Cardio-pulmonary function in preascitic (hypoxemic) or normal broilers inhaling ambient air or 100% oxygen

Cardio-Pulmonary Function in Preascitic (Hypoxemic) or Normal Broilers Inhaling Ambient Air or 100% Oxygen R. F. Wideman, Jr.,*,1 M. R. Fedde,† C. D. Tackett,* and G. E. Weigle† *Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 and †Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506-5602 tance was not reduced during 100% O2 inhalation. Cardiac output was higher in preascitic than in normal broilers before and after, but not during, 100% O2 inhalation. Mean arterial pressure and total peripheral resistance increased in the preascitic but not in the normal group during 100% O2 inhalation. Low coefficients of determination (R2) were obtained for linear regression comparisons of HbO2 vs pulmonary arterial pressure in both experiments. Overall, acute reversal of the systemic hypoxemia in preascitic broilers had little direct impact on pulmonary hypertension, providing no evidence of hypoxemic or hypoxic pulmonary vasoconstriction. Instead, acute reversal of the systemic hypoxemia primarily increased the total peripheral resistance and normalized the mean arterial pressure and cardiac output. A sustained reduction in cardiac output theoretically should attenuate pulmonary hypertension, but this was not observed because of the overriding influence of sustained pulmonary vascular resistance.

(Key words: pulmonary hypertension, systemic hypotension, hemoglobin saturation, hypoxemia, ascites) 2000 Poultry Science 79:415–425

ventilation per se. Instead, the hypoxemia has been attributed to an inability of the pulmonary vasculature to accommodate the requisite cardiac output. If blood flows too rapidly through the pulmonary vasculature, red blood cells may not reside at the gas exchange surfaces long enough to permit full saturation of the hemoglobin with oxygen, leading to a diffusion limitation attributed to inadequate time for blood-gas equilibration (Henry and Fedde, 1970; Peacock et al., 1989, 1990; Reeves et al., 1991; West, 1993; Wideman and Kirby, 1995a,b; Wideman et al., 1996a,b; Fedde et al., 1998). Specific hypertrophy of the right ventricle provides definitive evidence that increased work has been performed to maintain an elevated pulmonary arterial pressure. Necropsies performed on broilers exhibiting early cyanosis consistently reveal an elevated right:total (RV:TV) ventricular weight ratio. This ratio provides a sensitive index

INTRODUCTION Early during the pathophysiological progression leading to pulmonary hypertension syndrome (PHS, ascites), susceptible broilers exhibit a mild cyanosis indicative of arterial blood having a low (≤ 80%) saturation of hemoglobin with oxygen (HbO2). This hypoxemia serves as a reliable indicator that broilers are preascitic and subsequently will develop ascites (Peacock et al., 1989, 1990; Reeves et al., 1991; Julian and Mirsalimi, 1992; Wideman and Kirby, 1995a,b; Kirby et al., 1997; Roush et al., 1996, 1997; Wideman et al., 1997, 1998c). Preascitic hypoxemia cannot be attributed to low atmospheric oxygen (hypoxia), anemia, intracardiac right to left shunts, or hypo-

Received for publication July 16, 1999. Accepted for publication September 30, 1999. 1 To whom correspondence should be addressed: Robert F. Wideman, Jr., Ph.D., Professor of Poultry Science, O-402 Poultry Science Center, Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701; e-mail: [email protected].

Abbreviation Key: HbO2 = hemoglobin oxygen; RV:TV = right:total ventricular weight ratios.

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ABSTRACT We evaluated the influence of the percentage saturation of hemoglobin with oxygen (HbO2) on the pulmonary arterial pressure in normal and preascitic (hypoxemic) broilers breathing ambient air or 100% O2. In Experiment 1, unanesthetized preascitic broilers (right:total ventricular weight ratios [RV:TV] = 0.32 ± 0.02) breathing ambient air had initial values of 67% for HbO2 and 32 mm Hg for pulmonary arterial pressure. The HbO2 increased to ≥96.6% during inhalation of 100% O2; however, pulmonary arterial pressure was not reduced. In Experiment 2, anesthetized normal (RV:TV = 0.23; HbO2 = 88%) and preascitic broilers (RV:TV = 0.28; HbO2 = 76%) were compared. The groups did not differ in body weight or respiratory rate, but preascitic broilers had lower values for mean arterial pressure, total peripheral resistance, and partial pressure of O2 in arterial blood and had higher values for pulmonary arterial pressure. Inhaling 100% O2 increased HbO2 to 99.9% in both groups; however, pulmonary arterial pressure remained higher in preascitic than in normal broilers, and the pulmonary vascular resis-

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2 Vet/Ox Model 4403 using the universal C-sensor, Sensor Devices, Inc., Waukesha, WI 53188.

elevated blood flow through the lungs, regardless of the sustained hypoxemia attributed to a persistent diffusion limitation (Wideman et al., 1996a,b, 1997, 1998a). Currently, the incidence of ascites induced by various experimental models appears primarily to be correlated with the associated increment in the RV:TV ratio and, thus, the magnitude of pulmonary hypertension rather than the magnitude of the accompanying hypoxemia. In contrast to our consistent ability to experimentally dissociate systemic hypoxemia from concurrently induced pulmonary hypertension, exposure to low atmospheric oxygen (hypoxia) reliably triggers a rapid onset of pulmonary hypertension that, when sustained chronically, leads to the development of ascites in domestic fowl (Burton et al., 1968; Besch et al., 1971; Cueva et al., 1974; Besch and Kadono, 1978; Sillau and Montalvo, 1982; Peacock et al., 1990; Owen et al., 1990, 1994, 1995a,b,c). Pulmonary vascular resistance and pulmonary arterial pressure of domestic fowl increase within minutes after acute exposure to normobaric or hypobaric hypoxia, and the restoration of normoxia causes an equally rapid return to essentially normal pulmonary hemodynamic values (Besch and Kadono, 1978; Owen et al., 1995b). The present study was designed to compare the cardiopulmonary function of normal broilers and preascitic (hypoxemic) broilers. We tested the hypothesis that, if either intrapulmonary hypoxia or intravascular hypoxemia cause pulmonary vasoconstriction contributing to pulmonary hypertension early in the pathogenesis leading to ascites, then permitting preascitic broilers to inhale 100% oxygen should alleviate the preexisting pulmonary hypertension. Alternatively, if hypoxemia primarily contributes to pulmonary hypertension by reducing the total peripheral resistance and elevating the cardiac output, then breathing 100% oxygen should increase the total peripheral resistance, reduce the cardiac output, and normalize the blood pressure in preascitic broilers.

