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Am J Physiol Lung Cell Mol Physiol 294: L1197-L1205, 2008. First published March 21, 2008; doi:10.1152/ajplung.00199.2007
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Effects of chronic L-NAME treatment lung tissue mechanics, eosinophilic and extracellular matrix responses induced by chronic pulmonary inflammation

Patrícia Angeli,1 Carla M. Prado,1 Débora G. Xisto,2 Pedro L. Silva,2 Caroline P. Pássaro,2 Hugo D. Nakazato,1 Edna A. Leick-Maldonado,1 Milton A. Martins,1 Patricia R. M. Rocco,2 and Iolanda F. L. C. Tibério1

1Department of Medicine, School of Medicine, University of Sao Paulo, São Paulo; and 2Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Ilha do Fundão, Centro de Ciências da Saúde, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Submitted 17 May 2007 ; accepted in final form 20 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The importance of lung tissue in asthma pathophysiology has been recently recognized. Although nitric oxide mediates smooth muscle tonus control in airways, its effects on lung tissue responsiveness have not been investigated previously. We hypothesized that chronic nitric oxide synthase (NOS) inhibition by N{omega}-nitro-L-arginine methyl ester (L-NAME) may modulate lung tissue mechanics and eosinophil and extracellular matrix remodeling in guinea pigs with chronic pulmonary inflammation. Animals were submitted to seven saline or ovalbumin exposures with increasing doses (1~5 mg/ml for 4 wk) and treated or not with L-NAME in drinking water. After the seventh inhalation (72 h), animals were anesthetized and exsanguinated, and oscillatory mechanics of lung tissue strips were performed in baseline condition and after ovalbumin challenge (0.1%). Using morphometry, we assessed the density of eosinophils, neuronal NOS (nNOS)- and inducible NOS (iNOS)-positive distal lung cells, smooth muscle cells, as well as collagen and elastic fibers in lung tissue. Ovalbumin-exposed animals had an increase in baseline and maximal tissue resistance and elastance, eosinophil density, nNOS- and iNOS-positive cells, the amount of collagen and elastic fibers, and isoprostane-8-PGF2{alpha} expression in the alveolar septa compared with controls (P < 0.05). L-NAME treatment in ovalbumin-exposed animals attenuated lung tissue mechanical responses (P < 0.01), nNOS- and iNOS-positive cells, elastic fiber content (P < 0.001), and isoprostane-8-PGF2{alpha} in the alveolar septa (P < 0.001). However, this treatment did not affect the total number of eosinophils and collagen deposition. These data suggest that NO contributes to distal lung parenchyma constriction and to elastic fiber deposition in this model. One possibility may be related to the effects of NO activating the oxidative stress pathway.

experimental models of asthma; lung parenchyma constriction; elastic fibers; oxidative stress; N{omega}-nitro-L-arginine methyl ester

ALTHOUGH ASTHMA IS DEFINED as a chronic airway inflammatory disease, recent investigations have emphasized the importance of lung tissue alterations in the pathophysiology of this syndrome. In recent years, the significance of lung parenchyma mechanical properties has been characterized as one of the major determinants of physiological function (36, 40). Additionally, current investigations have shown that patients who died of asthma presented important alterations in lung parenchyma (14, 20) that could also be reproduced in animal models of experimental asthma (15, 46). Considering that lung parenchymal strips have long been used to study the behavior of the peripheral lung, they are commonly used to evaluate the mechanical and pharmacological properties of lung periphery (39).

Nitric oxide (NO) is an important modulator of inflammatory diseases such as asthma (22, 26, 28, 33). In the respiratory tract, NO can be produced by residential and also by inflammatory cells and is generated via oxidation of L-arginine, which is catalyzed by the enzyme NO synthase (NOS). NO derived from the constitutive isoforms of NOS [neuronal NOS (nNOS) and endothelial NOS] and other NO-adduct molecules (nitrosothiols) are able to modulate bronchomotor tone. On the other hand, NO derived from inducible NOS (iNOS) seems to be a molecule with immunomodulatory effects (32, 34).

Several lines of evidence suggest a role of NO in the bronchodilator response, particularly in larger airways, since the nitrergic innervation decreases throughout the bronchial tree (5). Prado et al. (27) demonstrated that the inhibition of NO by chronic N{omega}-nitro-L-arginine methyl ester (L-NAME) treatment amplified both respiratory system resistance and elastance responses and that it also interfered with the extracellular matrix airway remodeling in an experimental model of chronic lung inflammation. Considering that the respiratory system elastance responses are related to alterations in distal airways and lung tissue, the authors suggested that NO could also be involved in the modulation of lung tissue constriction. In addition, Dupuy et al. (5) proposed that inhaled NO only affects distal airways at high doses, suggesting that, although less intensive, NO can also modulate distal airways and/or lung tissue responses.

