The online version of this article can be found at:
2007 35: 890 Toxicol Pathol
Juliana F. Cardoso, Bruna R. Souza, Thaís P. Amadeu, Samuel S. Valença, Luís Cristóvào M. S. Porto and Andréa M. A.
Effects of Cigarette Smoke in Mice Wound Healing is Strain Dependent
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Toxicologic Pathology, 35:890–896, 2007
Copyright C ?by the Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
Effects of Cigarette Smoke in Mice Wound Healing is Strain Dependent
JULIANA F. CARDOSO, BRUNA R. SOUZA, THA´ ıS P. AMADEU, SAMUEL S. VALENC ¸A,
LU´ ıS CRIST´ OV˜ AO M. S. PORTO, ANDR´ EA M. A. COSTA
Histology and Embryology Department, State University of Rio de Janeiro, Rio de Janeiro, Brazil
It has been clinically and experimentally shown that cigarette smokers suffer from impaired wound healing, but the mechanisms that lead to the
alterations are not well understood. The aim of this study was to investigate if the effects of cigarette smoke exposure on excisional cutaneous wound
healing are different depending on the strain (Swiss, BALB/c and C57BL/6 mice) studied. Male mice were exposed to smoke of nine whole cigarettes
per day, 3 times/day, daily, for 10 days. In the 11th day a full-thickness excisional wound was performed. Control group was sham-exposed and also
had a full-thickness excisional wound. The cigarette smoke exposure protocol was performed until euthanasia. Animals were euthanatized 14 days
and immunostained for alpha-smooth muscle actin. Smoke exposed animals presented delay in wound contraction, in fibroblastic and inflammatory
cells recruitment and in myofibroblastic differentiation; those alterations were strain dependent. Cigarette smoke exposure also affected mast cells
recruitment and neoepidermis thickness. In conclusion, the present study demonstrated that the effects of cigarette smoke in mice cutaneous wound
healing are related to mice strain studied.
Cigarette smoke; wound healing; myofibroblasts; mast cells; connective tissue.
Cutaneous wound healing is a complex and dynamic pro-
cess that involves many coordinated events including inflam-
mation, cell migration, cell proliferation, angiogenesis, ma-
trix deposition, and remodeling (Singer and Clark, 1999).
such as keratinocytes, fibroblasts, and inflammatory and en-
dothelial cells (Martin, 1997) which are influenced by many
effects on wound healing were first reported by Mosely and
Finseth (Mosely and Finseth, 1977) that observed impaired
ter this study, epidemiological and experimental studies have
pointed to a significant correlation between cigarette smoke
and alterations in the process of tissue remodeling, such as
Manassa et al., 2003; Silverstein, 1992).
Cigarette smoke is a complex mixture of chemicals con-
taining more than 4,000 different constituents, but those of
greatest interest are nicotine, carbon monoxide and hydro-
effects of smoking is important because it provides another
tool for counseling patients on the dangers of smoking and
appearance than about the potential internal damage associ-
ated with smoking (Smith and Fenske, 1996).
Most published reports were based on retrospective clin-
ical analyses; few prospective controlled studies have been
conducted investigating the actions of cigarette smoke on
Address correspondence to Dr. Andr´ ea Monte Alto Costa, State Univer-
Professor Manuel de Abreu, 444, 3◦andar, 20550-170, Rio de Janeiro, RJ,
Brazil; e-mail: firstname.lastname@example.org
wound healing (Reus et al., 1992; Manassa; Hertl and Ol-
brisch, 2003). In animal models, studies are mainly focused
in investigate which phases of wound healing are compro-
mised by cigarette smoke (Lawrence et al., 1984; Craig and
A large variety of mice strains are used in experimental
studies. Different strains present a large range of sensitiv-
ity to behavioral effects of nicotine (the major constituent of
the particulate phase of tobacco smoke) (Collins and Marks,
1991) and physiological effects of cigarette smoke exposure
(Bartalesi et al., 2005; Guerassimov et al., 2004). The choice
on cutaneous excisional wound healing is an important tool
nisms at the cellular and molecular level. The purpose of this
study was to investigate the effects of cigarette smoke expo-
sure on wound healing in three mice strains (Swiss, BALB/c
MATERIAL AND METHODS
Use and Care of Animals and were approved by the Ethical
Committee for Animal Use of State University of Rio de
Male mice (10 weeks of age) of three different strains:
Swiss, BALB/c and C57BL/6 were used. Animals were
cycle, temperature and humidity) and had free access to food
and water during the experiment.
