| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
IMMUNOLOGY, HEALTH, AND DISEASE |
Department of Poultry Science, University of Arkansas, Fayetteville 72701
1 Corresponding author: gferf{at}uark.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Key Words: ascites • chicken • microparticle • macrophage • reverse transcription-polymerase chain reaction
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
The PAH response to MP entrapment has been attributed, in part, to the synthesis and release of vasoactive compounds produced and released by vascular endothelial cells, thrombocytes, and leukocytes (Wideman, 2001; Wideman et al., 2007). Nitric oxide (NO) is one of the principal vasodilators known to modulate the onset of PAH (Grabarevic et al., 1997). Nitric oxide is synthesized by the enzyme nitric oxide synthase (NOS) from the oxidation of L-arginine in the presence of O2, nicotinamide adenine dinucleotide phosphate (NADPH), and other cofactors (Zapol et al., 1994). Three isoforms of NOS have been identified so far: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS; Moncada et al., 1997). The inducible isoform, iNOS, is expressed by avian macrophages following activation by lipopolysaccharides (LPS), cytokines, and other factors (Hauschildt et al., 1991; Nathan, 1992) resulting in the production of large quantities of NO (Chang et al., 1996; Hussain and Qureshi, 1997). The competitive inhibitor N-nitro-L-arginine methyl ester (L-NAME) inhibits all NOS isoforms, and injecting L-NAME into broilers results in an acute increase in pulmonary arterial pressure (PAP) that can be counteracted by injecting a NOS-independent NO donor (Weidong et al., 2002; Chapman and Wideman, 2006). When broilers were preinjected with L-NAME the increase in PAP elicited by a subsequent MP injection was amplified 2-fold when compared with the responses of control broilers. Similarly, the mortality triggered within 48 h after injecting MP was more than doubled when L-NAME was combined with microparticle injection doses that otherwise caused relatively low mortality in the absence of L-NAME (Wideman et al., 2005). The modulatory efficacy of NO likely reflects its ability to dilate the vasculature directly, as well as its ability to inhibit the synthesis or release of key vasoconstrictors such as serotonin and endothelin-1 (Wideman et al., 2007).
The MP entrapped in the lungs initiate a vigorous, focal inflammatory response. Within minutes thrombocytes aggregate around MP lodged in pulmonary arterioles. This is followed by infiltration and aggregation of mononuclear cells (monocytes/macrophages and lymphocytes) in the perivascular region surrounding MP-occluded arterioles (Wideman et al., 2002, 2007; Wang et al., 2003). It is our hypothesis that activation of monocytes/macrophages leads to increased iNOS expression, resulting in increased NO synthesis, and that this focally produced NO assists in dilating (relaxing) the adjacent vascular smooth muscle as well as in modulating the release of vasoconstrictors. Subsequent to the acute PAH response induced by MP entrapment, the expression and activation of iNOS in recruited macrophages should serve as the major source of NO production contributing to the broilers’ ability to survive MP-induced PAH (Wideman et al., 2007). Accordingly, macrophages recruited to MP-occluded vessels in the lung would be expected to be activated and express iNOS, and the macrophages of broilers that are resistant to PHS may be infiltrating sooner, in greater numbers, or express more iNOS, or both, than the macrophages of broilers that are susceptible to PHS. Hence, the objectives of this study were to demonstrate the time course of pulmonary macrophage infiltration, iNOS mRNA expression and NO production following i.v. injection of MP in PHS-susceptible and PHS-resistant broiler lines.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Newly hatched male broiler chicks were transported from the hatchery to the Poultry Environmental Research Lab at the University of Arkansas Poultry Research Farm, Fayetteville. The broilers were progeny from the PHS-resistant (RES) and PHS-susceptible (SUS) lines that were developed by divergent selection in a hypobaric chamber (Pavlidis et al., 2007). At the time of this study the lines were at their ninth generation of selection, and when reared under conditions of hypobaric hypoxia they exhibited ascites mortalities of 7.5 and 75% for RES and SUS lines, respectively. One hundred male chicks per line were wing-banded and reared together on fresh wood shavings in environmental chambers (8 m2 of floor space). Chicks were brooded at 33°C on d 1 to 5, at 29°C on d 6 to 10, and at 23.8°C, d 11 onwards. Chicks were fed corn-soybean meal broiler ration formulated to meet National Research Council (1994) recommendations for all the ingredients. Feed and water was provided ad libitum. Lights were on 24 h/d through d 5, 23 h/d through d 19, and 16 h/d from d 20 onwards.
