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Transpulmonary Pressure Gradient Verifies Pulmonary Hypertension is Initiated by Increased Arterial Resistance in Broilers
Transpulmonary Pressure Gradient Verifies Pulmonary Hypertension is Initiated by Increased Arterial Resistance in Broilers A. G. Lorenzoni,1 N. B. Anthony, and R. F. Wideman Jr. Department of Poultry Science, University of Arkansas, Fayetteville 72701
Key words: broiler, pulmonary hypertension, transpulmonary pressure gradient 2008 Poultry Science 87:125–132 doi:10.3382/ps.2007-00178
which can be substituted into equation 1 to generate equation 2: (PAP − downstream pressure) = (CO × PVR). Equation 2 can be rearranged as equation 3: PAP = [(CO × PVR) + downstream pressure] to demonstrate that PAP must increase in direct multiplicative proportion to independent or combined increases in CO and PVR, and in 1 to 1 direct additive proportion in case of isolated increases in downstream pressure (Chemla et al., 2002). To establish the physiological basis for differences in PAP between PHS-susceptible and PHS-resistant broilers, contemporaneous measurements of PAP, downstream pressure, CO, and PVR must be obtained. Estimation of the downstream pressure is essential for differentiating between pulmonary venous hypertension (PVH) and pulmonary arterial hypertension (PAH) (Benza and Tallaj, 2006). Pulmonary venous hypertension is a consequence of an increased resistance to pulmonary blood flow downstream from the capillaries (through the pulmonary veins or left atrium) and is commonly caused by left-sided valvular or myocardial diseases (Simonneau et al., 2004). Thus PVH ensues whenever the left atrio-ventricular valve is incompetent to direct the complete stroke volume (SV) through the aorta or when myocardial disease prevents the weakened left ventricle from pumping all of the
INTRODUCTION Previous hemodynamic evaluations demonstrated that pulmonary arterial pressure (PAP) is higher in broilers that are susceptible to pulmonary hypertension syndrome (PHS, ascites) than in broilers that are resistant to PHS (Wideman et al., 2000, 2006; Chapman and Wideman, 2001; Bowen et al., 2006). These differences in PAP may be caused by differences in cardiac output (CO), pulmonary vascular resistance (PVR), or the pressure gradient across the pulmonary circulation (transpulmonary pressure gradient, TPG) as defined by equation 1: TPG = (CO × PVR). The TPG or pressure drop (⌬P) between pulmonary arteries and pulmonary veins is defined as the difference between precapillary pressure (PAP) and postcapillary (downstream) pressure caused by resistance to blood flow through the pulmonary veins into the left atrium. Accordingly, TPG or ⌬P equals (PAP − downstream pressure),
©2008 Poultry Science Association Inc. Received May 1, 2007. Accepted October 4, 2007. 1 Corresponding author: [email protected]
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catheterized to measure pressures in the wing vein, right atrium, right ventricle, pulmonary artery, and pulmonary veins (WP, wedge pressure). The transpulmonary pressure gradient (TPG) was calculated as (PAP-WP), with PAP quantifying precapillary pressure and WP approximating postcapillary pulmonary venous pressure. When compared with resistant and relaxed broilers, PAP values in susceptible broilers were ≥10 mmHg higher, TPG values were ≥8 mmHg higher, and WP values were ≤2 mmHg higher, regardless of sex. The combined hemodynamic criteria (elevated PAP and PVR combined with a proportionally elevated TPG) demonstrate that susceptibility to PHS can be attributed primarily to pulmonary arterial hypertension associated with increased precapillary (arteriole) resistance rather than to pulmonary venous hypertension caused by elevated postcapillary (venous and left atrial) resistance.
ABSTRACT Previous hemodynamic evaluations demonstrated that pulmonary arterial pressure (PAP) is higher in broilers that are susceptible to pulmonary hypertension syndrome (PHS, ascites) than in broilers that are resistant to PHS. We compared key pulmonary hemodynamic parameters in broilers from PHS-susceptible and PHS-resistant lines (selected for 12 generations under hypobaric hypoxia) and in broilers from a relaxed (control) line. In experiment 1 the PAP was measured in male broilers in which a flow probe positioned on one pulmonary artery permitted the determination of cardiac output and pulmonary vascular resistance (PVR). The PAP and relative PVR were higher in susceptible broilers than in relaxed and resistant broilers, whereas absolute and relative cardiac output did not differ between lines. In experiment 2 male and female broilers from the 3 lines were
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blood returning from the lungs (e.g., left-sided congestive heart failure). The resulting accumulation of excessive blood volume and pressure in the left atrium and pulmonary veins would force the right ventricle to increase the
MATERIALS AND METHODS Exposure to hypobaric hypoxia was used to select PHSsusceptible and PHS-resistant lines of broilers (Pavlidis
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Figure 1. Body weight, right: total ventricle weight (RV:TV), pulmonary arterial pressure (PAP), venous pressure (VP), relative pulmonary vascular resistance (PVR/BW), and relative cardiac output (CO/BW) for male broilers in experiment 1 from pulmonary hypertension syndrome (PHS)-susceptible (n = 14), PHS-resistant (n = 15) and relaxed selection (n = 15) lines during blood flow experiments. Closed circles represent means ± SEM. a,bLetters represent differences (P ≤ 0.05) between the broiler lines within a variable.
PAP to overcome the increased downstream (postcapillary) resistance and propel the requisite CO through the lungs. Because the increase in PAP is normally proportional to the increase in downstream pressure (HermoWeiler et al., 1998), PVH is characterized by elevated pulmonary arterial, pulmonary venous and left atrial pressures combined with a normal or reduced TPG and a normal PVR. In contrast, PAH occurs when the right ventricle must elevate PAP to overcome increased resistance to flow through the pulmonary arterioles upstream of the pulmonary capillaries and veins. The typical hemodynamic characteristics of PAH include elevated PAP, PVR, and TPG combined with normal pulmonary venous and left atrial pressures (Chemla et al., 2002). The present study was conducted to establish the hemodynamic basis for differences in PAP between PHS-susceptible, PHS-resistant, and relaxed broiler lines selected for 12 generations under conditions of hypobaric hypoxia (Pavlidis et al., 2007). The PAP, CO, and PVR were measured as described previously (Guthrie et al., 1987; Wideman et al., 1996, 2005; Wideman, 1999). Downstream pressure (essentially postcapillary pulmonary venous pressure) was estimated by measuring the wedge pressure (WP; Chapman and Wideman, 2001). Wedge pressures are obtained by advancing a catheter through the pulmonary arterial branches until the tip of the catheter reaches an artery of a similar or smaller diameter, causing the catheter’s tip to wedge in and thereby occlude blood flow from the upstream arterial tree. The pressure in small arterioles and capillaries connected to the occluded artery drops as some of the blood contained in these vessels drains into the capillaries and veins. A motionless column of blood then remains between the catheter tip and the downstream vasculature. This column transmits the blood pressure from the venous region, allowing the measurement of the postcapillary downstream blood pressure via the wedged catheter. Increases in the WP can be indicative of increased venous resistance, increased left atrial pressure, or both, which are symptoms of PVH (Zidulka and Hakim, 1985). Measuring the WP allows TPG to be calculated as PAP − WP (Chemla et al., 2002; Benza and Tallaj, 2006), and WP can be substituted for downstream pressure in equation 3 to derive equation 4: PAP = [(CO × PVR) + WP]. Equation 4 illustrates that if CO remains constant during the onset of pulmonary hypertension, then PAP must increase in direct 1 to 1 additive proportion to WP when the underlying cause is PVH, or in direct multiplicative proportion to PVR when the underlying cause is PAH. A previous study did not include measurements of CO but did implicate PAH rather than PVH as the cause of pulmonary hypertension in broilers, based on the absence of differences in WP in spite of very large differences in PAP for males from 3 different lines that were evaluated following exposure to thermoneutral or cool environmental temperatures (Chapman and Wideman, 2001).