MATERIALS AND METHODS Male broiler chicks were reared on fresh wood shavings in environmental chambers (8 m2 floor space). They were brooded at 32 and 30 C during Weeks 1 and 2, respectively, and thereafter the temperature was maintained at 14 C (Experiment 1) or 24 C (Experiment 2). They were fed a 23% CP corn-soybean meal-based broiler ration formulated to meet or exceed the minimum NRC (1984) standards for all ingredients. Feed and water were provided for ad libitum consumption. Lights were on for 24 h/d through Day 5 and for 23 h/d thereafter.

Experiment 1 Hypoxemic broilers, 34 to 36 d of age on March 6, 1997 to March 8, 1997 (1,633 ± 55 g body weight, mean ± SEM), were selected from a population of 50 individuals. Birds were lightly restrained in lateral recumbency, and the sensor of a pulse oximeter2 was positioned on the right wing to illuminate the tissue between the radius and ulna.

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of pulmonary arterial pressure and serves as a convenient benchmark for comparing animals of different ages and sizes, because normalizing the weight of the right ventricle for differences in total ventricular weight also serves to effectively compensate for correlated variability in cardiac output and body mass (Wideman et al., 1998c; Wideman, 1999; Wideman and French, 1999a,b). Under a wide variety of conditions, clinically healthy domestic fowl with normal pulmonary arterial pressures have RV:TV ratios averaging below 0.27, whereas RV:TV ratios averaging ≥0.28 reflect sustained pulmonary hypertension (Burton and Smith, 1967; Burton et al., 1968; Cueva et al., 1974; Huchzermeyer and DeRuyck, 1986; Huchzermeyer et al., 1988; Hernandez, 1987; Peacock et al., 1989; Julian, 1993; Wideman and Bottje, 1993; Lubritz et al., 1995; Forman and Wideman, 1999; Wideman and Tackett, 1999). The relationship between hypoxemia and pulmonary arterial pressure during the pathogenesis of ascites is complex. Hypoxemia theoretically can initiate pulmonary hypertension by reducing the total peripheral resistance (hypoxemic systemic vasodilation) or by increasing the pulmonary vascular resistance (hypoxemic pulmonary vasoconstriction) (Wideman and Bottje, 1993). Hypoxemic vasodilation of the systemic resistance vessels serves to increase blood flow and thus oxygen delivery to the tissues. The resulting increase in venous return to the heart forces the right ventricle to perform additional work to propel the additional blood flow through the pulmonary vasculature. When chronically sustained, hemodynamic changes accompanying systemic vasodilation in broilers should include a reduced total peripheral resistance, a low systemic arterial pressure, an elevated cardiac output, increases in the pulmonary arterial pressure and RV:TV ratio, and further amplification of an incipient diffusion limitation (Forman and Wideman, 1999; Wideman, 1999). A direct vasoconstrictive influence of hypoxemia on the pulmonary vasculature has not previously been demonstrated in broilers and has been questioned on the basis of discontinuities between blood oxygenation and pulmonary hypertension. For example, the degree of hypoxemia induced by occluding one extrapulmonary primary bronchus equals or exceeds that induced by occluding one pulmonary artery; however, unilateral pulmonary artery occlusion triggers a greater increase in pulmonary arterial pressure, a larger RV:TV ratio, and a higher incidence of ascites when compared with unilateral bronchial occlusion (Wideman and Kirby, 1995a, 1996; Wideman et al., 1996a,b, 1997; Kochera-Kirby et al., 1999a,b; Ruiz-Feria et al., 1999; Wideman and French, 1999a,b). Additional evidence indicates flow-dependent pulmonary vasodilation reduces the onset of pulmonary hypertension in broilers and Giant Jungle Fowl by minimizing the increment in pulmonary arterial pressure needed to propel an

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Experiment 2 Forty male broilers, 41 to 44 d of age on December 9, 1998 to December 12, 1998, were screened with the pulse oximeter for inclusion in the normal (range = 85 to 93% HbO2) or preascitic (range = 64 to 80% HbO2) groups. Ten birds in each category were prepared using surgical protocols described previously (Wideman and Kirby, 1995b; Wideman et al., 1996a,b, 1998a,b; 1999a,b). They were anesthetized to a light surgical plane with intramuscular injections of allobarbital (5,5-diallyl-barbituric acid; 25 mg kg/BW) and were fastened in dorsal recumbency on a surgical board thermostatically regulated to maintain a surface temperature of 30 C. The thoracic inlet was opened, a Transonic ultrasonic flowprobe9 was positioned on the left pulmonary artery, the probe was connected to a Transonic T206 blood flow meter9 to confirm signal acquisition, then the skin of the thoracic inlet was sealed with surgical wound clips. Silastic威 Tubing4 filled with heparinized saline was inserted through the left ulnar

3

Interstate Drug Exchange, Inc., Amityville, NY 11701. Konigsberg Instruments, Inc., Pasadena, CA 91107-3294. World Precision Instruments, Sarasota, FL 34230. 6 Biopac Systems, Inc., Goleta, CA 93117. 7 Nominally 20.9% oxygen. 8 Air Products, Inc., Fayetteville, AR 72701. 9 Transonic Systems Inc., Ithaca, NY 14850. 4 5

FIGURE 1. Experiment 1, percentage saturation of hemoglobin with oxygen (HbO2; upper panel), pulmonary arterial pressure (PAP; second panel), heart rate (HR; third panel), and respiratory rate (lower panel) when nine hypoxemic male broilers were breathing ambient air (sample intervals A and B), 100% oxygen (sample intervals C to J), or were restored to breathing ambient air (sample intervals L to Q). Sample intervals were 1.5 min each. Asterisks denote mean values (±SEM) that differ (P ≤ 0.05) from control sample intervals A and B.