Experimental Design and Tissue Collection
Fifty male broilers per line were injected with MP as described previously (Wideman and Erf, 2002; Wideman et al., 2002; Wang et al., 2003). Heparinized saline was prepared by dissolving 150 units of ammonium heparin (Sigma-Aldrich Inc., St. Louis, MO) per milliliter of 0.9% NaCl. Microgranular CM-32 ion exchange cellulose (Fisher Scientific, St. Louis, MO) having 30-µm average particle diameter was suspended at the rate of 0.02 g/mL in the heparinized saline solution. This suspension was vortexed continuously on a magnetic stirring plate to keep the MP evenly distributed and was injected to the broilers via the left wing vein at the dose of 0.30 mL/broiler using a 1-mL tuberculin syringe (Becton Dickinson, Franklin Lakes, NJ) attached to a 22-ga needle (Becton Dickinson). Lungs collected from 10 broilers per line that had not been injected with MP were designated as 0 h (uninjected control) samples. Lungs also were collected from 10 broilers per line at 2, 24, and 48 h post-MP injection.
Lung tissues were used for histology, immunohistochemistry, NADPH diaphorase histochemical staining, and iNOS mRNA gene expression analysis. At each time point 10 broilers per line were exsanguinated by decapitation. The portion of the right lung between the first and second anterior rib indentation was collected aseptically and was embedded in Tissue-Tek OCT freezing medium (Sakura Finetek Inc., Torrance, CA), snap frozen in liquid nitrogen, and stored at –80°C until analysis. An adjacent segment from each lung was immersed in 10% buffered formalin for histological evaluation. This tissue was embedded in paraffin, sectioned at 5 µm, stained with hematoxylin and eosin, and assessed microscopically to confirm that each of the lungs collected at 2, 24, and 48 h postinjection had entrapped substantial numbers of MP.
Immunohistochemistry for Macrophages
Immunohistochemical staining of the lung tissue for macrophages was done as described previously (Wang et al., 2003). Briefly, a 5-µm-thick transverse section of frozen lung tissue was cut using a cryostat (Microm Laborgeräte GmbH, Waldorf, Germany) at –22°C, mounted on poly-L-lysine-coated slides, fixed in acetone (Burdick and Jackson, Muskegon, MI) for 5 min, and air-dried. The lung sections were incubated overnight at room temperature (RT) with blocking buffer containing 10% horse serum, and 90% PBS (0.01 M, pH 7.2) in a humid chamber to inhibit nonspecific binding of immunoreagents. After overnight incubation, the lung sections were washed 5 times with PBS (this wash step was repeated after each incubation step) and the sections were incubated with 150 µL of primary antibody (1:50 dilution in blocking buffer) for 30 min at RT. The primary antibody was a mouse monoclonal antibody specific for chicken monocytes/macrophages (KUL01, Southern Biotechnology Associates, Birmingham, AL). After the 30 min incubation, the sections were washed with PBS and incubated with 150 µL of secondary antibody (1:100 dilution in blocking buffer) for 30 min at RT. The secondary antibody was biotinylated polyclonal horse anti-mouse IgG (Vector Laboratories Inc., Burlingame, CA). To detect binding of the primary and secondary antibody, the sections were incubated in 150 µL of ABC reagent for 30 min at RT. The ABC (Avidin and biotinylated-horseradish-peroxidase complex) reagent was prepared by adding 10 µL of reagent A and 10 µL of reagent B (Vector Laboratories Inc.) in 1 mL of PBS according to the manufacturer’s instructions. Finally, the sections were incubated with 150 µL of substrate 3,3'-diaminobenzidine (Sigma-Aldrich Inc.) for 5 to 10 min depending on the color development. The substrate was charged by adding 3.3 µL of 0.3% H2O2 per milliliter of substrate prior to adding to the sections. After sufficient color development, the tissue sections were washed with PBS, counterstained with Methyl-Green stain (EMD Chemicals Inc. Gibbstown, NJ), dehydrated, and covered with aqueous mounting medium (Aquamount, Lerner Laboratories, Pittsburgh, PA) and a cover slip (VWR International, West Chester, PA) for microscopic observations. Immunostained lung sections were examined microscopically for macrophages using a computerized image analysis system comprised of a Cool SNAP cf digital camera (Image Processing Solutions Inc., North Reading, MA) and Image Pro Plus software (Media Cybernetics, Silver Spring, MD). A minimum of 6 microscope fields per section were evaluated at 40x magnification using an Olympus BX50 microscope (Olympus America Inc., Center Valley, PA). Macrophage counts were expressed as the percentage of the total tissue area examined that was occupied by immunostained (brown) cells (macrophages, % area).