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et al., 2007). After the eighth generation, the resistant and susceptible broilers exhibited 26.0 and 98.6% ascites mortality, respectively, when exposed to hypobaric conditions (Pavlidis, 2003). The progeny from the 12th generation of these lines, and from an unselected relaxed commercial pedigree line, were transported on the day of hatch (June 30, 2006) to the Poultry Environmental Research Laboratory at the University of Arkansas Poultry Research Farm. Birds were wing-banded and placed on fresh wood shavings litter in environmental chambers (8 m2 floor space). The birds were brooded at 33°C from d 1 to 3, 31°C from d 4 to 6, 29°C from d 7 to 10, 26°C from d 11 to 14, and 24°C thereafter. Birds were fed a 23% CP corn-soybean meal-based diet formulated to meet the National Research Council (1994) standards for all ingredients. Feed and water were provided ad libitum. Feed was provided as crumbles throughout the experiment. Lights were on for 24 h/d through d 4, and 16 h/d thereafter.
Experiment 1. Evaluation of Pulmonary Blood Flow Between 34 and 42 d of age male broilers from the 3 lines were weighed and anesthetized using intramuscular injections of allobarbital (5, 5-diallyl-barbituric acid,
Sigma Chemical Co., St. Louis, MO; 16 mg/kg of BW) and ketamine HCl (40 mg/kg of BW). They were fastened in dorsal recumbency on a surgical board. After a subcutaneous injection of 1 cc of 2% lidocaine (Interstate Drug Exchange Inc., Amityville, NY) HCl, an incision was made to open the thoracic inlet, and a Transonic 3SB or 4SB ultrasonic flow probe (Transonic Systems Inc., Ithaca, NY) connected to a Transonic T206 blood flow meter (Transonic Systems Inc.) was positioned on the left pulmonary artery. The skin of the thoracic inlet was sealed air-tight with surgical wound clips. Lidocaine was injected subcutaneously around the basilica vein, then the proximal end of a Silastic catheter (0.012 in inside diameter, 0.025 in outside diameter, Dow Corning Corp., Midland, MI) filled with heparinized saline (200 IU of heparin/mL of 0.9% NaCl) was inserted into the basilica vein. The distal end of the catheter was attached to a blood pressure transducer (World Precision Instruments, Sarasota, FL) interfaced through a Transbrige preamplifier (World Precision Instruments) to a Biopac MP100 data acquisition system using Acqknowledge software (Biopac Systems Inc., Goleta, CA). Venous pressure (VP) was recorded with the tip of the catheter inserted approximately 2 cm into the basilica vein, then the catheter was slowly advanced into the main trunk of the pulmonary artery where PAP was recorded as described previously (Guthrie et al., 1987; Wideman et al., 1996; Wideman, 1999).
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Figure 2. Blood pressure (BP) recordings from apparently clinically healthy broilers from pulmonary hypertension syndrome (PHS)-resistant and PHS-susceptible lines (upper and lower panels, respectively). The figure illustrates pressure measurements in experiment 2 as a catheter was inserted in the basilica vein (VP) and advanced through the right atrium (RA), right ventricle (RV), main trunk of the pulmonary artery (PAP), and through branches of the pulmonary artery (PA) until the cannula wedged (wedge) in a terminal arterial branch, followed by the recording of the pressures in the reverse order as the catheter was withdrawn.
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Experiment 2. Measurement of Wedge Pressure Between 42 and 50 d of age male and female broilers from the 3 lines were anesthetized and fastened in dorsal recumbency as described above. The basilica vein was cannulated with Silastic tubing and pressures were recorded as the catheter was slowly advanced through the basilica vein (VP), right atrium (RA: right atrial pressure), right ventricle (RV: right ventricular pressure), main trunk of the pulmonary artery (PAP), major branches of the pulmonary artery (PA), and onward until the tip of the catheter became wedged (WP), which was indicated
by a sudden drop in the PAP. A sudden rise in the PAP when the catheter was carefully withdrawn from its position confirmed the recording of WP. At the termination of each experiment, birds were euthanized with a 10mL i.v. injection of 0.1 M KCl. The heart was removed, dissected, and weighed for calculation of the right/total ventricular weight ratio (RV:TV) as an index of pulmonary hypertension (Burton et al., 1968).
Data Acquisition and Statistical Analyses The Biopac MP 100 data acquisition system continuously recorded 2 primary data channels, including PAP
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Figure 3. Open symbols represent the mean blood pressures ± SEM in experiment 2 of (A) males from the pulmonary hypertension syndrome (PHS)-susceptible (n = 15), PHS-resistant (n = 15), and relaxed (n = 16) lines; and (B) females from the PHS-susceptible (n = 16), PHS-resistant (n = 16), and relaxed (n = 16) lines. The pressures were recorded as a catheter advanced through the wing vein (VP), right atrium (RA), right ventricle (RV), and main trunk of the pulmonary artery (PAP), until wedge pressure was recorded (wedge). Closed symbols represent the transpulmonary gradient (TPG, the arithmetical subtraction of the wedge pressure from the PAP in each broiler) ± SEM. Data were analyzed using 2-way ANOVA. a,bLetters represent differences (P ≤ 0.05) between the broiler lines at a single anatomical location. w–zLetters represent differences (P ≤ 0.05) within each line between the different anatomical locations.
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INCREASED ARTERIAL RESISTANCE IN BROILERS Table 1. Absolute values for cardiac output (CO), pulmonary vascular resistance (PVR), heart rate (HR), and stroke volume (SV), and relative SV (SV/BW) for male broilers in experiment 1 from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed lines selection during blood flow experiments1 Susceptible (n = 14)
Variable CO (mL/min) PVR (relative units) HR (beats/min) SV (mL/beat) SV/BW (mL/beat × kg of BW)
310.5 0.114 343.2 0.91 0.44
± ± ± ± ±
Resistant (n = 15)
16.9 0.01a 9.2 0.05 0.02
296.7 0.088 341.8 0.87 0.45
± ± ± ± ±
Relaxed (n = 15)
22.0 0.01ab 7.1 0.06 0.03
286.4 0.081 326.4 0.89 0.48
± ± ± ± ±
16.4 0.01b 6.5 0.06 0.03
Superscripts designate differences (P ≤ 0.05) between broiler lines within a variable. Values represent mean ± SEM.
a,b 1
the F test from the 1-way ANOVA was declared significant (P < 0.05). The SigmaStat linear regression procedure was used to evaluate relationships between WP and PAP, and WP and RV:TV.
RESULTS Experiment 1. Evaluation of Pulmonary Blood Flow Body weight, RV:TV, PAP, VP, PVR normalized for BW, and CO normalized for BW are shown in Figure 1. Broilers from the resistant and relaxed lines did not differ for any of these variables, whereas broilers from the susceptible line had higher values for all variables with the exceptions of BW which was higher compared with the relaxed line but not different from the resistant line, and CO/BW that did not differ for any of the lines. Values for absolute CO and PVR, HR, SV, and SV normalized for BW were not different between lines except for PVR that was higher in the susceptible line compared with the relaxed line but not different from the resistant line (Table 1).
