AJP - Lung Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 295: L670-L679, 2008. First published August 29, 2008; doi:10.1152/ajplung.00465.2007
1040-0605/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/4/L670    most recent
00465.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vieira, R. P.
Right arrow Articles by Carvalho, C. R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vieira, R. P.
Right arrow Articles by Carvalho, C. R. F.

Aerobic conditioning and allergic pulmonary inflammation in mice. II. Effects on lung vascular and parenchymal inflammation and remodeling

Rodolfo P. Vieira,1 Vanessa F. de Andrade,2 Anna Cecília S. Duarte,2 Ângela B. G. dos Santos,1 Thaís Mauad,1 Milton A. Martins,3 Marisa Dolhnikoff,1 and Celso R. F. Carvalho2

Departments of 1Pathology, 2Physical Therapy, and 3Medicine, School of Medicine, University of São Paulo, São Paulo, Brazil

Submitted 8 November 2007 ; accepted in final form 14 August 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent evidence suggests that asthma leads to inflammation and remodeling not only in the airways but also in pulmonary vessels and parenchyma. In addition, some studies demonstrated that aerobic training decreases chronic allergic inflammation in the airways; however, its effects on the pulmonary vessels and parenchyma have not been previously evaluated. Our objective was to test the hypothesis that aerobic conditioning reduces inflammation and remodeling in pulmonary vessels and parenchyma in a model of chronic allergic lung inflammation. Balb/c mice were sensitized at days 0, 14, 28, and 42 and challenged with ovalbumin (OVA) from day 21 to day 50. Aerobic training started on day 21 and continued until day 50. Pulmonary vessel and parenchyma inflammation and remodeling were evaluated by quantitative analysis of eosinophils and mononuclear cells and by collagen and elastin contents and smooth muscle thickness. Immunohistochemistry was performed to quantify the density of positive cells to interleukin (IL)-2, IL-4, IL-5, interferon-{gamma}, IL-10, monocyte chemotatic protein (MCP)-1, nuclear factor (NF)-{kappa}B p65, and insulin-like growth factor (IGF)-I. OVA exposure induced pulmonary blood vessels and parenchyma inflammation as well as increased expression of IL-4, IL-5, MCP-1, NF-{kappa}B p65, and IGF-I by inflammatory cells were reduced by aerobic conditioning. OVA exposure also induced an increase in smooth muscle thickness and elastic and collagen contents in pulmonary vessels, which were reduced by aerobic conditioning. Aerobic conditioning increased the expression of IL-10 in sensitized mice. We conclude that aerobic conditioning decreases pulmonary vascular and parenchymal inflammation and remodeling in this experimental model of chronic allergic lung inflammation in mice.

asthma; aerobic training; cytokines; pulmonary inflammation; pulmonary remodeling

EXPERIMENTAL MODELS OF ALLERGIC asthma are characterized by the predominance of eosinophils, Th2 lymphocytes, increased secretion of Th2 cytokines, and also by structural changes known as lung remodeling (61, 66). These alterations are present in large and small airways, in the lung parenchyma, and also in the pulmonary arteries (30, 50, 52, 54, 56, 61, 66). In murine models of asthma, the inflammatory and remodeling processes involving pulmonary vessels include eosinophilia, increased deposition of extracellular matrix such as collagen fibers, increased thickness of vascular smooth muscle, and proliferation of endothelial cells (30, 50, 52, 56, 54). Peripheral lung tissue inflammation and remodeling has also been associated with changes in tissue mechanics and with an increased response to bronchoconstrictor stimuli in experimental models of chronic allergic lung inflammation (66, 27). Although large and small airways and distal parenchyma inflammation and remodeling are well-established alterations in asthmatic patients, only few human studies have reported inflammatory and structural changes in pulmonary and bronchial arteries in patients that died of asthma (48, 16). Additionally, in asthmatic patients, small airway and lung parenchyma inflammation and remodeling have been related to asthma severity (4, 12, 58, 59).

Aerobic training has been recommended as an effective adjuvant treatment in the management of asthmatic patients (3, 6). The performance of aerobic training in asthmatic patients triggers several beneficial effects such as the improvement in physical fitness and ventilatory capacity and a decrease in asthma-related symptoms, exercise-induced bronchospasm, and daily use of inhaled steroids (7, 13, 36, 46). Aerobic training has been shown to modulate the immune response in healthy individuals; however, its effect depends on training intensity (42). In general, moderate training intensity improves the immune system, observed by the increased neutrophil and macrophage phagocytosis capacity and a reduction in reactive oxygen species production, whereas high-intensity exercise leads to opposite effects (8, 26, 65, 68). Aerobic exercise also has an important role in angiogenesis and reducing the vascular oxidative and remodeling process induced by cardiovascular disorders (5, 23, 64).

In previous studies, our and other groups have observed that low- and moderate-intensity aerobic exercise specifically decreases airway eosinophilic inflammation and remodeling (40, 41, 61). Recently, we demonstrated that low- and moderate-intensity aerobic training decreases airway inflammation and remodeling in an experimental model of chronic allergic lung inflammation in mice and that these effects are not mediated by increased Th1 response but could be partially mediated by increased interleukin (IL)-10 expression (61). Nuclear transcription factor-{kappa}B (NF-{kappa}B), a transcription factor that regulates the development of adaptive immunity in asthma, is correlated with increased Th2 cytokine expression and eosinophilic inflammation (25). Monocyte chemotatic protein 1 (MCP-1) is a chemokine that plays an important role in asthma, in the activation of the inflammatory cascade, bronchial hyperresponsiveness, and airway remodeling (47). However, the effects of different intensities of aerobic exercise on the pulmonary NF-{kappa}B and MCP-1 expression are poorly understood. Insulin-like growth factor-I (IGF-I) also presents an increased pulmonary expression in experimental models of allergic lung inflammation, and its effects are linked with airway inflammation and remodeling, but the effects of aerobic exercise on these models remain unknown (33, 62).

