A novel nitric oxide scavenger decreases liver injury and
improves survival after hemorrhagic shock
John
Menezes1,
Christian
Hierholzer1,
Simon C.
Watkins2,
Valerie
Lyons3,
Andrew B.
Peitzman1,
Timothy R.
Billiar1,
David J.
Tweardy4,5,6, and
Brian G.
Harbrecht1
Departments of 1 Surgery,
2 Cell Biology and Physiology,
3 Pathology,
4 Medicine, and
5 Molecular Genetics and
Biochemistry, and 6 University of
Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh,
Pennsylvania 15213
 |
ABSTRACT |
We tested the ability of a nitric oxide (NO)
scavenger to reduce tissue injury in a rodent model of hemorrhagic
shock. Rats were hemorrhaged to a mean arterial blood pressure (MAP) of
40 mmHg and then resuscitated when either 30% of their shed blood had
been returned (group 1) or after 100 min of
continuous shock (group 2). Selected animals
were treated with the NO scavenger NOX (30 mg · kg
1 · h
1)
infused over 4 h. Hemorrhaged rats had a lower MAP after resuscitation compared with sham-shock control rats. NOX treatment significantly increased MAP after resuscitation from hemorrhage. Hemorrhagic shock
also increased liver injury as reflected by elevated ornithine carbamoyltransferase (OCT) plasma levels, and NOX treatment
significantly reduced OCT release. In addition, NOX was associated with
significantly decreased hepatic neutrophil infiltration and improved
24-h survival (n = 8 of 9) compared
with saline-treated shock animals (n = 3 of 9). These data suggest that excess NO mediates shock-induced tissue injury and that suppression of NO availability with NO scavengers may reduce the pathophysiological sequelae of severe hemorrhage.
nitric oxide synthase; trauma; kidney; multiple organ failure
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INTRODUCTION |
HEMORRHAGIC SHOCK contributes to both short-term and
long-term morbidity and mortality after traumatic injury (17). The specific mechanisms involved in the pathophysiology of hemorrhage have
been incompletely defined. Hemorrhagic shock results in an oxidative
stress to cells and in the induction of the inflammatory response, with
an increased expression of a number of proinflammatory mediators and
cytokines (22). Hemorrhage thus results in a combined oxidative and
inflammatory insult to tissues that can contribute to cellular
dysfunction, produce tissue injury, and profoundly alter organ function.
Nitric oxide (NO) has been shown to combine with superoxide to form the
highly toxic peroxynitrite radical and produce oxidative injury (26,
29). NO also plays a role in proinflammatory cell signaling, altering
cellular gene expression, enzyme activity, and transcription factor
activation (18, 21). In addition, NO regulates vasodilation, platelet
and neutrophil aggregation, and local organ blood flow (18). Therefore,
NO may influence cell and tissue function following hemorrhage in a
variety of ways. Hemorrhagic shock decreases endothelial constitutive
nitric oxide synthase (ecNOS) activity (27, 34) but increases inducible nitric oxide synthase (iNOS) expression and activity (8, 30). Previous
work has demonstrated that providing exogenous NO donors early in shock
is beneficial (2, 25) and that inhibiting ecNOS activity during shock
is harmful (2, 3). Other authors have shown that inhibition of NOS
activity reduces organ injury and improves survival, suggesting that
the role of NO in the pathophysiology of hemorrhagic shock is complex
(35). We have shown that selective iNOS inhibition after hemorrhage
reduces lung injury, transcription factor activation, and
proinflammatory cytokine expression (4). This body of evidence supports
the hypothesis that distinct NOS isoforms may regulate different
metabolic and physiological aspects of the response to hemorrhage and
that excess NO from iNOS promotes inflammation and tissue injury.
The above hypothesis suggests that removal of excess NO while basal
beneficial levels of NO are preserved could improve outcome after
resuscitation from hemorrhagic shock. Most studies investigating the
use of NOS inhibitors in hemorrhagic shock, however, are limited by the
use of inhibitors that are either more selective for ecNOS or are
nonselective, inhibiting both ecNOS and iNOS isoforms (1, 36). Even
highly selective iNOS inhibitors can potentially interfere with ecNOS
activity at high doses or high tissue concentrations. Problems with NOS
isoform selectivity and dose-response relationships complicate the
potential therapeutic utility of NOS inhibitors in shock states.