Therefore, we hypothesized that NOS inhibition modulates lung parenchyma responses in a guinea pig model of chronic pulmonary inflammation. We evaluated the effects of chronic L-NAME administration, an unspecific inhibitor of NO production, on the modulation of lung tissue mechanics, eosinophilic inflammation, and extracellular matrix tissue remodeling in this experimental model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals received humane care in compliance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health publication 85–23, revised 1985), and the Local Ethical Committee approved the study.

Experimental model of pulmonary allergic inflammation. Chronic airway inflammation was induced as previously described (10, 17). Male Hartley guinea pigs (300–400 g) were placed in a box coupled to an ultrasonic nebulizer (US-1000; ICEL), and an aerosol of ovalbumin (grade V, Sigma-Aldrich Chemical) diluted in 0.9% NaCl (normal saline) was generated for 15 min or until respiratory distress occurred. Respiratory distress was defined as the onset of sneezing, coryza, cough, and/or in drawing of the thoracic wall, and the observer who made the decision to withdraw the animals from the inhalation box was blinded to the treatment status of the animal. The animals received seven inhalations during 4 wk, with increasing ovalbumin concentrations (1~5 mg/ml) to counteract tolerance (Fig. 1). Control animals received aerosolized normal saline (0.9% NaCl).


Figure 1
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  		Fig. 1. Time line of the experimental protocol. The guinea pigs were submitted to 7 inhalations (2x/wk during 4 wk) with aerosols of normal saline or ovalbumin solution with increasing doses of antigen. For the 1st to the 4th inhalations, the dose used was 1 mg/ml of ovalbumin (2 wk). In the 5th and 6th inhalations (3rd wk), animals inhaled 2.5 mg/ml of ovalbumin and in the 7th inhalation (beginning of the 4th wk) a 5 mg/ml dose of antigen was used. The solution of ovalbumin or normal saline was continuously aerosolized for 15 min or until respiratory distress occurred (sneezing, coryza, cough, or retraction of the thoracic wall). After the 7th inhalation (72 h), all guinea pigs were anesthetized and exsanguinated, and lungs were removed and submitted to the experimental protocol of oscillatory mechanics. We analyzed the lung tissue elastance and resistance in baseline and after a challenge with 0.1% of ovalbumin in the bath.

 
Chronic L-NAME treatment. Chronic L-NAME treatment (60 mg·kg–1·guinea pig–1·day–1) was carried out as previously described (27, 28). Briefly, guinea pigs received L-NAME dissolved in the drinking water ad libitum (OVA-L or NS-L groups, see below), beginning 24 h after the fourth inhalation of either ovalbumin or normal saline to avoid interference with the sensitization (Fig. 1). Control animals received sterile drinking water.

Experimental groups. Four groups of guinea pigs were used in the experimental protocol: 1) the first group received inhalations with normal saline and sterile drinking water (NS group, n = 8); 2) the second group received ovalbumin aerosols and sterile drinking water (OVA group, n = 10); 3) the third group received inhalation with normal saline and L-NAME diluted in the drinking water (NS-L group, n = 7); 4) the fourth group received ovalbumin aerosols and L-NAME diluted in the drinking water (OVA-L group, n = 8).

Mechanics of lung parenchymal strips. After the last inhalation (72 h), animals were sedated with diazepam (1 mg ip), anaesthetized with pentobarbital sodium (50 mg/kg), and tracheostomized. After that, the thorax was opened, and the animals were exsanguinated. The lungs were removed en bloc and placed in a modified Krebs-Henseleit (K-H) solution (in mM: 118.4 NaCl, 4.7 KCl, 1.2 K3PO4, 25 NaHCO3, 2.5 CaCl2·H2O, 0.6 MgSO4·H2O, and 11.1 glucose) at pH = 7.40 and 6°C (36). Strips (3 x 3 x 10 mm) were cut from the periphery of the left lung and suspended vertically in a K-H organ bath maintained at 37°C, continuously bubbled with a mixture of 95% O2-5% CO2. Lung strips were weighed, and their unloaded resting lengths (L0) were determined with a caliper. Lung strip volume was measured by simple densitometry, as: volume = {Delta}F/{delta}, where {Delta}F is the total change in force before and after strip immersion in K-H solution and {delta} is the mass density of K-H solution (17, 18, 35–39). Parenchyma strips were suspended vertically in a K-H organ bath (30 ml internal volume) maintained at 37°C and continuously bubbled with 95% O2-5% CO2 as previously described (39). Briefly, one end of the strip was attached to a force transducer (LETICA TRI-110; Scientific Instruments, Barcelona, Spain), whereas the other one was fastened to a lever arm actuated by means of a modified woofer driven by the signal generated by a computer and analog-to-digital converted (AT-MIO-16-E-10, National Instruments, Austin, TX). A sidearm of this rod was linked to a second force transducer (LETICA TRI-110; Scientific Instruments) by means of a silver spring of a known Young's modulus, thus allowing the measurement of displacement (Fig. 2). Neither amplitude dependence (<0.1% change in stiffness) nor phase changes with frequency were detected in the 0.01- to 14-Hz range. The hysteresivity of the system (<0.003) was frequency independent


