Phẫu Thuật Bỏng: Tiếp Cận Và Phương Pháp Điều Trị Từ Trung Quốc (Chinese Burn Surgery)

89
Z. Yang, Chinese Burn Surgery, DOI 10.1007/978-94-017-8575-4_5,
© Springer Science+Business Media Dordrecht and People’s Medical Publishing House 2015
The pathological mechanisms underlying the development
of multiple organ dysfunction syndrome (MODS) in
critically ill patients remain poorly understood. Nevertheless,
the most widely held view is that uncontrolled systemic
activation of the infl ammatory response somehow leads to
cellular dysfunction and/or destruction at locations distant
from the initiating site(s) of injury or infection. Complications
that occur during sepsis and MODS are ultimately the
consequence of an overreaction of host defense mechanisms
to bacterial infection. The reaction is the so-called general-
ized infl ammatory response, which is an essential reaction of
the host to both burns/trauma and septic episode [ 1 ]. When
overactivated, the systemic infl ammatory response can be
against the host in a self-destructive manner. While the initial
infl ammatory response can be initiated both by extensive
burns and sepsis, the perpetuation of the response is usually
associated with bacterial component. One of the central prin-
ciples by which bacteria induce the infl ammatory response
and subsequent organ dysfunction is via bacterial toxins, of
which endotoxin or lipopolysaccharide (LPS) from gram-
negative bacteria is the most prominent. Endotoxin, a cell
wall component of gram-negative bacteria, is known to acti-
vate the cytokine pathways that lead to the release of proin-
fl ammatory mediators, which results in a wide range of
pathophysiological effects. Recent advances in molecular
biology and mostly immunology have greatly contributed to
our understanding of the pathophysiology of sepsis not only
by endotoxin but also by other microbial products, including
exotoxin mainly from gram-positive bacteria [ 2 ]. Infections
begin when bacteria penetrate host barriers such as the skin
and mucosa, sometimes overwhelming host defenses and
releasing toxic bacterial products that activate plasma factors
(complement and clotting molecules) as well as cells of the
immune system. The result is that the host’s infl ammatory
response contributes substantially to the development of
sepsis and MODS secondary to severe burns/trauma.
5.1
Endotoxemia in Burns
5.1.1
Endotoxemia and Bacterial
Translocation After Burn Injury
Today, the gut is not only regarded as an organ responsible
for nutrient absorption but also as an important metabolic
and immunological system, functioning as an effective bar-
rier against the residing luminal microfl ora and associated
toxic by-products. In the past decades, there has been increas-
ing recognition of the impact that severe burns and critical
illness have upon the gastrointestinal tract and, in turn, the
infl uence that this complex organ can exert on metabolic and
infl ammatory responses. Although the potential role of bac-
terial/endotoxin translocation remains controversial, many
lines of evidence support the notion that the gut may be the
reservoir for systemic sepsis and subsequent MODS in a
number of pathophysiological states [ 1 , 3 , 4 ].
In general, impaired gut barrier function can be caused
either during the shock period by decreased intestinal blood
fl ow and reduced oxygen delivery, which results during
reperfusion in a stage of increased intestinal blood fl ow, or at
a later stage again by reduced fl ow. A variety of physiologi-
cal stresses such as hemorrhagic shock, trauma, and thermal
injury have been shown to cause failure of the gut mucosal
barrier, with translocation of bacteria/endotoxin from the
gastrointestinal tract into the mesenteric lymph nodes, and
under conditions of suffi cient ongoing stress, translocation
into remote organs and the systemic circulation [ 3 , 5 , 6 ].
This invasion of bacteria/endotoxin into extraintestinal sites
has led to the description of the gut as the motor of MODS,
the so-called gut hypothesis. Support for the gut-origin
hypothesis comes from several lines of investigation. Fine
et al. initially proposed that in hemorrhagic shock the gut
becomes the source of endotoxin and invasive gram-negative
bacteria and in the presence of a defective reticuloendothelial
Bacterial Endotoxin and Exotoxin
in Severe Burns
Yongming Yao
5

90
system leads to systemic infection. In a series of studies,
Deitch and his coworkers later demonstrated that enteric
bacteria can escape from the gut and induce both nonlethal
and lethal systemic infections [ 7 ]. It has been shown in a rat
model of hemorrhagic shock that the duration of hypotension
is related to mucosal injury, the magnitude of bacterial trans-
location, and the mortality rate. This phenomenon has been
noted after hemorrhage/burns in different species, including
rats, rabbits, dogs, and baboons.
Endotoxemia is relatively common following shock,
burns, and mechanical trauma and is associated with various
conditions leading to sepsis as well as MODS. In addition,
endotoxin has been shown to impair host defenses, increase
gut mucosal permeability, and enhance the process of bacte-
rial translocation. Thus, endotoxemia, in turn, aggravates the
disturbance of gut barrier function, thereby generating a
recurrent circle of gut-derived infection. Studies in rats sub-
jected to hemorrhagic shock indicated that endotoxemia was
present in 33 % of animals after only 30 min of shock and in
88 % of animals after 2 h of shock [ 8 ]. Experiments also
documented the signifi cance of the early appearance of
endotoxin in the systemic blood following hemorrhage and/
or trauma in both rodents and primates. The rat studies
revealed a marked, albeit transient, increase in plasma endo-
toxin concentrations, both in the portal and systemic circula-
tions following hemorrhagic shock, suggesting that the
endotoxin translocated from the gut at the early stage might
be transported, at least in part, to the liver via the portal route.
In rabbits, we found abnormally high endotoxin levels in the
systemic blood samples as a result of hypotensive insult, but
the change was much more pronounced in animals with
MODS when compared with those without MODS at the end
of shock and thereafter [ 9 ]. Likewise, elevated plasma endo-
toxin levels were found in hemorrhagic/traumatic shock
baboons at the end of a 3 h shock period and 1 h after reinfu-
sion. This fi nding was further confi rmed in a subchronic
model in baboons consisting of oxygen debt-controlled hem-
orrhage together with an infusion of zymosan-activated
plasma.
The results of our serial experiments indicate that severe
thermal injury could not only cause a signifi cant elevation of
endotoxin levels in both the portal and systemic circulations
but also lead to translocation of indigenous bacteria from the
gut to the mesenteric lymph nodes (MLNs) as well as various
organs. Meanwhile, there was a marked overgrowth of the
gram-negative enteric bacilli population in mucosal fl ora,
which paralleled the increased fecal endotoxin contents in
the intestine. Moreover, mucosal diamine oxidase (DAO)
activity, which served as a useful marker of intestinal dam-
age, was signifi cantly decreased following burn injury [ 10 ].
In another study, the tissue endotoxin levels were measured
by the chromogenic limulus amoebocyte lysate (LAL) assay
modifi ed by perchloric acid (PCA) treatment of samples to
remove nonspecifi c activators or inhibitors of the lysate. Our
data revealed that endotoxin levels in the liver, spleen, and
lungs reached a peak at 12 h after thermal injury. Endotoxin
levels in the liver and spleen then declined rapidly to baseline
thereafter, whereas the endotoxin in the lungs maintained a
high level till 24 h. There was no signifi cant difference in
endotoxin levels in the kidneys at various time point post-
burn [ 11 ] (Table 5.1 ). In the recombinant bactericidal/
permeability- increasing protein (rBPI 21 ), a well-described
bactericidal and endotoxin-neutralizing protein-treated
group, signifi cant reduction in serum amino leucine transfer-
ase levels and elevation in intestinal DAO activities were
found compared to those in the control group. These data
confi rm and extend previous studies suggesting that gut-
derived endotoxemia and/or bacterial translocation may
occur early as a result of acute insults to the gut barrier,
which in turn contributes to post-injury sepsis and MODS [ 1 ,
12 ]. Although the precise mechanism underlying endotoxin/
bacterial translocation due to thermal injury remains to be
clarifi ed, our data provided evidence that both gram-negative
bacterial overgrowth in mucosal fl ora and endotoxin pool
expansion within the gut might be important contributory
factors in the development of postburn sepsis. Therefore,
therapeutic measures aimed at controlling such translocation
by use of selective decontamination of the digestive tract
(SDD), which selectively reduced both potential pathogens
and fecal endotoxin contents, may be helpful in the preven-
tion of many burn injury-associated abnormalities and
mortality.
The above mentioned concept is also supported by our
observations in extensively burn patients, among whom we
demonstrated that endotoxemia occurred early and com-
monly, and circulating endotoxin was strongly associated
Table 5.1 Endotoxin levels in the liver, spleen, kidneys, and lungs in rats after thermal injury (EU/g, x
SE
±
)
Postburn hours
Organs
Control group
12
24
48
72
Liver
1.39 ± 0.31
17.34 ± 3.69*
1.60 ± 0.12
2.81 ± 0.31*
3.46 ± 1.72
Spleen
1.32 ± 0.08
10.85 ± 4.62*
1.85 ± 0.24
2.62 ± 0.32*
1.83 ± 0.27
Lungs
4.51 ± 1.14
9.65 ± 2.40
7.12 ± 3.97
6.32 ± 1.59
6.94 ± 1.72
Kidneys
1.52 ± 0.18
1.55 ± 0.16
2.04 ± 0.44
2.17 ± 0.41
0.99 ± 0.26
Compared to the controls:* p < 0.01
5 Bacterial Endotoxin and Exotoxin in Severe Burns

91
with development of MODS and mortality after major burns
[ 13 ]. In the clinical study, the normal plasma endotoxin con-
centrations of 22 healthy volunteers were 74 ± 23 endotoxin
units (EU)/L, which were signifi cantly different from the
values in burn patients within the fi rst day after injury
( p < 0.05). Endotoxin concentrations could be identifi ed in
two peaks: the fi rst was observed on the third day after injury
and the second at 2–3 weeks postburn. Of the samples from
35 patients with burns over 30 %, endotoxemia was detect-
able in 58.0 % (109/188 blood samples). The mean value of
plasma
endotoxin
in
septic
patients
( n = 15)
was
541 ± 233 EU/L (range, 94–1712 EU/L), whereas it was
279 ± 188 EU/L (range, 75 ± 672 EU/L) in those without sep-
sis ( n = 20, p < 0.01). Moreover, a correlation between the
development of MODS and circulating endotoxin concentra-
tion was noted. As the mean endotoxin values exceeded
500 EU/L, the incidence of MODS in burn patients was 75 %
(6/8 cases) (Table 5.2 ). When a comparison was made
between the survivors and those with a fatal outcome follow-
ing polytrauma and major operation, it was found that the
mean plasma endotoxin concentrations of nine non-survivors
were signifi cantly higher than that of 16 survivors on day 1
and 7 post injury [ 12 ].
5.1.2
Effect of Endotoxemia on Cytokine
Formation Following Burns
It has repeatedly been documented that translocation of
endotoxin and bacteria from the bowel very often ensues
after severe trauma, burns, shock, and major operation. This
endotoxemia that occurs as a result of disruption of intestinal
barrier function might elicit disorderly immunity response
and damage to cells both locally and systemically.
Proinfl ammatory cytokine including tumor necrosis factor
(TNF)-α, which is released by macrophage/monocyte sys-
tem, is supposed to be one of the pivotal damage responses
to tissues [ 14 ]. Our animal experimental study demonstrated
that after a major hemorrhagic shock or burns, there is a
rapid rise in endotoxin in portal/systemic circulation, and the
trend of changes in endotoxin levels in the portal blood was
similar to that of TNF-α in peripheral blood, though the peak
value of the former appeared obviously earlier [ 15 ]. Further,
it was proved that change in expression of TNF-α mRNA in
the liver after hemorrhagic shock was similar to that of
plasma TNF-α level. The result also showed that half an hour
after an insult of infection or endotoxemia, there was expres-
sion and release of TNF-α, which peaked at 1.5 h.
Coincidentally, clinical observations revealed that within
24 h there was an elevation of endotoxin content in the
plasma of patients with either major burns or serious trauma,
and its magnitude was positively correlated with plasma
TNF-α level, especially in those who were complicated by
sepsis. The results of animal experimental and clinical obser-
vations suggested that there was a close relationship between
an early enterogenous endotoxemia and the induction of
cytokines, such as TNF-α, in the body after thermal injury
and shock. In order to elucidate the relationship between
enterogenous endotoxemia and release of TNF-α, polymyxin
B or monoclonal antibody against core LPS was used to
either neutralize or abolish endotoxin. The results showed
that the treatment prevented the occurrence of the early
endotoxemia, while blood TNF-α level fell, and the peak
expression of TNF-α mRNA was only 47.8 % of the con-
trols, proving further the crucial role of enterogenous endo-
toxemia in eliciting the production of TNF-α [ 12 , 16 ].
Therefore, it was suggested that enterogenous endotoxin
could be one of the main causative factors of systemic tissue
damage in the development of sepsis and possibly MODS.
LPS invasion is able to produce rapid activation of the
innate immune system, leading to an excessive release of not
only early proinfl ammatory cytokines, such as TNF-α and
interleukin (IL)-1, but also late infl ammatory cytokines,
especially high-mobility group box 1 protein (HMGB1).
Recent evidence reveals that HMGB1 plays a role as a late
mediator of delayed endotoxin morbidity and lethality [ 17 –
19 ]. Studies using in vitro systems demonstrated that LPS or
mediators induced by LPS could induce the release of
HMGB1 [ 20 ]. Since our previous studies have shown that
burn injury per se could result in translocation of gut-derived
endotoxin, which was accumulated in various tissues, we
examined the infl uence of endotoxin translocated from the
gut on tissue HMGB1 mRNA expression after thermal
injury. It was found in the present study that when endotoxin
levels in organs were attenuated by rBPI 21 treatment, tissue
HMGB1 mRNA expression also signifi cantly decreased,
suggesting that local endotoxin may play an important role
in upregulating HMGB1 expression after acute insults [ 21 ].
Table 5.2 The relationship between circulating endotoxin levels and the incidence of MODS and clinical outcome in burn patients

Patients
Cases
Mean LPS levels (EU/ml)
(No.)
MODS (No.)
Non-MODS (No.)
Death (No.)
<0.20
18
0
18
0
0.20~
9
1
8
1
0.50~
6
4
2
3
1.00~
2
2
0
2
5.1 Endotoxemia in Burns

92
These observations are in agreement with a report showing
that serum HMGB1 concentrations were signifi cantly
increased after systemic LPS exposure. In addition to LPS,
in vitro studies with cultured pituicytes and macrophages
have shown that TNF-α and IL-1β could also lead to the
release of HMGB1 in a time- and dose-dependent manner.
Moreover, this release was synergistically enhanced by inter-
feron gamma (IFN-γ). Since these early occurring acute
cytokines might function as upstream regulators of HMGB1
release, the upregulation of HMGB1 after thermal injury
might be due to the direct and/or indirect modulation of
endotoxin. In a recent clinical study, no signifi cant correla-
tions were found between HMGB1 levels and the sizes of
total body surface area, while the plasma HMGB1 levels
were positively correlated with endotoxin concentrations on
days 3, 5, 7, and 21 after major burns [ 22 ]. In addition, there
were high positive correlations between hepatic HMGB1
mRNA and serum amino leucine transferase and aspartate
aminotransferase levels and also between pulmonary
HMGB1 mRNA and myeloperoxidase (MPO) activities.
Taken together, these fi ndings indicate that thermal injury
per se can markedly enhance HMGB1 gene expression in
various organs. Upregulation of HMGB1 expression may be
involved in the pathogenesis of endogenous endotoxin-
mediated multiple organ damage secondary to major burns.
If invading enteric endotoxin is indeed the reason for the
development of the systemic infl ammation response syn-
drome, then therapy directed at preservation of barrier func-
tion and/or elimination of potential endotoxin translocation
would be expected to decrease the incidence of organ dys-
function and subsequent mortality. There are several mea-
sures that control or eliminate translocated bacteria/endotoxin,
which have been used in experimental studies to evaluate the
potential role of translocation events in the pathogenesis of
sepsis and MODS associated with acute insults. In a pro-
longed hemorrhagic shock model (mean arterial pressure at
30–35 mmHg for 3 h) in rats, animals were treated with
rBPI 21 . Treatment with rBPI 21 neutralized systemic endotox-
emia, attenuated the damage in vital organs, and reduced 48 h
mortality in rats after severe hemorrhage [ 5 ]. With the same
model, administration of an anti-core endotoxin monoclonal
antibody (WN1 222-5) also provided noticeable protection of
organ damage and signifi cantly improved the survival rate
from 21 to 71 % [ 23 ]. This observation is in accordance with
reports in which Re-lipopolysaccharide antiserum, poly-
myxin B, and SDD were found to reduce the incidence of
translocation and improve the survival rate in animals suffer-
ing from severe injury. Boermeester et al. [ 24 ] reported that in
partially hepatectomized rats, a systemic infl ammatory
response and its clinical hemodynamic/biochemical sequelae
triggered by endogenous endotoxemia were blunted by the
administration of rBPI 23 . Taken together, these results suggest
that endotoxin translocation may play an important role in the
development of multiple organ dysfunction resulting from
trauma/hemorrhage. Gut-derived endotoxemia has been
incriminated as a prime factor for the release of cellular medi-
ators that may in turn activate or further perpetuate systemic
infl ammatory response to injury [ 1 , 2 ]. In view of the above
fi ndings, early and effective abolishment of endotoxin trans-
location would be benefi cial in inhibiting an excessive induc-
tion of cytokine production, such as TNF-α and HMGB1,
preventing thus at least partially the deleterious development
of uncontrollable systemic infl ammatory response.
In a rat hemorrhage model, signifi cant increases in plasma
TNF-α level and IL-1 activity of peritoneal macrophages
were shown in the controls after resuscitation, but markedly
prevented by the administration of polymyxin B before
induction of shock. In another experiment, we found that the
signifi cant rise in TNF-α level in the portal vein occurred
prior to the elevated levels in the systemic blood after hemor-
rhage, indicating gut-derived cytokine formation, for which
the gut-associated macrophages in the lamina propria of the
intestine can be considered a potential source [ 25 ]. New evi-
dence shows that the gut generates infl ammatory cytokines,
including TNF-α and IL-6, in response to shock and an
infl ammatory stimulus and that proinfl ammatory gene
expression occurs in Peyer’s patches both during and after
hemorrhage/resuscitation. More recently, an experimental
study has shown that an increase in whole blood monocyte
TNF-α response after hemorrhagic shock is mediated via the
endotoxin/CD14 pathway. Collectively, it has been postu-
lated that gut-associated macrophages constitute the major
source for cytokine formation at the early stage of shock and
that the Kupffer cells stimulated by endotoxin transported to
the liver via portal blood serve as the important cellular
source for the circulating cytokine at a later stage. In contrast
to these data, failure to detect enteric endotoxin translocation
has been reported by other authors. Studies by Rhee et al.
[ 26 ] had shown that hemorrhage in mice and rats seemed to
induce an early cytokine release, but without any detectable
amount of endotoxin in blood. The lack of detectable endo-
toxin in those experiments could probably have been the
result of several factors: (a) systemic samples rather than
portal samples used for assay, (b) endotoxin level below the
detection limit of the method, and (c) rapid clearance from
the systemic circulation by the reticuloendothelial system.
Nonetheless, in the setting of shock and burns, hypoxia may
also contribute to the induction of cytokines.
5.1.3
The Role of Lipopolysaccharide-
Binding Protein and CD14 Pathway
in Promoting Endotoxin
Responsiveness Associated with Burns
A growing body of circumstantial evidence has been accu-
mulated that after a variety of insults (such as severe trauma,
burns, and shock), endotoxin can traverse the gut wall to
5 Bacterial Endotoxin and Exotoxin in Severe Burns

93
enter into the systemic circulation. Furthermore, both in vitro
and in vivo studies indicate clearly that endotoxin triggers
infl ammatory cells to generate and release numerous endog-
enous mediators, such as cytokines and reactive oxygen spe-
cies. This cascade of events may eventually lead to
life-threatening
complications
including
sepsis
and
MODS. However, it has been found during in vitro study that
the concentration of LPS required to activate cells is greater
than 0.1 μg/ml. On the other hand, it is surprising to fi nd that
the usual plasma endotoxin level in septic patients is approx-
imately 100 times less than this amount. Therefore, it has
been postulated that there might be a mechanism through
which the infl ammatory cells are sensitized so that only a
low level of LPS is required to provoke activation.
A number of investigators have proved that host sensitiv-
ity to endotoxin was increased markedly after ischemia/
reperfusion (I/R), hemorrhagic shock, and thermal injury.
Our data indicated that LPS injection after I/R resulted in
marked hemodynamic dysfunction and multiple organ dam-
age, while no signifi cant or only minor changes in organ
function parameters were observed in both I/R and LPS
groups. In whole blood, monoclonal antibody to CD14 sig-
nifi cantly blocked the release of TNF-α at low LPS concen-
tration (≤10 ng/ml), and this response was dose dependent.
Moreover, TNF-α release in LPS-stimulated whole blood
obtained after I/R was inhibited to a signifi cantly greater
degree than that in baseline blood samples or sham-operated
group [ 27 ]. These fi ndings suggest that splanchnic artery
occlusion followed by reperfusion could lead to increased
sensitivity to the subsequent LPS challenge, which may be
associated with a shift toward CD14-dependent mechanisms.
Similarly, an increased dependence on CD14-mediated
mechanisms of activation was observed after hemorrhagic
shock. Likewise, alveolar macrophages on day 3 were 11.1
times greater than LPS-stimulated alveolar macrophages in
the sham group [ 28 ]. A similar priming effect has been
reported in tissue macrophages of other organs; LPS-
stimulated adherent splenocytes have increased TNF-α on
postburn days 8 and 10, and bone marrow macrophages have
a similar response on postburn day 1. Taken together, the
above reports indicate clearly that severe burns/hemorrhage
could lead to increased sensitivity of host cells to subsequent
LPS challenge.
During the past decade, great progress has been made in
identifying LPS recognition and signaling molecules. The
discovery of lipopolysaccharide-binding protein (LBP) and
CD14 led to the discovery of unanticipated mechanisms of
LPS-induced cell activation [ 29 ]. Binding LPS with high
affi nity, LBP and CD14 lower dramatically the threshold
stimulatory concentrations of LPS by >1,000-fold and
enhance markedly the rate of cytokine production.
Meanwhile, antibody-mediated blockade of LBP or CD14
prevents the in vitro LPS-induced cytokine release by cells
harboring CD14 on their surface. Recently, several Toll-like
receptors (TLRs) have been identifi ed, and studies have
demonstrated that TLRs act as transmembrane coreceptors
for CD14 in the cellular response to LPS. Engagement of
LPS by CD14 is still widely believed to be the fi rst step in the
LPS signaling pathway [ 30 ]. Moreover, clinical investiga-
tions revealed that plasma levels of LBP and CD14 were
increased signifi cantly in septic, polytraumatized, and
severely burn patients and were associated with clinical out-
come [ 31 ]. However, the role of LBP and CD14 in promoting
LPS responsiveness associated with extensive burns has not
been studied in vivo. We hypothesized that LBP/CD14 would
present a pair of key molecule in pathophysiological altera-
tions induced by low concentrations of endotoxin (pg/ml-ng/
ml) after burns.
Our fi ndings demonstrated that LBP mRNA was detected
in various tissues following thermal injury. LBP was fi rst
thought to be produced only by the liver, but reverse tran-
scription polymerase chain reaction (RT-PCR) results
revealed LBP production by extrahepatic tissues, such as
pulmonary tissue. Furthermore, LBP mRNA expression in
the liver and kidneys increased markedly after burns. Martin
et al. found elevated levels of LBP in BAL fl uids from acute
respiratory distress syndrome (ARDS) patients and sug-
gested that they might be local in origin. We verifi ed these
previous fi ndings by showing that extrahepatic LBP expres-
sion was increased postburn. Likewise, detectable levels of
CD14 mRNA were seen in all four organs (liver, intestine,
kidneys, and lungs) under normal conditions, although rela-
tively lower in the kidneys and intestine. After thermal injury
CD14 mRNA expression increased rapidly, particularly in
the pulmonary tissue [ 32 ]. Therefore, increased amounts of
LBP and CD14 produced in various organs as a result of sys-
temic insult might possibly participate directly in the early
phases of immune cell activity to LPS.
Previous studies, both in vitro and in vivo, have shown
that LPS or mediators induced by LPS can modulate CD14
expression. Herein, we examined the infl uence of endotoxin
translocated from the gut on tissue LBP/CD14 mRNA
expression after thermal injury. It was found in this study
that when endotoxin levels in organs were attenuated by
rBPI 21 treatment, tissue LBP and CD14 mRNA expression
also signifi cantly decreased, suggesting that local endotoxin
may play an important role in upregulating LBP/CD14
expression after acute insults [ 6 ]. These observations are in
agreement with a report showing that pulmonary LBP and
CD14 mRNA were upregulated after either systemic or local
LPS exposure. The resultant high levels of LBP and CD14
produced locally may in turn contribute to over- amplifi cation
of immune response in the presence of minute amounts of
LPS, thereby putting host at much greater risk of sustaining
prolonged infl ammatory responses.
In order to demonstrate that upregulation of CD14 expres-
sion might enhance the responsiveness of monocytes to LPS,
the relationship between tissue LBP/CD14 mRNA expression
5.1 Endotoxemia in Burns

