Accepted Article
Received Date : 15-Aug-2014 Revised Date : 23-Oct-2014 Accepted Date : 26-Oct-2014 Article type
: Regular Paper
Alveolar-capillary Adaptation to Chronic Hypoxia in the Fatty Lung
Cuneyt Yilmaz1, Priya Ravikumar1, Dipendra Gyawali1, Roshni Iyer1, Roger H. Unger2, and Connie C.W. Hsia1 1
Pulmonary and Critical Care Medicine
2
Touchstone Diabetes Center
Department of Internal Medicine
Corresponding author : [email protected]
University of Texas Southwestern Medical Center, Dallas, TX 75390-9034
Running Title: Adaptation to hypoxia in obese diabetic rats
Address Correspondence to Connie C.W. Hsia, MD. Dept. of Internal Medicine Pulmonary and Critical Care Medicine University of Texas Southwestern Medical Center
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/apha.12419 This article is protected by copyright. All rights reserved.
Accepted Article
5323 Harry Hines Blvd., Dallas, TX 75390-9034 TEL: 214-648-3426; FAX 214-648-8027
Abstract Aim: Obese diabetic (ZDF fa/fa) rats with genetic leptin resistance suffer chronic lipotoxicity associated with age-related lung restriction and abnormal alveolar ultrastructure. We hypothesized that these abnormalities impair adaptation to ambient hypoxia. Methods: Male fa/fa and lean (+/+) ZDF rats (4-months old) were exposed to 21% or 13% O2 for 3 weeks. Lung function was measured under anesthesia. Lung tissue was assayed for DNA damage and ultrastructure measured by morphometry. Results: In normoxia, lung volume, compliance, and diffusing capacity were lower while blood flow was higher in fa/fa than +/+ rats. In hypoxia, fa/fa animals lost weight, circulating hematocrit rose higher, and lung volume failed to increase compared to +/+. In fa/fa, the hypoxia-induced increase in postmortem lung volume was attenuated (19%) vs. +/+ (39%). Alveolar ducts were 35% smaller in normoxia but enlarged two-fold more in hypoxia compared to +/+. Hypoxia induced broad increases (90-100%) in the volumes and surface areas of alveolar septal components in +/+ lungs; these increases were moderately attenuated in fa/fa lungs (58-75%), especially that of type-II epithelium volume (16% vs. 61% in +/+). In fa/fa compared to +/+ lungs, oxidative DNA damage was greater and hypoxia induced efflux of alveolar macrophages. Harmonic mean thickness of the diffusion barrier was higher, indicating higher structural resistance to gas transfer. Conclusion: Chronic lipotoxicity impaired hypoxia-induced compensatory lung expansion and growth with disproportionate effect on resident alveolar progenitor cells. The moderate structural impairment was offset by physiological adaptation primarily via a higher hematocrit.
Key words: High altitude adaptation, lung structure, lung function, metabolic syndrome, morphometry, obesity
Introduction Obesity is associated with chronic lipid infiltration of extra-adipocyte tissues leading to a proinflammatory state and end-organ damage (Unger and Zhou, 2001, Unger and Scherer, 2010). In the lung, obesity causes abnormal ventilatory control and restrictive mechanics (O'Donnell et al., 2000); the impairment in lung function is independent of physical activity or fitness (Leone et al., 2009, Lin et al., 2006, Steele et al., 2009, van Huisstede et al., 2013). Obesity often co-exists with type-2 diabetes mellitus; both reflect a state of tissue hypoxia (Pasarica et al., 2009, Girgis et al., 2012) that heightens susceptibility to tissue injury and may aggravate existing organ dysfunction (Watz et al., 2009). Because normal alveolar microvascular reserves are extensive, significant alveolar lipotoxicity
This article is protected by copyright. All rights reserved.