NADPH Diaphorase Histochemistry
The NADPH diaphorase activity has been used as a histochemical marker for NOS enzyme localization (Mitchell et al., 1992; Beesley, 1995). The NADPH diaphorase histochemistry was performed as described by Moreno de Sandino and Hernandez (2003). Briefly, three 5-µm-thick sections per tissue were cut from frozen lung tissue using a cryostat, mounted on poly-L-lysine-coated slides, and were fixed in freshly prepared 2% buffered paraformaldehyde (TCI, Portland, OR) in PBS with 0.5% glucose (EMD Chemicals Inc.) for 10 min. Sections were then washed with wash buffer containing 0.1 M Tris HCl (Shelton Scientific Inc., Shelton, CT), and 0.05% Triton X-100 (Sigma-Aldrich Inc.). Three tissue sections (1 each for stain proper, negative control, and positive control) per lung tissue were incubated with 150 µL of negative control, positive control, and stain proper solution, for 60 min at 37°C in a humid chamber. Stain proper solution had 0.25 mg/mL of nitroblue tetrazolium, and 1 mg/mL of NADPH (Sigma-Aldrich Inc.) in 0.05% Triton X-100 in 0.1 M Tris buffer. The negative control solution was stain proper solution without NADPH, and the positive control solution contained stain proper solution and 15 mM p-nitrophenylphosphate (Southern Biotechnology Associates). After 60 min, the sections were washed with wash buffer, air dried, and covered with cover slips after addition of aqueous mounting medium. The tissue sections were then evaluated for NOS localization as evidenced by blue staining.
Real-Time Reverse Transcription-PCR
RNA Isolation and Quantification. Ten to fifteen 100-µm-thick frozen lung sections/lung (24 to 26 mg of tissue) were used for RNA isolation. The RNA was isolated using RNeasy mini kit including an additional DNA digestion step for 15 min with RNase-free DNase (Qiagen Inc., Valencia, CA).
The concentration of isolated RNA was determined using the Ribogreen RNA quantitation kit (Molecular Probes, Eugene, OR) following the high range assay protocol (20 ng/mL to 1 µg/mL of RNA). Briefly, the standards for high range assay were prepared as shown in Table 1
. The RNA samples were diluted 1:200 in 1x Tris EDTA buffer. Blanks, standards, and samples (100 µL each) in duplicate were pipetted into their designated wells in a 96-well microtiter plate. Ribogreen reagent (100 µL; 1:200 dilutions) was then added to each well, and the plate was incubated at RT for 5 min. The fluorescence was recorded in a fluorescence microplate reader (BioTek Instruments Inc., Winooski, VT) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The concentration of RNA was calculated based on the standard curve (fluorescence vs. RNA concentration) generated from the set of RNA standards included in each plate.
|
Real Time PCR and Quantification of Relative mRNA Expression. Real-time PCR was performed using the Taqman PCR Core Reagent Kit (Applied Biosystems) and an ABI PRISM 7700 sequence detection system (Applied Biosystems). The PCR was performed in a reaction volume of 25 µL containing the following reagents at final concentration: 1x Taqman buffer A, 5.5 mM MgCl2, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400 µM dUTP, forward primer 200 nM, reverse primer 200 nM, Probe 100 nM, 0.01 unit/µL of AmpErase UNG, 0.025 unit/µL of Amplitaq Gold DNA polymerase, and 2 µL of cDNA sample. The cycling profiles used were one cycle at 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s, and 60°C for 60 s.