94
and TNF-α mRNA expression was studied. Our data showed
that while LBP and CD14 mRNA expression increased
markedly after acute injury, TNF-α mRNA levels were also
signifi cantly upregulated in vital organs. Furthermore, when
treatment with rBPI 21 signifi cantly decreased tissue LBP and
CD14 mRNA expression in various organs, TNF-α mRNA
expression in the liver, kidneys, and lungs was also markedly
inhibited [ 6 , 32 ]. The above observations suggest that upreg-
ulation of LBP and CD14 mRNA expression may potentiate
synthesis and release of TNF-α by monocytes stimulated by
endotoxin.
In a clinical study, we reported that sCD14 was found in
serum in physiological and pathophysiological conditions.
22 patients with burns covering more than 70 % of body sur-
face area were included in this study. Serum sCD14 levels of
the MODS group were signifi cantly higher than those of
non-MODS group on days 7, 14, and 21 postburn ( p < 0.05–
0.01) (Table 5.3 ). The mean values of sCD14 were 10.9 μg/
ml in the MODS patients and 6.1 μg/ml in the non-MODS
patients, and the difference was signifi cant ( p < 0.05).
Additionally, serum sCD14 and endotoxin levels were posi-
tively correlated in patients who developed MODS on days
7, 14, and 21 postburn ( p < 0.05–0.01). Therefore, sequential
measurements of sCD14 in blood may be useful to predict
the possible occurrence of MODS [ 31 ]. Our data suggest that
sCD14 elevation is associated with the development of
MODS following major burns, and endotoxin release may be
involved in the activation of sCD14 in critically ill patients.
The pathophysiological signifi cance of elevated sCD14
levels in septic patients is not clear, but it is associated with
increased incidence of MODS and mortality. The possible
mechanism appears to be that sCD14 promotes LPS binding
to endothelial cells, resulting in massive upregulation of
adhesion molecules and synthesis of cytokines such as IL-8,
as well as enhanced procoagulant activity on endothelial cell
surface. Therefore, these changes induced by LPS-sCD14
could lead to increased endothelial permeability and coagu-
lation response related to endotoxemia, which might contrib-
ute to activation of the host’s systemic infl ammatory
response, subsequently resulting in multiple organ dysfunc-
tion [ 33 ]. The mechanism for sCD14 release from cells is
currently investigated. The stimulus for sCD14 liberation
may be LPS itself or a cytokine. In this respect, we found
that sCD14 was positively correlated with plasma endotoxin
levels, but not with TNF-α level. In vitro studies with cul-
tured monocytes have shown that endotoxin leads to the
release of sCD14 into the culture supernatant. The same
mechanisms may also account for the increased sCD14
in vivo during endotoxemia following major burns.
We demonstrate here that LBP and CD14 pathway
appears to be of importance in the development of sepsis and
MODS by mediating synthesis and release of proinfl amma-
tory mediators, such as TNF-α and HMGB1, stimulated by
endogenous endotoxin. Upregulation of LBP and CD14
expression, therefore, seems to be the major molecular
mechanism of increasing sensitivity to endogenous endo-
toxin response after major burns. Based on the work of previ-
ous investigations and our own, we have developed a
hypothesis of pathophysiological basis of sepsis and MODS
following severe thermal injury (Fig. 5.1 ) [ 31 , 32 ]. Our
hypothesis is that initial insult (such as trauma, burns, or
shock) per se can markedly upregulate LBP/CD14 expres-
sion, priming the host with increased sensitivity to endo-
Table 5.3 Comparison of serum sCD14 levels in burn patients with and without MODS (μg/ml, x
s
±
)
Postburn days
Groups
1
3
7
14
21
28
MODS ( n = 9)
3.77
5.43
10.76*
14.72**
16.88**
8.91
±0.60
±1.61
±1.78
±4.46
±8.33
±4.96
Non-MODS
( n = 13)
4.15
4.92
7.25
7.93
6.61
4.50
±0.87
±0.98
±2.64
±3.42
±2.89
±1.83
Compared to non-MODS group, * p < 0.05, ** p < 0.01
Initial insults
Increased intestinal permeability
Endotoxin translocation
Endotoxin accumulated in local sites
+
Upregulation of tissue
LBP/CD14 expression
Synthesis and release of
proinflammatory mediators
Uncontrolled inflammation
Sepsis, MODS
Fig. 5.1 Paradigm of sepsis and MODS as a result of increased sus-
ceptibility to endotoxin mediated by lipopolysaccharide-binding pro-
tein (LBP) and CD14 after severe trauma/burns

5 Bacterial Endotoxin and Exotoxin in Severe Burns

95
toxin. Meanwhile, initial insult can also result in translocation
of gut-derived endotoxin, which is accumulated in various
tissues. Excessive LBP/CD14 expression would lead to an
amplifi ed response to the subsequent insult from endogenous
endotoxin, resulting in enhanced synthesis and release of
TNF-α as well as HMGB1 [ 34 ]. On the other hand, endo-
toxin and cytokines may play important roles in further
upregulating LBP/CD14 expression after acute insults. Thus,
the aforementioned events collectively lead to sustained
infl ammatory responses, thereby causing uncontrolled sys-
temic infl ammation, culminating in end-organ failure.
5.1.4
The Relationship Between Endotoxemia
and Impairment of Cell- Mediated
Immunity in Thermal Injury
A number of elegant studies have clearly demonstrated trans-
location of bacteria/endotoxin from the gut in a variety of
pathological conditions, such as thermal injury, hemorrhage,
and trauma. However, the infl uence of endotoxemia on sys-
temic immunity in the setting of major burns is poorly under-
stood. Our studies provide delineation of one potential
mechanism involving interactions between gut-derived
endotoxin translocation and cell-mediated immunosuppres-
sion, which may contribute to the profound immune abnor-
malities and subsequent lethal sepsis seen in the burned host
[ 9 , 35 ].
The results clearly indicated that thermal injury per se
resulted in reductions in lymphocyte proliferative response
to T-cell mitogen, IL-2 production, and CD4/CD8 T-cell
ratio. The marked alterations in cell-mediated immunity
(CMI) were associated with bacterial/endotoxin translocation
which occurred after acute insult. Prophylactic treatment
with SDD signifi cantly decreased the incidences of bacterial
translocation and endotoxemia and prevented suppressive
mitogenic response and inadequate IL-2 production
( p < 0.05–0.01), but did not affect the abnormal ratio of CD4
to CD8 T lymphocytes in blood [ 35 ]. These observations are
in agreement with previous studies showing that transloca-
tion induced by gut overgrowth with E. coli was correlated
with suppression of lymphocyte blastogenesis in vitro and of
delayed hypersensitivity responsiveness in vivo. Similarly,
the enteral administration of an elemental diet for 7 days was
associated with disruption of the gut microfl ora, bacterial
translocation, and the development of an immunocompro-
mised state, which could be reversed by refeeding the ani-
mals a normal diet. All these fi ndings imply that translocation
of bacteria/endotoxin might play a role in the pathogenesis
of the postburn immune alterations and even distant organ
dysfunction [ 1 , 9 ]. It should be pointed out that many con-
tributory factors are involved in the pathogenesis of posttrau-
matic immunodefi ciency, and translocation of endotoxin
may be regarded as one of them because prevention of
translocation by SDD treatment was able to reverse, at least
in part, the cell-mediated immunosuppressive effects present
as a result of severe burns.
Several lines of evidence support the potential effects of
endotoxemia on the pathophysiological changes in host
response. First, intestinal endotoxin has been documented to
suppress both the nonspecifi c and specifi c immune functions
and to induce suppressor T-cell activation in gut-associated
lymphatic tissue, which subsequently migrates to systemic
lymphoid organs, including the spleen. Second, there is a
marked increase in circulating endotoxin concentrations in
both animal and humans after burns. Neutralization of endo-
toxin by treatment with low dose of polymyxin B restores
lymphocyte blastogenesis in thermally injured mice,
enhances in vitro lymphocyte recognition of antigens and
response to stimulation with IL-2, and increases the helper/
suppressor cell ratio in burn patients [ 36 ]. Third, endotoxin
is a potent activator to stimulate the macrophage/monocyte
to release TNF-α, IL-1, and prostaglandin E 2 , which may be
suffi cient to induce the prolonged state of immunodepres-
sion. In this respect, our previous studies had shown that
antiendotoxin agents were effective in dramatically reducing
the hemorrhage-induced translocation rate and excessive
TNF-α, IL-1, and prostaglandin E 2 formation, thereby
decreasing the susceptibility to systemic infection [ 9 , 16 ].
Fourth, it has been reported that infusion of live E. coli into
the portal vein resulted in more suppression of host immu-
nity when compared with systemic infusion of the same
numbers of E. coli or gram-positive organisms. Elevated
populations of E. coli in the small bowel lumen were also
associated with the similar suppressive effect. Therefore, it
seems that a close relationship exists between gut-derived
bacteria/endotoxin and acquired cell immunocompromised
state after thermal injury. That is, the translocation of bacte-
ria/endotoxin may predispose the host to subsequent infec-
tious complications by downregulating the CMI under
certain conditions.
In spite of modern therapeutic advances, overwhelming
sepsis remains the most common cause of death in patients
resuscitated after major thermal injuries. This has been
attributed to a number of immunological abnormalities,
including both cellular and humoral systems in the postburn
course [ 37 ]. The monitoring of essential immune functions
in the early postburn course might permit us to identify an
increased risk of infection in burn patients. A great deal of
evidence shows that cellular immune responses are associ-
ated with increased in vitro and in vivo excretion of neop-
terin, a low molecular weight pteridine compound, which is
produced as a metabolite in the biosynthetic pathway from
guanosine triphosphate to tetrahydrobiopterin [ 38 ]. Thus,
measurement of neopterin concentrations in human body fl u-
ids such as serum and urine would provide valuable insight
5.1 Endotoxemia in Burns

96
into the activation of cell-mediated immune reactions in a
variety of diseases. Although the measurement of neopterin
is nowadays frequently used for monitoring diseases associ-
ated with cellular immune activation, little is known about
whether neopterin may serve as a valuable marker to differ-
entiate between septic and non-septic patients and whether
endotoxemia could be involved in enhancement of neopterin
induction following severe burns. A clinical study was con-
ducted to determine the relationship between levels of neop-
terin and endotoxin in the circulation and whether the
neopterin level was related to the development of severe sep-
sis after extensive burns [ 39 ]. This prospective study included
35 patients with burn size greater than 30 % (30–98 %) and
22 healthy volunteers who served as a comparison group.
Neopterin levels increased in most patients on day 3 post-
burn, but they were not signifi cantly correlated with the
extent of the burn surface ( p > 0.05). A high serum neopterin
level was found in patients with sepsis ( n = 15) and a marked
elevation persisted throughout the observation period. The
difference between septic and non-septic patients ( n = 20)
became signifi cant on 14 and 28 days postburn. Although the
presence of early endotoxemia did not infl uence the altera-
tions in serum neopterin, patients with endotoxemia had
much higher neopterin values than those who showed no
endotoxemia from the second week onward ( p < 0.05–0.01).
In addition, circulating endotoxin and neopterin levels were
positively correlated in patients who developed endotoxemia
on day 14 ( r = 0.368, p < 0.05) and day 21 ( r = 0.439, p < 0.01)
after major burns. These results suggest that thermal injury
can lead to an elevation of serum neopterin independent of
the burn surface area. The initial increase in the neopterin
level may be a part of the acute-phase response to tissue
injury itself, whereas the endotoxin release in the circulation
may be responsible for the continuous induction of neopterin
during the late stage [ 12 ]. In addition, the presence of a con-
stant high neopterin level is associated with a critical event in
the development of severe burn sepsis.
5.1.5
Clinical Signifi cance of Circulating
Endotoxin Detection in Burns
Methods to detect and quantify levels of endotoxin in the
plasma of septic patients have been described for over 40 years.
The most sensitive assay described is the LAL assay. Increased
understanding of the complex biological properties of both
endotoxin and the LAL reagent has been achieved as a result of
numerous investigations during the last 20 years. Several modi-
fi cations to the assay have been made in an attempt to allow the
quantitative measurement of endotoxin and its application to
blood samples. As an assay for endotoxin, the LAL assay has
several desirable properties, which include its sensitivity, speci-
fi city, and potential for adaptation to a quantitative format [ 40 ].
Early endotoxemia in proportion to the burn size and
corresponding to the timetable of increased intestinal perme-
ability has been reported peaking commonly at 3–4 days
after burn injury. However, limited information is available
on the relative importance of endotoxemia in the pathogen-
esis of the postburn septic syndrome. The correlation
between plasma endotoxin levels and the progression of
MODS, as well as the clinical outcome following thermal
injury, remains controversial. Clarifi cation of this contro-
versy would be important from both the diagnostic and ther-
apeutic viewpoints. A new method has been developed in our
laboratory to measure plasma endotoxin concentration. We
used the modifi ed endotoxin-specifi c limulus tests (endo-
specy test) and pretreatment of plasma samples by a new per-
chloric acid method, which is more sensitive and specifi c for
endotoxin than those previously reported. In a clinical study,
we measured serial changes in the plasma levels of endo-
toxin in burn patients and investigated the relationship of
endotoxemia to the development of MODS (Fig. 5.1 ) and the
clinical outcome.
A prospective cohort study of 17 patients admitted with
burns covering more than 70 % of body surface area was
undertaken. Circulating endotoxin concentrations were
measured by modifi ed LAL assay in serial samples of
plasma [ 13 ]. Seven out of 17 burn patients developed MODS
according to multiple criteria. The plasma endotoxin con-
centrations of patients with MODS were 0.512–1.127 EU/
ml, which were signifi cantly higher than that of ten patients
without MODS (0.216–0.553 EU/ml), on 3, 14, 21, and
28 days postburn ( p < 0.05–0.01) (Table 5.4 ). A signifi cantly
higher incidence of positive endotoxin tests (≥0.120 EU/ml)
was found in patients who developed MODS as compared to
those patients who did not develop MODS during the obser-
vation period ( p < 0.05). As the mean endotoxin levels
increased, the incidence of MODS and mortality rate also
increased, and persistent endotoxemia carried a poor prog-
nosis. Six out of eight burn patients developed MODS when
the mean plasma endotoxin values exceeded 0.50 EU/ml,
and among them fi ve patients died. When a comparison was
made between the survivors and those with a fatal outcome,
Table 5.4 Comparison of plasma endotoxin levels in extensively burn
patients with and without MODS (EU/ml, x
s
±
)

Plasma endotoxin levels (EU/ml)
Postburn days
MODS ( n = 7)
Non-MODS ( n = 10)
1
0.512 ± 0.174
0.407 ± 0.232
3
1.016 ± 0.492*
0.553 ± 0.310
7
0.586 ± 0.382
0.366 ± 0.135
14
0.732 ± 0.321**
0.301 ± 0.214
21
1.127 ± 0.528**
0.284 ± 0.173
28
0.620 ± 0.251**
0.216 ± 0.202
Compared to non-MODS group * p < 0.05, ** p < 0.01
5 Bacterial Endotoxin and Exotoxin in Severe Burns

97
it was found that the mean plasma endotoxin level was
0.813 EU/ml in six non-survivors and 0.306 EU/ml in 11
survivors, respectively, and the difference was signifi cant
( p < 0.01). However, the initial values (within 7 days post-
burn) of circulating endotoxin were not markedly different
in survivors and those dying of burns ( p > 0.05). The present
investigation provides further evidence that endotoxemia in
severely burn patients commonly occurs. Circulating endo-
toxin has also been found to be strongly associated with the
development of MODS and mortality following major burn
injury.
Similar results had been shown in studies on surgical
intensive care patients, systemic meningococcal disease, and
human septic shock elsewhere. Aasen and his coworkers
observed that the chances for developing MODS in a group
of septic patients were 3.5 times higher in patients with a
positive endotoxin test than in patients with a negative test
for plasma endotoxin during the fi rst week after inclusion in
the study. In a series of studies in rabbits, our data suggested
that endotoxemia may play an important role in the patho-
genesis of MODS and endotoxin core antiserum may exert a
prophylactic effect on the course of sepsis and organ dys-
function [ 41 ]. In animals with MODS, plasma endotoxin lev-
els were signifi cantly elevated throughout the observation
period following shock, and there was a signifi cant differ-
ence between MODS and non-MODS groups from 24 h post
injury on ( p < 0.01). Moreover, plasma endotoxin levels
might be a good predicator of the development of MODS, as
the accuracies of prediction were as high as 80.0 and 90.5 %
at the end of shock and 24 h after shock, respectively [ 42 ].
Our studies differed from that of Munster et al. in which they
demonstrated that early postburn endotoxemia did not infl u-
ence the cytokine cascade, clinical manifestation of sepsis,
and mortality rate. The results of their study might be due to
a high incidence of gram-positive bacteremia due to line
infection, so that the sepsis score remained high despite a
lowering of the plasma endotoxin content. Endo et al. [ 43 ]
have reported that a raised endotoxin level was rare during
the week immediately after the injury, including the period
of burn shock, but later in the clinical course, endotoxin lev-
els were correlated signifi cantly with the burned area and
with the prognosis. Taken together, the above reports indi-
cate that sequential analyses of circulating endotoxin levels
may be useful to predict the possible development of MODS
and prognosis of clinical outcome.
5.1.6
Potential Therapeutic Approaches
Targeting Endotoxin in Burns
Over the years research efforts in sepsis and MODS have
been heavily involved with therapies targeted at modulating
the host response. Several pathways and mechanisms have
been studied in clinical trials. Unfortunately, very little
success has been found in these endeavors, including the use
of antibodies directed against the lipid A component of
endotoxin, TNF-α monoclonal antibody, IL-1 receptor
antagonist, and soluble TNF receptor. The failure of these
therapies led to a reappraisal of the pathophysiology, poten-
tial therapeutic targets, and clinical trial design in severe sep-
sis. In contrast to experimental studies where animals are
pretreated or treatment begins shortly after the onset of endo-
toxemia, treatment of patients with antiendotoxin therapies
is less feasible. In many cases, by the time patients are seen
and antiendotoxin treatment is considered, the hyperinfl am-
matory reactions may already be established. Moreover, it
has never been fully delineated whether antiendotoxin strate-
gies are directed at the primary gram-negative infectious
agent or whether the treatment is aimed at interfering with
the subsequent bacteremia or endotoxemia resulting from
bacterial translocation. In the latter case, antiendotoxin ther-
apies might be an appropriate indication for treatment.
5.1.6.1 Subtherapeutic Dose of Polymyxin B
Polymyxin B is a cyclic cationic polypeptide antimicrobial
agent known to bind endotoxin via its lipid A moiety. It was
used successfully as an antiendotoxin drug in a number of
in vivo experiments, but its high toxicity severely limits its
clinical use. Therefore, polymyxin B therapy can only be
used in treatment where it does not reach high blood levels,
such as with a hemofi ltration system. In the past decade, low-
dose intravenous polymyxin B had been demonstrated to
reduce serum endotoxin levels, reverse the unfavorable CD4/
CD8 ratio, and improve natural killer cell function, as well as
lymphocyte responsiveness to antigens in burn patients with-
out causing nephrotoxicity or hepatotoxicity [ 36 ]. Our
experiments were conducted to further determine whether
treatment with a subtherapeutic dose of polymyxin B sulfate
could prevent endotoxin/bacterial translocation and lower
mortality and, if so, to infl uence TNF-α and IL-1 formation
in animals subjected to hemorrhagic shock.
The study demonstrated that hemorrhagic shock and
resuscitation could lead to a signifi cant elevation of endo-
toxin concentration in both portal and systemic circulations.
A much higher endotoxin level was also found in the portal
vein than that in systemic blood throughout the experiment.
Meanwhile, bacterial translocation to the MLNs, as well as
viscera, took place in rats subjected to hypovolemic injury.
Treatment, however, with a subtherapeutic dose of poly-
myxin B could not only be able to curtail plasma endotoxin
in both circulations but also affected a remarkable reduction
in incidence of bacterial translocation induced by shock
[ 16 ]. These observations imply that endotoxemia/bactere-
mia resulting from translocation from the gut occurs early in
the ischemic episode and also likely plays an important role
in the pathogenesis of shock-induced sepsis, which may
5.1 Endotoxemia in Burns

98
result in organ failure. Furthermore, our data provide
additional evidence to support the idea that the vicious cir-
cle of endotoxin/bacterial translocation can be broken by
the action of polymyxin B in low dose. The fi ndings are in
agreement with previous studies suggesting that the
enhanced bacterial translocation from the gut in
Pseudomonas aeruginosa burn wound-colonized mice
could be lowered by a low dosage treatment of polymyxin
B. Although the exact mechanism by which endotoxin
would promote bacterial translocation has not yet been elu-
cidated, an increasing body of circumstantial evidence has
been accumulated that endotoxemia can initiate a self-sus-
taining cycle of endotoxin absorption from the gut and pro-
mote translocation of indigenous bacteria in a number of
pathophysiological states, such as hemorrhage, trauma, and
burns [ 3 ]. In the compromised host, endotoxin-mediated
alterations may be accentuated and contribute to the devel-
opment septic responses, vital organ damage, and death.
Therefore, therapeutic measures aim at early recognition
and control of the process of endotoxin/bacterial transloca-
tion may afford benefi cial effects on many hemorrhagic
shock-associated abnormalities and mortality [ 4 , 5 ].
The results revealed that polymyxin B treatment nearly
abolished the hemorrhage-induced elevation in circulating
TNF-α and IL-1 release by peritoneal macrophages in vitro.
Moreover, a low dose of intramuscular polymyxin B also
reduced the mortality resulting from shock, compared with
those of untreated rats. Because the reduction of many
infl ammatory mediators (e.g., TNF-α, IL-1, IL-6, and pros-
taglandin E 2 ) is correlated with a signifi cant improvement of
Kupffer cell capacity to present antigen and express major
histocompatibility complex class II, polymyxin B, with its
ability to inhibit the release of TNF-α and IL-1, may be a
useful adjuvant in the clinical setting for the immunomodu-
lation of postburn immunodepression and for reducing the
susceptibility to septic challenge [ 16 ]. Munster et al. also
documented that administration of polymyxin B success-
fully reduced the plasma concentration of endotoxin during
the early postburn phase, but without any impact of IL-6
level, the clinical sepsis score, and outcome. Nevertheless,
the fi ndings might be caused, at least in part, by a high inci-
dence of gram-positive bacteremia as a result of line infec-
tion in their series, so that the sepsis score remained high
despite lower circulating endotoxin. These data, together
with others, have led us to conclude that after an initial sur-
gical stress (such as shock and burns), translocation of
endotoxin/bacteria may trigger a cascade of deleterious
events via interaction of various mediators (e.g., TNF-α,
IL-1, and IL-6). Intramuscular administration of a subthera-
peutic dose of polymyxin B is effective to reduce markedly
hemorrhage- induced endotoxin/bacterial translocation from
the gastrointestinal tract and excessive TNF-α and IL-1
release.
5.1.6.2 Bactericidal/Permeability-Increasing
Protein
A naturally occurring compound with potent antiendotoxin
activity that has been studied as a potential therapeutic agent
in the treatment of sepsis is bactericidal/permeability-
increasing protein (BPI). BPI has a high-affi nity binding
domain for the lipid A domain of endotoxin. In addition to
avidly binding free endotoxin and suppressing endotoxin-
mediated cellular activation, BPI also has potent antibacte-
rial properties specifi c for gram-negative bacteria. Similarly,
during in vitro studies, recombinant BPI proteins bind endo-
toxin, inhibit cytokine formation, and exhibit direct bacteri-
cidal activity. An increasing body of circumstantial evidence
has been accumulated that BPI and its recombinant
N-terminal fragments could provide protection against chal-
lenge with bacteria or purifi ed bacterial endotoxin in several
animal models and, more recently, in human clinical trials
[ 44 ]. Moreover, we reported that rBPI 21 (a 21 kD amino-
terminal fragment of BPI) might be a useful therapeutic
agent against endogenous bacteria or endotoxin-related dis-
orders in severe hemorrhagic shock [ 5 ]. However, the defi -
nite mechanism(s) underlying benefi cial effect of rBPI 21 is as
yet fully elucidated. Since BPI and LBP share signifi cant
sequence identity and immuno-cross-reactivity, it has been
suggested that rBPI 21 could compete with LBP for binding
endotoxin and gram-negative organisms, and the potent anti-
endotoxin activity of rBPI 21 might be due to an inability of
LPS-LBP complexes to bind the CD14 receptors.
Our data have shown that treatment with rBPI 21 was effec-
tive in preventing gut-origin endotoxin translocation second-
ary to severe burns. The early increase in hepatic endotoxin
levels was signifi cantly attenuated in rats treated with rBPI 21 ,
approximating the baseline values. Similarly, levels of endo-
toxin in the lungs have been shown to decrease nearly one
third compared with the controls. Treatment with rBPI 21
resulted in about 20 % decrease in numbers of bacterial
translocation to the MLN compared with control animals,
although the total incidence of bacterial translocation was
not affected by rBPI 21 . Meanwhile, tissue LBP, CD14, and
TNF-α mRNA expression in various organs were markedly
inhibited by rBPI 21 secondary to acute insults ( p < 0.05–0.01).
Furthermore, signifi cant reduction in serum amino leucine
transferase levels and elevation in intestinal diamine oxidase
activities in the rBPI 21 -treated group were found compared to
those in the control group ( p < 0.05–0.01) [ 6 ]. In a rat model
of prolonged hemorrhagic shock (30–35 mmHg for 180 min)
followed by adequate resuscitation, we found that immedi-
ately after resuscitation, plasma endotoxin levels in the
control group (61.0 ± 16.3 pg/ml) were almost neutralized
by rBPI 21 treatment (13.8 ± 4.8 pg/ml, p < 0.05). Plasma
TNF-α levels were not signifi cantly infl uenced by rBPI 21
treatment. The 48 h survival rate was 68.8 % in the treatment
group versus 37.5 % in the control group. Microscopic
5 Bacterial Endotoxin and Exotoxin in Severe Burns