Although few studies have investigated the effects of low- and moderate-intensity aerobic exercise on allergic immune responses, particularly on vascular and parenchymal inflammation, only Pastva et al. (40, 41) evaluated these pulmonary compartments. Therefore, this study evaluated the hypothesis that low and moderate aerobic exercise could decrease perivascular and parenchymal inflammation and vascular tissue remodeling in a mouse model of chronic allergic lung inflammation (61, 62). In the present study, we evaluated the vascular and parenchymal inflammation and remodeling in the same experimental animals used to explore airway changes (61, 62).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was approved by the review board for human and animal studies of the School of Medicine of the University of Sao Paulo, protocol number 503/05. All animals in the study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85-23, revised 1985).

Animals and experimental groups. Our experimental protocol was described previously (1). Briefly, 48 male Balb/c mice (20–25 g) were divided in the following six groups (n = 8 each): nonsensitized and nontrained group (control group); nonsensitized and low-intensity aerobic training (Low group); nonsensitized and moderate-intensity aerobic training (Mod group); ovalbumin (OVA)-sensitized and nontrained (OVA group); OVA-sensitized and low-intensity aerobic training (OVA + Low group); and OVA-sensitized and moderate-intensity aerobic training (OVA + Mod group).

OVA sensitization. Balb/c mice were sensitized by an intraperitoneal injection of OVA (20 µg/mouse) adsorbed with aluminum hydroxide on days 0, 14, 28, and 42 or with saline solution, used as control in nonsensitized mice. After the first intraperitoneal OVA or saline injection (21 days), the mice were challenged with aerolized OVA (1%) or with saline solution three times a week until the 50th day (61, 62). The OVA aerosol was always performed between 1700 and 1800. Figure 1 shows the time line of the experimental protocol .


Figure 1
View larger version (3K):
[in this window]
[in a new window]

  		Fig. 1. Time line of the experimental protocol. Mice received ip injections of either ovalbumin solution (20 µg/animal) or vehicle on days 0, 14, 28, and 48 (open triangles). Aerosol challenges with either 1% ovalbumin solution or vehicle (NaCl 0.9%) were performed 3 times/wk starting on day 21 (filled triangles) until day 51. A maximal exercise capacity test was performed on days 18 and 51 (open circles). Physical training (5 times/wk) was started on day 22 (filled circle) and performed until day 50. Animals were killed and studied on day 53 (open square).

 
Aerobic training and exercise test. Animals were initially adapted to the treadmill for 3 days (15 min, 25% inclination, 0.2 km/h). After that, a maximal exercise capacity test was performed with a 5-min warm-up (25% inclination, 0.2 km/h) followed by an increase in treadmill speed (0.1 km/h every 2.5 min) until animal exhaustion, i.e., until they were not able to run even after 10 gentle mechanical stimuli. The test was repeated after 30 days (before death). Maximal aerobic capacity (100%) was established as the maximal speed reached by each animal. Mice were trained with low- or moderate-intensity exercise (respectively, 50 or 75% of maximal speed), 60 min/day, 5 days/wk during 4 wk. Aerobic conditioning started on the 1st day after OVA or saline inhalation (61). The exercise bout was performed always between 1000 and 1200 and OVA exposure was always performed between 1700 and 1800.

Anesthesia and death of animals. After the last day of inhalation (72 h), animals were anesthetized by an intramuscular injection of ketamine (50 mg/kg) and xylazine (40 mg/kg), tracheostomized, cannulated, and killed by exsanguination.

Lung histology, immunohistochemistry, and morphometry. All eight animals for each group were analyzed through histological and immunohistochemical technique. Lungs were fixed in formalin and embedded in paraffin. Thick (5 µm) sections were stained with hematoxylin and eosin for lung structure analysis, with Luna staining for detecting eosinophils (33, 61, 62), with Picrosirius for collagen fibers (27, 61, 62), and with Weigert's Resorcin-Fuchsin with oxidation for elastic fibers (61, 62, 66).

Immunohistochemistry was performed with the following antibodies: anti-IL-4, anti-IL-5, anti-interferon-{gamma} (IFN-{gamma}), anti-IL-2, anti-MCP-1, anti-NF-{kappa}B p65, anti-IGF-I (Santa Cruz), and anti-IL-10 (R&D Systems) through the biotin-streptavidin peroxidase method. With a 50-line and 100-point grid connected to the ocular of the microscope, the perivascular and parenchymal density of eosinophils, mononuclear cells, and IGF-I-positive cells and inflammatory IL-4, IL-5, IFN-{gamma}, IL-2, MCP-1, NF-{kappa}B p65, and IL-10-positive cells were assessed using a point-counting technique (27, 61, 62). Counting was performed in 5 pulmonary arteries (in peribronchiolar space) and 15 alveolar parenchyma fields (area of alveolar septa, excluding areas of pulmonary capilar) in each animal at x1,000 magnification, for a blinded investigator. The results were expressed as cells per square millimeter (27, 61, 62). The perivascular edema index was established as the number of points hitting an edema area divided by the number of intercepts (lines of ocular grid) that crossed the arterial adventitia (53). The arterial muscle thickness index was assessed as the number of points hitting muscle areas divided by the number of intercepts that crossed the external limit of the muscular wall. The volume proportion of collagen and elastic fibers in the pulmonary artery was determined by dividing the number of points hitting the collagen or elastin by the number of points hitting the arterial area. In the alveolar tissue, the volume proportion was determined by dividing the number of points hitting collagen or elastin by the total number of points hitting the alveolar septa. Results of volume proportion of collagen and elastin in the parenchyma and pulmonary artery were expressed as a percentage (27, 61, 62).