vein and was advanced into the right pulmonary artery. The distal end of the cannula was attached to a blood pressure transducer interfaced through a Transbridge preamplifier5 to a Biopac MP 100 data acquisition system using AcqKnowledge software.6 The left brachial artery was cannulated with PE-50 polyethylene tubing filled with heparinized saline; the cannula was advanced to a position near the descending aorta and was attached to a blood pressure transducer for continuous monitoring of systemic arterial pressure. Intravenous infusions were not administered. When surgical preparations were complete and a stabilization period of 20 min had elapsed, control data were collected for 20 min, and two arterial blood samples were collected 5 and 15 min into the control period (sample intervals A and B, Figure 3). An inhalation mask was used to administer gas consisting of 100% O2. Previous studies indicated that administering compressed air alone has no effect on cardio-pulmonary function (Wideman et al., 1999b). The 100% O2 was released through the inhalation mask at 10 psi and a rate of approximately 14

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The percentage saturation of HbO2 was recorded, and 9 individuals with HbO2 values ≤80% (67 ± 3%, mean ± SEM; range: 50 to 80%) were identified as being hypoxemic. General anesthesia was not employed. A 2% (wt/ vol) Lidocaine3 solution was infiltrated intracutaneously as a local anesthetic along the left cutaneous ulnar vein, which was cannulated with Silastic威 Tubing (0.012 in I.D., 0.037 in O.D.)4 filled with heparinized saline. The distal end of the cannula was attached to a blood pressure transducer5 interfaced through a Transbridge preamplifier5 to a Biopac MP 100 data acquisition system using AcqKnowledge software.6 The cannula was advanced into the pulmonary artery as judged by pressure tracings (Guthrie et al., 1987; Owen et al., 1995b). Birds were placed in an upright posture, and, when they were calm, pulmonary arterial pressure, HbO2, and heart rate were recorded while the birds breathed ambient air7 (sample intervals A and B in Figure 1; approximately 1.5 min each). An inhalation mask was placed on the bird’s head to direct gas toward the beak and nostrils while allowing ample open space for excess gas to freely escape. Gas8 consisting of 100% O2 was released through the mask, and sample intervals C to J (Figure 1) were recorded. The mask was removed to allow the birds to breathe ambient air while sample intervals L to Q (Figure 1) were recorded. At the end of the experiment, the birds were killed with a 10mL i.v. injection of 0.1 M KCl and were dissected to obtain heart weights for calculating the RV:TV ratio as an index of pulmonary hypertension (Burton et al., 1968; Cueva et al., 1974; Huchzermeyer et al., 1988).

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10

Radiometer America Inc., Westlake, OH 44145.

sure, respectively (Wideman et al., 1996a,b, 1998a,b), the relationships between pressure gradients, flow rates, and resistances were summarized by the respective equations: pulmonary arterial pressure = cardiac output × pulmonary vascular resistance, and mean systemic arterial pressure = cardiac output × total peripheral resistance. Thus, pulmonary vascular resistance was calculated in relative resistance units as pulmonary arterial pressure (mm Hg) divided by cardiac output (mL/min), and total peripheral resistance was calculated in relative resistance units as mean systemic arterial pressure (mm Hg) divided by cardiac output (mL/min) (Besch and Kadono, 1978; Sturkie, 1986; Wideman et al., 1996a,b, 1998a,b). Respiratory rate (breaths/min) was obtained by counting the wave cycles associated with respiratory movement that comprise an integral part of the pulmonary arterial pressure recordings (Sturkie, 1986).

Statistical Analysis Data were analyzed within a group over time (across sample intervals) using the SigmaStat威 repeated measures analysis of variance procedure, and means were differentiated by the Student-Newman-Keuls method (Jandel Scientific, 1994). Within a single sample interval across groups, the SigmaStat威 t-test was used to assess significant (P ≤ 0.05) differences among means. The SigmaStat威 linear regression procedure was used to evaluate relationships among cardiopulmonary variables.

RESULTS Experiment 1 The initial values for broilers breathing ambient air (Sample intervals A and B combined) were 67.8 ± 3.6% for HbO2, 32.0 ± 3.1 mm Hg for pulmonary arterial pressure, 346 ± 10 beats/min for heart rate, and 55.1 ± 3.1 breaths/min for respiratory rate (mean ± SEM). The RV:TV ratios averaged 0.32 ± 0.02. As shown in Figure 1, the HbO2 rapidly increased to a stable plateau (≥96.6%) when the broilers were breathing 100% O2 (sample intervals C to J). The HbO2 returned to hypoxemic levels (75 ± 2%) within 3 min after ambient air again was provided (sample intervals M to Q), and these final HbO2 values did not differ from the initial values. Pulmonary arterial pressure did not decline below the initial level while the broilers were breathing 100% O2 (ANOVA over time: P = 0.956; ANOVA on ranks over time: P = 0.892). The heart rate did not change over the course of the experiment (ANOVA over time: P = 0.194; ANOVA on ranks over time: P = 0.172). The respiratory rate did not decline below the initial level during 100% O2 inhalation but thereafter was higher (P < 0.05) during sample intervals N and O than during intervals H to J (Figure 1). When data for all birds and across the entire experiment were pooled, very low coefficients of determination (R2) were obtained for linear regression analyses of pulmonary arterial pressure vs HbO2 (R2 = 0.0481; P = 0.213) and pulmonary arterial

