Therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-α Therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-� A. Charlotte P. Sewing,1 Crystal Kantores,1 Julijana Ivanovska,1 Alvin H. Lee,1 Azhar Masood,1,3 Amish Jain,1,3,4 Patrick J. McNamara,1,3,4 A. Keith Tanswell,1,3,4 and Robert P. Jankov1,2,3,4 1Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; 2Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario, Canada; 3Department of Physiology, University of Toronto, Toronto, Ontario, Canada; and 4Division of Neonatology, Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada Submitted 21 February 2012; accepted in final form 2 May 2012 Sewing AC, Kantores C, Ivanovska J, Lee AH, Masood A, Jain A, McNamara PJ, Tanswell AK, Jankov RP. Therapeutic hyper- capnia prevents bleomycin-induced pulmonary hypertension in neo- natal rats by limiting macrophage-derived tumor necrosis factor-�. Am J Physiol Lung Cell Mol Physiol 303: L75–L87, 2012. First published May 11, 2012; doi:10.1152/ajplung.00072.2012.—Bleomy- cin-induced lung injury is characterized in the neonatal rat by inflam- mation, arrested lung growth, and pulmonary hypertension (PHT), as observed in human infants with severe bronchopulmonary dysplasia. Inhalation of CO2 (therapeutic hypercapnia) has been described to limit cytokine production and to have anti-inflammatory effects on the injured lung; we therefore hypothesized that therapeutic hypercapnia would prevent bleomycin-induced lung injury. Spontaneously breath- ing rat pups were treated with bleomycin (1 mg/kg/d ip) or saline vehicle from postnatal days 1–14 while being continuously exposed to 5% CO2 (PaCO2 elevated by 15–20 mmHg), 7% CO2 (PaCO2 elevated by 35 mmHg), or normocapnia. Bleomycin-treated animals exposed to 7%, but not 5%, CO2, had significantly attenuated lung tissue macrophage influx and PHT, as evidenced by normalized pulmonary vascular resistance and right ventricular systolic function, decreased right ventricular hypertrophy, and attenuated remodeling of pulmo- nary resistance arteries. The level of CO2 neither prevented increased tissue neutrophil influx nor led to improvements in decreased lung weight, septal thinning, impaired alveolarization, or decreased num- bers of peripheral arteries. Bleomycin led to increased expression and content of lung tumor necrosis factor (TNF)-�, which was found to colocalize with tissue macrophages and to be attenuated by exposure to 7% CO2. Inhibition of TNF-� signaling with the soluble TNF-2 receptor etanercept (0.4 mg/kg ip from days 1–14 on alternate days) prevented bleomycin-induced PHT without decreasing tissue macro- phages and, similar to CO2, had no effect on arrested alveolar development. Our findings are consistent with a preventive effect of therapeutic hypercapnia with 7% CO2 on bleomycin-induced PHT via attenuation of macrophage-derived TNF-�. Neither tissue macro- phages nor TNF-� appeared to contribute to arrested lung develop- ment induced by bleomycin. That 7% CO2 normalized pulmonary vascular resistance and right ventricular function without improving inhibited airway and vascular development suggests that vascular hypoplasia does not contribute significantly to functional changes of PHT in this model. carbon dioxide; inflammation; neonatal lung injury BRONCHOPULMONARY DYSPLASIA (BPD) is a chronic pneumopathy affecting extremely premature infants who frequently require supplemental oxygen and/or mechanical ventilation. The inci- dence of BPD is inversely proportional to the gestational age at which infants are born, with an incidence approaching 60% in the smallest survivors (4). The cardinal feature of BPD, as observed in the current era, is an inhibition or arrest of alveolar and vascular growth (29). The underlying pathogenesis of BPD appears to be multifactorial, but upregulation of inflammatory mediators leading to, or caused by, infiltration of inflammatory cells, including macrophages and neutrophils, is believed to play a major role (61, 62, 66). Pulmonary hypertension (PHT) is common in those infants that are most severely affected (15, 58, 67), which signals greatly increased morbidity and mortal- ity related, in major part, to consequent right heart failure (33). Putative factors contributing to progressive and fatal PHT in BPD include sustained vasoconstriction, arterial wall remod- eling, and pruning of peripheral resistance arteries (58), but the upstream mediators and relative contributions of these changes to chronically increased pulmonary vascular resistance (PVR) and right ventricular dysfunction remain poorly understood. Management strategies employed in an attempt to prevent or ameliorate BPD have thus far met with limited success but include avoidance or minimization of mechanical ventilation and high inspired O2 concentrations to reduce the potential for volutrauma and oxygen toxicity (1). With avoidance of exces- sive mechanical ventilation has come a necessary shift in clinical practice towards “permissive hypercapnia” (acceptance of “higher-than-normal” PaCO2 levels) becoming the norm (28), although evidence of the effectiveness in reducing the inci- dence and severity of BPD remains lacking (76). Possible explanations for a lack of benefit in clinical trials (49) may be that the target PaCO2 was less than required or that harmful effects of hypoventilation and small tidal volumes in the suboptimally recruited lung may have counteracted any bene- fit. Despite ongoing uncertainty about the utility of permissive hypercapnia in the neonate (35), there is growing experimental evidence to indicate that inhaled or exogenous CO2 (so-called “therapeutic hypercapnia”) may protect the lung from diverse injuries, including those secondary to systemic ischemia rep- erfusion (38), endotoxin (37, 72), high tidal volume mechani- cal ventilation (7, 65, 69), and chronic exposure to hyperoxia (45) or hypoxia (31, 56). Putative mediators of lung injury shown to be attenuated by therapeutic hypercapnia in these models have included inflammatory cell influx (37, 45, 72), proinflammatory cytokines (14, 38, 72), and oxidative stress (31, 38, 52, 72). However, several studies have also indicated the potential for hypercapnia to worsen lung injury (41, 53, 54) and to potentially cause an increase in inflammation in the Address for reprint requests and other correspondence: R. P. Jankov, Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute, 555 Univ. Ave, Toronto, Ontario, Canada M5G 1X8 (e-mail: robert.jankov@sickkids.ca). Am J Physiol Lung Cell Mol Physiol 303: L75–L87, 2012. First published May 11, 2012; doi:10.1152/ajplung.00072.2012. 