Previously published primers and probes for 28S and chicken iNOS (Smith et al., 2005) were used for PCR. The primer and probe sequences are given in Table 2. In each plate, a no template control (no cDNA, master mix only), a calibrator sample, cDNA samples, a set of 5 standards for iNOS (dilutions: 100, 10–1, 10–2, 10–3, and 10–4), and 28S (dilutions: 10–2, 10–3, 10–4, 10–5, and 10–6) were used. The 28S was used as an endogenous control. The cDNA used for standards was synthesized from RNA isolated from an LPS-stimulated chicken macrophage cell line (MQ-NCSU) 6 h post-LPS stimulation. The relative quantification of iNOS mRNA expression was carried out by using the cycle threshold value by relative standard curve method as described in ABI PRISM 7700 sequence detection system, user bulletin no. 2 (Applied Biosystems).
|
For the macrophage count (% area) and the relative expression level of iNOS mRNA, 1-way ANOVA was carried out to determine line difference within a time point and also the time point difference within a line using SYSTAT statistical software (version 10.2, Systat Software Inc., San Jose, CA). Differences among the group means were determined by Fisher’s LSD multiple mean comparison test. Data were expressed as mean ± SEM, and the differences were considered to be significant at P 
0.05.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
In the lungs of uninjected control (0 h) broilers, immunohistochemical staining with the KUL01 antibody revealed only few evenly distributed monocytes/macrophages (Figure 1A). Following MP injection, immunohistochemistry revealed macrophage infiltration in the vicinity of entrapped MP in all of the lung tissues (Figure 1A). By 2 h the MP-occluded pulmonary arterioles were outlined by very strong KUL01 staining, which may be due to monocytes surrounding the particle inside the vessels as well as macrophages infiltrating the perivascular region of the occluded arterioles. Moreover, small macrophage foci were clearly evident in the perivascular region of the occluded arterioles. By 24 h the infiltration of macrophages was more intense, and many more macrophages were seen in the perivascular area of MP-occluded arterioles. By 48 h, the macrophage infiltration became more generalized and incorporated almost the whole area of the lung sections (Figure 1A).
|
). For the RES line, the macrophage count increased steadily from a control level of 4.61 ± 0.16% at 0 h to 8.60 ± 0.14% at 48 h, whereas for the SUS line the macrophage count increased from 3.58 ± 0.14% at 0 h to 6.13 ± 0.15% at 24 h but did not increase further by 48 h (5.78 ± 0.16%; Figure 2). The present study demonstrates the time-course of macrophage infiltration following MP injection and confirms the results of a previous study (Wang et al., 2003) demonstrating that cellulose MP entrapped in the pulmonary vasculature acutely initiate marked, focal inflammatory responses within the lung parenchyma of broilers. The higher macrophage count and the sustained increase in macrophage infiltration in the RES line, compared with the SUS line over the 48-h time period examined, suggests that the RES line’s ability to better cope with and control the MP-induced PAH may be due, in part, to an inherent superiority in mounting a fast and appropriate inflammatory response able to modulate the vascular challenge initiated by the i.v. MP injection.
|
In the context of MP-induced PAH, NO produced by NOS may be critical for counteracting the MP-induced vasoconstriction. The NOS activity in the lung tissue of MP-injected broilers from the RES and SUS line was examined by NADPH diaphorase histochemistry as described by Moreno de Sandino and Hernandez (2003). In this assay, blue color development (seen as dark staining in the black and white pictures) indicates the location of NOS activity, which was the primary objective using this staining method (Figure 1B
). The NOS activity was not observed at 0 h in uninjected controls, but within 2 h after MP injection dark staining could be observed around the vessels occluded with the MP. The staining was also observed in the area of the perivascular macrophage infiltrate, but the intensity of the stain was highest in the immediate vicinity of the occluded vessels. Furthermore, the intensity as well as the area involved in NOS activity increased consistently from 2 to 48 h (Figure 1B). The NADPH diaphorase histochemistry is a marker for the localization of NOS enzyme, rather than a quantitative estimate of NOS activity (Mitchell et al., 1992; Beesley, 1995), and therefore could not be used for quantitative comparison between RES and SUS birds in this study. Additionally, this staining procedure does not identify a specific NOS isoform. However, this staining procedure clearly showed that the location, intensity, and extent of NOS activity coincided with the location and amount of macrophage infiltration (% area) from 0 to 48 h and hence would be primarily indicative of iNOS activity.
