Intravenous micro-particle injection and pulmonary hypertension in broiler chickens: cardio-pulmonary hemodynamic responses

Intravenous Micro-Particle Injection and Pulmonary Hypertension in Broiler Chickens: Cardio-Pulmonary Hemodynamic Responses1 R. F. Wideman, Jr.,2 and G. F. Erf Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701

(Key words: broiler, pulmonary hypertension, vascular occlusion, immunology, ascites) 2002 Poultry Science 81:877–886

revealed an incipient pulmonary hemodynamic insufficiency that predisposes broiler chickens to pulmonary hypertension (an elevated pulmonary arterial pressure), hypoxemia (reduced oxygen content of arterial blood), and the pathophysiological progression leading to pulmonary hypertension syndrome (ascites) (Wideman and Bottje, 1993; Wideman, 2000, 2001). For example, during rapid increases in cardiac output (volume of blood pumped by one ventricle of the heart per min) most broilers are incapable of reducing their pulmonary vascular resistance by dilating the primary resistance vessels or recruiting reserve vascular channels. Consequently, increases in cardiac output must be accompanied by proportional increases in the pulmonary arterial pressure and the rate at which red blood cells flow through the pulmonary gas exchange capillaries (Henry and Fedde, 1970; Powell et al., 1985; Peacock et al., 1989; Wideman and Kirby, 1995; Wideman et al., 1996a, 2000; Wideman and Tackett, 2000; Chapman and Wideman, 2001). These pulmonary hemodynamic insufficiencies previously have been exposed in susceptible broilers by tightening a snare surgically positioned around one pulmonary artery. Acute unilateral pulmonary artery occlusion instantaneously doubles the pulmonary vascular

INTRODUCTION Anatomical studies have demonstrated that the lungs of domestic fowl are relatively hyperperfused with blood (reduced lung volume relative to body weight; increased blood:air capillary volume ratio) and have a reduced efficiency of diffusive gas exchange (thicker blood-gas barrier; lower gas exchange surface area; lower oxygen diffusing capacity) when compared with non-domesticated avian species (Duncker, 1972; Abdalla et al., 1982; Vidyadaran et al., 1990). Physiological evaluations have consistently

2002 Poultry Science Association, Inc. Received for publication December 18, 2001. Accepted for publication February 14, 2002. 1 U.S. patent pending (File No. 09-913,774) protects the exclusive rights of the University of Arkansas to all uses of the intravenous microparticle injection technology within the context of evaluating or affecting pulmonary vascular capacity, pulmonary vascular resistance, pulmonary hypertension, cardio-pulmonary hemodynamics, and susceptibility to pulmonary hypertension and pulmonary hypertension syndrome (ascites) in domesticated animal species. 2 To whom correspodence should be addressed: rwideman@mail. uark.edu.

877

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

in cardiac output but rather was attributed to hypoxemic vasodilation of the systemic vasculature (reduced total peripheral resistance). Preliminary histological evaluations revealed micro-particles lodged in inter- and intraparabronchial arterioles, surrounded by aggregates of thrombocytes and mononuclear leukocytes within 30 min post-injection. These observations infer that intravenously injected micro-particles are carried to the lungs by the returning venous blood, where trapping of the micro-particles by the pulmonary vasculature triggers acute responses (increased pulmonary vascular resistance, pulmonary hypertension, systemic hypoxemia, systemic hypotension) that mirror those previously observed following acute occlusion of one pulmonary artery. Additional studies will be required to determine the extent to which the focal immune response to trapped micro-particles promotes local vasoconstriction that amplifies the pulmonary hypertension attributable to direct physical obstruction of precapillary arterioles.

