, 20]. EMSA was performed using a commercial kit (Gel Shift
Assay System; Promega, Madison, Wis, USA) following the methods in our laboratory. The NF-κB oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′)
was end-labeled with [γ-32P] ATP (Free Biotech, Beijing, China). EMSA
was performed according to our previous study [14, 20].


## 2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of inflammatory mediators
were quantified using specific ELISA kits for rats according to the
manufacturers’ instructions (TNF-α from Diaclone Research, Besançon, France; IL-1β, IL-6 from Biosource Europe SA, Nivelles, Belgium)
and our previous studies [14, 20]. Values were expressed as ng/g protein.


## 2.5. Immunohistochemical Study

Immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded sections. The rabbit-antirat
monoclonal antibody of ICAM-1 (diluted 1 : 100, Santa Cruz Biotechnology,
Calif, USA) was used. Immunohistochemical assay was performed
according to our previous study [14]. The positive cells were identified, counted, and analyzed
under the light microscope by an investigator blinded to the grouping. The number of
positive microvessels in each section was counted in 10 microscopic fields (at
× 100 magnifications) and averaged for the positively immunostained vessel
number of per visual field.


## 2.6. Brain Water Content

Brain edema was determined using the wet/dry method as
previously described where the
percentage of brain water = [(wet weight − dry weight)/wet weight]×
100% [21]. Briefly, brains were rapidly removed from the skull and the
bilateral brains were separated. Both were placed separately into preweighed
and labeled glass vials and weighed. The vials were then placed in an oven for
72 hours at 100°C and then reweighed to obtain dry weight content. The number
of animals used in each group for brain edema study was as follows: for SHAM (n = 5), for TBI (n = 6), and for
TBI-NAC (n = 6).


## 2.7. Blood-Brain Barrier Permeability

Blood-brain barrier (BBB) permeability was determined by
Evans blue (EB) extravasation at 3 days after TBI. Briefly, 2% Evans blue was
injected IV at a dose of 2 mL/kg. Animals were then reanesthetized after 1 hour
with urethane (1000 mg/kg) and perfused using saline to remove intravascular EB dye. Animals were then
decapitated; the brains were removed and homogenized in phosphate buffered
saline. Trichloroacetic acid was then added to precipitate protein, and the
samples were cooled and centrifuged. The resulting supernatant was measured for
absorbance of EB at 610 nm using a spectrophotometer. The number of animals
used in each group for BBB permeability study was as follows: for SHAM (n = 5), for TBI (n = 6), and for TBI-NAC
(n = 6).


## 2.8. TUNEL Staining and Quantitation  of Apoptotic Cells

The formalin-fixed tissues were embedded in paraffin and
sectioned at 4μm thickness with a microtome. The sections were detected for
apoptotic cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL) method. TUNEL: in situ cell death detection kit POD (ISCDD, Boehringer Mannheim, Germany)
was used. The procedures were according to the protocol of the kit and our previous study
[14]. The positive cells
were identified, counted, and analyzed under the light microscope by an
investigator blinded to the grouping. The extent of brain damage was evaluated
by apoptotic index which was the average number of TUNEL-positive cells in
each section counted in 10 microscopic fields (at ×200 magnifications).


## 2.9. Statistical Analysis

All data were presented as mean ± SD. SPSS 12.0 was used for
statistical analysis of the data. All data were subjected to one-way ANOVA. Differences between experimental groups were determined by Fisher's LSD
posttest. Statistical  significance was inferred atP < .05.


## 3. Results

### 3.1. EMSA for NF-κB

EMSA autoradiography of NF-κB DNA binding activity of the injured brain samples was
shown in Figure 2. Low NF-κB binding activity (weak EMSA autoradiography) was found in the SHAM
group. Compared with SHAM group, NF-κB binding activity in the injured brain was significantly
increased (P < .01) in TBI group. In TBI-NAC group, the NF-κB binding activity was significantly downregulated (P < .01) in the brain area surrounding the injury site after TBI.Figure 2
NF-κB activity in the brain area surrounding the injury site in SHAM
group (n = 5), TBI group (n = 6), and TBI-NAC group (n = 6). (Upper) EMSA
autoradiography of NF-κB DNA binding activity. (Bottom) levels of NF-κB DNA binding activity quantified by computer-assisted densitometric
scanning and expressed as arbitrary densitometric units (ADUs). As compared with SHAM group, NF-κB binding activity measured by EMSA was significantly increased
in TBI group. Compared to TBI group, NAC significantly suppressed NF-κB activation in TBI-NAC
group. P**<.01  versus SHAM group, P##<.01 versus
TBI group.