Accepted Article
remains subclinical under basal conditions and is under-recognized as a pathological entity. However, lipotoxicity diminishes alveolar-capillary reserves, which may interfere with adaptation to additional challenges such as ageing, concurrent lung disease, cardio-renal co-morbidity or hypoxic stress, thereby causing or exacerbating morbidity. For example, obesity increases the risk of acute mountain sickness in human subjects (Ri-Li et al., 2003, Wu et al., 2007), suggesting the failure to acclimatize to a hypoxic environment. On the other hand, hypoxia is a primitive and universal stimulus for the growth and remodeling of gas exchange organs. While extreme hypoxia suppresses metabolic function and causes cell death, postnatal exposure to moderate hypoxia (equivalent to 3,100-3,800m altitude) accelerates alveolar tissue and capillary growth and acinar remodeling in various mammals (Reinke et al., 2011, Lechner and Banchero, 1980, Burri and Weibel, 1971b, Johnson et al., 1985, Hsia et al., 2005) as a compensatory mechanism for preserving O2 uptake. The interactions between obesity and hypoxia on the structure and function of the distal lung have not been examined.
To characterize the effects of lipotoxicity on alveolar-capillary structure and function during hypoxia challenge, we utilized the Zucker diabetic fatty (ZDF) rat model (Yilmaz et al., 2010, Foster et al., 2010), which originated in outbred Zucker fatty (fa/fa) rats carrying the fa mutation, an amino acid substitution in the extracellular domain of the leptin receptor that renders the animal insensitive to leptin, leading to hyperphagia, diet-induced obesity, extra-adipocyte fat infiltration, systemic proinflammatory stress, end-organ damage and a shortened life span (Unger and Zhou, 2001, Unger and Scherer, 2010). The type-2 diabetic trait develops in an inbred substrain of obese fa/fa males [ZDF/Drt-fa] receiving at least 6% dietary fat; hyperglycemia develops by age ~12 weeks as insulin production and peripheral insulin sensitivity decline (Shimabukuro et al., 1997, Shimabukuro et al., 1998). The lungs of fa/fa animals show baseline restrictive changes (Yilmaz et al., 2010, Yilmaz et al., 2014) resembling that in clinical metabolic syndrome. The distal lung exhibits age-related thickening of alveolar septa and capillary basement membrane, cellular hyperplasia, elevation of triglyceride content, infiltration of lipid-laden macrophages, increased connective tissue elements, and altered surfactant protein profiles (Foster et al., 2010). Here we tested the hypothesis that these obesityassociated abnormalities impair alveolar adaptation during exposure to ambient hypoxia.
Materials and methods Animals and exposure: The Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center approved the protocol. Male ZDF diabetic fatty (fa/fa, n=7) and lean ZDF control (+/+, n=8) rats (4mo old), bred in the Unger laboratory, were fed rodent chow containing 6.5% fat (Formulab diet 5008, Purina, St. Louis, Missouri) ad libitum and had free access to water. The diabetic phenotype was confirmed by tail vein blood glucose level (557±50 and 102±13 mg.dL-1 in fa/fa and +/+, respectively). The animals were exposed to 13% ambient O 2 (hypoxia, equivalent to ~3,800m high altitude) in an environmental chamber (Biospherix, Lacona, NY) for 3 weeks. The chamber was opened for no more than 20min each day for animal feeding and maintenance. The buildup of CO2 and moisture was prevented using soda lime. Body weight was measured each week. Hematocrit was measured from tail vein blood. At the end of exposure, the animal was fasted 3 hr,
This article is protected by copyright. All rights reserved.