Table 2. Pressures and heart parameters in experiment 2 for male and female broilers from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed lines1 Males Variable n BW (g) VP (mmHg) RA (mmHg) RV (mmHg) PAP (mmHg) WP (mmHg) RV (g) LV+S (g) TV (g) RV:TV (g:g) RV/BW (g/kg) LV+S/BW (g/kg) TV/BW (g/kg)
Susceptible 15 2,140.8 ± 10.15 ± 9.07 ± 25.23 ± 36.60 ± 12.36 ± 1.92 ± 6.03 ± 7.95 ± 0.24 ± 0.90 ± 2.82 ± 3.72 ±
46.5 0.65a 0.57a 1.67a 2.24a 0.85a 0.13 0.21 0.28 0.01a 0.06 0.09 0.12
Resistant 2,073.1 7.36 6.20 18.93 26.20 9.98 1.61 5.94 7.54 0.21 0.78 2.87 3.65
15 ± ± ± ± ± ± ± ± ± ± ± ± ±
54.0 0.33b 0.35b 0.66b 1.06b 0.66ab 0.08 0.20 0.26 0.01ab 0.04 0.08 0.11
Females Relaxed 16 2,009.8 ± 6.58 ± 5.86 ± 16.85 ± 22.97 ± 9.07 ± 1.57 ± 5.87 ± 7.44 ± 0.21 ± 0.77 ± 2.92 ± 3.69 ±
53.4 0.35b 0.29b 0.55b 0.69b 0.58b 0.12 0.23 0.33 0.01b 0.05 0.06 0.10
Susceptible 16 2,098.9 ± 10.33 ± 8.93 ± 25.76 ± 34.82 ± 12.11 ± 1.78 ± 5.32 ± 7.11 ± 0.25 ± 0.85 ± 2.55 ± 3.41 ±
54.8b 0.70a 0.46a 1.34a 2.12a 0.60a 0.10 0.16 0.24 0.01a 0.04a 0.07 0.11a
Resistant 2,189.8 7.79 6.69 18.59 24.34 9.89 1.59 5.50 7.09 0.22 0.73 2.51 3.24
16 ± ± ± ± ± ± ± ± ± ± ± ± ±
32.9ab 0.47b 0.35b 0.72b 1.23b 0.55b 0.07 0.15 0.20 0.01b 0.03b 0.06 0.08ab
Relaxed 2,297.6 7.54 6.66 17.46 21.18 9.56 1.59 5.36 6.95 0.23 0.69 2.33 3.02
16 ± ± ± ± ± ± ± ± ± ± ± ± ±
31.5a 0.25b 0.25b 0.52b 1.03b 0.47b 0.09 0.20 0.28 0.01ab 0.04b 0.08 0.10b
Superscripts designate differences (P ≤ 0.05) between broiler lines within a variable and sex. Data are presented as mean ± SEM. VP = venous pressure; RA = right atrium; RV = right ventricle; PAP = pulmonary arterial pressure; WP = wedge pressure; LV+S = left ventricle plus septum; TV = total ventricle. a,b 1
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(mmHg) and blood flow through the left pulmonary artery (mL per min). Data were measured and averaged electronically over 10-s intervals from the Biopac recording. Based on the assumption that CO normally is divided approximately equally between 2 lungs of equal size, CO was calculated as 2 × blood flow. Heart rate (HR, beats per min) was obtained by counting systolic peaks over time in the PAP recording. Stroke volume (mL per beat) was calculated as CO/HR. Assuming that atrial pressure remains close to zero and has little impact on vasoconstrictive responses of pulmonary arterioles (Wideman and Bottje, 1993; Chapman and Wideman, 2001) the PVR was calculated in relative resistance units as PAP/CO (Sturkie, 1986; Wideman et al., 1998). The TPG (mmHg) was calculated as PAP − WP (Chemla et al., 2002; Benza and Tallaj, 2006). Cardiac output, PVR, and SV were calculated as absolute and relative (BW normalized) values to compensate for differences in BW when comparing birds of different size or age. Data in experiment 1 were analyzed using the SigmaStat 1-way ANOVA. In experiment 2 the SigmaStat 2-way ANOVA was used to evaluate sex (male, female) × line (susceptible, relaxed, resistant) interactions among pressure recorded at each anatomical location (Jandel Scientific, 1994). Means were separated by the Tukey test when
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LORENZONI ET AL. Table 3. Regression of wedge pressure vs. pulmonary arterial pressure in experiment 2 for male and female broilers from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed selection lines Line Susceptible Resistant Relaxed Susceptible Resistant Relaxed
Sex
n
Male Male Male Female Female Female
15 15 16 16 16 16
Experiment 2. Measurement of Wedge Pressure
Y Y Y Y Y Y
= = = = = =
0.12X + 7.93 −0.15X + 13.84 0.30X + 2.17 0.15X + 7.07 0.24X + 4.00 0.16X + 6.20
r2
P-value
0.10 0.06 0.13 0.27 0.29 0.12
0.25 0.40 0.18 0.04 0.03 0.19
from VP within any line and sex comparison, except for males from the relaxed line in which VP was slightly lower than WP. Wedge pressure was not different from RA in the susceptible line, but it was slightly higher than the RA in the resistant and relaxed lines within each sex. The TPG, calculated as PAP − WP, was markedly greater in males and females from the susceptible line when compared with their respective counterparts in the resistant and relaxed lines. The TPG did not differ between the resistant and relaxed lines. Body weight, weights of the RV, right and left ventricle plus septum (total ventricle, TV), left ventricle plus septum (LV+S), RV:TV, BW-normalized RV (RV/BW), BWnormalized LV+S (LV+S/BW), and BW-normalized TV (TV/BW) are shown for males and females in Table 2. The BW did not differ for males of the 3 lines, but in females the BW was higher in the relaxed line than in the susceptible line, whereas the BW in the resistant line was not different from the other lines. The RV did not differ between lines for males or females; however, males from the susceptible line tended to have values higher than the resistant and relaxed lines (P = 0.056). The RV:TV ratio was higher in males from the susceptible line than in the relaxed line, whereas males from the resistant line did not differ from the other lines. In females the resistant line had lower values for RV:TV than the susceptible line and the relaxed line was not different from the other lines. The RV/BW did not differ between the resistant and relaxed lines but it was higher in the susceptible line in females; in contrast, there were no differences for RV/ BW in males. The TV/BW did not differ between lines in males, but females from the susceptible line had a higher TV/BW than females from the relaxed line, and TV/BW values in the resistant line did not differ from the other lines. Values for LV+S, TV, and LV+S/BW did not differ between lines within sexes. The linear regression comparisons for WP vs. PAP within each sex and line were only significant for females from the susceptible
Table 4. Regression of wedge pressure vs. right to total ventricle weight ratio in experiment 2 for male and female broilers from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed selection lines Line Susceptible Resistant Relaxed Susceptible Resistant Relaxed
Sex
n
Male Male Male Female Female Female
15 15 16 16 16 16
Equation Y Y Y Y Y Y
= = = = = =
17.72X + 8.09 −1.20X + 10.24 29.41X + 2.97 18.34X + 7.54 35.24X + 2.01 −5.11X + 10.73
r2
P-value
0.05 0.00 0.16 0.05 0.15 0.01
0.42 0.97 0.12 0.40 0.14 0.80
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A typical recording for a clinically healthy broiler from the resistant line is shown in the upper panel of Figure 2 (RV:TV= 0.23; PAP = 27 mmHg), whereas the lower panel of Figure 2 illustrates the recording from an apparently clinically healthy broiler from the susceptible line (RV:TV= 0.28; PAP = 44.2 mmHg). After the catheter was inserted approximately 2 cm into the basilica vein, VP averaged 8.6 and 8.3 mmHg in the resistant and susceptible birds, respectively. The pressure averaged 7.2 mmHg in both birds when the catheter tip entered the right atrium, and rose to 20.8 and 26.5 mmHg in the resistant and susceptible birds, respectively, when the catheter reached the right ventricle. When the catheter entered the trunk of the pulmonary artery the PAP values averaged 27 and 44.2 mmHg in the resistant and susceptible birds, respectively. The arterial pressure subsided slightly as the catheter advanced through major branches of the PA. In both birds the pressure dropped dramatically when the catheter’s tip wedged inside a terminal branch of the pulmonary arterial tree, with WP averaging 11.64 and 13 mmHg in the resistant and susceptible birds, respectively. Figure 2 also shows the pressures recorded as the cannula was withdrawn (PAP, RV, RA, and VP). Group means for VP, RA, RV, PA, and WP are shown in Figure 3. No sex × line interactions (P < 0.05) were detected for the pressures measured at any of the anatomical locations. There were no differences in pressure within any of these anatomical locations when males (Figure 3A) or females (Figure 3B) from the resistant and relaxed lines were compared. In contrast, broilers from the susceptible line consistently had higher pressures at all anatomical locations than did their counterpart from the resistant and relaxed lines (Figure 3A). Within each line regardless of sex, PAP was higher than RV pressure, which in turn was higher than WP. The WP did not differ
Equation
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and resistant lines (Table 3). None of the regression comparisons for WP vs. RV:TV within the separate lines were significant, regardless of sex (Table 4).