ABSTRACT Experiments were conducted to determine whether intravenous injections of micro-particles, having a size suitable to be trapped by the pulmonary precapillary arterioles, could be used to increase the pulmonary vascular resistance and thereby trigger an acute increase in the pulmonary arterial pressure (pulmonary hypertension). Anesthetized male broilers injected intravenously with inorganic (silica gel, polystyrene) or organic (cellulose, Sephadex) micro-particles developed an immediate pulmonary hypertension in proportion to the cumulative quantities of micro-particles injected. Micro-particle occlusion of a portion of the pulmonary arterioles forced the cardiac output to flow at a higher rate through the remaining vascular channels, thereby exposing a diffusion limitation characterized by undersaturation of the systemic arterial blood with oxygen (hypoxemia). The concurrent onset of systemic hypotension (reduced systemic arterial blood pressure) was not due to a reduction

878

WIDEMAN AND ERF

3

Hubbard ISA, Hot Springs, AR. Sigma Chemical Co., St. Louis, MO. 5 Konigsberg Instruments, Inc., Pasadena, CA. 6 World Precision Instruments, Sarasota, FL. 7 Biopac Systems, Inc., Santa Barbara, CA. 8 Fisher Scientific, St. Louis, MO. 4

MATERIALS AND METHODS Animal Management Broilers were obtained from a commercial hatchery3 on the day of hatch (Day 1), transported to the Poultry Environmental Research Lab at the University of Arkansas Poultry Research Farm, wing-banded, and placed on fresh wood shavings litter in environmental chambers (8 m2 floor space). They were brooded at 33 C on Days 1 to 5, 29 C on Days 6 to 10, and 21 to 24 C thereafter. 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 ad libitum. Lights were on for 24 h/d through Day 5 and for 23 h/d thereafter.

Evaluation of Silica Gel At 30 d of age, three male broilers (1,416 ± 81 g BW) were prepared for pulmonary arterial pressure recordings. The surgical protocol has been described previously (Wideman and Kirby, 1995; Wideman et al., 1996a,b, 1998a, 1999a,b). A surgical plane of anesthesia was induced with intramuscular injections of allobarbital (5,5-diallyl-barbituric acid;4 25 mg kg/BW), and the birds were fastened in dorsal recumbency on a surgical board thermostatically regulated to maintain a surface temperature of 30 C. An incision was made to open the thoracic inlet, and the left pulmonary artery was exposed. A 27-ga × 1-inch needle, bent to a 90° angle midway along its length, was scored and snapped off adjacent to the hub. The resulting blunt end was pressure-fit into a 30 cm length of Silastic tubing5 (0.012-inch i.d., 0.037-inch o.d.) filled with heparinized saline. The point of the needle was inserted into the left pulmonary artery, a 4-cm loop of the Silastic tubing was coiled inside the thoracic inlet to provide strain relief during respiratory motion, and the thoracic inlet was sealed with stainless steel wound clips (Forman and Wideman, 2000). The distal end of the Silastic tubing was attached to a blood pressure transducer interfaced through a Transbridge preamplifier6 to a Biopac MP 100 data acquisition system7 using AcqKnowledge software.7 The left cutaneous ulnar vein (wing vein) was cannulated with PE-50 polyethylene tubing for systemic i.v. injections, and the left anterior tibial vein (leg vein) was cannulated with polyethylene tubing for i.v. injections into the renal portal system. Ongoing intravenous infusions were not administered. When surgical preparations were complete and a stabilization period of 20 min had elapsed, control pulmonary arterial pressure data were recorded. Silica gel8 (32- to 63-µm size range) was suspended at 0.1 g/mL in heparinized saline (150 U ammonium heparin2/mL of 0.9% NaCl), and the mixture was vortexed continuously on a magnetic stirring plate to keep the particles in suspension. The mixture was injected through the leg vein and wing vein cannulae at 0.1 to 0.5 mL (0.01 to 0.05 g) while the pulmonary arterial pressure was recorded. At the end of the experiment, the birds were euthanized with an overdose of anesthetic.