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L/min for 20 min, and two arterial blood samples were collected 5 and 15 min into the gas inhalation period (sample intervals C and D, Figure 3). Five minutes after collecting sample D, the gas flow was shut off, the inhalation mask was removed, and three arterial samples were collected 5, 15, and 25 min into the recovery period (sample intervals E, F, and G; Figure 3). Arterial blood (1 mL) was withdrawn anaerobically and injected within 30 s into a Radiometer ABL 330 Acid-Base Laboratory.10 Appropriate functioning of the blood gas analyzer was assessed by periodically injecting Blood Gas Qualicheck威 reference standards.10 The primary arterial blood values for pH, partial pressure of O2, and partial pressure of CO2 were generated by the ABL330 operating at a sample chamber temperature of 37 C and were recalculated by the ABL330 for a temperature of 41 C to match the normal body temperature of domestic fowl (Fedde, 1986). A portion of the arterial blood was used for duplicate hematocrit determinations using heparinized capillary tubes and a microhematocrit centrifuge. Ten minutes after sample G was collected, the birds were killed with a 10 mL intraarterial injection of 0.1 M KCl and were dissected to obtain heart weights for calculating the RV:TV ratio. The Biopac MP 100 data acquisition system recorded three primary data channels, including systemic arterial pressure in millimeters of mercury (mm Hg), pulmonary arterial pressure (mm Hg), and blood flow through the left pulmonary artery (mL/min). Average values for these parameters were measured electronically during representative intervals at the start of the experiment (sample interval S, Figures 4 to 6), immediately preceding (sample intervals A1, B1, C1, etc., Figures 4 to 6) and following (sample intervals A2, B2, C2, etc., Figures 4 to 6) withdrawal of each arterial blood sample, immediately preceding the beginning of 100% O2 inhalation (sample interval B3, Figures 4 to 6), and during the last 30 s of the experiment (sample interval L, Figures 4 to 6). The protocol used for data averaging previously was demonstrated to accurately compensate for the influences of pulse pressure and respiratory cycles on pulmonary and systemic arterial pressures (Wideman et al., 1996a,b). These primary values were used to calculate cardiac output, stroke volume, pulmonary vascular resistance, and total peripheral resistance. Based on the assumption that cardiac output (mL/min) normally is divided equally between the two lungs, cardiac output was calculated as 2 × blood flow through the left lung. The cardiac output is the product of heart rate × stroke volume (mL/beat), consequently stroke volume was calculated as cardiac output divided by heart rate. Heart rate (beats/min) was obtained by counting systolic peaks over time in the pulmonary arterial pressure recording coincident with each sample interval. Assuming the pressure gradients across the pulmonary and systemic circulations are essentially equal to pulmonary arterial pressure and systemic arterial pres-

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= 0.334 to 0.494; P = 0.052 to 0.002). In each case for which a significant correlation with pulmonary arterial pressure existed, the coefficient of determination for heart rate exceeded that for HbO2, supporting a dominant role for cardiac output (Figure 2).

Experiment 2

pressure vs heart rate (R2 = 0.0117; P = 0.011). Because of the widely variable pulmonary arterial pressure responses of individual broilers during 100% O2 inhalation, linear regression analyses was conducted on an individual bird basis (Figure 2). There was no correlation (P > 0.11) between pulmonary arterial pressure and heart rate in four of the nine broilers, and the same broilers did not exhibit a correlation between pulmonary arterial pressure and HbO2. These nonresponding broilers had widely varied initial HbO2 values (range: 80 to 50%) and RV:TV ratios (range: 0.26 to 0.36). Five of nine birds exhibited a positive correlation between pulmonary arterial pressure and heart rate (R2 range = 0.386 to 0.887; P = 0.02 to 0.0001), and four of nine birds exhibited a negative correlation between pulmonary arterial pressure and HbO2 (R2 range

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FIGURE 2. Experiment 1, coefficients of determination (R2) for linear regression of pulmonary arterial pressure vs heart rate, or vs the saturation of hemoglobin with oxygen (HbO2), in nine hypoxemic broilers. Asterisks denote individual variables for which the regression slope was significant (P ≤ 0.05); variables from the same broiler are connected by a dashed line.

The groups did not differ in body weight, left ventricle + septum weight, or total ventricle weight, but preascitic broilers did have heavier (P = 0.07) right ventricles and higher RV:TV ratios than normal broilers (Table 1). When compared with normal broilers while breathing ambient air, the preascitic broilers had similar values for hematocrit and respiratory rate but lower values for the partial pressure of O2 and the HbO2 calculated by the blood gas analyzer or measured with the pulse oximeter. Preascitic broilers had higher values for the partial pressure of CO2, bicarbonate concentration, and hydrogen ion concentration when compared with normal broilers (Table 1). Inhaling 100% O2 increased the partial pressure of oxygen and HbO2 to equivalent values of >400 mm Hg and 99.9%, respectively, for both groups (Figure 3). The subsequent return to breathing ambient air restored the initial group differences in the partial pressure of oxygen and HbO2. Within each group, inhaling 100% O2 did not change the partial pressure of CO2 (Figure 3), hydrogen ion concentration (Figure 3), bicarbonate concentration (P ≥ 0.449; not shown), or respiratory rate (Table 2) when compared with the initial values. At the end of the experiment, the hematocrit had declined to 32.8 ± 0.7% (P ≥ 0.02) for the normal group and remained at 34.2 ± 0.9% (P ≥ 0.38) for the preascitic group (Table 2). Hemodynamic variables for the systemic and pulmonary circulations are shown in Table 3, and Figures 4 to 6. Initially, the groups did not differ in heart rate, stroke volume, or pulmonary vascular resistance. The preascitic broilers had lower initial values for mean systemic arterial pressure and total peripheral resistance and a higher pulmonary arterial pressure when compared with normal broilers (Table 3; Figures 4 to 6). Pulmonary arterial pressure remained higher in preascitic than in normal broilers when compared within contemporaneous sample intervals throughout the experiment. In both groups, the pulmonary arterial pressures recorded during and after 100% O2 inhalation (sample intervals C1 to L) were not lower than during sample intervals B1 to B3 immediately preceding 100% O2 inhalation (Figure 4). The pulmonary vascular resistance was not higher in preascitic than in normal broilers during any sample interval, and in both groups the pulmonary vascular resistance during sample intervals C1 to L was not lower than during sample intervals B1 to B3 immediately preceding 100% O2 inhalation (Figure 4). Blood flow through the left pulmonary artery was higher in preascitic than in normal broilers preceding (sample intervals B1 to B3) and following (sample intervals E1, F1, F2, G2), but not during, 100% O2 inhalation (Figure 4). Cardiac output was higher in preascitic than in normal broilers preceding (sample intervals B1 to B3)