1040-0605/12 Copyright © 2012 the American Physiological Societyhttp://www.ajplung.org L75 Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. noninjured lung (2, 45), highlighting the need for further study. Our aim therefore was to examine effects of therapeutic hy- percapnia in a new rat model with similarities to human BPD (47), secondary to bleomycin exposure. Bleomycin sulfate is a chemotherapeutic agent that produces a dose-dependent pulmonary inflammatory and fibrotic re- sponse in humans when administered systemically and in adult rodents when instilled as a single intratracheal dose (32) or by repeated intraperitoneal injection (6). The early phases of bleomycin-induced lung injury are characterized by increased expression of proinflammatory cytokines (19), a marked in- flammatory cell influx that is dominated by macrophages (27), increased apoptosis (20), changes in growth factors, such as transforming growth factor-� (3, 12), “emphysematous” lung morphology, and severe PHT (63, 77). In neonatal rats, selec- tive decrease in lung growth, arrested alveolarization and vascular hypoplasia are prominent features (47, 74). Herein, we report that exposure to CO2 led to dose-dependent preventive effects on bleomycin-induced PHT without af- fecting lung growth, arrested alveolarization, or vascular hypoplasia. Tumor necrosis factor (TNF)-� was found to be increased by bleomycin and to colocalize with tissue mac- rophages, both of which were attenuated by 7% CO2. Inhibition of TNF-� signaling with a soluble TNF-2 recep- tor, etanercept, also prevented the hemodynamic and struc- tural changes of chronic PHT, suggesting that the benefits of CO2 on the pulmonary vasculature were mediated, in major part, via this pathway. MATERIALS AND METHODS Materials. Bleomycin sulfate was purchased from Calbiochem (San Diego, CA). Etanercept (Enbrel) was from Amgen (Thousand Oaks, CA). Plexiglas animal exposure chambers and automated O2/ CO2 controllers were from BioSpherix (Lacona, NY). Acids, alcohols, organic solvents, paraformaldehyde, Permount, and Superfrost/Plus microscope slides were from Fisher Scientific (Whitby, ON, Canada). DMEM, trypsin, and FBS were from Wisent Bioproducts (St-Bruno, Quebec, Canada). Avidin-biotin-peroxidase complex immunohisto- chemistry kits, 3, 3=-diaminobenzidine staining kits, DAPI fluorescent mounting medium, and normal goat serum were from Vector Labo- ratories (Burlingame, CA). Weigert’s resorcin-fuchsin stain was from Rowley Biochemical (Danvers, MA). Terminal deoxyuridine triphos- phate (dUTP) nick-end labeling (TUNEL) assay kits were from Roche (Laval, Québec, Canada). Alexa Fluor 488-conjugated isolectin B4 (cat no. I21411), derived from Griffonia Simplicifolia, Alexa Fluor 488-conjugated goat anti-mouse (cat no. A-10680), and AlexaFluor 546-conjugated anti-goat secondary antibody (cat no. A-11056) were from Life Technologies (Burlington, ON, Canada). Anti-glyceralde- hyde-3-phosphate dehydrogenase (GAPDH; cat no. sc-25778), anti- tumor necrosis factor (TNF)-� (cat no. sc-1349, used for fluorescent immunostaining), and goat anti-rabbit and -mouse IgG-biotin antibod- ies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- cluster of differentiation (CD)68 (cat no. MCA341R) was from Serotec (Raleigh, NC). Anti-myeloperoxidase (MPO; cat no. A0398) was from DAKOCytomation (Mississauga, ON, Canada). Anti- TNF-� (cat no. HP8001, used for Western blotting) was from Hycult Biotech (Uden, The Netherlands). Goat anti-rabbit and anti-mouse IgG-peroxidase antibodies were from Cell Signaling Technology (Beverly, MA). Unless otherwise specified, all other chemicals and reagents were from Bioshop Canada (Burlington, ON, Canada). Animal exposures and interventions. All procedures involving animals were performed in accordance with the standards of the Canadian Council on Animal Care and were approved by the Animal Care Committee of the Hospital for Sick Children Research Institute. Commencing on the day after birth, pups received bleomycin sulfate 1 mg/kg in 0.9% saline (5 �l/g body wt by 27-G needle in the right iliac fossa) or 0.9% saline (vehicle control) daily intraperitoneally for 14 days, as previously described (47), while being concurrently exposed to either normocapnia (� 0.5% environmental CO2) or 5% or 7% CO2 (O2 21%, balance N2) (31). In separate experiments, bleo- mycin-exposed pups received an additional intraperitoneal injection of etanercept (0.4 mg/kg in 0.9% saline; 5 �l/g body wt ip) on alternate days commencing on postnatal day 1. The dose of etanercept used was the same as that recently described to be effective in preventing PHT in monocrotaline-exposed adult rats (71). Each litter was maintained at n � 10 –12 pups to control for nutritional effects. At the end of each 14-day exposure period, pups were either killed by pentobarbital overdose or exsanguinated after anesthesia. Arterial blood gas measurement. Pups were lightly anesthetized with ketamine (40 mg/kg ip) and xylazine (6 mg/kg ip), and the neck was dissected to expose the external carotid artery. After a 15-min recovery, while the animals breathed the appropriate concentrations of CO2, the artery was transected and blood was immediately collected