Effects of Cigarette Smoke in Mice Wound Healing is Strain Dependent

Abstract

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 after wounding. Wound contraction was evaluated 7 and 14 days after lesion. Sections were stained with hematoxylin-eosin, Sirius red or toluidine blue 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.

Introduction

Cutaneous wound healing is a complex and dynamic process that involves many coordinated events including inflammation, cell migration, cell proliferation, angiogenesis, matrix deposition, and remodeling (Singer and Clark, 1999). These events are managed by a number of different cell types such as keratinocytes, fibroblasts, and inflammatory and endothelial cells (Martin, 1997) which are influenced by many factors. The relationship between smoking and its detrimental effects on wound healing were first reported by Mosely and Finseth (Mosely and Finseth, 1977) that observed impaired healing of a wound hand in a smoker with arteriosclerosis. After 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 delayed and abnormal wound healing (Frick and Seals, 1994; Manassa et al., 2003; Silverstein, 1992).

Cigarette smoke is a complex mixture of chemicals containing more than 4,000 different constituents, but those of greatest interest are nicotine, carbon monoxide and hydrogen cyanide (Silverstein, 1992). Knowledge of the cutaneous effects of smoking is important because it provides another tool for counseling patients on the dangers of smoking and provides them an effective motivation to quite smoking, especially those who may be more concerned about their outward appearance than about the potential internal damage associated with smoking (Smith and Fenske, 1996).

Most published reports were based on retrospective clinical analyses; few prospective controlled studies have been conducted investigating the actions of cigarette smoke on wound healing (Reus et al., 1992; Manassa; Hertl and Olbrisch, 2003). In animal models, studies are mainly focused in investigate which phases of wound healing are compromised by cigarette smoke (Lawrence et al., 1984; Craig and Rees, 1985).

A large variety of mice strains are used in experimental studies. Different strains present a large range of sensitivity 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 of a good experimental model to study cigarette smoke effects on cutaneous excisional wound healing is an important tool to understand the cigarette smoke pathophysiological mechanisms at the cellular and molecular level. The purpose of this study was to investigate the effects of cigarette smoke exposure on wound healing in three mice strains (Swiss, BALB/c and C57BL/6) and point out the differences among the strains.

Material and Methods

Animals

All animal procedures are in accordance with the Guide for Use and Care of Animals and were approved by the Ethical Committee for Animal Use of State University of Rio de Janeiro.

Male mice (10 weeks of age) of three different strains: Swiss, BALB/c and C57BL/6 were used. Animals were housed under controlled environmental conditions (light/dark 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 chamber, to a smoke-air mixture of commercial filtered Virginia cigarette, 3 times/day, 7 days/week, during the entire experiment, 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 experiment. 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.

Macroscopic analysis

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 tissue 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 neoepidermis thickness and inflammatory infiltrate, with Sirius 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 (Olympus BH-2, Olympus Optical Co. Ltd; Tokyo, Japan) in middle region of granulation tissue. The amount of mast cells was evaluated in toluidine blue stained sections. Six random fields (0.119 mm²), located in the superficial region of granulation tissue, were counted using the same objective and microscope. The percentage of degranulating mast cells was also evaluated. Results are expressed as mean of cells per field. All analyses were repeated without significant difference among them.

Epidermal and neoepidermal thickness

Epidermis and neoepidermis thickness were evaluated in hematoxylin-eosin stained sections using Zeiss image-processing system KS400 (Zeiss-Vision; Oberkochen, Germany). Epidermis and neoepidermis thickness was measured from dermo-epidermal junction to the outermost extent of the 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 each wound.

Immunohistochemistry

Myofibroblasts expressing alpha-smooth muscle actin were localized by immunohistochemistry. To allow the use of a mice monoclonal antibody in mouse tissue, some modifications in standard technique were performed. Sections (5 μm) were deparaffinized, hydrated and after washing in PBS (phosphate buffered saline) sections were incubated with EnVision system (DAKO; Carpinteria, CA) for 50 minutes to allow anti-mouse IgG to bind to tissue, then peroxidase (endogenous and polymer-linked) was inhibited by incubation in 3% H₂O₂ in methanol, for 30 minutes. After washing, sections were incubated with a solution of a monoclonal antibody against alpha-smooth muscle actin (DAKO) (1:200) and Envision (1:20) in PBS/BSA (PBS/bovine serum albumin) 1% overnight. Diaminobenzidine was used as chromogen. Sections were counterstained with Delafield’s hematoxylin. Negative controls were performed replacing primary antibody by PBS/BSA and no labeling was observed.