99
histopathological examination revealed relatively minor
damage to various organs in the treatment group [ 5 ]. More
recently, postburn administration of rBPI 23 has been shown
to reduce the incidence of bacterial translocation after burn
injury in mice. The fi ndings of other authors, taken together
with the observations in the present study, indicate clearly
that early treatment with rBPI 21 could block bacterial or
endotoxin translocation after acute insult.
Endotoxin-stimulated cytokine response is governed
by the balance between factors that potentiate this pro-
cess, such as LBP and CD14, and factors that attenuate
this process, such as BPI. Concentrations of plasma LBP
are signifi cantly increased in both endotoxemic volun-
teers and patients with sepsis. However, the endogenous
response of plasma BPI in patients with sepsis is weak.
Plasma LBP levels were 250- fold higher than plasma BPI
levels during experimental endotoxemia in healthy volun-
teers and about 3,000-fold higher during sepsis. Because
both LBP and BPI bind to LPS, it could be anticipated that
endogenously derived plasma levels of BPI are likely to
be inadequate to compete with much more abundant LBP
in the circulation to bind LPS. Therefore, it seems reason-
able to conclude that the magnitude of endogenous BPI
response to LPS is insuffi cient to attenuate an ongoing
endotoxemic challenge. To achieve a therapeutic goal in
the treatment of sepsis, a reasonably high supply of exog-
enous BPI is most probably necessary to nullify the sensi-
tizing effect of LBP [ 44 , 45 ]. Our data indicate that the
therapeutic strategies that block endotoxin translocation
secondary to acute insults inhibited the effects of LBP/
CD14 pathway and excessive infl ammatory response,
therefore, decreasing sensitivity to endotoxin and prevent-
ing progression of organ dysfunction following burns.
Early treatment with rBPI 21 is effective on attenuating
multiple organ damage which resulted from gut-origin
endotoxin translocation which might be associated with
the downregulatory effects of tissues LBP and CD14 gene
expression by use of rBPI 21 .
5.1.6.3 Selective Decontamination
of the Digestive Tract
Since life-threatening sepsis can originate from the gut, sev-
eral groups of investigators have attempted to reduce the
incidence of systemic infections in high-risk patients by the
use of SDD: a regimen of oral administration of nonabsorb-
able, small-spectrum antibiotics, which are directed against
gram-negative bacilli and yeasts. These antibiotics are bacte-
ricidal for gram-negative aerobic bacilli, such as
Enterobacteriaceae, Pseudomonadaceae, and Acinetobacter
spp., and yeasts, but have no effect on the naturally occur-
ring microbiological fl ora [ 46 , 47 ]. This sparing of the nor-
mal, predominant anaerobic fl ora prevents overgrowth of
pathogens. It is postulated that the use of SDD to maintain
colonization resistance and to eliminate potential pathogens
selectively in the gastrointestinal tract could reduce exposure
of the host to translocate bacteria and/or endotoxemia in cir-
cumstances in which gut permeability becomes abnormally
high following major burns. However, the opinion in regard
to clinical effi cacy of selective decontamination in critical
illness remains controversial. There are reports alleging that
SDD is effective in reducing the incidence of secondary col-
onization and acquired infection; on the other hand, debate is
continuing about its benefi cial effect on outcome and its
limitation in eradicating multiresistant microorganisms. In
rats following 40 % full-thickness scald injury, we evaluated
the effectiveness of the technique of SDD in preventing
endotoxin absorption and bacterial translocation from the
gut, as well as its potential effect on mortality in rats infl icted
with thermal injury.
Marked elevations of plasma endotoxin concentrations
in both the portal and systemic circulations were found in
untreated rats throughout the observation period, but were
prevented by pretreatment with SDD (except on postburn
day 3 in the portal vein: 0.189 ± 0.102 EU/ml). The mean
endotoxin contents in sham controls were 8.86–15.60 μg/g
of feces; they were signifi cantly increased in fecal mate-
rial from different segments of the intestinal tract at
1–5 days post injury. Furthermore, the increased fecal
endotoxin levels paralleled the overgrowth of gram-nega-
tive bacilli in the intestine. Following intragastric admin-
istration of the selective antibiotics, there was a reduction
in the mean endotoxin levels of fecal supernatants to less
than 0.5 % (0.04–0.45 %) of the nontreatment group.
Mucosal DAO activity was similar in both groups in base-
line value and was signifi cantly decreased 24 h postburn
in both groups. However, when SDD was instituted the
mean DAO activity returned to baseline on day 5; it was
then much higher than that of the controls ( p < 0.05).
Moreover, there were no statistically signifi cant differ-
ences in survival rates between the two groups at 1 and
3 days after injury (SDD vs. control: 96.7 % vs. 90.0 %,
93.3 % vs. 73.3 %, respectively), but the 5-day survival
rate was markedly improved from 63.3 % (19/30) in the
control group to 90.0 % (27/30) in the SDD-treated group
( χ 2 = 4.57, p < 0.05). These data suggested that SDD was
effective in preventing gut-origin endotoxemia and bacte-
rial translocation and improving the survival rate in rats
following severe thermal injuries [ 10 ].
Recently, it has been reported that the process of endo-
toxin translocation may contribute to systemic immune dys-
function, thereby increasing susceptibility of the host to
invasive infection with fatal outcome. However, it remains to
be proven that measures aimed at controlling translocation
events would prevent burn injury-associated derangement
of host immunity. Therefore, our study was to investigate
the infl uence of pretreatment with SDD on systemic
5.1 Endotoxemia in Burns

100
immunosuppression and the relationship between bacterial/
endotoxin translocation and abnormalities of CMI in a rat
model of thermal injury [ 35 ]. The results showed that ther-
mal injury resulted in marked reduction in splenocyte prolif-
erative response to T-cell mitogens, IL-2 production, and T
helper/suppressor cell (CD4/CD8) ratio. Prophylactic treat-
ment with SDD signifi cantly decreased the incidences of
bacterial translocation and endotoxemia and prevented sup-
pressive mitogenic response and inadequate IL-2 production.
To our knowledge, the fi ndings in this experiment represent
the fi rst report of a successful intervention with SDD to pre-
vent inadequate IL-2 production and suppressive mitogen
responsiveness to both phytohemagglutinin (PHA) and con-
canavalin A after thermal injury. We have not correlated
abnormalities of immune competence with susceptibility to
sepsis in this study. Yet, that our previous studies with the
same animal model revealed a signifi cant increase in 5-day
survival in the SDD-treated group suggests that reversal of
the impairment of cellular immune functions would contrib-
ute to the improvement of outcome in rats subjected to a seri-
ous insult. Thus, it is apparent that selective antibiotic
prophylaxis may blunt postburn immune dysfunction with
subsequent septic states by preventing bacterial/endotoxin
translocation from the gut.
Our studies confi rmed that the SDD regimen might pro-
vide a prophylactic effect on prevention of endotoxin/bacte-
rial translocation and mortality in rats with severe thermal
injury. Therefore, it was supposed that SDD might be of
value in decreasing the risk of endogenous-derived sepsis
associated with MODS in patients with extensive injuries.
Although it was shown to improve the outcome in rats receiv-
ing antibiotics for SDD, it should be pointed out that the
regimen was started 3 days prior to the present experimental
thermal injury. Thus, further studies are required to test the
therapeutic advantage of this regimen when it is started after
burn injury.
In summary, interventions in the infl ammatory response
induced by severe burns can be performed at several levels,
such as inhibition of the inducer, interference with induction,
control of early and late cytokines, and blockade at the tar-
gets. Among the possibilities of intervention, binding and
inactivating bacterial toxins is the most attractive because it
occurs at the top of the infl ammatory network such that it
avoids downstream multiple mediator induction and does not
directly interfere with the immune system, minimizing
unpredictable side effects and blockade of favorable host
responses. Nevertheless, the development of new therapeutic
strategies is based on a better understanding of the biology of
infl ammation, including new information on the intracellular
signaling response to extracellular challenge, the molecular
responses to infl ammation, and a new understanding of the
mechanisms of postburn sepsis as well as organ failure [ 37 ,
48 , 49 ].
5.2
Bacterial Exotoxins in Burns
In the last two decades, the incidence of gram-positive
infection has progressively increased. To date, up to 50 %
of severe sepsis or septic shock cases in modern intensive
care units were caused by gram-positive organisms, and
among them, Staphylococcus aureus ( S. aureus ) ranked
foremost as the causative organism of postburn or post-
trauma sepsis. We should be also alerted that S. aureus is
acquiring antibiotic resistance with remarkable profi ciency,
and vancomycin remains the only effective therapeutic
agent for many of its strains [ 2 , 50 ]. More recently, the
alarming emergence of S. aureus with reduced sensitivity
to vancomycin further highlights the diffi culty in the treat-
ment of S. aureus infection. Therefore, further investigation
of the pathogenesis of sepsis and search for new therapeutic
strategy against postburn S. aureus infection would
undoubtedly be necessary.
Compared to the well-studied gram-negative bacteria, in
which the LPS component is believed to be the primary ini-
tiator of septic shock, the virulence of S. aureus is multifac-
torial, as it is mediated by a variety of extracellular toxins
and the cell wall components. It has been widely accepted
that S. aureus does not possess LPS in the outer cell wall;
its toxic manifestations are attributable to the presence of
exposed peptidoglycan and lipoteichoic acids on the sur-
face of the bacteria. While their bioactivities seem to be
less than those of gram-negative LPS, the signals of the sur-
face structures are transduced via the same receptor (CD14)
as LPS. Therefore, they seem to possess some activities
similar to LPS. Moreover, S. aureus may also secrete a fam-
ily of exotoxins, such as staphylococcal enterotoxins (SEs)
and toxic shock syndrome toxin-1 (TSST-1), and these exo-
toxins were demonstrated to be potent activators of the
immune system [ 51 ]. In vitro studies showed that staphylo-
coccal toxins, including staphylococcal enterotoxin (SE)
A, SEB, SEC, SED, SEE, and TSST-1, could polyclonally
activate a large proportion of T cells (5–30 %) at picomolar
concentrations, whereas a conventional antigen could stim-
ulate less than 0.01 % of the T-cell population. Owing to
these facts, a term “superantigen” was coined to describe
these toxins.
Different from conventional antigens, staphylococcal
superantigens have unique interactions with host cells. For
example, they bind to major histocompatibility complex
(MHC) class II molecules of antigen-presenting cells outside
the conventional antigenic peptide-binding groove and exert
their effect as an intact molecule without being “processed.”
Therefore, their presentation to T cells is not MHC II
restricted. Furthermore, the recognition of superantigen by
the T-cell receptor (TCR) is dependent only on the variable
region of β-chain (Vβ) of the TCR, but not on the combina-
tion of TCR-α and TCR-β chain as in the case of conventional
5 Bacterial Endotoxin and Exotoxin in Severe Burns

101
peptide antigens. Because the amino acid is highly conserva-
tive in Vβ chain of TCR, many subtypes of T cells may pos-
sess the same TCR Vβ domain. Thus, a single member of
superantigen may induce massive proliferation of multiple
T-cell clones (Fig. 5.2 ). Just because of such special affi nities
of superantigens for TCR and MHC II molecule, they could
readily stimulate both T cells and antigen- presenting cells,
such as monocytes and macrophages. This trimolecular
interaction will lead to uncontrolled release of various proin-
fl ammatory cytokines especially IFN-γ, TNF- α, and inter-
leukins (ILs) [ 52 ]. These cytokines, in turn, may be critical
for initiating superantigen-mediated toxic shock. Thus, the
presence of superantigens ends in a release of cytokines,
similar to gram-negative sepsis. It is assumed that actions
other than cytokine production may be responsible for supe-
rantigen actions in toxic shock syndrome (TSS); for exam-
ple, superantigen may amplify the effects of LPS [ 53 ].
Therefore, inhibition of the effect of staphylococcal superan-
tigens might be of therapeutic value in preventing the conse-
quences of S. aureus infection. However, up to now, the
signifi cance of changes in tissue SE distribution and its
potential role in the development of postburn S. aureus sep-
sis remains to be elucidated.
5.2.1
The Role of Staphylococcal Enterotoxin
B in Postburn Staphylococcus Aureus
Sepsis
As it is well known, enterotoxins produced by the ubiquitous
S. aureus are 23–29 kD single-chain proteins with potent
immunomodulation properties, which exert a number of
pathological effects in human beings as well as susceptible
animals. Early in the 1980s, several studies had demonstrated
that enterotoxins were closely associated with menstrual
TSS or TSS-like symptoms in human beings, causing fever,
skin rash, hypotension, shock, and even death. Rabbits
receiving SEs also exhibited histopathological changes simi-
lar to those in human TSS cases. More recently, accumulat-
ing evidence showed that a large amount of nonmenstrual
life-threatening TSS or TSS-like symptoms were also related
to SEs. Among these toxins, SEB is notorious because of its
universality and its potent immunostimulating ability. In our
previous work, we found that intravenous injection of SEB
could induce multiple organ dysfunction in rabbits [ 54 ].
Other studies further demonstrated that intraperitoneal
administration of SEB to rats also resulted in leukocytosis,
fever, hypotension, and lethal shock. However, up to now,
the immunopathogenesis of SEB-involved sepsis was not
fully elucidated yet, especially those of postburn or post-
trauma sepsis. Thus, a better understanding of the in vivo
effects of SEB in postburn S. aureus infection is quite in
need.
In the present study, we found that, in postburn septic rats,
plasma SEB level transiently elevated, peaked at 0.5–6 h
after S. aureus challenge, and declined rapidly at 12 h.
However, SEB levels in the liver, kidneys, lungs, and heart
continuously increased up to 24 h, especially in the liver and
kidneys [ 55 ]. These results indicated that the disappearance
of circulating SEB might be due to the distribution of SEB
in local tissues. Owing to the fact that SEB levels in the liver
and kidneys were much higher than those in other organs, we
hypothesized that the liver and kidney might play more
important roles in accumulating and, probably, inactivating
T cell
Antigen-presenting cell
T-cell receptor
MH Cclass II
T cell
Conventional
antigen
Superantigen
TCR Vβ
ng/ml
Antigen-presenting cell
Fig. 5.2 Schematic representation
of the interaction of superantigens
with the MHC class II molecule
and T-cell receptor (TCR)

5.2 Bacterial Exotoxins in Burns

102
SEB. Such opinion was supported by an in vivo study which
showed that SEB could be fi ltered easily by the glomerulus
and then rapidly and completely be reabsorbed from the
tubular urine by the cells of the proximal renal tubule.
Furthermore, SEB could bind to the glycosphingolipids of
kidney proximal tubular cells with high affi nity, and this
component might be a putative receptor for SEB. In addition
to the kidneys, the liver appears to be another important
detoxicating organ, where many toxins are taken up and
inactivated by Kupffer cells. Thus, in our study, the large
amount of SEB deposited in the liver might be a result of the
uptake of SEB by Kupffer cells.
In order to investigate the potential role of SEB in the
pathophysiology of postburn sepsis, we next investigated
the relationship between local SEB contents and organ dys-
function indexes [ 55 ]. It was shown that, in scald control
animals, serum alanine aminotransferase, MB isoenzyme of
creatine kinase (CK-MB), and creatinine levels and pulmo-
nary MPO activity were higher than those of normal con-
trols. These indexes were further elevated at 0.5 h after S.
aureus administration and remained in high values till 24 h,
indicating a progressive injury of the liver, heart, kidneys,
and lungs. Statistical analysis showed that CK-MB, alanine
aminotransferase, and creatinine levels were positively cor-
related with SEB contents in corresponding organs, suggest-
ing that SEB might be involved in the development of
multiple organ damage. To confi rm this assumption, a spe-
cifi c monoclonal antibody to SEB-MAb2D 1 was used in our
study to block the activity of SEB. The results showed that
pretreatment with MAb2D 1 0.5 h prior to S. aureus chal-
lenge could effectively reduce the levels of SEB in plasma
as well as tissues. Meanwhile, the elevation of serum ala-
nine aminotransferase and CK-MB values was also signifi -
cantly mitigated at 6 h, comparing to those without MAb2D 1
treatment. These results demonstrated that SEB might be
involved in the development of septic response after burns
combined with SEB-producing S. aureus infection. Early
treatment with specifi c SEB-MAb2D 1 could partially pro-
tect rats against S. aureus infection and improve the progno-
sis of postburn sepsis. Furthermore, it was interesting to fi nd
that the mortality of postburn S. aureus septic rats was
markedly decreased in MAb2D 1 -pretreated animals, being
17.64 % (3/17) at 6 h [ p < 0.05, compared to the 55.55 %
(20/36) in postburn sepsis group]. Consistent to our results,
LeClaire et al. reported that SEB-specifi c antibodies could
effectively suppress the in vitro T-cell proliferation elicited
by SEB and totally protect mice against SEB. Likewise,
Stiles and Visvanathan et al. demonstrated that vaccination
of recombinantly attenuated SEB or conserved peptide of
SEB to mice could elicit systemic and mucosal antibodies to
SEB, and it also protected mice against SEB-induced lethal
shock [ 56 ].
5.2.2
The Mechanisms Underlying
SEB- Mediated Postburn Sepsis
and Multiple Organ Dysfunction
As it is mentioned above, the mechanisms underlying
SEB- mediated multiple organ dysfunction were not fully
understood yet. Since the dual affi nity of SEB to MHC II
molecules and selected TCR Vβ enables it to potently
activate both macrophages and T cells, it is assumed that
cytokines produced by both types of cells may be involved in
the pathophysiology of sepsis. Previously, several research
groups had reported that SEB, injected either intraperitoneally
or intravenously, could induce excessive formation of TNF-α
and IFN-γ, which are supposed to be “critical mediators” in
the development of septic shock [ 2 , 56 ]. In the animal study,
we found that burn injury combined with S. aureus infection
could also signifi cantly increase TNF-α and IFN-γ levels
both in plasma and various tissues, whereas early treatment
with SEB-specifi c MAb2D 1 partially inhibited such response.
These results further demonstrated that SEB might be
involved in the induction of TNF-α and IFN-γ in postburn
septic rats. Herein, it should be emphasized that SEB-
induced lethal shock is T-cell dependent, and the polyclonal
activation of T cells is an important characteristic of SEB-
mediated sepsis. IFN-γ, which is produced mainly by T cells,
is a potent immunomodulating factor and plays an important
modulating effect on macrophage and endothelium. It has
been reported that TNF-α mRNA in the spleen of SEB-
treated mice showed a biphasic response. In the fi rst phase,
the early appearance of small quantity of TNF-α mRNA was
T-cell derived, while the larger amount of TNF-α mRNA in
the second phase was expressed mainly in macrophage.
However, the latter was T-cell dependent. That is, only under
the co-stimulation of SEB and IFN-γ from T cells could
macrophages produce massive amount of cytokines, such as
TNF-α. This is quite different from the kinetic of cytokines
in gram-negative bacterial infection [ 2 ]. We learned from
other reports that SEB-induced elevation of TNF-α and
IFN-γ in galactosamine-sensitized mice was closely associ-
ated with organ injury. TNF-α- or IFN-γ-specifi c MAb treat-
ment could obviously improve the prognosis of the septic
mice. Moreover, Stiles et al. demonstrated with TNF recep-
tor 1 (TNFR1)-defi cient mice that TNF-α played a key role
in SEB-induced shock, and transgenic knockout mice lack-
ing TNFR1 were protected against the lethal effect of
SEB. These results, taken together with ours, suggested that
TNF-α and IFN-γ were critical factors in mediating the toxic
effects of SEB, and multiple organ dysfunction induced by S.
aureus infection might be associated with SEB-induced
TNF-α/IFN-γ overproduction [ 57 ]. Therefore, the therapeutic
effect of SEB-MAb2D 1 in our experiment might be attrib-
uted to their inhibition on cytokine formation. However, it
5 Bacterial Endotoxin and Exotoxin in Severe Burns

103
should be emphasized that the inhibitory activity of
monoclonal antibody depends not only on its affi nity to cer-
tain antigen but on the special epitope to which it binds as
well. Our preliminary results, herein, suggested that SEB-
MAb2D 1 might be of signifi cance in preventing the develop-
ment of postburn S. aureus sepsis. The potential therapeutic
effect of SEB-specifi c antibody on sepsis needs further
investigation.
HMGB1, once known as a nuclear protein and an impor-
tant architectural chromatin-binding factor, now has been
identifi ed as a late cytokine that can be released with a lag of
12–18 h by activated monocytes, macrophages, neutrophils,
and platelets and in turn mediate infl ammation and enhance
cell motility in sepsis [ 17 , 58 ]. In addition to early cyto-
kines, such as TNF-α and IFN-γ, we further investigated
changes in HMGB1 in postburn S. aureus infection and its
potential role in the pathogenesis of sepsis. 70 male Wistar
rats were randomly divided into three groups as follows:
normal control group, scald control group, and postburn
sepsis group. Tissue samples from the liver and lungs were
collected to determine HMGB1 and LBP mRNA expres-
sion; SEB and endotoxin levels in these tissues were also
measured. After S. aureus challenge, HMGB1 mRNA
expressions were upregulated markedly in both the liver and
lungs, peaking at 6–12 h ( p < 0.05–0.01) and keeping at high
levels up to 24 h. It was also shown that LBP gene expres-
sions were signifi cantly positively correlated with hepatic
and pulmonary HMGB1 gene expressions ( r = 0.800,
p = 0.031 and r = 0.942, p = 0.002, respectively). These data
demonstrated that thermal injury combined with S. aureus
challenge could upregulate HMGB1 expressions, which
may serve as a “late” mediator involved in the pathogenesis
of postburn sepsis [ 59 ]. Taken together, SEB is a superanti-
gen with potent immunostimulating activity. It could induce
massive production of both early and late infl ammatory
cytokines and play a potential role in the development of
postburn S. aureus sepsis and subsequent MODS. Early
treatment with a specifi c monoclonal antibody to SEB-
MAb2D1 appears to be effective in ameliorating the multi-
ple organ damage induced by postburn sepsis, which might
be associated with its inhibitory effect on the formation of
infl ammatory mediators.
5.2.3
The Signal Transduction Pathway
of Postburn Staphylococcus Aureus
Sepsis
Signifi cant progress has been made over the past decade in
our understanding of the intracellular events that couple
receptor-ligand binding to subsequent infl ammatory and
immune responses of the cell. Signaling cascade is a very
important pathway which widely participates in cell
biological activities in both humans and animals. From
humans to fl ies, the Janus kinase (JAK) and signal transduc-
ers and activators of transcription (STAT), mitogen-activated
protein kinase (MAPK), and nuclear factor-κB (NF-κB)
pathways transduce a multitude of signals for development
and homeostasis and affect a wide array of cytokines and
growth factors. Because proinfl ammatory events regulated
by the JAK/STAT, MAPK, and other signaling cascades have
been implicated in the pathogenesis of both sepsis and
MODS, it is probable that in the upcoming years such signal-
ing mediators will enter common parlance in the ICU as both
diagnostic biomarkers and therapeutic targets [ 49 , 60 , 61 ].
JAK/STAT pathway has been demonstrated to be involved
in signal transduction of both proinfl ammatory and anti-
infl ammatory mediators. For example, IFN-γ can magnify
proinfl ammatory effect of many infl ammatory mediators,
such as LPS, TNF-α, and IL-1; the signaling is partly chan-
neled through JAK/STAT pathway. On the other hand, IL-10
and IL-4 partially depend on JAK/STAT pathway to accom-
plish their anti-infl ammatory effect. Recent fi ndings also
show that JAK/STAT signaling may regulate expressions of
many infl ammatory cytokines induced by LPS. LPS can
quickly activate JAK/STAT pathway in cardiac myocytes
and endothelial cells of the cerebral vessel in rats, and this is
shown to be highly involved in engendering the expression
of IFN-γ, TNF-α, and IL-2. Mutation of STAT binding sites
on the promoters of these cytokines results in dramatically
abating expression of these cytokines induced by bacterial
toxins [ 62 ]. However, the signal transduction mechanisms in
regulation of cytokine expression and release during post-
burn S. aureus sepsis are far from being elucidated.
Our study was conducted to observe the activation of
JAK2/STAT3 in postburn S. aureus infection and the poten-
tial effect of treatment with JAK/STAT inhibitors on the
development of postburn sepsis. In addition, the infl uence of
MAPK and NF-κB inhibitors on STAT3 activation was
investigated to elucidate the possible crosstalk among these
signal transduction pathway in vivo [ 63 , 64 ]. Ninety-seven
male Wistar rats were randomly divided into eight groups as
follows: (a) normal control group; (b) scald control group,
rats were subjected to a 20 % total body surface area (TBSA)
III scald injury; (c) postburn sepsis group, rats infl icted with
20 % TBSA III° scald followed by SEB-produced S. aureus
challenge and were further divided into 0.5, 1, 2, 6, 12, and
24 h groups, respectively; (d) AG490 (inhibitor of JAK2)
treatment group ( n = 10), intraperitoneal injection of a dose
of 8 mg/kg AG490 just prior to S. aureus challenge, being
further divided into 0.5 and 2 h groups, respectively; (e)
RPM (rapamycin, inhibitor of STAT3) treatment group,
intraperitoneal injection of a dose of 0.4 mg/kg prior to S.
aureus challenge, being further divided into 0.5 and 2 h
5.2 Bacterial Exotoxins in Burns

