Inammation, Vol. 36, No.

6, December 2013 ( # 2013)

DOI: 10.1007/s10753-013-9701-4

Vagal Efferent Fiber Stimulation Ameliorates Pulmonary

Microvascular Endothelial Cell Injury by Downregulating
Inammatory Responses
Chao Chen,1 Ying Zhang,1 Zhaohui Du,1,2 Min Zhang,1 Li Niu,1 Yanlin Wang,1 and Jianguo Li1

AbstractElectrical stimulation of the vagus nerve may have positive effects on many inammatory
diseases. This study determined the benecial effects of vagus nerve stimulation and the mechanisms by
which it attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI). Rats were intraperitoneally injected with 10 mg/kg LPS to induce ALI. The results showed that vagus nerve stimulation could
improve lung injury, as evidenced by remarkable reductions in lung edema (wet-to-dry weight ratio),
neutrophil inltration (myeloperoxidase activity), and pulmonary permeability [total number of cells and
protein concentrations in bronchoalveolar lavage uid (BALF)]. In addition, vagus nerve stimulation not
only decreased the expressions of Src-suppressed C kinase substrate and E-selectin proteins in lung
tissue but also effectively attenuated the concentrations of the proinammatory cytokines tumor necrosis
factor-, interleukin-1, and interleukin-6 in BALF. These suggest that vagus nerve stimulation is a
suitable treatment for LPS-induced ALI and indicate that it helps ameliorate pulmonary microvascular
endothelial cell injury by downregulating inammatory responses.
KEY WORDS: vagal stimulation; lipopolysaccharide; acute lung injury; Src-suppressed C kinase substrate;
E-selectin.

INTRODUCTION
Acute lung injury (ALI) and its severe form, acute
respiratory distress syndrome (ARDS), are the most
common complications of sepsis. Despite extensive investigations on new strategies for treatment, the morbidity and
mortality of sepsis-induced ALI in critically ill patients
remain unacceptably high [1]. The pathophysiological
mechanism of ALI involves alveolar damage caused by
the proliferation of inammation, increased endothelial cell
spacing, increased capillary permeability, alveolar edema,
inammatory cells in the circulation and uid extravasation into the alveoli, and hyaline membrane formation [2].
1

Department of Anesthesia, Critical Care Medicine & Emergency

Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan, 430071,
Hubei Province, Peoples Republic of China
2
To whom correspondence should be addressed at Department of
Anesthesia, Critical Care Medicine & Emergency Medicine Center,
Zhongnan Hospital, Wuhan University, Wuhan, 430071, Hubei Province,
Peoples Republic of China. E-mail: drdu1969@163.com

Vascular endothelial cells are not only passive target cells

in inammatory responses but also effector cells [3], and
changes in their morphology and functions are crucial to
the evolution of sepsis [4].
Lipopolysaccharide (LPS), the major constituent of
the outer cell wall of Gram-negative bacteria, exerts its
toxic effects on the lungs through direct injury to
endothelial cells and indirect activation of neutrophils and
macrophages, thereby releasing proinammatory cytokines, such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and IL-6. The complex network of these
proinammatory cytokines initiates, amplies, and perpetuates inammatory responses, severely altering gas exchange and inducing refractory hypoxemia. Therefore, an
anti-inammatory therapeutic strategy may be crucial in
the management of LPS-induced ALI.
Studies have conrmed that electrical stimulation of
the vagus nerve may have positive effects on septic shock
[5], inammatory bowel disease [6], postoperative ileus
[7], and renal ischemiareperfusion [8]. However, the

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0360-3997/13/0600-1567/0 # 2013 Springer Science+Business Media New York

1568
protective potential of vagus nerve stimulation against
pulmonary microvascular endothelial cell injury has yet
to be reported. We hypothesized that vagus nerve
stimulation suppresses LPS-induced ALI.