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

resistance by halving the pulmonary vascular capacity, thereby forcing the right ventricle to develop an elevated pulmonary arterial pressure to propel the returning venous blood through the fully engorged vasculature of the unoccluded lung. The increased blood flow rate through pulmonary gas exchange capillaries creates a diffusion limitation (hypoxemia) when the increased speed at which erythrocytes transit the gas exchange capillaries provides too little residence time for the hemoglobin to become fully saturated with oxygen (Henry and Fedde, 1970; Powell et al., 1985; West, 1993; Wideman et al., 1996a, 2000; Wideman, 2000; Wideman and Tackett, 2000). Surgical techniques for increasing the pulmonary vascular resistance have proven highly effective for exposing the pulmonary hemodynamic limitations of susceptible broilers; however, less time consuming and minimally invasive methodologies are desirable for genetic selection programs in which large numbers of individuals must be evaluated (Wideman and French, 1999, 2000; Wideman, 2000). On a theoretical basis, micro-particles having a size suitable to be trapped in the pulmonary vasculature could be injected into a systemic vein and then would be carried to the lungs by the returning venous blood. Vascular occlusion in proportion to the number of particles injected should provide the means for efficiently creating a controllable, sustained increase in pulmonary vascular resistance. Indeed, the capacity of blood-borne micro-particles to lodge within the systemic or pulmonary micro-vasculature serves as the basis for using intravascularly injected microspheres of various sizes (15- to 52-µm diameter) and compositions (cross-linked dextran, polystyrene) to measure the distribution of blood flow to various organs and tissues (Boelkins et al., 1973; Scheid and Holle, 1978; Wolfenson et al., 1978; Wolfenson, 1983; Brackenbury et al., 1990; Bernard et al., 1996). A pulmonary vascular trapping efficiency of close to 100% has been validated for 15- to 52-µm diameter microspheres in several avian species (Boelkins et al., 1973; Sheid and Holle, 1978; Brackenbury et al., 1990). Only trace quantities of microspheres are injected during blood flow studies, specifically to avoid vascular occlusion and the consequent alteration of micro-vascular hemodynamics. It also has been recognized that microspheres injected into venous blood flowing into the avian liver or kidneys will become trapped within the hepatic and renal portal sinusoids, thereby preventing those microspheres from reaching the lungs (Wolfenson et al., 1978; Wolfenson, 1983). The experiments reported herein were designed to evaluate the acute cardio-pulmonary hemodynamic responses caused by intravenous injections of micro-particles in quantities sufficient to occlude precapillary arterioles and trigger an increase in pulmonary vascular resistance.

MICRO-PARTICLE INJECTIONS AND ACUTE CARDIO-PULMONARY RESPONSES

Evaluation of Dextran Polymer

Evaluation of Polystyrene Four male broilers (1,811 ± 37 g BW) were anesthetized with allobarbital at 36 to 37 d of age, cannulated for wing vein injections, and prepared for recording the pulmonary and systemic arterial pressures, as described in the Sephadex protocol above. In addition, to measure pulmonary blood flow, an incision was made to open the thoracic inlet, 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, and then the skin of the thoracic inlet was sealed with surgical wound clips. Polystyrene microspheres10 (15-µm diameter) coated with calcium phosphate to improve aqueous miscibility were suspended at 0.02 g/mL in heparinized saline (200 U ammonium heparin/mL of 0.9% NaCl). Appropriate volumes (0.1, 0.2, 0.4, and 0.6 mL) of the suspension were injected via the wing vein. The birds were euthanized with a 10-mL i.v. injection of 0.1 M KCl at the end of the experiment.

Evaluation of Cellulose Seven male broilers (2,691 ± 22 g BW) were anesthetized with allobarbital at 46 to 47 d of age, cannulated for wing

9

Transonic Systems Inc., Ithaca, NY. Bangs Laboratories, Inc., Fishers, IN. Radiometer America Inc., Westlake, OH.