104
groups, respectively; (f) AG126 (inhibitor of MAPK)
treatment group, intraperitoneal injection of a dose of 4 mg/
kg prior to S. aureus challenge, being further divided into 0.5
and 2 h groups, respectively; (g) PDTC (pyrrolidine dithio-
carbamate, inhibitor of NF-κB) treatment group, intraperito-
neal injection of a dose of 50 mg/kg prior to S. aureus
challenge, being further divided into 0.5 and 2 h groups,
respectively; and (h) DMSO (dimethyl sulfoxide, solvent of
drugs) treatment group, intraperitoneal injection of a dose of
4 ml/kg prior to S. aureus challenge. Blood and tissue sam-
ples from the liver, kidneys, and lungs were collected to
determine transcription factors, cytokines, as well as organ
functional indexes.
Following thermal injury combined with S. aureus chal-
lenge, TNF-α, IFN-γ, and IL-10 gene and protein expression
in various tissues were markedly upregulated, which were
closely associated with changes in serum alanine transami-
nase, aspartate transaminase, creatinine, and blood urea
nitrogen levels. These results demonstrated that the exces-
sive release of pro- and anti-infl ammatory cytokines might
be involved in the pathogenesis of multiple organ dysfunc-
tion in rats secondary to postburn S. aureus sepsis.
Meanwhile, STAT3 in various tissues could be activated at
early stage following S. aureus challenge, which appeared to
be related to the direct stimulation of SEB. In the treatment
with selected JAK2 inhibitor AG490 and STAT3 serine
inhibitor rapamycin, STAT3 activation was markedly inhib-
ited in the liver, lungs, and kidneys at 0.5 h. Concomitantly,
TNF-α, IFN-γ, and IL-10 mRNA and protein expression
declined together with decreases in alanine transaminase and
aspartate transaminase levels at 2 h after AG490 or rapamy-
cin treatment [ 64 ]. These data suggested that direct inhibi-
tion of JAK2/STAT3 pathway might ameliorate systemic
infl ammation and prevent the development of multiple organ
dysfunction in rats with postburn sepsis. In addition, treat-
ment with pyrrolidine dithiocarbamate, an inhibitor of
NF-κB, could inhibit STAT3 activation to a certain extent in
S. aureus sepsis. The results implied that there might exist a
crosstalk between NF-κB and STAT3 signal transduction
pathways, and STAT3 seems to be a key point in the signal
network. On the other hand, our data showed that treatment
with AG126, an inhibitor of MAPK, was not markedly infl u-
enced by STAT3 activation, indicating that little interaction
could be evident between MAPK and STAT3 in postburn S.
aureus sepsis.
A new family of suppressors of cytokine signaling
(SOCS), induced by cytokines activating the JAK/STAT
pathway, has been recently identifi ed as a negative feedback
loop of intracellular signaling [ 65 ]. Overexpression of some
SOCS (SOCS-3, CIS, and SOCS-2) has been reported to
inhibit the JAK/STAT pathway stimulated by growth hor-
mone. Thus, we further observed the changes in SOCS
expression in postburn S. aureus infection, to evaluate their
potential role in the pathogenesis of postburn sepsis. Wistar
rats were infl icted with 20 % TBSA III scald followed by S.
aureus challenge, and then SOCS1, SOCS2, and SOCS3
mRNA expressions and IFN-γ levels in the liver and lungs
were determined at 0.5, 2, 6, 12, and 24 h following S. aureus
challenge. After S. aureus challenge, the levels of IFN-γ in
the liver and lungs increased signifi cantly, peaking at 0.5–6 h
( p < 0.05–0.01), and then declined to baselines at 12 h.
Accordingly, SOCS gene expressions in the lungs were also
upregulated rapidly, especially SOCS2 and SOCS3, which
were obviously higher than those of control animals at 0.5 h
after S. aureus challenge, were robust at 2 and 6 h, and then
returned to basal levels. In contrast, SOCS1 gene expression
in the liver was much slow, which increased markedly at 2 h
and remained high till 24 h. SOCS2 and SOCS3 gene expres-
sion only showed a trend of upregulation. Pretreatment of
monoclonal antibody to SEB could effectively inhibit pul-
monary IFN-γ production, and the gene expression of SOCSs
in the lungs was also suppressed [ 66 ]. These data indicated
that thermal injury combined with S. aureus challenge could
upregulate the gene expressions of SOCSs in local tissues,
which, acting as negative regulators of cytokine signaling,
might play a role in regulating the balance of immunosup-
pression and infl ammation in postburn gram-positive sepsis.
Intracellular signaling involves precisely timed, compart-
mentalized, specifi c, and reversible interactions between
proteins and underlies biological phenomena of health and
disease alike, ranging from cellular proliferation to immu-
nity to sepsis. Increasingly, the potential of cellular signaling
molecules as diagnostic markers and therapeutic targets is
being realized [ 37 , 49 ]. A basic understanding of the princi-
ples of signal transduction and of the important cellular sig-
naling cascades is necessary to evaluate current research in
major burns and may be of increasing use to the clinical
practice.
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5 Bacterial Endotoxin and Exotoxin in Severe Burns

107
Z. Yang, Chinese Burn Surgery, DOI 10.1007/978-94-017-8575-4_6,
© Springer Science+Business Media Dordrecht and People’s Medical Publishing House 2015
6.1
Introduction
Since the early 1960s, modern immunology has gradually
been involved in every department of clinical medicine.
There is no exception for burn medicine. Several burn units
in Chongqing, Shanghai, and Beijing of China began to
research the immunological consequences of burn clinically
and experimentally in the late 1970s [ 1 – 4 ]; their studies have
been continued over 30 years [ 5 – 8 ]. Relevant research in this
fi eld has been carried out by institutions in other regions in
recent years. Overall, the study of burn immunology in China
has its own characteristics and advantages with the unremit-
ting efforts of many scholars in the last decades. Our studies
in several aspects, including the initiating factors in the
pathogenesis of immune dysfunction and molecular mecha-
nism of the immune cell dysfunction after burns, have come
up to the international advanced level. Therefore, the phe-
nomena of immune function alterations, etiological analysis,
pathogenesis, clinical consequences, and modulating mea-
sures of postburn immune dysfunction are summarized as
follows, and the future directions in the study of burn immu-
nology and related research are also analyzed.
6.2
Phenomenon of Postburn Immune
Dysfunction
Extensive and deep burns have widespread and profound
impact on the various cells and molecules of the innate and
adaptive immune systems. These immunological conse-
quences after thermal injury are not always depressed, and
many of them are also overactivated. The hypothesis of post-
burn immune dysfunction was put forward from these results
of severely burn patients and animals [ 1 , 2 , 5 ]. The major
characteristics of postburn immune dysfunction are hyperin-
fl ammatory response and hypoimmune response for innate
and adaptive immunity, respectively.
The changes in innate immune response after burns occur
at the following aspects, such as myelopoiesis, phagocyte
function, complement, and acute phase proteins. The myelo-
poiesis in the bone marrow is activated and more neutrophils
are produced after burns particularly when combined with
infection [ 6 ]. The leukocytes infi ltrate to the burn wounds
and remote tissues and organs [ 9 ]. The phagotrophic and
bactericidal function of neutrophils is inhibited in both
burn patients and rats [ 2 – 4 , 10 ]. Macrophages after scald
produce a large number of proinfl ammatory cytokines such
as TNF-α but produce relatively insuffi cient anti-
infl ammatory cytokines such as IL-10 (Fig. 6.1 ) [ 11 ].
Macrophages also act as professional antigen-presenting cell
in triggering the immune response. However, their antigen-
presenting function is continuously inhibited from early
hours to late several days postburn [ 5 ]. The fragments of acti-
vated complements are produced rapidly within several min-
utes after thermal injury and subsequently aggravate
infl ammatory response by increasing the proinfl ammatory
cytokine production from macrophages [ 12 , 13 ]. More NO
and PGE 2 are also produced by macrophages from scalded
mice [ 7 ]. Burn injury signifi cantly prolongs acute phase
response. Proinfl ammatory cytokines stimulate the liver and
cause the increased levels of some acute phase proteins in
blood, such as haptoglobin (HP) and α 1 acid glycoprotein
(AG) [ 14 , 15 ]. These proteins and small inorganic molecules
have inhibitory effect on various immune cell functions and
become the immunosuppressive factors in burn serum [ 7 ,
15 ]. We think, therefore, that the changes of innate immune
response after burn would be mainly characterized by hyper-
infl ammatory
response
with
depressed
function
of
phagocytes.
Severe burn injury can cause the marked changes of cel-
lular and humoral immunity in adaptive immune response
[ 1 – 3 , 16 – 20 ]. The total T-cell number and the CD4/CD8
ratio in peripheral blood are decreased, and the proliferations
of T cells from the blood, thymus, and spleen are inhibited
Advances in Burn Immunology
Daizhi Peng
6

108
under the stimulation of various mitogens after burn. The
immune-stimulating cytokines such as interleukin-2 (IL-2)
secreted by T cells are depressed continuously for a longer
time, while the immunosuppressive cytokines (such as
IL-10) produced by T cells are relatively increased. B cells
also synthesize and secrete less amounts of immunoglobu-
lins following thermal injury. Obviously, the main properties
of aberrant adaptive immune response after burn are the
depressed cell-mediated immunity with the shift of Th (T
helper) cells from Th 1 type to Th 2 type.
Many researchers at home and abroad found that the func-
tion of various immune cells is in an inhibited state and
describe the postburn changes of immune function as immu-
nosuppression [ 21 ]. Through comprehensive and systematic
analysis of various immune cells and molecules, we fi nd that
postburn changes of immune function are complicated.
Some are always upregulated and downregulated, and others
exhibit two-phase changes with earlier increase and later
decrease or vice versa. Even in one type of immune cell, one
of their functions may be hyperfunction and the other may be
hypofunction at the same time. We summarized this compli-
cated immunological phenomenon following thermal injury
as the postburn immune dysfunction in 1994 [ 5 ]. Now it has
been recognized and widely used by most scholars [ 22 ].
6.3
Etiological Analysis of Postburn
Immune Dysfunction
Burn causes acute wound pain and massive wound necrotic
tissue. Hypovolemic shock and wound infection or even sys-
temic infection would occur subsequently. These events, act-
ing as stressors, can stimulate the central nervous system and
sequentially activate the autonomic nervous system and the
hypothalamic-pituitary-adrenal axis (HPAA). The immune
response is regulated directly by neuronal activation or
indirectly through the release of catecholamines, glucocorti-
coids, nerve tachykinins, and various neuropeptides [ 3 , 16 ].
Denatured and necrotic tissue of burn wound is the main
reason of postburn immune dysfunction. Injured tissue can
not only directly activate the coagulation, fi brinolytic, kinin,
and complement systems in the circulating blood, which
subsequently produces a large number of infl ammatory
mediators [ 23 ], but also can release cellular components
with powerful proinfl ammatory effects, such as DNA, high-
mobility group protein B1 (HMGB1) (Fig. 6.2 ), and even
burn eschar toxin [ 24 , 25 ]. These infl ammatory mediators
are leaked out into burn wound and stored in subeschar tissue
edema fl uid. Burn wound is the critical source and reservoir
of these infl ammatory mediators.
Severely burn patients usually undergo hypovolemic
shock with cellular oxygen insuffi ciency. It has been proved
that hypoxia markedly promotes macrophages to produce
proinfl ammatory cytokines, such as TNF-α, IL-1, and IL-6,
and continuously inhibits IL-2 production of T cells.
Systemic hypoxia can also induce a rapid infl ammatory
response. Hypoxia combined with burn serum can increase
the expressions of TNF-α and IL-1 mRNA in cardiac myo-
cyte [ 26 ]. The components of bacteria during infection such
as endotoxin and bacterial DNA released into the blood-
stream are proinfl ammatory mediators. The burn wound con-
tamination and intestinal bacteria translocation that often
occur after severe burns can lead to endotoxemia and signifi -
cantly increase the levels of proinfl ammatory cytokines in
serum, such as HMGB1 and TNF-α [ 5 , 25 – 27 ] (Fig. 6.2 ).
For severe burn patients, fl uid and electrolyte imbalance
and acid-base disorders usually occur in fl uid resuscitation
and infection period. Nutrient defi ciency or loss is derived
from hypermetabolism and wound exudates. Antimicrobial
agents, anesthetic agents, and blood products for repetitious
*
#
#
#
#
#
#
#
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
2
6
12
24
48
PBH
Relative expression
TNF–a
IL–10
Fig. 6.1 The expressions of
TNF-α and IL-10 mRNA in
macrophages after burn. Relative
expression represents the ratio
between TNF-α or IL-10 and
β-actin mRNA expression,
respectively. # p < 0.05, *
p < 0.01, compared with 0
postburn hour ( PBH )

6 Advances in Burn Immunology

109
operation are often administered during the long-term
treatment. These therapeutics-associated regimens have
different effect on immune cells and molecules. The massive
tissue necrosis, severe stress reaction, shock, infection,
malnutrition, and various therapeutic procedures after major
burns can alter the microenvironment of the immune cells
and molecules in which they reside and sequentially result in
immune dysfunction (Fig. 6.3 ). These burn-derived events
and therapeutics- associated factors under a variety of
circumstances can contribute to the development of immune
dysfunction following thermal injury. They have become the
main reasons in the pathogenesis of postburn immune
dysfunction.
6.4
Mechanism and Clinical
Consequences of Postburn Immune
Dysfunction
Postburn immune dysfunction is chiefl y characterized by
hyperinfl ammatory response and hypoimmune response.
The imbalance between excessive infl ammatory mediators
and relatively insuffi cient anti-infl ammatory mediators
results in hyperinfl ammatory response or systemic infl am-
matory reaction syndrome (SIRS). The sustained activated
state of transmembrane signal transduction system during
the early postburn period and obviously activated intracel-
lular signal pathway in macrophages play an important
role in the molecular mechanism of excessive infl amma-
tory mediators secreted by macrophages [ 11 , 28 , 29 ]. The
cytokines secreted by T cells following burns are trans-
formed from Th 1 -type cytokines into Th 2 -type cytokines.
The transmembrane signal transduction system of T cells
has been in an inhibited state for a long time after burn,
and most of intracellular signal transduction pathways in
1
2
3
4
5
6
7
8
9
10
Fig. 6.2 The change of HMGB1 level in serum after burn and burn
wound sepsis. Lanes 1–5 in Western blotting represent the sera from 3
days before burn and 4, 7, 11, and 14 days after burn, respectively;
lanes 6–9 represent the sera from 3 days before infection and 4, 7, and
9 days after infection, respectively; lane 10 represents the molecular
weight marker
……
Burns
Shock
Infection Hypermetabolism
Tissue injury
Denatured proteins
Necrotic cells
Apoptosis cells
Patient factors:
Altered microenvironment
Therapeutic factors:
Anaesthesia
Fluid resuscitation
Genetic background
Antimicrobial agent
Immune cells & molecules
Immune dysfunction
……
Age
Gender
Stress
Fig. 6.3 Etiological factors of
postburn immune dysfunction
(this fi gure is adapted from the
textbook of Pediatric Burns,
Cambria Press, New York, USA,
2010)


6.4 Mechanism and Clinical Consequences of Postburn Immune Dysfunction

110
T cells are also continuously suppressed. These consti-
tute the molecular basis of decreased T-cell proliferation,
diminished Th 1 -type cytokines, and increased Th 2 -type
cytokines [ 18 , 19 , 30 ].
Severe burns usually activate and overactivate macro-
phages. Subsequently, antigen-presenting capacity of macro-
phages is continuously depressed; and infl ammatory
cytokines and immunosuppressive are excessively produced
by macrophages. They can act on neutrophils and lympho-
cytes, which cause hyperinfl ammation and cell-mediated
immunity suppression, respectively [ 5 , 7 , 15 ]. Therefore,
macrophage is a critical initiating factor of postburn immune
dysfunction (Fig. 6.4 ). Compared with an earlier occurred
hyperinfl ammation response, a latter anti-infl ammatory
response can limit infl ammatory response. A large number
of anti-infl ammatory mediators, acting as immunosuppres-
sive factors, may also directly induce the resulting hypoim-
mune response.
Severe burns result in hyperinfl ammatory response,
which causes large numbers of neutrophils infi ltrating in
the tissues. The sequestrated neutrophils along with the
infl ammatory mediators can result in tissue and organ
damage. The depressed phagocyte function and adaptive
immunity enhance the susceptibility of host to pathogens.
Extensive wound and epithelial tissue injury of the respira-
tory and alimentary tract result in bacterial and endotoxin
translocation as well as endotoxemia. The invasive wound
infection, pneumonia, and systemic infections are easily
caused by conditional pathogens. Infection itself can inten-
sify the infl ammatory response and hypoimmune response.
Hypoimmune response could also delay the rejection of
the body and prolong the survival of skin allograft.
Severely burn patients, thus, are vulnerable to infections
and organ damage, which are common lethal complica-
tions (Fig. 6.4 ).
6.5
Preventive and Therapeutic Measures
of Postburn Immune Dysfunction
The preventive and therapeutic measures of postburn immune
dysfunction include three aspects, i.e., the measures aiming
at the etiological factors, the application of immunomodula-
tor acting on the altered parameters of immune function, and
the administration of drugs targeting the critical molecules of
abnormal signal transduction in immune cells (Fig. 6.5 ). It
has been proved by extensive evidence from clinical and
experimental studies that prompt wound coverage [ 19 , 31 ],
proper fl uid resuscitation with cytoprotective drugs [ 32 ],
appropriate antibiotic regimen considering its releasing
effect on pathogen components [ 33 , 34 ], enteral nutrition
support combined with immune nutrients and parenteral
growth hormone [ 35 ], and the medication reducing the stress
response [ 8 ] have the effect of relieving the immune
Neutrophils
Inflammatory
Cytokines ↑
Innat
Immunity
(such as TNF-α, IL-6)
(mostly refers to hyperinflammation)
Burns
Acute Phase Proteins ↑
(such as HP, AG)
Immunosuppressive
Factors ↑
Adaptive
Immunity ↓
Systemic
Infection
(such as PGE2, NO)
Antigen-presenting ↓
Lymphocytes
(Th1 → Th2 type)
Organ
Damage
Macrophages
Fig. 6.4 General pattern and
clinical consequences of burn
immune dysfunction

6 Advances in Burn Immunology

111
dysfunction for burn patients. These measures all directly
aim at the etiological factors.
The parameters of immune dysfunction in burned animals
have been improved by a variety of Chinese and Western
immunomodulators, such as Flos Lonicerae, Polygonum cus-
pidatum , Radix Ginseng, thymosin, ibuprofen, cimetidine,
and granulocyte colony-stimulating factor (G-CSF) [ 36 , 37 ].
The immunomodulative effects of thymosin and G-CSF have
also been proved in burn patients. The cutting edge of immu-
nomodulator study in the fi eld of postburn immune dysfunc-
tion has been focused on the drugs targeting the critical
molecules in aberrant signal transduction of immune cells.
The preliminary results from our institute show that panax
notoginseng saponin has a biphasic regulative effect on
nuclear factor-κB (NF-κB) of postburn macrophages [ 11 ]. It
might be an optimal candidate drug for ameliorating the
uncontrolled infl ammatory response. In terms of postburn
depressed cell-mediated immunity, the immunomodulators
aiming at the key molecules in T-cell signal transduction
could be selected from protein kinase C agonists and Ca 2+
channel activators [ 30 ].
6.6
Prospectives in the Field of Burn
Immunology Research
The studies of burn immunology have shown that severe
burns have extensive impact on the various cells and mole-
cules of the immune system. Most of clinical and experimen-
tal researches on the burn immunology remain in observing
the phenomenon of postburn immune dysfunction, and some
of them have begun to understand its mechanism at the cel-
lular and molecular levels of immune function. There are
considerably complicated interactions between immune
cells and molecules. Various indexes of immune function
represent the different profi les of the immune system. It is
diffi cult to evaluate clinical effi cacy of immunomodulators
in a few clinical trials of burn patients.
It is evident that the research of burn immunology has
come into a very crucial period. In order to transform the
understanding of postburn immune dysfunction into clinical
practice, continually deepening the connotation and extend-
ing the denotation on burn immune research have to be
emphasized. Various causes and their relationship resulting
in postburn immune dysfunction should be analyzed glob-
ally. The interactions and balances between several impor-
tant molecules in the pathogenesis of postburn immune
dysfunction should be further explored. These will provide
a comprehensive clinical protocol for preventing and ame-
liorating the postburn immune dysfunction. Along with the
rapid development of modern immunology, new theories
and techniques are emerging in the areas of molecular
immunology, transplantation immunology, and infection
immunity and could be used to solve the critical immuno-
logical issues in burn medicine, such as immune tolerance
induction of skin allograft and comprehensive treatment of
sepsis [ 38 ]. Through our long-term efforts in these direc-
tions, we expect to make a breakthrough in burn immunol-
ogy and a new contribution for the development of burn
medicine in China.
Acknowledgments This article was supported by grants from the
National Natural Science Foundation of China (Grand Program, No.
39290700-01; programs of No. 39500149, 30240024, and 3047679),
National Key Basic Research and Development Project of China (973
Project, No. 2005CB522605), and National High Technology Research
and Development Project of China (863 Project, No. 2006AA02A121).
Drugs targeting at
abnormal activation
of cells and molecules
Measures aiming at the
etiological factors
Drugs acting on immune
function indexes
Immune molecules
Immune dysfunction
Thermal injury
Abnormal microenvironment
Immune cells
Aberrant signal transduction
Fig. 6.5 Preventive
and therapeutic strategies
of burn immune dysfunction

6.6 Prospectives in the Field of Burn Immunology Research

112
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subeschar tissue fl uid and plasma in burned guinea pigs. Chin J
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6 Advances in Burn Immunology

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Z. Yang, Chinese Burn Surgery, DOI 10.1007/978-94-017-8575-4_7,
© Springer Science+Business Media Dordrecht and People’s Medical Publishing House 2015
7.1
Escharectomy En Masse
and Autologous-Allogeneic
Intermingle Skin Graft for Extensive
Full-Thickness Burn
Zhenjiang Liao
Escharectomy is an operation to excise a full-thickness burn
wound. Then the wound is covered with allogeneic or xeno-
geneic skin. Intermingled skin grafting is an operation that
shears 0.3 × 0.3 cm to 1.0 × 1.0 cm windows of allogeneic or
xenogeneic skin with scissors some days post-escharectomy,
and then the corresponding sized pieces of split thickness
autologous skin are put into the windows to protect the wound
from exposure during wound healing. Escharectomy en
masse with autologous–allogeneic intermingled skin grafting
for extensive full-thickness burns is one of the successes of
burn surgery based on the development of critical care and
immunology in the 1960s. Because of the different character-
istics of immune-competence, when the allo- epidermis was
rejected, the allo-dermal layer was preserved on the wound
bed. Then, auto-epidermal were creep on allograft dermal
scaffold . This measure can ensure small pieces of autologous
skin to repair the large area of the full- thickness burn wounds
[ 1 ]. Because the later operation requires a huge amount of
manual work, it has been seen as the sword of age of cold
steel glisten till now . Undoubtedly, this measure which is
invited by the Chinese, the RuiJin Hospital of Shanghai, has
extracted the long and profound background of Chinese
culture and wisdom. Such surgical techniques soaked up the
sweat of our older generation, encouraging us to make
sacrifi ces for human medical science.
7.1.1
Background of Intermingled Skin
Grafting
Before the 1960s, the majority of burn surgeons focused on
the fl uid resuscitation from burn shock. In the 1950s, Evans
and his colleagues recommended a fl uid resuscitation
formula relating the burn surface to body weight during the
fi rst 48 h. Then, some formulas according to the clinical and
pathophysiological research results, such as that presented
by RuiJin Hospital in Shanghai and South-West Hospital of
the Third Military Medical University, help many patients to
survive shock in the initial phase. With the development of
skills in fl uid resuscitation, mortality from hypovolemic
shock and its complications decreased. Burn wound infec-
tion or burn wound-induced sepsis then became the major
cause of mortality of patients with extensive burns. At that
time many burn surgeons turn their consideration to the
importance of the excision of necrotic tissue and the wound
bed covered by cadaver skin or xenogeneic skin. However,
both perioperative preparations and the technique of skin
grafting were not resolved at that moment.
Effi cacious topical antimicrobial agents, even disinfec-
tant, or traditional Chinese medicine, were widely used to
prevent wounds from infection and to promote wound
healing until the 1960s. For full-thickness burn wounds, burn
surgeons usually used the abovementioned measures to dis-
solve necrotic tissue and form granulation tissue on the burn
wound bed. Surgeons then grafted small pieces of auto-skin
on the wound [ 2 ]. The risk factor for this method was sepsis
resulting from wound infection during the dissolved necrotic
tissue and wound bed bearing, which resulted in evaporative
fl uid loss and infection. Repeat anesthetic and bleeding dur-
ing the operation increased the incidence of immunosuppres-
sion and infection. Research into trauma, especially with
models of burns, advocated that wound infection or wound-
induced sepsis and repeat operation can be described as “sec-
ond or secondary hate” to burns patients. Before the 1970s
many burns surgeons began to try escharectomy to excise the
full-thickness burn wound with the understanding that the
existence of the burn wound is the reasons for the patients’
death. Because every excision was smaller than 10 % body
surface area (BSA), escharectomy at that time was so-called
“timely (phasic) escharectomy and auto-skin graft.”
An extensive burns patient would be required to undergo
Burn Wound Care
Bi Chen, Zhenjiang Liao, Zhenrong Guo,
Xiaoyuan Huang, and Yiping Zhou
7