Relative iNOS mRNA Expression
To specifically address the involvement of iNOS in the response to MP lodged in the pulmonary vasculature, the relative iNOS mRNA expression in the broiler lung tissue was examined by real time RT-PCR methodology, and the results were expressed as fold change. Line differences were observed in the relative iNOS mRNA expression at 24 h post-MP injection (P = 0.001) but not at the 0, 2, and 48 h sampling intervals (Figure 3). For the RES line the iNOS mRNA expression increased consistently from 0 h (2.70 ± 0.08) to 48 h (24.90 ± 6.47), but for the SUS line iNOS mRNA expression was biphasic and increased at 2 h (10.80 ± 2.58), decreased to base line value at 24 h (2.38 ± 0.62), and increased again at 48 h (26.20 ± 4.40) (Figure 3). This study demonstrates for the first time in situ iNOS mRNA expression in lungs within 2 h post i.v. MP injection. Maximal levels in iNOS expression were achieved at 48 h post-MP injection for both the RES and SUS lines. In an in vitro study, Hussain and Qureshi (1997) examined the expression of iNOS by cells from the MQ-NCSU macrophage cell line and cephadex-elicited abdominal macrophages from 3 different chicken strains [Cornell K strain, GB1 (B13B13), and GB2 (B6B6)] following in vitro stimulation with LPS. They reported that iNOS expression in the macrophages peaked at 6 h post-LPS stimulation and that the amount of NO produced by the macrophages was directly proportional to iNOS mRNA expression (i.e., high in high NO-producing Cornell K-strain and MQ-NCSU macrophages; low in low NO-producing GB1 and GB2 macrophages). In an another study, Bowen et al. (2007) reported that plasma NO concentrations in response to i.v. LPS injection in male broiler chickens peaked between 5 to 6 h post-LPS injection; however, following i.v. MP injection plasma NO concentrations did not increase throughout the 12-h study period. The difference in the plasma NO concentration following the i.v. injection of LPS compared with MP may be due to the fact that LPS is able to systemically activate monocytes/macrophages, whereas the MP act locally within the vicinity of the obstructed pulmonary microvasculature. Hence, the extent of the local MP-induced iNOS activation in the lung may be sufficient to elicit a biological response but not sufficient to cause an increase in plasma NO concentrations. Previous studies suggest that NO produced in the immediate vicinity of pulmonary vascular smooth muscle can have a significant biological impact by dilating the local vasculature without necessarily elevating total plasma NO concentrations (Chapman and Wideman, 2006; Bowen et al., 2007). Moreover, as the MP are entrapped in the pulmonary vasculature the stimulus of entrapped MP persists for a long time and macrophage recruitment and activation continue for > 48 h, whereas, for LPS, the single i.v. dose likely dissipates and thus the stimulus for iNOS activation subsides rapidly. For the 12 h sampling window used by Bowen et al. (2007), the local pulmonary iNOS expression and macrophage recruitment in response to the MP injection was in the initial stages, and hence NO production was likely not sufficiently high to be noted in the circulation. Whether local pulmonary NO production in MP injected broilers may be reflected in the plasma at later time points when peak macrophage infiltration is in progress remains to be determined.
|
|
ACKNOWLEDGMENTS |
|---|
Received for publication November 20, 2007. Accepted for publication December 26, 2007.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Bowen, O. T., G. F. Erf, M. E. Chapman, and R. F. Wideman Jr. 2007. Plasma nitric oxide concentrations in broilers after intravenous injections of lipopolysaccharide or microparticles. Poult. Sci. 86:2550–2554.
Chang, C. C., C. C. McCormick, A. W. Lin, R. R. Dietert, and Y. J. Sung. 1996. Inhibition of nitric oxide synthase gene expression in vivo and in vitro by repeated doses of endotoxin. Am. J. Physiol. 271:G539–G548.[Web of Science][Medline]
Chapman, M. E., and R. F. Wideman. 2006. Evaluation of total plasma nitric oxide concentrations in broilers infused intravenously with sodium nitrite, lipopolysaccharide, aminoguanidine, and sodium nitroprusside. Poult. Sci. 85:312–320.
Grabarevic, Z., M. Tisljar, B. Artukovic, M. Bratulic, P. Dzaja, S. Seiwerth, P. Sikiric, J. Peric, D. Geres, and J. Kos. 1997. The influence of BPC 157 on nitric oxide agonist and antagonist induced lesions in broiler chicks. J. Physiol. (Paris) 91:139–149.[CrossRef][Web of Science][Medline]
Hauschildt, S., P. Scheipers, and W. G. Bessler. 1991. Inhibitors of poly (ADP-ribose) polymerase suppress lipopolysaccharide-induced nitrite formation in macrophages. Biochem. Biophys. Res. Commun. 179:865–871.[CrossRef][Web of Science][Medline]
Hussain, I., and M. A. Qureshi. 1997. Nitric oxide synthase and mRNA expression in chicken macrophages. Poult. Sci. 76:1524–1530.
