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MATERIALS AND METHODS

By Gregory Richardson,2014-08-12 12:48
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MATERIALS AND METHODS ...

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    MATERIALS AND METHODS

    Animal treatments

    Nineday old SpraqueDawley rat pups (Charles River Laboratories, Wilmington,

    MA) were injected subcutaneously either with 60 mg/kg of monocrotaline (Sigma, St. Louis,

    1,2MO) or an equal volume of phosphatebuffered saline (PBS). Pups remained with their

    mothers and were not weaned during the experiments. The litters that were treated with 20 ppm inhaled NO at FiO 0.21 were maintained in 40 L acrylic chambers in the same room 2

    as those that did not breathe NO gas. The levels of inhaled NO and oxidative products of NO were measured using chemiluminescence (Model 14A, Thermo Environmental Instruments Inc., Franklin, MA) and were carefully controlled using sodalime and high fresh

    3gas flows of air, O, and NO gas, as previously described. 2

    Pulmonary hemodynamics

    Pulmonary and systemic artery pressures were determined using 6 to 7 pups in each experimental group, one and two weeks after treatment with monocrotaline or PBS. After a pup was anesthetized with 36 mg/kg ketamine IP, the trachea was cannulated, and the

    lungs were ventilated with air at a peak inspiratory pressure of 10 cm HO (Harvard Small 2

    Animal Ventilator, Model 683, South Natick, MA). A cannula (0.61 mm OD) was secured in the right carotid artery via an arteriotomy to measure systemic blood pressure and arterial pHand gas tensions. Ventilator rates of 90110 breaths per min produced an arterial pH of

    7.45. Through a thoracotomy, a 30 gauge needle was secured in the pulmonary artery using cyanoacrylate ester (Super Glue). The mean pulmonary (PAP) and systemic artery pressures (SAP) were measured using previously calibrated, fluidfilled membrane trans-

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    ducers (Argon, Athens, TX), instrumentation amplifiers (Hewlett Packard 8805C, Andover, MA), and a data acquisition system (DI 220, Dataq Instruments, Akron, OH). The frequency response of the pressure measurement system was measured using the sinusoidal

    4 and a blood pressure calibration pump (Bio-Tek Instruments, Burlington, VT) method

    triggered by a function generator (Tektronix Instruments, Beaverton, OR). Since a 50 mmHg systolic pressure signal injected into the transduceramplifier system at a rate of 10

    Hz resulted in a measured systolic pressure that was decreased by < 2%, good precision in the hemodynamic values were obtained using the instrumentation.

    In situ pulmonary artery flow and pressure relationships were determined using the lungs of 6 to 7 additional pups in each experimental group at one and 1.5 weeks after they were treated with either monocrotaline or PBS. Immediately after a pup was killed with 200 mg / kg sodium pentobarbital IP, the trachea was cannulated. The lungs were ventilated with 21% O, 6% CO and 73% N at a rate of 100 breaths per minute, peak inspiratory 222

    pressure of 10 cm H0 and end expiratory pressure of 2 cm HO. Following injection of 10 22

    U heparin into the right ventricle, a cannula (0.61 mm OD) was advanced from the right ventricle through the pulmonary valve and secured in the main pulmonary artery. The lungs were perfused in situ, in a heated and humidified chamber (Hugo-Sachs Elektronik, March-Hugstetten, Germany), using a peristaltic pump and Hanks' Balanced Salt Solution, pH 7.357.45, containing 30 µmol/L indomethacin, 5% albumin, and 5% dextran at 37?C. The gas mixture and ventilator rate caused a PCO of 3545 mmHg in the pulmonary 2

    effluent. The pump was calibrated before each experiment using a stop watch and graduated cylinder. An incision in the left atrium permitted drainage of the perfusate. The pulmonary

    5artery pressures were recorded in Zone 2 conditions (i.e., P>P>P) while the arteries PAAWLA

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    were perfused at 40, 50, and 60 ml/kg (whole body weight) • min and the airways were

    O pressure. statically distended with the gas mixture at 2 cm H2

    Right ventricular weight

    The right ventricular weight was measured using 8 pups in each experimental group at

    6one and two weeks after monocrotaline or PBS treatment and the method of Fulton et al.

    After each pup was killed, the heart was fixed in 4% formaldehyde at 4?C for three days. Subsequently, the atria were removed, and the right ventricular free wall was dissected from the left ventricle and interventricular septum. The right ventricle and the left ventricle with septum were weighed separately. The ratio of the right ventricular weight to the weight of the left ventricle with septum (RV / LV+S) weight was calculated.