Accepted Article
then deeply anesthetized with an intraperitoneal injection of ketamine (100 mg/kg), xylazine (10 mg/kg) and acepromazine (2.0 mg/kg) to suppress spontaneous respiration. The trachea was cannulated via a midline neck incision, and the cannula tied securely with silk suture. Mechanical ventilation (CIV 101, Columbus Instruments, Columbus, OH) was maintained (tidal volume 10 mL/kg, respiratory rate 90 breaths per min). Heart rate and transcutaneous O2 saturation were monitored via a tail probe (Vet/Ox G2, West Yorkshire, UK). Cardiopulmonary function was measured followed by euthanasia and lung harvest (described below). Results were compared to age- and gendermatched control animals exposed to normoxia (21% O2, fa/fa n=18, +/+ n=12) (Table 1). All animal procedures were performed within a span of 6 months by the same personnel using the same methods. Postmortem tissue analysis was first completed in the normoxia groups (+/+ and fa/fa); these results (lung function and morphometry) have been published (Foster et al., 2010, Yilmaz et al., 2010). Due to personnel constraints and other laboratory priorities, data analysis of hypoxiaexposed groups (+/+ and fa/fa) was completed later and the results reported here.
Apparatus: As described previously (Yilmaz et al., 2010), the tracheal cannula was attached via a manifold to the ventilator, a 10-20 mL glass syringe, and 2 three-way computer-controlled solenoid valves (GH3315, Hans Rudolph, Kansas City, MO). Total apparatus dead-space was 0.5 mL. Airway pressure was recorded using a pressure transducer (Amplifier 1 Model 1100, Hans Rudolph, Kansas City, MO) calibrated against a manometer before each experiment. Data were acquired (100 Hz) using a laptop computer and LabView software (National Instruments, Austin, TX).
Static airway pressure–lung volume (PV) relationship: A glass syringe containing the desired volume of air was used to inflate the lungs from end-expiration. Each inflation was held for ~6 s before exhalation and resumption of mechanical ventilation for 10 to 15 s before initiating the next inflation step. Mean airway pressure during the 3rd second after reaching peak pressure was recorded. The maneuver was repeated at incremental inflation volumes (10 mL/kg steps) until airway pressure exceeded 30 cmH2O then repeated in stepwise volume decrements. Duplicate measurements at each volume were averaged.
Rebreathing measurements: A test gas mixture (0.3% CO, 0.5% Ne, 0.8% C2H2, 40 or 90% O2 in balance of N2) was drawn into a Mylar reservoir bag and the initial gas concentrations measured by a gas chromatograph (CP-4900 Micro GC, Varian Inc, CA) with 2 columns (M5AHIBF and PPUHI) and a thermal conductivity detector using 100% high purity helium as a carrier gas. A volume of the test gas equal to the inflation volume at 30cmH2O of airway pressure (11.8±1.1 and 15.3±2.3 mL in fa/fa and +/+ animals, respectively, mean±SD) was drawn into the glass syringe and attached to the manifold. The ventilator was stopped to allow exhalation and valve switching. Rebreathing was conducted by gently pumping the syringe at 60 strokes per minute to mix the test gas with resident lung gas for 5, 7 or 9 s in separate maneuvers in random order. The precise rebreathing duration was determined from the change in airway pressure signals with valve opening or closing. Final gas concentrations in the syringe were measured immediately. Duplicate measurements were made at each inspired O2 tension in random order. The interval between successive rebreathing maneuvers
This article is protected by copyright. All rights reserved.
Accepted Article
was at least 2 min. At the end, CO backpressure was measured by rebreathing 30 mL of 100% O2 for 60 s.
Euthanasia, open chest PV curves, and tissue harvest: Following the above procedures, the thoracic cavity was opened to expose the lungs and heart, and the PV measurements repeated as described above. Blood was drawn by cardiac puncture to measure hematocrit. The right lung was clamped at the hilum, and the heart stopped by an intracardiac overdose injection of Euthasol™. The left lung was perfused with oxygenated PBS, removed and snap frozen in liquid nitrogen. The clamp was removed and the right lung fixed via tracheal instillation of 2.5% buffered glutaraldehyde at 25cmH2O of hydrostatic pressure. Liver and kidney were removed as control organs.
