We used male Spaque-Dawley (SD) rats, 12-14 wk-old, weighing 360-380 g. The animals were obtained from the National Animal Center and housed in the University Laboratory Animal Center with good environment control. The animal experiment was approved by the University Committee of Laboratory Animal Care and Use, and followed the guidelines of the National Animal Research Center. The room temperature was maintained at 21 ± 1°C under a 12/12 hr light/dark regimen. Food and water were provided ad libitum.
Isolation and perfusion of the lung in Situ
We followed the procedures for the preparation of isolated and perfused rat's lungs in situ [18, 19]. Rats were anesthetized with an intraperitioneal injection of pentobarbital (40 mg/kg) and intubated with an endotracheal tube. A rodent ventilator provided ventilation with a mixture of 95% room air and 5% carbon dioxide. The respiratory rate and tidal volume were 60-65 breaths/min and 2-3 ml, respectively. The inspiratory and expiratory pressures were 5 and 1 cm H2O. A vertical incision was made along the midline of the thorax. Heparin (1 U/g) was then injected into the right ventricle. An afferent line (silicon tubing) was then inserted into the pulmonary arterial trunks via the right ventricle, while an efferent line (silicon tubing) was inserted into the left atrium via the left ventricle. Blood (10-15 ml) was collected for later use.
The pulmonary trunk and the aorta were then tied off. The isolated perfused lungs were left in situ, and the whole rat was placed on an electronic balance. The digital signals of the electronic balance were converted to analog signals by a digital-to-analog converter and were recorded on a polygraph recorder. Weight changes were precalibrated on the electronic balance before preparation for the experiment. In this isolated lung preparation, we have verified that the changes in body weight (BW) reflect the lung weight (LW) changes [18, 19].
The isolated lungs were perfused with heparinized blood (10 ml) with Krebs-Heseleit balanced salt solution (5 ml). The perfusion system included a venous reservoir and a roller pump. The perfusate was circulated via the roller pump to maintain a constant flow. The venous blood was diverted via the efferent line into the reservoir. The later was placed in a 38°C water bath for constant temperature.
Pulmonary arterial pressure (PAP) and venous pressure (PVP) were measured from sideports connected to the afferent and efferent tubings. The lungs were perfused with a constant flow (10-14 ml/min). Flow rate was adjusted to maintain the initial PAP at 15-16 mmHg. The PVP was kept at 0-1 mmHg by adjusting the height of efferent outflow tubing. The changes in PAP at a constant-flow condition reflect the changes in pulmonary vascular resistance.
Lung weight (LW), LW/body weight ratio (LW/BW) and LW gain (LWG)
Lung weight (LW) was obtained from 20 euthanized rats by an intravenous sodium pentobarbital (100 mg/kg). The initial LW was estimated from an equation relating to the body weight (BW) [18
]. The LW was then plotted against BW for a regression equation:
LWG was obtained by the increase in LW and also calculated as:
Microvascular permeability (Kfc)
Capillary filtration coefficient (Kfc) as an index of microvascular permeability was calculated from the increase in LW produced by an elevation in PVP. The Kfc was defined as the initial weight gain rate (g/min) divided by PVP (10 cm H2O) and LW, expressed as g/min/cmH2O/100 g. During the experiment, PVP was rapidly elevated by 10 cm H2O for 7 mins to measure Kfc. This hydrostatic challenge elicited a biphasic increase in LW: an initial rapid component, followed by slow and steady component. The slow component of the weight gain was plotted on a semilog scale as a function of time. The capillary filtration rate was obtained by extrapolating the slow component of the weight gain back to zero time [18, 19].
An increase in exhaled NO concentration has been used as an early marker of lung inflammation or injury [20, 21]. We measured the NO concentration in expired air. A specimen of exhaled air (300 ml in 30 min) was suctioned into a gas purge chamber preciously evacuated to remove oxygen. The NO concentration was rapidly determined after air collection. The measurement of NO with a chemiluminescence analyzer (Sievers 270B NOA; Sievers Institute, Denver, CO, USA) was based on the principle that NO interacts with ozone to generate chemiluminescent light. The chemiluminescence is directly proportional to the NO level. In addition to a photomultiplier tube, an ozone generator and a gas chamber were included. The ozone generator was used to produce ozone internally. The exhaled NO was measured every 30 min after introduction of PMA into the lung perfusate. It reached its peak depending on the experimental conditions. The peak value was taken as the NO concentration.