114
operation fi ve to ten times and its coverage using various
techniques. Obviously, repeat operations increase the risk of
mortality and morbidity of sepsis for a patient with extensive
burns [ 3 ]. At the beginning of the 1950s, burns surgeons
tended to reach a consensus: to reduce mortality, the number
of operations should be decreased and prevent wound expo-
sure during the healing process.
Escharectomy and allo-auto intermingled skin grafting
are two pieces of interactively developed surgical technol-
ogy. Escharectomy en masse is not a diffi cult operation in
itself, but related problems such as the patient’s tolerance
and perioperative resuscitation, are more complicated. How
to grasp the balance between the speed of allo-grafting and
the growth of the auto-epidermis or the size of the auto-skin
that can be supplied for autografting is another important
question that we thought about. There is no doubt that the
quantity of auto-skin is a key point in surviving extensive
burns. At that time burn wounds had to be covered fi nally in
auto-skin. However, for a patient with extensive full-
thickness burns, they always lack auto-skin. Burns surgeons
should face how to utilize the limited auto-skin to repair a
large area of full-thickness burn wounds.
Undoubtedly, Shih Tsi-siang, Yang Chih-chun, and their
colleagues (Shanghai RuiJin Hospital) were pioneers in
wound excision and skin grafting in China. Without expo-
sure of the wound bed, their creative methods allowed sur-
geons to cure patients with extensive full-thickness burns
with a small area of normal skin as a donor site to repair a
large area of full-thickness burn wounds. Following on from
Jackson’s ideas, Jackson from the USA and Janzekovic from
the Former Republic of Yugoslavia who showed earlier that
excision and skin grafting can decrease mortality and length
of hospital stay in patients with extensive burns as early
1954. Jackson and some other burns surgeons are the advo-
cates of the “mixed skin graft” at that time. They covered the
wound bed post-excision with the 1.5-cm-wide auto-skin
sheets and the same wide allo-skin sheets in the interval.
However, Jackson’s method can only cover a limited area of
full-thickness burn wounds. For the patients who suffered
from full-thickness burn wounds, their destinies were pitiful.
Restricted by medical developments such as pathophysiol-
ogy, histology, and immunology, people were unable do
more profound research. In any case, his method was a bright
torch in the darkness at that time. Inspired by Jackson’s
method, Shih Tsi-siang and Yang Chih-chun from Shanghai
RuiJin Hospital developed the auto-allo-skin graft in a chi-
meric measure like a Chinese-style brick. It provided the
possibility of repairing a large area of full-thickness burn
wounds with limited auto-skin in the 1960s.
In 1966, a major advancement in allo-auto-skin grafting
was developed to decrease the mortality and morbidity of
wound-induced sepsis in extensive full-thickness burns
patient by Shih Tsi-siang and Yang Chih-chun. First, they
tried a larger escharectomy and to cover the wound bed
postoperatively in cadaver skin. Three to seven days after
escharectomy, they began to shear 0.3 × 0.3 cm to 1.0 × 1.0 cm
windows into allo-skin, and then graft the corresponding
sized pieces of split thickness auto-skin into the windows in
a timely fashion. This method makes the best of the exten-
sion of epidermal (Eb) cells creep over the borderline of graft
auto-skin. It is convenient to fi nd that the smaller piece of
auto-skin with a relatively longer circumference has a large
expansive area and a faster work. Thus, we could use a small
area of auto-skin to repair a large area full- thickness burn
wound. It was observed that if the area of auto-skin is greater
than 0.3 × 0.3 cm (0.09 cm 2 ), the expansion ratio of auto- and
allo-skin can be achieved up to 1:10 (expanded nine times).
In other words we can use 10 % of the normal autologous
skin of the BSA to repair a 90 % BSA wound. We then found
that the scalp can be shaved repeatedly as a donor site called
a “body skin bank”. In 1977, Yang Chih-chun and Liao
Zhenjiang’s group advocated the practice of the scalp donor
site and successfully cured a woman with 100 % burns of the
total body surface area (TBSA), whose third-degree burn
wound was 94 % BSA. The name “intermingled skin graft-
ing” was proposed in that fi rst case report. Since then, inter-
mingled skin grafting became one of the mainstream
measures for curing extensive full-thickness burns patients
in the last four decades. Because of a signifi cant decrease in
mortality and morbidity of wound-induced sepsis, this mea-
sure gave Shih Tsi-siang, Yang Chih-chun, and their Chinese
colleagues an opportunity to establish their position as world
leaders in burns care.
7.1.2
Escharectomy En Masse
and Intermingled Skin Grafting
for Extensive Full-Thickness Burns
Patients
Excision of necrotic tissue and coverage by allo-skin as a
temporary substitute had became a necessary choice for a
patient to survive an extensive full-thickness burn after
escharectomy. A large area of the wound bed would be
exposed if the allo-skin were rejected. Exposure of the
wound bed results in further pathological progress such as
wound infection and burn wound-induced sepsis. Burns sur-
geons then tried to fi nd a way to prevent exposure of a full-
thickness burn wound post- allo- and auto-skin graft by
extension of epidermal cell creep at the borderline of the
auto-skin while the allo-epidermis is rejected, and intermin-
gled skin grafting came to be the only method of preventing
patients from acquiring sepsis because of the large wound
area.
7.1.2.1 Choice of Operative Phase
Before the 1990s, one of the most important consensuses
was that an extensive excision should be carried out between
7 Burn Wound Care

115
the 5th and the 7th days post-burn. There was a concern that
extensive burns patients had to overcome the risk of hypovo-
lemic shock at that time. The patients’ systemic situation
returned to relative stability. However, the interstitial space
in both injured and uninjured tissues, especially under
eschar, accumulated a large quantity of proteinaceous exu-
dates lost from blood vessels because of the consequence of
damage or increased permeability of capillary endothelium.
Within a few days, burn-induced systemic infl ammatory
response syndrome (SIRS) or sepsis, so-called re-absorp-
tion-induced sepsis, could result from the re-absorption of
this huge quantity of protein or proinfl ammatory factor-rich
exudate fl uid.
Nowadays, we have come to understand that so-called
re-absorption- induced sepsis would not always result from
bacterial infection. Many researchers have reported a large
quantity of fl uid under necrotic skin and the interstitial tissue
became an excellent environment for the proliferation of pro-
infl ammatory cytokines and products of necrotic burn tissue
or endotoxin of bacteria. These may lead to excessive infl am-
matory response and substantial immunosuppression. In the
1990s of last century, some burns surgeon proposed that
excision of necrotic wounds should begin between the fi rst
and the second 24 h post-burn with development of anesthe-
siology and critical care medicine.
Sheng Zhiyong and some Chinese burns surgeons and
researchers advocated in clinical practice and research about
the excision of burn wounds in fi rst 48 h post-burn. They
reported that wound excision in that phase could decrease
the length of the hospital stay and mortality in extensively
burned patients.
Randomized prospective studies conducted in the USA
have shown a decrease in mortality in extensively burned
adults with third-degree burns when they were treated with
early excision of the total burn wound as opposed to conser-
vative treatment. Herndon et al. also reported that massively
burned children with 95 % TBSA burns may have a 50 %
survival rate after early burn wound excision if a burns center
has developed the conditions for anesthesia and resuscita-
tion. As described by Herndon, burn wound excision during
an earlier phase can be carried out as soon as possible. It can
decrease the risk of wound-induced sepsis mortality in
patients with extensive full-thickness burns.
Because of decreased resistance to fl uid accumulation or
edema production and cardiac compliance in the elderly,
some measures should be applied to improve the circulatory
and cardiac and even pulmonary functions. The same
approach to wound care used in younger patients applies to
the elderly. As older patients can tolerate full-thickness burn
wound excision, a conservative approach is not warranted.
Escharectomy during the earlier phase can decrease the mor-
tality and morbidity of wound-induced sepsis in the elderly.
We have found that early wound excision has been one of the
most effective measures in decreasing mortality in extensive
full-thickness burns patients. When we reviewed our work
over the past two decades, certain questions were raised. We
had discussed the operation during the resuscitation phase.
Questions had been raised, such as whether carrying out this
operation with a short interval after the original burns injury
could come to be a superimposed hate and what kinds of
systemic response and dynamic changes in hemodynamics
and homeostasis would occur. Even if wound excision dur-
ing the resuscitation phase had been believed to have more
benefi cial effects, we would have to discuss which operative
phases are suitable for implementation during the fi rst 3 days
post-burn. In our study we found that there are dynamic sys-
temic changes and responses that take place during the fi rst
8 h and fi rst 24 h or the second 24 h post-burn. Even if a large
area is burned during the fl uid resuscitation, and each time
the status of the circulatory system is also completely differ-
ent, what is the best time for the operation? Unfortunately,
we can only say “the sooner the better” to describe wound
excision during the resuscitation phase. Next, we began to
study which operative phase is more suitable for regular sys-
temic responses. We tried to propose a guideline on the oper-
ative phase of wound excision and the management during
the perioperative phase.
7.1.2.2 Escharectomy in Large Deep Burns
Currently, after the vital signs have been stabilized, initial
escharectomy–allogeneic skin transplantation can be per-
formed for extensive full-thickness burns patients 1–5 days
post-burn [ 13 ]. Our experience is that escharectomy should
fi rst removed necrotic tissue in the four limbs. According to
the current techniques of anesthesia care and the progress of
peri-operative treatment, escharectomy involving >60 % of
the TBSA is not always recommended. Before escharec-
tomy, a tourniquet should be used on the four limbs. Driving
blood from the distal to the proximal end of a limb when the
tourniquet is applied should be forbidden, to prevent the
release of infl ammatory cytokines, bacteria, or bacterial tox-
ins from the interstitium into the circulation. In general,
escharectomy starts at both the upper and lower edges of
burn wounds in the limb. In consideration of the future
appearance of the healed wound and for the convenience of
hemostasis during operation, excision of the upper and lower
edges of the eschar should be performed obliquely until
punctuate bleeding of the dermis is noted. Thereafter, the
eschar is slit at the lateral line of the limb from top-to-bottom
and perpendicular to the wound [ 14 ]. If escharectomy had
been performed for decompression, the decompression
incision line should not be used for subsequent surgical pro-
cedures given the risk of infection, and a new incision should
be made nearby. Escharectomy is generally performed in the
plane of the deep fascia. The eschar is fi rst cut open to iden-
tify the surgical dissecting plane and an assistant uses an
Allis clamp to clip the eschar to lift and excise it along the
dissecting plane of the deep fascia.
7.1 Escharectomy En Masse and Autologous-Allogeneic Intermingle Skin Graft for Extensive Full-Thickness Burn

116
In the scenarios in which muscles are thought to be
involved in the full-thickness burns of limbs, or no
decompression incision is made in the circumferential
full- thickness burns, the fascia should be incised for explo-
ration. After excision of the eschar, the count and the kinds
of bacteria under the eschar need to be identifi ed and quanti-
fi ed, which could more accurately indicate likely pathogens
for invasive infections, and can guide the selection of anti-
microbials [ 15 ].
The escharectomy wounds are covered by a warm saline
dressing, and then bandaged from the distal-to-proximal
ends of the limb. The temperature of warm saline should not
generally exceed 55 °C; thus, the extrinsic coagulation path-
way is activated to achieve homeostasis. The tourniquets on
the four limbs cannot be released at the same time, and the
anesthesiologist should be informed before releasing
the tourniquet. Under the guidance of the anesthesiologist,
the tourniquets are depressurized in sequence as the simulta-
neous release of the tourniquets may result in the sequestra-
tion of blood in the limbs, leading to a dramatic decrease in
the blood volume of circulation. After the release of the tour-
niquets, the bandages should be removed from the proximal
to the distal end, which can avoid profuse bleeding in the
surgical fi eld due to obstructed venous fl ow to the heart
because of incomplete release of the proximal ends. After
removal of the bandages, warm saline gauzes should not be
removed without fi rst being rinsed with warm saline to avoid
re- bleeding of the wound surface as a result of the disruption
of blood clots by the abrupt removal of the wound gauzes.
After removal of warm saline gauzes from the wound, bleed-
ing is mainly located at the upper and lower edges of the cut
and around the knee. If bleeding is visible, blood vessels
should be ligated. If it is punctate bleeding, electrocoagula-
tion can be applied for hemostasis. If it is suffused oozing,
epinephrine in normal saline can be applied to cover the
wound. 30 mg of epinephrine are dissolved in 1,000 ml of
normal saline and applied to the wound surface. The wound
is pressed and dressed from the distal to the proximal end.
After 3–5 min, the bandage is removed from the proximal to
the distal end to the heart, and the gauze is rinsed with nor-
mal saline, which is gently lifted from the wound for more
complete hemostasis. In order to avoid contamination of the
wound surface by bacteria in the eschar, in general, chlorhex-
idine (1:2,000 solution) can be used to clean and moisturize
the wound after the bleeding has been completely controlled.
We have studied the amount of bleeding during the operation
to estimate the approximate amount of blood for transfusion
in adults during escharectomy of burn wounds in the limbs.
Each upper limb requires approximately 400 ml of whole
blood, and each lower limb requires approximately 600 ml of
whole blood. In general, escharectomy for all limbs requires
approximately 1,600 ml of blood. In order to shorten
the operative time, the nurses or physicians need to start
the preparation of allo-skin during hemostasis after the
escharectomy. If the allo-skin is cryopreserved, after
warming the skin graft is washed with saline and gently
rubbed until there is no fl occulation in the cleaning basin.
The skin graft is punctured depending on the needs. If the
skin is simply for drainage, a pointed blade can be used for
puncture. Generally, the window size of the allo-skin is
determined together with the size of the autologous skin
graft. Currently, there are small windows that can punch
0.3–1.0 cm in size in two longitudinal rows 1 cm apart. If
there is no skin stapler, the large sheets of skin allo-grafts to
be transplanted are sutured together according to the circum-
ference, length, and width of the recipient limb.
Allo-skin transplantation occasionally fails. The reasons
for allo-skin graft failure include wound infections, wound
hematomas, and improperly fi xed skin grafts [ 16 ]. Wound
infections are mainly due to necrotic tissue remaining in the
basal wound or adipose tissue separated from the deep fas-
cia. The latter is frequently the cause of wound infection, and
therefore it should not be ignored during escharectomy. The
deep fascia is selected as the surgical plane for escharec-
tomy; usually, there is little necrotic tissue, but repeated elec-
trocoagulation and electric knife cutting generate new
necrotic tissues, which need to be cleaned up. In general,
especially under general anesthesia, the tension of the arte-
rial circulatory pressure system is relatively lower, and the
small blood vessels, particularly small veins, may transiently
appear hemostatic, even though occult bleeding or the poten-
tial for overt bleeding exists. When the blood pressure returns
to normal after completion of surgery, the bleeding in these
areas becomes more serious and creates subcutaneous hem-
orrhage in the allograft. Therefore, compulsive attention to
hemostasis should be practiced during the operation. If a
hematocele is found under the allogeneic skin graft after sur-
gery, the allo-skin should be incised immediately to stop
bleeding and evacuate the hematocele, and then the wound
should be appropriately bandaged. An improperly fi xed skin
graft leads to the allo-skin slipping and poor vascularization
between the basement membrane and the wound-bed, result-
ing in the failure of allogeneic skin transplantation. Therefore,
it is very important to secure the allogeneic skin graft on the
escharectomized burn wound, and the allo-skin should be
tightly attached to the escharectomized wound bed. If the
injury involves four limbs, use of thick gauze should be
avoided initially. Gauze should be placed on the hollow part
of the wound and then packed gradually to fi x the bandage.
To wrap joint parts, a cross-dressing, cross-joint fi xation
should be used to ensure that the joints will not have fl exure
movement to prevent the skin graft from slipping.
7.1.3
Method of Intermingled Skin Graft
The autologous skin graft is embedded into the punctured
allo-skin 5–7 days after escharectomy and allogeneic skin
7 Burn Wound Care

117
grafting [ 17 ], and can be repeated once every 5–7 days based
on the situation of the patient. RuiJin Hospital in Shanghai
has developed special burrowing scissors based on long-term
experience. The burrow scissors are approximately 15 cm in
length, the cutter bar of the knife shaft is 3 cm from the tip,
and the head is slightly curved. The scissors are easy to use,
and also reduce the handiwork required to puncture a
windows.
The pre-operative preparation and whole body monitor-
ing are similar to those in the escharectomy. When punctur-
ing windows in the skin graft, the bleeding is obvious;
intermingled grafting surgery is minor and the major bleed-
ing area is the donor site. The amount of skin bleeding for
every 1 % body surface area is approximately 15–20 ml.
Therefore, the amount of bleeding during surgery should be
estimated according to the skin area. Volume expansion due
to anesthesia during operation and surgical trauma during
escharectomy should be considered for the additional vol-
ume of blood required for transfusion. Complete muscle
relaxation should be achieved by anesthesia for the window-
puncturing procedure; otherwise, the embedded skin graft is
easily mobile, resulting in prolonged surgical time and an
increase in surgical trauma.
The size of the donor site and the wound area that can be
covered by auto-allo-skin are estimated before surgery. The
skin grafts are 1 cm apart. An accurate estimation can avoid
puncturing excessive unneeded windows on the allo-skin,
which jeopardize the integrity of the skin. Because the allo-
skin at the junction of the escharectomized and non-
escharectomized wound edges is easily dissolved, the
windows are always punctured 5 cm from the edge. Window
puncture refers to the cutting off of a corner or one side of
previously cut and equidistant allo-skin; cutting off a square
window in the allograft allows an autologous skin graft of
identical size to be embedded. No matter what kind of cut-
ting strategy is employed, when pulling the corner of alloge-
neic skin, do not pull the other part of allo-skin, otherwise it
will cause a massive separation between the allogeneic skin
graft and the basal membrane of the wound, or result in dis-
placement of the implanted autologous skin graft. The gen-
eral size of the autologous skin is 0.3 × 0.3 cm to 1 × 1 cm,
and there is now a “cutting machine” available to make dif-
ferent sized auto-skin pieces. When the machine is used, fi ne
mesh gauze greased with Vaseline is fi rst placed on a Bakelite
board and the autologous skin graft is then placed on the
Bakelite plate with the dermis facing upward. If the cutting
knife is far from the Bakelite and the Vaseline-greased gauze
is not cut off, there is diffi culty in picking up the transplanted
autologous skin graft. If Vaseline-greased gauze is used for
skin grafting, it should be removed as it increases the risk of
infection as a foreign body embedded in the allogeneic skin
graft. After the auto-skin graft has been transplanted, the
limb under surgery is gently lifted, the operative site is cov-
ered with thick mesh Vaseline-greased gauzes and wrapped
with a bandage impregnated with antimicrobial agents, and
then dressed with gauze to fi x in place. After the burn wound
in the limbs has undergone intermingled punctured skin
grafting, the limbs should be well-immobilized. Figure-of-
eight bandages are used to stabilize the joints and plaster is
used to prevent the bending of joints. The movement of joints
can cause skin slippage and displacement. The immobiliza-
tion of limb joints requires the immobilization of three joints
(the elbow, wrist, and hand joint in the upper limb, or the
knee, ankle, and foot joint in the lower limb). This method
achieves satisfactory immobilization. In the recovery stage
after anesthesia, the patients are easily agitated, which may
lead to bleeding or displacement of the skin graft. Sedatives
can be used when the patients have recovered from anesthe-
sia, with caution, as excessive use of sedatives may result in
respiratory depression.
7.1.4
Post-operative Dressing and Outcome
of Intermingled Skin Grafting
The fi rst dressing change for skin graft wounds can be
arranged on the 2nd day after the surgery. If there is a marked
increase in body temperature, wound pain, or noticeable
signs of infection in the dressing exudates or at the time of
skin grafting, the dressings should be promptly changed and
the wound should be inspected. Sedatives and analgesic
drugs can be used during the dressing change, which is con-
ducted on the ward at 28 °C with humidity <50 %. Use of
electric fans should be forbidden to prevent the spread of
bacteria. If a patient is placed in a turning frame (invented by
Dr. Stryker at the University of Michigan between 1936
and1939), the patient must fi rst be placed in the prone posi-
tion for a dressing change on the back. The dressings on the
limbs are not changed at this time to avoid irritating
the patient, which can result in movement of the limbs. After
the dressing on the back is completely changed, the patient is
placed in the supine position, and the outer layers of the
dressing on the limbs are opened. The sterilized paraffi n oil
should be used to moisturize the inner layers of the dressing,
and then the inner dressings are removed. In order to prevent
autologous skin grafts from being removed, the dressing is
gently lifted off from one corner to nearly 180° from and
nearly parallel to the wound surface, and is slowly separated.
If some auto-skin pieces adhere to the dressing during
removal, they should be returned to their original position.
After the dressing is fully opened, normal saline gauze is
used to draw out the wound exudation. Gauze should be
applied to swab the wound. Ten percent mafenide solution
can be used as a topical antimicrobial agent. There is a new
type of inner dressing containing nano-silver particles, which
can reduce the frequency of dressing changes and has a supe-
rior antibacterial effect. In general, if the wound dressing
does not have a fetid odor and infected exudates, the dressing
7.1 Escharectomy En Masse and Autologous-Allogeneic Intermingle Skin Graft for Extensive Full-Thickness Burn

118
can be changed every other day and even every 2 days. After
the change, the wound is then immobilized and dressed.
The adhesion of auto-skin grafts to the wound bed should
be noted when the dressings are changed 1–2 days post-
operatively. On the second day following surgery, the skin
graft adheres to the wound bed adhesion with no change in
color. The main work at this stage is to protect the skin and
wound substrate from separation. Three to 4 days after sur-
gery, the autologous skin graft appears pale pink and adheres
solidly to the wound substrate. A histological examination
reveals ongoing neoangiogenesis in the skin graft and the
wound bed. On day 7 after the operation, all the autologous
skin grafts embedded in the wound become slightly red, and
there is no separation between the autologous and allogeneic
skin grafts, indicating survival of the autologous skin graft.
Around day 14 after surgery, rejection of the allo-skin grafts
tend to be observed, which is manifested as a partial separa-
tion of the epidermis from the dermis with exfoliation of
some of the allogeneic epidermis. Thereafter, the autologous
epidermis extends onto the allogeneic dermis. Approximately
21 days after surgery, most of the allo-epidermis falls off, but
the allogeneic dermis adheres well to the graft bed because
of its lower immunogenicity. The autologous epidermis
expands and covers the allogeneic dermis that has shed its
epidermis, but has not yet developed into an integrated skin
patch. On about day 28, almost all of the allogeneic epider-
mis has fallen off and is substituted with fully integrated
autologous skin, and the wound heals. The presence of allo-
geneic dermis in the wound after wound healing can improve
the quality of wound healing and provides a certain degree of
elasticity and softness.
7.1.5
Infections and Rejection
of Intermingled Grafts
The speed of proliferation and migration of newly generated
epidermis at the edge of the autologous skin graft in the
intermingled skin graft wound bed is basically identical to
the speed of the rejection and shedding of the allo-skin. As a
result, the wound surface is not exposed during healing.
However, the allo-skin as a foreign body may also be rejected
or infected during the wound healing process [ 18 ]. Rejections
generally begin about 2 weeks after allograft transplantation.
The allo-epidermis fi rst becomes blistered, the allo-dermis
then becomes thickened, and part or all of the allograft
begins to fall off. The wound is susceptible to infection dur-
ing this process. Rejection and infection lead to an increase
in body temperature and marked allograft infl ammation. The
exudate increases in volume in the allo-skin and becomes
putrid. It may cause persistent high fevers. The rejected allo-
geneic skin graft should be removed at this time or during the
dressing change, and the wound should be covered with
antibiotics- soaked dressings for autologous or allogeneic
skin grafting.
7.1.6
Appropriate Management During
the Perioperative Period
Although the development of intensive care techniques has
led to breakthroughs in the surgical phase and methods, the
operation was still considered as a “second hate ” to extensive
burns patients, which is one of the most important risk fac-
tors resulting in SIRS or sepsis. Therefore, perioperative
management holds the key to a successful outcome in patient
management.
If surgery needs to be carried out within 48 h post-burn,
the microcirculation should remain perfused. Currently,
except for surgical operation or hemofi ltration, which has a
marked impact on blood circulation, invasive monitoring
methods that have been widely encouraged are not always
being practiced. Under this circumstance, the heart rate
(HR), mental status, urinary output, and arterial blood gas
analysis become the main parameters for monitoring. If
patients have undergone uneventful resuscitation after a
extensive burn, and if there is no inhalation injury, combined
injury, or full-thickness circumferential burns on the chest
and limbs, the HR can generally be maintained at between
100 and 120 beats/min. Patients normally need no sedation,
urine output is maintained at 0.5–1 ml/kg, the peripheral cir-
culation is well fi lled, and the dorsalis pedis pulse is strong.
In addition, there is no serious acidosis or increase in resid-
ual alkali, which indicates that microcirculatory perfusion is
improved. If the HR is >140 beats/min, and when respiratory
and other factors are excluded, it should be considered fi rst
that the circulatory perfusion is not adequate and there is a
need to increase the resuscitation quantity of colloids and
crystalloids. If the amount of crystalloids is increased, and
the HR increases while the dorsalis pedis pulse remains
strong, sedative drugs can be considered. We do not consider
the use of artifi cial colloids in the resuscitation stage of burn
shock. Some literature reports that artifi cial colloids may
reduce exudation from the capillary; however, the metabolic
pathway for artifi cial colloids and how they maintain the
circulatory volume during massive exudation of plasma into
the interstitium during the initial phase in extensive full-
thickness burn remains unknown.
The respiratory system is one of the commonly damaged
organs post-extensive burns because of inhalation injury or
systemic infl ammatory response syndrome and sepsis.
Changes in the respiratory system are not only associated
with depression of respiratory movement by circumferential
burn eschar in the body trunk, but are also affected by micro-
circulation perfusion, the respiratory function reserve, and
the presence or absence of inhalation injury. Patients with
7 Burn Wound Care

