Clinical Science (2004) 107, 137–143 (Printed in Great Britain) 137

R E V I E W

Pathophysiology of acute lung injury in

combined burn and smoke inhalation injury

Perenlei ENKHBAATAR and Daniel L. TRABER

Department of Anesthesiology, University of Texas Medical Branch, 610 Texas Ave, Galveston, TX 77555, U.S.A.

A B S T R A C T

In the U.S.A., more than 1 million burn injuries occur every year. Although the survival from
burn injury has increased in recent years with the development of effective fluid resuscitation
management and early surgical excision of burned tissue, the mortality of burn injury is still high.
In these fire victims, progressive pulmonary failure and cardiovascular dysfunction are important
determinants of morbidity and mortality. The morbidity and mortality increases when burn injury
is associated with smoke inhalation. In the present review, we will describe the pathophysiological
aspects of acute lung injury induced by combined burn and smoke inhalation and examine various
therapeutic approaches.

ACUTE LUNG INJURY AND BRONCHIAL
CIRCULATION
ARDS (acute respiratory distress syndrome) is one of
the major complications of thermal injury. There are
several factors that affect the pulmonary function. In
patients with extensive cutaneous burns in which the
burned area exceeds 30 % of the TBSA (total body surface
area), capillary hyperpermeability occurs not only at
the injured site, but also in regions distant from the
injury [1,2]. Vascular hyperpermeability leads to a large
amount of fluid flux from the circulating plasma to the
interstitial spaces. This lung oedema formation is even
more severe when the thermal injury is associated with
smoke inhalation [3]. The investigators in our laboratory
have shown previously that both smoke inhalation alone
[4,5] and combined burn and smoke inhalation injury
[3] causes pulmonary microvascular hyperpermeability
to both fluid and protein. Pulmonary oedema formation
could be affected by changes in both systemic and pul-
monary blood supply. Bronchial blood flow enters into
the pulmonary vasculature through the various bronchio-
pulmonary anastomoses. Airway (trachea) blood flow
increases approx. 20-fold in sheep with combined burn
and smoke inhalation injury [3,6]. Abdi et al. [7] reported
that bronchial blood flow was increased approx. 8-fold
after smoke inhalation injury in sheep. The bronchial
artery occlusion either by ligation or ethanol injection
after smoke inhalation injury improved pulmonary fun-
ction markedly. Reduced bronchial blood flow reversed
the fall in pulmonary gas exchange and an increase in
lung lymph flow and lung water content seen in sheep
with smoke inhalation injury [8]. We have described
previously [3,6] the pathophysiological responses to the
combined burn and smoke inhalation injury in sheep.
Acute lung injury in this model is characterized by an

Key words: acute lung injury, burn and smoke inhalation, cytokine, nitric oxide (NO), pathophysiology; poly(ADP-ribose)
polymerase (PARP).
Abbreviations: ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; COX, cyclo-oxygenase; HPV,
hypoxic vasoconstriction; IL-1, interleukin-1; IL-8, interleukin-8; LPS, lipopolysaccharide; NO, nitric oxide; NOx, nitrite and
nitrate; NOS, NO synthase; eNOS, endothelial NOS; iNOS, inducible NOS; MPO, myeloperoxidase; nNOS, neuronal NOS;
NF-κB, nuclear factor κB; O2 − , superoxide; ONOO− , peroxynitrite; Pao2 /F i o2 , arterial partial pressure of O2 /inspired fraction
of O2 ; PARP, poly(ADP-ribose) polymerase; RNS, reactive nitrogen species; TNF-α, tumour necrosis factor-α; TPA, tissue
plasminogen activator.
Correspondence: Professor Daniel L. Traber (email dltraber@utmb.edu).