with a heparinized capillary tube and analyzed (ABL800; Radiometer, Copenhagen, Denmark). Cardiac ventricular weights, lung wet/dry weights, and brain weight. Right ventricular hypertrophy (RVH) was quantified by mea- suring the right ventricle (RV) to left ventricle and septum (LV � S) weight ratio (Fulton index), as previously described (25). Lung wet-to-dry weight ratio was determined by measuring left lung weight immediately upon harvest and again following baking at 65°C for 48 h. Brain wet weight included the cortex and cerebellum only after removal of the brainstem and spinal cord. Two-dimensional echocardiography-derived measurements of pul- monary hemodynamics. Pulmonary hemodynamics were evaluated noninvasively using two-dimensional echocardiography and Doppler ultrasound (Vivid 7 cardiac ultrasound system and I13L linear probe; GE Medical Systems, Milwaukee, WI), as previously described in detail (31, 47). Animals were lightly anesthetized with ketamine/ xylazine and spontaneously breathing at the time of the study. For estimation of PVR, a short axis view at the level of the aortic valve was obtained and the pulmonary artery was identified by color flow Doppler. The pulmonary arterial acceleration time (PAAT) was mea- sured as the time from the onset of systolic flow to peak pulmonary outflow velocity and the RV ejection time (RVET) as the time from onset to completion of systolic pulmonary flow. Pulmonary vascular resistance was estimated using the formula: RVET/PAAT. For esti- mation of RV stroke volume, the PA diameter was measured by color flow Doppler at the hinge point of the pulmonary valve leaflets. From the same Doppler interrogation of the pulmonary artery used to measure PAAT and RVET, RV output was calculated using the formula: (PA diameter/2)2 � 3.14 � PA velocity time integral � heart rate (beats/min). The PA velocity time integral was measured by tracing the leading edge of the velocity time graph from the onset to completion of systolic pulmonary flow. RV output (ml/min) was corrected for body weight to derive a RV performance index (RVI; ml·min�1·kg�1). Left ventricular (LV) shortening fraction was mea- sured from the parasternal short axis view according to the formula: [(LV end-diastolic diameter � LV systolic diameter)/LV end-dia- stolic diameter] � 100. Histological studies. Lungs from four animals from each group (2 from each of 2 separate litters) were air inflated and perfusion fixed at a constant pressure, embedded in paraffin, sectioned, immunostained for CD68 (to identify macrophages) and myeloperoxidase (MPO; to identify neutrophils), and stained with hematoxylin and eosin, or for elastin, as previously described (31, 45, 57). Paraffin-embedded cortical brain sections (5 �m) were labeled for apoptotic nuclei using a commercially available fluorescein TUNEL enzymatic assay kit (Roche). L76 CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. Morphometric analyses. For all analyses, measurements were car- ried out on four left lung sections per animal and four animals (representing 2 litters) per treatment group by observers blinded to group identity. For assessment of percentage arterial medial wall thickness (%MWT), pulmonary arteries were identified by the pres- ence of both inner and outer elastic lamina using Hart’s elastin stain, as previously described in detail (31). Analyses of tissue macrophage (CD68-positive) and neutrophil (MPO-positive) cell numbers and distal airway structure, including mean linear intercept (MLI; using hematoxylin and eosin-stained sections), tissue fraction (hematoxylin and eosin), secondary crest numbers (identified by positive staining for elastin at their tips), and counts of peripheral arteries (identified as vessels of external diameter between 20 and 65 �m with both internal and external elastic laminae visible) were conducted as previously described in detail (44, 45, 57) from 10 random nonoverlapping fields captured from each section. Staining of frozen sections. Lungs were inflated and snap frozen in optimum cutting temperature compound, as previously described (22) and then cut by cryostat in to 10-�m sections, which were mounted and stored at �80°C until analysis. Slides were fixed in ice-cold acetone and incubated with Isolectin B4 (diluted 1:100), according to the manufacturer’s instructions, known to label “stimulated” or clas- sically activated macrophages by binding to cell membrane glycocon- jugates bearing terminal �-D-galactose (42). For colabeling studies, anti-CD68 and -TNF-� were applied (both diluted 1:50, 1 h at room temperature), followed by appropriate secondary antibodies (each diluted to 1:300 with blocking solution, at room temperature in the dark for 30 min), before aqueous mounting with DAPI. Images were digitally captured using an epifluorescent microscope with appropriate filter sets. Identical images acquired with different filter sets were merged using Image-Pro Plus software (version 7.0, Media Cybernetics, Bethesda, MD). Quantitative PCR. RNA was extracted (Absolutely RNA Miniprep kit; Agilent/Stratagene, La Jolla, CA) and reverse transcribed (Affini- tyScript; Agilent) from lung tissue samples stored in RNALater (Applied Biosystems, Streetsville, ON, Canada). Quantitative (q)PCR was performed on a Stratagene MX3000P qPCR system using SYBR Green qPCR Master Mix (SA Biosciences, Frederick, MD). Cycling conditions were 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. A standard curve for each primer set was run, using a rat lung cDNA standard (Agilent), to ensure that reaction efficiency was equivalent to primers for housekeeping genes. Primer sequences are listed in Table 1. A dissociation curve was run for each set of samples to exclude nonspecific product formation and reaction contamination. Samples were run in duplicate, and expression of the gene of interest, where reported, was normalized to �-actin and GAPDH. Fold or fraction