Recently, a number of compounds have been developed that are designed
to scavenge excess extracellular NO. Because NO scavengers bind NO that
diffuses from the site of production (13), they may potentially reduce
toxicity from excessive iNOS activity. Poor water solubility may limit
the potential use of some scavengers in biological systems (16). The
compound NOX, however, is a dithiocarbamate that is water
soluble and effectively binds NO in vivo (11, 20, 31, 33). We therefore
tested the ability of NOX to ameliorate tissue injury after hemorrhagic shock. Our data demonstrate that NOX infusion reduced liver injury, improved systemic blood pressure after hemorrhage, and improved survival following hemorrhagic shock.
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MATERIALS AND METHODS |
Hemorrhagic shock model.
The model of hemorrhagic shock used in these studies has been
previously described (2, 3). Briefly, male Sprague-Dawley rats
(250-310 g) were fasted overnight but allowed free access to water
before the experiment. After being anesthetized with pentobarbital (30 mg/kg ip), the rats were mechanically ventilated with room air, and
catheters were placed in the right jugular vein and left carotid artery
for infusion therapy and monitoring of mean arterial blood pressure
(MAP), respectively. After a stabilization period of 20 min, baseline
hemodynamic data were recorded and a blood sample was obtained. The
rats were then hemorrhaged to a MAP of 40 mmHg and were maintained at
that level by the withdrawal or reinfusion of shed blood as needed.
Once the endpoint for resuscitation was reached, the rats were
resuscitated with all the remaining shed blood plus 2× the
maximum shed-blood volume as lactated Ringer solution. Sham-shock
animals were subjected to anesthesia and instrumentation for a period
of time identical to that of shock rats but were not hemorrhaged. When
not receiving test solutions, all animals received a maintenance
infusion of 0.9% NaCl at 1.5 ml/h. Animal care was in accord with the
guidelines of the University of Pittsburgh Animal Care and Use
Committee and followed guidelines prescribed by the National Institutes
of Health.
The animals were studied in two separate experimental protocols. In the
first (group 1; Fig.
1A),
the rats were hemorrhaged, and the point of vascular decompensation
(end of compensated shock, CE) was recorded. Vascular decompensation is
defined as the point at which shed blood must be reinfused to maintain
the MAP at 40 mmHg. Once 30% of the total shed-blood volume had been
reinfused, the rats were resuscitated and observed during the 4 h after
resuscitation, at which point blood was collected for analysis.
Randomly selected animals received either saline or NOX (30 mg · kg
1 · h
1;
provided by Dr. Ching-San Lai, Medinox, San Diego, CA) by intravenous infusion, beginning at CE and continuing for 4 h.


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Fig. 1.
Hemorrhagic shock protocol. Rats were hemorrhaged to a mean arterial
pressure (MAP) of 40 mmHg and were given either saline or the nitric
oxide (NO) scavenger NOX beginning at end of compensated shock (CE,
A) or after 60 min of shock
(B). NOX was continued for 4 h after
resuscitation.
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|
In the second experimental protocol (group 2;
Fig. 1B), the rats were maintained
at a MAP of 40 mmHg for a total of 100 min and then were resuscitated
as described above. Randomly selected animals received either saline or
NOX (30 mg · kg
1 · h
1)
by continuous intravenous infusion beginning at 60 min of shock and
continuing until 4 h after resuscitation. At this time, a blood sample
was obtained, the vascular catheters were removed, and the animals were
returned to their cages and allowed to recover. Survival was recorded
24 h after shock, and blood and tissues were collected from surviving
animals for analysis. Sham-shock and sham-shock plus NOX (sham + NOX)
animals for each separate experimental protocol were used as controls.
Tissue injury.