    Morphometric analysis of pulmonary arteries

    The pulmonary artery muscularization was determined in 4 pups in each experimental group at 7 days after treatment with PBS or after treatment with monocrotaline with and

    7,8without 20 ppm continuously inhaled NO gas, using the technique of Reid and coworkers.

    Our previous studies demonstrated that inhalation of this level of NO does not change the

    3muscularization of the peripheral pulmonary arteries of pups breathing air. After a pup was

    killed, the airway and main pulmonary artery were cannulated, and the pulmonary veins were ligated. The pulmonary artery and airways were distended to 100 and 23 cm HO 2

    pressure, respectively, with 4% phosphatebuffered formaldehyde and fixed at 4?C for 3

    days. The lungs were embedded in plastic (Historesin Plus, Leica, Heidelberg), and 2 µm

    9thick sections were treated with Miller’s stain, to permit identification of elastin, and

    toluidine blue. Because the infused fixative washes blood from the pulmonary arteries into the veins, the lung arteries were identified by the elastin staining of their walls and the

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    absence of blood in their lumen. These arteries were classed as muscular if two elastic laminae were identified in their wall. Vessels without two elastic lamina that were greater than or equal to 15 µm in external diameter were classed as nonmuscular; whereas those less than 15 µm in external diameter were considered capillaries and were not analyzed. In a section from the left, diaphragmatic and cardiac lobes from each pup, the percentage of muscular arteries in the first 25 true or obliquely sectioned alveolar duct or wall pulmonary arteries observed in distended lung areas was determined.

    Detection of DNA synthesis in pup lungs

    The numbers of nuclei of peripheral pulmonary artery cells synthesizing DNA was determined in 4 pups in each experimental group at 5 days after treatment with PBS or monocrotaline with and without 20 ppm inhaled NO gas, using bromodeoxyuridine (BrdU;

    10,11 Pups were injected with 50 mg / kg Sigma) labeling and direct immunohistochemistry.

    BrdU IP one hour before they were killed. After the pulmonary veins were ligated, the pulmonary arteries were perfused with a 60?C emulsion of barium sulfate in gelatin and

    12buffered phenol at 100 cm HO pressure. Subsequently, the airways were perfused with 2

    4% formaldehyde at 23 cm HO pressure, the whole lungs were fixed in the formaldehyde 2

    for 3 days at 4?C, and 5 mmthick lung sections were embedded in paraffin. Lung sections (4 µm) were treated with 0.3% HO in methanol, 2 mol/L HCl, and protease XXIV (Sigma), 22

    and the newly synthesized DNA was detected using an antiBrdU antibody conjugated with

    peroxidase (Boehringer Mannheim, Indianapolis, IN) and tetramethylbenzidine (TMB; Kirkegaard & Perry Laboratories, Gaithersburg, MD). The sections were counterstained

    with nuclear fast red stain. The filling of the pulmonary arteries with bariumgelatin and

    darkfield microscopy of the lung sections permitted identification of arteries in the lung

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    periphery. BrdUlabeled and nonlabeled nuclei in the walls of 25 alveolar duct or wall arteries from the left and diaphragmatic lobes of the pups were counted.

    ; smooth muscle actin expression and DNA synthesis in pup lungs Detection of

    Pulmonary artery cells expressing ; smooth muscle actin and/or synthesizing DNA

    five days after exposure to monocrotaline were identified using anti; smooth muscle actin

    and BrdU antibodies, and indirect and direct immunohistochemistry, respectively. The pups were treated with BrdU IP, and the pulmonary arteries and airways were perfusedfixed

    with formaldehyde as described above. Paraffinembedded lung sections (4 µm) were

    treated with 0.3% HO in methanol, and ; smooth muscle actin expression was detected 22

    13using a monoclonal murine antibody (Cone 1A4 IgG, Sigma), biotinylated antimurine 2a

    ?antibody, avidinbiotinalkaline phosphatase, and Vector red stain (Vector Laboratories,

    Burlingame, CA). After treating the lung with 2 mol/L HCl, protease XXIV, and 1% goat serum, the newly synthesized DNA was detected using an antiBrdU antibody conjugated

    with peroxidase and 3, 3’–diaminobenzidine (DAB; Vector). The lung sections were counterstained with hematoxylin before examination using bright field microscopy. Detection of cytokine and adhesion molecule expression and platelet aggregation

    Pulmonary IL1, Eselectin, and ICAM1 expression was determined using RNA

    blot hybridization and randomprimed cDNA templates. RNA was isolated from the lungs of 2 to 3 pups in each experimental group 7 days after exposure to monocrotaline or PBS and the lungs of mice 6 hours after treatment with LPS or PBS using guanidine

    14isothiocyanate and centrifugation on a CsCl cushion. The IL1 probe was generated

    using the polymerase chain reaction, sense (5’-atg aaa gac ggc aca ccc ac-3’) and antisense

    (5’-ccc aca cgt tga cag ct-3’) primers, and cDNA generated from reverse transcription using

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    2 µg of RNA isolated from LPStreated mouse lungs, Moloney murine leukemia virus

    reverse transcriptase, RNasin, and the antisense primer. The Eselectin probe was generated

    from the Pvu II digest of rat Eselectin cDNA cloned into pBluescribe (provided by Dr.