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138 P. Enkhbaatar and D. L. Traber
increase in transpulmonary fluid flux (lung lymph flow),
lung water content and a fall in the Pao2 /F i o2 (arterial
partial pressure of O2 /inspired fraction of O2 ) ratio
with increased pulmonary shunt fraction. Tissue injury
is also shown by histological changes. These injured
animals showed approx. 10-fold increase in lung lymph
flow and the Pao2 /F i o2 ratio was below 200 48 h after
the injury. All abnormal changes were associated with
marked airway obstruction with subsequent increase in
ventillatory pressures (peak and pause airway pressures).
Although the exact mechanisms of these patholo-
gical changes are not completely clear, several mechan-
istical aspects have been investigated. One of the possible
candidates is NO (nitric oxide).
ROLE OF NO IN ACUTE LUNG INJURY
It is well known that NO plays an important role in
the pathogenesis of conditions that are frequently com-
plicated by ARDS, such as sepsis and multiple trauma.
We have reported previously [9,10] an up-regulation of
NO in burn and smoke inhalation injury. Plasma NOx
(nitrite and nitrate), a stable metabolite of NO, was in-
creased approx. 2- to 2.5-fold.
NO is formed from arginine by the enzyme NOS (NO
synthase). There are three isoforms of NOS: nNOS (neur-
onal NOS), eNOS (endothelial NOS) and iNOS (in-
ducible NOS). nNOS and eNOS are referred as to cNOS
(constitutive NOS). The other isoform is induced by
cytokines and bacterial products [11,12]. These cytokines
and bacteria are present in multiple traumas, including
the combination of burn and smoke inhalation. With
combined thermal and inhalation injuries, cytokines, such
as IL-1 (interleukin-1), are up-regulated in lung tissue
[13]. In addition, translocation of endotoxin and bacteria
from the intestine into the systemic circulation has been
reported to occur after burn plus smoke inhalation injury
[14,15]. Both IL-1 and endotoxin activate NF-κB (nuclear
factor κB), which induces the synthesis of iNOS. iNOS
catalyses the production of large amounts of NO and,
under conditions of substrate or cofactor limitation, may
also synthesize O2 − (superoxide) [16]. The formation of
NO in the lung results in the loss of HPV (hypoxic
vasoconstriction), the physiological process that diverts
blood flow from alveoli that are not being ventilated
and perfuses the alveoli that are being ventilated [17,18].
When the loss of HPV is present, vasodilation occurs
in the low or non-ventilated areas of the lung, leading
to ventilation/perfusion mismatching, which, in turn,
results in poor oxygenation [19]. At high concentrations,
NO becomes a potential pro-inflammatory and cytotoxic
factor by reacting with O2 − to form the toxic product
ONOO− (peroxynitrite) [20], which can oxidize/nitrate
other molecules or decay and produce even more dama-
ging species, such as the hydroxyl radical [21]. ONOO−


C 2004 The Biochemical Society
Figure 1 Role of NO in acute lung injury
When excessive NO is present, a loss of pulmonary vasoconstriction occurs,
resulting in ventilation/perfusion mismatching. ONOO− , a toxic product of NO,
causes tissue injury resulting in pulmonary dysfunction.
may damage the alveolar capillary membrane [22], result-
ing in additional pulmonary oedema (Figure 1). We
have reported previously [9] the presence of ONOO−
in the airway and parenchyma of the lung of sheep
subjected to burn and smoke inhalation injury. We have
also reported [23] that the plasma concentration of
l-arginine is markedly depleted after burn and smoke
inhalation injury. In conditions where arginine is dep-
leted, NOS produces O2 − , which also can cause tissue
injury. Exogenous arginine treatment was shown [23] to
ameliorate the pulmonary function in sheep subjected
to combined burn and smoke inhalation injury. Recently,
our laboratory [9] has demonstrated the involvement
of iNOS in the pathogenesis of acute lung injury in
sheep with combined burn and smoke inhalation injury.
This evidence was initially obtained with l-NAME
(NG -nitro-l-arginine methyl ester), a non-selective NOS
inhibitor, and subsequently with MEG (mercaptoethyl-
guanidine), a specific iNOS inhibitor. A key role of
iNOS-derived NO in the pathogenesis of acute lung in-
jury in combined burn and smoke inhalation injury model
was described in our recent study [6] using the truly
selective and potent iNOS dimerization inhibitor BBS-2,
which would selectively inhibit de novo synthesized
iNOS. The selectivity of this compound for iNOS is
1500 and 620 times higher than for eNOS and nNOS
respectively [24]. The iNOS inhibitor significantly
improved pulmonary status [i.e. pulmonary gas exchange
and pulmonary vascular permeability (Figure 2), and
histological changes]. Thus the role of iNOS-derived NO
in the pathogenesis of acute lung injury induced by burn
and smoke inhalation is significant.