Statistical analysis. Parametric and nonparametric data are expressed as means ± SD or median ± 95% confidence interval, respectively. Comparisons between groups were carried out by two-way ANOVA followed by the Student-Newman-Keul's post hoc test (parametric data) or by one-way analysis of variance on ranks followed by Dunn's post hoc test (nonparametric data); the significance level was adjusted to 5% (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Physical exercise capacity. Table 1 shows the initial time, final time, and the final less initial time reached in the physical test in all groups. All trained groups, regardless of the exercise intensity, presented an increase in physical exercise capacity when compared with the nontrained groups (control and OVA groups) (P < 0.001) and no difference was found among the trained groups (P > 0.05).


View this table:
[in this window]
[in a new window]

  		Table 1. Time in minutes reached in the initial, final, and difference between the final and initial physical tests in all experimental groups

 
Perivascular and parenchyma inflammation. Figure 2, A–D, shows the perivascular and parenchyma density of eosinophils and mononuclear cells. Nonsensitized animals submitted to low- or moderate-intensity training (Low and Mod groups, respectively) did not present changes in the perivascular density of eosinophils and mononuclear cells when compared with the control group (nonsensitized and nontrained). Low- and moderate-intensity exercise training led to an increased density of mononuclear cells in the alveolar tissue when compared with the control group (P < 0.01). The OVA group presented an increased perivascular and parenchymal density of eosinophils when compared with all groups (P < 0.01). OVA group also presented an increased density of perivascular density of mononuclear cells when compared with control, Low, and Mod groups (P < 0.01) and in the parenchyma when compared with the control group (P < 0.01). OVA + Low and OVA + Mod groups presented a decreased density of eosinophils in perivascular tissue and in lung parenchyma (P < 0.001), but no changes in the density of mononuclear cells were observed. Additionally, low- and moderate-intensity exercise training did not present changes in the perivascular edema when compared with the control group (control -3.73 ± 1.0 vs. Low -3.81 ± 0.8 vs. Mod -3.33 ± 1.44). The OVA group presented increased perivascular edema when compared with all groups (P < 0.001) (OVA -5.58 ± 1.33 vs. control -3.73 ± 1.0 vs. Low -3.81 ± 0.8 vs. Mod -3.33 ± 1.44 vs. OVA + Low 2.82 ± 1.12 vs. OVA + Mod 3.77 ± 0.96). OVA + Low and OVA + Mod groups presented a decreased perivascular edema when compared with the OVA group (P < 0.001) (OVA -5.58 ± 1.33 vs. OVA + Low 2.82 ± 1.12 vs. OVA + Mod 3.77 ± 0.96).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. Box plots representing the perivascular density of eosinophils (A), mononuclear cells (B), and the parenchymal density of eosinophils (C) and mononuclear cells (D). Boxes show interquartile range, whiskers show range, and horizontal lines represent median values. A and C: *P < 0.001 when compared with all groups. B: *P < 0.01 when compared with control and low (Low)- and moderate (Mod)-intensity aerobic training groups. D: *P < 0.01 when compared with the control group.

 
Perivascular expression of cytokines, chemokine, NF-{kappa}B p65, and IGF-I. Figure 3, A–H, respectively shows the perivascular expression of positive inflammatory cells to IL-4, IL-5, IL-2, IFN-{gamma}, MCP-1, IGF-I, IL-10, and NF-{kappa}B p65. Low and Mod groups did not present changes in the perivascular density on the expression of any investigated protein type by inflammatory cells. The OVA group presented increased perivascular density of IL-4, IL-5, MCP-1, IGF-I, and NF-{kappa}B p65 positive cells when compared with all groups (P < 0.001). The OVA group also presented increased perivascular density of IL-10 when compared with control, Low, and Mod groups (P < 0.001). The OVA group did not present changes in the perivascular density of IL-2 and IFN-{gamma} (P < 0.05). OVA + Low and OVA + Mod groups presented a decreased perivascular density of IL-4, IL-5, MCP-1, IGF-I, and NF-{kappa}B p65 when compared with the OVA group (P < 0.001). OVA + Low and OVA + Mod groups presented an increased perivascular density of IL-10 when compared with all groups (P < 0.01). OVA + Low and OVA + Mod groups did no present changes in the perivascular density of IL-2 and IFN-{gamma} (P < 0.05). Figure 4, A–C, shows the representative photomicrographs of positive inflammatory cells to MCP-1 in control, OVA, and OVA + Low groups at x400 magnification. Figure 4D shows the representative photomicrographs of positive inflammatory cells to MCP-1 in OVA at x1,000 magnification.


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Box plots representing the perivascular density of interleukin (IL)-4-positive cells (A), IL-5-positive cells (B), IL-2-positive cells (C), interferon-{gamma} (IFN-{gamma}; D), monocyte chemotatic protein (MCP)-1-positive cells (E), insulin-like growth factor I (IGF-I)-positive cells (F), IL-10-positive cells (G), and nuclear factor-{kappa}B (NF-{kappa}B; H). A, B, E, F, and H: *P < 0.001 when compared with all groups. G: *P < 0.001 when compared with control, Low, and Mod groups.

 

Figure 4
View larger version (108K):
[in this window]
[in a new window]

  		Fig. 4. Representative photomicrographs of perivascular inflammatory MCP-1-positive cells. A: control group at x400 magnification. B: ovalbumin (OVA) group at x400 magnification. C: OVA + Low group at x400 magnification. D: OVA group at x1,000 magnification. Scale bars = 25 µm.

 
Parenchymal expression of cytokines, chemokine, NF-{kappa}B p65, and IGF-I. Figure 5, A–H, shows the parenchymal expression of positive inflammatory cells to IL-4, IL-5, IL-2, IFN-{gamma}, MCP-1, IGF-I, IL-10, and NF-{kappa}B p65. Neither OVA-induced allergic inflammation nor exercise training changed the expression of IFN-{gamma} and IGF-I in the parenchyma (P > 0.05). Low and Mod groups did not present changes in the parenchymal density of all outcomes (P > 0.05). The OVA group presented increased parenchymal density of IL-4, IL-5, MCP-1, and NF-{kappa}B p65 when compared with all groups (P < 0.001). The OVA group also presented increased parenchymal density of IL-10 when compared with control, Low, and Mod groups (P < 0.001). OVA + Low and OVA + Mod groups presented a decreased parenchymal density of IL-4, IL-5, MCP-1, and NF-{kappa}B p65 when compared with the OVA group (P < 0.001). OVA + Low and OVA + Mod groups also presented an increased parenchymal density of IL-2 when compared with all groups (P < 0.01). OVA + Low and OVA + Mod groups also presented an increased parenchymal density of IL-10 when compared with all groups (P < 0.01).