change in expression relative to control samples was calculated by the 2� Ct method using Stratagene MxPro software (v 4.01). Western blot analyses. Lung tissues from four animals per group (2 from each of 2 separate litters) were lysed in RIPA buffer containing protease inhibitors, fractionated by SDS-PAGE, transferred to poly- Table 1. Rat primer sequences for quantitative PCR Gene (RefSeq Accession No.) Forward 5=-3= Reverse 5=-3= �-Actin (NM_031144) CTGGGTATGGAATCCTGTGG TAGAGCCACCAATCCACACA CCL2 (MCP-1) (NM_031530) CTGTAGCATCCACGTGCTGT TGAGGTGGTTGTGGAAAAGA CCL3 (MIP-1�) (NM_013025) CCACCGCTGCCCTTGCTGTT CACCCGGCTGGGAGCAAAGG CCL4 (MIP-1�) (NM_053585) CTCTCTCCTCCTGCTTGTGG CACAGATTTGCCTGCCTTTT CCL5 (RANTES) (NM_031116) CTGCTGCTTTGCCTACCTCT CGAGTGACAAAGACGACTGC CCL7 (MCP-3) (NM_001007612) AACCAGATGGGACCAATTCA CACAGACTTCCATGCCCTTT CCR1 (NM_020542) ACCTGTTCAACCTGGCTGTC AGGGAAAACACTGCATGGAC CCR2 (NM_021866) CTGCCCCTACTTGTCATGGT GGCCTGGTCTAAGTGCATGT CCR3 (NM_053958) CAGCAGAGCATACACCTGGA CGCCAGGAAGGAATGAAATA CCR5 (NM_053960) AAAGTCTGGCAATGGTGAGC CTCCCAGTAAACCTCCCACA CXCL1 (CINC-1) (NM_030845) AGACAGTGGCAGGGATTCAC GGGGACACCCTTTAGCATCT CXCL2 (MIP-2�) (NM_053647) ACCAACCATCAGGGTACAGG GGCTTCAGGGTTGAGACAAA CXCR1 (NM_019310) GCTATGAGGTCCTGGGTGAA GAGTGTCCGAGAGCAGAACC CXCR2 (NM_017183) GATTCTTGGCTTCCTCCACA GGAGGTGTTCGCTGAAGAAG GAPDH (NM_017183) CCATGTTTGTGATGGGTGTG GGCATGGACTGTGGTCATGA Interleukin-1� (NM_022194) TCGGGAGGAGACGACTCTAA GAAAGCTGCGGATGTGAAGT Interleukin-1� (NM_017008) CTGTGACTCGTGGGATGATG GGGATTTTGTCGTTGCTTGT Interleukin-1 Receptor Antagonist (NM-017019) GAAAAGACCCTGCAAGATGC GATGCCCAAGAACACATTCC Interleukin-6 (NM_012589) GCCAGAGTCATTCAGAGCAA GGTTTGCCGAGTAGACCTCA Tumor necrosis factor-� (NM_012675) CAGCAGATGGGCTGTACCTT CTGGAAGACTCCTCCCAGGT Tumor necrosis factor receptor-1 (NM_013091) GTCAAAGAGGTGGAGGGTGA TTACAGGTGGCACGAAGTTG Tumor necrosis factor receptor-2 (NM_130426) GTCATCCCCAAGCAAGAGTC CATCCTTTGGAGACCCTGAA Table 2. Body weight, lung weight and arterial blood gas values on day 14 Parameter/Group Vehicle � Normocapnia Bleomycin � Normocapnia Vehicle � 5% CO2 Bleomycin � 5% CO2 Vehicle � 7% CO2 Bleomycin � 7% CO2 BW, g 28.7 1.2 29.5 1.8 31.3 2.3 28.5 1.4 30.0 1.9 27.6 1.6 LW, mg 517 39 360 24* 490 41 375 31* 534 33 318 37* LW/BW � 103 18.3 0.3 12.5 0.4* 16.0 0.03 12.9 0.02* 17.8 0.04 12.1 0.05* pH 7.33 0.03 7.30 0.06 7.30 0.03 7.32 0.01 7.20 0.02† 7.20 0.04† PaCO2, mmHg 49.0 4.1 55.5 9.1 62.2 7.1 72.5 4.2† 80.2 4.8† 90.5 8.0† PaO2, mmHg 69.9 6.5 56.8 13.4 128.9 9.4† 103.1 5.3† 159.1 46† 131.2 15.8† HCO3 �, mmol/l 25.7 0.9 24.1 1.8 29.7 1.1† 36.1 1.7‡ 29.7 1.2† 29.9 3.1† Values represent means SD; n � 8 –20 animals per group for weight data and 5– 8 animals per group for blood gas data. LW, lung weight; BW, body weight. *P � 0.01, by ANOVA, compared with respective vehicle-treated group. †P � 0.05, by ANOVA, compared with normocapnia-exposed groups. ‡P � 0.01, by ANOVA, compared with all other groups. L77CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. vinylidene difluoride membranes, and blotted, and band densities were measured as previously described (23). Differences in protein loading were compensated for by reblotting for GAPDH, the expres- sion of which was found, in preliminary studies, to be unaffected by chronic exposure to bleomycin or to CO2. Dilutions of primary antisera were 1:200 for TNF-� and 1:5,000 for GAPDH. Protein bands were identified using enhanced chemiluminescent substrate (Immobilon; Millipore), and images were digitally captured using a MicroChemi chemiluminescent image analysis system (DNR Bio- imaging Systems, Jerusalem, Israel). Bands were quantified by digital densitometry of nonsaturated images with background density re- moved (ImageJ, NIH, Bethesda, MD). Data presentation and analysis. All values are expressed as means SE. Statistical significance (P � 0.05) was determined by one-way ANOVA followed by pair-wise multiple comparisons using the Tukey test (SigmaStat; Systat Software, San Jose, CA). Fig. 1. Exposure to 7% CO2 prevents bleomycin- induced pulmonary hypertension. Pups were treated from postnatal days 1–14 with vehicle (open bars) or bleomycin (1 mg·kg�1·day�1; closed bars) while receiving concurrent exposure to normocapnia (� 0.5%) or 5% or 7% CO2. All values represent means SE. A: pulmonary vascular resistance (PVR; left), as estimated by the right ventricular ejection time (RVET)/pul- monary arterial acceleration time (PAAT) ratio and right ventricular index (right), as a marker of right-ventricular systolic function (n � 6 – 8 ani- mals/group). B: right ventricle (RV)/left ventricle � septum (LV � S) dry weight ratios as a marker of RV hypertrophy (n � 10 –12 animals/group) and percent arterial medial wall thickness (n � 4 animals/group) as a marker of pulmonary arterial remodeling. *P � 0.01, by ANOVA, compared with bleomycin-treated normocapnia-exposed group. #P � 0.01, by ANOVA, compared with vehicle-treated groups. C: representative photo- micrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall; bar length � 25 �m) demonstrat- ing medial wall thickening in bleomycin-exposed animals (bleomycin), which was largely pre- vented by concurrent exposure to 7% CO2 (bleo- mycin � 7% CO2). L78 CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. Fig. 2. Exposure to CO2 does not improve abnormal distal airway morphology. Pups were treated from postnatal days 1–14 with vehicle (open bars) or bleomycin (1 mg·kg�1·day�1; closed bars) while receiving concurrent exposure to normocapnia (� 0.5%) or 5% or 7% CO2. Representative low-power photomicrographs of hematoxylin and eosin-stained sections (A) demonstrating marked distal airway simplification and septal thinning in bleomycin-treated animals and elastin-stained sections (B) demonstrating a marked decrease in numbers of small peripheral arteries (outlined by dark brown stain for elastin) in bleomycin-treated animals, both of which were