Cigarette smoke exposure and wounding
In each strain, animals were separated in control (n =
15) and smoke exposed groups (n = 21). Smoke exposed
groups had the hole-body exposed, in an inhalation cham-
ber, to a smoke-air mixture of commercial filtered Virginia
Vol. 35, No. 7, 2007
CIGARETTE EXPOSURE AND WOUND HEALING891
cigarette, 3 times/day, 7 days/week, during the entire ex-
periment, as already described (Valenca et al., 2004). The
cigarette smoke exposure protocol started 10 days before the
excisional wound and continued until the end of the experi-
ment. The control group was sham-exposed.
On day 0 (10 days after the beginning of smoke or sham
exposition), the animals were anaesthetized with ketamine
(5 mg/kg, ip) and xylazine (2 mg/kg, ip). The dorsal surface
was shaved and a full-thickness excisional wound (1 cm2)
was performed. The wound was not sutured or covered and
healed by second intension.
To evaluate wound contraction, a transparent plastic sheet
was placed over the wound and the wound margins were
traced. After digitalization, the wound area was evaluated
using Zeiss image-processing system KS400 (Zeiss-Vision;
Oberkochen, Germany) (Souza et al., 2005).Wound area was
measured soon after wounding and 7 and 14 days later. Data
are expressed as a percentage of the initial wound area.
Tissue harvesting and staining
Mice were sacrificed 14 days after wounding in a CO2
chamber. Fragments of wound with adjacent normal skin tis-
sue were formalin-fixed (pH 7.2) and paraffin-embedded.
Sections (5 µm) were stained with hematoxylin-eosin, for
general evaluation of wound and normal skin, epidermis and
ius red for analysis of collagen fibers arrangement and with
toluidine blue for analysis and quantification of mast cells.
Inflammatory and Mast Cells Quantification
The amount of inflammatory cells was evaluated in
hematoxylin-eosin stained sections. Ten random fields
(0.119 mm2) were counted using the ×40 objective (Olym-
region of granulation tissue. The amount of mast cells was
(0.119 mm2), located in the superficial region of granulation
tissue, were counted using the same objective and micro-
scope. The percentage of degranulating mast cells was also
analyses were repeated without significant difference among
Epidermal and neoepidermal thickness
Epidermis and neoepidermis thickness were evaluated
in hematoxylin-eosin stained sections using Zeiss image-
processing system KS400 (Zeiss-Vision; Oberkochen, Ger-
granular layer. Three random fields were analyzed using the
x40 objective, with three measures in each field (Olympus
BH-2). Data are expressed as the mean of measurements in
Myofibroblasts expressing alpha-smooth muscle actin
were localized by immunohistochemistry. To allow the use
of a mice monoclonal antibody in mouse tissue, some mod-
ifications in standard technique were performed. Sections
(5 µm) were deparaffinized, hydrated and after washing in
allow anti-mouse IgG to bind to tissue, then peroxidase (en-
dogenous and polymer-linked) was inhibited by incubation
in 3% H2O2in methanol, for 30 minutes. After washing, sec-
against alpha-smooth muscle actin (DAKO) (1:200) and En-
vision (1:20) in PBS/BSA (PBS/bovine serum albumin) 1%
overnight. Diaminobenzidine was used as chromogen. Sec-
PBS/BSA and no labeling was observed.