Triglyceride content analysis: Lung and liver tissue (50–100mg) were homogenized in 2:1 chloroform-methanol, and lipids were extracted for 1 h at room temperature with intermittent vortexing. Volume of distilled water (2X) was vortexed into the homogenates and the mixture centrifuged (2,000g). The lipid extract was withdrawn, and 50–600μL was dried completely. The dried samples were mixed with 30μL of t-butanol, 20μL of 1:1 Triton X-100-methanol, and 1mL of a 4:1 mixture of Free Glycerol Reagent: Triglyceride Reagent (Sigma-Aldrich, St. Louis, MO), and the absorbance read (540nm). Triglyceride content was calculated from a glycerol standard curve (Sigma-Aldrich) and expressed in each animal as the mean milligrams triglyceride per gram of tissue in duplicate samples.
DNA damage: Lung 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was measured as a marker of oxidative DNA damage. DNA was extracted using DNAzol (Life Technologies, Grand Island, NY), precipitated in 100% ethanol, washed with 70% ethanol, suspended in 8mM NaOH, and the 8-OHdG concentration determined by ELISA (OxiSelect™ Oxidative DNA damage Cell BioLabs, San Diego, CA) compared with that of a standard curve.
Estimation of lung fluid: Lung tissue (~100mg) was weighed and transferred to a platinum ashing crucible and placed on a hot plate set at 100°C under a heat lamp for 2h. Then, the dry samplecontaining crucible was weighed to determine the sample dry weight and the dry-to-wet-weight ratio. The crucible was placed overnight in the ash oven at 600°C, removed and weighed to determine the ash weight. The ash was dissolved in 2 mL of HCl and the sodium content measured by flame photometry. Sodium-to-dry weight ratio serves as a surrogate marker for the relative amount of interstitial fluid (extracellular fluid sodium concentration 135-140 mM; intracellular sodium concentration 10-15 mM) (Ravikumar et al., 2014).
Lung volume estimation and sampling: Volume of the intact fixed right lung was measured by saline immersion (Yan et al., 2003). The lung was serially sectioned (3mm intervals) starting with a random
This article is protected by copyright. All rights reserved.
Accepted Article
orientation; the cut surfaces were imaged using a digital camera. Volume of the sectioned lung was estimated from the images using the Cavalieri principle (Yan et al., 2003); this tension-free volume was used in subsequent morphometric calculations. Lung slices were divided into roughly equal cranial and caudal regions. Using a systematic sampling scheme with a random start, 2 tissue blocks were sampled from each region (total of 4 blocks per lung), post-fixed with 1% osmium tetroxide in 0.1M cacodylate buffer, treated with 2% uranyl acetate, dehydrated through graded alcohol, and embedded in Spurr (Electron Microscopy Sciences, Hatfield, PA). Each block was sectioned (1μm) and stained with toluidine blue.
Morphometric analysis: A stratified analytical scheme was used (Foster et al., 2010, Hsia et al., 2010): low-power light microscopy (LM; 275x), high power LM (550x) and transmission electron microscopy (TEM; ~19,000x). One section per block was overlaid with a test grid at the appropriate magnification. From a random start, at least 20 non-overlapping microscopic fields per block (80 per lung) were systematically examined at 275x magnification. Using point counting, conducting structures larger than 20µm in diameter, i.e., bronchioles and blood vessels, were excluded from the reference space to estimate the volume density of fine parenchyma, alveolar ducts and sacs per unit lung volume. At 550x magnification, 15 non-overlapping microscopic fields per block (60 per lung) were systematically imaged to estimate the volume density of alveolar septa per unit lung volume. For TEM, 2 blocks per region (4 blocks per lung) were sectioned (70nm thickness), mounted on copper grids, and examined at ~19,000x (JEOL EXII). Thirty non-overlapping EM fields per grid were systematically sampled (total 120 per lung). The volume densities of epithelium, interstitium, and endothelium were estimated by point counting. Alveolar epithelial and capillary surface densities per unit septum volume were estimated by intersection counting. About 300 points/intersections were counted per grid with a coefficient of variation
Received Date : 15-Aug-2014 Revised Date : 23-Oct-2014 Accepted Date : 26-Oct-2014 Article type
: Regular Paper
Alveolar-capillary Adaptation to Chronic Hypoxia in the Fatty Lung
Cuneyt Yilmaz1, Priya Ravikumar1, Dipendra Gyawali1, Roshni Iyer1, Roger H. Unger2, and Connie C.W. Hsia1 1
Pulmonary and Critical Care Medicine
2
Touchstone Diabetes Center
Department of Internal Medicine
Corresponding author : [email protected]
University of Texas Southwestern Medical Center, Dallas, TX 75390-9034
Running Title: Adaptation to hypoxia in obese diabetic rats
Address Correspondence to Connie C.W. Hsia, MD. Dept. of Internal Medicine Pulmonary and Critical Care Medicine University of Texas Southwestern Medical Center
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/apha.12419 This article is protected by copyright. All rights reserved.