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WIDEMAN ET AL. TABLE 1. Body weight, heart values, hematocrit, respiratory rate, and blood gas values for normal and preascitic broilers breathing ambient air during initial sample intervals1 Variable Body weight, g Right ventricle (RV) weight, g Left ventricle + septum weight, g Total ventricle (TV) weight, g RV:TV ratio Hematocrit, % Respiratory rate, breaths/min Partial pressure of O2, mm Hg HbO2, % (blood gas analyzer) HbO2, % (pulse oximeter) Partial pressure of CO2, mm Hg Bicarbonate concentration, mM Hydrogen ion concentration (Eq/L × 10−8) [pH]

Normal (n = 10) 2,375 ± 2.40 ± 7.97 ± 10.40 ± 0.23 ± 33.6 ± 51.0 ± 96.0 ± 96.2 ± 88.4 ± 34.2 ± 24.3 ± 3.30 ± [7.48]

Preascitic (n = 10) 98 0.17 0.44 0.52 0.01 0.8 2.3 2.2 0.3 0.9 1.4 0.5 0.08

2,780 ± 2.91 ± 7.57 ± 10.50 ± 0.28 ± 35.0 ± 46.8 ± 81.4 ± 92.5 ± 75.9 ± 43.1 ± 26.5 ± 3.87 ± [7.41]

P 33 0.20 0.32 0.47 0.01 0.9 3.5 2.8 0.9 1.6 1.9 0.8 0.14

0.67 0.07 0.47 0.88 0.02 0.25 0.32 0.001 0.001 0.0001 0.001 0.03 0.002

Data are means ± SEM.

1

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FIGURE 3. Experiment 2, partial pressure of O2 (PO2; upper panel), percentage saturation of hemoglobin with oxygen (HbO2; second panel), partial pressure of CO2 (PCO2; third panel), and hydrogen ion concentration (lower panel) in the arterial blood of normal (circles; mean ± SEM; n = 10) and preascitic (squares; mean ± SEM; n = 10) broilers during an initial 20-min period while all broilers breathed ambient air (sample intervals A and B), during a 20-min period when 100% oxygen was supplied (sample intervals C and D), and during a 30-min recovery period when all broilers again breathed ambient air (sample intervals E to G). Different letters (a,b) designate differences (P ≤ 0.05) between the groups within a sample interval. Asterisks denote values that differ (P ≤ 0.05) from both control sample intervals A and B.

and following (sample intervals E1, F1, F2, G2), but not during, 100% O2 inhalation (Figure 5). Blood flow and cardiac output during 100% O2 inhalation were not lower than during the initial sample intervals B1 to B3 in preascitic broilers (Figures 4 and 5). Stroke volume and heart rate did not change over the course of the experiment (Figure 5). Mean arterial pressure increased in the preascitic but not in the normal group during 100% O2 inhalation (sample intervals B1 to B3 vs C1 to D2) (Figure 6). Total peripheral resistance was lower in preascitic than in normal broilers preceding (sample intervals B1 to B3) and following (sample intervals E1, F1, F2, G2), but not during, 100% O2 inhalation. The total peripheral resistance of preascitic broilers increased during 100% O2 inhalation (sample intervals B1 to B3 vs C1 to D2) (Figure 6). Linear regression analysis was used to evaluate the possibility that changes in the partial pressure of O2 or HbO2 triggered subtle changes in other cardio-pulmonary variables. Coefficients of determination for comparisons of the partial pressure of O2 vs cardio-pulmonary variables were poor on a group basis, as well as on an individual bird basis (R2 ≤ 0.05 for all regressions of partial pressure of O2 vs pulmonary arterial pressure, pulmonary vascular resistance, mean arterial pressure, and total peripheral resistance). Presumably the high partial pressures of O2 in arterial blood during 100% O2 inhalation (≥400 mm Hg) disproportionately skewed the regression analysis. Low coefficients of determination (R2) also were obtained for group comparisons of HbO2 vs pulmonary arterial pressure (normal group: R2 = 0.00575; preascitic group: R2 = 0.0984) and pulmonary vascular resistance (normal group: R2 = 0.000453; preascitic group: R2 = 0.0524). However, preascitic broilers did exhibit significant positive correlations between HbO2 vs mean arterial pressure (normal group: R2 = 0.00643, P = 0.2985; preascitic group: R2 = 0.307, P = 0.0001) and total peripheral resistance (normal group: R2 = 0.0109, P = 0.1715; preascitic group: R2 = 0.275, P = 0.0001). Linear regression analyses also were conducted on an individual bird basis (Figure 7). The HbO2 was negatively correlated (P ≤ 0.02) with pulmonary arterial pressure in 3 of 10 normal (R2

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BROILERS BREATHING AIR OR 100% OXYGEN TABLE 2. Respiratory rate and hematocrit for normal and preascitic broilers breathing ambient air (sample intervals A and B), 100% oxygen (sample intervals C and D), and ambient air (sample intervals E to G)1 Sample intervals Variable

Group

A

B

C

D

E

F

G

Respiratory rate (breaths/min)

Normal Preascitic

50.8 ± 2.3 46.5 ± 3.6

51.7 ± 2.6 47.0 ± 3.9

51.3 ± 2.4 43.8 ± 3.6

50.8 ± 2.5 42.7 ± 3.4

53.0 ± 2.9 46.3 ± 4.0

52.3 ± 2.7 46.3 ± 3.1

51.5 ± 3.1 47.2 ± 3.5

Hematocrit (%)

Normal Preascitic

33.6 ± 0.8ab 35.0 ± 1.0

33.5 ± 0.8ab 35.1 ± 0.9

33.9 ± 0.8a 35.2 ± 0.9

33.3 ± 0.8ab 35.0 ± 1.0

33.1 ± 0.7b 34.9 ± 1.0

33.2 ± 0.7b 34.5 ± 0.9

32.8 ± 0.7b 34.2 ± 0.9

Means with different superscripts differ at P ≤ 0.05. Data are means + SEM, n = 10 per group.

