The Journal of TRAUMA威 Injury, Infection, and Critical Care

Effect of Nitric Oxide Synthase Inhibitor on Experimentally

Induced Burn Wounds
Müfide Nuran Akçay, MD, Önder Özcan, MD, Cemal Gündoğdu, MD, Güngör Akçay, MD, PhD,
Ahmet Balık, MD, Kamil Köse, MD, and Durkaya Ören, MD

Background: Nitric oxide (NO) may
have an important role in the healing of
burn wounds. This study investigated the
effect of NO on experimentally induced
burn wounds by preventing NO synthesis.
Methods: A total of 40 mice weigh-
ing 25 to 30 g were used in this study. The
shaved skin on the back of the mice was
immersed in 100°C water for 10 seconds
to achieve a partial-thickness scald burn.
The mice were divided into two groups of
20. In group I (control group), 17.5 mg/kg
of serum physiologic (placebo) was in-
jected intraperitoneally two times a day
for 15 days. In group II (study group),
17.5 mg/kg of aminoguanidine (NO syn-
thase inhibitor) was injected intraperito-
neally two times a day for 15 days. On day
15 of the burn, the animals were killed
and the burn areas were investigated his-
tologically. Histologic changes such as ep-
ithelial proliferation, abscess, collagen,
and granulation tissue were evaluated.
Results: Epithelial proliferation, for-
mation of collagen, and granulation tissue
with rich capillaries observed in the con-
trol group were statically significantly
higher than those observed in the study
group (z ⴝ ⴚ2.022, p < 0.05; z ⴝ ⴚ2.02,
p < 0.05; and z ⴝ ⴚ2.022, p < 0.05; re-
spectively).
Conclusion: We concluded that heal-
ing of the burn wound is delayed by pre-
venting NO synthesis.
Key Words: Burn wound, Nitric ox-
ide synthase inhibitor
J Trauma. 2000;49:327–330.

N
itrogen oxides are biomolecules with diverse physi-
ologic functions. Some of these compounds are free
radicals and potent oxidizing agents, and others
serve as important homeostatic agents and may modulate
immune function.1 The production of nitric oxide (NO)
depends on the induction of NO synthase, which can be
inhibited by two known inhibitors: N-monomethyl-argi-
nine and aminoguanidine.2
Thermal injury results in significant alteration of multi-
ple mediator pathways. NO is produced by macrophages, and
the production of NO by these cells is stimulated by mito-
gens, endotoxin, and cytokines after thermal injury.1,3 In
many experiments, NO was found to prevent postburn myo-
cardial dysfunction and mediate the lymphoproliferative
response.4 In this study, we intended to investigate the effect
of NO on experimentally induced burn wounds by preventing
NO synthesis.
MATERIALS AND METHODS
A total of 40 mice weighing 25 to 30 g were used
throughout the study. Animals were quarantined for 1 week
in standard laboratory conditions so that they could adapt to
the environment. After fasting mice were anesthetized with
ketamine HCl, body hair on their backs was shaved. The
Submitted for publication February 23, 1999.
Accepted for publication May 5, 2000.
Copyright © 2000 by Lippincott Williams & Wilkins, Inc.
From the Departments of General Surgery (M.N.A., Ö.Ö., A.B., K.K.,
D.Ö.), Pathology (C.G.) and Division of Endocrinology (G.A.), Department
of Internal Medicine, Atatürk University Medical School, Erzurum, Turkey.
Address for reprints: Müfide Nuran Akçay, MD, Atatürk Üniversitesi
Postanesi, P. K. 18, 25171 Erzurum, Turkey.
shaved skin on the back was immersed in 100°C water for 10
seconds to achieve a partial-thickness scald burn.
The mice were divided into two groups of 20. In group I
(control group), 17.5 mg/kg of serum physiologic (placebo)
was injected intraperitoneally two times a day for 15 days. In
group II (study group), 17.5 mg/kg of aminoguanidine was
injected intraperitoneally two times a day for 15 days.
No animal died during the study. There were bullae on
all burn wounds, which were removed to accelerate healing.
On day 15 of the burn, the animals were killed, burn areas
were excised, and tissue samples were obtained showing
whole lesions (the centers and edges of the wounds) and fixed
in formalin. After being dehydrated by alcohol of various
degrees, the tissue samples were embedded in paraffin
blocks, cut into 5-␮m sections for histologic staining with
hematoxylin-eosin and Masson’s trichrome, and investigated
by light microscopy. Formation of collagen was graded mild,
moderate, or severe in the sections stained with Masson’s
trichrome. Histologic grading was based on a standardized
assessment of epithelial proliferation, abscess formation of
collagen, and granulation tissue. Formation of granulation
tissue was assessed according to the absence or presence of
the granulation tissue. Epithelial proliferation was assessed
according to the absence or presence of the epithelization and
the number of layers of epithelial tissue. Abscess was graded
1 if it was absent and 2 if it was present.
Tests for significant differences between histologic
changes, such as epithelial proliferation, abscess, and forma-
tion of collagen and granulation tissue, observed in the con-
trol and study groups were carried out with the Wilcoxon
matched-pairs signed-ranks test. A p value of ⬍ 0.05 was
accepted as significant.