Accepted Article
5323 Harry Hines Blvd., Dallas, TX 75390-9034 TEL: 214-648-3426; FAX 214-648-8027
Abstract Aim: Obese diabetic (ZDF fa/fa) rats with genetic leptin resistance suffer chronic lipotoxicity associated with age-related lung restriction and abnormal alveolar ultrastructure. We hypothesized that these abnormalities impair adaptation to ambient hypoxia. Methods: Male fa/fa and lean (+/+) ZDF rats (4-months old) were exposed to 21% or 13% O2 for 3 weeks. Lung function was measured under anesthesia. Lung tissue was assayed for DNA damage and ultrastructure measured by morphometry. Results: In normoxia, lung volume, compliance, and diffusing capacity were lower while blood flow was higher in fa/fa than +/+ rats. In hypoxia, fa/fa animals lost weight, circulating hematocrit rose higher, and lung volume failed to increase compared to +/+. In fa/fa, the hypoxia-induced increase in postmortem lung volume was attenuated (19%) vs. +/+ (39%). Alveolar ducts were 35% smaller in normoxia but enlarged two-fold more in hypoxia compared to +/+. Hypoxia induced broad increases (90-100%) in the volumes and surface areas of alveolar septal components in +/+ lungs; these increases were moderately attenuated in fa/fa lungs (58-75%), especially that of type-II epithelium volume (16% vs. 61% in +/+). In fa/fa compared to +/+ lungs, oxidative DNA damage was greater and hypoxia induced efflux of alveolar macrophages. Harmonic mean thickness of the diffusion barrier was higher, indicating higher structural resistance to gas transfer. Conclusion: Chronic lipotoxicity impaired hypoxia-induced compensatory lung expansion and growth with disproportionate effect on resident alveolar progenitor cells. The moderate structural impairment was offset by physiological adaptation primarily via a higher hematocrit.
Key words: High altitude adaptation, lung structure, lung function, metabolic syndrome, morphometry, obesity
Introduction Obesity is associated with chronic lipid infiltration of extra-adipocyte tissues leading to a proinflammatory state and end-organ damage (Unger and Zhou, 2001, Unger and Scherer, 2010). In the lung, obesity causes abnormal ventilatory control and restrictive mechanics (O'Donnell et al., 2000); the impairment in lung function is independent of physical activity or fitness (Leone et al., 2009, Lin et al., 2006, Steele et al., 2009, van Huisstede et al., 2013). Obesity often co-exists with type-2 diabetes mellitus; both reflect a state of tissue hypoxia (Pasarica et al., 2009, Girgis et al., 2012) that heightens susceptibility to tissue injury and may aggravate existing organ dysfunction (Watz et al., 2009). Because normal alveolar microvascular reserves are extensive, significant alveolar lipotoxicity
This article is protected by copyright. All rights reserved.