MATERIALS AND METHODS

Experimental Animals
Male SpragueDawley rats (Wuhan University Center for Animal Experiment) weighing 200 to 250 g were
used for the experiment. All animals were maintained in a
sterile, standard laboratory supplied with food and water as
needed. The rats were placed in individual ventilated cages
under specic pathogen-free conditions in the animal
facility of Wuhan University. All protocols were approved
by the Wuhan University of Science and Technology
Animal Care and Use Committee. In addition, the animals
received humane care in compliance with the Principles of
Laboratory Animal Care.
Reagents
LPS from Escherichia coli serotype 055:B7 was
purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Rat TNF-, IL-1, and IL-6 enzyme-linked
immunosorbent assay (ELISA) kits were purchased from
R&D systems (Minneapolis, MN, USA). Kits used to
determine myeloperoxidase (MPO) were obtained from
Nanjing Jiancheng Bioengineering Institute (Nanjing,
China). Polyclonal anti-Src-suppressed C kinase substrate
(SSeCKS) antibody, polyclonal anti-E-selectin antibody
and SYBRGreen/Fluorescent qPCR Master Mix (2)
were purchased from Santa Cruz Biotechnology (California,
USA).

Chen, Zhang, Du, Zhang, Niu, Wang, and Li

vagus nerve trunk. The samples were collected at 6 h after
LPS treatment.
Electrical Vagal Nerve Stimulation
The rats were anesthetized with 40 mg/kg sodium
pentobarbital. The left vagus nerve trunk was placed across
bipolar platinum electrodes connected to a stimulation
module and controlled by an acquisition system. Stimuli
(5 V, 1 Hz, 2 ms) were applied to the left cervical vagus for
20 min after LPS injection. For the control and LPS groups,
a cervical midline incision was made, the bilateral vagal
trunks were exposed and isolated from the surrounding
tissue but not transected, after which the wound was
covered with sterile, moist gauzes for 12 min. The rats were
anesthetized and paralyzed throughout the experiment.
Histopathological Evaluation of Lung
Lungs were harvested immediately after the rats (n=8,
respectively) were sacriced and specimens were embedded
in parafn. The lung tissues were deparafnized, rehydrated,
and stained with hematoxylin and eosin (H&E). The tissues
were evaluated blindly without prior knowledge of the
animal background. Ten randomly selected elds from each
specimen were observed by light microscopy.
Lung Wet-to-Dry Weight Ratio
The lung wet-to-dry (W/D) ratio was used as a
parameter of lung edema induced by LPS and calculated
at 6 h in the rats that were not subjected to histological
examination(n=8, respectively). After the rats were
sacriced, their lungs were removed, weighed, and then
dried in an oven at 80 C for 48 h. The dried lungs were
weighed again to calculate pulmonary W/D ratios.

Experiment Protocols

Determination of Bronchoalveolar Lavage Protein,

Cell Counts, and Proinammatory Cytokines

The rats were divided randomly into four groups,

namely, control, LPS, vagotomy (VGX group), and
electrical stimulation (STM group), with eight rats in
each group. In the LPS group, the rats were injected
intraperitoneally with 10 mg/kg LPS dissolved in 1 mL
sterile saline. In the control group, the rats were treated
with saline in the same manner as that in the LPS group.
The VGX group was subjected to bilateral cervical
vagotomy after LPS injection. The STM group was
subjected to bilateral cervical vagotomy after LPS
injection, followed by electrical stimulation of the left

The animals (n=8, respectively) were sacriced at

6 h after the LPS challenge. The lung was lavaged three
times through a tracheal cannula with 5 mL of autoclaved
phosphate buffer saline (PBS) instilled up to a total
volume of 13.5 mL. Bronchoalveolar lavage uid
(BALF) samples were centrifuged at 3,000 rpm for
10 min at 4 C. The sediment cells were washed and
resuspended in PBS, after which the total number of cells
in BALF was counted double-blindly using a hemocytometer. The concentrations of cytokine TNF-, IL-1,
and IL-6 in the supernatant were measured by ELISA

1569

Vagal Efferent Fiber Stimulation

according to the manufacturers instructions, and the
concentrations of protein in the supernatants of the BALF
were quantied according to the method of Hartree [9].

densitometry and expressed as mean area density using

the accompanied software. Mean area density was
expressed for all protein blots relative to -actin
expression.