### 3.2. Concentrations of IL-1β, TNF-α, and IL-6  in the Injured Brains

Concentrations of IL-1β, TNF-α, and IL-6 were low in the rat
brains of SHAM group (6.16±1.63, 0.58±0.20, and 0.19±0.03 ng/g protein, resp.) (see Figure 3). Compared with SHAM group, cortical levels of the three
inflammatory cytokines were greatly induced after TBI (P < .01). As shown in
Figure 3, NAC administration after TBI
could lead to significantly decreased IL-1β and TNF-α concentrations, but had
no significant effect on the IL-6 concentration (P > .05) in rat brain tissue.Figure 3
Changes of inflammatory mediators in the injured brains as determined by ELISA in SHAM group
(n = 5), TBI group (n = 6), and TBI-NAC group
(n = 6). TBI could induce the significantly increased concentrations of IL-1β,
TNF-α, and IL-6 in the rat brain surrounding the injury site. In TBI-NAC group, the cortical
concentrations of IL-1β and TNF-α but not IL-6 were markedly downregulated as
compared with that of TBI group. P**<.01 versus SHAM group;
P#<.05 and P##<.01 versus TBI group.


### 3.3. ICAM-1 Expression in the Vessels of Injured Brain

As shown in Figure4(a), few ICAM-1-immunostained cerebral
microvessels were observed in the SHAM group rat brain. In TBI group, the
number of ICAM-1-positive vessels was significantly increased as compared with
that in the SHAM group (P < .01) (see
Figures 4(b) and 4(d)). In TBI-NAC group, when compared with the TBI group, the
number of ICAM-1-positive vessels was significantly decreased (P < .05) (see Figures 4(c) and 4(d)).
The results showed that systemic injections of NAC could significantly
downregulate the ICAM-1 immunoreactivity in the vessels of injured brain (see Figure
4(d)).ICAM-1 immunohistochemistry in the injured cortex in SHAM group (n = 5), TBI
group (n = 6), and TBI-NAC group (n = 6). (a)
SHAM rats showing few ICAM-1 positive vessels. (b) TBI rats showing strong
ICAM-1 positive vessels stained as brown. (c) TBI-NAC rats showing less ICAM-1
positive vessels than TBI rats (scar bar, 50 μm). (d) Administration of NAC
remarkably inhibited TBI-induced upregulation of ICAM-1 expression on
cerebrovascular endothelia. P**<.01 versus SHAM group; P#<.05 versus TBI group.
(a)(b)(c)(d)


### 3.4. Brain Water Content

Significant increase (P < .01) in water content was detected in the brain samples of injured side at
3 days after TBI when compared with sham-operated rats (see Figure 5). The mean
value of brain water content in the injured side was decreased by NAC
administration (P < .01) as
compared with TBI group. For the uninjured side, the mean values of brain water content had no significant
difference among SHAM, TBI, and TBI-NAC groups. The
results suggested that post-TBI NAC treatment could attenuate brain edema in this rat TBI model.Figure 5
Alterations in brain water content in SHAM group (n=5), TBI group
(n = 6), and TBI-NAC group (n = 6). The brain water
content of the injured side was increased significantly at 3 days after TBI.
NAC treatment markedly reduced brain water content. No difference of brain
water content was detected in the uninjured side among the three groups. P**<.01 versus SHAM group; P##<.01 versus TBI group.


### 3.5. Blood-Brain Barrier Permeability

The pattern of Evans blue extravasation following TBI is
shown in Figure6. Rats in TBI group demonstrated a significant increase (P <. 01) in BBB permeability to Evans
blue relative to rats of SHAM group. Administration of NAC significantly
inhibited Evans blue extravasation (P < .05), indicating a reduced BBB opening in response to NAC treatment.Figure 6
Alterations in Evans blue extravasation in SHAM group (n = 5), TBI group
(n = 6), and TBI-NAC group (n = 6).
TBI could induce a marked increase of BBB extravasation in the rat brain
compared with SHAM group. After NAC administration, the Evans blue
extravasation was significantly reduced as compared with TBI group.
P**<.01 versus SHAM group, P#<.05 versus
TBI group.