Figure 2
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  		Fig. 2. Schematic setup used for tissue mechanics measurements.

 
Cross-sectional, unstressed area (A0) of the strip was determined from volume and unstressed length, according to A0 = vol/L0. Basal force (FB) for a stress of 0.1 N/cm2 was calculated as FB (N) = 10 (N/cm2) x A0 (cm2) and adjusted by vertical displacement of the force transducer, as previously described (8). The displacement signal was then set to zero. Once basal force and displacement signals were adjusted, the length between bindings (LB) was measured by means of a precision caliper. Instantaneous length during oscillation around LB was determined by adding the value of LB to the measured value of displacement at any time. Instantaneous average cross-section area (Ai) was determined as Ai = vol/Li (cm2), where Li is instantaneous length.

Instantaneous stress ({sigma}i) was calculated by dividing force (F; in g) by Ai (cm2), i.e., {sigma}i = F/Ai. Strain was calculated as {Delta}{varepsilon} = (L – LB)/LB. After the basal force was adjusted to 0.5 x 10–2 N, each parenchyma strip was preconditioned by sinusoidal oscillation of the tissue during 30 min (frequency = 1 Hz, which is a large enough amplitude to reach a final force of 1 x 10–2 N). Thereafter, the amplitude was adjusted to 5% L0, and the oscillation was maintained for another 30 min, or until a stable length-force loop was reached. The isometric stress adaptation period resulted in a final force of 0.5 x 10–2 N. After preconditioning, the strips were oscillated at a frequency (f) of 1 Hz (36, 46) and with a constant force of 0.5 x 10–2 N. The bath solution was renewed regularly (every 20 min) with 37°C K-H solution.

All mechanical parameters were measured cycle by cycle. Tissue resistance (R) was determined from the enclosed area of force length loops: R = (4 x H)/[{pi} x {omega} x ({Delta}{varepsilon})2], where H is the stress-strain hysteresis area, {omega} is the angular frequency [{omega} = 2{pi}f (rad/s), where f is frequency], and {Delta}{varepsilon} is the normalized strain or peak-to-peak change in length divided by LB. Tissue dynamic E was determined as: E = ({Delta}{sigma}{iota}/{Delta}{varepsilon})cos{theta}, where {Delta}{sigma}{iota} is the peak-to-peak change in force, and {theta} is the phase lag between force and displacement ({theta} = sin–1{4 x H/[{pi}({Delta}{sigma}{iota} x {Delta}{varepsilon})]}). The {eta}, which is an empirically determined variable that quantifies the dependence of dissipative processes on elastic processes (7), was calculated as {eta} = tan{theta}.

Tissue resistance (R), elastance (E), and hysteresivity ({eta}) were calculated at baseline condition and after ovalbumin challenge (dose of 0.1% of ovalbumin) (7, 40).

Morphometric analysis. Lung tissue strips were fixed at –70°C by immersion in Carnoy's fixative [solution of ethanol-chloroform-acetic acid (60:30:10)]. After 24 h, the lungs were kept at –20°C, and the concentration of ethanol was progressively increased to reach that of absolute ethanol (41). To assure the homogeneity of the strip samples used, slices were cut (5 µm thick), stained with hematoxylin-eosin, and analyzed through an integrating eyepiece with a coherent system made of a 100-point grid consisting of 50 lines of known length coupled to a light microscope. Sections were examined at x400 magnification, and the fractional areas of alveolar wall (AW), blood vessel wall (BVW), and bronchial wall (BW) were determined by the point-counting technique (43). All points falling on these components were counted and divided by the total number of points. This analysis was performed in 10 random, nonoverlapping fields in each strip. BVW and BW were counted when a point fell on the endothelial layer, the epithelial layer, the smooth muscle, or associated connective tissue. Points falling on alveolar air spaces, blood vessel lumen, and bronchial lumen were excluded.