MPO Activity in Lung Tissue

MPO activity was assessed as an index of neutrophil
inltration in lung tissues and measured at 6 h in the
rats that were subjected to W/D ratio(n=8, respectively).
Lung tissues were homogenized in 0.5 % hexadecyltrimethylammonium bromide solution using a homogenizer. The homogenate was then centrifuged at 12,000 rpm for
15 min at 4 C. The supernatant obtained was used for
assays of MPO activity according to the kit instructions.
The absorbance was measured at 460 nm with a
spectrometer. MPO activity was expressed in units per
gram of tissue.
Transmission Electron Microscopy of Pulmonary
Microvascular Endothelial Cell
Transmission electron microscopy was performed
on rats (n = 8, respectively) that were subjected to
histopathological evaluation. The 2-mm sections of the
lung were washed and xed with 4 % glutaraldehyde for
2 h, post-xed with 1 % osmium tetroxide, embedded in
Epon 812, and then thinly sliced. The thinly sliced tissues
were cut, stained with uranyl acetate and lead citrate, and
then examined in an H-600 (Hitachi, Japan) transmission
electron microscope(TEM) operated at 75 kV.
Tissue Nuclear Protein Extraction
The nuclear extracts were prepared following a
previous protocol [5]. The homogenates were centrifuged
at 14,000 rpm for 5 min, and the supernatant nuclear extracts
were then harvested and stored at 70 C. The extracted
proteins were quantied by LowryKalckar assays [10].
Western Blot Analysis of Lung Tissue
Western blot analysis was carried out to measure the
protein expressions of SSeCKS and E-selectin. Equal
amounts of each extract were electrophoresed in 6 %
sodium dodecyl sulfatepolyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene diuoride
membranes (Bio-Rad), and then incubated with the
primary antibody overnight at 4 C and with the
secondary antibodies for 90 min at room temperature.
The bands were detected by enhanced chemiluminescence, and the band intensities were quantied by

Real-time PCR
A tissue block measuring approximately 333 mm
was taken from the right lower lobe of the lung,
homogenized with Trizol (1 ml), gently inverted with
200 l of chloroform several times for mixing, incubated
for 5 min, and then centrifuged at 12,000 rpm for 15 min
at 4 C. The upper aqueous phase (~400 l) was
transferred into a new EP tube (1.5 ml), mixed with
isopropanol (400l), incubated at room temperature for
10 min, and then centrifuged at 12,000 rpm for 10 min at
4 C. The supernatant uid was removed. The precipitate
was washed three times with cold 70 % ethanol, dried
for 510 min in the air, and dissolved in 20 l of DEPC
water. RNA concentrations were measured using a
spectrophotometer.
The expression of -actin gene was considered as
internal reference. The following SSeCKS and -actin
primers were synthesized by the Nanjing Jinsirui Company
using Primer Premier5: SSeCKS, R:5-TGACTTC
AGGAACTTCAAGGCTC-3 and F:5-AAGTGCTGG
CTTCGGAGAAAG-3 (amplied fragment=132 bp); actin, R:5-TAAAGACCTCTATGCCAACACAGT-3 and
F:5-CACGATGGAGGGGCCGGACTCATC-3 (amplied fragment=240 bp). The reaction system was composed of 20 l of reaction volume, 4 l of cDNA (10
diluted), 10 l of SYBR Green/Fluorescent qPCR Master
Mix (2 diluted), and 0.4 l of each 100 mol/L primer.
The following reaction conditions were observed: 50 C
for 2 min, 95 C for 10 min, and then 40 cycles of 95 C for
30 s and 60 C for 30 s. The uorescence signal was
collected in the annealing stage and analyzed using the
real-time quantitative analysis software that came with the
ABI 7900 real-time uorescence quantitative PCR system.
The expression of SSeCKS gene was measured by
the level of SSeCKS/-actin under the same conditions.
Statistical Analysis
Data were presented as mean SEM and compared
through one-way analysis of variance (ANOVA). The
StudentNewmanKeuls q test was used for statistical
analysis to compare the data among all groups. A
signicant difference was presumed when P<0.05.