Protein concentration in bronchoalveolar lavage (PCBAL) and Evans blue leakage
After the experiment, lungs were lavaged twice with saline (2.5 ml per lavage). Lavage samples were centrifuged at 1,500 g at room temperature for 10 min. The PCBAL was determined with a spectrophotometer by measuring the change in absorbance at 630 nm after the addition of bromocresol green [18, 22]. Five mins before the end of the experiment, Evans blue dye (1 mg) was given into the lung perfusate. The Evans blue content in lung tissue was determined spectrophotometrically at an optical density 620 nm .
Nitrate/nitrite, methyl guanidine, tumor necrosis factorα and interleuin-1β
Samples (0.5 ml) were taken from the lung perfusate 1 hr before and at various time points after PMA administration. The samples were centrifuged at 3,000 g for 10 mins. The supernatant was used for determination of nitrate/nitrite with high-performance chromatography [23, 24]. The formation of methyl guanidine (MG) has been identified as an index of hydroxyl radical production . It was determined with its fluorescence spectrum (Jasco 821-FP, Spectroscopic Co., Tokyo, Japan). The emission maximum was set at 500 nm and the excitation maximum at 398 nm. The assay was calibrated with authentic MG (Sigma M0377). Tumor necrosis factorα (TNFα) and interleukin-1β (IL-1β) were measured with antibody enzyme-linked immunosorbent assays (ELISAs) with a commercial antibody pair, recombinant standards, and a biotin-streptavdin-peroxidase detection system (Endogen, Rockford, IL, USA). All agents, samples, and working standards were prepared at room temperature according to the manufacturer's directions. The optical density was measured at 450/540 nm wavelengths by automated ELISA readers.
Neutrophil elastase, myeloperoxidase, and malondialdehyde activity
The neutrophil elastase (NE) was determined with a synthetic substrate, N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as described previously [15, 18]. In brief, samples were incubated in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5 M Nacl and 1 mM substrate at 37°C for 24 hrs. After incubation, p-nitroanilide release was measured spectrophotometrically at 450 nM and considered NE activity.
To measure the myeloperoxidase (MPO) activity in lung perfusate, the samples were mixed with 2 ml of potassium phosphate buffer (50 mM, pH 6.0) containing 0.5% cetyltrimethylammonium bromide and were centrifuged at 2,500 g for 10 mins at 4°C. The supernatant was diluted with dilution buffer, then mixed with an assay buffer composed of 0.00107% H2O2 in potassium phosphate buffer and o-dianisidine. The reaction mixture was incubated at room temperature. The change in absorbance at 450 nm over 1 min was detected spectrophotometrically. The MPO activity was expressed as units per ml of lung perfusate using the absorbance of MPO standard (Elastine Products, Detroit, MC., USA). The procedures were basically followed those by Kinoshita et al. .
Malondialdehyde (MDA) was measured by thiobarbituric and reaction. The principle of the method depends on the development of pink color produced by the interaction of barbituric acid with MDA as a result of lipid peroxidation. Tetraetoxypropane was used as standard [27, 28].
Lungs were harvested after the experiments. A BioOrbit ATP Assay kit (Bio-Orbit Oy, Turku, Finland) was used to determine the lung ATP content with bioluminescence technique. The assay was based on quantitative measurement of a stable level of light as a result of an enzyme reaction catalysed by luciferase. Under the effect of luciferase, the luminescence evoked by ATP and luciferin interaction was recorded photometrically after amplification by a photomultiplier. The sensitivity of ATP was in a nanomolar range. The luciferin-luciferase reagent was used according to the manufacturer's manual. ATP served as the standard. The test procedures have been described previously [11, 29].