DISCUSSION
pulmonary hemodynamic changes observed in PHS-susceptible broilers (e.g., elevated PVR, elevated TPG) do not conform to the criteria necessary for a diagnosis of PVH caused by left-sided valvular or myocardial diseases (low TPG, normal PVR, and elevated pulmonary venous and left atrial pressures). Indeed, a diagnosis of PVH would require direct 1 to 1 proportionality between increments in WP and PAP, accompanied by a normal TPG (Zidulka and Hakim, 1985; Chemla et al., 2002; Benza and Tallaj, 2006). For example, acute obstruction of the mitral valve in sheep causes corresponding increases of approximately 1 mmHg in PAP per each mmHg increased in the left atrial pressure (Hermo-Weiler et al., 1998). Similarly, replacing damaged mitral valves in humans reduces WP and PAP by a proportionality of 1 to 1 (Fawzy et al., 1996). In the present study marked elevations in PAP were associated with very modest increases in WP in broilers from the susceptible line, which may reflect an elevated pressure dissipation profile transmitted along the length of the pulmonary vasculature due to the large upstream increase in PAP, or modest increases in postcapillary resistance to blood flow through the pulmonary venous drainage of PHS-susceptible broilers (e.g., venoconstriction; Zidulka and Hakim, 1985). Finally, when a dilated and engorged right ventricle having an elevated end diastolic volume pushes the ventricular septum into the chamber of the left ventricle, the extent to which the left ventricular wall can expand against the pericardium and fill with blood may be limited. This form of left ventricular diastolic dysfunction caused by right ventricular dilation would cause the returning venous blood to pool in the left atrium and pulmonary veins, leading to a modest increase in WP. However, WP values were not significantly correlated with RV:TV ratios by linear regression analysis for males or females within any line, and any effect of right ventricular dilation on cardiopulmonary hemodynamics would only be anticipated after the evolution of severe pathological changes in the heart. Such changes clearly cannot serve as a primary or initiating event in the pathogenesis of PHS. We conclude that neither increases in downstream resistance, left myocardial failure nor mitral valve degeneration are involved in the initial pathogenesis of PHS in broilers. Consistently, in both studies, we observed greater PAP in the susceptible line when compared with the resistant and relaxed lines. This is in agreement with previous studies (Chapman and Wideman, 2001; Bowen et al., 2006) and indicates that the genetic selection for ascites susceptibility has been successful. The RV:TV values tended to be higher in the susceptible line within all groups; however, the RV:TV did not differ between the susceptible and resistant males in experiment 2 and between the susceptible and relaxed females in experiment 2. A similar situation occurred for comparisons of the RV/BW values in experiment 2, when females of the susceptible line had values higher than those of females in the resistant and relaxed lines, whereas for males there were no differences between lines. Because we selected clinically healthy birds for the cardiopulmonary evalua-
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The present study was designed to evaluate key pulmonary hemodynamic characteristics of broilers from PHSsusceptible, PHS-resistant, and relaxed lines to determine the origin of PHS (e.g., PAH vs. PVH) as well as to provide a complete characterization of the pressures within the pulmonary circulation in these lines. Pulmonary hemodynamic characteristics can be used as selection criteria for broiler lines (Wideman and French, 1999). To determine the early (initiating) hemodynamic differences between these lines, all broilers were reared under moderate conditions (e.g., thermoneutral temperatures, 16L:8D photoperiod, feed provided as crumbles) and only broilers that appeared to be clinically healthy (e.g., noncyanotic, nonascitic individuals) were used for pulmonary blood flow and pressure measurements. In both experiments the PAP was higher in broilers from the PHS-susceptible line than in broilers from the relaxed and PHS-resistant lines. Data collected in experiment 1 demonstrated that the higher PAP in broilers from the susceptible line cannot be attributed to increases in the absolute or relative CO when compared with broilers from the relaxed or resistant lines. Instead, the pulmonary hypertension in broilers from the susceptible line was caused by an elevated PVR normalized for BW. In experiment 2 we measured the WP and calculated the TPG to differentiate between PAH and PVH as the primary underlying cause of pulmonary hypertension in broilers. The combined data from both experiments demonstrated that, when compared with broilers from the PHS-resistant and relaxed lines, broilers from the PHS-susceptible line had elevated PAP and PVR values combined with directly proportional increments in TPG and minimal increases in WP. These pulmonary hemodynamic characteristics must be attributed primarily to PAH rather than to PVH, based on the relationships established in equation 4 (see Introduction; Chemla et al., 2002; Benza and Tallaj, 2006). A modestly higher WP was observed when birds from the susceptible line were compared with birds from the relaxed and resistant lines. However, these modest increases in WP values in susceptible males and females did not account for the much larger increases in PAP and TPG values. Overall the PAP values in susceptible broilers were ≥10 mmHg higher and TPG values were ≥8 mmHg higher when compared with relaxed and resistant broilers, whereas the WP was ≤2 mmHg higher in susceptible broilers when compared with relaxed or resistant broilers. The association between WP and PAP was not significant by linear regression analysis for males within any of the lines or for females from the relaxed line, but WP and PAP were significantly correlated in females from the susceptible and resistant lines. These modest increases in WP cannot account for the much greater increases in PAP for PHS-susceptible broilers, and the accompanying
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ACKNOWLEDGMENTS This research was supported by USDA/CSREES/NRI grant 2003-35204-13392.
REFERENCES Benza, R. L., and J. A. Tallaj. 2006. Pulmonary hypertension out of proportion to left heart disease. Adv. Pulm. Hypertens. 5:21–29. Bowen, O. T., G. F. Erf, N. B. Anthony, and R. F. Wideman. 2006. Pulmonary hypertension triggered by lipopolysaccharide in ascites-susceptible and -resistant broilers is not amplified by aminoguanidine, a specific inhibitor of inducible nitric oxide synthase. Poult. Sci. 85:528–536. Burton, R. R., E. L. Besh, and A. H. Smith. 1968. Effect of chronic hypoxia on the pulmonary arterial blood pressure of the chicken. Am. J. Physiol. 214:1438–1442. Chapman, M. E., and R. F. Wideman. 2001. Pulmonary wedge pressures confirm pulmonary hypertension in broilers is initiated by an excessive pulmonary arterial (precapillary) resistance. Poult. Sci. 80:468–473. Chemla, D., V. Castelain, P. Herve´, Y. Lecarpentier, and S. Brimioulle. 2002. Haemodynamic evaluation of pulmonary hypertension. Eur. Respir. J. 20:1314–1331. Fawzy, M. E., L. M. Mimish, V. Sivanandam, J. Lingamanaicker, A. Patel, B. Khan, and C. M. Duran. 1996. Immediate and long-term effect of mitral ballon valvotomy on severe pulmonary hypertension in patients with mitral stenosis. Am. Heart J. 131:89–93. Guthrie, A. J., J. A. Cilliers, F. W. Huchzermeyer, and V. M. Killeen. 1987. Broiler pulmonary hypertension syndrome. II. The direct measurement of right ventricular and pulmonary artery pressures in the closed chest domestic fowl. J. Vet. Res. 54:599–602. Hermo-Weiler, C. I., T. Koizumi, R. Parker, and J. H. Newman. 1998. Pulmonary vasoconstriction induced by mitral valve obstruction in sheep. J. Appl. Physiol. 85:1655–1660.
Jandel Scientific. 1994. SigmaStat威 Statistical Software User’s Manual. Jandel Scientific Software, San Rafael, CA. Liu, X. 2001. Effect of cold stress or bronchus challenge on ascites resistant or susceptible lines of chickens. MSc Diss. Univ. Arkansas, Fayetteville. Lorenzoni, A. G. 2006. Effects of alpha-tocopherol and L-arginine on cardiopulmonary function in broilers. MSc Diss. McGill Univ., Montreal. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Pavlidis, H. O. 2003. Correlated responses to divergent selection for ascites in broilers. MS Thesis. Dep. Poult. Sci., Univ. Arkansas, Fayetteville. 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. Simonneau, G., N. Galie`, L. J. Rubin, D. Langleben, W. Segger, G. Dominighetti, S. Gibbs, D. Lebrec, R. Speich, M. Beghetti, S. Rich, and A. Fishman. 2004. Clinical classification of pulmonary hypertension. J. Am. Coll. Cardiol. 43:5S–12S. Sturkie, P. D. 1986. Heart and circulation: Aanatomy, hemodynamics, blood pressure, blood flow. Pages 130–166 in: Avian Physiology. 4th ed. P. J. Sturkie, ed. Springer-Verlag, New York, NY. Wideman, R. F. 1999. Cardiac output in four, five, and six-weekold broilers, and hemodynamic responses to intravenous injections of epinephrine. Poult. Sci. 78:392–403. Wideman, R. F., and W. G. Bottje. 1993. Current understanding of the ascites syndrome and future research directions. Pages 1–20 in Nutr. Technical Symp. Proc. Novus Int. Inc., St. Louis, MO. Wideman, R. F., O. T. Bowen, G. F. Erf, and M. E. Chapman. 2006. Influence of aminoguanidine, an inhibitor of inducible nitric oxide synthase, on the pulmonary hypertensive response to microparticle injection in broilers. Poult. Sci. 85:511–527. 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., G. F. Erf, and M. E. Chapman. 2005. Nω-NitroL-arginine methyl ester (L-NAME) amplifies the pulmonary hypertensive response to microparticle injections in broilers. Poult. Sci. 84:1077–1091. Wideman, R. F., M. R. Fedde, C. D. Tackett, and G. E. Weigle. 2000. Cardio-pulmonary function in preascitic (hypoxemic) or normal broilers inhaling ambient air or 100% oxygen. Poult. Sci. 79:415–425. Wideman, R. F., M. F. Forman, J. D. Hughes, Y. K. Kirby, N. Marson, and N. B. Anthony. 1998. Flow-dependent pulmonary vasodilation during acute unilateral pulmonary artery occlusion in Jungle Fowl. Poult. Sci. 77:615–626. Wideman, R. F., and H. French. 1999. Broiler survivors of chronic unilateral pulmonary artery occlusion produce progeny resistant to pulmonary hypertension syndrome (ascites) induced by cool temperatures. Poult. Sci. 78:404–411. Wideman, R. F., Y. K. Kirby, C. D. Tackett, N. E. Marson, and R. W. McNew. 1996. Cardio-pulmonary function during acute unilateral occlusion of the pulmonary artery in broilers fed diets containing normal or high levels of arginine-HCl. Poult. Sci. 75:1587–1602. Wideman, R. F., P. Maynard, and W. Bottje. 1999. Venous blood pressure in broilers during acute inhalation of five percent carbon dioxide or unilateral pulmonary artery occlusion. Poult. Sci. 78:1443–1451. Zidulka, A., and T. S. Hakim. 1985. Wedge pressure in large vs. small pulmonary arteries to detect pulmonary venoconstriction. J. Appl. Physiol. 59:1329–1332.