Blood samples were collected at the indicated time points, the plasma
was separated by centrifugation, and samples were stored at
70°C until analyzed. Plasma samples were analyzed for the liver-specific urea-cycle enzyme ornithine carbamoyltransferase (OCT)
as a marker of hepatic injury (2, 3). Plasma OCT levels were determined
by the method of Oshita et al. (19). As an index of renal damage,
plasma creatinine levels were measured with the use of an automated
analyzer. Excised tissue samples were placed in 10% buffered Formalin
and stored at
4°C. Tissue injury was assessed histologically
by staining with hematoxylin and eosin (H + E). Tissue
polymorphonuclear cell (PMN) infiltration was quantitated by staining
tissues for myeloperoxidase (MPO) (5). Tissue nitrotyrosine
immunoreactivity was measured as an index of peroxynitrite formation by
immunohistochemistry and fluorescence microscopy (28). To perform these
microscopic analyses, 5-µm cryosections were cut and prepared for H + E and MPO staining by standard methods. For immunocytochemical
detection of nitrotyrosine-nonspecific antibody, binding to sections
was blocked with 10% BSA in TBS (50 mM Tris, pH 7.0; 150 mM NaCl) for
30 min, and cells were then washed with 0.25% BSA in TBS. Sections
were incubated with monoclonal mouse anti-nitrotyrosine antibody
(1:200) (Transduction Laboratories, Lexington, KY) in 0.25% BSA in
PBS for 1 h, the sections were washed, and a secondary
antibody, Alexa 488-conjugated anti-mouse (1:100) (Molecular Probes,
Eugene, OR) antibody, was added for 1 h. After additional washing, the
nuclei of cells were labeled with a specific DNA dye (Hoechst 24232)
(Sigma), mounted with Gelvatol (Monsanto, St. Louis, MO), and viewed
with an Olympus Provis microscope with the use of a ×100
objective and epifluorescence optics. Images in perfect registration
were collected with the use of appropriate cubes for each color.
Individual color images were then added together with Adobe Photoshop
5.0 without further filtration or processing. To perform the
quantitative analyses, 10 randomly chosen fields were examined blindly
and scored for the number of positive cells in each section.
NO-hemoglobin.
To establish that NOX scavenged excess NO, the quantity of NO bound to
hemoglobin (NO · Hb) was measured in 4-h blood
samples from group 2 animals. Red
blood cells (RBCs) were separated from plasma, washed twice, and
resuspended in 10 mM Tris · HCl buffer (pH 7.4)
containing 0.15 M NaCl. After sonication and centrifugation, the
supernatant containing Hb was passed through a Sephadex G-25 column to
remove nitrite and nitrate, and the eluate was analyzed by
diazotization (23).
RT-PCR amplification.
Liver samples were obtained immediately after killing, frozen in liquid
nitrogen, and stored at
80°C until analyzed. Total cellular
RNA was isolated, and RT-PCR was performed as described (4). Briefly,
total RNA (2.5 µg) was subjected to first-strand cDNA synthesis with
the use of oligo(dT) primer and Moloney murine leukemia virus (MMLV)
RT. PCR primers and conditions were as previously described (4). RAW
264.7 murine macrophages stimulated with endotoxin were used as
positive controls, with water as a negative control. PCR products were
separated on a 10% polyacrylamide gel and were exposed to a
PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA), and
radioactivity was determined by scanning densitometry (4).
Statistical analysis.
Data are presented as means ± SE. For the analysis of survival,
statistical significance was determined by
2. For all other analyses,
significance was determined by ANOVA followed by Fisher's least
significant difference test. A P value of <0.05 was considered statistically significant.