Accepted Article
remains subclinical under basal conditions and is under-recognized as a pathological entity. However, lipotoxicity diminishes alveolar-capillary reserves, which may interfere with adaptation to additional challenges such as ageing, concurrent lung disease, cardio-renal co-morbidity or hypoxic stress, thereby causing or exacerbating morbidity. For example, obesity increases the risk of acute mountain sickness in human subjects (Ri-Li et al., 2003, Wu et al., 2007), suggesting the failure to acclimatize to a hypoxic environment. On the other hand, hypoxia is a primitive and universal stimulus for the growth and remodeling of gas exchange organs. While extreme hypoxia suppresses metabolic function and causes cell death, postnatal exposure to moderate hypoxia (equivalent to 3,100-3,800m altitude) accelerates alveolar tissue and capillary growth and acinar remodeling in various mammals (Reinke et al., 2011, Lechner and Banchero, 1980, Burri and Weibel, 1971b, Johnson et al., 1985, Hsia et al., 2005) as a compensatory mechanism for preserving O2 uptake. The interactions between obesity and hypoxia on the structure and function of the distal lung have not been examined.
To characterize the effects of lipotoxicity on alveolar-capillary structure and function during hypoxia challenge, we utilized the Zucker diabetic fatty (ZDF) rat model (Yilmaz et al., 2010, Foster et al., 2010), which originated in outbred Zucker fatty (fa/fa) rats carrying the fa mutation, an amino acid substitution in the extracellular domain of the leptin receptor that renders the animal insensitive to leptin, leading to hyperphagia, diet-induced obesity, extra-adipocyte fat infiltration, systemic proinflammatory stress, end-organ damage and a shortened life span (Unger and Zhou, 2001, Unger and Scherer, 2010). The type-2 diabetic trait develops in an inbred substrain of obese fa/fa males [ZDF/Drt-fa] receiving at least 6% dietary fat; hyperglycemia develops by age ~12 weeks as insulin production and peripheral insulin sensitivity decline (Shimabukuro et al., 1997, Shimabukuro et al., 1998). The lungs of fa/fa animals show baseline restrictive changes (Yilmaz et al., 2010, Yilmaz et al., 2014) resembling that in clinical metabolic syndrome. The distal lung exhibits age-related thickening of alveolar septa and capillary basement membrane, cellular hyperplasia, elevation of triglyceride content, infiltration of lipid-laden macrophages, increased connective tissue elements, and altered surfactant protein profiles (Foster et al., 2010). Here we tested the hypothesis that these obesityassociated abnormalities impair alveolar adaptation during exposure to ambient hypoxia.
Materials and methods Animals and exposure: The Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center approved the protocol. Male ZDF diabetic fatty (fa/fa, n=7) and lean ZDF control (+/+, n=8) rats (4mo old), bred in the Unger laboratory, were fed rodent chow containing 6.5% fat (Formulab diet 5008, Purina, St. Louis, Missouri) ad libitum and had free access to water. The diabetic phenotype was confirmed by tail vein blood glucose level (557±50 and 102±13 mg.dL-1 in fa/fa and +/+, respectively). The animals were exposed to 13% ambient O 2 (hypoxia, equivalent to ~3,800m high altitude) in an environmental chamber (Biospherix, Lacona, NY) for 3 weeks. The chamber was opened for no more than 20min each day for animal feeding and maintenance. The buildup of CO2 and moisture was prevented using soda lime. Body weight was measured each week. Hematocrit was measured from tail vein blood. At the end of exposure, the animal was fasted 3 hr,
This article is protected by copyright. All rights reserved.