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 5. Box plots representing the parenchymal density of IL-4-positive cells (A), IL-5-positive cells (B), IL-2-positive cells (C), IFN-{gamma} (D), MCP-1-positive cells (E), IGF-I-positive cells (F), IL-10-positive cells (G), and NF-{kappa}B (H). A, B, E, and H: *P < 0.001 when compared with all groups. C and G: *P < 0.01 when compared with all groups.

 
Vascular and parenchymal remodeling. Figure 6 shows the volume proportion of collagen and elastic fibers and the smooth muscle thickness in the pulmonary artery. Low and Mod groups (nonsensitized animals submitted to low- or moderate-intensity aerobic training) did not present changes either in vascular collagen and elastic fiber content or in the artery smooth muscle thickness when compared with the control group (nonsensitized and nontrained, Fig. 6, A–C). The OVA group presented an increased arterial volume proportion of collagen and elastic fibers and in the smooth muscle thickness when compared with the other groups (P < 0.01, Fig. 6, A–C). Sensitized animals submitted to low- or moderate-intensity aerobic training (OVA + Low and OVA + Mod groups) presented a decrease in vascular collagen and elastic contents and smooth muscle thickness when compared with the OVA group (P < 0.01, Fig. 6, A–C). Figure 7, A–D, shows the representative photomicrographs of collagen fibers in the pulmonary artery in control, OVA, OVA + Low, and OVA + Mod groups. No changes were found in the volume proportion of collagen and elastic fibers in the alveolar parenchyma. Neither aerobic training nor OVA exposure modified the volume proportion of collagen and elastic fibers in the alveolar parenchyma.


Figure 6
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 6. Box plots representing the vascular collagen (A), elastic (B), and smooth muscle thickness index (C). A–C: *P < 0.01 when compared with all groups.

 

Figure 7
View larger version (82K):
[in this window]
[in a new window]

  		Fig. 7. Representative photomicrographs of collagen fibers in the pulmonary artery. A: control group. B: OVA group. C: OVA + Low group. D: OVA + Mod group. Scale bars = 25 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study shows that aerobic training at low or moderate intensity decreases OVA-induced perivascular edema and increases density of eosinophils and also of positive inflammatory cells to IL-4, IL-5, MCP-1, NF-{kappa}B p65, and IGF-I in the perivascular and in the alveolar parenchyma compartments. Aerobic training also reduced OVA-induced vascular remodeling evaluated by smooth muscle thickness and collagen and elastic fiber contents. Therefore, our data present evidence supporting the extensive benefits of aerobic training to decrease chronic allergic lung inflammation.

The beneficial effects of aerobic training for asthmatic patients have been recognized by the American Association of Cardiovascular and Pulmonary Rehabilitation and the British Thorax Society (3, 6). These effects have been mainly attributed to an improvement in the ventilatory capacity and physical fitness as well as a decrease in dyspnea, exercise-induced bronchospasm severity, and use of corticosteroids (7, 13, 36, 46). These clinical benefits have also suggested a possible anti-inflammatory effect of aerobic training on asthma. Only few studies have investigated the anti-inflammatory effects of aerobic training on asthma, using an allergic murine model (40, 41, 61). In accordance with these studies, the anti-inflammatory effects of aerobic exercise could be attributed to decreased levels of IL-4, IL-5, IgE and also an increase in the anti-inflammatory cytokine IL-10 (40, 41, 61). Although these studies verified the leukocytes infiltration in the airways, they did not evaluate the remodeling in the pulmonary vessels and parenchyma, and only Pastva et al. (40) evaluated the perivascular infiltration by leucocytes (4, 12, 16, 27, 30, 48, 50, 52, 54, 56, 58, 59, 61, 66). In the present study, we observed that eosinophilic inflammation in the perivascular and parenchymal compartment was inhibited by aerobic conditioning, similarly to what was observed in the eosinophilic inflammation in the airways (61).

In healthy subjects, aerobic conditioning increases the Th1 response (8, 26, 42, 65, 68); however, its effect on the Th2 response is poorly understood. Postulated immune mechanisms underlying allergic disease suggest that, in the general population, allergy should be less common in individuals with improved Th1 response, since T lymphocytes would respond to antigen-presenting cells tending toward a Th1 subtype. We then hypothesized that anti-inflammatory effect due to aerobic training in chronic allergic airway inflammation could be mediated by improved Th1 response and consequent reduced Th2 response (8, 26, 32, 42, 65, 68). However, in our experimental animal model, the OVA-induced increase in the expression of Th2 cytokines (IL-4 and IL-5) was reduced by aerobic training, although the expression of Th1 cytokines (IL-2 and IFN-{gamma}) remained unchanged, suggesting that the anti-inflammatory effects of aerobic training in a murine model of lung allergic inflammation may occur due to a direct effect on the Th2 response. These results are in agreement to what we found in the previous study evaluating the airways (61). However, other pathways could be involved as possible mechanisms of anti-inflammatory effects of aerobic training.

First, we evaluated the effects of aerobic training on the anti-inflammatory cytokine IL-10. Some studies present that the effects of aerobic training on the immune system are mediated by increased release of IL-10 (34, 43, 55). Some recognized sources of IL-10 release during aerobic training are activated skeletal muscle and lymphocytes, as part of the anti-inflammatory effects of aerobic training in cardiovascular disease and type 2 diabetes (34, 43, 55). IL-10 also presents anti-inflammatory effects in experimental models of chronic allergic lung inflammation (14, 35). Recently, we demonstrated that, in the airways, aerobic exercise decreased Th2 cytokine expression, effects that seem mediated partially by exercise-increased IL-10 expression (61). As observed in the airway response, the results of the present study show that aerobic exercise training in sensitized animals increases the expression of IL-10 by inflammatory cells also in perivascular and alveolar parenchyma, reinforcing a possible mechanism of the exercise-induced decrease in allergic inflammation.