### 3.6. Apoptosis in the Injured Cortex

Few TUNEL-positive apoptotic cells were found in the SHAM
group rat brain (see Figure7(a)). In TBI group, the apoptotic index in the cortex surrounding the injured
site was found to be significantly increased compared with that in SHAM animals (P<.01) (see Figures 7(b) and 7(d)).
In TBI-NAC group, when compared with that in the TBI group, the apoptotic index in the studied cortex was
significantly decreased (P < .01)
(see Figures 7(c) and 7(d)). The result showed that NAC administration
following TBI could lead less cell death in the brain surrounding the cortical
contusion and was potential to ameliorate the secondary brain damage following
TBI.TUNEL immunohistochemistry staining of the injured cortex in SHAM group
(n = 5), TBI group (n=6), and TBI-NAC
group (n=6). (a) SHAM group rats showing few TUNEL apoptotic cells. (b) TBI
group rats showing more TUNEL apoptotic cells stained as brown. (c) TBI-NAC
group rats showing less TUNEL apoptotic cells than TBI group (scar bar, 50 μm).
(d) Administration of NAC significantly decreased the apoptotic index in rat injured brain following TBI.
P**<.01 versus SHAM group; P##<.01 versus
TBI group.
(a)(b)(c)(d)


## 3.1. EMSA for NF-κB

EMSA autoradiography of NF-κB DNA binding activity of the injured brain samples was
shown in Figure 2. Low NF-κB binding activity (weak EMSA autoradiography) was found in the SHAM
group. Compared with SHAM group, NF-κB binding activity in the injured brain was significantly
increased (P < .01) in TBI group. In TBI-NAC group, the NF-κB binding activity was significantly downregulated (P < .01) in the brain area surrounding the injury site after TBI.Figure 2
NF-κB activity in the brain area surrounding the injury site in SHAM
group (n = 5), TBI group (n = 6), and TBI-NAC group (n = 6). (Upper) EMSA
autoradiography of NF-κB DNA binding activity. (Bottom) levels of NF-κB DNA binding activity quantified by computer-assisted densitometric
scanning and expressed as arbitrary densitometric units (ADUs). As compared with SHAM group, NF-κB binding activity measured by EMSA was significantly increased
in TBI group. Compared to TBI group, NAC significantly suppressed NF-κB activation in TBI-NAC
group. P**<.01  versus SHAM group, P##<.01 versus
TBI group.


## 3.2. Concentrations of IL-1β, TNF-α, and IL-6  in the Injured Brains

Concentrations of IL-1β, TNF-α, and IL-6 were low in the rat
brains of SHAM group (6.16±1.63, 0.58±0.20, and 0.19±0.03 ng/g protein, resp.) (see Figure 3). Compared with SHAM group, cortical levels of the three
inflammatory cytokines were greatly induced after TBI (P < .01). As shown in
Figure 3, NAC administration after TBI
could lead to significantly decreased IL-1β and TNF-α concentrations, but had
no significant effect on the IL-6 concentration (P > .05) in rat brain tissue.Figure 3
Changes of inflammatory mediators in the injured brains as determined by ELISA in SHAM group
(n = 5), TBI group (n = 6), and TBI-NAC group
(n = 6). TBI could induce the significantly increased concentrations of IL-1β,
TNF-α, and IL-6 in the rat brain surrounding the injury site. In TBI-NAC group, the cortical
concentrations of IL-1β and TNF-α but not IL-6 were markedly downregulated as
compared with that of TBI group. P**<.01 versus SHAM group;
P#<.05 and P##<.01 versus TBI group.


## 3.3. ICAM-1 Expression in the Vessels of Injured Brain

As shown in Figure4(a), few ICAM-1-immunostained cerebral
microvessels were observed in the SHAM group rat brain. In TBI group, the
number of ICAM-1-positive vessels was significantly increased as compared with
that in the SHAM group (P < .01) (see
Figures 4(b) and 4(d)). In TBI-NAC group, when compared with the TBI group, the
number of ICAM-1-positive vessels was significantly decreased (P < .05) (see Figures 4(c) and 4(d)).
The results showed that systemic injections of NAC could significantly
downregulate the ICAM-1 immunoreactivity in the vessels of injured brain (see Figure
4(d)).ICAM-1 immunohistochemistry in the injured cortex in SHAM group (n = 5), TBI
group (n = 6), and TBI-NAC group (n = 6). (a)
SHAM rats showing few ICAM-1 positive vessels. (b) TBI rats showing strong
ICAM-1 positive vessels stained as brown. (c) TBI-NAC rats showing less ICAM-1
positive vessels than TBI rats (scar bar, 50 μm). (d) Administration of NAC
remarkably inhibited TBI-induced upregulation of ICAM-1 expression on
cerebrovascular endothelia. P**<.01 versus SHAM group; P#<.05 versus TBI group.
(a)(b)(c)(d)