Accepted Article
then deeply anesthetized with an intraperitoneal injection of ketamine (100 mg/kg), xylazine (10 mg/kg) and acepromazine (2.0 mg/kg) to suppress spontaneous respiration. The trachea was cannulated via a midline neck incision, and the cannula tied securely with silk suture. Mechanical ventilation (CIV 101, Columbus Instruments, Columbus, OH) was maintained (tidal volume 10 mL/kg, respiratory rate 90 breaths per min). Heart rate and transcutaneous O2 saturation were monitored via a tail probe (Vet/Ox G2, West Yorkshire, UK). Cardiopulmonary function was measured followed by euthanasia and lung harvest (described below). Results were compared to age- and gendermatched control animals exposed to normoxia (21% O2, fa/fa n=18, +/+ n=12) (Table 1). All animal procedures were performed within a span of 6 months by the same personnel using the same methods. Postmortem tissue analysis was first completed in the normoxia groups (+/+ and fa/fa); these results (lung function and morphometry) have been published (Foster et al., 2010, Yilmaz et al., 2010). Due to personnel constraints and other laboratory priorities, data analysis of hypoxiaexposed groups (+/+ and fa/fa) was completed later and the results reported here.
Apparatus: As described previously (Yilmaz et al., 2010), the tracheal cannula was attached via a manifold to the ventilator, a 10-20 mL glass syringe, and 2 three-way computer-controlled solenoid valves (GH3315, Hans Rudolph, Kansas City, MO). Total apparatus dead-space was 0.5 mL. Airway pressure was recorded using a pressure transducer (Amplifier 1 Model 1100, Hans Rudolph, Kansas City, MO) calibrated against a manometer before each experiment. Data were acquired (100 Hz) using a laptop computer and LabView software (National Instruments, Austin, TX).
Static airway pressure–lung volume (PV) relationship: A glass syringe containing the desired volume of air was used to inflate the lungs from end-expiration. Each inflation was held for ~6 s before exhalation and resumption of mechanical ventilation for 10 to 15 s before initiating the next inflation step. Mean airway pressure during the 3rd second after reaching peak pressure was recorded. The maneuver was repeated at incremental inflation volumes (10 mL/kg steps) until airway pressure exceeded 30 cmH2O then repeated in stepwise volume decrements. Duplicate measurements at each volume were averaged.
Rebreathing measurements: A test gas mixture (0.3% CO, 0.5% Ne, 0.8% C2H2, 40 or 90% O2 in balance of N2) was drawn into a Mylar reservoir bag and the initial gas concentrations measured by a gas chromatograph (CP-4900 Micro GC, Varian Inc, CA) with 2 columns (M5AHIBF and PPUHI) and a thermal conductivity detector using 100% high purity helium as a carrier gas. A volume of the test gas equal to the inflation volume at 30cmH2O of airway pressure (11.8±1.1 and 15.3±2.3 mL in fa/fa and +/+ animals, respectively, mean±SD) was drawn into the glass syringe and attached to the manifold. The ventilator was stopped to allow exhalation and valve switching. Rebreathing was conducted by gently pumping the syringe at 60 strokes per minute to mix the test gas with resident lung gas for 5, 7 or 9 s in separate maneuvers in random order. The precise rebreathing duration was determined from the change in airway pressure signals with valve opening or closing. Final gas concentrations in the syringe were measured immediately. Duplicate measurements were made at each inspired O2 tension in random order. The interval between successive rebreathing maneuvers
This article is protected by copyright. All rights reserved.
Accepted Article
was at least 2 min. At the end, CO backpressure was measured by rebreathing 30 mL of 100% O2 for 60 s.
Euthanasia, open chest PV curves, and tissue harvest: Following the above procedures, the thoracic cavity was opened to expose the lungs and heart, and the PV measurements repeated as described above. Blood was drawn by cardiac puncture to measure hematocrit. The right lung was clamped at the hilum, and the heart stopped by an intracardiac overdose injection of Euthasol™. The left lung was perfused with oxygenated PBS, removed and snap frozen in liquid nitrogen. The clamp was removed and the right lung fixed via tracheal instillation of 2.5% buffered glutaraldehyde at 25cmH2O of hydrostatic pressure. Liver and kidney were removed as control organs.