Second, we investigated the expression of the chemokine MCP-1. Chemokines present an important role in the physiopathology of asthma, such as activation of the inflammatory cascade, bronchial hyperresponsiveness, and airway remodeling (2, 22, 45, 51). In accordance with current literature, our mouse model of allergic lung inflammation presented an increased expression of MCP-1 by inflammatory cells, both in the pulmonary arteries and lung parenchyma (Figs. 3E and 5E). It is possible that peribronchial and perivascular compartments communicate during the recruitment of cells in the lung inflammation process, and it is known that blood-bone cells such as eosinophils and neutrophils need to cross the vascular endothelium for an eventual transmigration to the injured sites (16, 37, 38, 39). The effects of aerobic conditioning on the vascular response have been extensively investigated except in the lung tissue (1, 23, 57). Aerobic training seems to exert its anti-inflammatory effects on diseases such as metabolic syndrome and cardiovascular disorders by reducing the levels of MCP-1 (1, 57). Our results show that aerobic exercise at low or moderate intensities reduces OVA-induced expression of MCP-1 in perivascular and parenchymal compartments, suggesting its involvement as a mechanism in the reduction of cellular migration to the perivascular and lung parenchyma compartments in this animal model.

At last, we investigated the expression of NF-kB p65. NF-kB is a major transcription factor that plays an essential role in the development of innate and adaptive immunity, and it is considered a major mediator of many genetic diseases such as asthma (25). NF-kB is activated in asthmatic patients and in animal models, and its activation has been related to an exacerbatory response due to allergens, ozone, particulate matters, and viral infection exposure, suggesting that NF-kB activation mediates Th2 cytokine synthesis and lung eosinophilic inflammation (17, 18, 20, 24, 25, 44). The levels of NF-kB in response to aerobic training in asthmatic patients have not been previously evaluated. In nonasthmatic patients, some studies demonstrate that high intense and exhaustive aerobic training increases NF-kB activation, effects that seem mediated by increased oxidative stress (11, 15, 60). An experimental study by Pastva et al. (4) demonstrated that aerobic training decreases NF-kB expression in the airways of OVA-sensitized animals. Our results demonstrated that aerobic training performed at either low or moderate intensities was capable of reducing the expression of NF-kB by inflammatory cells in the lung perivascular and alveolar parenchyma compartments (Figs. 3H and 5H). Therefore, we hypothesize that the suppression of NF-kB and MCP-1 followed by aerobic training together with aerobic training-increased expression of IL-10 could help to explain the anti-inflammatory effects of aerobic training in our model of chronic allergic lung inflammation.

The pulmonary remodeling in the asthmatic patients achieves mainly the airways, but some evidence shows that pulmonary vessels (16, 48) and parenchyma are also affected (4, 58, 59). The increased deposition of extracellular matrix in pulmonary vessels and parenchyma in asthma presents a significant clinical implication and has an important correlation with asthma severity, including airflow limitation and bronchial hyperresponsiveness (4, 16, 21, 31, 48, 49, 58, 59). Although there are differences between human asthma and murine models of asthma, we are aware that animals do not develop asthma and that there are several limitations in animal studies. However, many of the growing advances in the asthma pathophysiology and treatment are based on animal models (27, 30, 50, 52, 54, 56, 61, 66). For this purpose, we evaluated in the present study the distribution of collagen and elastic fibers in pulmonary artery and parenchyma and also the arterial smooth muscle thickness. In our animal model, no changes in collagen and elastic fiber contents were found in the lung parenchyma in all groups. Contrarily, our results demonstrated that the OVA group presented an increase in vascular remodeling, characterized by increased deposition of collagen and elastic fibers as well as in smooth muscle thickness (Fig. 6, A–C). The results also demonstrated these effects were completely inhibited by aerobic training at low and moderate intensities (Fig. 6, A–C). These results are similar to our previous data obtained in airways where OVA sensitization increased collagen and elastic fiber deposition and smooth muscle thickness in the airways, which were completely inhibited by aerobic conditioning (61). Taking that perivascular inflammation could be involved in the pathogenesis of structural alterations and influence the vascular remodeling to be a source of activated inflammatory cells, IL-4 and IL-5 cytokines, and IGF-I (Figs. 2, A and B, and 3, A, B, and F), we could speculate that the inhibitory effects of aerobic training on the arterial collagen and elastic fiber deposition and smooth muscle thickness could have been mediated by aerobic training, decreased vascular inflammatory cell recruitment, and IL-4 and IL-5 expression. We also add that, one time that IGF-I is an important mediator of synthesis of collagen, elastic fibers, and also of smooth muscle proliferation (9, 10, 62), these inhibitory effects of aerobic training also could have been mediated by a inhibited expression of IGF-I by inflammatory cells in the lung perivascular compartment in sensitized animals submitted to aerobic training of low and moderate intensities (Fig. 3F).