119
severe burns combined with severe inhalation injury undoubt-
edly need a tracheostomy. Patients with extensive burns
involving the face and neck, although the burns are superfi -
cial, sometimes need a tracheostomy or nasal intubation after
sedation because increased capillary permeability can lead to
massive swelling of the face and neck, thus compromising
ventilation. Patients with full-thickness circumferential chest
or abdominal burns need an emergency escharectomy to
reduce tension and remove the limitation of respiratory
movements, and indirectly reduce the respiratory work. If
the respiratory frequency is above 35 times/min, the HR is
above 140 beats/min, and the arterial blood gas analysis
should be promptly examined. The PCO 2 is more important
than the PO 2 as a clinical indicator in this situation because
an increased respiratory rate leads to hyperventilation, and
PO 2 may not be decreased and may be even higher, while the
decline in PCO 2 is more signifi cant. If the PO 2 is <5.7 kPa,
mechanical ventilation should be considered. Because of the
presence of various degrees of pulmonary interstitial edema
in patients with severe deep burns, the continuous positive
airway pressure (CPAP) mode is often preferred. Oxygen
fl ow may be set at 40 % initially, and adjusted based on
changes in the arterial blood gas analysis. Ventilation pres-
sure is a more important parameter and the inspiration pres-
sure can be gradually increased from 15 cm H 2 O. If there is
no serious obstacle to recycling, the positive end-expiratory
pressure (PEEP) mode can also be used. PEEP can be set at
6–10 cm H 2 O fi rst. PEEP <12 cm H 2 O reduces the chance of
ventilator-induced lung injury. If ventilation and diffusion of
the respiratory system are well-maintained, the patient will
gradually calm down, the HR will stabilize at 100–120 beats/
min, and the respiratory rate will be 20–24 times/min. This
will not interfere with autonomous respiration; moreover,
man–machine antagonism will generally not occur, which is
the chief advantage of the CPAP mode.
Many reports confi rmed the early use of sedative drugs to
regulate the stress response to critically traumatic patients.
After the problems of microcirculation and ventilation, or
when perfusion and diffusion issues have been excluded, if
patients are still agitated, hypnotics or sedative drugs can be
used. Colloid instillation is needed before using hibernation
mixture at the fl uid resuscitation stage to prevent the vasodi-
latation caused by the use of hibernation mixture, leading to
hypovolemic shock. In adults hibernation mixtures are pre-
pared by dissolving meperidine (100 mg), phenergan
(50 mg), and chlorpromazine (50 mg) in 5 % glucose solu-
tion (250 ml). Patients initially receive a rapid infusion of
50–80 ml of hibernation mixture at an infusion rate of 600–
800 ml/h, and gradually calm down at this time. However,
the HR gradually increases, and respiration becomes shallow
and fast. If the volume is well-supported before administra-
tion of the hibernation mixture, the HR can be maintained
<160 beats/min, Hibernation mixture can be continued, but
the infusion rate can be reduced to one-half of the initial rate.
The total infusion volume administered is about 100 ml, and
the patient enters a deep sleep. The speed can be maintained
slowly at about 100 ml per hour. The total volume of hiber-
nation mixture used for the fi rst time is about 150 ml. In
order to prevent the tongue being swallowed in patients with-
out a tracheotomy, an oropharyngeal airway should be pre-
pared at the bedside before infusion, and if there are
inspiratory stridors or snoring, an oropharyngeal airway
should be placed in time. If inhalation occurs, or if wheezing
or snoring occurs, an oropharyngeal airway should be placed.
If respiratory depression occurs during the administration of
hibernation mixture, the airway should be maintained fi rst. If
an oropharyngeal airway has already been placed, ventila-
tion assistance can be carried out. Mechanical ventilation
can also be used in patients who had received a tracheotomy.
Some new ventilation apparatus, especially throat covers, is
often used to maintain effective ventilation in the absence of
a tracheostomy or when an oropharyngeal airway is in use so
that medical personnel have time to calmly carry out a tra-
cheostomy. Some new types of anesthetic drugs, such as pro-
pofol, are used to reduce the patient response, but the overall
effect of these kinds of drugs, particularly on the circulation,
needs to be further investigated. Midazolam is also often
used for sedation, but because it does not work as an endor-
phin, it does not function very well in patients with relatively
intense reactions.
There is no specialized anesthetist for burns management;
therefore, an anesthetist often does not anticipate critical
events during the operation in patients with severe burn inju-
ries. At present, endotracheal intubation in combination with
intravenous anesthesia is mainly used for burns patients, and
there have been few respiratory accidents during the surgical
process. However, most anesthetics cause vascular dilation,
and can easily cause hypotension. Therefore, the main risk of
escharectomy in large severe burns or the skin graft is the
decline in effective blood volume and vascular tension. The
combined use of certain anesthetics and drugs that reduce the
HR may mask the increase in the HR from ineffective blood
volume perioperatively. If the arterial pressure or central
venous pressure or pulmonary wedge pressure cannot be
determined, the urine volume can refl ect the capacity of
microcirculatory perfusion. Hourly urine volume during sur-
gery without diuretics needs to be maintained at about
0.5–1 ml/(h·kg) in adults or 1–2 ml/(h·kg) in children.
Anesthesiologists believe that it is necessary to fully increase
the blood volume before surgery or during surgery, and thus
it is easy to control blood volume during surgery. However,
burns patients with severe infl ammatory responses often suf-
fer from poor arterial perfusion and high venous pressure,
which easily lead to pulmonary edema postoperatively.
Postoperative care for the respiratory system is therefore
very important. If bilateral wheezing appears in the early
7.1 Escharectomy En Masse and Autologous-Allogeneic Intermingle Skin Graft for Extensive Full-Thickness Burn

120
postoperative stages, aminophylline and diuretic drugs can
be used. If large volumes of liquids have already been
infused, the urine volume is small, and wheezing in the lungs
is obvious, the patient should be continuously given small
doses of norepinephrine via intravenous micro-pump at 1
and 0.5 mg/h in the fi rst and second/third hours after surgery
respectively [ 19 ]. If the vascular tension problem is con-
fi rmed, use of diuretic drugs is considered 2 h after norepi-
nephrine infusion. Then, the excess blood volume can be
thinned out by urinary output, reducing the possibility of
pulmonary edema after operation. Patients often appear irri-
tated as they awake from anesthesia, which can easily lead to
allo-skin slippage and wound bleeding. Sedatives could be
prescribed, but respiration should be carefully monitored in
the case of respiratory suppression.
If bleeding is observed on the surface of the wound dress-
ing after the operation, the boundary of bleeding should fi rst
be marked by a red pen. If the area of bleeding expands after
a short period of time, it is necessary to open the dressing to
stop the bleeding and remove the blood clot under the
allo-skin.
After the donor site has healed, autologous skin grafts are
repeated using the method above. However, repeated opera-
tions often lead to decreased immunity and increased sus-
ceptibility of infection in extensive full-thickness burns
patients. Therefore, the duration of each skin graft operation
is one of the key factors in reducing complications and mor-
tality. There is no experience draw upon and the clinical situ-
ation is the important basis for evaluating whether patients
can withstand the operation, primarily their mental status,
the presence or absence of abdominal distension, respiratory
status, and other factors. If patients have symptoms of sepsis,
the operation should be performed after the systemic situa-
tion of the patients is in a stable condition to reduce the risks
associated with surgery. Eight to 18 h after each operation is
considered to be the period in which the patients respond to
the operation. The patients show rapid shallow breathing, the
HR is >140 beats/min, and wheezing is heard during the
chest auscultation. In severe cases, abdominal distension and
even urinary output reduction are seen. These problems are
often due to the stress following operations that aggravate
the infl ammatory response, which manifests as pulmonary
interstitial edema and small gastrointestinal ischemia. The
treatment should focus on maintaining the visceral blood
supply and ventilation support. Use of sedatives may exacer-
bate hypotension, and thus use of appropriate pressor agents
is often a better choice. If diffuse wheezing is detected via
lung auscultation, aminophylline can be applied via intrave-
nous injection; if the PCO 2 is <5.7 kPa, mechanical ventila-
tion should be applied rather than passively waiting for a
decrease in the PO 2 .
After escharectomy for extensive full-thickness burns is
performed, which is followed by embedding a small
autologous skin graft in the punctured holes, the wound can
heal without exposure to the external environment, and the
success rate of critically ill burns patients can be improved.
That is why this operation is still widely used in China, and
it can establish a solid foundation for developing a new
method of skin graft operation. Burns experts sum up inter-
mingled skin grafting as a candle in the dark, lighting the
way for the development of burns medicine. In recent years,
research into the intermingled skin grafting mechanism has
provided a concept for a series of issues, such as the immune
response and the systemic response to surgical organ trans-
plantation, and has highlighted burns and trauma operations
as role models.
7.1.7
Mechanisms of Immune Rejection
of Intermingled Skin Grafts
Intermingled skin grafts have components of both autolo-
gous and allogeneic grafts and induce different immune
rejection responses. (1) The autologous epidermis in the
graft is shielded from immune rejection because it shares an
identical MHC background with the recipient tissue. Its
growth is accelerated in the presence of an allogeneic der-
mis. (2) The fi broblasts (FBs) in the allogeneic dermis elicit
almost no host immune response because they have weak
immunogenicity. Thus, these cells remain in the recipient’s
body for a long time and become the scaffold for auto-
epidermis in the wound repair process. (3) The allogeneic
epidermis is the main target of acute rejection, which could
be blunted by intermingled allogeneic and autologous epi-
dermis. In intermingled skin grafts, subclinical rejection
(with exfoliation of the epithelial cells from the wound sur-
face) occurs mainly in the allogeneic skin. The muted inten-
sity of graft rejection is currently attributed to the development
of tolerance as a result of intermingled autologous and allo-
geneic skin grafting [ 23 ].
7.1.8
Studies on the Systemic Immune State
After Intermingled Skin Grafting
A important scientifi c project, Mechanisms of Wound
Healing , funded by the National Natural Science Foundation,
carried out under the auspices of Dr. Jixiang Shi and Dr. Ao
Li in the 1990s, has led to the fi nding that the host’s response
to allogeneic skin grafts is not only alloantigen-specifi c but
also nonspecifi c. Changes in the status of systemic immunity
following severe extensive burns also affect the host’s
response to allogeneic skin grafts. Hufnagel et al. observed
in 1989 that under sham-burn conditions, the main immuno-
logical factors protecting intermingled skin grafts from
rejection were not outside the graft. Reports from Shanghai
7 Burn Wound Care

121
RuiJin Hospital studied the systemic immune response to
allogeneic skin grafts, intermingled skin grafts, and no skin
grafts (control group) in a rat model of an extensive full-
thickness burn. The percentages of CD4 + and CD8 + T cells
and T cell proliferation (mixed leukocyte reaction [MLR])
were signifi cantly reduced in the three groups compared
with sham burn rats 4 days after burn injury. The number of
CD4 + T cells increased rapidly in the two groups of skin graft
recipients on day 7, and the proportion of CD8 + T cells
increased on day 14. The response of recipient T cells to the
allogeneic graft was 16,487 (in cpm) in normal rats and
9,474 (in cpm), 11,390, and 12,571 in the control group (the
negative control) 4, 7, and 14 days after burn injury respec-
tively; It suggests that suppression had occurred. The
response of allogeneic skin graft recipients (the positive
control; 19,031, 20,625, and 25,368 respectively) was
signifi cantly different from that of the control group, indicat-
ing that the immune response to the allogeneic skin graft was
strong enough to mediate rejection. Meanwhile the response
to the intermingled skin graft was intermediate between the
responses of the negative control and the positive control
groups. The early responses to the intermingled skin grafts
(13,429 and 19,384 cpm on day 4 and day 7, respectively)
and allogeneic skin grafts (19,031 and 20,625 cpm) were
very approximative. However, the response to intermingled
skin grafts after 14 days postoperatively was less than the
response to allogeneic skin grafts, but close to that of the
control group (Fig. 7.1 ). This suggested that T cell recogni-
tion of alloantigen in the intermingled skin decreased to a
certain extent 2 weeks after skin grafting. However, Fig. 7.2
shows that the cytocidal activity of the cytotoxic T lympho-
cyte (CTL) cells that dominate the specifi c immune response
increased signifi cantly compared with that in the control
group from day 7 to day 21 after intermingled skin grafting,
but tended to decline until day 28. This suggests that the sys-
temic immune response induced by the intermingled skin
graft, particularly immune cytocidal effects, remained strong
during the 3 weeks after skin grafting and did not fall below
a level that was too low to mediate graft rejection. In other
words, the difference between immune rejection in intermin-
gled skin graft recipients and in allogeneic skin graft recipi-
ents is not suffi cient to explain the difference between the
rapid rejection of simple allogeneic skin grafts and the sub-
clinical rejection of intermingled skin grafts observed in
clinical practice. This suggests that the decline in the sys-
temic immune status after burn injury helps prevent acute
rejection of the intermingled skin graft, but it does not play a
decisive role in the early stages [ 24 ].
0
5,000
10,000
15,000
20,000
25,000
30,000
0 day
4 days
7 days
14 days
21 days
28 days
Allogeneic skin grafts
Intermingled skin graft
Control group
Fig. 7.1 Allo- or intermingled
skin affects mixed leukocyte
reaction (MLR)
0
5
10
15
20
25
30
35
7 days
14 days
21 days
28 days
Graft group
Control group
Fig. 7.2 Activity of cytotoxic T
lymphocyte (CTL) cells
infl uenced by intermingled skin
graft


7.1 Escharectomy En Masse and Autologous-Allogeneic Intermingle Skin Graft for Extensive Full-Thickness Burn

122
Findings of a parallel animal experiment supported the
aforementioned conclusion. Peripheral blood lymphocytes
sensitized to transplant donor spleen cells were reactive to a
similar extent in the simple allogeneic skin graft group and
the intermingled skin graft group [ 25 ]. Thus, in the early
stages after an extensive burn injury, the systemic immune
response was not impaired at least, and yet it was less able to
mediate intermingled skin graft rejection than allogeneic
graft rejection. Apparently, a small number of autologous
skin islets in the intermingled skin graft have some as yet
undefi ned local protective effects. Thus, systemic immuno-
suppression after a burn injury cannot explain completely the
delay in intermingled skin graft rejection. Mechanisms of
local immune suppression including immune tolerance may
play a role.
7.1.9
Studies of Local Immune Tolerance
Induced by the Grafts
of Intermingled Skin
An early study found lymphoid cell infi ltration around the
blood vessels in the allogeneic dermis 1 week after place-
ment of intermingled skin grafts. Immune response to the
intermingled skin graft was suggested by the presence of
desquamation of the allogeneic skin around 3 weeks, and
accumulation of a large number of mononuclear cells at the
junction of the allogeneic epidermis and dermis. We con-
ducted a follow-up cytological analysis in recipients of inter-
mingled skin grafts and found that a large number of T
lymphocytes remained in the graft from 20 days to 2 years
after graft placement. Moreover, the number of infi ltrating T
cells was higher in the autologous skin islets than in the
allografts, and even the number of T cells infi ltrating in the
deep dermis layer was higher in the autologous region than
in the allograft region. This evidence led us to ask whether
the presence of autologous skin islets induced local immune
tolerance, which protected the graft from attack by the recip-
ient’s immune system.
Kistter et al. found that the number of Th and Ts cells
increased signifi cantly in the autologous skin islets of inter-
mingled skin grafts, suggesting that the rejection delay
might be due to immune suppression mediated by the autol-
ogous skin islets. In addition, they found that Langerhans
(Lh) cells could be detected in the newly formed epidermis
of the transplant, but in below normal numbers, even after
many years, suggesting that donor Lh cells might play a
relatively weak role in transplant rejection [ 26 ]. In the
absence of immune suppression or tolerance, recipient Lh
cells and dendritic cells (DCs) in the skin migrate to the der-
mis after they take up donor antigen at the graft site and
present the MHC I and MHC II molecules of the graft and
other tissue-specifi c antigens to the CD4 + and CD8 + T cells
in the draining lymph nodes. With the help of those antigen-
presenting cells (APC), T cells recognize the alloantigen
and are activated, resulting in the secretion of multiple cyto-
kines such as interleukin (IL)-2 and interferon (IFN)-γ. At
the same time the activated APCs also secrete cytokines
(such as IL-2) that cooperatively boost the functions of
effector cells (CD4 + and CD8 + T cells) through feedback
amplifi cation. The recirculating effector cells return to the
transplant bed, killing their targets (cells expressing alloge-
neic MHC I and MHC II antigens, including graft Eb cells,
capillary endothelial cells (ECs), ECs in sweat glands, and
hair follicle cells).
To mimic the rejection response of transplant recipients to
exogenous epidermis in vitro, lymphocytes from transplant
recipients and Eb cells from the donor were intermingled to
form mixed epidermal cell–lymphocyte cultures (MELC),
and the mixed epidermal cell-lymphocyte reaction (MECLR)
was monitored.
Table 7.1 showed that the proliferation rate of the donor
lymphocytes (La) in response to allogeneic Eb cells was at a
high level (14,040 cpm and 11,155 cpm respectively, in two
independent experiments). However, the proliferation rate of
the La in response to autologous epidermal cells (control
group) increases only slightly (581 and 801 cpm), and the
stimulation index (SI, the ratio of the two) was 24.1 and 13.9
respectively. Therefore, it was confi rmed that the MECLR
refl ects the intensity of graft rejection by recipients. At the
same time, some of the important parameters of the MELC
system were studied. For example, an MELC system
containing lymphocytes and Eb cells from the same source
(a patient with burn injury) revealed no signifi cant difference
in the proliferation response of recipient peripheral blood
leukocytes (PBL) to donor allogeneic lymphocytes (La + Lb)
from that of healthy people. Second, Table 7.1 showed that
Eb cells cultured alone (Ea + Eb) did not proliferate; it sug-
gests that allogeneic Eb cells might be immunogenic.
We also used the MELC system to study the effects of
autologous Eb cells. Autologous Eb cells from patients with
burn injury were added to a mixed culture of recipient lym-
phocytes and donor Eb cells to simulate the local environ-
ment of the autologous skin islets in intermingled skin grafts.
There were three types of cells with different functions in the
mixed culture (La + Eb + Ea, corresponding to reactive,
Table 7.1 Epidermal cell and
lymphocyte proliferation in
mixed epidermal cell–
lymphocyte cultures (MELC)
Experiment
La + Ea
La + Eb
La + Lb
Ea + Eb
Stimulation index
1
581 ± 156
14,040 ± 1,757
12,516 ± 1,988
158 ± 35
24.1
2
801 ± 61
11,155 ± 1,926
12,074 ± 1,653
325 ± 92
13.9
7 Burn Wound Care

123
stimulatory, and regulatory cells respectively). It was found
that the emergence of the regulatory cells of donor origin
inhibited the proliferation of lymphocytes responding largely
to allogeneic Eb cells. This assessment was usually based on
comparison with an MELC without the addition of regula-
tory cells (i.e., La + Eb). To rule out the possibility that the
cause of suppression was increased cell density due to the
addition of regulatory cells, the same number of regulatory
cells was added to all control wells. However, those regula-
tory cells were from the donor, and thus the control group
became La + Eb + Eb, which ensured that the overall cell
densities in the experimental and control wells were the
same.
At this time, we also found that autologous and allo-
geneic Eb cells caused different regulatory effects on the
MECLR. Figure 7.3 shows that the addition of the regula-
tory cell Eb from the donor in the MELC (control group)
slightly increased the proliferation response of alloge-
neic cells from 11,300 cpm (Eb = 0) to about 12,100 cpm
(Eb = 0.5). However, this response was not changed signifi -
cantly by increasing the Eb number. On the other hand, the
T cell proliferation response decreased dramatically and the
rate of this decline increased in a Ea-dose dependent manner
from 58.3 % (Ea = 0.1) to 91.5 % (Ea = 1.5) gradually after
the addition of the same number of regulatory cells from
the recipient (Ea). These results suggested that the signifi -
cant suppression of allogeneic lymphocyte proliferation was
mediated by autologous Eb cells introduced in this experi-
ment to simulate the grafting of intermingled skin.
These results reveal that the ability of Eb cells to act as a
regulating component of MECLR suppression depends on
their genetic background. It was also suggested that Eb cells
interact with lymphocytes in the body, and this interaction is
restricted by their genetic background. As Eb cells are non-
professional antigen-presenting cells (non-professional
APCs), they can express MHCII molecules upon stimulation
or in response to cytokines such as IFN-γ. Under stress, they
may also act as APCs and activate T lymphocytes.
When chloroquine-pretreated autologous Eb cells were
added to the MELC system, the suppression of the prolifera-
tion response of allogeneic lymphocytes disappeared almost
completely if Ea = 0.5. However, treatment with the same
dose of chloroquine had no effect on the T cell proliferation
rate (Fig. 7.4 ). This suggests that the immunosuppression
blocking effect of chloroquine might not have been due to its
toxicity, thereby supporting the previous assumption that the
suppression of autologous Eb cells in the MECLR is related
to their participation in antigen processing and presentation
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
1:1:1.5
1:1:1
1:1:0.5
1:1:0.1
La+Eb…
La+Eb…
Fig. 7.3 Immune activity
depressed by auto-epidermal
cells
0
2,000
Control group
Chloroquine group
4,000
6,000
8,000
10,000
12,000
14,000
La + Eb + Eb
La + Eb + Ea
Fig. 7.4 Chloroquine impacted
on immune depression induced
by epidermal cells


7.1 Escharectomy En Masse and Autologous-Allogeneic Intermingle Skin Graft for Extensive Full-Thickness Burn

124
[ 29 ]. The treatment of Ka and Kb with chloroquine (for
1–2 h before the cells were washed and added to the MELC)
proved that inhibition by autologous keratinocytes is related
to their function as APCs [ 30 ].
We then further studied Th cell polarization and the effect
of the cytokine IL-10 on local immune tolerance. First of all,
we found that the expression profi le of the Th1/Th2-related
cytokines changes after the addition of autologous keratino-
cytes to the MELC. We also found a large amount of Th cell
infi ltration in intermingled skin grafts, suggesting the impor-
tance of Th cells in the induction of local immune tolerance.
Graft rejection is typically mediated by cellular immunity.
The use of cytokines to down-regulate the activity of Th1
cell subsets may help to protect the graft from acute
rejection.
We added autologous keratinocytes to the MELC system
and analyzed the expression of several key Th1/Th2-related
cytokines in control (La + Eb) and experimental (La + Eb +
Ka) mixed cultures. Massive amounts of IL-2 and IFN-γ
secreted by Th1 cells were detected (1,079 and 690 pg/ml
respectively at 78 h) in the MELC system without autolo-
gous keratinocytes (Fig. 7.5 ). The levels of the Th 2 -type
cytokines, IL-4 and IL-10, were below the lower limits of
detection by the kit. This suggests that the Th1 subset was
strongly activated, which is consistent with the development
of strong allograft rejection in the MELC culture [ 32 ].
However, although only small amounts of IFN-γ and IL-2
were secreted early after the addition of autologous keratino-
cytes (Fig. 7.6 ), the levels of IL-10 and IL-4 gradually
increased 32 h after the addition of autologous keratinocytes
and reached 310 pg/ml and 500 pg/ml at 78 h. The level of
IFN-γ and IL-2 secretion decreased and fi nally disappeared
completely. This suggests that the introduction of autologous
keratinocytes might have induced a shift from Th1 to Th2
cells, and that this shift might be a critical event in the local
immune tolerance induced by the grafting of intermingled
skin [ 33 ].
After these works, we tried to determine which Th2-
related cytokine, IL-10 or IL-4, plays a more important
role. IL-10 (aka the cytokine synthesis inhibitory factor
[CSIF])
is
a
highly
effective
immunosuppressant.
Introduction of a certain concentration of monoclonal anti-
bodies to IL-10 into a MELC system containing autologous
keratinocytes (Fig. 7.6 ) with anti-IL-4 monoclonal antibod-
ies at the same time partially reduced the inhibition induced
by keratinocytes [ 34 ]. The addition of anti-IL-4 alone had
no such effect. However, the addition of anti-IL-10 alone
signifi cantly alleviated inhibition before 32 h. Figure 7.7
shows that only IL-10 but not IL-4 appears before 32 h,
which is consistent with the pattern of response to the
experimental combination, La + Eb + Ka. These fi ndings
indicated that IL-10 plays a far greater role than IL-4 in the
negative regulation of MECLR by keratinocytes. Later
studies also showed that IL-10 can not only inhibit the
secretion of IL-2, IFN-γ, and tumor necrosis factor (TNF)-α
of the Th1 cell subset, but can also inhibit the antigen pre-
sentation of MHC II molecules and B 7 costimulatory mol-
ecules by down-regulating their expression on the surface
of APCs.
Under certain conditions such as UV irradiation or
Leishmania infection, keratinocytes secrete large amounts of
IL-10 and thereby induce immunosuppression. To confi rm
this viewpoint, we examined sites of cytokine expression
within keratinocytes attached to the wall of the MELC sys-
tem and we found that IL-10 is expressed by autologous
keratinocytes. This is consistent with the report in the litera-
ture that states that keratinocytes can secrete IL-10 in
response to trauma or allergic reaction. Expression of inhibi-
tory cytokines such as IL-10 seems to be a characteristic of
the feedback regulation of the skin immune response.
Compared with wild-type mice, IL-10 gene knockout mice
showed an accelerated and longer skin infl ammatory
Fig. 7.5 Massive amounts of IL-2 and IFN-γ secreted by Th1 cells
were detected in the MELC system without autologous keratinocytes
Fig. 7.6 The levels of the Th2-type cytokines secreted early after the
addition of autologous keratinocytes
Fig. 7.7 The changes of the Th2-type cytokines levels



7 Burn Wound Care

125
response after exposure to allergens, proving that IL-10 is an
essential factor in the maintenance of skin immune
homeostasis.
Currently, the immunological principles underlying the
success of intermingled skin grafting remain only prelimi-
narily explored. With in-depth studies, its mechanism will be
understood and this understanding will contribute to the fur-
ther development of transplantation immunology.
7.2
Excision of Eschar and Skin Grafting
During the Shock Phase
of an Extensive Burn
Zhenrong Guo
Excision of the necrotic tissue of the wound surface of a deep
burn and graft autologous skin or allogeneic skin plus autol-
ogous skin is one of the critical steps toward curing patients
with severe burns. The clinical experiences confi rmed that
carrying out excision of eschar and skin grafting in the early
stages results in earlier healing of the wound surface, milder
systemic reaction, and better curative effect. Over several
decades, many hospitals have set the time of the fi rst eschar
excision at 4–7 days after the burn; however, 304 Hospital
considered that, for the patients with a stable systemic condi-
tion, the excision of the eschar and skin grafting might be
conducted during the shock phase (within 48 h after the
burn).
7.2.1
Hazards of the Wound Surface
of a Deep Burn
Although the pathophysiological changes in the early stages
of an extensive burn are very complicated, the most important
changes should be the hypovolemic ischemic/hypoxic dam-
age resulting from the loss of a large quantity of body fl uid
and the infection and intoxication of the necrotic tissue of the
burn; in short, the most important changes are shock and
infection. Both shock and infection are closely related to the
existence of the wound surface of a deep burn.
The wound surface of a deep burn is the main route for
loss of body fl uid; eschar has a direct cytotoxic effect; the
wound surface is the nutrient medium for bacteria; thus, it
becomes the main origin of the burn infection. The skin burn
can result in a polyreaction generating toxic polymer burn
toxin mainly composed of lipid protein complex (LPC). The
manifestations of the hazard of this kind of toxin are:
1. Resulting in systemic physiological and pathophysiologi-
cal changes.
2. Inducing lymphocytes and mononuclear macrophages to
produce and release many kinds of mediators of infl am-
mation, which is the main reason for the resulting sys-
temic infl ammatory reaction syndrome, and the severe??
may result in the secondary diseases: sepsis and multiple
organ dysfunction syndrome.
3. Inducing hypermetabolism and inhibiting the synthesis of
liver starch.
4. Resulting in systemic resistance mechanism disorders
and having a signifi cant inhibitory effect on the functions
of both humoral immunity and cellular immunity.
5. Causing marrow depression.
Although many problems about burn toxins have not been
resolved and studies on its nature and pathopoiesis have been
carried out, there are still many researchers who accept the
existence of burn toxins and many clinical pictures are
closely related to it. The wound surface should be removed
as early as possible since it is the ringleader of various etio-
logical factors.
7.2.2
The Necessity of Excision of Eschar
and Skin Grafting During
the Shock Phase
7.2.2.1 Breaking the Cyclic Model
of “Exudation–Infusion–Reexudation–
Reinfusion”
The presence of burnt tissue determines the increase in vas-
cular permeability, exosmosis of plasma fractions, and the
decrease in blood volume; therefore, the main measures for
resuscitation during the shock phase are intravenous infu-
sion, which results in the circulation of infusion and exuda-
tion. To excise the wound surface of the deep burn in the
peak period of acute exudation during the shock phase can
essentially or predominantly block the route of exudation to
reduce the loss of fl uid and decrease the amount of infusion.
The reduction of exudation also decreases the production of
excessive oxygen radicals resulting from a large amount of
reperfusion, which alleviates the damage of oxygen radicals
in the early stages .
7.2.2.2 Reducing the Routes of Infection
The wound surface of the burn is one of the most common
routes of infection, and the measurements of subeschar bac-
terial content increase within 1 week, which shows signifi -
cant direct correlation with the burn surface area and the time
after the burn. It is known that the peaks of endotoxin pre-
sented at 7–12 h and 4 days after the burn. The excision of
eschar and skin grafting in the early stages can not only
decrease the incidence of endotoxemia but also reduce the
responses of the lung and the whole body to endotoxin. It is
doubtless that eliminating the infection in the early stages is
the optimal choice for eliminating the risk of wound
infection.
7.2 Excision of Eschar and Skin Grafting During the Shock Phase of an Extensive Burn