Figure 2 Effects of iNOS inhibitor (BBS-2) and TPA on the
pulmonary permeability index
Pulmonary permeability index was calculated using a standard equation. Each
group includes six animals. ∗ P < 0.05 compared with control (injured and non-
treated animals); †P < 0.05 compared with BBS-2 (injured animals treated with
BBS-2), §P < 0.05 compared with TPA (injured animals treated with TPA). Data
are means +− S.E.M.
Currently, there is a significant debate over whether
the inhibition of NO is beneficial or harmful and, if the
excessive amount of NO should be suppressed, what
isoform should be targeted. Recently, it was reported [25]
that the non-selective NOS inhibitor 546C88 increased
mortality in patients with septic shock. Non-specific
NOS inhibitors inhibit all three isoforms of NOS,
including eNOS, which adversely affects cardiovascular
function. We have hypothesized that certain NOS iso-
forms could be expressed at certain time points depending
on the type of injury. More targeted and selective
inhibition of NOS isoforms at their maximal activity
could be beneficial in the management of multiple organ
failure in this model. Our recent studies suggest that
iNOS may dominate the pathophysiology of the acute
lung injury in a combined burn and smoke inhalation
model. There are studies that have indicated that nNOS
may be induced in pathological conditions [26]. We have
also reported [27] a possible role of nNOS-derived NO
in the pathogenesis of acute lung injury in sheep with
sepsis. A specific nNOS inhibitor, 7-nitroindazole, was
shown to reverse the pathological changes in sheep with
smoke inhalation and pneumonia [27]. If cNOS-derived
NO is harmful, it may present a therapeutic target similar
to iNOS. To test this hypothesis, further studies should
investigate the effects of a truly selective nNOS inhibitor
on a model of combined burn and smoke inhalation
injury. Recently, we have reported [27a] that a non-
steroidal anti-inflammatory agent (ketorolac) improved
pulmonary function in sheep subjected to burn and
smoke inhalation by inhibiting an excessive NO. There
are a number of studies showing that NOS and
Acute lung injury in combined burn and smoke inhalation injury 139
Figure 3 Role of PARP in acute lung injury
ONOO− -induced activation of PARP causes cell energy depletion, neutrophil
activation and excessive NO production.
COX (cyclo-oxygenase) are co-localized in a variety
of inflammation disorders [27b]. The interplay between
excessive NO and COX should be investigated in the
future studies.
ROLE OF PARP [POLY (ADP-RIBOSE)-
POLYMERASE] IN ACUTE LUNG INJURY
It has been proposed that RNS (reactive nitrogen species)-
mediated injury is related to DNA damage and the
consequential activation of the nuclear enzyme PARP
[28]. PARP is a chromatin-bound enzyme constitutively
expressed in most cell types [29] and is involved in DNA
repair [30]. The overactivation of PARP in response to
oxidant-mediated DNA single-strand breaks leads to a
fall in ATP and NAD + , resulting in cellular dysfunction
and ultimately to necrotic cell death.
PARP has been shown recently [31] to be involved
in the regulation of inflammatory processes, being func-
tionally associated with important transcription factors,
most notably NF-κB. Liaudet et al. [32] reported that
the activation of PARP-1 is a central mechanism of LPS
(lipopolysaccharide)-induced acute lung inflammation
and that PARP-1 suppression resulted in the reduction
of proinflammatory cytokines, such as TNF-α (tumour
necrosis factor-α), and chemokines, such as MIP-1
(macrophage inflammatory protein-1). The authors [32]
also reported that PARP-1 inhibition resulted in sup-
pressed MPO (myeloperoxidase) activity in lung tissue
of mice. Pulido et al. [33] have shown that inhibition of
PARP prevented the decrease in lung ATP levels and
attenuated pulmonary dysfunction induced by LPS. It
was shown that ONOO− -dependent activation of PARP
caused energy depletion and increased permeability in
human pulmonary epithelial cells in vitro (Figure 3).
PARP inhibition has also be shown [34,35] to result in