In conclusion, we demonstrated that the vascular and parenchymal inflammation and remodeling in our experimental model of chronic allergic lung inflammation was substantially inhibited by aerobic conditioning. These effects seem to be mediated by the reduction of Th2 response and might be explained by the increase of IL-10 and decreases of NF-kB, MCP-1, and IGF-I expression.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by: Laboratórios de Investigação Médica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação da Amparo à Pesquisa do Estado de São Paulo (FAPESP; Grant Nos. 2005/04413-1 and 2002/08422-7), and Conselho Nacional de Desenvolvimento Científico e Tecnológico. R. P. Vieira is a postdoctoral fellow from FAPESP, Grant No. 2007/01026-2.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. R. Fernandes de Carvalho, School of Medicine, Univ. of São Paulo, Av. Dr. Arnaldo, 455, 1° andar, sala 1216, 01246-903, São Paulo, SP, Brazil (e-mail: cscarval{at}usp.br)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adamopoulos S, Parissis J, Kroupis C, Georgiadis M, Karatzas D, Karavolias G, Koniavitou K, Coats AJ, Kremastinos DT. Physical training reduces peripheral markers of inflammation in patients with chronic heart failure. Eur Heart J 22: 791–797, 2001.[Abstract/Free Full Text]
  2. Alam R, York J, Boyars M, Stafford S, Grant JA, Lee J, Forsythe P, Sim T, Ida N. Increased MCP-1, RANTES, and MIP-1alpha in bronchoalveolar lavage fluid of allergic asthmatic patients. Am J Respir Crit Care Med 153: 1398–1404, 1996.[Abstract]
  3. American Association of Cardiovascular & Pulmonary Rehabilitation.Guidelines of Pulmonary Rehabilitation Programmes (2nd ed.). Champaign, IL: Human Kinetics, 1998.
  4. Balzar S, Wenzel SE, Chu HW. Transbronchial biopsy as a tool to evaluate small airways in asthma. Eur Respir J 20: 254–259, 2002.[Abstract/Free Full Text]
  5. Bloor CM. Angiogenesis during exercise and training. Angiogenesis 8: 263–271, 2005.[Medline]
  6. BTS Statement. Pulmonary rehabilitation. Thorax 56: 827–834, 2001.[Free Full Text]
  7. Caroll N, Sly P. Exercise training as an adjunct to asthma management. Thorax 54: 190–191, 1999.[Free Full Text]
  8. Ceddia MA, Voss EW, Woods JA. Intracellular mechanisms are responsible for the exhaustive exercise-induced suppression of macrophage antigen presentation. J Appl Physiol 88: 804–810, 2000.[Abstract/Free Full Text]
  9. Chetty A, Cao GJ, Nielsen HC. Insulin-like Growth Factor-I signaling mechanisms, type I collagen and alpha smooth muscle actin in human fetal lung fibroblasts. Pediatr Res 60: 389–394, 2006.[CrossRef][Web of Science][Medline]
  10. Cohen P, Noveral JP, Bhala A, Nunn SE, Herrick DJ, Grunstein MM. Leukotriene D4 facilitates airway smooth muscle cell proliferation via modulation of the IGF axis. Am J Physiol Lung Cell Mol Physiol 269: L151–L157, 1995.[Abstract/Free Full Text]
  11. Cuevas MJ, Almar M, García-Glez JC, García-López D, De Paz JA, Alvear-Ordenes I, González-Gallego J. Changes in oxidative stress markers and NF-kappaB activation induced by sprint exercise. Free Radic Res 39: 431–439, 2005.[CrossRef][Web of Science][Medline]
  12. de Magalhaes Simoes S, dos Santos MA, da Silva Oliveira M, Fontes ES, Fernezlian S, Garippo AL, Castro I, Castro FF, de Arruda Martins M, Saldiva PH, Mauad T, Dolhnikoff M. Inflammatory cell mapping of the respiratory tract in fatal asthma. Clin Exp Allergy 35: 602–611, 2005.[CrossRef][Web of Science][Medline]
  13. Fanelli A, Cabral ALB, Neder JA, Martins MA, Carvalho CRF. Exercise training on disease control and quality of life in asthmatics children. Med Sci Sports Exerc 39: 1474–1480, 2007.[Web of Science][Medline]
  14. Fu CL, Chuand YH, Chau LY, Chiang BL. Effects of adenovirus-expressing IL-10 in alleviating airway inflammation in asthma. J Gene Med 8: 1393–1399, 2006.[CrossRef][Web of Science][Medline]
  15. García-López D, Cuevas MJ, Almar M, Lima E, De Paz JA, González-Gallego J. Effects of eccentric exercise on NF-kappaB activation in blood mononuclear cells. Med Sci Sports Exerc 39: 653–664, 2007.[CrossRef][Web of Science][Medline]
  16. Green FH, Butt JC, James AL, Carroll NG. Abnormalities of the bronchial arteries in asthma. Chest 130: 1025–1033, 2006.[CrossRef][Web of Science][Medline]
  17. Hancox RJ, Stevens DA, Adcock IM, Barnes PJ, Taylor DR. Effects of inhaled beta agonist and corticosteroid treatment on nuclear transcription factors in bronchial mucosa in asthma. Thorax 54: 488–492, 1999.[Abstract/Free Full Text]
  18. Hart L, Lim S, Adcock I, Barnes PJ, Chung KF. Effects of inhaled corticosteroid therapy on expression and DNAbinding activity of nuclear factor kappaB in asthma. Am J Respir Crit Care Med 161: 224–231, 2000.[Abstract/Free Full Text]
  19. Henderson WR Jr, Chiang GK, Tien YT, Chi EY. Reversal of allergen-induced airway remodeling by cyslt1 receptor blockade. Am J Respir Crit Care Med 173: 718–728, 2006.[Abstract/Free Full Text]
  20. Henderson WRJ, Chi EY, Eo JL, Guyen C, Ahn M. A small molecule inhibitor of redox-regulated NF-kappa B and activator protein-1 transcription blocks allergic airway inflammation in a mouse asthma model. J Immunol 169: 5294–5299, 2002.[Abstract/Free Full Text]