All data are presented as mean ± standard error of mean
(SEM). Data concerning lesion area, inflammatory and mast
cells amount, epidermis and neoepidermis thickness were
3.01 (GraphPad Software Inc.; CA, USA).
In Swiss mice, smoke exposure did not altered wound
contraction 7 or 14 days after wounding. In BALB/c mice
smoke exposure delayed wound contraction 7 and 14 days
after wounding (p < 0.01 and p < 0.05, respectively). In
C57BL/6 mice 7 and 14 days after wounding (p < 0.01 and
p < 0.001, respectively). (Figure 1).
Seven and fourteen days after wounding comparison be-
tween strains showed that BALB/c strain always presented
larger wound than Swiss and C57BL/6 strains in both con-
trol and smoke exposed groups (p < 0.001, in all cases). No
differences were observed in wound contraction when Swiss
and C57BL/6 strains were compared.
FIGURE 1.—Wound contraction evaluation in Swiss, BALB/c and C57BL/6
mice strains (mean ± SEM).
892 CARDOSO ET AL.
inflammatory (2.52 ± 0.3 cells/field) and fibroblastic cells
were observed in granulation tissue; in the smoke exposed
group the amount of inflammatory (1.73 ± 0.2 cells/field; p
< 0.05) and fibroblastic cells was reduced. In BALB/c con-
trol group, 14 days after wounding, a reduced amount of
inflammatory (1.68 ± 0.3 cells/field) and fibroblastic cells
were observed compared to smoke exposed group in granu-
lation tissue (2.45 ± 0.3 inflammatory cells/field; p < 0.05).
C57BL/6 control and smoke exposed groups presented the
same pattern of inflammatory and fibroblastic cells distribu-
tion observed in counterpart of BALB/c animals (control:
matory cells/field; p < 0.05) (Figure 2).
In all groups mast cells were sparse and localized in the
superficial region of granulation tissue. The majority of mast
cells were ovoid but some tadpole-shaped cells were also
Data concerning mast cell quantification are presented
in Figure 3. In Swiss animals, smoke exposed group pre-
sented higher amount of mast cells (+117%) than control
group (p < 0.001). BALB/c smoke exposed group showed
a lower amount of mast cells (−78%) than control group
(p < 0.0001). On the other hand, C57BL/6 smoke exposed
group also presented higher amount of mast cells (+214%)
compared to control group (p < 0.05).
When the amount of mast cells present in lesion was com-
pared among control groups of mice strains, we observed
that BALB/c mice presented a higher amount of mast cells
(+ 48%) than Swiss mice (p < 0.0001) and (+76%) than
C57BL/6 mice (p < 0.0001). Control groups of C57BL/6
and Swiss strains did not present difference in amount of
mast cells. Comparison between smoke exposed groups
showed a different response pattern; Swiss mice presented
a higher amount of mast cells (+80%) compared to BALB/c
(p < 0.0001), but did not presented difference compared
to C57BL/6. Comparison between C57BL/6 and BALB/c
smoke exposed group showed a higher amount of mast cells
(+71%) in C57BL/6 mice (p < 0.001).
Cigarette smoke exposure did not affect mast cell degran-
ulation in any strain. Among strains no differences were ob-
served in the amount of degranulating mast cells.
Epidermis and neoepidermis thickness
Epidermal thickness was not altered by cigarette smoke
exposure in Swiss strain. In BALB/c smoke exposed animals
epidermis was thinner (9.1 ± 2.7 mm) than control animals
(p < 0.05). In C57BL/6 smoke exposed animals epidermis
was thicker than in control animals (p = 0.01). (Figure 4a).
In Swiss mice, control animals presented a thicker neo-
epidermis than smoke exposed animals (p < 0.0001). In
BALB/c and C57BL/6 cigarette smoke exposure did not af-
fect neo-epidermis thickness (Figure 4b).