126
7.2.2.3 Reducing the Response of the Body
to Intoxication
Many studies have shown that burn toxins produced by the
skin and hypoderm after a heat injury, such as high molecu-
lar low molecular proteins, can lead to autointoxication. The
changes in vascular permeability, disorders of the metabo-
lism and immune function, and marrow depression, are all
related to burn toxins. It is known that the subeschar tissue
fl uid inhibited the regulation function of cellular immunity,
and the growth of lymphocytes was completely inhibited
when the fl uid was injected into the culture medium of lym-
phocytes from healthy subjects. It is thus clear that burn tis-
sue is the origin of autointoxication and eliminating it as
early as possible removes the harm to the patients.
7.2.2.4 Increasing the Recovery Rate
Eliminating the lesion as early as possible and enclosing the
wound surface in advance reduce the disturbance of infec-
tion and decrease the incidence of complications of internal
organs so that the recovery rate increases. Herndon studied
41 adult patients (18–54 years old) with a burn surface area
of >30 % and third-degree burns >20 % without inhalation
injury, and the comparison between the eschar excision per-
formed at 72 h after the burn and that performed 2 weeks
after the burn indicated that the mortality of the former was
24 % and the latter was 60 %, which showed the signifi cance
of early eschar excision in relation to the increased recovery
rate. The increased recovery rate, advanced healing, and the
shorter period of hospitalization all greatly decreased the
economic burden of the patients.
7.2.3
Feasibility of Eschar Excision During
the Shock Phase
Some researchers worry that the hemodynamics is not stable
during the shock phase and performing anesthetization and
extensive eschar excision at this time may induce or aggra-
vate shock; thus, the traditional concept does not advocate
performing eschar excision during the shock phase but hopes
to perform the operation when the conditions of the patients
are stable. In reality, the body cannot achieve the real stable
status during the “waiting” period although this suggestion is
reasonable, and the eschar has become the origin of infection
during this period. The invasive infection of bacteria, the
release of various mediators of infl ammation, and disorders
of the internal environment all constitute threats to patients
with burns.
The hemodynamic stability is critical to the fact that is a
general concern to surgeons??, i.e., whether patients with
burns can tolerate eschar excision during the shock phase. In
304 Hospital, we monitored the hemodynamic indexes via a
Swan–Ganz catheter in 21 patients with extensive burns of
the total burn surface area of 63.2 ± 18.1 % and third-degree
burns of 35.9 ± 19.6 %. We continuously monitored the right
atrial pressure (RAP), mean pulmonary arterial pressure
(MPAP), pulmonary arterial wedge pressure (PAWP), blood
pressure (BP), HR, and cardiac output (CO), and calculated
the cardiac index (CI). Stroke volume index (SVI), left ven-
tricular stroke work index (LVSWI), right ventricular stroke
work index (RVSWI), systemic vascular resistance (SVR),
and pulmonary vascular resistance (PVR) were calculated
according to the formulas. The results indicated that intra-
operative and post-operative RAP, PAP, PAWP, BP, CO, and
CI all maintained comparatively high levels. The results sug-
gested that it might be safe to perform eschar excision during
the shock phase with the support of satisfactory circulation.
The increases in CI, SVI, LVSWI, and RVSWI indicated that
the patients’ hearts have the potential to receive the chal-
lenge of increased peripheral oxygen consumption.
In this group, the operations were performed at
24.1 ± 13.9 h after the burn. The excision of eschar, combin-
ing large allogeneic skin plus micro-particle autologous skin,
was applied in each patient, and the area of the fi rst eschar
excision was 32.3 ± 6.7 % of the TBSA. The patients showed
no adverse effect during the operation. The skin graft healed
well. The systematic condition was stable and all healed. The
practice confi rmed that, for the large eschar excision during
shock phase, each hemodynamic index maintained stable
during the intra-operative and post-operative periods when
the blood volume had been supplemented suffi ciently.
Therefore, it is considered that to perform excision of eschar
and skin grafting during shock phase is safe and feasible.
7.2.4
Advantages of Eschar Excision During
Shock Phase
7.2.4.1 Eschar Excision During Shock Phase Can
Decrease the Quantities of Fluid Infusion
and Blood Transfusion
To indicate the advantages of eschar excision during shock
phase, 60 cases of eschar excision during shock phase and 48
cases of eschar excision during non-shock phase were sum-
marized in Table 7.2 . The 48 patients in the control group
treated with eschar excision during non-shock phase did not
receive eschar excision during shock phase due to the limita-
tion of economic condition. However, the other conditions
were similar. There were no signifi cant differences for the
age, total burn surface area and area of III° burn between
these two groups, thus, these two groups were comparable.
The quantity of infusion was larger in the eschar excision
during shock phase group due to the synchronization of fl uid
infusion during shock phase and eschar excision, the total
quantity of infusion was 13,141 ml (intravenous 12,420 ml,
oral 721 ml) within 24 h after burn, and the quantity of
7 Burn Wound Care

127
infusion in the eschar excision during non-shock phase was
11,743 ml (intravenous 10,841 ml, oral 902 ml) within 24 h
after burn. The quantities of infusion in these two groups
were substantially equal in the second 24 h after the burn,
and on the 4th day after the burn, the quantity of infusion in
the eschar excision during the shock phase group decreased
to 8,262 ml. The quantity in the eschar excision during the
nonshock phase group was still 10,272 ml; however, the
urine volume in the eschar excision during the nonshock
phase group was lower than that in the eschar excision dur-
ing shock phase group (Table 7.3 ).
At about 24 h after burn, the wound surface presents no
obvious vascular dilation caused by infl ammatory reaction
and the layers are clear and easy to peel off with the presence
of subcutaneous dropsy; thus, eschar excision at this time
results in less bleeding, and each 1 % of eschar excised
required 49 ± 9.7 ml of blood transfusion; however, each 1 %
of eschar excised during the nonshock phase group required
56.2 ± 16.2 ml of blood transfusion. The blood transfusion in
the eschar excision during the shock phase within 24 h after
the burn was higher, which recovered to the normal level on
the 3rd day after the burn. The accumulative amount of blood
transfusion in the eschar excision during the shock phase
group within 2 weeks was about 700 ml less than that in the
eschar excision during the nonshock phase group.
The comparatively higher hemoglobin and hematocrit
levels were maintained, although the amount of blood trans-
fusion decreased. Both groups presented hemoconcentration
at admission, and hemoconcentration in the eschar excision
during the shock phase was corrected on the third day after
the burn, and the levels of hemoglobin and hemoconcentra-
tion were both slightly higher than those in the eschar exci-
sion during the nonshock phase group on the 14th day
(Table 7.4 ).
7.2.4.2 Eschar Excision During the Shock Phase Is
Benefi cial for the Control or Reduction
of Infection
1. The degree of systematic infection is mild during the
shock phase: excising most of the eschar during the
shock phase reduces the routes of infection by eliminat-
ing the origin of burn toxins and bacterial infection so
that the wound surface is well covered; thus, the infec-
tion from the wound surface is decreased and the inci-
dence of complications of systematic infection also
decreases accordingly. In the 38 patients with extensive
burns in the eschar excision during the shock phase
group, the eschar excision was performed before the
presence of SIRS and only 6 patients had SIRS (15.6 %);
however, in the 46 patients in the eschar excision during
the nonshock phase group, 8 patients (18.6 %) had SIRS
before the operation and 23 patients after the operation;
its incidence was as high as 53.5 %. The other complica-
tions of infections in the eschar excision during the shock
phase group were also lower than those in the eschar
excision during the nonshock phase group. The positive
Table 7.2 Comparison of general conditions between the two groups
Number
of cases Age
Total burn
surface area
Third-degree
area
Time of operation
after burn (h)
Area of the fi rst
eschar excision (%)
Eschar excision during the shock phase
60
32.14 ± 7.26 70.03 ± 17.75
41.24 ± 14.72 24.47 ± 11.76
34.64 ± 10.01
Eschar excision during the nonshock phase
48
36.26 ± 4.10 68.41 ± 20.06
40.32 ± 21.27 112.71 ± 29.17
33.96 ± 11.05
Table 7.3 Quantity of infusion and urine volume of the patients in the two groups
Group
Intake and output volume
Time after burn (h)
16
24
48
72
96
Shock phase
Intravenous infusion (ml)
8,362 ± 568
12,420 ± 1,404
9,020 ± 545
5,450 ± 364
4,550 ± 216
Eschar excision group
Oral administration (ml)
232 ± 21
721 ± 102
1,070 ± 132
2,056 ± 240
3,712 ± 212
Urine volume (ml/h)
50 ± 19
50 ± 19
96 ± 24
104 ± 29
110 ± 36
Nonshock phase
Intravenous infusion (ml)
7,682 ± 732
10,841 ± 1,096*
8,845 ± 402
6,056 ± 315
8,946 ± 381
Eschar excision group
Oral administration (ml)
212 ± 30
902 ± 213
1,230 ± 211
2,012 ± 363
1,326 ± 292
Urine volume (ml/h)
48 ± 18
92 ± 16
96 ± 19
95 ± 14
84 ± 14
Note: Comparison between the two groups: * p < 0.05
Table 7.4 Changes in hemoglobin and hemoconcentration of the patients in the two groups
Group
Hemoglobin (g/L)
Hemoconcentration
At admission 3 days
7 days
14 days At admission 3 days
7 days
14 days
Eschar excision during the shock phase group
159 ± 16
132 ± 37 * 116 ± 33 128 ± 17 0.53 ± 0.12
0.47 ± 0.12 0.36 ± 0.08 0.45 ± 0.03
Eschar excision during the nonshock phase group 159 ± 27
155 ± 22 107 ± 20 120 ± 16 0.54 ± 0.09
0.53 ± 0.08 0.36 ± 0.07 0.43 ± 0.08
Note: * p < 0.05
7.2 Excision of Eschar and Skin Grafting During the Shock Phase of an Extensive Burn

128
rate of bacterial culture on the intravenous catheter was
decreased owing to the advanced healing of the wound
surface and the shortened duration of infusion. The inci-
dence of sepsis, multisystem organ failure (MSOF), and
osteomyelitis, as well the positive rate of blood culture
were all decreased because of the decreased degree of
infection. Only the incidence of pneumonia in the two
groups was similar, which was due to the fi ve cases with
severe inhalation injuries in the eschar excision during
the shock phase group (Table 7.5 ).
2. The curative effect of eschar excision during the shock
phase is satisfactory: in the eschar excision during the
shock phase group, the time of enclosure of the wound
surface was advanced, the infection was reduced, there
were fewer types of applied antibiotics, the time of the
application of antibiotics was short, the mortality was
lower, and the time of healing was shortened; thus, the
effect was signifi cantly better than that in the eschar
excision during the nonshock phase group (Table 7.6 ).
3. The levels of endotoxin and mediators of infl ammation:
(a) Endotoxin (LPS): detect LPS of 22 blood donors to
obtain the normal control value (0.074 ± 0.023) EU/
ml. In the patients with extensive burns, the bacteria
in the intestinal tract and LPS enter into the blood
owing to the destruction of the function of intestinal
barrier in the early stages, which even results in endo-
toxemia. The plasma LPS presents the fi rst peak at
7–12 h after the burn, which is mainly from the intes-
tinal tract; and the second peak is at 3–4 days, which
is mainly from the wound surface. Eschar excision
during the shock phase blocks the second peak and
makes the level of LPS decrease signifi cantly since
the 7th day after the burn; however, affected by the
second peak, the level of LPS before the operation in
the eschar excision during the non-shock phase group
has already increased to 0.5 EU/ml and remains at
this level until the 21st day.
(b) Tumor necrosis factor: LPS induces the mononuclear
phagocyte system to release TNF in “pulse patter.”
In the detection of plasma TNF of the 22 blood
donors, 12 cases were not detected and 10 cases were
measured as (0.03 ± 0.01) ng/ml. TNF increases
signifi cantly after extensive burn. Some researchers
considered that TNF be the most critical mediator to
cause systematic infl ammatory reaction and eschar
excision during the shock phase can reduce the sys-
tematic infl ammatory reaction.
(c) IL-6 and IL-8: the factors such as stress after a burn,
the absorption of burn toxins, bacterial infection, etc.,
may stimulate the mononuclear macrophages, ECs,
etc., to synthesis and secrete IL-6 and IL-8. IL-6 is
one of the critical cytokines mediating immune
response and acute infl ammatory reaction. IL-8 has
the function of regulating neutrophilic granulocytes
and T lymphocytes, which increases during severe
infection. Detection of reference values for IL-6 and
IL-8 performed in 20 blood donators indicated that
they were lower than the highest sensitivities of
0.1 ng/ml and 0.15 ng/ml. The IL-8 in the eschar
excision during the shock phase group began to be
lower than that in the eschar excision during the non-
shock phase group since the 3rd day after the burn,
and the IL-6 in the eschar excision during the shock
phase group began to be lower than that in the eschar
excision during the non-shock phase group since the
7th day. The tendencies were essentially synchronous
Table 7.5 Incidence of the complications of infection in patients with burns in the two groups (%)
Group
Number
of cases Pneumonia Sepsis
MSOF Osteomyelitis
Positive bacterial
culture of the
intravenous catheter
Positive bacterial
culture of the
blood culture
Eschar excision during the shock phase
38
5 a (13.6)
6 (15.8)
2 (5.3)
0 (0)
2 (5.3)
3 (7.9)
Eschar excision during the nonshock phase 41
5 b (12.2)
12 (29.3) 4 (9.8)
2 (4.9)
7 (17.1)
4 (9.8)
MSOF multisystem organ failure
a All with severe inhalation injuries
b Four patients with severe inhalation injuries
Table 7.6 Curative effects of the patients in the two groups
Group
Number of cases
Application of antibiotics
Death
Time until healing (days)
Category
Time (days)
n
%
Eschar excision during the shock phase
38
5.0 ± 2.0
24.9 ± 14.3
2
5.3
32.4 ± 4.6
Eschar excision during the nonshock phase
41
7.6 ± 2.7
29.6 ± 11.2
6
14.7
39.7 ± 14.1
7 Burn Wound Care

129
with LPS and TNF, which indicated that the eschar
excision during the shock phase is benefi cial to
decreased infl ammatory reaction.
4. Eschar excision during the shock phase can eliminate the
basis for bacteria proliferation as early as possible: the
wound surface of the burn is a kind of fi ne culture medium
for the growth and proliferation of bacteria, and the sub-
eschar bacteriological investigations indicated that the
detective subeschar bacterial content increases within
1 week. It is obvious that to excise eschar as early as pos-
sible can decrease the wound surface area and subeschar
bacterial content. At about 24 h after the burn, the sube-
schar tissue from the eschar excision should be sent for
bacterial culture and the calculated bacterial content is
(4.6 ± 2.2) × 10 2 cfu/g; however, at 4~5 days after burn, the
subeschar
bacterial
content
greatly
increases
to
(8.36 ± 7.43) × 10 2 cfu/g.
In minipigs with a 35 % third-degree burns area, exposure
to the natural condition without any drugs, the subeschar
bacterial content at 8, 24, 48, 72, and 96 h after the burn were
detected and the results also indicated that the bacterial con-
tent increased with prolongation of time after the burn.
These results indicated that the delay in the treatment
established the bacteriological basis for secondary infection.
If the systemic conditions permit, the earlier and more thor-
ough eschar excision will decrease the basis for bacterial pro-
liferation so that the disturbance of infection will also be
reduced.
7.2.4.3 Eschar Excision During the Shock Phase Is
Benefi cial to Improve the Immune
Function of the Body
Extensive deep burn severely inhibits the immune function
of the body and the hypofunction of the immune system is an
important factor resulting in a high incidence of infection
and mortality.
The increased microvascular permeability of the wound
of a deep burn leads to subeschar tissue edema, one of the
characteristics of edema fl uid is to inhibit a cellular immune
function. The in vitro experiment confi rmed that the edema
fl uid can inhibit the mitosis of lymphocytes during in vitro
culture, and the inhibitory effect presents at 9 h after burn.
The burnt tissue transplanted into the normal mice could also
lead to the inhibition of immune function mediated by T
cells. Therefore, excising the burnt tissue as early as possible
can signifi cantly improve the immune function.
At 304 Hospital, we analyzed the dynamic changes of the
subgroups of T lymphocytes in patients with extensive burns.
We found that both the total number of T lymphocytes (CD 3 + )
and T helper cells (CD 4 + ) decreased signifi cantly, the T sup-
pressor cells (CD 8 + ) increased, and the CD 4 + /CD 8 + decreased.
However, the postoperative CD 3 + , CD 4 + , and CD 4 + /CD 8 + on 3,
7, 14, and 12 days in the eschar excision during the shock phase
group were all higher than those in the eschar excision during
the nonshock phase group, and the CD 8 + was lower than that in
the eschar excision during the nonshock phase group (Tables 7.6
and 7.7 ). It is obvious that eschar excision during the shock
phase is benefi cial to the immune function of the body.
7.2.4.4 Eschar Excision During the Shock Phase
Can Alleviate Hypermetabolism
Hypermetabolism presents extensively after severe burn, and
the metabolic rate increases with the aggravation of severe
burn and decreases with the healing of the wound surface.
We utilized the metabolic cart (MedGraphic Company, St
Paul, MN, USA) to monitor the resting energy expenditure
(REE) of the patients with extensive burns in these two
groups. The total burn surface area and area of third-degree
burn in the eschar excision during the shock phase group (15
patients) were 60.6 ± 13.4 % and 39.5 ± 15.1 % respectively,
and the eschar excision was performed 1.2 days after the
burn; the total burn surface area and area of third degree burn
in the eschar excision during the nonshock phase group (six
patients) were 56.3 ± 11.7 %, and 37.8 ± 16.0 % respectively,
and the eschar excision was performed 5.1 days after the
burn. The oxygen consumption and carbon dioxide produc-
tion in unit time and REE of the patients in the two groups
were measured using the helmet method on 1, 2, 3, 5, 7, 14,
21 and 28 days after burn (Table 7.8 ). Moreover, the REE
levels of 13 cases of male volunteers were measured
(38.5 ± 2.1 kcal/[h·m 2 ]) as the normal control.
Table 7.9 showed that the REE levels of the patients in the
two groups were signifi cantly higher than in the normal con-
trols; meanwhile, the REE levels in the eschar excision dur-
ing the shock phase group were signifi cantly lower than
those in the eschar excision during the nonshock phase
group, and REE started to decrease gradually until 3 weeks
after the burn owing to the healing of most of the wound
surface, and the REE levels in the two groups were similar
only at this time.
The results indicated that eschar excision during the
shock phase signifi cantly reduced the hypermetabolism,
although it had not reversed the hypermetabolism com-
pletely, which may be related to the remaining wound sur-
face. This phenomenon is related to two factors: one is that
the advanced healing of most of the wound surface decreases
the exudation and evaporation of body fl uid and body heat
loss so that the energy consumption is decreased; the other is
that the deep wound surface was removed before the hyper-
metabolism, which reduces the routes of infection, decreases
the poisoning of burns toxins, and alleviates the systemic
infl ammatory reaction.
7.2 Excision of Eschar and Skin Grafting During the Shock Phase of an Extensive Burn

130
Table 7.7 Changes in CD3+ and CD4+ of each group %, x
s
±
(
)
Group
CD 3 +
CD 4 +
Reference
value
Preoperative 3
7
14
21
Reference
value
Preoperative
3
7
14
21
Eschar excision
during the shock
phase
70.4 ± 6.1
53.4 ± 5.79
54.7 ± 5.11
49.2 ± 4.35 48.7 ± 5.47 53.9 ± 6.71 47.2 ± 5.3
37.3 ± 4.25 ▲
36.8 ± 4.31 ▲
32.4 ± 3.30 ▲ 34.63 ± 3.18 ▲
34.4 ± 4.71 ▲
Eschar excision
during the
non-shock phase
70.4 ± 6.1
47.2 ± 5.76
36.1 ± 7.32*
42.7 ± 6.70 48.5 ± 5.85 46.7 ± 6.54 47.2 ± 5.3
31.2 ± 4.52 ▲
24.3 ± 2.71 #,△△ 27.4 ± 3.65 ▲ 26.9 ± 2.89 ▲△ 25.4 ± 3.66 #,△
Comparison between the two groups: * p <0.05, △ p <0.01; compared with those before the burn: ▲ p <0.05; compared with those before operation: # p <0.05
Table 7.8 Changes of CD 8 + , CD 4 + /CD 8 + %,
x
s
±
(
)
Group
CD 3 +
CD 4 +
Reference
value
Preoperative
3
7
14
21
Reference
value
Preoperative 3
7
14
21
Eschar excision
during the shock
phase
23.4 ± 2.9
53.4 ± 2.15
31.6 ± 3.87 ▲
32.4 ± 4.37 ▲
30.1 ± 4.21 ▲ 37.0 ± 4.02*, # , 1.71 ± 5.3
1.39 ± 0.53
1.18 ± 0.71 ▲
1.04 ± 0.61 ▲
1.45 ± 0.06
1.19 ± 0.44 ▲
Eschar excision
during the
non-shock phase
23.4 ± 2.9
32.7 ± 4.16 ▲
42.6 ± 5.12*, #,△
40.2 ± 4.25*, #,△ 37.2 ± 4.15 #
35.1 ± 4.36 ▲
1.71 ± 0.53
1.06 ± 0.14 ▲ 0.57 ± 0.06*, #△ 0.68 ± 0.15*, # 0.72 ± 0.14 #,△ 0.81 ± 0.20 ▲
Comparison between the two groups: * p <0.05, △ p <0.01; compared with those before the burn: ▲ p <0.05; compared with those before operation: # p <0.05
7 Burn Wound Care

131
7.2.4.5 Protective Effect of Eschar Excision
on the Body During the Shock Phase
A group of animal experiments suggested that eschar exci-
sion during the shock phase might have a protective effect on
the body. Sixteen minipigs were subjected to 35 % third-
degree burns with 3 % napalm and treated with routine resus-
citation and fl uid infusion were assigned to one of two
groups: eschar excision during the shock phase group (S
group, eight animals) in which eschar excision was per-
formed at 24 h after the burn, and the eschar excision during
the nonshock phase group (N group, eight animals) in which
eschar excision was performed at 96 h after the burn. Then,
the allogeneic skin under cryopreservation was grafted after
the excision of all the eschar. The animals were killed on the
12th day after the burn and the internal organs were taken for
histological examination. In the experimental studies, we
found that eschar excision during the shock phase had a sig-
nifi cant protective effect on the body:
1. It improved the hemorheological indexes: the hemorheo-
logical abnormalities generally present after the burn, the
blood viscosity reaches its peak value at 8 h after the burn,
and the plasma viscosity and erythrocyte aggregation
index reach peak values at 4 h after the burn; however, the
red blood cell deformation index decreases to the minimal
value at 4 h after the burn, which reverts with resuscita-
tion through fl uid infusion. The indexes in the S group
recover to the normal levels over 24–48 h after the burn,
but the hemorheological changes in the N group present
peaks again at 96 h after the burn and the red blood cell
deformation index decreases to the minimal value.
These results indicated that eschar excision during the
shock phase can improve hemorheological indexes,
which is benefi cial to the smooth microcirculation.
However, the blood samples in the eschar excision during
the nonshock phase group collected on the morning
before 96 h after the burn had passed had already shown
increased blood viscosity, enhanced erythrocyte aggrega-
tion, thickness of blood, low speed of blood fl ow, and bad
tissue perfusion, which aggravated ischemia and hypoxia.
2. It alleviates EC damage: the coexistence of vasocon-
striction and increased permeability after severe burns is
the main reason for ischemic and hypoxic damage
resulting from bad blood perfusion, in which endothelin
(ET) and nitric oxide (NO) participate in the early
pathopoiesis.
Endothelin is synthesized and secreted by ECs, which
is the most powerful vasoexcitor material as far as we
know, and it also has powerful contraction effects on tra-
cheal and gastrointestinal smooth muscle. As a kind of
autocrine/paracrine hormone, it can also regulate the endo-
thelium-dependent relaxing factor NO released by the ECs
themselves through feedback. Vascular ECs are not only
the main secretory cells for synthesis and the secretion of
ET, but also the target cell with the surface receptors bind-
ing with ET, which results in EC damage to increase per-
meability so that the exudation is increased, the tissue
shows edema, the blood volume is decreased, and the
shock is aggravated. The secretion of NO after a burn also
increases, but the extent is lower than that for ET; thus, the
ET/NO ratio increases and the blood vessels contract.
In the animal experiment of burns using minipigs, the
levels of ET/NO of the two groups both increased signifi -
cantly at 8 h after the burn. The ET in the S group at 96 h
after the burn was signifi cantly lower than that in the N
group; however, the NO was higher than that in the N
group, and the ratio of ET/NO was signifi cantly lower
than that in the N group, which recovered to the level
before the burn. It is obvious that eschar excision during
the shock phase removes the reasons that result in a con-
tinuous increase in ET and reduces EC damage so that the
edema and microcirculation disturbance are alleviated.
3. It increases oxygen delivery, improves oxygen consump-
tion, and alleviates metabolic acidosis. The purpose of
resuscitation during the shock phase is not only to correct
the blood volume defi ciency and hemodynamic abnor-
malities, but also to improve hypoxia and normalize the
oxygen delivery and consumption of the tissues.
The oxygen delivery (DO 2 ) in the two groups decreased
to the minimal level at 8 h after the burn; however, the
oxygen delivery in the S group was signifi cantly higher
than that in the N group. With the decrease in DO 2 within
8 h after the burn, the oxygen consumption (VO 2 ) also
decreased, the VO 2 in the S group started to increase at
48 h after the burn and recovered to the normal level on
the 7th day, but the VO 2 in the N group was still higher
than the normal level on the 12th day. The oxygen uptake
Table 7.9 Changes in the resting energy expenditure (REE) of the patients in the two groups [kcal/(h·m 2 )]
Time after burn
That day
1
2
3
5
7
14
21
28
Eschar excision
during the shock
phase group
39.2 ± 1.8
45.7 ± 2.3 # 43.3 ± 2.0* 48.5 ± 3.1 #, * 56.8 ± 3.3 ##, *
65.6 ± 4.1 ##, *
74.2 ± 3.6 ##, *
75.3 ± 4.2 ## 59.8 ± 3.6 ##
Eschar excision
during the
nonshock phase
group
–
46.1 ± 1.9 # 48.9 ± 2.4 ## 57.6 ± 2.7 ##
64.5 ± 2.9 ##
73.8 ± 3.9 ##
82.4 ± 3.9 ##
77.1 ± 4.6 ## 61.2 ± 4.1 ##
Compared with a healthy person # p < 0.05, ## p < 0.01; comparison of the same time-point in the two groups * p < 0.05
7.2 Excision of Eschar and Skin Grafting During the Shock Phase of an Extensive Burn