10 11

vein injections, and prepared for continuously recording the pulmonary arterial pressure, systemic arterial pressure, and blood flow through the left pulmonary artery, as described above. When surgical preparations were complete and a stabilization period of 10 min had elapsed, control data were collected for 10 min, and an arterial blood sample was collected from the brachial artery cannula within 5 min after the start of data recording (blood sample A). One milliliter of heparinized saline was injected i.v. (volume control), data were collected for 10 min, and an arterial blood sample was collected within 5 min after the start of the Volume Control period (blood sample B). Microgranular CM-32 ion exchange cellulose8 (30-µm average particle diameter) was suspended at 0.02 g/mL in heparinized saline (150 U ammonium heparin/mL of 0.9% NaCl), and the cellulose was injected through the wing vein cannula in volumes of 0.1, 0.2, and 0.2 mL (0.002, 0.004, and 0.004 g, respectively). Data were recorded for 10 min after each injection, and arterial blood samples were collected within 5 min after each injection (blood samples C, D, and E). Each cellulose injection was immediately followed by 0.8 mL of heparinized saline to flush any residual cellulose through the wing vein cannula. Arterial blood (1 mL) was withdrawn anaerobically and injected within 30 s into a Radiometer ABL 330 Acid-Base Laboratory.11 Appropriate function of the blood gas analyzer was assessed by periodically injecting Blood Gas Qualicheck reference standards.11 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). At the end of the experiment, the birds were euthanized with a 10-mL i.v. injection of 0.1 M KCl, dissected to obtain heart weights for calculating the right to total ventricular weight ratio ratio, and the lungs were fixed by immersion in 10% phosphate-buffered formalin.

Data Calculation The primary channels recorded by the Biopac MP 100 data acquisition system included systemic arterial pressure (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 data collection (start), immediately preceding (sample intervals A1, B1, C1, D1, E1) and following (sample intervals A2, B2, C2, D2, E2) withdrawal of each arterial blood sample, immediately preceding each volume control or cellulose injection (sample intervals volume control pre, 0.1 mL pre, 0.2 mL pre, and 0.2 mL pre), and during the maximum (peak) pulmonary arterial pressure recorded within 60 s after each volume control or cellulose injection (sample intervals volume control peak, 0.1 mL peak, 0.2 mL peak, and 0.2 mL peak). 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

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

Four male broilers (1,080 + 64 g BW) were anesthetized with allobarbital at 28 to 32 d of age and prepared for surgery as described above. Silastic tubing (0.012-inch i.d., 0.037-inch o.d.) filled with heparinized saline was inserted through the left cutaneous ulnar vein, advanced into the right pulmonary artery as described previously (Owen et al., 1995; Wideman et al., 1996a), and the distal end of the cannula was attached to a blood pressure transducer for continuous monitoring of pulmonary arterial pressure. The right cutaneous ulnar vein was cannulated with PE-50 polyethylene tubing for systemic i.v. injections. 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 as described previously (Wideman et al., 1996a). Baseline data were collected for 10 to 20 min, and then a suspension of dextran polymer (Sephadex,4 G25-40 or G25-80, particle sizes from 10 to 80 µm; 0.02 g/mL in heparinized saline) was injected through the wing vein cannula at 0.1 to 0.4 mL (0.002 to 0.008 g) per injection, with approximately 10 min allowed to elapse between injections. At the end of the experiment, the birds were euthanized with a 10-mL i.v. injection of 0.1 M KCl, and the lungs were fixed by immersion in 10% phosphate-buffered formalin.

879

880

WIDEMAN AND ERF

Statistical Analysis Data were analyzed over time (across sample intervals) using the SigmaStat repeated measures analysis of variance procedure, and means were differentiated by the StudentNewman-Keuls method (Jandel Scientific, 1994).