 |
RESULTS |
In the first experimental protocol, rats were maintained at a MAP of 40 mmHg until 30% of their shed-blood volume was returned, and then they
were resuscitated. NOX treatment was begun at CE. As shown in Table
1, there were no differences in baseline
MAP between groups. All rats responded to hemorrhage in a similar manner with no differences in the time to reach the point of vascular decompensation or in the total volume of blood removed to maintain the
MAP of 40 mmHg (Table 1). However, once the NOX infusion was
instituted, NOX-treated rats required a less-rapid rate of return of
shed blood to maintain the MAP at 40 mmHg compared with saline-treated
animals (P < 0.05, Table 1). There
were no differences in MAP between sham and sham + NOX animals in this
experiment (data not shown). The MAP for hemorrhaged rats was
significantly lower than the MAP for sham-shock rats at 120, 180, 210, and 240 min after resuscitation (data not shown). After hemorrhage,
NOX-treated rats had a significantly greater MAP 120 min after
resuscitation than saline-treated rats (shock alone, 94 ± 9 mmHg;
shock + NOX, 115 ± 5 mmHg; P < 0.05). NOX-treated rats had a higher MAP at later time points after
resuscitation that approached but did not reach statistical
significance (at 180 min: shock alone, 83 ± 10 mmHg, shock + NOX
101 ± 5 mmHg, P = 0.070; at 210 min: shock alone 76 ± 11 mmHg, shock + NOX 98 ± 7 mmHg,
P = 0.077). NOX treatment alone had no
effect on plasma OCT levels in sham-shock rats. Despite having a longer
total time in shock (Table 1), NOX-treated rats that were hemorrhaged
had lower OCT plasma levels than saline-treated rats when measured 4 h
after resuscitation (Fig. 2).

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Fig. 2.
Liver injury in group 1 animals. Rats
were bled to a MAP of 40 mmHg as described in
MATERIALS AND METHODS, and plasma
samples were collected 4 h after resuscitation. Crosshatched bar, sham;
hatched bar, sham + NOX; solid bar, shock; open bar, shock + NOX.
* P < 0.05 compared with sham.
** P <0.05 compared with
shock.
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A second experimental protocol was undertaken to standardize the total
duration of shock. Here, rats were subjected to 100 min of continuous
shock at MAP of 40 mmHg with either saline or NOX given beginning at 60 min. As shown in Table 2, there were no
differences in baseline MAP between groups. Shock and shock + NOX rats
were also comparable in the amount of time required to reach CE and in
the total volume of shed blood removed to maintain the MAP at 40 mmHg
(Table 2). Once the NOX infusion was begun at 60 min, the NOX-treated
animals again appeared to tolerate the hypotensive state better than
saline-treated animals, requiring only 12% of their shed-blood volume
to maintain their MAP at 40 mmHg over the next 40 min, compared with
29% for saline-treated animals (Table 2). Both sham-shock rats and
sham + NOX rats had a stable MAP throughout the experimental period
(Fig. 3). Shock rats had a significantly
lower MAP compared with sham animals after resuscitation at all time
points. The MAP following resuscitation was significantly higher in
NOX-treated rats compared with saline-treated rats (Fig. 3).

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Fig. 3.
Blood pressure in group 2 animals.
Group 2 rats were subjected to sham
shock (open symbols) or shock (filled symbols) as described in
MATERIALS AND METHODS, and MAP was
recorded. Animals received either saline (circles) or NOX (squares).
* P < 0.05 compared with sham + saline.
# P < 0.05 compared with shock + saline.
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To confirm the ability of NOX to scavenge excess NO after hemorrhage,
we measured NO · Hb as an index of extracellular NO release. RBCs from sham and sham + NOX animals liberated little NO · Hb (Fig. 4). RBCs
from shocked rats liberated increased quantities of NO, consistent with
previous findings demonstrating increased NOS activity after shock (8,
27). RBCs from shock + NOX rats had significantly less
NO · Hb than saline-treated rats, consistent with the
NO-scavenging effect of NOX after hemorrhage.

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Fig. 4.
NOX decreases NO bound to hemoglobin (Hb). Red blood cells in
group 2 animals from blood samples
obtained 4 h after resuscitation were analyzed for Hb-bound NO as
described in MATERIALS AND METHODS.
Crosshatched bar, sham; hatched bar, sham + NOX; solid bar, shock; open
bar, shock + NOX. * P < 0.05 compared with shock alone.