  21. Horvath G, Wanner A. Inhaled corticosteroids: effects on the airway vasculature in bronchial asthma. Eur Respir J 27: 172–187, 2006.[Abstract/Free Full Text]
  22. Ip WK, Wong CK, Lam CW. Interleukin (IL)-4 and IL-13 up-regulate monocyte chemoattractant protein-1 expression in human bronchial epithelial cells: involvement of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 and Janus kinase-2 but not c-Jun NH2-terminal kinase 1/2 signalling pathways. Clin Exp Immunol 145: 162–172, 2006.[CrossRef][Web of Science][Medline]
  23. Kojda G, Hambrecht R. Molecular mechanisms of vascular adaptations to exercise. Physical activity as an effective antioxidant therapy? Cardiovasc Res 67: 187–197, 2005.[Abstract/Free Full Text]
  24. Kumar A, Lnu S, Malya R, Barron D, Moore J, Corry DB, Boriek AM. Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma. FASEB J 17: 1800–1811, 2003.[Abstract/Free Full Text]
  25. Kumar A, Takada Y, Boriek AM, Aggarwal BB. Nuclear factor-kB: its role in health and disease. J Mol Med 82: 434–448, 2004.[Web of Science][Medline]
  26. Lakier Smith L. Overtraining, excessive exercise, and altered immunity: is this a T helper-1 versus T helper-2 lymphocyte response? Sports Med 33: 347–364, 2003.[CrossRef][Web of Science][Medline]
  27. Lancas T, Kasahara DI, Prado CM, Tiberio IF, Martins MA, Dolhnikoff M. Comparison of early and late responses to antigen of sensitized guinea pig parenchymal lung strips. J Appl Physiol 100: 1610–1616, 2006.[Abstract/Free Full Text]
  28. Larsen GL, Holt PG. The concept of airway inflammation. Am J Respir Crit Care Med 162: S2–S6, 2000.[Free Full Text]
  29. Leigh R, Ellis R, Wattie JN, Hirota JA, Matthaei KI, Foster PS, O'Byrne PM, Inman MD. Type 2 cytokines in the pathogenesis of sustained dysfunction and airway remodeling in mice. Am J Respir Crit Care Med 169: 860–867, 2004.[Abstract/Free Full Text]
  30. Leino MS, Alenius HT, Fyhrquist-Vanni N, Wolff HJ, Reijula KE, Hintikka EL, Salkinoja-Salonen MS, Haahtela T, Makela MJ. Intranasal exposure to Stachybotrys chartarum enhances airway inflammation in allergic mice. Am J Respir Crit Care Med 173: 512–518, 2006.[Abstract/Free Full Text]
  31. Li X, Wilson JW. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 156: 229–233, 1997.[Abstract/Free Full Text]
  32. Lowder T, Padgett DA, Woods JA. Moderate exercise early after influenza virus infection reduces the Th1 inflammatory response in lungs of mice. Exerc Immunol Rev 12: 97–111, 2006.[Web of Science][Medline]
  33. Luna LG. AFIP Manual of Histologic Staining Methods. New York, NY: McGraw Hill, 1986.
  34. Moldoveanu AI, Shephard RJ, Shek PN. The cytokine response to physical activity and training. Sports Med 3: 115–144, 2001.
  35. Nakagome K, Dohi M, Okunishi K, Komagata Y, Nagatani K, Tanaka R, Miyazaki J, Yamamoto K. In vivo IL-10 gene delivery suppresses airway eosinophilia and hyperreactivity by down-regulating apc functions and migration without impairing the antigen-specific systemic immune response in a mouse model of allergic airway inflammation. J Immunol 174: 6955–6966, 2005.[Abstract/Free Full Text]
  36. Neder JA, Nery LE, Silva AC, Cabral AL, Fernandes AL. Short-term effects of aerobic training in the clinical management of moderate to severe asthma in children. Thorax 54: 202–206, 1999.[Abstract/Free Full Text]
  37. O'Byrne PM, Postma DS. The many faces of airway inflammation: asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 159: S41–S66, 1999.[Web of Science][Medline]
  38. Pabst R, Tschernig T. Perivascular capillaries in the lung: an important but neglected vascular bed in immune reactions? J Allergy Clin Immunol 110: 209–214, 2002.[CrossRef][Web of Science][Medline]
  39. Pabst R. The periarterial space in the lung: its important role in lung edema, transplantation, and microbial or allergic inflammation. Pathobiology 71: 287–294, 2004.[CrossRef][Web of Science][Medline]
  40. Pastva A, Estell K, Schoeb TR, Atkinson TP, Schwiebert LM. Aerobic exercise attenuates airway inflammatory responses in a mouse model of atopic asthma. J Immunol 172: 4520–4526, 2004.[Abstract/Free Full Text]
  41. Pastva A, Estell K, Schoeb TR, Schwiebert LM. RU486 blocks the anti-inflammatory effects of exercise in a murine model of allergen-induced pulmonary inflammation. Brain Behav Immun 19: 413–422, 2005.[CrossRef][Web of Science][Medline]
  42. Pedersen BK, Hoffman-Goetz L. Exercise and the immune system: regulation, integration, and adaptation. Physiol Rev 80: 1055–1081, 2000.[Abstract/Free Full Text]
  43. Petersen AMW, Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol 98: 1154–1162, 2005.[Abstract/Free Full Text]
  44. Poynter ME, Irvin CG, Janssen-Heininger YM. Rapid activation of nuclear factor-kappaB in airway epithelium in a murine model of allergic airway inflammation. Am J Pathol 160: 1325–1334, 2002.[Abstract/Free Full Text]
  45. Qiu Z, Fujimura M, Kurashima K, Nakao S, Mukaida N. Enhanced airway inflammation and decreased subepithelial fibrosis in interleukin 6-deficient mice following chronic exposure to aerosolized antigen. Clin Exp Allergy 34: 1321–1328, 2004.[CrossRef][Web of Science][Medline]
  46. Ram FS, Robinson SM, Black PN, Picot J. Physical training for asthma. Cochrane Database Syst Rev 4: CD001116, 2005.[Medline]