1570
RESULTS
Effects of Vagus Nerve Stimulation on LPS-Mediated
Lung Histopathologic Changes
Histological changes after vagus nerve stimulation in
LPS-exposed rats were determined to evaluate the effects
of vagus nerve stimulation on lung injury. Histological
examination of specimens from the LPS group revealed
signicantly damaged lung tissues, with inammatory cell
inltration into alveolar spaces and the interstitium,
thickening of the alveolar wall, interstitial edema, and
hemorrhage. Bilateral cervical vagotomy aggravated these
histological changes, but electrical vagus nerve stimulation
attenuated these responses (Fig. 1).
Effects of Vagus Nerve Stimulation on Lung W/D
Ratio and MPO Activity
W/D ratio and MPO activity were evaluated 6 h after
LPS induction. In the LPS and VGX groups, the W/D ratio

Chen, Zhang, Du, Zhang, Niu, Wang, and Li

were markedly increased compared with the control group
(*P<0.05, #P<0.05). Vagus nerve stimulation ameliorated
LPS-induced lung edema (*P<0.05, #P<0.05). Compared
with the control group, MPO activity was higher in the LPS
and VGX groups (*P<0.05, #P<0.05). Vagus nerve
stimulation caused a signicant reduction in MPO
activity (*P<0.05, #P<0.05, Fig. 2).

Effects of Vagus Nerve Stimulation on LPS-Induced

Inammatory Cell Accumulation and Total Protein
Concentration in BALF
Total cell numbers and protein concentrations in
BALF indicate the extent of lung leakage, which denotes
the severity of lung injury. Administration of LPS and VGX
massively recruited a large number of cells and proteins in
BALF (*P<0.05, #P<0.05). Vagus nerve stimulation
signicantly reduced the total cell numbers and protein
concentrations in BALF (*P<0.05, #P<0.05, Fig. 3).

Fig. 1. Effects of vagus nerve stimulation on LPS-mediated lung histopathologic changes. Lungs(n=3) from each experimental group were processed for
histologic evaluation 6 h after LPS challenge: lung section of the a control group, b LPS group, c STM group, and d VGX group. Representative
histological sections of the lungs were stained with H&E at 400 magnication.

Vagal Efferent Fiber Stimulation

Fig. 2. Effects of vagus nerve stimulation on lung W/D ratio and MPO
activity. The lung W/D ratio (a) and MPO activity (b) were determined 6 h
after LPS administration. The values are presented as mean SEM (n=8).
*P<0.05, vs. the LPS group; #P<0.05, vs. the VGX group.

1571

Fig. 3. Effects of vagus nerve stimulation on LPS-induced inammatory cell accumulation and total protein concentration in BALF. The total
number of cells (a) and total protein concentrations (b) in BALF were
determined 6 h after LPS induction. BALF was prepared from rats 6 h
after LPS injection. The values are presented as mean SEM (n=8).
*P<0.05, vs. the LPS group; #P<0.05, vs. the VGX group.