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tions, these observations strongly suggest that the earliest changes associated with pulmonary hypertension are detected through direct measurements of PAP. Differences in RV weights in birds undergoing different degrees of pulmonary hypertension presumably develop after differences in PAP have been sustained more chronically (Wideman et al., 2000; Liu, 2001; Lorenzoni, 2006). Blood pooling in large systemic veins may be one of the earliest hemodynamic changes that indicates the onset of rightsided congestive heart failure, occurring before the RV undergoes work hypertrophy as it propels the requisite CO through the constricted pulmonary vasculature (Wideman et al., 1999). The early onset of a mild rightsided congestion would explain the consistent increase in VP and not in RV mass in the susceptible line. Under the conditions of this study, the resistant and relaxed broiler lines did not differ for any of the cardio-pulmonary parameters studied. Nevertheless it is expected that under chronic exposure to periods of PHS-inducing stressful conditions (e.g., cool temperatures, faster growth, >20 h/d photoperiod) the unselected line would present hemodynamic values (especially for PAP and PVR) intermediate to the values of the susceptible and resistant lines, as was demonstrated previously (Chapman and Wideman, 2001; Bowen et al., 2006; Wideman et al., 2007).
Key words: broiler, pulmonary hypertension, transpulmonary pressure gradient 2008 Poultry Science 87:125–132 doi:10.3382/ps.2007-00178
which can be substituted into equation 1 to generate equation 2: (PAP − downstream pressure) = (CO × PVR). Equation 2 can be rearranged as equation 3: PAP = [(CO × PVR) + downstream pressure] to demonstrate that PAP must increase in direct multiplicative proportion to independent or combined increases in CO and PVR, and in 1 to 1 direct additive proportion in case of isolated increases in downstream pressure (Chemla et al., 2002). To establish the physiological basis for differences in PAP between PHS-susceptible and PHS-resistant broilers, contemporaneous measurements of PAP, downstream pressure, CO, and PVR must be obtained. Estimation of the downstream pressure is essential for differentiating between pulmonary venous hypertension (PVH) and pulmonary arterial hypertension (PAH) (Benza and Tallaj, 2006). Pulmonary venous hypertension is a consequence of an increased resistance to pulmonary blood flow downstream from the capillaries (through the pulmonary veins or left atrium) and is commonly caused by left-sided valvular or myocardial diseases (Simonneau et al., 2004). Thus PVH ensues whenever the left atrio-ventricular valve is incompetent to direct the complete stroke volume (SV) through the aorta or when myocardial disease prevents the weakened left ventricle from pumping all of the
INTRODUCTION Previous hemodynamic evaluations demonstrated that pulmonary arterial pressure (PAP) is higher in broilers that are susceptible to pulmonary hypertension syndrome (PHS, ascites) than in broilers that are resistant to PHS (Wideman et al., 2000, 2006; Chapman and Wideman, 2001; Bowen et al., 2006). These differences in PAP may be caused by differences in cardiac output (CO), pulmonary vascular resistance (PVR), or the pressure gradient across the pulmonary circulation (transpulmonary pressure gradient, TPG) as defined by equation 1: TPG = (CO × PVR). The TPG or pressure drop (⌬P) between pulmonary arteries and pulmonary veins is defined as the difference between precapillary pressure (PAP) and postcapillary (downstream) pressure caused by resistance to blood flow through the pulmonary veins into the left atrium. Accordingly, TPG or ⌬P equals (PAP − downstream pressure),
©2008 Poultry Science Association Inc. Received May 1, 2007. Accepted October 4, 2007. 1 Corresponding author: [email protected]
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catheterized to measure pressures in the wing vein, right atrium, right ventricle, pulmonary artery, and pulmonary veins (WP, wedge pressure). The transpulmonary pressure gradient (TPG) was calculated as (PAP-WP), with PAP quantifying precapillary pressure and WP approximating postcapillary pulmonary venous pressure. When compared with resistant and relaxed broilers, PAP values in susceptible broilers were ≥10 mmHg higher, TPG values were ≥8 mmHg higher, and WP values were ≤2 mmHg higher, regardless of sex. The combined hemodynamic criteria (elevated PAP and PVR combined with a proportionally elevated TPG) demonstrate that susceptibility to PHS can be attributed primarily to pulmonary arterial hypertension associated with increased precapillary (arteriole) resistance rather than to pulmonary venous hypertension caused by elevated postcapillary (venous and left atrial) resistance.
ABSTRACT Previous hemodynamic evaluations demonstrated that pulmonary arterial pressure (PAP) is higher in broilers that are susceptible to pulmonary hypertension syndrome (PHS, ascites) than in broilers that are resistant to PHS. We compared key pulmonary hemodynamic parameters in broilers from PHS-susceptible and PHS-resistant lines (selected for 12 generations under hypobaric hypoxia) and in broilers from a relaxed (control) line. In experiment 1 the PAP was measured in male broilers in which a flow probe positioned on one pulmonary artery permitted the determination of cardiac output and pulmonary vascular resistance (PVR). The PAP and relative PVR were higher in susceptible broilers than in relaxed and resistant broilers, whereas absolute and relative cardiac output did not differ between lines. In experiment 2 male and female broilers from the 3 lines were
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blood returning from the lungs (e.g., left-sided congestive heart failure). The resulting accumulation of excessive blood volume and pressure in the left atrium and pulmonary veins would force the right ventricle to increase the
MATERIALS AND METHODS Exposure to hypobaric hypoxia was used to select PHSsusceptible and PHS-resistant lines of broilers (Pavlidis
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Figure 1. Body weight, right: total ventricle weight (RV:TV), pulmonary arterial pressure (PAP), venous pressure (VP), relative pulmonary vascular resistance (PVR/BW), and relative cardiac output (CO/BW) for male broilers in experiment 1 from pulmonary hypertension syndrome (PHS)-susceptible (n = 14), PHS-resistant (n = 15) and relaxed selection (n = 15) lines during blood flow experiments. Closed circles represent means ± SEM. a,bLetters represent differences (P ≤ 0.05) between the broiler lines within a variable.