PARP activity in the harvested lung tissue was measured with a commercially available assay kit (Genzyme Diagnostics, Cambridge, MA, USA). Lung tissue samples were placed on ice in 2 mL buffer containing 50 mM Tris Cl (pH 8.0), 25 mM MgCl2 and 0.1 mM phenylmethylsulfonyl fluoride. The samples were homogenized for 30 s and then sonicated for 20 s using an ultrasonic homogenizer. The suspension was centrifuged at 3,000 × g for 5 min at 4°C. Supernatant containing 20 μg protein, PARS buffer, 1 mM NAD, 2 μCi 32P-labelled NAD (1 μCi/μL) and distilled water was mixed in a microcentrifuge tube. The reaction was allowed to continue at room temperature for 1 min and was stopped by adding 900 μL of tricarboxylic acid. Enzyme activity was determined by measuring the incorporation of radiolabelled NAD as PARP catalysed the poly (ADP) ribosylation of proteins. The labeled ADP was determined by scintillation counting after tricarboxylic acid precipitation onto a filter. The procedures and calculation of PRRP activity were carried out according to those described by Pulido et al. .
Detection of iNOS and eNOS mRNA in lung tissue
Real-time polymerase chain reaction (RT-PCR) was employed for the detection of iNOS and eNOS mRNA expression. PCR primers and TaqMan-MGB probes were designed using Primer Express V.2.0 software (Applied Biosystems Inc., Foster, CA, USA) based on the sequences from GenBank. TaqMan-MGB probes were labeled with 6-carboxy-fluorescein (FAM) as the reporter dye. Real-time PCR was performed in a two-step process: In the first step, sample RNA (100 ng) was reverse-transcribed with 50 ng random hexamers in a volume of 20 μl using 200 U of Superscript III reverse transcriptase and 40 U of RNaseOUT recombinant RNase inhibitor (both from Invitrogen, Carlsbad, CA, USA). In the second, real-time PCR was carried out in a MicroAmp Optical 96-well plate using TaqMan Master Mix (Applied Biosystems Inc.), with 5 μl cDNA in each well. PCR reactions were monitored in real time using the ABI PRISM 7000 Sequence Detector (Applied Biosystems Inc. Foster, CA, USA). The thermal cycling conditions for real-time PCR were a) 50°C for 2 mins, b) 95°C for 10 mins, c) 40 cycles of melting (95°C, 15 sec) and d) annealing/extension (60°C, 60 sec).
Immunofluorescent staining was used to detect the activities of nitrotyrosine and iNOS in lung tissue using specific polyclonal antibodies as described previously [31, 32].
Lung pathology and immunohistochemical examinations
Lung tissue was fixed in 10% formaldehyde for 24 hrs and then rinsed with tap water to remove formaldehyde. For light microscopic examination, lung tissue was dehydrated with graded alcohol and then embedded in paraffin at 60°C. A series of microsections (5 μm) was stained with hematoxylin and eoxin. For quantification of lung injury score, we employed a modified grading method reported previously [21, 28]. Various degree of lung injury score (LIS) were assessed as follows: degree 0, 1, 2 and 3 for no, mild, moderate and severe edema, respectively. For inflammatory cell infiltration, the scoring was similar to the edema extent: 0-3 for no, mild, moderate and severe cell infiltration. The histopathological assessment was performed in a blind fashion by several laboratory assistants. The individual scores were added together to obtain a final score, ranging from 0-6.
Antigen retrieval immunohistochemical stain was used to identify the source of inducible NO synthase (iNOS) in lung cells [33, 34].
Administration of PMA and niacinamide (NAC)
PMA (4 μg/g) and NAC at various doses were added into the lung perfusate via the venous reservoir [35, 36]. NAC was given simultaneously with PMA [20, 37, 38]. Examinations of biochemical factors were taken before administration of PMA and NAC, and at the end of the experiments.
A total of 60 isolated lungs were randomized into 6 groups to receive Vehicle (DMSO 100 μg/g), and PMA 4 μg/g (lung weight) cotreatment with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. The doses of PMA and NAC were used in accordance with previous studies [12, 13, 20]. The experiments were observed for 120 mins.
All data were expressed as mean ± SEM. Comparisons within and among groups were made using one-way analysis of variance with repeated measures, followed a post hoc comparison with Newman-Keuls test. Differences were considered to be statistically significant at p < 0.05.