Quantification of eosinophil density. Slices (5 µm thick) of lung strips were stained with Luna's eosinophil granule stain for the detection of eosinophils (19, 42). By conventional morphometry, we analyzed the density of eosinophils within the alveolar septa of lung strips. Using a 100-point grid with a known area (104 µm2 x1,000 magnification) attached to the microscope ocular, we counted the number of points hitting alveolar tissue in each field and the number of eosinophils within the alveolar septa. Eosinophil density was determined as the number of eosinophils in each field divided by tissue area. Measurements are expressed as cells per 104 µm2. Counting was performed in 10 fields of lung strip in each animal.

Immunohistochemistry for nNOS detection. Histological sections of 5 µm were stained with specific monoclonal IgG2a antibody to nNOS (nNOS/NOS type I-N31020; BD Transduction Laboratories, San Diego, CA) to detect nNOS expression. Immunohistochemistry was performed as previously described (28). Briefly, sections were initially incubated in a humid chamber (30 min at room temperature) with a blocking solution containing normal mouse serum followed by the primary monoclonal antibody anti-nNOS from mouse (overnight at room temperature). After three washes in TBS for 5 min, sections were incubated with a streptavidin-alkaline phosphatase conjugate (LSAB + AP-streptavidin AP; Dako, Carpinteria, CA) for 30 min at 37°C, followed by incubation with Fast Red TR substrate for 6 min and light hematoxylin counterstaining (Merck) for 1 min. We counted eosinophils, epithelial cells, and macrophages that were positive for nNOS. These cells were determined as the number of positive cells in each field divided by tissue area, at a magnification of x1,000, and were expressed as distal lung cells positive for nNOS expression (104 µm2).

Immunohistochemistry for iNOS detection. For iNOS detection, we used the same sections employed at the morphometric evaluation of nNOS expression. Immunohistochemistry was performed as previously described (3). Subsequently, the sections were incubated seven times for 5 min at room temperature with a blocking solution containing 3% hydrogen peroxide. Polyclonal antisera raised in rabbit against iNOS (cod. RB-9242-P; LabVision, NeoMarkers, Fremont, CA) (26) were used as primary antisera [incubation overnight in a humid chamber (refrigerator) at 4–8°C, 1:400 dilution in BSA]. After three 5-min washes in PBS, sections were incubated with a secondary antibody [Vector ABCElite, horseradish peroxidase (HRP), cod. PK-6101; Vector Laboratories, Burlingame, CA] at 37°C in a humidity chamber. Slides were given three more 5-min washes in PBS and revealed with 3,3'-diaminobenzidine (DAB) (cod. K3468; DakoCitomation). Slides were given abundant washes in tap water. This was followed by Harry's hematoxylin counterstaining for 1 min. A total of 10 fields was analyzed per lung as described above. We counted eosinophils, epithelial cells, and macrophages that were positive for iNOS. These cells were determined as the number of positive cells in each field divided by tissue area, at a magnification of x1,000, and were expressed as distal lung cells positive for iNOS expression in cells per unit area (104 µm2) (27).

Quantification of collagen and elastic fiber density. Lung strips were also stained with Picrosirius, a specific method for collagen detection and Weigert's Resorcin-Fuchsin method for elastic fibers (4). From each strip, 20 different microscopic fields were randomly selected to quantify collagen and elastic fibers. Quantification (x400 magnification) was carried out by the same method described above. The volume proportion of collagen and elastic fibers in the alveolar tissue of lung strips was determined by dividing the number of points hitting collagen or elastin by the total number of points hitting the alveolar septa. Results were expressed as percentage of area.

Immunohistochemistry for actin. Immunohistochemical staining was performed using monoclonal antibody to {alpha}-smooth muscle actin (Dako) at a 1:500 dilution. Sections were deparaffinized, and 0.5% peroxidase in methanol solution was applied for 10 min to inhibit endogenous peroxidase activity. Antigen retrieval was performed with a citrate solution for 30 min. Sections were incubated with anti-human smooth muscle actin (1A 4; Dako) overnight at 4°C. LSAB Plus-HRP kit (K-0690; DAKO) was used as secondary antibody, and DAB (Sigma Chemical, St. Louis, MO) was used as chromogen. The sections were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany). The analysis was performed in the slides stained for actin by applying the same point-counting technique described above. We determined the volume proportion of smooth muscle-specific actin in the alveolar septa as the relation between the number of points falling on actin-stained and non-actin-stained tissue (5). Measurements were carried out at x400 in each slide.