Effects of Vagus Nerve Stimulation

on the Ultrastructure of Pulmonary Microvascular
Endothelial Cell in LPS-Treated Rats
The ultrastructure of pulmonary microvascular endothelial cells in the lung was observed by TEM to determine
the inuence of vagus nerve stimulation on LPS-induced
pulmonary microvascular endothelial cell injury. The
pulmonary microvascular endothelial cells and basement
membrane morphology were normal in the control group.
Greater lung microvascular endothelial cells swelling,
basement membrane defects, as well as necrosis and
cavitation, a wider perinuclear space, margination, and
apoptosis were observed in the LPS group. Bilateral
cervical vagotomy aggravated these morphological alterations, whereas electrical vagus nerve stimulation signicantly attenuated them (Fig. 4).
Effects of Vagus Nerve Stimulation on the Expressions
of SSeCKS and E-selectin in LPS-Exposed Rats
As shown in Fig. 5a, the expressions of SSeCKS
mRNA and protein in lung tissue signicantly increased

in the LPS and VGX groups compared with the control

group (*P<0.05, #P<0.05). Vagus nerve stimulation
signicantly decreased the expressions of them
(*P<0.05, #P<0.05).
As shown in Fig. 5b, the nuclear expression of Eselectin increased in the LPS and VGX groups (*P<0.05,
#
P<0.05). Vagus nerve stimulation signicantly attenuated
the expressions of E-selectin (*P<0.05, #P<0.05).
Effect of Vagus Nerve Stimulation on LPS-Induced
Proinammatory Mediators
TNF-, IL-1, and IL-6 in BALF were observed to
assess the inuence of vagus nerve stimulation on LPSinduced proinammatory mediators. Figure 6 shows that
TNF-, IL-1, and IL-6 were only minimally expressed in
the control group. LPS and VGX markedly increased TNF-

1572

Chen, Zhang, Du, Zhang, Niu, Wang, and Li

Fig. 4. Effects of vagus nerve stimulation on LPS-mediated pulmonary microvascular endothelial cell ultrastucture. Lungs (n=3) from each experimental group were processed for histological evaluation 6 h after LPS challenge: lung sections from the a control group, b LPS group, c STM group, and d
VGX group are shown. Arrows indicate the location of the pulmonary microvascular endothelial cells. The scale bar represents 1,900 nm.

Fig. 5. Effects of vagus nerve stimulation on expressions of lung SSeCKS and E-selectin. a Lung SSeCKS mRNA (a) and protein (b) were determined
6 h after LPS administration. b Effects of vagus nerve stimulation on expression of lung E-selectin. Lung E-selectin protein was determined 6 h after LPS
administration. Protein samples were analyzed by western blot analysis, and -actin was used as an internal control. Similar results were obtained among
three independent experiments, and the results from one of which are shown. *P<0.05, vs. control group. #P<0.05, vs. LPS group.

Vagal Efferent Fiber Stimulation

Fig. 6. Effects of vagus nerve stimulation on the concentrations of inflammatory cytokines in rat BALF with ALI. BALF was collected 6 h
following LPS challenge to analyze the inammatory cytokines TNF-
(a), IL-1 (b), and IL-6 (c). The values are presented as mean SEM
(n=8). *P<0.05, vs. the LPS group; #P<0.05, vs. the VGX group.

, IL-1, and IL-6 concentrations (*P<0.05, #P<0.05).

Administration of vagus nerve stimulation downregulated
TNF-, IL-1, and IL-6 production (*P<0.05, #P<0.05).

DISCUSSION
To the best of our knowledge, this study is the rst to
demonstrate that vagus nerve stimulation signicantly
protects rats against LPS-induced ALI by ameliorating