## 3.4. Brain Water Content

Significant increase (P < .01) in water content was detected in the brain samples of injured side at
3 days after TBI when compared with sham-operated rats (see Figure 5). The mean
value of brain water content in the injured side was decreased by NAC
administration (P < .01) as
compared with TBI group. For the uninjured side, the mean values of brain water content had no significant
difference among SHAM, TBI, and TBI-NAC groups. The
results suggested that post-TBI NAC treatment could attenuate brain edema in this rat TBI model.Figure 5
Alterations in brain water content in SHAM group (n=5), TBI group
(n = 6), and TBI-NAC group (n = 6). The brain water
content of the injured side was increased significantly at 3 days after TBI.
NAC treatment markedly reduced brain water content. No difference of brain
water content was detected in the uninjured side among the three groups. P**<.01 versus SHAM group; P##<.01 versus TBI group.


## 3.5. Blood-Brain Barrier Permeability

The pattern of Evans blue extravasation following TBI is
shown in Figure6. Rats in TBI group demonstrated a significant increase (P <. 01) in BBB permeability to Evans
blue relative to rats of SHAM group. Administration of NAC significantly
inhibited Evans blue extravasation (P < .05), indicating a reduced BBB opening in response to NAC treatment.Figure 6
Alterations in Evans blue extravasation in SHAM group (n = 5), TBI group
(n = 6), and TBI-NAC group (n = 6).
TBI could induce a marked increase of BBB extravasation in the rat brain
compared with SHAM group. After NAC administration, the Evans blue
extravasation was significantly reduced as compared with TBI group.
P**<.01 versus SHAM group, P#<.05 versus
TBI group.


## 3.6. Apoptosis in the Injured Cortex

Few TUNEL-positive apoptotic cells were found in the SHAM
group rat brain (see Figure7(a)). In TBI group, the apoptotic index in the cortex surrounding the injured
site was found to be significantly increased compared with that in SHAM animals (P<.01) (see Figures 7(b) and 7(d)).
In TBI-NAC group, when compared with that in the TBI group, the apoptotic index in the studied cortex was
significantly decreased (P < .01)
(see Figures 7(c) and 7(d)). The result showed that NAC administration
following TBI could lead less cell death in the brain surrounding the cortical
contusion and was potential to ameliorate the secondary brain damage following
TBI.TUNEL immunohistochemistry staining of the injured cortex in SHAM group
(n = 5), TBI group (n=6), and TBI-NAC
group (n=6). (a) SHAM group rats showing few TUNEL apoptotic cells. (b) TBI
group rats showing more TUNEL apoptotic cells stained as brown. (c) TBI-NAC
group rats showing less TUNEL apoptotic cells than TBI group (scar bar, 50 μm).
(d) Administration of NAC significantly decreased the apoptotic index in rat injured brain following TBI.
P**<.01 versus SHAM group; P##<.01 versus
TBI group.
(a)(b)(c)(d)