Organization and distribution of collagen fibers
Collagen organization was evaluated in tissue sections
skin collagen fibers were yellow-red, thick and presented a
basket-like pattern in all experimental groups. Fourteen days
after wounding, collagen fibers, in superficial region of scar,
were mainly greenish, some yellow-greenish and yellow-red
fragmented collagen fibers were always present. Collagen
fibers were loosely arranged and parallel to surface. In deep
region of scar collagen fibers were arranged in bundles (data
not shown). There were no significant differences in colla-
gen arrangement in all animal groups (control or exposed),
neither among strains.
Analysis of myofibroblasts
Myofibroblasts were fusiform, parallel to surface and ho-
mogeously distributed in granulation tissue of all strains and
sity of myofibroblasts in smoke exposed groups was higher
than in control group of all strains (Figure 5).
This study was the first to show that cigarette smoke ex-
posure affects mice wound healing in different ways depend-
ing on mice strain studied (Swiss, BALB/c or C57BL/6).
cigarette smoke exposure, in BALB/c and C57BL/6 strains
there was a delay in wound contraction. The inflammatory
infiltrate, mast cells mobilization, neo-epidermis thickness
were affected by cigarette smoke exposure in a different way
according to strain studied. Myofibroblastic differentiation
was delayed by cigarette smoke in all strains. However, col-
first related by researches who observed impaired healing of
a hand wound in a smoker with arteriosclerosis (Mosely and
Finseth, 1977). From this observation many clinic studies
were developed, but the use of animal models are still an
important tool to investigate the effects of cigarette smoke
exposure on wound healing. However, it is very important
the definition of what experimental model is more appro-
priate. Studies, mainly involving behavioral aspects, showed
that different strains respond in different ways to nicotine
(Garg, 1969; Marks et al., 1983b), this fact has stimulated
tine, the main component of cigarette smoke (Hatchell and
Collins, 1977; Mohammed, 2000). For example Marks et al.
(1983) studied four strains (BALB/c, C57BL/6, DBA/2 and
C3H/2) to observe behavioral and physiological effects of
nicotine and showed that C57BL/6 and DBA/2 strains were
more sensitive to nicotine depressive effects while C3H/2
strain showed more sensitivity to nicotine stimulant effects.
However, BALB/c strain showed more diverse effects, being
sometimes more and others less sensitive to nicotine effects,
evidencing nicotine effects in this strain are not regulated by
an unique mechanism; the authors also suggested that nico-
tine metabolism is not important in controlling genotype-
dependent differences in sensitivity to the drug (Marks et al.,
Swiss strain seems to be more resistant to nicotine behav-
ioral depressive effects requiring a higher dose to present the
same depressive effects presented by others strains (Tizabi
et al., 1998). We can speculate that the absence of effects
in wound contraction in Swiss smoke exposed mice may be
Vol. 35, No. 7, 2007
CIGARETTE EXPOSURE AND WOUND HEALING 893
FIGURE 2.—Granulation tissue on wound area of different mice strains 14 days after wounding. Swiss control group (a) presented a higher amount of inflammatory
and fibroblast-like cells than smoke exposed group (b). BALB/c control group (c) presented a lower amount of inflammatory and fibroblast-like cells than smoke
exposed group (d). C57BL/6 control group (e) presented a lower amount of inflammatory and fibroblast-like cells than smoke exposed group (f). Hematoxylin-Eosin.
Bar 30 µm.
due to a resistance to nicotine. This resistance to nicotine is
probably due to genetic background since studies published
still had not succeed to demonstrate alterations on nicotine
metabolism in different strains (Marks; Burch and Collins,
Cigarette smoke have been associated with increase of the
neutrophils infiltrate (Chow et al., 1997) as well as with
alterations in its functions (chemotaxis and phagocytosis)
(Kenney et al., 1977; Seymour, 1991; Lannan et al., 1992).