Immunohistochemistry for 8-iso-PGF2{alpha} detection. Immunohistochemical staining was performed using antibody to anti-8-epi-PGF2{alpha} (Oxford Biomedical Research, Rochester Hills, MI) at a 1:500 dilution (23). Sections were deparaffinized and washed seven times for 5 min with 3% H2O210V to inhibit endogenous peroxidase activity. After washes in PBS and water, the antigen retrieval was performed with tripsine for 20 min. Afterward, three washes in PBS were performed for 3 min each. Sections were incubated with anti-8-epi-PGF2 diluted in BSA overnight. After washes in PBS, ABCKit Vectastain (Vector Elite PK-6105) was used as secondary antibody, and DAB (Sigma Chemical) was used as chromogen. The sections were counterstained with Harris hematoxylin (Merck). The analysis was performed in the slides stained for 8-isoprostane-PGF2{alpha} by applying the same point-counting technique described above. We determined the volume proportion of 8-iso-PGF2{alpha} in the alveolar septa as the relation between the number of points falling on 8- iso-PGF2{alpha}-stained and nonstained tissue. Measurements were carried out at x400 in each slide (23). All morphometric analyses described in this section were performed by individuals blinded to the protocol design.

Statistical analysis. Values are expressed as means ± SE. Statistical analysis was performed using the SigmaStat 2.0 statistical software package (Jandel, San Raphael, CA). Data were evaluated by two-way analysis of variance, and multiple comparisons were made using the Holm-Sidak method. A P value of <0.05 was considered significant (47).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mechanics of lung parenchymal strips. Values of oscillatory pulmonary mechanics in baseline condition and after antigen challenge are shown in Fig. 3. Tissue resistance and elastance were higher in ovalbumin-exposed animals compared with saline-exposed ones in baseline condition (P < 0.05 for all comparisons). Tissue resistance and elastance were higher in ovalbumin-exposed animals compared with saline-exposed ones after challenge (P < 0.05 for all comparisons). L-NAME treatment in ovalbumin-exposed animals (OVA-L group) reduced all these responses compared with ovalbumin-exposed and vehicle-treated animals (P < 0.01). Hysteresivity was similar in all groups in baseline [NS: 0.04 ± 0.00; NS-L: 0.05 ± 0.00; OVA: 0.05 ± 0.00; OVA-L: 0.05 ± 0.00 {eta}] and after OVA challenge [NS: 0.04 ± 0.00; NS-L: 0.05 ± 0.00; OVA: 0.05 ± 0.00; OVA-L: 0.05 ± 0.00 {eta}].


Figure 3
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  		Fig. 3. Baseline and postovalbumin challenge (0.1%) values of tissue resistance (A) and elastance (B) in guinea pigs exposed to 7 inhalations with ovalbumin or normal saline and chronically treated with N{omega}-nitro-L-arginine methyl ester (L-NAME) or vehicle. Values are expressed as means ± SE. *P < 0.05 compared with baseline and postchallenge values of the normal saline (NS) group; **P ≤ 0.01 compared with the ovalbumin (OVA) group.

 
Morphometric analysis. All groups showed similar anatomical composition of lung strips with ~60–70% of AW.

Eosinophil density in lung tissue is shown in Fig. 4. We observed that there was an increase in the eosinophil density in ovalbumin-exposed animals compared with saline-exposed ones (P < 0.05). L-NAME treatment did not affect this response. Figure 5 shows the number of nNOS- and iNOS-positive distal lung cells in alveolar tissue. There was a significant increase in both nNOS- and iNOS-positive cells in the OVA group compared with controls (P < 0.05). L-NAME treatment reduced the number of both nNOS- and iNOS-positive cells in ovalbumin-exposed animals (P < 0.001). There was no difference between NS and NS-L groups.


Figure 4
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  		Fig. 4. Eosinophil density in lung tissue of guinea pigs exposed to 7 inhalations with ovalbumin or normal saline and treated with L-NAME or vehicle. Values are expressed as means ± SE. *P < 0.05 compared with NS and NS + L-NAME (NS-L) groups.

 

Figure 5
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  		Fig. 5. Neuronal (nNOS) and inducible (iNOS) nitric oxide synthase-positive distal lung cells (eosinophils, macrophages, and epithelial cells in alveolar septa) of guinea pigs previously exposed to 7 inhalations with ovalbumin or normal saline and treated with L-NAME or vehicle. Values are expressed as means ± SE. *P < 0.05 compared with NS and NS-L groups; **P < 0.001 compared with OVA group.

 
The amount of collagen (Fig. 6A) and elastic fibers (Fig. 6B) in the alveolar septa is shown in Fig. 6. Both collagen and elastic fiber contents were higher in ovalbumin-exposed animals compared with the saline-exposed ones (P < 0.001). Ovalbumin-exposed animals that received chronic L-NAME treatment presented lower elastic fiber content in the alveolar septa compared with those that received vehicle (P < 0.001). The L-NAME treatment did not modify collagen fiber deposition in lung tissue.