RESULTS Histological evaluations of lungs fixed after Cellulose and Sephadex injections revealed particles lodged in the inter- and intra-parabronchial arterioles (Abdalla and King, 1975; King et al., 1978). Quantitative estimates, obtained by us, suggest that fewer than 10% of the arterioles in the gas exchange region were occluded by cellulose or Sephadex particles at the 0.2-mL injection dose. There was preliminary evidence of very rapid inflammation-associated changes in the lung parenchyma. Trapped particles were surrounded by aggregates of thrombocytes and mononuclear leukocytes within 30 min post-injection. Swelling of endothelial cells and congestion of capillaries with erythrocytes also were evident. These preliminary observations demonstrate the induction of a dynamic inflammatory response following blood-borne particle presentation to the lungs of broilers, detailed assessments of which will be published separately. Silica gel rapidly settled out of suspension when aspirated into a syringe prior to i.v. injection and tended to

clog needles of 23 ga or smaller. Figure 1 illustrates the typical response to silica gel injections. The initial pulmonary arterial pressure averaged 22 mm Hg. Injecting 0.4 mL (0.04 g) silica gel into the leg vein flowing into the renal portal system had no impact on pulmonary arterial pressure. The subsequent 0.3-mL (0.03 g silica gel) injection into the wing vein increased the pulmonary arterial pressure to a peak of 25 mm Hg, with the pressure subsiding to 23 mm Hg within 1 min. A portion of the particles remained visible in the wing vein cannula, and flushing the cannula with 1.0 mL of heparinized saline triggered a secondary peak in the pulmonary arterial pressure. A final injection of 0.4 mL (0.04 g silica gel) into the wing vein cannula caused the pulmonary arterial pressure to increase to 31.5 mm Hg (Figure 1), and this level of pulmonary hypertension was sustained until the experiment was terminated 1 h post-injection (not shown). Cumulative wing vein injections of ≥0.4 mL (0.04 g) of silica gel caused sustained increases in pulmonary arterial pressure in all broilers evaluated, whereas leg vein (renal portal) injections and 0.5-mL bolus injections of heparinized saline had no detectable impact on pulmonary arterial pressure (not shown). Responses representative of intravenous dextran polymer injections are shown in Figure 2. The pulmonary arterial pressure averaged 25 mm Hg during the 20 min preinjection control period, increased by an average of 3 mm Hg after the first dextran polymer injection, and then increased to 30 mm Hg following the second dextran polymer injection. The systemic arterial pressure tended to decline after each injection (Figure 2). Qualitatively similar responses were observed in all four of the broilers evaluated (not shown). Figure 3 illustrates typical responses to i.v. injections of polystyrene microspheres. The pulmonary arterial pressure averaged 26 mm Hg during the control interval and did not increase after the initial 0.2 mL injection of microspheres (not shown). The subsequent 0.4- and 0.6-mL injections of the microsphere suspensions caused the pulmonary arterial pressure to increase to 31 and then 35 mm Hg. The mean arterial pressure and cardiac output were unaffected by these cumulative injection doses of polystyrene microspheres, and the pulmonary arterial pressure rapidly returned toward the pre-injection baseline following the microsphere injections (not shown). Responses of an individual broiler to cellulose injections are shown in Figure 4. The pulmonary arterial pressure initially averaged 23 mm Hg, and the 1-mL volume control injection did not influence any of the physiological variables. The 0.1-mL cellulose injection increased the pulmonary arterial pressure to 25 mm Hg. The pulmonary arterial pressure increased to 28 mm Hg following the first 0.2-mL cellulose injection and then to 30 mm Hg after the second 0.2-mL cellulose injection. Both 0.2-mL cellulose injections tended to cause contemporaneous reductions in the mean systemic arterial pressure and cardiac output (Figure 4). The responses of seven male broilers to cellulose injections are summarized in Figures 5 to 8. The pulmonary arterial pressure initially averaged 22 mm Hg, did not increase

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

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 approximately equally between both lungs, cardiac output was calculated as 2 × blood flow. 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 pressure, respectively (Wideman et al., 1996a,b, 1998a,b), then the relationships between pressure gradients, flow rates, and resistances are 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, 1998a). 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).

MICRO-PARTICLE INJECTIONS AND ACUTE CARDIO-PULMONARY RESPONSES

881

FIGURE 2. Physiograph recording of systemic arterial pressure (upper) and pulmonary arterial pressure (lower) in a male broiler during repeated 0.2-mL wing vein injections of dextran polymer (Sephadex; 0.02 g/mL of heparinized saline).