PAP to overcome the increased downstream (postcapillary) resistance and propel the requisite CO through the lungs. Because the increase in PAP is normally proportional to the increase in downstream pressure (HermoWeiler et al., 1998), PVH is characterized by elevated pulmonary arterial, pulmonary venous and left atrial pressures combined with a normal or reduced TPG and a normal PVR. In contrast, PAH occurs when the right ventricle must elevate PAP to overcome increased resistance to flow through the pulmonary arterioles upstream of the pulmonary capillaries and veins. The typical hemodynamic characteristics of PAH include elevated PAP, PVR, and TPG combined with normal pulmonary venous and left atrial pressures (Chemla et al., 2002). The present study was conducted to establish the hemodynamic basis for differences in PAP between PHS-susceptible, PHS-resistant, and relaxed broiler lines selected for 12 generations under conditions of hypobaric hypoxia (Pavlidis et al., 2007). The PAP, CO, and PVR were measured as described previously (Guthrie et al., 1987; Wideman et al., 1996, 2005; Wideman, 1999). Downstream pressure (essentially postcapillary pulmonary venous pressure) was estimated by measuring the wedge pressure (WP; Chapman and Wideman, 2001). Wedge pressures are obtained by advancing a catheter through the pulmonary arterial branches until the tip of the catheter reaches an artery of a similar or smaller diameter, causing the catheter’s tip to wedge in and thereby occlude blood flow from the upstream arterial tree. The pressure in small arterioles and capillaries connected to the occluded artery drops as some of the blood contained in these vessels drains into the capillaries and veins. A motionless column of blood then remains between the catheter tip and the downstream vasculature. This column transmits the blood pressure from the venous region, allowing the measurement of the postcapillary downstream blood pressure via the wedged catheter. Increases in the WP can be indicative of increased venous resistance, increased left atrial pressure, or both, which are symptoms of PVH (Zidulka and Hakim, 1985). Measuring the WP allows TPG to be calculated as PAP − WP (Chemla et al., 2002; Benza and Tallaj, 2006), and WP can be substituted for downstream pressure in equation 3 to derive equation 4: PAP = [(CO × PVR) + WP]. Equation 4 illustrates that if CO remains constant during the onset of pulmonary hypertension, then PAP must increase in direct 1 to 1 additive proportion to WP when the underlying cause is PVH, or in direct multiplicative proportion to PVR when the underlying cause is PAH. A previous study did not include measurements of CO but did implicate PAH rather than PVH as the cause of pulmonary hypertension in broilers, based on the absence of differences in WP in spite of very large differences in PAP for males from 3 different lines that were evaluated following exposure to thermoneutral or cool environmental temperatures (Chapman and Wideman, 2001).
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et al., 2007). After the eighth generation, the resistant and susceptible broilers exhibited 26.0 and 98.6% ascites mortality, respectively, when exposed to hypobaric conditions (Pavlidis, 2003). The progeny from the 12th generation of these lines, and from an unselected relaxed commercial pedigree line, were transported on the day of hatch (June 30, 2006) to the Poultry Environmental Research Laboratory at the University of Arkansas Poultry Research Farm. Birds were wing-banded and placed on fresh wood shavings litter in environmental chambers (8 m2 floor space). The birds were brooded at 33°C from d 1 to 3, 31°C from d 4 to 6, 29°C from d 7 to 10, 26°C from d 11 to 14, and 24°C thereafter. Birds were fed a 23% CP corn-soybean meal-based diet formulated to meet the National Research Council (1994) standards for all ingredients. Feed and water were provided ad libitum. Feed was provided as crumbles throughout the experiment. Lights were on for 24 h/d through d 4, and 16 h/d thereafter.
Experiment 1. Evaluation of Pulmonary Blood Flow Between 34 and 42 d of age male broilers from the 3 lines were weighed and anesthetized using intramuscular injections of allobarbital (5, 5-diallyl-barbituric acid,
Sigma Chemical Co., St. Louis, MO; 16 mg/kg of BW) and ketamine HCl (40 mg/kg of BW). They were fastened in dorsal recumbency on a surgical board. After a subcutaneous injection of 1 cc of 2% lidocaine (Interstate Drug Exchange Inc., Amityville, NY) HCl, an incision was made to open the thoracic inlet, and a Transonic 3SB or 4SB ultrasonic flow probe (Transonic Systems Inc., Ithaca, NY) connected to a Transonic T206 blood flow meter (Transonic Systems Inc.) was positioned on the left pulmonary artery. The skin of the thoracic inlet was sealed air-tight with surgical wound clips. Lidocaine was injected subcutaneously around the basilica vein, then the proximal end of a Silastic catheter (0.012 in inside diameter, 0.025 in outside diameter, Dow Corning Corp., Midland, MI) filled with heparinized saline (200 IU of heparin/mL of 0.9% NaCl) was inserted into the basilica vein. The distal end of the catheter was attached to a blood pressure transducer (World Precision Instruments, Sarasota, FL) interfaced through a Transbrige preamplifier (World Precision Instruments) to a Biopac MP100 data acquisition system using Acqknowledge software (Biopac Systems Inc., Goleta, CA). Venous pressure (VP) was recorded with the tip of the catheter inserted approximately 2 cm into the basilica vein, then the catheter was slowly advanced into the main trunk of the pulmonary artery where PAP was recorded as described previously (Guthrie et al., 1987; Wideman et al., 1996; Wideman, 1999).
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Figure 2. Blood pressure (BP) recordings from apparently clinically healthy broilers from pulmonary hypertension syndrome (PHS)-resistant and PHS-susceptible lines (upper and lower panels, respectively). The figure illustrates pressure measurements in experiment 2 as a catheter was inserted in the basilica vein (VP) and advanced through the right atrium (RA), right ventricle (RV), main trunk of the pulmonary artery (PAP), and through branches of the pulmonary artery (PA) until the cannula wedged (wedge) in a terminal arterial branch, followed by the recording of the pressures in the reverse order as the catheter was withdrawn.
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Experiment 2. Measurement of Wedge Pressure Between 42 and 50 d of age male and female broilers from the 3 lines were anesthetized and fastened in dorsal recumbency as described above. The basilica vein was cannulated with Silastic tubing and pressures were recorded as the catheter was slowly advanced through the basilica vein (VP), right atrium (RA: right atrial pressure), right ventricle (RV: right ventricular pressure), main trunk of the pulmonary artery (PAP), major branches of the pulmonary artery (PA), and onward until the tip of the catheter became wedged (WP), which was indicated
by a sudden drop in the PAP. A sudden rise in the PAP when the catheter was carefully withdrawn from its position confirmed the recording of WP. At the termination of each experiment, birds were euthanized with a 10mL i.v. injection of 0.1 M KCl. The heart was removed, dissected, and weighed for calculation of the right/total ventricular weight ratio (RV:TV) as an index of pulmonary hypertension (Burton et al., 1968).
Data Acquisition and Statistical Analyses The Biopac MP 100 data acquisition system continuously recorded 2 primary data channels, including PAP
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Figure 3. Open symbols represent the mean blood pressures ± SEM in experiment 2 of (A) males from the pulmonary hypertension syndrome (PHS)-susceptible (n = 15), PHS-resistant (n = 15), and relaxed (n = 16) lines; and (B) females from the PHS-susceptible (n = 16), PHS-resistant (n = 16), and relaxed (n = 16) lines. The pressures were recorded as a catheter advanced through the wing vein (VP), right atrium (RA), right ventricle (RV), and main trunk of the pulmonary artery (PAP), until wedge pressure was recorded (wedge). Closed symbols represent the transpulmonary gradient (TPG, the arithmetical subtraction of the wedge pressure from the PAP in each broiler) ± SEM. Data were analyzed using 2-way ANOVA. a,bLetters represent differences (P ≤ 0.05) between the broiler lines at a single anatomical location. w–zLetters represent differences (P ≤ 0.05) within each line between the different anatomical locations.
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INCREASED ARTERIAL RESISTANCE IN BROILERS Table 1. Absolute values for cardiac output (CO), pulmonary vascular resistance (PVR), heart rate (HR), and stroke volume (SV), and relative SV (SV/BW) for male broilers in experiment 1 from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed lines selection during blood flow experiments1 Susceptible (n = 14)
Variable CO (mL/min) PVR (relative units) HR (beats/min) SV (mL/beat) SV/BW (mL/beat × kg of BW)
310.5 0.114 343.2 0.91 0.44
± ± ± ± ±
Resistant (n = 15)
16.9 0.01a 9.2 0.05 0.02
296.7 0.088 341.8 0.87 0.45
± ± ± ± ±
Relaxed (n = 15)
22.0 0.01ab 7.1 0.06 0.03
286.4 0.081 326.4 0.89 0.48
± ± ± ± ±
16.4 0.01b 6.5 0.06 0.03
Superscripts designate differences (P ≤ 0.05) between broiler lines within a variable. Values represent mean ± SEM.
a,b 1
the F test from the 1-way ANOVA was declared significant (P < 0.05). The SigmaStat linear regression procedure was used to evaluate relationships between WP and PAP, and WP and RV:TV.