Figure 6
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  		Fig. 6. Collagen (A) and elastic fiber (B) content in guinea pigs exposed to 7 inhalations with ovalbumin or normal saline and chronically L-NAME or vehicle treated. Values are expressed as means ± SE. Results are expressed as percentage. *P < 0.001 compared with NS and NS-L groups; **P < 0.001 compared with OVA group.

 
Volume proportion of smooth muscle-specific actin was similar in all groups (OVA group: 11.25 ± 042; NS group: 10.39 ± 0.48; OVA-L group: 9.96 ± 0.48; NS-L group: 11.47 ± 0.48%).

Volume proportion of 8-iso-PGF2{alpha} in alveolar septa is shown in Fig. 7. Ovalbumin-exposed animals showed higher values of 8-isoprostane-PGF2{alpha} density compared with saline-exposed ones (P < 0.001). Ovalbumin-exposed animals that received chronic L-NAME treatment (OVA-L group) presented lower content of 8-iso-PGF2{alpha} positive tissue compared with those that received vehicle (OVA group, P < 0.001). There was no significant difference between saline-exposed animals.


Figure 7
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  		Fig. 7. Volume proportion of 8-isoprostane-PGF2{alpha} in alveolar septa of guinea pigs exposed to 7 inhalations with ovalbumin or normal saline and chronically L-NAME or vehicle treated. Values are expressed as means ± SE. Results are expressed as percentage. *P < 0.001 compared with NS and NS-L groups; **P < 0.001 compared with OVA group.

 
To evaluate if the strip procedures in the bath interfere with immunohistochemistry evaluation, we also performed the immunohistochemistry to detect the 8-iso-PGF2{alpha} in the lung that was not submitted to the strip evaluation. We observed that the ovalbumin group (OVA group: 25.24 ± 1.90) presented a higher volume proportion of 8-isoprostane-PGF2{alpha} positive tissue compared with saline-exposed ones [(NS group: 4.81 ± 2.55; NS-L group: 7.83 ± 1.61), P < 0.001]. Ovalbumin-exposed animals that received chronic L-NAME treatment (OVA-L group: 3.81 ± 1.61) presented a lower volume proportion of 8-iso-PGF2{alpha} positive tissue compared with those exposed to ovalbumin that received only vehicle (P < 0.001). There was no significant difference between saline-exposed animals.

Figure 8 shows representative photomicrographs of lung parenchyma stained with Luna (A–C), Picrosirius (D–F), Weigert's Resorcin-Fuchsin (G–I), nNOS-stained eosinophils (J–L), and 8-iso-PGF2{alpha} (M–O) in guinea pigs that received saline inhalation (A, D, G, J, and M) or those chronically exposed to ovalbumin with vehicle (B, E, H, K, and N) or chronic treatment with L-NAME (C, F, I, L, and O). Lung tissue of ovalbumin-exposed animals presented eosinophil infiltration with collagen and elastic fiber deposition within the alveolar septa. Chronic L-NAME treatment reduced the amount of elastic fibers in ovalbumin-exposed animals as well as the number of nNOS-positive eosinophils and 8-iso-PGF2{alpha} positive tissue.


Figure 8
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  		Fig. 8. Guinea pig lung tissue samples obtained from controls, OVA-exposed vehicle, and OVA-exposed L-NAME treated, stained with Luna (A–C, magnification x1,000), Picrosirius (D–F, x400), Weighert Resorcin-Fuchsin (G–I, x400), immunohistochemistry to nNOS (J–L, x1,000) and 8-isoprostane PGF2{alpha} detection (M–O, x400). Control groups show scanty amounts of collagen fibers in alveolar tissue sections, coincident with the maintenance of the histoarchitecture of the alveolar septa, and a scarce number of eosinophils (A) and those positive to nNOS (J). In contrast, distal lung parenchyma of OVA-exposed vehicle-treated animals show intense eosinophilic infiltration (B) within the alveolar septa, including those positive to nNOS expression (K), an increase in the amount of collagen (E) and elastic fibers (H), and in the content of 8-isoprostane-PGF2{alpha}-positive tissue (N). Chronic L-NAME treatment in ovalbumin-exposed animals did not interfere with the number of eosinophils (C) and the collagen content present in the alveolar septa. However, this treatment reduced elastic fiber content (I), the number of eosinophils positive to nNOS in alveolar septa (L), and the amount of lung tissue positive to 8-iso-PGF2{alpha} (O).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have data suggesting that NO is involved in the mechanical responses of distal lung parenchyma. Using an animal model of chronic lung inflammation in guinea pigs, we showed that NOS inhibition attenuated lung tissue constriction, which was associated with a reduction in isoprostanes (8-iso-PGF2{alpha}) generation and elastic fiber deposition in alveolar septa. Notwithstanding a significant lung tissue mechanical attenuation, eosinophilic infiltration and collagen fiber deposition in alveolar septa were not modified by NOS inhibition.