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140 P. Enkhbaatar and D. L. Traber
the reduction of oedema formation in septic and non-
septic models of lung inflammation. We have shown
[34] that PARP inhibition results in a significant im-
provement in pulmonary function, as evidenced by
improved oxygenation, reduced pulmonary vascular
permeability and reduced lung water content in sheep
with combined burn and smoke inhalation injury.
Post-treatment with a PARP inhibitor was also shown
[35] to improve cardiopulmonary dysfunction in sheep
with smoke inhalation and pneumonia-induced sepsis.
The pathological changes seen in injured (burn/smoke)
non-treated animals were associated with a marked in-
crease in PARP activation in lung tissue. PARP inhibition
in these animals resulted in a marked decrease in plasma
NOx levels. Liaudet et al. [32] reported that LPS-in-
duced increase in BALF (bronchoalveolar lavage fluid)
NOx was significantly reduced by PARP inhibition. The
authors also showed that PARP-deficient mice have
significantly less NOx compared with wild-type mice
[32].
Activation of NF-κB causes an up-regulation of iNOS,
thus accelerating the formation of NO and RNS. As
mentioned above, RNS are potent stimulants of the
PARP activation because of their high potential to damage
DNA. Thus activation of iNOS and PARP may be linked.
To test this hypothesis, further studies are needed to
determine PARP activation in sheep subjected to burn
and smoke inhalation injury and treated with iNOS
inhibitor.
It is particularly interesting that the accumulation of
neutrophils in the lung tissue in sheep with burn and
smoke inhalation injury, evaluated by measuring MPO
activity, was attenuated by a PARP inhibitor [34]. Histo-
logical examination revealed extravasated neutrophils in
lung tissue of these sheep. A large amount of neutrophils
were found in the lung lymph and airways (in the form
of an obstructive cast). It is well known that oxygen radi-
cals and elastase released from activated neutrophils, if
sufficiently extensive, can cause tissue injury [36,37].
Thus the inhibition of excessive PARP activation could
improve pulmonary function through (i) the inhibition
of excessive NO, and (ii) the activation of neutrophils by
interacting with transcriptional factors, such as NF-κB
(Figure 3).
ROLE OF AIRWAY OBSTRUCTION IN ACUTE
LUNG INJURY
One of the major causes of progressively worsening pul-
monary gas exchange is airway obstruction. In a previous
study, we have shown [38] that there was significant
airway obstruction with a mean reduction in cross-
sectional area of approx. 29 % in bronchi, 11 % in bron-
chioles and 1.2 % in respiratory bronchioles in sheep


C 2004 The Biochemical Society
48 h after being subjected to combined burn and smoke
inhalation injury. Obstructive cast material occludes the
lumen of the airway, resulting in hypoventilation or
focal loss of ventilation. The blood vessels in the under-
ventilated areas fail to constrict normally, causing a
perfusion/ventilation mismatch. This transfer of blood
from a ventilated area to a non-ventilated part results in
poor oxygenation of arterial blood, which leads to hy-
poxaemic changes in organs. In addition, obstruction of
part of the bronchial tree results in the hyperventilation
of the non-occluded parts, thus increasing airway pres-
sure when volume-controlled mechanical ventilation is
given [39]. The over-stretching of the alveoli by high
pressure results in mechanical trauma or volutrauma of
the non-obstructed alveoli, thus worsening oxygenation
[40]. This over-stretching of the alveoli also induces
synthesis and secretion of pro-inflammatory chemokines,
such as IL-8 (interleukin-8) [41], which is a major chemo-
tactic factor for neutrophils. IL-8 has been shown [42]
to mediate smoke inhalation-induced lung injury. It
was also reported that inflammatory mediators, such as
TNFα and IL-1, are induced by mechanical ventilation
[43]. Previously, we have shown [38] that airway ob-
structing material is composed of fibrin, neutrophils,
mucus and epithelial cell debris, leading us to hypothesize
that pathogenic therapy targeting those factors could
represent a more effective form of airway management. In
our laboratory, we tested the hypothesis that a reduction
of a fibrin clot in the airway could reduce the degree of
airway obstruction. For our experiments, we studied the
effects of two different types of anticoagulants: those
that prevented fibrin formation and those that lysed
already formed fibrin clots. We reported that the pre-
vention of fibrin clot formations in the airways using
anticoagulants (delivered via nebulization), such as hep-
arin [44] and antithrombin [45], was beneficial in treating
sheep subjected to smoke inhalation and pneumonia.
Interestingly, these anticoagulants failed to ameliorate
the lung injury in sheep with combined burn and smoke
inhalation injury. Although the exact mechanism of this
discrepancy is not completely understood, Murakami
et al. [46] reported that antithrombin concentration in
BALF in sheep with smoke inhalation and pneumonia
was much higher than in sheep with burn and smoke
inhalation injury. Their findings may explain why heparin
is effective in one case and not in the other. The adminis-
tration of antithrombin improved pulmonary function
in sheep with burn and smoke inhalation injury only
when combined with heparin. It is a well-known fact that
antithrombin exerts a much more potent anticoagulant
effect as an antithrombin–heparin complex. The exact
mechanism by which antithrombin nebulization alone
ameliorates acute lung injury in a smoke inhalation and
pneumonia model, but not in burn and smoke inhalation
model, remains unclear. At the present time, we can
only speculate that the anti-inflammatory property of