1573
pulmonary microvascular endothelial cell injury. The
protective effects of vagus nerve stimulation are associated with the downregulation of inammatory responses.
ALI is characterized by interstitial edema, neutrophil
accumulation, epithelial integrity disruption, and protein
leakage into the alveolar space, severely altering gas
exchange [11, 12]. In this study, vagus nerve stimulation
decreased the wet-to-dry ratio of the lungs, lung MPO
activity, and the total number of cells and protein
concentrations in bronchoalveolar lavage uid. Histological analysis further indicated that vagus nerve stimulation signicantly attenuated lung tissue injury.
Some studies have conrmed that LPS can damage
vascular endothelial cells [13], induce endothelial cell
contraction, increase cell spacing, and enhance microvascular permeability [14, 15]. SSeCKS, a cytoskeletal protein
located in vascular endothelial cells, regulates microvascular permeability by regulating F-actin [16, 17]. It is also an
inammatory reaction protein directly related to the severity
of the inammation [18]. Therefore, the level of SSeCKS in
lung tissue can represent the degree of vascular endothelial
cell injury. Cheng [19] found that endothelial cell adhesion
is regulated by SSeCKS. The adhesion between vascular
endothelial cells and leukocytes in early inammation is
mainly mediated by E-selectin, which is only expressed in
activated vascular endothelial cell surfaces [20]. Therefore,
the level of E-selectin in lung tissue can represent the degree
of vascular endothelial cell injury. The results of our study
demonstrated that vascular endothelial cells underwent
contraction and were injured as well as that the activities
of SSeCKS and E-selectin were signicantly elevated 6 h
after LPS exposure. These changes were even worse in the
VGX group. However, their production signicantly
decreased after vagus nerve stimulation. Therefore, vagus
nerve stimulation may have protective effects against LPSinduced vascular endothelial cell injury.
Excessive cytokine-mediated inammation may play
a fundamental role in the pathogenesis of ALI/ARDS [21].
Studies have shown that increased levels of TNF-, IL-1,
and IL-6 in BALF can be observed in ARDS patients and
that persistent elevation of proinammatory cytokines in
patients with ALI or sepsis has very poor prognosis [22].
TNF- and IL-1 amplify the inammatory cascade,
cause inammatory injury, and recruit neutrophils into
the lung [23]. You [24] showed that SSeCKS plays an
important role in vascular permeability increases induced
by TNF- and IL-1. Plasma IL-6 is known to be a
signicant predictor of morbidity and mortality in patients
with ARDS [25]. As some cytokines are potent mediators
of potentially damaging tissue responses, several mecha-

1574
nisms exist to ensure that the effects of these cytokines are
restricted [26]. In this study, we found that the levels of
TNF-, IL-1, and IL-6 in lung homogenate dramatically
increased after LPS administration. However, their production signicantly decreased after vagus nerve stimulation. These results indicate that vagus nerve stimulation
limits inammatory responses, thus ameliorating pulmonary microvascular endothelial cell injury.
In the development of ALI and ARDS, inltration of
numerous neutrophils into alveolar spaces is another wellknown pathogenic mechanism [27]. In the present study,
MPO activity, which reects neutrophil inltration into the
lung parenchyma or alveolar spaces, was signicantly
elevated after LPS exposure, and vagus nerve stimulation
signicantly reduced the MPO activity. Therefore, vagus
nerve stimulation may inhibit neutrophil inltration into the
lung parenchyma or alveolar spaces in LPS-induced ALI.
These results suggest that inhibition of neutrophil inltrationassociated lung injury maybe another pathway through which
vagus nerve stimulation attenuates LPS-induced lung injury.
However, the precise mechanisms underlying the
inhibitory effects of inammatory responses still need to
be further studied, including the effects of vagal efferent
ber stimulation on upriver genes, the complex mechanisms involved in signaling pathway in the antimicrobial
host defense, such as high-mobility group box 1, NF-B,
Toll-like receptors, MyD88, and so forth.
In conclusion, we have shown that vagus nerve
stimulation attenuates LPS-induced ALI. The mechanisms
behind the benecial effects of vagus nerve stimulation are
correlated with the downregulation of inammatory responses, remarkably ameliorating pulmonary microvascular
endothelial cell injury. The data suggest that vagal efferent
ber stimulation administration could serve as a potential
treatment for the early stages of lung injury and presents a
novel strategy for the modulation of inammatory responses.

ACKNOWLEDGMENTS
This work was nancially supported by the Natural
Science Foundation of Hubei Province of China
(NO.2011CDB518).

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