The possible mechanism by which occurs the increase of
894 CARDOSO ET AL.
FIGURE 3.—Mast cells/field in wound area of control and smoke exposed
groups. (mean ± SEM).
neutrophils amount would be the stimulation of interleukin
8 (IL-8) release, which is powerful chemotactic factor to
leukocytes and is produced by many types of cells such as
monocytes, keratinocytes, lymphocytes, eosinophils, fibrob-
IL-8 first exerts its effect in neutrophils (Hebert and Baker,
1993; Graves and Jiang, 1995) and that the cigarette smoke
Zhu and Cho, 2000). These effects of the cigarette smoke on
neutrophils may explain the higher amount of inflammatory
FIGURE 4.—Epidermis (a) and neo-epidermis (b) thickness in control and
smoke exposed groups (mean ± SEM).
cells found in granulation tissue of C57BL/6 and BALB/c
mice strains exposed to cigarette smoke. And the prolon-
gation of inflammatory phase explains the delay in wound
Our study showed an alteration in mast cells recruitment
according to the strain studied. It is known that mast cells
have an important role in the inflammatory phase of wound
healing, mainly by neutrophils recruitment through the re-
lease of chemotactic factors (Abraham and Malaviya, 1997;
cess of mast cells mediators (such as chymase) may impair
epithelial cell migration (Ebihara et al., 2005). A previous
study showed that cigarette smoke stimulated mast cells de-
granulation (Thomas et al., 1992) while another one showed
that mast cells increase fibroblasts-mediated collagen gel re-
traction (Moyer et al., 2004). No alteration in mast cells de-
granulation was observed in our study. The higher amount
of mast cells in Swiss and C57BL/6 mice strains exposed to
tial steps of wound healing process leading to an impairment
on recruitment of these cells to the granulation tissue. Sur-
prisingly the amount of mast cells was reduced in BALB/c
mice exposed to cigarette smoke, but it should be considered
that the amount of mast cells was higher in control BALB/c
mice compared to the other strains (80% higher than Swiss
and 240% higher than C57BL/6). So we can argue that in
BALB/c mast cells did not need to be mobilized, mast cells
already present in tissue degranulated and exerted functions,
and as mast cells degranulated the amount of mast cells was
wound healing process through the inhibition of recruitment
and migration of fibroblasts (Nakamura et al., 1995); (Wong
and Martins-Green, 2004; Wong et al., 2004). The higher
amount of myofibroblasts observed in our study 14 days af-
recruitment that induced a delay in myofibroblasts differen-
tiation. This delay in myofibroblast differentiation probably
is the cause of the delay in wound contraction since myofi-
contraction (Serini and Gabbiani, 1999; Wong et al., 2004)
can lead to development of fibrosis. It is well known that in
hypertrophic scars (a typical example of excessive scarring)
fibrosis is due to accumulation of myofibroblasts that do not
disappear by apoptosis and are continuously secreting extra-
cellular matrix (Desmouliere et al., 1995; Costa et al., 1999;
Lorena et al., 2002; Linge et al., 2005). It will be important
to evaluate, in future studies, if the delay in cutaneous wound
The differences between mice strains and cigarette smoke
exposure observed in our study suggest that the genetic pre-
disposition is an important factor that contributes for the use
(Collins et al., 1988) and for the effects of tobacco, as well as
acteristics of the cigarette (Zacny et al., 1987; Bridges et al.,
and should be considered in future studies (experimental and
Vol. 35, No. 7, 2007
CIGARETTE EXPOSURE AND WOUND HEALING 895
FIGURE 5.—Myofibroblast distribution on wound area of different mice strains 14 days after wounding. Swiss control group (a) presented a lower amount of
myofibroblasts than smoke exposed group (b). BALB/c control group (c) presented a lower amount of myofibroblasts than smoke exposed group (d). C57BL/6
control group (e) presented a lower amount of myofibroblasts than smoke exposed group (f). Immunohistochemistry against alpha-smooth muscle actin. Bar 30 µm.
This study was partially supported by CNPq and
FAPERJ. BR Souza and JF Cardoso held a graduate
fellowship from PIBIC-CNPq and PIBIC-UERJ respec-
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