unaffected by exposure to CO2. Bar lengths � 200 �m. C: morphometric analyses of mean linear intercept (Lm), tissue fraction, and secondary crests or peripheral arteries per field, corrected for tissue fraction. Values represent means SE for n � 4 animals/group. *P � 0.01, by one-way ANOVA, compared with respective vehicle-treated groups. #P � 0.05, by one-way ANOVA, compared with respective vehicle-treated group. L79CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. L80 CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. RESULTS Body weights, lung weights, and arterial blood gas measurements. Values are shown in Table 2. Neither treatment with bleomycin nor exposure to either level of CO2 for 14 days had any significant effect on body weight, relative to vehicle-treated or normocapnia-exposed controls. In contrast, lung weights and lung weight-to-body weight ratios were significantly decreased in bleomycin-treated animals, whether or not they were ex- posed to CO2. Treatment with bleomycin and/or exposure to CO2 had no significant effects on lung wet/dry weight ratios (P � 0.5; data not shown). Exposure of bleomycin-treated animals to 5% CO2 increased PaCO2 by � 15–20 mmHg and exposure to 7% CO2 by � 35 mmHg above levels measured in normocapnia-exposed controls. Only exposure to 7% CO2 caused significant persistent acidosis at 14 days due to partial metabolic correction, as reflected in lower pH and higher HCO3 �HCO3 levels than normocapnic controls, whereas ani- mals exposed to 5% CO2 had evidence of complete metabolic correction by day 14. Interestingly, and as previously reported in hypoxia-exposed animals (31), exposure to either level of CO2 significantly increased PaO2 in both vehicle- and bleomy- cin-treated animals. Effects of CO2 on abnormal pulmonary hemodynamics. To distinguish acute from chronic effects of hypercapnia, prelim- inary measurements were first obtained while animals contin- ued to breathe CO2 and were repeated following a 15-min period of recovery in room air. PVR index was found to be unchanged (P � 0.5 by ANOVA; data not shown) by removal from CO2; therefore, all reported data were obtained while animals were breathing room air. As previously reported (47), treatment with bleomycin for 14 days led to a significantly increased the PVR index (Fig. 1A, left) and decreased RVI (Fig. 1A, right), relative to vehicle-treated controls. Bleomy- cin-induced increase in PVR index and decrease in RVI were normalized by exposure to 7% CO2 (Fig. 1A), whereas expo- sure to 5% CO2 had no significant effect (P � 0.05). Neither treatment with bleomycin, nor exposure to CO2, caused any significant change in LV fractional shortening (P � 0.5 by ANOVA; data not shown). Effects of CO2 on structural changes of pulmonary hypertension. As previously reported (47), treatment with bleomycin for 14 days led to significant RVH (Fig. 1B) and increased %MWT (Fig. 2B) in pulmonary resistance arteries. Exposure to 7% CO2 significantly (P � 0.05) attenuated RVH (Fig. 2A) and %MWT in bleomycin-treated animals (Fig. 1B). Exposure to 5% CO2 caused no change in RVH (P � 0.05) and a smaller, but significant (P � 0.05) decrease in %MWT (Fig. 1B). Relative differences in medial wall thickness between groups are illustrated by high-power images of elastin-stained pulmo- nary arteries (Fig. 1C). Changes in distal airway morphology secondary to bleomycin. As shown in representative low-power hematoxylin and eosin- stained sections (Fig. 2A) and by low-power elastin-stained sections (Fig. 2B), the lung structure of bleomycin-treated animals was characterized by septal thinning, arrested alveo- larization (manifesting as “emphysematous” distal airspaces), and vascular rarefaction as quantified by increased MLI and by decreased tissue fraction, secondary crest counts and peripheral artery counts (Fig. 2C). Neither level of CO2 had any effects on these changes in either vehicle- or bleomycin-treated animals (Fig. 2C). Effects of CO2 on tissue inflammatory cells. As illustrated by CD68 immunostaining (Fig. 3A) and by isolectin B4 binding (Fig. 3B), large classically activated macrophages were present in greatly increased numbers in the lungs of animals treated with bleomycin for 14 days (Fig. 3C). Concurrent exposure to 7% CO2 significantly (P � 0.05) attenuated macrophage num- bers in the bleomycin-treated lung, whereas 5% CO2 had no significant effect (P � 0.05; Fig. 3C). As also shown in Fig. 3C, treatment with bleomycin led to significantly (P � 0.05) increased numbers of MPO-positive neutrophils, which were unaffected by exposure to either level of CO2. Changes in expression of proinflammatory cytokines and chemokines (listed in Table 1) were screened by qPCR on day 14. Genes for which significant (P � 0.05) changes were found are listed in Table 3. The neutrophil chemokine CXCL1 (also known as CINC-1) and its receptor, CXCR1, were greatly increased by bleomycin but were unaffected by exposure to 7% CO2 (Table 3). TNF-�, a proinflammatory cytokine that may recruit mac- rophages to the lung (18, 46) or be tissue macrophage derived (59), was increased in bleomycin-exposed lungs and was normalized by concurrent exposure to 7% CO2 (Table 3). Similar to findings in qPCR, lung TNF-� protein content was significantly (P � 0.05) increased by treatment with bleomycin and completely normalized by exposure to 7%, but not by 5%, CO2 (Fig. 3D). Double labeling for immunoreactive CD68 and TNF-� in lungs of bleomycin-treated animals revealed that TNF-� colocalized with macrophages (Fig. 3E), implicating Fig. 3. Exposure to 7% CO2 normalizes lung tissue macrophage number and tumor necrosis factor (TNF)-� content. Pups were treated from postnatal days 1–14 with vehicle (open bars) or bleomycin (1 mg·kg�1·day�1; closed bars) while receiving concurrent exposure to normocapnia (� 0.5%) or 5% or 7% CO2. Representative medium-power photomicrographs of CD68-immunostaining (large