RESULTS Experiment 1. Evaluation of Pulmonary Blood Flow Body weight, RV:TV, PAP, VP, PVR normalized for BW, and CO normalized for BW are shown in Figure 1. Broilers from the resistant and relaxed lines did not differ for any of these variables, whereas broilers from the susceptible line had higher values for all variables with the exceptions of BW which was higher compared with the relaxed line but not different from the resistant line, and CO/BW that did not differ for any of the lines. Values for absolute CO and PVR, HR, SV, and SV normalized for BW were not different between lines except for PVR that was higher in the susceptible line compared with the relaxed line but not different from the resistant line (Table 1).
Table 2. Pressures and heart parameters in experiment 2 for male and female broilers from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed lines1 Males Variable n BW (g) VP (mmHg) RA (mmHg) RV (mmHg) PAP (mmHg) WP (mmHg) RV (g) LV+S (g) TV (g) RV:TV (g:g) RV/BW (g/kg) LV+S/BW (g/kg) TV/BW (g/kg)
Susceptible 15 2,140.8 ± 10.15 ± 9.07 ± 25.23 ± 36.60 ± 12.36 ± 1.92 ± 6.03 ± 7.95 ± 0.24 ± 0.90 ± 2.82 ± 3.72 ±
46.5 0.65a 0.57a 1.67a 2.24a 0.85a 0.13 0.21 0.28 0.01a 0.06 0.09 0.12
Resistant 2,073.1 7.36 6.20 18.93 26.20 9.98 1.61 5.94 7.54 0.21 0.78 2.87 3.65
15 ± ± ± ± ± ± ± ± ± ± ± ± ±
54.0 0.33b 0.35b 0.66b 1.06b 0.66ab 0.08 0.20 0.26 0.01ab 0.04 0.08 0.11
Females Relaxed 16 2,009.8 ± 6.58 ± 5.86 ± 16.85 ± 22.97 ± 9.07 ± 1.57 ± 5.87 ± 7.44 ± 0.21 ± 0.77 ± 2.92 ± 3.69 ±
53.4 0.35b 0.29b 0.55b 0.69b 0.58b 0.12 0.23 0.33 0.01b 0.05 0.06 0.10
Susceptible 16 2,098.9 ± 10.33 ± 8.93 ± 25.76 ± 34.82 ± 12.11 ± 1.78 ± 5.32 ± 7.11 ± 0.25 ± 0.85 ± 2.55 ± 3.41 ±
54.8b 0.70a 0.46a 1.34a 2.12a 0.60a 0.10 0.16 0.24 0.01a 0.04a 0.07 0.11a
Resistant 2,189.8 7.79 6.69 18.59 24.34 9.89 1.59 5.50 7.09 0.22 0.73 2.51 3.24
16 ± ± ± ± ± ± ± ± ± ± ± ± ±
32.9ab 0.47b 0.35b 0.72b 1.23b 0.55b 0.07 0.15 0.20 0.01b 0.03b 0.06 0.08ab
Relaxed 2,297.6 7.54 6.66 17.46 21.18 9.56 1.59 5.36 6.95 0.23 0.69 2.33 3.02
16 ± ± ± ± ± ± ± ± ± ± ± ± ±
31.5a 0.25b 0.25b 0.52b 1.03b 0.47b 0.09 0.20 0.28 0.01ab 0.04b 0.08 0.10b
Superscripts designate differences (P ≤ 0.05) between broiler lines within a variable and sex. Data are presented as mean ± SEM. VP = venous pressure; RA = right atrium; RV = right ventricle; PAP = pulmonary arterial pressure; WP = wedge pressure; LV+S = left ventricle plus septum; TV = total ventricle. a,b 1
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(mmHg) and blood flow through the left pulmonary artery (mL per min). Data were measured and averaged electronically over 10-s intervals from the Biopac recording. Based on the assumption that CO normally is divided approximately equally between 2 lungs of equal size, CO was calculated as 2 × blood flow. Heart rate (HR, beats per min) was obtained by counting systolic peaks over time in the PAP recording. Stroke volume (mL per beat) was calculated as CO/HR. Assuming that atrial pressure remains close to zero and has little impact on vasoconstrictive responses of pulmonary arterioles (Wideman and Bottje, 1993; Chapman and Wideman, 2001) the PVR was calculated in relative resistance units as PAP/CO (Sturkie, 1986; Wideman et al., 1998). The TPG (mmHg) was calculated as PAP − WP (Chemla et al., 2002; Benza and Tallaj, 2006). Cardiac output, PVR, and SV were calculated as absolute and relative (BW normalized) values to compensate for differences in BW when comparing birds of different size or age. Data in experiment 1 were analyzed using the SigmaStat 1-way ANOVA. In experiment 2 the SigmaStat 2-way ANOVA was used to evaluate sex (male, female) × line (susceptible, relaxed, resistant) interactions among pressure recorded at each anatomical location (Jandel Scientific, 1994). Means were separated by the Tukey test when
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LORENZONI ET AL. Table 3. Regression of wedge pressure vs. pulmonary arterial pressure in experiment 2 for male and female broilers from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed selection lines Line Susceptible Resistant Relaxed Susceptible Resistant Relaxed
Sex
n
Male Male Male Female Female Female
15 15 16 16 16 16
Experiment 2. Measurement of Wedge Pressure
Y Y Y Y Y Y
= = = = = =
0.12X + 7.93 −0.15X + 13.84 0.30X + 2.17 0.15X + 7.07 0.24X + 4.00 0.16X + 6.20
r2
P-value
0.10 0.06 0.13 0.27 0.29 0.12
0.25 0.40 0.18 0.04 0.03 0.19
from VP within any line and sex comparison, except for males from the relaxed line in which VP was slightly lower than WP. Wedge pressure was not different from RA in the susceptible line, but it was slightly higher than the RA in the resistant and relaxed lines within each sex. The TPG, calculated as PAP − WP, was markedly greater in males and females from the susceptible line when compared with their respective counterparts in the resistant and relaxed lines. The TPG did not differ between the resistant and relaxed lines. Body weight, weights of the RV, right and left ventricle plus septum (total ventricle, TV), left ventricle plus septum (LV+S), RV:TV, BW-normalized RV (RV/BW), BWnormalized LV+S (LV+S/BW), and BW-normalized TV (TV/BW) are shown for males and females in Table 2. The BW did not differ for males of the 3 lines, but in females the BW was higher in the relaxed line than in the susceptible line, whereas the BW in the resistant line was not different from the other lines. The RV did not differ between lines for males or females; however, males from the susceptible line tended to have values higher than the resistant and relaxed lines (P = 0.056). The RV:TV ratio was higher in males from the susceptible line than in the relaxed line, whereas males from the resistant line did not differ from the other lines. In females the resistant line had lower values for RV:TV than the susceptible line and the relaxed line was not different from the other lines. The RV/BW did not differ between the resistant and relaxed lines but it was higher in the susceptible line in females; in contrast, there were no differences for RV/ BW in males. The TV/BW did not differ between lines in males, but females from the susceptible line had a higher TV/BW than females from the relaxed line, and TV/BW values in the resistant line did not differ from the other lines. Values for LV+S, TV, and LV+S/BW did not differ between lines within sexes. The linear regression comparisons for WP vs. PAP within each sex and line were only significant for females from the susceptible
Table 4. Regression of wedge pressure vs. right to total ventricle weight ratio in experiment 2 for male and female broilers from pulmonary hypertension syndrome (PHS)-susceptible, PHS-resistant, and relaxed selection lines Line Susceptible Resistant Relaxed Susceptible Resistant Relaxed
Sex
n
Male Male Male Female Female Female
15 15 16 16 16 16
Equation Y Y Y Y Y Y
= = = = = =
17.72X + 8.09 −1.20X + 10.24 29.41X + 2.97 18.34X + 7.54 35.24X + 2.01 −5.11X + 10.73
r2
P-value
0.05 0.00 0.16 0.05 0.15 0.01
0.42 0.97 0.12 0.40 0.14 0.80
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A typical recording for a clinically healthy broiler from the resistant line is shown in the upper panel of Figure 2 (RV:TV= 0.23; PAP = 27 mmHg), whereas the lower panel of Figure 2 illustrates the recording from an apparently clinically healthy broiler from the susceptible line (RV:TV= 0.28; PAP = 44.2 mmHg). After the catheter was inserted approximately 2 cm into the basilica vein, VP averaged 8.6 and 8.3 mmHg in the resistant and susceptible birds, respectively. The pressure averaged 7.2 mmHg in both birds when the catheter tip entered the right atrium, and rose to 20.8 and 26.5 mmHg in the resistant and susceptible birds, respectively, when the catheter reached the right ventricle. When the catheter entered the trunk of the pulmonary artery the PAP values averaged 27 and 44.2 mmHg in the resistant and susceptible birds, respectively. The arterial pressure subsided slightly as the catheter advanced through major branches of the PA. In both birds the pressure dropped dramatically when the catheter’s tip wedged inside a terminal branch of the pulmonary arterial tree, with WP averaging 11.64 and 13 mmHg in the resistant and susceptible birds, respectively. Figure 2 also shows the pressures recorded as the cannula was withdrawn (PAP, RV, RA, and VP). Group means for VP, RA, RV, PA, and WP are shown in Figure 3. No sex × line interactions (P < 0.05) were detected for the pressures measured at any of the anatomical locations. There were no differences in pressure within any of these anatomical locations when males (Figure 3A) or females (Figure 3B) from the resistant and relaxed lines were compared. In contrast, broilers from the susceptible line consistently had higher pressures at all anatomical locations than did their counterpart from the resistant and relaxed lines (Figure 3A). Within each line regardless of sex, PAP was higher than RV pressure, which in turn was higher than WP. The WP did not differ
Equation
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and resistant lines (Table 3). None of the regression comparisons for WP vs. RV:TV within the separate lines were significant, regardless of sex (Table 4).