a,b 1

DISCUSSION Increases in the pulmonary arterial pressure, RV:TV ratio, and hematocrit and decreases in the HbO2, mean systemic arterial pressure (systemic hypotension) and heart rate (bradycardia) serve as characteristic symptoms of the pathophysiological progression leading to ascites (Peacock et al., 1989; Roush et al., 1996, 1997; Kirby et al., 1997; Wideman et al., 1998c; Forman and Wideman, 1999). The HbO2, RV:TV ratios, and pulmonary arterial pressures of the preascitic broilers in Experiments 1 and 2 are entirely indicative of the early pathophysiological progression leading to ascites. In Experiment 2, the preascitic broilers exhibited pulmonary hypertension, systemic hypotension, hypoxemia, hypercapnia (excessive CO2 reten-

tion in arterial blood), and respiratory acidosis when compared with normal broilers; however, the average RV:TV ratio (0.28) for preascitic broilers was marginal for use as an independent index of pulmonary hypertension. Furthermore, the hematocrit was not elevated, nor was the heart rate depressed when preascitic broilers were compared with normal broilers in Experiment 2. Apparently, the elevated pulmonary arterial pressure and hypoxemia had not been maintained long enough to further amplify the RV:TV ratio and hematocrit, respectively. Similarly, bradycardia is a poor predictor of the earliest stages in the pathogenesis of ascites (Wideman et al., 1998c). The reduction in heart rate reported for some preascitic and ascitic broilers presumably reflects a proportionally longer ventricular filling cycle associated with advanced cardiac dilation (Roush et al., 1996, 1997; Kirby et al., 1997; Forman and Wideman, 1999, Wideman, 1999). Cumulatively, these observations indicate that pulmonary hypertension, the onset of a diffusion limitation (hypoxemia, hypercapnia), and an associated hypoxemic vasodilation (reduced total peripheral resistance, systemic hypotension, and elevated cardiac output) are among the earliest changes occurring during the pathogenesis of ascites. The novel observation that preascitic broilers have an elevated cardiac output when compared with normal broilers contrasts with previous assumptions that normal and ascitic broilers may have a similar cardiac output because their relative left and total ventricular weights did not differ (Wideman et al., 1998c; Wideman, 1999; Wideman and French, 1999a,b). This assumption was based on the strong positive correlation between cardiac output (or stroke volume) and heart size (or ventricular

TABLE 3. Systemic and pulmonary hemodynamic values for normal and preascitic broilers breathing ambient air during initial sample intervals1 Variable

Normal (n = 10)

Cardiac output, mL/min Heart rate, beats/min Stroke volume, mL/beat Mean systemic arterial pressure, mm Hg Total peripheral resistance, standard units Pulmonary arterial pressure, mm Hg Pulmonary vascular resistance, standard units

471 338 1.41 105.8 0.230 22.1 0.048

Data are means ± SEM.

1

Preascitic (n = 10) ± ± ± ± ± ± ±

24 9 0.11 3.3 0.014 2.2 0.005

520 330 1.60 93.1 0.179 29.5 0.057

± ± ± ± ± ± ±

P 13 7 0.05 4.8 0.009 1.7 0.004

0.09 0.51 0.13 0.04 0.01 0.02 0.18

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range: 0.251 to 0.459) and 4 of 10 preascitic (R2 range: 0.339 to 0.610) broilers. The HbO2 was negatively correlated (P ≤ 0.02) with pulmonary vascular resistance in 2 of 10 normal (R2 range: 0.318 to 0.333) and 4 of 10 preascitic (R2 range: 0.406 to 0.492) broilers. Five of 10 normal broilers and 8 of 10 preascitic broilers exhibited positive correlations between HbO2 and mean arterial pressure (normal R2 range for significance: 0.258 to .574; preascitic R2 range for significance: 0.411 to 0.823). Four of 10 normal broilers and 9 of 10 preascitic broilers exhibited positive correlations between HbO2 and total peripheral resistance (normal R2 range for significance: 0.275 to 0.585; preascitic R2 range for significance: 0.448 to 0.902). Four of 10 normal broilers and 8 of 10 preascitic broilers exhibited negative correlations between HbO2 and cardiac output (normal R2 range for significance: 0.252 to 0.503; preascitic R2 range for significance: 0.240 to 0.684) (Figure 7).

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FIGURE 4. Experiment 2, pulmonary arterial pressure (PAP; upper panel), pulmonary vascular resistance (PVR; second panel), and blood flow through the left pulmonary artery (lower panel) for normal (circles; mean ± SEM; n = 10) and preascitic (squares; mean ± SEM; n = 10) broilers during an initial 20-min period while all broilers breathed ambient air (sample intervals S to B3), during a 20-min period when 100% O2 was supplied (sample intervals C1 to D2), and during a 30-min recovery period when all broilers again breathed ambient air (sample intervals E1 to L). Different letters (a,b) designate differences (P ≤ 0.05) between the groups within a sample interval; ns designates not significant.

weight) for a wide variety of avian species (Wideman, 1999). However, ventricular mass reflects the total work performed by each ventricle, which is proportional to the pressure gradient developed by the ventricles to propel the blood, as well as the volume of blood pumped per minute (the cardiac output). The pressure gradient for the left ventricle is essentially equal to the systemic arterial pressure, assuming right atrial pressure is close to zero. In the present study, the systemic arterial pressure was lower in preascitic than in normal broilers, yet the left ventricle plus septum and total ventricular weights did not differ between the groups. Accordingly, the hearts of preascitic broilers pumped a higher cardiac output at a lower systemic arterial pressure and, thereby, performed the same left ventricular work as the hearts of normal broilers pumping a lower cardiac output at a higher systemic arterial pressure. The capacity of the preascitic heart to perform work at a level similar to that of the normal heart contradicts the possibility that a cardiomyopathy

FIGURE 5. Experiment 2, cardiac output (CO; upper panel), heart rate (HR; second panel), and stroke volume (SV; lower panel) for normal (circles; mean ± SEM; n = 10) and preascitic (squares; mean ± SEM; n = 10) broilers during an initial 20-min period while all broilers breathed ambient air (sample intervals S to B3), during a 20-min period when 100% O2 was supplied (sample intervals C1 to D2), and during a 30-min recovery period when all broilers again breathed ambient air (sample intervals E1 to L). Different letters (a,b) designate differences (P ≤ 0.05) between the groups within a sample interval; ns designates not significant.