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When the effect of NOX on liver injury was examined, sham-shock rats
given either saline or NOX had low plasma levels of OCT throughout the
experiment. Hemorrhaged rats given saline alone had elevated plasma OCT
levels at both 4 and 24 h that were significantly decreased by NOX
(Fig.
5A). All
sham-shock and sham + NOX animals survived 24 h. The number of rats
that survived for 24 h was significantly greater in
hemorrhaged rats treated with NOX than in saline-treated rats
(n = 8 of 9 vs. 3 of 9 24-h survivors,
NOX vs. saline, respectively; P < 0.05 by
2).


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Fig. 5.
Liver and kidney injury is decreased by NOX. Group
2 rats were subjected to sham shock or shock and given
either saline or NOX as described in MATERIALS AND
METHODS. Blood samples were obtained at baseline (0 h)
and 4 and 24 h after resuscitation and analyzed for ornithine
carbamoxyltransferase (OCT; A) and
creatinine (B). Crosshatched bar,
sham; hatched bar, sham + NOX; solid bar, shock; open bar, shock + NOX.
* P < 0.05 compared with sham.
** P < 0.05 compared with
shock alone.
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Plasma creatinine levels were measured in group
2 animals as an index of renal injury (Fig.
5B). Sham and sham + NOX rats had
low plasma creatinine at all time points measured. Shocked rats treated
with saline had low baseline creatinine levels that were increased at 4 h and elevated further at 24 h (Fig.
5B). However, shocked rats treated
with NOX had plasma creatinine levels that were similar to the low
baseline values at each time point.
Increased PMN infiltration into the liver contributes to the hepatic
injury produced by hemorrhagic shock and hepatic
ischemia-reperfusion (6, 15, 32). Selective iNOS inhibition
after hemorrhage decreased PMN infiltration into the lung (4). We
therefore assessed the degree of PMN infiltration into liver and kidney utilizing MPO staining. Sham-shock rats had low numbers of MPO-positive cells in both liver and kidney sections, and this low number was unaffected by NOX treatment (data not shown). Liver sections from shocked rats had significantly increased numbers of MPO-positive cells
compared with sham-shock controls. The number of MPO-positive cells was
significantly reduced by NOX treatment after shock (Fig. 6). Hemorrhagic shock induced
increased PMN infiltration into kidney sections compared with
sham-shock animals, but this influx was not as marked as that seen in
liver sections. Increased PMN infiltration was chiefly localized to the
glomeruli. In addition, glomerular erythrocyte casts and altered
mesangial cell morphology were seen in saline-treated hemorrhaged rats
with H + E staining. These changes were difficult to quantitate because
of the lower number of infiltrating cells in kidney compared with liver
sections, but qualitatively the shock-induced changes in renal
histology were diminished with NOX treatment.


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Fig. 6.
NOX decreases neutrophil infiltration into the liver. Livers of animals
were fixed in Formalin, sectioned, and stained for myeloperoxidase
(MPO). Representative fields (magnification, ×400) are shown for
sham (A), shock
(B), and shock + NOX
(C). Arrows demonstrate intensely
staining MPO-positive cells. D: ten
randomly selected fields were blindly scored for number of MPO-positive
cells per ×400 field. * P < 0.05 compared with sham.
** P < 0.05 compared with
shock.
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Increased tissue injury after shock has been proposed to be due, in
part, to NO-mediated oxidative injury through the formation of
peroxynitrite (26, 29). To determine if a reduction in peroxynitrite
formation with NOX contributed to the changes in tissue injury seen in
these experiments, we measured hepatic nitrotyrosine immunoreactivity
in liver samples collected at 24 h. Sham-shock rats had little
detectable nitrotyrosine immunoreactivity and no changes were seen in
sham + NOX rats (data not shown). Increased nitrotyrosine staining was
present in hemorrhaged rats, and NOX infusion reduced immunoreactivity
in hemorrhaged rats to the levels present in sham animals (Fig.
7).

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Fig. 7.
Nitrotyrosine immunoreactivity after hemorrhage. Liver sections were
analyzed for nitrotyrosine immunoreactivity as described in
MATERIALS AND METHODS. Representative
fields (magnification, ×20) are shown for shock
(A), shock + NOX
(C), sham
(D), and negative control
(E).