## 4. Discussion

The main findings of this study are that (1) as common inflammation-related factors
to TBI, NF-κB, proinflammatory cytokines, and ICAM-1
were upregulated after
TBI and could be suppressed when treated with NAC and (2) after NAC
administration, the brain edema, BBB permeability, and apoptotic cell death were ameliorated. These
findings suggest for the first time that NAC attenuates the TBI-induced cerebral
inflammatory response and alleviates secondary brain damage following primary trauma in the rat
TBI model.There have been several studies focusing on the neuroprotective effects of NAC in TBI. As
mentioned by Ellis etal. in their literature, NAC could direct scavenge
radicals and stimulate glutathione peroxidase activity in a cat TBI model [22].
Their results suggested that NAC might be useful for treatment of oxygen-free
radical-mediated brain injury. Another research indicated that NAC administered
postinjury at an early stage could effectively restore TBI-induced
mitochondrial dysfunction and the protective effect of NAC might be related to
its restoration of glutathione levels in the brain [3]. More recent data revealed by
Hicdonmez etal. was that
NAC treatment after trauma was effective in lipid peroxidation, antioxidant
enzyme activity, and neuronal protection in cerebral injury following closed
head trauma [4]. In this current study, we found that NAC administration
following TBI could reduce cerebral edema, BBB permeability, and apoptotic cell death which played
important roles and were the major part in the secondary brain injury following
TBI. However, despite the demonstrated mechanisms of NAC in neuroprotection, none of the previous
studies focused on cerebral inflammatory response that might facilitate the development of
secondary brain damage following primary trauma.Brain inflammation represents only one of the numerous processes activated after TBI;
it has not been fully elucidated due to the complexity of the large number of
molecules shaping a sophisticated circuitry. The major effectors in this
cascade are the proinflammatory cytokines that are usually released within
minutes after challenge because they are stored intracellularly as precursor
proteins eventually modified into active molecules. TBI damages the blood-brain
barrier, then the blood cells such as neutrophils and macrophages accumulate in
the brain and further sustain the cerebral inflammatory cascade [23]. Numerous immune
mediators such as IL-1β, TNF-α, and IL-6 released within minutes of the primary injury can initiate
the infiltration of inflammatory cells into the brain by activating ICAM-1 and
other adhesion molecules [14]. The functional
importance of NF-κB in acute inflammation is based on its ability to regulate the promoters of a
variety of genes whose products, such as IL-1β, TNF-α, IL-6, ICAM-1, and acute
phase proteins, are critical to inflammatory processes [11]. NF-κB activation enhances the
transcription of proinflammatory cytokines, and the cytokines are known to in
turn activate NF-κB [24]. The positive  feedback is believed to serve to amplify inflammatory
signals. In our previous studies, it has been found that there were an  early concomitant and persistent upregulation
of NF-κB binding activity, TNF-α, IL-6, and ICAM-1 expression in the injured brain
after cortical contusion trauma [13, 14].A number of studies have demonstrated that NAC could modulate inflammatory factors
production such as NF-κB, proinflammatory cytokines, and ICAM-1 [25]. Carroll
etal. examined the influence of NAC on cerebral NF-κB activity after temporary
middle cerebral artery occlusion and indicated that activated NF-κB was
significantly increased 15 minutes after reperfusion in the affected hemisphere
and could be abolished with NAC treatment [25]. Li etal. investigated effects
of NAC on NF-κB activation and proinflammatory cytokines production in protein
malnutrition using an animal septic shock model and their results suggested
that supplementation of NAC could normalize lipopolysaccharide-induced NF-κB
activation and proinflammatory cytokines production during early rehabilitation
of protein malnourished mice [26]. Our study showed that cortical levels of
NF-κB, TNF-α, IL-1β, IL-6, and ICAM-1 were significantly induced by TBI at 3 days
after trauma. Post-TBI NAC administration could repress the expressions of most
of these inflammatory agents that accompanied TBI. Although our data have proved
that NAC could downregulate the cerebral inflammatory factors after TBI,
changes about the signaling pathways of inflammation and oxidative stress in the brain
remain unknown. It is assured that further ingenious studies are needed and will be conducted
in our laboratory.Apoptosis in the traumatically injured brain occurs not only at the impact site but also as a
result of secondary brain insults such as intracranial hypertension, hypoxia,
or disturbances of microcirculation [27]. Studies regarding the effect of NAC on neural apoptosis after
TBI have not been found to date. Nevertheless, several previous studies have demonstrated that NAC
could prevent apoptotic death of neuronal cells in vitro [28, 29]. NAC also
inhibited procaspase-9 processing and the activation of enzymatic activity of caspases induced by
acrolein. Inhibition of acrolein-induced apoptosis using NAC was confirmed
morphologically by diminished condensation of nuclear chromatin, as evaluated
by fluorescence microscopy [30]. In this present study, we found that systemic NAC
administration could inhibit apoptotic cell death in the injured brain and was
potential to reduce the secondary brain damage following TBI. However, it is
still unclear whether NAC can modulate the apoptotic signals and the whole mechanisms
involved call for further research.In conclusion, to the best of our knowledge, this is the first study to
demonstrate the effect of NAC on the inflammatory response in the injured brain after TBI. We found
that TBI could upregulate the expressions of NF-κB, IL-1β, TNF-α, and ICAM-1 in the surrounding
brain of injured site, which could be markedly inhibited by NAC administration. The therapeutic benefit
of post-TBI NAC administration might be due to its salutary effect on modulating cerebral
inflammation secondary to TBI.



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*Source: 716458-2008-05-06.xml*