Figure 4 Airway obstruction-related pulmonary dysfunc-
tion
Occluded part of the alveola is hypo- or non-ventilated, resulting in pulmonary
shunt fraction, which leads to ventilation/perfusion mismatch. Open alveolae are
overstretched when mechanical ventilation is present, leading to barotraumas.
Overstretching of the alveolar wall induces cytokine release, such as IL-8, a
leucocyte chemoattractant.
the antithrombin may be dominated in sheep with
sepsis induced by smoke inhalation and pneumonia. The
anticoagulants we used were nebulized starting 2 h after
the injury; however, fire victims are often admitted to
specialized burn units well after 2 h after sustaining
their injuries. In those patients, it is possible that fibrin
could already have begun to form clots in the airway
in which case the use of fibrinolytic agents is the only
choice available to physicians. Considering this fact, we
tested the effect of TPA (tissue plasminogen activator,
which converts plasminogen into plasmin, leading to
break of both fibrinogen and fibrin) on burn and smoke
inhalation-induced pathologies in sheep. Nebulization of
TPA starting at 4 h after the injury markedly reduced
the degree of the airway obstruction, resulting in im-
proved oxygenation and microvascular permeability
(Figure 1). Following pulmonary epithelial injury, plasma
exudes into the distal airway. Extravascular plasma ra-
pidly clots because of the procoagulant properties of
the pulmonary epithelium and alveolar macrophages.
Fibrinolytic activity of the lavage fluid in patients with
ARDS is reduced in contrast with increased PAI-1
(plasminogen activator inhibitor-I). In the alveolar space,
fibrin or fibrinogen can impair surfactant function [47],
thereby contributing to atelectasis. Both fibrin and fibri-
nogen provide adhesion sites for inflammatory cells that
are recruited to the site of tissue damage [48]. Thus
direct delivery of anticoagulants including fibrinolytic
agents into the airways might be an effective treatment
strategy.
As mentioned previously, airway blood flow increases
dramatically in sheep with combined burn and smoke
inhalation injury. We have reported that iNOS inhi-
Acute lung injury in combined burn and smoke inhalation injury 141
bitors or PARP inhibitor significantly reduced the increa-
sed airway blood flow. The decrease in airway blood
flow in these treated animals was associated with signi-
ficantly lower airway obstruction scores. Consequently,
ventilatory pressures were also lower in these sheep. Thus
it is possible that effective airway management based on
this treatment strategy could reduce the degree of acute
lung injury in cases of thermal injury associated with
smoke inhalation (Figure 4).
CONCLUSION
In this review, we have attempted to summarize some
of the pathophysiological aspects of the acute lung
injury induced by thermal injury associated with smoke
inhalation. Although the exact mechanism of acute lung
injury in patients with thermal injury remains unclear, we
have described the important roles played by excessive
NO, PARP activation and airway obstruction in this
process.
We believe that future studies should seek to investigate
in detail the interplay between PARP activation and
iNOS. Because the link between iNOS and COX has
been well described in previous studies, the effect of
specific COX inhibitors should be tested. We also suggest
that the inhibition of excessive NO, especially iNOS-
derived NO, and PARP activation with effective airway
management using various aerosolized anticoagulants
might be an option of the effective strategy for treating
patients with combined burn and smoke inhalation
injury.
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Received 5 May 2004/14 May 2004; accepted 19 May 2004

Published as Immediate Publication 19 May 2004, DOI 10.1042/CS20040135

C 2004 The Biochemical Society