B: shock field (magnification,
×100).
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We have previously demonstrated that inhibition of iNOS with a
selective inhibitor reduced transcription factor activation and
proinflammatory cytokine production in the lung (4). To assess whether
NOX decreased proinflammatory cytokine production in these experiments,
we measured tumor necrosis factor (TNF)-
and interleukin (IL)-1
mRNA expression in the liver by RT-PCR. Sham-shock rats had low levels
of TNF-
and IL-1
mRNA present in the liver, and these low levels
were unaffected by NOX treatment. Rats subjected to hemorrhagic shock
had increased hepatic TNF-
and IL-1
mRNA expression, and the
expression of both proinflammatory cytokines was reduced by NOX
treatment (Figs. 8 and
9).

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Fig. 8.
NOX decreases hepatic tumor necrosis factor (TNF)- mRNA expression.
TNF- expression was determined by RT-PCR in samples of liver
collected 24 h after hemorrhage. PCR products were separated in a 10%
polyacrylamide gel and developed with the use of a PhosphorImager
(A). HS, hemorrhagic shock; ,
negative control; +, positive control.
B: mRNA was quantitated by scanning
densitometry.
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Fig. 9.
NOX decreases hepatic interleukin (IL)-1 expression. Liver samples
were collected 24 h after hemorrhage, total RNA was isolated, and
IL-1 mRNA expression was determined by RT-PCR as described in
MATERIALS AND METHODS. PCR products
were separated on a 10% polyacrylamide gel, developed with the use of
a PhosphorImager (A), and
quantitated by scanning densitometry
(B).
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 |
DISCUSSION |
NO plays an important role in a vast array of cellular functions by
mediating intracellular signaling pathways, modulating cellular
oxidative stress, regulating nuclear transcription factor activation,
and mediating gene expression (14, 18, 21, 24). NO can also profoundly
alter organ function by regulating regional blood flow and organ
perfusion (18). NO was initially thought to mediate hypotension and
vascular decompensation following hemorrhage through its effects on
vascular tone (10). However, as our knowledge of the numerous effects
of NO has increased, the complexity of the role of NO during
hemorrhagic shock has become more evident. Most studies examining the
role of NO in hemorrhagic shock have utilized NOS inhibitors that
either act predominantly against ecNOS or nonselectively inhibit both
ecNOS and iNOS. The activity of ecNOS is decreased following hemorrhage
(27, 34), and delivering NO with NO donors early after hemorrhage
improves blood pressure and short-term survival (25). Inhibition of NOS
with compounds that are nonselective or principally inhibit ecNOS
increases tissue injury, suggesting that ecNOS activity is essential in
maintaining organ and tissue perfusion during hypovolemia (2, 3, 35). Use of NOS inhibitors with greater selectivity toward iNOS, however, is
beneficial against shock-induced tissue injury (4). Therefore, maintaining the protective effects of NOS activity (ecNOS) while preventing the potentially harmful effects of NOS activity (excessive iNOS) is a significant obstacle in attempts to therapeutically manipulate NOS activity after hemorrhagic shock. Even the most selective iNOS inhibitors can potentially inhibit ecNOS if a sufficient dose is used, because the perfectly selective NOS inhibitor has yet to
be developed. Potentially narrow ranges of safe and effective doses of
these compounds, as well as altered tissue perfusion and
bioavailability in shock, further complicate this issue.