The effectiveness of L-NAME treatment in this animal model has been demonstrated previously (27, 28), and it was confirmed in the present study, since there was a significant reduction in both nNOS- and iNOS-positive distal lung cells of guinea pigs with chronic pulmonary inflammation that received treatment with L-NAME. In addition, we have previously shown (28) that chronic L-NAME treatment in this animal model reduced the exhaled NO by ~50% compared with controls. The L-NAME treatment began 24 h after the forth inhalation to avoid interference with the sensitization since in this period animals have already been sensitized (IgG1 title 1:320) (16). In fact we had previously evaluated the effects of this approach of L-NAME treatment in the sensitization process, and we observed that this treatment did not interfere with the sensitization (26, 27, 28).

Several studies demonstrated that the effects of NO are more pronounced in proximal airways (1, 5, 27, 28). All of these studies showed that NO has an important role in the control of smooth muscle acting as a bronchodilator in proximal airways. However, few authors have previously evaluated NO-related effects on lung parenchyma. This may be due to the fact that there is a progressive reduction of nitrergic nerves toward the bronchial tree (34). Dupuy et al. (5) showed that inhaled NO acted as a bronchodilator of distal airways only when high doses were administrated.

By analyzing the oscillatory mechanical responses, we observed that L-NAME treatment reduced both baseline and postchallenge tissue resistance and elastance in ovalbumin-exposed animals, suggesting that NO contributes to pulmonary parenchyma constriction in this experimental model.

In fact, in the present study, we used the same protocol of sensitization and the same approach of L-NAME treatment used in the study performed by Prado et al. (27, 28). Previously we noted that chronic L-NAME treatment induced a bronchoconstriction only in proximal airways, represented by resistance of respiratory system, both in physiological and after antigen challenge. The authors also observed that chronic L-NAME treatment reduced the baseline respiratory system elastance (Ers) and did not affect the Ers postantigen challenge, suggesting that the effects of NO in distal airways and/or lung parenchyma were different from those observed in proximal airways, and it could be due to a particular effect in lung parenchyma. In this regard, our findings are in accordance of those obtained by Prado et al. (28). Using a model of lung strip oscillatory mechanics that evaluate only the lung parenchyma, we also observed a reduction in lung tissue resistance and elastance after L-NAME treatment, suggesting that NO is involved in lung tissue constrictor effects induced by repeated ovalbumin exposures.

At least in our knowledge, there were no other studies that have evaluated the effects of NO in lung parenchyma. Although the exact mechanism involved in the effect of L-NAME treatment on reducing lung parenchyma constriction is unclear, some explanations can be suggested. Several authors have discussed that NO release by NOS activation also contributes to the oxidative stress, amplifying the deleterious and harmful effects on lungs (32). The potent oxidant peroxynitrite is formed by NO and superoxide interaction by a rapid isostoichiometric reaction (24, 29).

Peroxynitrite formation leads to lipid peroxidation and generation of isoprostanes (8-iso-PGF2{alpha}). Although previous studies have evaluated the effects of PGE2, which was more potent as a constrictor than PGF2{alpha}, the latter isoprostane is considered the predominant form generated during free radical attack of cell membranes (23). Jourdan et al. (13) showed that L-NAME treatment greatly inhibits 8-iso-PGF2{alpha}. Therefore, isoprostanes appear to induce airway and vascular smooth muscle contractions acting through tyrosine kinase, Rho, and Rho kinase, leading to decreased activity of myosin light chain phosphatase. The net response is associated with an increased level of phosphorylated myosin light chain and contraction (11). We observed in the present study that the attenuation of mechanical responses was associated with a significant decrease in 8-iso-PGF2{alpha} density in lung tissue, which corroborates the idea that NO-derived effects in distal parenchyma were more dependent on the oxidative stress pathway than on the eosinophilic recruitment.

In fact, chronic NOS inhibition by L-NAME treatment did not modify eosinophilic lung tissue infiltration induced by chronic lung inflammation. NO effects in eosinophil recruitment are still a matter of controversy (26, 27, 28, 33, 34, 6). Corroborating our findings, we previously showed that, in guinea pigs with chronic lung inflammation, eosinophilic airway recruitment was also not reduced by chronic L-NAME treatment (28). Although we have observed no effects on eosinophils, it is important to note that L-NAME could interfere with other cells (3, 32, 34) such as macrophages, mast cells, lymphocytes, neutrophils, and also pneumocytes that can be presented in lung parenchyma, which was not evaluated in this model since there are no specific antibodies commercially available to detect these cells in guinea pigs.