dark brown cells highlighted by arrows; A) and isolectin B4-labeled frozen sections (green fluorescent-labeled cells highlighted by arrows; B) demonstrating increased numbers of tissue macrophages in bleomycin-exposed animals (bleomycin), which was largely prevented by concurrent exposure to 7% CO2 (bleomycin � 7% CO2). Bar lengths � 100 �m. C: tissue macrophage and neutrophil counts per field normalized to tissue fraction. Values represent means SE for n � 4 animals/group. D: Western blot analyses of lung TNF-� content normalized to GAPDH. Representative immunoblots are shown with noncontiguous gel lanes demarcated by black lines. Values represent means SE for n � 4 samples/group. *P � 0.01, by one-way ANOVA, compared with vehicle-treated groups. #P � 0.01, by one-way ANOVA, compared with bleomycin-treated normocapnia-exposed group. E: representative photomicrographs of fluorescent immunolabeling for CD68 and TNF-�, demonstrating colocalization with tissue macrophages (bar length � 50 �m). Table 3. Changes in mRNA expression secondary to bleomycin Gene Bleomycin Vehicle � 7% CO2 Bleomycin � 7% CO2 CXCL1 9.7 2.4* 2.7 0.5* 13.3 1.7* CXCR1 2.3 0.2* 2.4 0.3* 2.1 0.2 Tumor necrosis factor-� 2.3 0.3* 1.3 0.1 1.0 0.2 Values are means SE of 4 samples per group relative to control (vehicle-treated normocapnia-exposed) group, which was assigned a value of 1. *P � 0.05, by ANOVA, compared with control. L81CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. this cell type as the major source of TNF-� in the bleomycin- exposed lung. Effects of TNF-� inhibition on bleomycin-induced pulmo- nary hypertension, macrophage influx, and abnormal distal lung morphology. Bleomycin-induced increases in PVR index, RVH and %MWT in pulmonary resistance arteries were all normalized by treatment with etanercept (Figs. 4, A and B), In contrast, treatment with etanercept had no effect on bleomycin- induced macrophage influx (Fig. 4C), indicating that upregu- lated TNF-� signaling does not contribute to increased num- bers of activated tissue macrophages in the bleomycin-exposed lung. As shown in representative low-power hematoxylin and eosin-stained sections (Fig. 5A), abnormal lung morphology in bleomycin-treated animals was unaffected by treatment with etanercept, as evidenced by MLI, tissue fraction, secondary crest counts, and peripheral artery counts that did not differ (P � 0.05) from animals treated with bleomycin and vehicle (Fig. 5B). Effects of bleomycin and/or 7% CO2 on brain, liver, and kidney weights and on apoptosis in brain cortex. As shown in Table 4, exposure to bleomycin led to no change in brain or liver weight, while causing a small, but statistically significant (P � 0.05), decrease in combined kidney weight. Exposure to 7% CO2 caused no change in brain, liver, or kidney weight in either vehicle- or bleomycin-treated animals (Table 4). In support of a lack of effect of a 14-day exposure to 7% CO2 on brain growth, no apparent increase in TUNEL-labeled nuclei was observed compared with normocapnic controls (Fig. 6). DISCUSSION The effects of chronic CO2 exposure on the neonatal lung and pulmonary vasculature have received limited attention in experimental models despite the widespread use of permissive hypercapnia in the newborn intensive care unit in the hope of limiting lung injury. We have previously shown that chronic inhalation of CO2 prevented PHT in chronic hypoxia-exposed neonatal rats (31), a model in which pulmonary inflammation is not apparent (47). Given the major putative role for lung inflammation in the pathogenesis of BPD (61, 62, 66) and evidence for anti-inflammatory effects of inhaled CO2 (37, 40, 45, 72), we were interested in examining the effects of thera- peutic hypercapnia on an alternative rat model in which in- flammation is a prominent early feature. The concentrations of CO2 examined in bleomycin-exposed animals were intended to reproduce what are generally considered, in the clinical setting, to represent moderate (5% CO2; mean elevation of PaCO2 15–20 mmHg) and severe (7% CO2; mean elevation of PaCO2 35 mmHg) levels of hypercapnia. Our findings were that 7% CO2 attenuated the hemodynamic and structural indexes of PHT secondary to bleomycin, whereas 5% CO2 had lesser or no effects on these parameters. These changes were associated with attenuated tissue macrophage influx and TNF-� expres- sion and content in the bleomycin-exposed lung. Our findings, which suggest a major role for tissue macrophages in the pathogenesis of chronic neonatal PHT, are in agreement with previous work from our group using a neonatal rat model of Fig. 4. TNF-� inhibition prevents bleomycin-induced pulmonary hypertension without affecting tissue macrophage number. Pups were treated from postnatal days 1–14 with 0.9% saline vehicle (Control; open bars), bleomycin alone (1 mg·kg�1·day�1; closed bars) or bleomycin and etanercept (0.4 mg/kg ip alternate days; grey bars). All values represent means SE. A, left: PVR, as estimated by the RVET/PAAT ratio (n � 6 animals/group). A, middle: RV/LV � S dry weight ratios as a marker of right-ventricular hypertrophy (n � 6 animals/group). A, right: percentage arterial medial wall thickness as a marker of pulmonary arterial remodeling (n � 3– 4 animals/group). *P � 0.01, by ANOVA, compared with all other groups. B: representative photomicrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall; bar length � 25 �m) demonstrating medial wall thickening in bleomycin-exposed animals (bleomycin), which was prevented by treatment with etanercept (bleomycin � etanercept). C: tissue macrophage counts per field normalized to tissue fraction. Values represent means SE for n � 4 samples or animals/group. *P � 0.01, by ANOVA, compared with other groups. L82 CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. BPD secondary to hyperoxia (24), in which chronic inhalation of CO2 was also shown to limit macrophage influx