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

FIGURE 1. Physiograph recording of pulmonary arterial pressure in a male broiler when a silica gel suspension (0.1 g silica gel/mL of heparinized saline) was injected into a leg vein flowing through the renal portal system (0.4 mL in leg vein) or into a wing vein returning to the right ventricle and thus the lungs (0.3 and 0.4 mL in wing vein). The “flush cannula” markers denote 1-mL injections of heparinized saline (150 U ammonium heparin/mL 0.9% NaCl) into the pulmonary artery cannula to reduce blood clot formation at the tip of the cannula (note diminishing pulse pressure prior to the first three flushes). The “flush particles out of wing vein cannula” marker denotes an injection of 1-mL heparinized saline through the wing vein cannula to rinse retained silica gel particles into the venous blood.

882

WIDEMAN AND ERF

FIGURE 4. Physiograph recording of systemic arterial pressure (upper), pulmonary arterial pressure (middle), and cardiac output (lower) in a male broiler during sequential wing vein injections of 1-mL heparinized saline (volume control), or of microgranular cellulose (0.02 g/mL of heparinized saline) in volumes of 0.1, 0.2, and 0.2 mL. The gaps in the systemic arterial pressure tracing correspond with blood sample collections (1 mL each) from the brachial artery cannula for blood gas determinations. Arterial samples were collected 5 min after the start to the experiment (sample A), and within 5 min following each of the injections (samples B to E). The cardiac output data represent a direct tracing from a flow probe on the left pulmonary artery, with the Y-axis values doubled in this figure to represent the cardiac output flowing through both pulmonary arteries.

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

FIGURE 3. Physiograph recording of systemic arterial pressure (upper), pulmonary arterial pressure (middle), and cardiac output (lower) in a male broiler during sequential wing vein injections of polystyrene microspheres (0.02 g/mL of heparinized saline) in volumes of 0.4, and 0.6 mL. The cardiac output data represent a direct tracing from a flow probe on the left pulmonary artery, with the Y-axis values doubled in this figure to represent the cardiac output flowing through both pulmonary arteries.

MICRO-PARTICLE INJECTIONS AND ACUTE CARDIO-PULMONARY RESPONSES

above control levels following the volume control and 0.1mL cellulose injections, but increased to values averaging 25 and then 29 mm Hg after the first and second 0.2-mL cellulose injections, respectively (Figure 5). These increases in pulmonary arterial pressure were sufficient to overcome contemporaneous increases in pulmonary vascular resistance; accordingly, the rate of blood flow through the left pulmonary artery did not decline (P > 0.1) when compared with pre-injection control values (Figure 5). Reductions in the mean systemic arterial pressure during sequential cellulose injections were associated with corresponding reductions in total peripheral resistance (systemic vasodilation) rather than reductions in cardiac output (Figure 6). Heart rate and stroke volume remained unchanged throughout the experiment (Figure 7). Cellulose trapping by a portion of the pulmonary vascular channels caused a modest diffusion limitation characterized by mild reductions in the arterial partial pressure of oxygen and the

FIGURE 6. Mean systemic arterial pressure (upper panel), total peripheral resistance (middle panel), and cardiac output (lower panel) for seven male broilers (mean ± SEM) at the start of data collection (Start), immediately preceding (sample intervals A1, B1, C1, D1, E1) and following (sample intervals A2, B2, C2, D2, E2) withdrawal of each arterial blood sample, immediately preceding each volume control or cellulose injection [sample intervals volume control (VC) pre, 0.1 pre, 0.2 pre, and 0.2 pre], and during the peak pulmonary arterial pressure response within 60 s following each volume control or cellulose injection (sample intervals VC Peak, 0.1 Peak, 0.2 Peak, and 0.2 Peak). Different letters (a,b,c) designate differences between means over time (P ≤ 0.05).

saturation of hemoglobin with oxygen (Figure 8). The partial pressure of carbon dioxide and the hydrogen ion concentration in arterial blood were unchanged by the cellulose injections, reflecting the substantially higher rate of diffusion for carbon dioxide crossing the gas exchange surfaces (Figure 8). The respiratory rate did not change over the course of the experiment, averaging (mean ± SEM) 51 ± 3, 53 ± 4, 53 ± 4, 51 ± 3, and 58 ± 4 breaths/min at blood sample intervals A to E, respectively.