Triglyceride content analysis: Lung and liver tissue (50–100mg) were homogenized in 2:1 chloroform-methanol, and lipids were extracted for 1 h at room temperature with intermittent vortexing. Volume of distilled water (2X) was vortexed into the homogenates and the mixture centrifuged (2,000g). The lipid extract was withdrawn, and 50–600μL was dried completely. The dried samples were mixed with 30μL of t-butanol, 20μL of 1:1 Triton X-100-methanol, and 1mL of a 4:1 mixture of Free Glycerol Reagent: Triglyceride Reagent (Sigma-Aldrich, St. Louis, MO), and the absorbance read (540nm). Triglyceride content was calculated from a glycerol standard curve (Sigma-Aldrich) and expressed in each animal as the mean milligrams triglyceride per gram of tissue in duplicate samples.
DNA damage: Lung 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was measured as a marker of oxidative DNA damage. DNA was extracted using DNAzol (Life Technologies, Grand Island, NY), precipitated in 100% ethanol, washed with 70% ethanol, suspended in 8mM NaOH, and the 8-OHdG concentration determined by ELISA (OxiSelect™ Oxidative DNA damage Cell BioLabs, San Diego, CA) compared with that of a standard curve.
Estimation of lung fluid: Lung tissue (~100mg) was weighed and transferred to a platinum ashing crucible and placed on a hot plate set at 100°C under a heat lamp for 2h. Then, the dry samplecontaining crucible was weighed to determine the sample dry weight and the dry-to-wet-weight ratio. The crucible was placed overnight in the ash oven at 600°C, removed and weighed to determine the ash weight. The ash was dissolved in 2 mL of HCl and the sodium content measured by flame photometry. Sodium-to-dry weight ratio serves as a surrogate marker for the relative amount of interstitial fluid (extracellular fluid sodium concentration 135-140 mM; intracellular sodium concentration 10-15 mM) (Ravikumar et al., 2014).
Lung volume estimation and sampling: Volume of the intact fixed right lung was measured by saline immersion (Yan et al., 2003). The lung was serially sectioned (3mm intervals) starting with a random
This article is protected by copyright. All rights reserved.
Accepted Article
orientation; the cut surfaces were imaged using a digital camera. Volume of the sectioned lung was estimated from the images using the Cavalieri principle (Yan et al., 2003); this tension-free volume was used in subsequent morphometric calculations. Lung slices were divided into roughly equal cranial and caudal regions. Using a systematic sampling scheme with a random start, 2 tissue blocks were sampled from each region (total of 4 blocks per lung), post-fixed with 1% osmium tetroxide in 0.1M cacodylate buffer, treated with 2% uranyl acetate, dehydrated through graded alcohol, and embedded in Spurr (Electron Microscopy Sciences, Hatfield, PA). Each block was sectioned (1μm) and stained with toluidine blue.
Morphometric analysis: A stratified analytical scheme was used (Foster et al., 2010, Hsia et al., 2010): low-power light microscopy (LM; 275x), high power LM (550x) and transmission electron microscopy (TEM; ~19,000x). One section per block was overlaid with a test grid at the appropriate magnification. From a random start, at least 20 non-overlapping microscopic fields per block (80 per lung) were systematically examined at 275x magnification. Using point counting, conducting structures larger than 20µm in diameter, i.e., bronchioles and blood vessels, were excluded from the reference space to estimate the volume density of fine parenchyma, alveolar ducts and sacs per unit lung volume. At 550x magnification, 15 non-overlapping microscopic fields per block (60 per lung) were systematically imaged to estimate the volume density of alveolar septa per unit lung volume. For TEM, 2 blocks per region (4 blocks per lung) were sectioned (70nm thickness), mounted on copper grids, and examined at ~19,000x (JEOL EXII). Thirty non-overlapping EM fields per grid were systematically sampled (total 120 per lung). The volume densities of epithelium, interstitium, and endothelium were estimated by point counting. Alveolar epithelial and capillary surface densities per unit septum volume were estimated by intersection counting. About 300 points/intersections were counted per grid with a coefficient of variation