The potential utility of reducing excess NO levels after hemorrhagic
shock and the limitations of the NOS inhibitors developed to date
combine to enhance the potential usefulness of compounds designed to
scavenge or bind excess NO. A NO scavenger must be given in a manner
that does not interfere with essential ecNOS function and yet
neutralizes excess iNOS-produced NO that may mediate tissue damage. In
these experiments, we have used a continuous infusion of the NO
scavenger NOX in two separate shock protocols. In both protocols, NOX
improved systemic MAP after resuscitation from hemorrhage and reduced
shock-induced liver injury compared with saline-treated controls. NOX
infusion also reduced renal dysfunction and improved 24-h survival
compared with saline-treated animals, although it had no apparent
noxious effect on either shock or sham animals. The reduction in injury
with NOX was associated with a reduction in hepatic PMN infiltration,
suggesting that NOX may reduce tissue injury, in part, by reducing the
shock-induced inflammatory cell infiltrate. We cannot exclude the
possibility, however, that the reduced PMN infiltrate is a result of
the decrease in tissue injury produced by NOX, as opposed to a direct
effect on PMN by NOX itself.
The ability of NOX to reduce shock-induced hepatic injury is similar to
our previous findings of reduced shock-induced tissue injury in rats
treated with the iNOS-selective inhibitor
L-N6-(1-iminoethyl)lysine
and in iNOS-knockout mice (4). In these animals, inhibiting iNOS after
shock was associated with reduced lung PMN infiltration and with a
downregulation in the inflammatory response, with decreased IL-6 and
granulocyte colony-stimulating factor expression and
decreased nuclear factor-
B and STAT3 activation (4). NOX treatment
likewise leads to a decreased expression of the proinflammatory
cytokines TNF-
and IL-1
after hemorrhage in these experiments.
This finding suggests that a reduction in the proinflammatory response
by NOX may contribute to its beneficial effect after hemorrhage. The
interaction of NO with superoxide to produce peroxynitrite has been
hypothesized to contribute to tissue injury after ischemia and
hemorrhage (26, 29). We detected reduced hepatic nitrotyrosine
immunoreactivity, an index of peroxynitrite formation, in hemorrhaged
rats treated with NOX. This finding suggests that the beneficial
effects of NOX on hepatic injury could also be due, in part, to reduced
peroxynitrite formation. Therefore, an infusion of NOX after hemorrhage
is associated with a number of potentially beneficial effects,
including a reduction in proinflammatory cytokine production,
peroxynitrite formation, tissue neutrophil infiltration, and
shock-induced hepatic and renal injury.
Other authors have hypothesized that NO mediates the vascular
decompensation associated with prolonged hemorrhagic shock and that
inhibiting NO may improve vascular tone and increase blood flow to
vital tissues (30). Although it remains to be proven whether NO is
responsible for vascular decompensation after hemorrhage, by
experimental design the NOX infusion was begun after vascular decompensation had occurred. NOX-treated rats had a greater systemic MAP than saline-treated rats in both experimental protocols. Therefore, if NO contributes to hemorrhage-induced vascular decompensation, it is
possible that NOX infusion may be improving vascular tone or tissue
perfusion. Determining whether NOX can prevent or delay vascular
decompensation after hemorrhage will require further study. NOX is a
dithiocarbamate and readily binds NO with high affinity (20, 31, 33).
Although some dithiocarbamates are poorly soluble in water, NOX is
quite hydrophilic and binds NO readily in vivo (20, 31, 33). NOX
therefore represents a promising compound for studying NO production
and the effect of NO synthesis in in vivo systems.
In conclusion, we have demonstrated that infusion of the NO-scavenging
compound NOX after hemorrhagic shock improves the hemodynamic response
to hemorrhage, reduces tissue injury, proinflammatory cytokine
expression, and peroxynitrite formation, and improves 24-h survival
compared with saline-treated control animals. These data support the
hypothesis that excessive NO contributes to hemorrhage-induced tissue
injury and that reducing excess NO production may be beneficial after
hemorrhage. The finding that trauma patients who have experienced hypotension express iNOS in liver tissue (7) suggests that additional
work on the role of NO in hemorrhagic shock is merited.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of General Medical
Sciences Grants P50-GM-53789 and GM-44100 and by Deutsche Forschungsgemeinshaft Grant HI-614/1-1.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. G. Harbrecht,
A1010 Presbyterian Univ. Hosp., DeSoto at O'Hara Sts., Pittsburgh, PA
15213 (E-mail: harbrechtbg{at}msx.upmc.edu).
Received 31 March 1998; accepted in final form 23 March 1999.
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