The present study showed that ovalbumin-exposed animals presented an increase in elastic fiber content in the alveolar septa. We also demonstrated that chronic L-NAME treatment in ovalbumin-exposed animals prevented the increment in the amount of elastic fibers in the alveolar septa. It was remarkable that these findings were associated with the attenuation of lung tissue mechanics observed in these animals. In addition, we did not observe any difference in the elastic fiber content in ovalbumin-exposed animals, animals treated with L-NAME, and the saline-exposed animals.

One crucial question was related to the mechanisms that linked NO to elastogenesis and/or elastolysis, which were, until now, poorly understood. Although we were not able to clarify the exact mechanism linking NO and elastic fibers in the present study, some hypothesis could be suggested. Pastor et al. (25) studying an experimental model of papain-induced lung emphysema, observed that an electrodense amorphous substance was ruptured and tended to disappear 24 h after papain instillation, and after 2 mo there was an accumulation of elastic fibers associated with collagen deposition, particularly in the wall alveolar duct. Cantor et al. (2), evaluating the effects of oxidants on elastase activity in vitro, suggested that hydrogen peroxide and other oxidants derived from inflammatory cells or from the environment act as the priming agent for elastase-mediated breakdown of elastic fibers with de novo resynthesis, which may sometimes occur in an abnormal morphological organization with abnormal functional properties (25). However, other authors (12) have suggested that the elastic fibers might be normal after the repair process.

The induction of chronic lung inflammation in guinea pigs resulted in an increase of oxidative stress. We evaluated the oxidative stress in this study by 8-iso-PGF2{alpha} staining (23). The 8-iso-PGF2{alpha} is the most well-characterized isoprostane that may act through a novel receptor closely related to, but distinct from, the tromboxane A2/PGH2 receptor, with a high specificity for 8-iso-PGF2{alpha} (45). Considering the physiological effects of isoprostanes, Quaggiotto and Garg (30) demonstrated that 8-iso-PGE2 produces physiological effects similar to 8-iso-PGF2{alpha}, but at a reduced potency. In addition, Wood et al. (44) showed that 8-iso-PGF2{alpha} is increased in persistent asthmatic patients three to four times more compared with the normal group. For these reasons, the evaluation of 8-iso-PGF2{alpha} represents a valuable indicator of the oxidative stress pathway (21, 23). Another important point is that 8-iso-PGF2{alpha} is a soluble and short-lived mediator. We also performed the evaluation of oxidative stress by immunohistochemistry in the lung that was not submitted to oscillatory mechanical evaluation. The findings were similar to those obtained in the lung strips.

Probably, the increase in elastic fiber content observed in ovalbumin-exposed animals can due to a repair process. Considering that L-NAME treatment reduced the oxidative stress, the elastic fiber injury/repair process was attenuated, as observed in the present study.

We observed that L-NAME treatment did not modify collagen fiber content in lung tissue of sensitized animals. Although previous study showed a profibrotic effect of NO (9, 10), the mechanisms involved in these responses have to be further investigated. In the same animal model, Prado et al. (28) have previously demonstrated that the inhibition of NO by chronic L-NAME treatment amplified the extracellular collagen airway remodeling. The variability in the response observed concerning collagen deposition in airways and lung tissue of this animal model may be related to the higher activity of arginase I and II in the peribronchial connective tissue (31), as well as the intensity of the inflammatory response that was greater in airways compared with that in the lung tissue (28, 41).

In conclusion, our results suggest that NO plays an important role in lung tissue constriction and elastic fiber deposition within the alveolar septa in this animal model of chronic pulmonary inflammation. The activation of the pulmonary oxidative stress pathway, mainly 8-iso-PGF2{alpha}, may contribute to these responses.


   ACKNOWLEDGMENTS
 
We are grateful to the Brazilian Scientific Agencies, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo à Pesquisa do Estado de São Paulo e do Rio de Janeiro, Programa de Núcleos de Excelência, and Laboratório de Investigação Médica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (LIM 20). We express our gratitude to Thais Mauad for help in isoprostane evaluation and to André Benedito da Silva and Jaqueline Lima do Nascimento for skilful technical assistance.

This study was presented in part at the International Meeting of the European Respiratory Society in Munich-2006.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. de Fátima Lopes Calvo Tibério, Departamento de Clínica Médica, Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455-Sala 1210, 01246-903, São Paulo, SP, Brazil (e-mail: iocalvo{at}uol.com.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
 REFERENCES
 

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