DISCUSSION
pulmonary hemodynamic changes observed in PHS-susceptible broilers (e.g., elevated PVR, elevated TPG) do not conform to the criteria necessary for a diagnosis of PVH caused by left-sided valvular or myocardial diseases (low TPG, normal PVR, and elevated pulmonary venous and left atrial pressures). Indeed, a diagnosis of PVH would require direct 1 to 1 proportionality between increments in WP and PAP, accompanied by a normal TPG (Zidulka and Hakim, 1985; Chemla et al., 2002; Benza and Tallaj, 2006). For example, acute obstruction of the mitral valve in sheep causes corresponding increases of approximately 1 mmHg in PAP per each mmHg increased in the left atrial pressure (Hermo-Weiler et al., 1998). Similarly, replacing damaged mitral valves in humans reduces WP and PAP by a proportionality of 1 to 1 (Fawzy et al., 1996). In the present study marked elevations in PAP were associated with very modest increases in WP in broilers from the susceptible line, which may reflect an elevated pressure dissipation profile transmitted along the length of the pulmonary vasculature due to the large upstream increase in PAP, or modest increases in postcapillary resistance to blood flow through the pulmonary venous drainage of PHS-susceptible broilers (e.g., venoconstriction; Zidulka and Hakim, 1985). Finally, when a dilated and engorged right ventricle having an elevated end diastolic volume pushes the ventricular septum into the chamber of the left ventricle, the extent to which the left ventricular wall can expand against the pericardium and fill with blood may be limited. This form of left ventricular diastolic dysfunction caused by right ventricular dilation would cause the returning venous blood to pool in the left atrium and pulmonary veins, leading to a modest increase in WP. However, WP values were not significantly correlated with RV:TV ratios by linear regression analysis for males or females within any line, and any effect of right ventricular dilation on cardiopulmonary hemodynamics would only be anticipated after the evolution of severe pathological changes in the heart. Such changes clearly cannot serve as a primary or initiating event in the pathogenesis of PHS. We conclude that neither increases in downstream resistance, left myocardial failure nor mitral valve degeneration are involved in the initial pathogenesis of PHS in broilers. Consistently, in both studies, we observed greater PAP in the susceptible line when compared with the resistant and relaxed lines. This is in agreement with previous studies (Chapman and Wideman, 2001; Bowen et al., 2006) and indicates that the genetic selection for ascites susceptibility has been successful. The RV:TV values tended to be higher in the susceptible line within all groups; however, the RV:TV did not differ between the susceptible and resistant males in experiment 2 and between the susceptible and relaxed females in experiment 2. A similar situation occurred for comparisons of the RV/BW values in experiment 2, when females of the susceptible line had values higher than those of females in the resistant and relaxed lines, whereas for males there were no differences between lines. Because we selected clinically healthy birds for the cardiopulmonary evalua-
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The present study was designed to evaluate key pulmonary hemodynamic characteristics of broilers from PHSsusceptible, PHS-resistant, and relaxed lines to determine the origin of PHS (e.g., PAH vs. PVH) as well as to provide a complete characterization of the pressures within the pulmonary circulation in these lines. Pulmonary hemodynamic characteristics can be used as selection criteria for broiler lines (Wideman and French, 1999). To determine the early (initiating) hemodynamic differences between these lines, all broilers were reared under moderate conditions (e.g., thermoneutral temperatures, 16L:8D photoperiod, feed provided as crumbles) and only broilers that appeared to be clinically healthy (e.g., noncyanotic, nonascitic individuals) were used for pulmonary blood flow and pressure measurements. In both experiments the PAP was higher in broilers from the PHS-susceptible line than in broilers from the relaxed and PHS-resistant lines. Data collected in experiment 1 demonstrated that the higher PAP in broilers from the susceptible line cannot be attributed to increases in the absolute or relative CO when compared with broilers from the relaxed or resistant lines. Instead, the pulmonary hypertension in broilers from the susceptible line was caused by an elevated PVR normalized for BW. In experiment 2 we measured the WP and calculated the TPG to differentiate between PAH and PVH as the primary underlying cause of pulmonary hypertension in broilers. The combined data from both experiments demonstrated that, when compared with broilers from the PHS-resistant and relaxed lines, broilers from the PHS-susceptible line had elevated PAP and PVR values combined with directly proportional increments in TPG and minimal increases in WP. These pulmonary hemodynamic characteristics must be attributed primarily to PAH rather than to PVH, based on the relationships established in equation 4 (see Introduction; Chemla et al., 2002; Benza and Tallaj, 2006). A modestly higher WP was observed when birds from the susceptible line were compared with birds from the relaxed and resistant lines. However, these modest increases in WP values in susceptible males and females did not account for the much larger increases in PAP and TPG values. Overall the PAP values in susceptible broilers were ≥10 mmHg higher and TPG values were ≥8 mmHg higher when compared with relaxed and resistant broilers, whereas the WP was ≤2 mmHg higher in susceptible broilers when compared with relaxed or resistant broilers. The association between WP and PAP was not significant by linear regression analysis for males within any of the lines or for females from the relaxed line, but WP and PAP were significantly correlated in females from the susceptible and resistant lines. These modest increases in WP cannot account for the much greater increases in PAP for PHS-susceptible broilers, and the accompanying
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ACKNOWLEDGMENTS This research was supported by USDA/CSREES/NRI grant 2003-35204-13392.
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tions, these observations strongly suggest that the earliest changes associated with pulmonary hypertension are detected through direct measurements of PAP. Differences in RV weights in birds undergoing different degrees of pulmonary hypertension presumably develop after differences in PAP have been sustained more chronically (Wideman et al., 2000; Liu, 2001; Lorenzoni, 2006). Blood pooling in large systemic veins may be one of the earliest hemodynamic changes that indicates the onset of rightsided congestive heart failure, occurring before the RV undergoes work hypertrophy as it propels the requisite CO through the constricted pulmonary vasculature (Wideman et al., 1999). The early onset of a mild rightsided congestion would explain the consistent increase in VP and not in RV mass in the susceptible line. Under the conditions of this study, the resistant and relaxed broiler lines did not differ for any of the cardio-pulmonary parameters studied. Nevertheless it is expected that under chronic exposure to periods of PHS-inducing stressful conditions (e.g., cool temperatures, faster growth, >20 h/d photoperiod) the unselected line would present hemodynamic values (especially for PAP and PVR) intermediate to the values of the susceptible and resistant lines, as was demonstrated previously (Chapman and Wideman, 2001; Bowen et al., 2006; Wideman et al., 2007).