    15 The ICAM1 probe was obtained from a Hind III digest of the murine Tucker Collins).

    expressed sequence tag fragment X15372, which had been cloned into pBluescript SK

    (63111; ATCC). Inspection of ethidium bromide stained electrophoresis gels confirmed that equal quantities of RNA were analyzed.

    16To identify platelet containing thrombi, GPIb; expression was examined in

    formaldehydefixed, paraffinembedded sections of pup lungs exposed to monocrotaline or PBS and thrombosed rat carotid arteries. Expression was analyzed using 10 µg/ml of a murine monoclonal antiGPIb; antibody (clone G28E5, kindly provided by Dr. Stefan

    17Janssens), biotinylated antimurine antibody, avidinbiotin conjugated to peroxidase

    (M.O.M. kit, Vector Labs), and Vector VIP. Tissues were counterstained with acidified methylgreen and bright field microscopy.

    RESULTS

    One week after treatment with monocrotaline, the pups were active, and they were indistinguishable in general appearance from those treated with PBS. However, two weeks after treatment, the pups treated with monocrotaline were less active and were unkempt. Although pups from all experimental groups grew during the course of the studies, those treated with monocrotaline gained less weight over the two weeks (PBStreated 59.5?4.5

    gm, monocrotalinetreated 48.7?8.0 gm; P<0.05). In addition, monocrotaline treatment

    decreased systemic oxygen levels two weeks after treatment. The PaO of the pups treated 2

    with PBS was 114?16 and 108?19 mmHg, after one and two weeks. One week after mono-

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    crotaline treatment the PaO was 95?9 mmHg and did not differ from that in the PBS2

    treated control group. However, two weeks after monocrotaline treatment, the PaO was 2

    decreased to 66?20 mmHg (P<0.05, vs. values one and two weeks after PBS treatment). Monocrotaline exposure causes pulmonary artery remodeling in the absence of hyper-tension.

    Although monocrotaline exposure causes pulmonary neomuscularization in rat pups

    2without right ventricular hypertrophy, it is unknown whether the lung remodeling occurs in the absence of hypertension. One week after treatment, when a threefold increase in pul-

    2monary artery neomuscularization is observed, monocrotaline did not cause pulmonary art-

    ery hypertension in pups. The mean pulmonary artery pressure was 14.0?2.6 mmHg after monocrotaline treatment and did not differ from the pressure of 14.0?1.5 mmHg measured in the PBStreated group (Fig. 1). Furthermore, monocrotaline did not cause an increase in pulmonary artery tone since acute inhalation of 20 ppm NO did not decrease the lung pressure in these pups (data not shown).

    Because cardiac output and, therefore, pulmonary vascular resistance could not accurately be measured in the pups, pulmonary artery pressureflow relationships and

    pulmonary vascular resistance (PVR) were determined using in situ ventilated and perfused

    pup lungs. The pressureflow relationships were similar in the monocrotaline and PBS

    treated groups one week after treatment (Fig. 2). In addition, one week after treatment with monocrotaline, the PVR did not differ from that in the PBStreated pups. The PVR in the

    lungs perfused at 50 ml/kg•min was 0.12?0.01 and 0.13?0.02 mmHg•kg•min/ml, in the monocrotaline and PBStreated pups, respectively, one week after treatment. Furthermore, after one week of treatment the right ventricular weight of the monocrotalinetreated pups

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    was the same as that of those treated with PBS (Fig. 3). Taken together, these data indicate that monocrotaline does not cause pulmonary hypertension in pups by one week after treatment.

    Two weeks after treatment, monocrotaline exposure caused pulmonary artery hyper-tension in the pups (Fig. 1). At this time, the mean pulmonary artery pressure in the mono-crotalinetreated pups was 25.3?5.4 mmHg and was increased by nearly 70% in compari-son with pups one week after monocrotaline administration and one and two weeks after PBS treatment (P<0.05). The systemic arterial pressure was increased two weeks after treatment with monocrotaline or PBS. At this time, monocrotaline exposure was associated with a 100% increase in the pulmonary arterial to systemic pressure ratio in comparison with PBS treatment (P<0.05). The pulmonary vascular resistance was also increased by 1.5 weeks in the monocrotalinetreated pups in comparison with control pups (PVR at 50

    ml/kg•min: PBS–treated 0.16?0.00, monocrotalinetreated 0.21?0.02 mmHg•kg•min/ml;