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contributes to the early pathogenesis of the ascites syndrome. Intracardiac right to left shunts previously had been discounted as a possible cause of the hypoxemia evident in preascitic and ascitic broilers (see Introduction). However, the possibility of intrapulmonary shunts previously had not been addressed in relation to the pathogenesis of ascites. Intrapulmonary shunts occur when blood passes through nonventilated regions of the lungs. Examples of shunts within the avian lung would include direct intrapulmonary arterio-venous anastomoses or vascular perfusion of unventilated parabronchi. When shunts exist, the resulting hypoxemia cannot be abolished by providing 100% oxygen, because blood shunted through an unventilated region is not exposed to the elevated partial pressure of oxygen (West, 1993). In the present study, the hypoxemia extant in preascitic broilers was fully reversed during inhalation of 100% oxygen, consequently shunts do not contribute to the onset or maintenance of hypoxemia early in the pathogenesis of ascites. Instead, the

BROILERS BREATHING AIR OR 100% OXYGEN

concurrent onset of hypoxemia and hypercapnia, coupled with the acute reversal of hypoxemia but not hypercapnia during 100% oxygen inhalation, are symptoms wholly consistent with the existence of a substantial diffusion limitation in preascitic broilers (West, 1993). The available evidence does not support the hypothesis that hypoxemia or localized intrapulmonary hypoxia directly increase the resistance of the pulmonary vasculature and thereby contribute to the onset of pulmonary hypertension in susceptible broilers reared near sea level (see Introduction). The systemic hypoxemia was acutely reversed when preascitic broilers inhaled 100% oxygen, yet pulmonary vascular resistance remained unaffected, and pulmonary arterial pressure was not reduced to normal levels. The use of regression analysis to detect subtle correlations between HbO2 and pulmonary arterial pressure or pulmonary vascular resistance was unrewarding. Fewer than half of the broilers in both experiments exhibited any correlation between HbO2 and pulmonary artery pressure, indicating a wide range of individual variability exists. If intrapulmonary hypoxic or hypoxemic vasoconstriction contributed to the pulmonary hypertension observed in preascitic broilers, then the mechanisms involved clearly cannot be acutely reversed by providing

100% oxygen in the inspired air or by fully saturating the arterial blood with oxygen at a partial pressure of 400 mm Hg. Similarly, normally occurring oxygen gradients along the length of the parabronchi in duck lungs do not significantly influence the distribution of parabronchial blood flow, and any inhomogeneity of vascular perfusion resulting from such a mechanism should reduce the efficiency of gas exchange in the avian lung (Scheid and Holle, 1978). As a caveat to this interpretation, preascitic and ascitic broilers consistently exhibit hypertrophied medial smooth muscle layers in their pulmonary arterioles, confirming an adaptive response of the resistance vessels to chronically sustained pulmonary hypertension (Cueva et al., 1974; Sillau and Montalvo, 1982; Huchzermeyer, 1985; Hernandez, 1987; Peacock et al., 1989; Maxwell, 1991; Enkvetchakul et al., 1995). Chronic remodeling of the primary resistance arterioles has been associated with altered smooth muscle responsiveness to vasoconstrictors and to endothelium-derived vasodilators in mammals. Consequently, the possibility exists that a more prolonged period of inhaling supplemental oxygen might be required to reverse putative hypoxemic pulmonary vasoconstriction in preascitic broilers. However, cattle chronically exposed to hypoxia at high altitudes also exhibit hypertrophy of the pulmonary arteriole medial muscle layer, coupled with an amplified pulmonary vascular responsiveness to changes in inspired oxygen levels (Grover et al., 1963; Alexander, 1965; Grover, 1965; Vogel et al., 1966; Stenmark et al., 1987; Tucker et al., 1975; Will et al., 1975). In contrast to the absence of acute pulmonary vasodilation during 100% O2 inhalation, the systemic resistance

FIGURE 7. Experiment 2, coefficients of determination (R2) for linear regression of the percentage saturation of hemoglobin with oxygen (HbO2) vs pulmonary arterial pressure (PAP), pulmonary vascular resistance (PVR), mean systemic arterial pressure (MAP), total peripheral resistance (TPR), and cardiac output (CO) for individual broilers in the normal (circles) or preascitic (squares) groups. There were 10 birds per group, and each symbol represents 17 sample intervals per bird. Asterisks denote individual variables for which the slope was significant (P ≤ 0.05).

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FIGURE 6. Experiment 2, mean systemic arterial pressure (MAP; upper panel) and total peripheral resistance (TPR; lower panel) for normal (circles; mean ± SEM; n = 10) and preascitic (squares; mean ± SEM; n = 10) broilers during an initial 20-min period while all broilers breathed ambient air (sample intervals S to B3), during a 20-min period when 100% O2 was supplied (sample intervals C1 to D2), and during a 30-min recovery period when all broilers again breathed ambient air (sample intervals E1 to L). Different letters (a,b) designate differences (P ≤ 0.05) between the groups within a sample interval; ns designates not significant. Asterisks denote values that differ (P ≤ 0.05) from sample intervals B1 to B3.

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ACKNOWLEDGMENTS This research was supported by a US-Israel Binational Agricultural Research and Development grant (BARD US-2736-96) and by a grant from Hubbard ISA, Walpole, NH 03608.

REFERENCES Alexander, A. F., 1965. Normal morphology and pathology of the bovine pulmonary circulation at high altitude. Ann. NY Acad. Sci. 127:640–645. Besch, E. L., R. R. Burton, and A. H. Smith, 1971. Influence of chronic hypoxia on blood gas tensions and pH in domestic fowl. Am. J. Physiol. 220:1379–1382. Besch, E. L., and H. Kadono, 1978. Cardiopulmonary responses to acute hypoxia in Domestic fowl. Pages 71–78 in: Respiratory Function in Birds Adult and Embryonic. J. Piiper, ed. Springer-Verlag, New York, NY. Burton, R. R., and A. H. Smith, 1967. The effect of polycythemia and chronic hypoxia on heart mass in the chicken. J. Appl. Physiol. 22:782–785. Burton, R. R., E. L. Besch, and A. H. Smith, 1968. Effect of chronic hypoxia on the pulmonary arterial blood pressure of the chicken. Am. J. Physiol. 214:1438–1442.