  47. Romagnani S. Cytokines and chemoattractants in allergic inflammation. Mol Immunol 38: 881–885, 2002.[CrossRef][Web of Science][Medline]
  48. Saetta M, DiStefano A, Rosina C, Thiene G, Fabbri LM. Quantitative structural analysis of peripheral airways and arteries in sudden fatal asthma. Am Rev Respir Dis 143: 138–143, 1991.[Web of Science][Medline]
  49. Salvato G. Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non-asthmatic subjects. Thorax 56: 902–906, 2001.[Abstract/Free Full Text]
  50. Singh B, Shinagawa K, Taube C, Gelfand EW, Pabst R. Strain-specific differences in perivascular inflammation in lungs in two murine models of allergic airway inflammation. Clin Exp Immuno 141: 223–229, 2005.[CrossRef][Web of Science][Medline]
  51. Sousa AR, Lane SJ, Nakhosteen JA, Yoshimura T, Lee TH, Poston RN. Increased expression of the monocyte chemoattractant protein-1 in bronchial tissue from asthmatic subjects. Am J Respir Cell Mol Biol 10: 142–147, 1994.[Abstract]
  52. Sur S, Wild JS, Choudhury BK, Sur N, Alam R, Klinman DM. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG Oligodeoxynucleotides. J Immunol 162: 6284–6293, 1999.[Abstract/Free Full Text]
  53. Tibério IF, Turco GM, Leick-Maldonado EA, Sakae RS, Paiva SO, do Patrocínio M, Warth TN, Lapa e Silva JR, Saldiva PH, Martins MA. Effects of neurokinin depletion on airway inflammation induced by chronic antigen exposure. Am J Respir Crit Care Med 155: 1739–1747, 1997.[Abstract]
  54. Tigani B, Cannet C, Quintana HK, Ble FX, Zurbruegg S, Schaeublin E, Fozard JR, Beckmann N. Lung inflammation and vascular remodeling after repeated allergen challenge detected noninvasively by MRI. Am J Physiol Lung Cell Mol Physiol 292: L644–L653, 2007.[Abstract/Free Full Text]
  55. Tilz GP, Domej W, Diez-Ruiz A, Weiss G, Brezinschek R, Brezinschek HP, Huttl E, Pristautz H, Wachter H, Fuchs D. Increased immune activation during and after physical exercise. Immunobiology 188: 194–202, 1993.[Web of Science][Medline]
  56. Tormanen KR, Uller L, Persson CG, Erjefalt JS. Allergen exposure of mouse airways evokes remodeling of both bronchi and large pulmonary vessels. Am J Respir Crit Care Med 171: 19–25, 2005.[Abstract/Free Full Text]
  57. Troseid M, Lappegard KT, Claudi T, Damas JK, Morkrid L, Brendberg R, Mollnes TE. Exercise reduces plasma levels of the chemokines MCP-1 and IL-8 in subjects with the metabolic syndrome. Eur Heart J 25: 349–355, 2004.[Abstract/Free Full Text]
  58. Tulic MK, Christodoulopoulos P, Hamid Q. Small airway inflammation in asthma. Resp Res 2: 333–339, 2001.[CrossRef]
  59. Tulic MK, Hamid Q. Contribution of the distal lung to the pathologic and physiologic changes in asthma: potential therapeutic target. Roger S Michell lecture. Chest 123: 348–355, 2003.[CrossRef]
  60. Vider J, Laaksonen DE, Kilk A, Atalay M, Lehtmaa J, Zilmer M, Sen CK. Physical exercise induces activation of NF-kappaB in human peripheral blood lymphocytes. Antioxid Redox Signal 3: 1131–1137, 2001.[CrossRef][Web of Science][Medline]
  61. Vieira RP, Claudino RC, Duarte ACS, Santos ABG, Perini A, Faria-Neto HCC, Mauad T, Martins MA, Dolhnikoff M, Carvalho CRF. Aerobic exercise decreases chronic allergic lung inflammation and airway remodeling in mice. Am J Respir Crit Care Med 176: 1–7, 2007.[Free Full Text]
  62. Vieira RP, Duarte ACS, Claudino RC, Perini A, Santos ABG, Moriya HT, Arantes-Costa FM, Martins MA, Carvalho CRF, Dolhnikoff M. Creatine supplementation increases airway inflammation, remodeling and hyperresponsiveness in sensitized mice. Am J Respiratory Cell Mol Biol 37: 660–667, 2007.[CrossRef]
  63. Vrugt B, Wilson S, Bron A, Holgate ST, Djukanovic R, Aalbers R. Bronchial angiogenesis in severe glucocorticoid-dependent asthma. Eur Respir J 15: 1014–1021, 2000.[Abstract]
  64. Wahl P, Bloch W, Schmidt A. Exercise has a positive effect on endothelial progenitor cells, which could be necessary for vascular adaptation processes. Int J Sports Med Nov 28: 374–380, 2006.[CrossRef][Web of Science][Medline]
  65. Woods JA, Lu Q, Ceddia MA, Lowder T. Exercise and neuroendocrine modulation of macrophage function. Immunol Cell Biol 78: 545–553, 2000.[CrossRef][Medline]
  66. Xisto DG, Farias LL, Ferreira HC, Picanço MR, Amitrano D, Lapa e Silva JR, Negri EM, Mauad T, Carnielli D, Silva LFF, Capelozzi VL, Faffe DS, Zin WA, Rocco PRM. Lung parenchyma remodeling in a murine model of chronic allergic inflammation. Am J Respir Crit Care Med 171: 829–837, 2005.[Abstract/Free Full Text]
  67. Yamashita N, Tashimo H, Ishida H, Matsuo Y, Arai H, Nagase H, Adachi T, Ohta K. Role of insulin-like growth factor-I in allergen-induced airway inflammation and remodeling. Cell Immunol 235: 85–91, 2005.[CrossRef][Web of Science][Medline]
  68. Zieker D, Zieker J, Dietzsch J, Burnet M, Northoff H, Fehrenbach E. CDNA-microarray analysis as a research tool for expression profiling in human peripheral blood following exercise. Exerc Immunol Rev 11: 86–96, 2005.[Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/4/L670    most recent
00465.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vieira, R. P.
Right arrow Articles by Carvalho, C. R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vieira, R. P.
Right arrow Articles by Carvalho, C. R. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.