Statistical Analysis

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 analyzed with non-parametric Mann-Whitney test. Statistical analysis was done using the software Graph Pad Instat version 3.01 (GraphPad Software Inc.; CA, USA).

Results

Macroscopic Analysis

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 the same way, smoke exposure delayed wound contraction 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 between strains showed that BALB/c strain always presented larger wound than Swiss and C57BL/6 strains in both control 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.

General Histology

In Swiss control group, 14 days after wounding, numerous 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 control 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 granulation 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 distribution observed in counterpart of BALB/c animals (control: 1.1 ± 0.2 inflammatory cells/field, exposed 1.8 ± 0.2 inflammatory cells/field; p < 0.05) (Figure 2).

Mast cells

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 observed.

Data concerning mast cell quantification are presented in Figure 3. In Swiss animals, smoke exposed group presented 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 compared 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 degranulation in any strain. Among strains no differences were observed 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 affect neo-epidermis thickness (Figure 4b).

Organization and distribution of collagen fibers

Collagen organization was evaluated in tissue sections stained with Sirius red observed under polarization. In normal 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 collagen arrangement in all animal groups (control or exposed), neither among strains.

Analysis of myofibroblasts

Myofibroblasts were fusiform, parallel to surface and homogeously distributed in granulation tissue of all strains and experimental groups. Fourteen days after wounding, the density of myofibroblasts in smoke exposed groups was higher than in control group of all strains (Figure 5).

Discussion

This study was the first to show that cigarette smoke exposure affects mice wound healing in different ways depending on mice strain studied (Swiss, BALB/c or C57BL/6). Whereas wound contraction in Swiss mice was not altered by 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, collagen arrangement was not affected by cigarette smoke in any strain.

The detrimental effects of smoking on wound healing were 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 appropriate. 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 many studies about genotype influence on sensitivity to nicotine, 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 nicotine metabolism is not important in controlling genotype-dependent differences in sensitivity to the drug (Marks et al., 1983a).

Swiss strain seems to be more resistant to nicotine behavioral 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 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, 1983b).

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 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, fibroblasts and endothelial cells (Wang et al., 2000). It is known that IL-8 first exerts its effect in neutrophils (Hebert and Baker, 1993; Graves and Jiang, 1995) and that the cigarette smoke can induce the release of IL-8 in cells of the bronchial epithelia (Mio et al., 1997) as well as in endothelial cells (Wang; Ye; Zhu and Cho, 2000). These effects of the cigarette smoke on neutrophils may explain the higher amount of inflammatory cells found in granulation tissue of C57BL/6 and BALB/c mice strains exposed to cigarette smoke. And the prolongation of inflammatory phase explains the delay in wound healing process.

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 release of chemotactic factors (Abraham and Malaviya, 1997; Schwartz and Austen, 1984) but it was also shown that the excess 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 degranulation (Thomas et al., 1992) while another one showed that mast cells increase fibroblasts-mediated collagen gel retraction (Moyer et al., 2004). No alteration in mast cells degranulation was observed in our study. The higher amount of mast cells in Swiss and C57BL/6 mice strains exposed to cigarette smoke could be explained by an alteration in the initial steps of wound healing process leading to an impairment on recruitment of these cells to the granulation tissue. Surprisingly 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 reduced.

Several studies have shown that cigarette smoke can impair 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 after wounding is a consequence of the impairment in fibroblast recruitment that induced a delay in myofibroblasts differentiation. This delay in myofibroblast differentiation probably is the cause of the delay in wound contraction since myofibroblasts are the main cellular component involved in wound contraction (Serini and Gabbiani, 1999; Wong et al., 2004) showed that cigarette smoke increase fibroblast survival what 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 extracellular 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 healing induced by cigarette smoke exposition is followed by excessive scarring.

The differences between mice strains and cigarette smoke exposure observed in our study suggest that the genetic predisposition is an important factor that contributes for the use (Collins et al., 1988) and for the effects of tobacco, as well as the velocity and the depth of the inhalation, sex, race and characteristics of the cigarette (Zacny et al., 1987; Bridges et al., 1986). All these aspects in joint could explain differences between individuals to the adverse effects of the cigarette smoke and should be considered in future studies (experimental and in humans).