DISCUSSION When injected into a wing vein in sufficient quantities, all micro-particles evaluated in the present study triggered an acute (within 30 s) increase in pulmonary arterial pressure. Injections into a leg vein did not elicit pulmonary hemodynamic responses, presumably because the microparticles became trapped within the renal portal sinusoids and thus were cleared from the venous blood (Wolfenson

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

FIGURE 5. Pulmonary arterial pressure (upper panel), pulmonary vascular resistance (middle panel), and blood flow through the left pulmonary artery (PA; lower panel) for seven male broilers (mean ± SEM) at the start of data collection (Start), immediately preceding (sample intervals A1, B1, C1, D1, E1) and following (sample intervals A2, B2, C2, D2, E2) withdrawal of each arterial blood sample, immediately preceding each volume control or cellulose injection [sample intervals volume control (VC) pre, 0.1 pre, 0.2 pre, and 0.2 pre], and during the peak pulmonary arterial pressure response within 60 s following each volume control or cellulose injection (sample intervals VC Peak, 0.1 Peak, 0.2 Peak, and 0.2 Peak). Different letters (a,b,c) designate differences between means over time (P ≤ 0.05).

883

884

WIDEMAN AND ERF

et al., 1978; Wolfenson, 1983). The physiological sequelae to systemic (nonportal) intravenous injections of cellulose micro-particles are consistent with the interpretation that particle trapping within pulmonary precapillary arterioles cumulatively increased the resistance to blood flow in proportion to the number of vascular channels affected. The increased pulmonary vascular resistance forced the right ventricle to develop a higher pulmonary arterial pressure to propel the cardiac output at a higher flow rate through the unobstructed vascular channels. Hypoxemia ensued when the increased speed at which erythrocytes transited the gas exchange capillaries exposed an incipient diffusion limitation known to be characteristic of broiler lungs (Wideman and Tackett, 2000; Wideman et al., 2000). The onset of hypoxemia apparently caused the systemic vasculature to dilate and reduce the total peripheral resistance as a means of increasing the blood flow and thus oxygen delivery to the tissues. The increased outflow of blood from the arterial pressure reservoir in turn caused a contemporaneous reduction in systemic arterial pressure, without initiating compensatory changes in heart rate, stroke volume, or cardiac output. Overall, the cardio-pulmonary hemodynamic perturbations caused by systemic i.v. micro-particle injections are directly analogous to those initiated by acute unilateral pulmonary artery occlusion, including increases in pulmonary vascular resistance and pulmonary arterial

FIGURE 8. Partial pressure of oxygen (upper panel), saturation of hemoglobin with oxygen (second panel), partial pressure of carbon dioxide (third panel), and hydrogen ion concentration (lower panel) in arterial blood from male broilers (mean ± SEM, n = 7) within 5 min after the start of data collection (sample A), and within 5 min following a volume control injection (sample B) or cellulose injections (samples C, D, E). Different letters (a,b) designate differences between means over time (P ≤ 0.05).

pressure, hypoxemia, and reductions in total peripheral resistance and systemic arterial pressure (Peacock et al., 1989, 1990; Reeves et al., 1991; Wideman and Kirby, 1995; Wideman et al., 1996a,b, 1998b, 1999b; Fedde et al., 1998; Forman and Wideman, 1999; Wideman, 2000, 2001). Additional studies will be required to determine the extent to which the observed increases in pulmonary vascular resistance caused by micro-particles reflect immunemediated responses within the pulmonary vasculature. The lungs normally perform the essential function of rapidly clearing the blood of particulate matter, such as aged red blood cells, micro- and macro-thromboemboli, bacteria, immune complexes, and cellular debris. This pulmonary vascular capacity to rapidly filter the blood serves to defend sensitive tissues such as the brain and heart; however, the process of physically trapping and immunologically responding to particulates can be profoundly deleterious to pulmonary hemodynamics and gas exchange (Malik, 1983; Dantzker, 1997; Heffner and Repine, 1997; Brain et al., 1999). Two interactive mechanisms may contribute to