Mitchell, J. A., K. L. Kohlhaas, T. Matsumoto, J. S. Pollock, U. Forstermann, T. D. Warner, H. H. Schmidt, and F. Murad. 1992. Induction of NADPH-dependent diaphorase and nitric oxide synthase activity in aortic smooth muscle and cultured macrophages. Mol. Pharmacol. 41:1163–1168.[Abstract]
Moncada, S., A. Higgs, and R. Furchgott. 1997. International Union of Pharmacology nomenclature in nitric oxide research. Pharmacol. Rev. 49:137–142.
Moreno de Sandino, M., and A. Hernandez. 2003. Nitric oxide synthase expression in the endothelium of pulmonary arterioles in normal and pulmonary hypertensive chickens subjected to chronic hypobaric hypoxia. Avian Dis. 47:1291–1297.[CrossRef][Web of Science][Medline]
Nathan, C. 1992. Nitric oxide as a secretory signal of mammalian cells. FASEB J. 6:3051–3064.[Abstract]
National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC.
Pavlidis, H. O., J. M. Balog, L. K. Stamps, J. D. Hughes Jr., W. E. Huff, and N. B. Anthony. 2007. Divergent selection for ascites incidence in chickens. Poult. Sci. 86:2517–2529.
Smith, C. K., P. Kaiser, L. Rothwell, T. Humphrey, P. A. Barrow, and M. A. Jones. 2005. Campylobacter jejuni-induced cytokine responses in avian cells. Infect. Immun. 73:2094–2100.
Wang, W., R. F. Wideman, T. K. Bersi, and G. F. Erf. 2003. Pulmonary and hematological inflammatory responses to intravenous cellulose micro-particles in broilers. Poult. Sci. 82:771–780.
Weidong, S., W. Xiaolong, W. Jinyong, and J. Ruiping. 2002. Pulmonary arterial pressure and electrocardiograms in broiler chickens infused intravenously with L-NAME, an inhibitor of nitric oxide synthase, or sodium nitroprusside (SNP), a nitric oxide donor. Br. Poult. Sci. 43:306–312.[CrossRef][Web of Science][Medline]
Wideman, R. F. 2001. Pathophysiology of heart/lung disorders: Pulmonary hypertension syndrome in broiler chickens. World’s Poult. Sci. J. 57:289–307.[CrossRef][Web of Science]
Wideman, R. F., M. E. Chapman, K. R. Hamal, O. T. Bowen, A. G. Lorenzoni, G. F. Erf, and N. B. Anthony. 2007. An inadequate pulmonary vascular capacity and susceptibility to pulmonary arterial hypertension in broilers. Poult. Sci. 86:984–998.
Wideman, R. F., and G. F. Erf. 2002. Intravenous micro-particle injection and pulmonary hypertension in broiler chickens: Cardiopulmonary hemodynamic responses. Poult. Sci. 81:877–886.
Wideman, R. F., G. F. Erf, and M. E. Chapman. 2005. Nomeganitro-L-arginine methyl ester (L-NAME) amplifies the pulmonary hypertensive response to microparticle injections in broilers. Poult. Sci. 84:1077–1091.
Wideman, R. F., G. F. Erf, M. E. Chapman, W. Wang, N. B. Anthony, and L. Xiaofang. 2002. Intravenous micro-particle injections and pulmonary hypertension in broiler chickens: Acute post-injection mortality and ascites susceptibility. Poult. Sci. 81:1203–1217.
Zapol, W. M., S. Rimar, N. Gillis, M. Marletta, and C. H. Bosken. 1994. Nitric oxide and the lung. Am. J. Respir. Crit. Care Med. 149:1375–1380.[Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  K. R. Hamal, R. F. Wideman, N. B. Anthony, and G. F. Erf Differential expression of vasoactive mediators in microparticle-challenged lungs of chickens that differ in susceptibility to pulmonary arterial hypertension Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2010; 298(1): R235 - R242. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