and to prevent the structural changes of PHT (45). Examination of arterial acid-base status revealed that a 14-day exposure to 7% CO2 caused significant acidosis, whereas 5% CO2 did not, due in part to differing degrees of metabolic correction. A possible implication of these observa- tions is that benefits of CO2 were related to acidosis, rather than to hypercapnia, in keeping with recent findings in adult animals by Christou et al. (9). Furthermore, in short-term lung injury models, normalizing pH by buffering has been shown to worsen injury (36) where inhaled CO2 has otherwise been protective (38, 64, 72). It was not possible to separate the effects of acidosis from hypercapnia by buffering in this chronic model, leaving the relative contributions of hypercap- nia and acidosis an open question. Interestingly, both levels of hypercapnia increased PaO2 levels, in keeping with previously reported observations made by our group (31) and others (19, 35). Possible mechanisms for this effect include increased cardiac output, improved ventilation-perfusion matching, and reduced tissue metabolic activity and O2 consumption (28). Macrophages are differentiated mononuclear phagocytes that may reside in tissues for several months or be recruited to injured tissue from circulating monocytes, which then change phenotype. Although macrophages are essential for tissue re- modeling and wound healing, when activated and present in Fig. 5. TNF-� inhibition does not improve abnormal distal airway morphology. Pups were treated from postnatal days 1–14 with 0.9% saline vehicle (Control; open bars), bleomycin alone (1 mg·kg�1·day�1; closed bars), or bleomycin and etanercept (0.4 mg/kg ip alternate days; grey bars). A: representative low-power photomicrographs of hematoxylin and eosin-stained sections demonstrating marked distal airway simplification and septal thinning in bleomycin-treated animals, which was unaffected by treatment with etanercept. Bar length � 200 �m. B: morphometric analyses of mean linear intercept (Lm), tissue fraction, and secondary crests or peripheral arteries per field, corrected for tissue fraction. Values represent means SE for n � 4 animals/group. *P � 0.01, by one-way ANOVA, compared with other groups. #P � 0.05, by one-way ANOVA, compared with other groups. L83CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. exaggerated numbers, they may produce large quantities of reactive oxygen (34) and nitrogen (21, 45) species, bioactive lipid peroxidation products (75), and cytokines (60). In human preterm infants with respiratory distress, macrophage numbers in bronchoalveolar lavage fluid increase early in the second week of life (48), remain elevated in infants who later develop clinical and radiological features of BPD, and decline in those who do not (10). Pulmonary macrophages are also central to the pathogenesis of PHT induced by monocrotaline injection in rats (50, 70) and in anti-platelet serum-induced PHT in sheep (51). We remain uncertain whether increased tissue macro- phages in the present model result from recruitment of circu- lating monocytes to the lung or whether they result from expansion of resident macrophages. However, our observation that expression of classical macrophage chemokines, including CCL2 (also known as monocyte chemoattractant protein-1), CCL3, and CCL4, were not increased in bleomycin-exposed animals suggests the latter possibility. Of a number of cytokines screened to determine whether a causative role may exist, TNF-� was the only candidate found to be upregulated by bleomycin at the time point (day 14) examined. That both macrophage numbers and TNF-� expres- sion/content were found to be attenuated by concurrent expo- sure to 7% CO2 suggested a causative role in PHT, with a lesser effect of 5% CO2 and colocalization indicating that macrophages were the major source of TNF-�. Lang et al. (39) have described inhibition of TNF-� secretion by CO2 in pulmonary macrophages activated by LPS in vitro. Exogenous administration of TNF-� is also known to increase pulmonary vascular reactivity in isolated rat lungs (68), and its overex- pression has been described to cause emphysematous lung structure and PHT in mice (16, 17), similar to bleomycin- induced lung injury. Furthermore, Sutendra et al. (71) recently reported that sustained inhibition of TNF-� signaling both prevented and reversed chronic PHT in monocrotaline-exposed rats. Similarly, a critical role for increased TNF-� signaling in PHT was confirmed in the present model using a soluble TNF-2 receptor/IgG fusion protein, etanercept, which prevents binding of the endogenous ligand to its receptors. Our group has used this approach successfully in the past to explore the roles of various growth factors in lung development and injury (8, 26). Our data do not provide insight into the downstream mediators of upregulated TNF-� signaling that led to PHT, but candidates implicated in other models include upregulation of endothelin-1 (43) and activation of Rho-kinase (46). Further, the upstream mechanisms causing a decrease in tissue macro- phages by 7% CO2 were also not elucidated in the present study; however, our data suggest there is no relationship Table 4. Brain, liver, and kidney weights on day 14 Parameter/Group Vehicle � Normocapnia Bleomycin � Normocapnia Vehicle � 7% CO2 Bleomycin � 7% CO2 Brain weight, mg 1,116 39 1,080 43 1,110 21 1,083 27 Brain weight/body weight � 103 35.5 1.6 36.9 1.4 34.4 1.4 36.9 1.8 Liver weight, mg 956 68 960 83 1033 76 912 24 Liver weight/body weight � 103 30.4 1.7 32.8 1.5 31.4 2.0 30.7 2.1 Kidney weights, mg 316 22 290 12 353 30 260 18* Kidney weights/body weight � 103 10.1 0.6 9.9 0.3 10.7 0.8 8.9 0.8* Values represent means SD; n � 5– 6 animals per group. *P � 0.01, by ANOVA, compared with vehicle-treated 7% CO2-exposed group. Fig. 6. Neither bleomycin nor exposure to 7% CO2 increases apoptosis in brain cortex. Rep- resentative