Cueva, S., H. Sillau, A. Valenzuela, and H. Ploog, 1974. High altitude induced pulmonary hypertension and right ventricular failure in broiler chickens. Res. Vet. Sci. 16:370–374. Enkvetchakul, B., J. Beasley, and W. Bottje, 1995. Pulmonary arteriole hypertrophy in broilers with pulmonary hypertension syndrome (ascites). Poultry Sci. 74:1676–1682. Fedde, M. R., 1986. Respiration. Pages 191–220 in: Avian Physiology. 4th ed. P. J. Sturkie, ed. Springer-Verlag, New York, NY. Fedde, M. R., G. E. Weigle, and R. F. Wideman, 1998. Influence of feed deprivation on ventilation and gas exchange in broilers: relationship to pulmonary hypertension syndrome. Poultry Sci. 77:1704–1710. Forman, M. F., and R. F. Wideman, 1999. Renal responses of normal and preascitic broilers to systemic hypotension induced by unilateral pulmonary artery occlusion. Poultry Sci. 78:1773–1785. Grover, R. F., 1965. Pulmonary circulation in animals and man at high altitude. Ann. NY Acad. Sci. 127:632–639. Grover, R. F., J. T. Reeves, D. H. Will, and S. G. Blount, Jr., 1963. Pulmonary vasoconstriction in steers at high altitude. J. Appl. Physiol. 18:567–574. 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. Onderstepoort J. Vet. Res. 54:599–602. Henry, J. D., and M. R. Fedde, 1970. Pulmonary circulation time in the chicken. Poultry Sci. 49:1286–1290. Hernandez, A., 1987. Hypoxic ascites in broilers: A review of several studies done in Colombia. Avian Dis. 31:171–183. Huchzermeyer, F. W., 1985. Waterbelly ‘altitude disease’. Poult. Int. May:62–66. Huchzermeyer, F. W., and A.M.C. DeRuyck, 1986. Pulmonary hypertension syndrome associated with ascites in broilers. Vet. Rec. 119:94. Huchzermeyer, F. W., A.M.C. DeRuyck, and H. Van Ark, 1988. Broiler pulmonary hypertension syndrome. III. Commercial broiler strains differ in their susceptibility. Onderstepoort J. Vet. Res. 55:5–9. Jandel Scientific, 1994. SigmaStat威 Statistical Software User’s Manual. Jandel Scientific Software, San Rafael, CA. Julian, R. J., 1993. Ascites in poultry. Avian Pathol. 22:419–454. Julian, R. J., and S. M. Mirsalimi, 1992. Blood oxygen concentration of fast-growing and slow-growing broiler chickens, and chickens with ascites from right ventricular failure. Avian Dis. 36:730–732. Kirby, Y. K., R. W. Mcnew, J. D. Kirby, and R. F. Wideman, Jr., 1997. Evaluation of logistic versus linear regression models for predicting pulmonary hypertension syndrome (ascites) using cold exposure or pulmonary artery clamp models in broilers. Poultry Sci. 76:392–399. Kochera-Kirby, Y. K., N. B. Anthony, J. D. Hughes, R. W. McNew, J. D. Kirby, and R. F. Wideman, 1999a. Electrocardiographic and genetic evaluation of clinically healthy broilers and giant jungle fowl following unilateral bronchus occlusion. Poultry Sci. 78:125–134. Kochera-Kirby, Y. K., R. W. McNew, N. B. Anthony, N. E. Marson, J. D. Hughes, J. D. Kirby, and R. F. Wideman, 1999b. Electrocardiographic evaluation of broilers following unilateral occlusion of an extrapulmonary primary bronchus. Poultry Sci. 78:242–254. Lubritz, D. L., J. L. Smith, and B. N. McPherson, 1995. Heritability of ascites and the ratio of right to total ventricle weight in broiler breeder male lines. Poultry Sci. 74:1237–1241. Maxwell, M. H., 1991. Red cell size and various lung arterial measurements in different strains of domestic fowl. Res. Vet. Sci. 50:233–239. National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC.

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vessels of preascitic broilers rapidly constricted in response to the improved saturation of arterial blood with oxygen. This direct relationship between HbO2 and total peripheral resistance was confirmed by significant regression analysis in 90% of the preascitic broilers, as shown in Figure 7. Increasing the total peripheral resistance reduced the outflow of blood from the arterial pressure reservoir, permitting preascitic broilers to elevate their systemic arterial pressure to normal levels while pumping a cardiac output that no longer exceeded the cardiac output of normal broilers during 100% O2 inhalation. A significant reduction in cardiac output would be expected to attenuate the pulmonary arterial pressure in preascitic broilers but did not do so in the present study. Evidently the sustained pulmonary vascular resistance counteracted the impact of modest reductions in cardiac output, as would be anticipated based on Poiseuille’s relationship in which the vascular pressure gradient is linearly related to flow but exponentially related to the radius of the blood vessel raised to the fourth power, r4 (Sturkie, 1986). The cardio-pulmonary hemodynamic interactions shown in Figures 3 to 7 provide the first direct evidence that the systemic hypotension previously observed in preascitic and ascitic broilers (Peacock et al., 1989; Forman and Wideman, 1999) is caused by profound hypoxemic systemic vasodilation that overwhelms the ability of the heart to elevate cardiac output sufficiently to maintain a normal blood pressure in rapidly growing broilers. The relative cardiac output values for normal and preascitic broilers demonstrate that “poor circulation” does not contribute to the early pathogenesis leading to ascites. Instead, the key feature of this pathogenesis is the primary inability of the lungs to accept the requisite cardiac output at pressures and flow rates low enough to avoid pulmonary hypertension and hypoxemia.

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