132
increases compensatively because of the decrease in
oxygen delivery. The oxygen uptake in the S group recov-
ered to the level before the burn at 48 h, which indicated
the dependent relationship between oxygen delivery and
oxygen uptake; however, the oxygen uptake in the N
group was always higher than the normal level.
The lactic acid in the arterial blood in the two groups
started to increase at 4 h after the burn, the level of lactic
acid reached the peak at 8 h after the burn when the levels
of DO 2 and VO 2 were at their minimal levels. The level of
lactic acid in the S group started to decrease at 24 h after
the burn and it had decreased to the level before the burn
by 96 h after the burn; however, the lactic acid in the N
group increased to the second peak at 96 h after the burn
and it had decreased to the normal level by the 12th day.
These results indicated that eschar excision during the
shock phase can improve oxygen delivery, decrease oxy-
gen consumption, and alleviate hypermetabolism and
metabolic acidosis resulting from anaerobic metabolism
and the accumulation of lactic acid.
4. It has a preventive and therapeutic effect on myocardial
damage: to maintain a satisfactory HR, the normal func-
tion of myocardial contraction is required in addition to
the effective blood volume and proper hemorheological
indexes. Some researchers found that the heart stroke had
started to decrease before the signifi cant decrease in
blood volume and it was considered to be due to the pres-
ence of myocardial depressant factor. However, there is
still controversy regarding the defi nite component of
myocardial depressant factor.
The detection of myocardial enzymes in minipigs dis-
covered that the creatine kinase (CK), isoenzyme of cre-
atine kinase (CK-MB), lactate dehydrogenase (LDH),
and
γ-hydroxybutyrate
dehydrogenase
(γ-HBDH)
increased immediately after the burn, and the levels of
these enzymes in the S group started to decrease at 24 h
after the burn; however, the levels of these enzymes in the
N group continued to increase until 96 h after the burn;
however, they were still higher than those in the S group,
and these enzymes in the S group did not recover to the
normal levels until the 12th day, and there was a signifi -
cant difference between the two groups.
Troponin T (TnT) is a kind of calmodulin among
myocardial constitutive proteins, and it cannot enter the
blood circulation through the membrane of myocardial
cells when the cell is integral; however, it tends to be dis-
sociated from the myofi bril and enters the blood circula-
tion when the myocardial cell is degenerated and necrotic.
Therefore, it is used as the very specifi c and sensitive
index for the judgment of myocardial damage. In the
animal experiment of burns in rats, TnT increased signifi -
cantly at 3 h after the burn and reached the peak
(3.53 ± 0.69) μg/L at 12 h (the reference value is
0.14 μg/L), but the TnT had not increased after the exci-
sion of all eschar and allogeneic skin grafting at 4 h after
the burn and it remained to be about 0.57 μg/L at 12 h.
The above-mentioned data indicated that the myocar-
dial damage presented rapidly after the burn, and a great
amount of enzyme proteins in the cytoplasm and organ-
elles entered the blood. CK-MB in particular also has the
specifi city to detect myocardial damage. The decomposi-
tion of myocardial constitutive proteins presents as a rapid
increase in TnT in the blood. These results all indicate that
the myocardial damage presents early after the burn, which
directly affects cardiac contractility; thus, it is diffi cult to
achieve satisfactory resuscitation by simple fl uid infusion
and expansion in the early stages of a burn. Only excising
the tissue of a deep burn as early as possible and removing
the eschar, resulting myocardial damage to the greatest
extent can most effectively improve blood circulation.
5. It has a protective effect on internal organs: the minipigs
were killed on the 12th day after the burn and the internal
organs were sampled for histological examination. It was
discovered that the pathological changes in the eschar
excision during the nonshock phase group were signifi -
cantly more severe than those in the eschar excision dur-
ing the shock phase group, which included cloudy
swelling and degeneration of hepatocytes, congestion in
the hepatic sinusoids, and severe infl ammatory cell infi l-
tration in the portal area. The alveolar wall thickened with
severe neutrophil infi ltration and pulmonary interstitial
edema. There was intravascular coagulation around the
renal glomerulus and infl ammatory cell infi ltration among
the renal tubules. The gastric mucosa presented necrosis
and was falling off, the lamina propria was congested,
and there was severe neutrophil infi ltration in the muscu-
lar layer. The epithelial cells of the jejunal mucosa also
had necrosis and were falling off, the submucous tissue
was edematous, and the capillaries congested with severe
infl ammatory cell infi ltration. The arrangement of cardiac
muscle fi bers was disordered with cloudy swelling,
degeneration, and disappearance of transverse striation
on the sarcolemma.
The reverse transcription- polymerase chain reaction
(RT-PCR) and immunohistochemical methods were applied
to observe the expressions of TNFα mRNA and three kinds
of adhesion molecules in the lung, including ICAM-1,
E-selectin, and P-selectin. The expression of these molecules
in the eschar excision group with operations performed at 8
or 24 h after the burn had recovered to the normal levels at
96 h after the burn, but the expression of these molecules in
the eschar excision group with operations performed at 96 h
after the burn had not recovered to the normal levels by the
7th day. These results indicated that excising eschar as early
as possible can prevent the synthesis of a large number of
adhesion molecules in the ECs, which alleviates further
damage to ECs resulting from the neutrophils mediated by
adhesion molecules.
7 Burn Wound Care

133
The pathological changes of the organs in the eschar exci-
sion during the shock phase group were not signifi cant,
which indicated that excising the wound surface of a deep
burn during the shock phase blocked the damage to the
organs by the burn toxins, infection, and various infl amma-
tory mediators, and reduced the incidence of infl ammatory
response syndrome and MODS. The results indicate that
eschar excision during the shock phase has a satisfactory
protective effect on the internal organs.
7.2.5
Clinical Indexes for the Selection
of Opportunity for Eschar Excision
During the Shock Phase
A large number of clinical practices have already confi rmed
that performing eschar excision during the shock phase
under the monitoring of perioperative hemodynamic indexes
using the Swan–Ganz catheter is safe and reliable.
Meanwhile, the data on each clinical index with a normal
hemodynamic status were also obtained.
The monitoring results of hemodynamic indexes in 60
patients with burns showed that the indexes had been almost
normal at 16 h after the burn and substantially normal at
24 h. Performing eschar excision and skin grafting within
this period can not only guarantee the intraoperative safety,
but also the postoperative stability (Table 7.10 ).
Meanwhile, as shown in Table 7.2 , the amount of infusion
within the fi rst 24 h was 13,141 ml (including the infusion
during operation) and the urine volume was 96 ml/h. The
clinical symptoms at admission, such as lethargy, restless-
ness, dipsia, nausea, etc. disappeared over 16–24 h. The HR,
hemoglobin (Hb), and hematocrit (Hct) also decreased with
the increase in the amount of infusion, which recovered to
normal levels at 24 h and was maintained at normal levels
after the operation (Table 7.11 ).
In the summary of this group of cases, we found that the
clinical manifestations, Hb and Hct, also synchronously
approached or recovered to the normal level when the hemo-
dynamic indexes recovered to normal levels over 16–24 h
after the burn. On this basis, we proposed clinical indexes for
eschar excision during the shock phase:
1. The amount of infusion within the fi rst 24 h after the burn
is (2.6–3.0) ml·kg −1 ·1 % −1 TBSA.
2. The urine volume is (80–100) ml/h.
3. Conscious.
4. The dipsia is signifi cantly alleviated, without vomiting or
nausea.
5. The HR is about 100–110 beats/min.
6. The Hb is ≤150 g/L.
7. The Hct is ≤0.50.
To verify the reliability of the above-mentioned clinical
indexes, we simply performed extensive eschar excision dur-
ing the shock phase according to the clinical indexes without
monitoring the hemodynamic indexes in 33 patients with
extensive burns and achieve success in all cases. This result
fully confi rmed that performing eschar excision and skin
grafting during the shock phase according to the clinical
indexes is safe and reliable, and may be referred to by the
hospitals without the condition of monitoring using the
Swan–Ganz catheter.
7.3
Large Sheet Skin Autograft for Small/
Medium-Sized Deep Burns
Juntao Han, Xiongxiang Zhu , and Bi Chen
7.3.1
Overview
In view of the progress in techniques, including early fl uid
resuscitation, eschar excision, infection control, and early
enteral nutrition, the majority of burned patients are able to
survive during the critical stage. Now, the rescue treatment is
not a critical challenge in the clinic for patients with small/
medium-sized burns (total burns area ≤50 % of the TBSA, or
deep burns area ≤30 % of the TBSA). This type of burn has
a signifi cant morbidity in terms of time to healing, infective
complications, and subsequent scarring. Conservative man-
agement leading to spontaneous healing usually involves
prolonged and painful dressing changes and the resultant
scar is invariably hypertrophic, leading to cosmetic and func-
tional debility. Thus, an early surgical approach that tries to
preserve the dermis and achieve prompt wound healing is
preferred. Furthermore, for the most of these patients, the
donor sites and available grafts are plentiful. Therefore, the
focus of treatment is to minimize donor site morbidity and to
maximize the functional and cosmetic outcome.
Skin autografting is a conventional scheme for the repair
of skin injuries as well as the gold standard for resurfacing
deep burns. Although modern burns surgery has gained solid
theories and techniques with regard to skin grafting, func-
tionally and cosmetically appropriate grafting still deserves
in-depth exploration.
The Burn Center of the Xijing Hospital, Fourth Military
Medical University, conducted a valuable study on the man-
agement of small/medium-sized deep burns. In the 592 cases
of small/medium-sized deep burns, the size of the deep burns
ranged from 1 to 30 % of the TBSA with an average of
11.2 ± 2.7 %. The burn wounds were treated with eschar
shaving and tangential excision, while large split-thickness
sheet skin grafts were harvested from the donor sites with a
drum dermatome, or an electric dermatome, or a Humby
knife. Sheet grafts, instead of meshed grafts, were employed
for cosmetic considerations and secured atraumatically with
7.3 Large Sheet Skin Autograft for Small/Medium-Sized Deep Burns

134
Table 7.10 Hemodynamic changes in 60 patients with eschar excision during the shock phase
Inspection item
Reference value
At admission
Time after burn (h)
8
16
24
36
48
72
96
RAP (mmHg)
1–10
1.06 ± 0.62
1.48 ± 0.52
2.20 ± 0.24*
3.28 ± 0.35 #
3.96 ± 0.43 #
3.88 ± 0.33 #
4.03 ± 0.42 #
4.32 ± 0.30 #
MPAP (mmHg)
10–20
8.76 ± 0.41
9.51 ± 3.52
11.90 ± 1.96*
12.34 ± 1.38*
12.45 ± 0.41*
130 ± 0.45*
1,296 ± 0.70 #
1,165 ± 1.24*
PAWP (mmHg)
5–15
5.20 ± 0.69
5.46 ± 0.60
6.85 ± 0.87*
7.10 ± 0.90*
8.94 ± 0.94 #
9.03 ± 0.86 #
9.10 ± 1.33 #
9.86 ± 1.30 #
CO (L/min)
4–8
3.11 ± 0.30
3.80 ± 0.34
3.92 ± 0.29
5.37 ± 0.30*
7.10 ± 0.38*
12.12 ± 0.41 #
13.39 ± 0.35 #
14.24 ± 0.92 #
CI (L/min/m 2 )
2.5–4.1
1.83 ± 0.28
2.14 ± 0.14
2.19 ± 0.022*
3.94 ± 0.30 #
4.16 ± 0.45 #
7.18 ± 0.57 #
7.90 ± 0.44 #
7.68 ± 0.80 #
SVI ml/(beat/m 2 )
40–60
13.09 ± 3.51
17.51 ± 3.25
18.10 ± 4.30
40.79 ± 4.37 #
46.30 ± 3.56
63.52 ± 3.25 #
80.07 ± 10.95 #
75.85 ± 11.04 #
RAP right atrial pressure, MPAP mean pulmonary arterial pressure, PAWP pulmonary arterial wedge pressure, CO cardiac output, CI cardiac index, SVI stroke volume index
Compared with those at admission: * p < 0.05, # p < 0.01
Table 7.11 Observations of the clinical indexes
Item
At admission
Time after burn (h)
8
16
24
36
48
72
96
Heart rate (beat/min)
136 ± 20
118 ± 19
120 ± 18
94 ± 16*
117 ± 15
112 ± 14
94 ± 16*
96 ± 18*
Hb (g/L)
174.4 ± 11.6
170.4 ± 23.2
162.9 ± 9.4
147.7 ± 14.4*
142.8 ± 12.7*
136.6. ± 11.9 #
131.1 ± 6.3 #
128.4 ± 6.8 #
Hct
0.567 ± 0.094
0.564 ± 0.016
0.557 ± 0.028
0.449 ± 0.046*
0.446 ± 0.039*
0.410 ± 0.049 #
0.390 ± 0.029*
0.372 ± 0.022
Hb hemoglobin, Hct hematocrit
Compared with those at admission: * p < 0.05, # p < 0.01
7 Burn Wound Care

135
3-0 or 6-0 silk sutures. The total rate of sheet graft use was
over 95 % and the percentage of 100 % success was about
92 % (544 out of 592). The 1-year follow-up data showed
that the burns patients had soft, smooth, complete skin with
mild pigmentation but no apparent hypertrophic scars at the
recipient sites, and fl exible limbs and bone joints, while the
donor sites were slightly rougher than normal, without appar-
ent pigmentation or hypertrophic scars.
It is indicated in this study that the large split-thickness
sheet grafts can enable functional and cosmetic healing of
small/medium-sized deep burns, based on the advanced
management of burn wounds and proper skin harvesting
techniques.
7.3.2
Techniques for the Repair of Burns
7.3.2.1 Management of Burn Wounds
Burn wounds that are not going to heal within 2 weeks
should be debrided and covered with autologous split skin
grafts to minimize hypertrophic scarring. The type of wound
excision depends on the state of the burn wound. When the
vital signs of burned patients are stable, burns that are
deemed to be of deep partial thickness are best tangentially
excised or shaved using a skin graft knife. In partial- thickness
wounds an attempt is made to preserve viable dermis,
whereas in full-thickness injury all necrotic and infected tis-
sue must be removed, leaving a viable wound bed. Full-
thickness excision can also be achieved using sharp excision
with a knife. The edges of the wound were also cut for pre-
cise suturing to the grafts. In general, most areas are excised
with a knife or a hand skin graft knife and the wound bed is
stopped from bleeding and fl ushed adequately for grafting.
7.3.2.2 Donor Site and Split-Thickness Skin
Grafts
Split skin grafts can be harvested either as split-thickness or
full-thickness grafts depending on the available donor site and
the wound bed. Split skin grafts can be harvested with a hand
knife or a drum dermatome or a powered dermatome. We
choose the back, lateral chest, abdomen, anterior and outer
aspects of the thigh, posterior and inner aspect of the thighs,
and the lower leg as donor sites in that order. The grafts are
harvested using a drum dermatome if the burn area is less than
10 % of the TBSA, while a drum dermatome in combination
with a Humby knife or an electric dermatome is used to har-
vest grafts if the burn area is over 10 % of the TBSA.
7.3.2.3 Skin Grafting
All skin grafts are transplanted as whole-sheet grafts instead
of being meshed or with a hole. The grafts are atraumatically
secured to the wounds with 3-0 or 6-0 silk sutures in a con-
tinual manner. Special burn areas such as the axilla, neck,
buttocks, and perineum are fi xed with a tie-over bolster
before the application of pressure dressings, while skin grafts
for the limbs require only pressure dressings. The dressings
are changed at 5–7 days post-grafting for the fi rst time
depending on the specifi c conditions of the wound, and the
sutures are removed at 8–10 days post-grafting.
7.3.3
Repair of Deep Facial Burns
The face and neck are areas that are both cosmetically and
functionally important. Conservative treatment tends to pre-
serve viable tissue and is preferred. Most facial burns that
heal within 3 weeks after injury often have desirable func-
tional and cosmetic outcomes, while others that take more
than 3 weeks to heal often leave hyperplastic scars, even
organ displacement or deformity. Deep burns of the face are
usually treated conservatively until a viable wound bed is
present. Third-degree burn wounds cannot heal without skin
grafting. However, surgical repair still poses a challenge with
regard to the cosmetically and functionally appropriate out-
comes for the surgeon.
7.3.3.1 Early Treatment
In the early stages, the facial burns surface is managed with
exposure therapy. The 1 % silver sulfadiazine is smeared on
the burn for anti-infection purposes. After debridement,
Silvadene is applied topically to the wound. The Silvadene is
changed once or twice daily. If the eyelids cannot close
because of facial swelling, chlorotetracycline eye ointment is
pasted on the eye balls, followed by covering of the eyes with
Vaseline dressings. The separate eschar is shaved in time to
semi-expose the burn surface.
7.3.3.2 Debridement
Delayed eschar excision is preferred. At 3 weeks post-burn,
the granulation tissues are completely removed or shaved or
tangentially excised according to wound bed conditions.
Repeated debridement due to incomplete clearing of necrotic
tissues is avoided to reduce bleeding during treatment for the
wound surface. The eyelids require special attention to pre-
vent corneal injury. Early excision and closure of eyelid
burns reduces the incidence of corneal injury secondary to
exposure and should be a priority. If eversion or contracture
of the eyelids is noted, the eyelids should be relaxed
appropriately.
7.3.3.3 Autografting
Sheet skin grafting should be used and placed on the facial
wound in cosmetic units such as the forehead, cheeks (2),
nasal dorsum, upper eyelids (2), lower eyelids (2), upper lip,
lower lip, and mandibular base. Medium to thick sheet
split- skin autograft should be used for the face and applied in
7.3 Large Sheet Skin Autograft for Small/Medium-Sized Deep Burns

136
cosmetic units to place marginal scars in natural skin crease
lines. If part of the cosmetic and functional unit heals, the
epithelialization areas are removed before intermediate split-
thickness skin grafting. The donor sites are selected opti-
mally from the area between the mammillary line and the
horizontal line at the navel level, the outer aspect of the thigh,
the lower abdomen or the back for optimal color and texture
match. Following facial autografting, the wound is managed
with elastic pressure dressings. The dressings are changed at
7–10 days post-burn for the fi rst time. When applying auto-
graft to the eyelids thick split-thickness graft should be used
and overcorrection should be performed putting more skin in
than seems to be needed as contraction can lead to corneal
exposure. Throughout the course of the burn injury the cor-
neas should protected by regular application of eye ointment
(Fig. 7.8a–c ).
a
b
c
Fig. 7.8 Sheet skin grafting on
the face regarding defi ned
cosmetic and functional units.
( a ) 23 days following facial and
neck burns. ( b ) Autografting
regarding defi ned cosmetic and
functional units. ( c ) Division of
cosmetic and functional units

7 Burn Wound Care

137
Whether to conduct eschar shaving and tangential exci-
sion in the early stages or in the delayed stages is always
controversial. The commonly reported loophole for early
facial eschar shaving is severe bleeding with an average vol-
ume of 331.4 ml/100 cm 2 ± 121.3 ml/100 cm 2 as opposed to
98.3 ml/100 cm 2 ± 51.6 ml/cm 2 for delayed eschar shaving,
showing a signifi cant difference between the two stages.
Additionally, eschar shaving in the early stages tends to
result in hematoma or seroma under the skin grafts that com-
promises the success rate of skin grafting and the overall
grafting effects.
The Xijing Hospital reported 54 cases of full-thickness
and deep partial burns on the face, of which 48 were man-
aged with delayed eschar shaving and tangential excision
and 6 with early shaving. Results demonstrated that delayed
eschar shaving and tangential excision reduced bleeding and
hematoma, leading to desirable surgical effects, including a
complete face with good skin elasticity. However, the early
eschar shaving resulted in undesirable outcomes, including
poor survival of local skin grafts. Hence, it is regarded that
the delayed eschar shaving and tangential excision are opti-
mal in similar conditions. For some third-degree full facial
burns or chemical burns, early eschar shaving is still a good
option owing to the long duration of the natural separation of
eschar.
7.3.3.4 Repair of Eyelid Burns
Before deep facial burns can be managed with delayed eschar
shaving and tangential excision, eyelid contracture often
occurs. Debridement and relaxation are recommended for
eyelid contracture for expanded skin grafting. Meanwhile, at
the edge of each pupil, a 5 × 5 mm fl ap is prepared from the
upper and lower eyelids and sutured after being fl ipped. New
wounds cross the palpebral fi ssure before skin grafting. A
skin bridge across the palpebral fi ssure following skin graft-
ing is maintained for 6 months to 1 year. The advantage of
ankyloblepharon with a skin bridge is that it maintains a pal-
pebral fi ssure of 3–5 mm wide following skin grafting. As
the technique does not affect patients’ vision and daily activ-
ities, they are more likely to show good compliance
(Fig. 7.9a–f ).
7.3.3.5 Post - operative Treatment
1. Patients are required to lie supine to fi x the head.
2. Airway obstruction should be prevented. The patients
should be nursed by designated health staff and emer-
gency equipment is prepared before the patient wakes up
after general anesthesia.
3. The patients are required to lie in bed for 3–5 days.
Conversation, mouth opening, and crying are prohibited.
4. Liquid food for nasal feeding or oral feeding is required
for 3 days and the oral cavity should be kept clean.
5. The nasal cavity is kept ventilated and clean.
6. Venous antibiotics are administered for 3–5 days.
7. Nutritional support is required.
8. Sutures are removed at 6–8 days post-surgery and the
dressings or exposure technique are considered with
regard to specifi c conditions.
7.3.3.6 Typical Case Report
The patient was a 35-year-old man with burns on the head,
face, neck, and all four limbs. The overall burns area was
approximately 51 % of the TBSA and the area of deep third-
degree
burns
was
approximately
38
%
of
the
TBSA. Autografting was conducted at 3 weeks post-burn on
the face and the dressings were changed at 7 days post-
surgery for the fi rst time. Five years after the burn, the eye-
brows were established through hair grafting. Eight years
after the burns in the follow-up period, he was found to have
complete and soft skin on the face. Hypertrophic scars fol-
lowing suture removal were not obvious. The facial topogra-
phy was generally acceptable (Fig. 7.10a–d ).
7.3.4
Repair of Deep Burns
of the Upper Limbs
The upper limbs are two of the most important functional
units for humans. Previous data indicated that the cases of
burns of the upper limbs accounted for 30 % of all cases of
burns. As the fat layer and the dermis of the upper limbs are
both thin, deep burns are often caused and the deformity rate
post-healing is as high as 50–70 %. Additionally, the upper
limbs are cosmetically important components owing to a
high exposure rate. Consequently, timely and appropriate
treatment for burns of the upper limbs is closely related to
functional and cosmetic outcomes post-healing and to
patients’ quality of life. Under conditions of adequate grafts,
the upper limbs should be repaired both functionally and
cosmetically.
7.3.4.1 Treatment Scheme
1. Treatment for the burn surface: early eschar shaving and
tangential excision are adopted. For the fresh deep burn
surface and the surface of the granulation tissues, the
necrotic tissues should be shaved or excised completely
under the tourniquet, including the basal lamina of the
granulation tissues and the superfi cial layer of fat or the
superfi cial layer of the deep fascia. Hematoma can be
minimized by meticulous hemostasis following wound
excision.
2. Skin harvesting: intermediate split-thickness skin grafts
are harvested using a drum dermatome or an electric der-
matome as above. Full-thickness skin grafts are har-
vested and the subcutaneous fat is excised in pediatric
patients.
7.3 Large Sheet Skin Autograft for Small/Medium-Sized Deep Burns