Volume 49 • Number 2 327

 The Journal of TRAUMA威 Injury, Infection, and Critical Care

those observed in the study group (z ⫽ ⫺2.022, p ⬍ 0.05; z ⫽

Table 1. Histologic changes observed in control and
⫺2.02, p ⬍ 0.05; and z ⫽ ⫺2.022, p ⬍ 0.05; respectively).
study groups
There was no difference between abscess observed in the
Study Group Control Group study and control groups (z ⫽ ⫺1.60, p ⬎ 0.5).
Formation of granulation tissue Histologic changes observed in the study and control
Absent (1) 16 6 groups are shown in Figures 2, 3, and 4. A partial thickness
Present (2) 4 14
burn heals by proliferation of epithelium from the skin ap-
Epithelial proliferation
Absent (1) 6 4 pendages or from the wound edges. As shown in Figure 2,
Single layered (2) 14 8 regeneration of epithelium in part from the underlying hair
Stratified (3) 0 8 follicles was seen in the control group. Figure 3 shows the
Formation of collagen more advanced level of epithelization that occurred in the
Mild (1) 8 2
control group. Here, stratified squamous epithelium formed,
Moderate (2) 8 10
Severe (3) 4 8 and collagenized tissue was seen under it. However, as shown
Abscess in Figure 4, chronic inflammatory mononuclear cell infiltra-
Absent (1) 8 14 tion and collagenized tissue and, on the left top corner,
Present (2) 12 6 stratified epithelial tissue belonging to the healthy tissue were
seen in the study group. Epithelial proliferation, one of the
RESULTS most important indicators of wound healing, is absent in
Histologic changes observed in the study and control Figure 4.
groups are shown in Table 1 and Figure 1. As shown in Table
1, formation of granulation tissue was absent in 16 mice and
present in 4 mice in the study group. Conversely, it was DISCUSSION
absent in 6 mice and present in 14 mice in the control group. Injury triggers an organized and complex cascade of
Epithelial proliferation was absent in 6 mice, single layered in cellular and biochemical events that result in a healed
14 mice, and stratified in none of the mice in the study group wound.5 Burn injury is associated with inflammatory re-
and absent in 4 mice, single layered in 8 mice, and stratified sponse and related hyperdynamic cardiovascular profile. In-
in 8 mice in the control group. Formation of collagen was creased production of NO, a potent endogenous vasodilator,
mild in 8 mice, moderate in 8 mice, and severe in 4 mice in has been reported in patients with inflammatory states, in-
the study group and mild in 2 mice, moderate in 10 mice, and cluding sepsis, but not after trauma other than burns.4
severe in 8 mice in the control group. Abscess was absent in Activation of wound macrophages results in the synthe-
8 mice and present in 12 mice in the study group and absent sis of NO, which has many functions, including antimicrobial
in 14 mice and present in 6 mice in the control group. In properties.5 Albina et al.6 showed that macrophages are ac-
Figure 1, only positive histologic findings obtained in the tivated during the early phase of healing to synthesize NO
subjects are shown. Epithelial proliferation, formation of col- and the hypoxic wound environment further enhances its
lagen, and granulation tissue with rich capillaries observed in expression. Recent studies demonstrated that NO synthesis is
the control group were statically significantly higher than reduced in impaired wound-healing models; conversely, in

Fig. 1. Positive histologic findings observed in the control and study groups.

328 August 2000


Fig. 2. Partial thickness burn in control group, showing regenera-
tion of epithelium in part from the underlying hair follicles (hema-
toxylin and eosin; original magnification, ⫻100).
Fig. 3. Collagenized tissue in control group under stratified squa-
mous epithelium (hematoxylin and eosin; original magnification,
⫻100).
vivo inhibition of NO synthesis in mice impairs wound
healing.5,6
NO release after human burn injury has not been well
studied. An experimental study on burn trauma in rats re-
ported an increase in NO production that was maximal at day
2 postburn.7 In other rat experiments, NO was found to
Volume 49 • Number 2
Nitric Oxide Synthase Inhibitor and Burn Wounds
Fig. 4. In study group, chronic inflammatory mononuclear cell in-
filtration and collagenized tissue and, on the left top corner, strat-
ified epithelial tissue belonging to the healthy tissue (hematoxylin
and eosin; original magnification, ⫻100).
prevent postburn myocardial dysfunction and mediate the
lymphoproliferative response.8 –10
In this study, formation of granulation tissue with rich
capillaries observed in the control group was statically sig-
nificantly higher than that observed in the study group, show-
ing the positive effects of NO on inflammatory cell infiltra-
tion, fibroblastic proliferation, and angiogenesis. Partial-
thickness wounds heal by the process of epithelization. A few
days after wounding, epithelial cells start proliferating from
wound edges or uninjured epithelial islands within the
wound.5,11,12 In the present study, epithelial proliferation may
have decreased because of the decrease of mitogenic activity
in the study group. We did not observe any stratified epithe-
lial proliferation in the study group. Collagen is the major
component of the extracellular matrix of all soft tissues.
During the second week of burn, there is continued accumu-
lation of collagen. In the control group, we observed a better
formation of collagen compared with the study group, indi-
cating that the healing of wounds was better in the control
group. There was no statistically significant difference ob-
served between abscesses in the study and control groups.
However, abscess formation in the study group was higher
than that in the control group, indicating that inflammatory
response was ineffective in the study group. Formation of the
abscess may have been attributable to the decreased epithelial
proliferation in the study group, and the abscess may have
decreased healing.
In another study, we investigated the effects of nitroglyc-
erin ointment on the healing of infected wounds. Nitroglyc-
erin is an organic nitrate that binds to protein receptors,
releasing NO. We concluded that topical nitroglycerin appli-
cation has a beneficial effect on the healing of wound
infection.13 It is possible that nitroglycerin ointment may
329
 The Journal of TRAUMA威 Injury, Infection, and Critical Care
assist healing of wound infection by inducing local vasodi-
latation, improving wound blood flow, and modulating im-
mune function.
In conclusion, the present study was undertaken to ex-
amine the importance of NO in the healing of burn wounds.
Our results indicate that the healing of the burn wound is
delayed by preventing NO synthesis.
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