    P<0.05 vs. one week and each other). The pressures measured in perfused lungs from monocrotalinetreated pups after 1.5 weeks were greater than those in lungs from pups treated with PBS at one or 1.5 weeks and in the lungs of monocrotalinetreated pups at one

    week (Fig. 2). Two weeks after treatment with monocrotaline, the right ventricular weight was nearly 45% greater than that observed in pups one and two weeks after PBS treatment (Fig. 3). Together, these data indicate that pulmonary artery remodeling in monocrotaline treated pups occurs in the absence of pulmonary hypertension.

    Monocrotaline does not induce pulmonary inflammation or thrombosis in rat pups.

    18,19Although monocrotaline exposure can induce pulmonary inflammation and

    20plateletrich thrombus formation in adult rats, it is unknown whether or not they are

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    observed in the remodeling pup lung. The lungs of pups 3.5 and 7 days after monocrotaline exposure did not exhibit increased adventitial or alveolar cellularity consistent with inflammation. Monocrotaline exposure also did not increase cytokine or adhesion molecule expression. The levels of IL1 and ICAM1 and Eselectin mRNA were not increased in

    monocrotaline exposed pup lungs (Fig. 4). In contrast, the mRNA levels of IL1 and

    ICAM1, which were not observed to have constitutive gene expression in the pup lung, were increased following LPS exposure in the mouse lung.

    Inspection of monocrotaline exposed pup lung sections did not reveal a decrease in the density of bariumgelatin filled pulmonary arteries that would be observed with thrombosis.

    17In addition, immunoreactivity for GPIb;, a glycoprotein observed in plateletrich thrombi,

    was not detected in monocrotalinetreated lung pulmonary arteries (data not shown). A high level of GPIb; expression, however, was detected in the thrombosed carotid arteries of rats. Inhaled NO prevents monocrotalineinduced pulmonary artery remodeling.

    The salutary effect of inhaled NO was tested in monocrotalinetreated pups that do

    not exhibit pulmonary artery hypertension, inflammation, and thrombosis. Monocrotaline caused neomuscularization of peripheral pulmonary arteries in rat pups one week after treatment. The proportion of muscularized arteries in the alveolar duct and wall of the pup lung increased nearly threefold one week after monocrotaline treatment (Fig. 5). Over this same period, continuous inhalation of NO gas protected the lungs from monocrotaline

    induced pulmonary artery remodeling. The percent muscularization of alveolar duct and wall pulmonary arteries was the same in the monocrotalinetreated pups that breathed NO

    as in the PBStreated pups.

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    Since monocrotaline exposure increases cell proliferation in adult rat pulmonary arter-

    21,22 and inhaled NO attenuated neomuscularization in monocrotalinetreated pups, we ies,

    examined the ability of inhaled NO to decrease pulmonary artery cell proliferation in mono-crotalineexposed pups. Because pilot studies revealed that the maximum increase in the fraction of BrdUlabeled cells in the pulmonary arteries occurred 3 to 5 days after monocro-taline treatment, the effect of inhaled NO on DNA synthesis was tested in pups 5 days after monocrotaline or PBS exposure. Monocrotalinetreatment increased the proliferation of

    cells in the walls of pulmonary arteries (Fig. 6). In comparison with the PBStreated pups,

    the fraction of proliferating cells and the number of proliferating cells per pulmonary artery were greater in the alveolar duct and wall arteries of monocrotalinetreated pups (P<0.05).

    Because previous studies indicate that monocrotaline exposure is associated with SMC

    21,22proliferation, experiments using doubleimmunolabeling of BrdU and ; smooth muscle

    actin were performed to identify proliferating pulmonary artery SMC. We observed that monocrotaline exposure was associated with proliferation of epithelial cells of airways and endothelial and adventitial cells in peripheral pulmonary arteries (Fig. 7). In contrast, no proliferating cells in the walls of monocrotalinetreated pulmonary arteries were observed to

    be expressing ; smooth muscle actin, which is indicative of a smooth muscle cell lineage. Although monocrotaline treatment increased cell multiplication, fewer cells per pulmonary artery where observed in the monocrotalinetreated pups than in the PBStreated ones

    (8.2?2.0 vs. 6.4?1.3; P<0.05). Since monocrotaline exposure increases cell proliferation and yet decreases the cell numbers, these data suggest that it increases cell turnover in peripheral pulmonary arteries.

    Inhaled NO protected the monocrotalinetreated pup lungs from increased cell turn-

    over. Continuous NO inhalation prevented the monocrotalineinduced increase in DNA

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