photomicrographs of terminal de- oxyuridine triphosphate nick-end-labeled sec- tions of brain cortex demonstrating no differ- ences in numbers of apoptotic nuclei between groups. Inset (top left): DNAse I-treated pos- itive control section. Bar length � 100 �m. L84 CO2 PREVENTS BLEOMYCIN-INDUCED PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2012 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. between inhibition of TNF-� and decrease in recruitment or activation of macrophages in the bleomycin-exposed lung. In contrast with our previous findings in 60% O2-exposed neonatal rats (45), therapeutic hypercapnia did not improve abnormal lung structure in the present model. Tourneux et al. (74) reported in bleomycin-exposed neonatal rats that inhaled nitric oxide prevented PHT and partially improved vascular rarefaction and lung structure, suggesting a major role for decreased endogenous NO. Our observation that PVR was normalized by 7% CO2, while arterial rarefaction was not, suggests that vascular hypoplasia was not the major factor accounting for raised PVR. These findings are in keeping with our previous work (47) that demonstrated that acute treatment with a potent vasodilator (Rho-kinase inhibitor) also normal- ized PVR, thus pointing to sustained vasoconstriction as the major contributor to raised PVR in bleomycin-exposed ani- mals, as described in other chronic PHT models (55). We observed that neutrophils, which were greatly increased in number in the lung tissue of bleomycin-exposed animals, were unaffected by exposure to CO2. These findings suggest that neutrophils alone are unlikely to be contributory to chronic PHT and that macrophages may not recruit neutrophils to the bleomycin-exposed lung. Indeed, work by others (73) suggests that neutrophils do not play a major role in bleomycin-induced lung injury, as least in adult animals, but our current observa- tions leave open the question of their involvement in arrested lung development in the bleomycin-exposed neonatal rat. Al- though there are many other factors in the bleomycin model that could contribute to arrested development, neutrophils have been suggested to play a part by producing proteases that disrupt elastin deposition, which is essential to secondary septation (5). We examined the effects of CO2 on inflammation and lung structure in control (vehicle-treated) animals and observed small, but nonstatistically significant, trends toward increased inflammatory cells, decreased MLI, and decreased secondary crest numbers with 7% CO2. In neonatal mice, chronic expo- sure to 8% CO2 from birth has been reported to induce changes in lung morphology consistent with accelerated maturation, including thinning of alveolar walls and increased secondary septation (13). Taken together, these observations sound a note of caution that the risks vs. benefits of chronic hypercapnia for long-term lung development, function, and susceptibility to lung injury remain unclear and require further study. In addi- tion, despite our present findings suggesting a lack of effect of 7% CO2 on brain growth, there remains the potential for harmful effects of hypercapnia, or hypercapnic acidosis, on the immature brain (28, 30). There are a number of limitations to this study. First, a major feature of bleomycin-induced lung injury is collagen deposi- tion (32), which is not a feature of modern BPD (11). Our use of this model for the present studies was based on the striking degree of arrested lung growth and development, which is disproportionate to effects on other organs despite systemic administration, and the fact that macrophage influx, known to be involved in the pathogenesis of pulmonary hypertension, is a prominent early feature. Second, our estimation of arterial blood gases in anesthetized animals introduced the likelihood of respiratory suppression, which may have confounded data on PaCO2, although we anticipate that all groups would have been affected equally. Our purpose in providing these data was to estimate the degree of change in PaCO2 and acid-base status secondary to provide some clinical context. In conclusion, our current findings indicate that therapeutic hypercapnia has a dose-dependent preventive effect on chronic bleomycin-induced PHT relating to inhibitory effects on mac- rophage influx and subsequent attenuation of TNF-�. We propose that bleomycin-induced lung injury may represent a potentially useful new model for mechanistic studies relating to arrested development and chronic PHT in the neonatal lung. GRANTS This work was supported by operating funding from the Canadian Institutes of Health Research (CIHR; MOP-93956 to R. P. Jankov and MOP-82703 to A. K. Tanswell) and by infrastructure funding from the Canada Foundation for Innovation (to R. P. Jankov). R. P. Jankov is a CIHR New Investigator. DISCLOSURES No conflicts of interest, financial or otherwise are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: A.C.P.S., A.K.T., and R.P.J. conception and design of research; A.C.P.S., C.K., J.I., A.H.L., A.M., and A.J. performed experiments; A.C.P.S., C.K., J.I., A.H.L., A.J., P.J.M., and R.P.J. analyzed data; A.C.P.S., A.J., P.J.M., A.K.T., and R.P.J. interpreted results of experiments; A.C.P.S., A.M., P.J.M., and R.P.J. prepared figures; A.C.P.S. drafted manuscript; A.C.P.S., C.K., J.I., A.H.L., A.M., A.J., P.J.M., A.K.T., and R.P.J. edited and revised manuscript; A.C.P.S., C.K., J.I., A.H.L., A.M., A.J., P.J.M., A.K.T., and R.P.J. approved final version of manuscript. REFERENCES 1. Abman SH, Groothius JR. Pathophysiology and treatment of broncho- pulmonary dysplasia. Current issues. Pediatr Clin North Am 41: 277–315., 1994. 2. Abolhassani M, Guais A, Chaumet-Riffaud P, Sasco AJ, Schwartz L. Carbon dioxide inhalation causes pulmonary inflammation. Am J Physiol Lung Cell Mol Physiol 296: L657–L665, 2009. 3. 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