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

FIGURE 7. Heart rate (upper panel) and stroke volume (lower panel) for seven male broilers (mean ± SEM) at the start of data collection (Start), immediately preceding (sample intervals A1, B1, C1, D1, E1) and following (sample intervals A2, B2, C2, D2, E2) withdrawal of each arterial blood sample, immediately preceding each volume control or cellulose injection [sample intervals volume control (VC) pre, 0.1 pre, 0.2 pre, and 0.2 pre], and during the peak pulmonary arterial pressure response within 60 s following each volume control or cellulose injection (sample intervals VC Peak, 0.1 Peak, 0.2 Peak, and 0.2 Peak).

MICRO-PARTICLE INJECTIONS AND ACUTE CARDIO-PULMONARY RESPONSES

pable of responding to vasoactive mediators (Abdalla and King, 1975; King et al., 1978). Such considerations must be evaluated in future studies designed to clarify the different pulmonary hemodynamic responses to particulates of varying sizes and compositions.

ACKNOWLEDGMENTS This research was supported by Hubbard ISA, BARD project No. US-2736-96, and an Arkansas Agricultural Experiment Station Animal Health—Research Initiation Grant.

REFERENCES Abdalla, M. A., and A. S. King. 1975. The functional anatomy of the pulmonary circulation of the domestic fowl. Respir. Physiol. 23:267–290. Abdalla, M. A., J. N. Maina, A. S. King, D. Z. King, and J. Henry. 1982. Morphometrics of the avian lung. I. The domestic fowl (Gallus gallus variant domesticus). Respir. Physiol. 55:291–278. Bernard, S. L., R. W. Glenny, H. H. Erickson, M. R. Fedde, N. Polissar, R. J. Basaraba, and M. P. Hlastala. 1996. Minimal redistribution of pulmonary blood flow with exercise in racehorses. J. Appl. Physiol. 81:1062–1070. 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. SpringerVerlag, New York. Boelkins, J. N., W. J. Mueller, and K. Hall. 1973. Cardiac output distribution in the laying hen during shell formation. Comp. Biochem. Physiol. 46A:735–743. Brackenbury, J. H., M. S. El-Sayed, and C. Darby. 1990. Effects of treadmill exercise on the distribution of blood flow between the hindlimb muscles and abdominal viscera of the laying fowl. Br. Poult. Sci. 31:207–214. Brain, J. D., R. M. Molina, M. M. DeCamp, and A. E. Warner. 1999. Pulmonary intravascular macrophages: their contribution to the mononuclear phagocyte system in 13 species. Am. J. Physiol. 276:L146–L154. Chapman, M. E., and R. F. Wideman. 2001a. Pulmonary wedge pressures confirm pulmonary hypertension in broilers is initiated by an excessive pulmonary arterial (precapillary) resistance. Poult. Sci. 80:468–473. Chapman, M. E., and R. F. Wideman. 2002. Hemodynamic responses of broiler pulmonary vasculature to intravenously infused serotonin. Poult. Sci. 81:231–238. Dantzker, D. R. 1997. Pulmonary embolism. Pages 1599–1607 in The Lung: Scientific Foundations. 2nd ed. R. G. Crystal, J. B. West, P. J. Barnes, E. R. Weibel, ed. Lippincott-Raven, Philadelphia. Duncker, H. R. 1972. Structure of avian lungs. Respir. Physiol. 22:1–19. Faltin, D. L., A. Weber, J. S. Lacroix, M. Jorge-Costa, and D. R. Morel. 1996. Lung mechanics and pulmonary but not systemic vascular responses to ET-1 are Tx and infusion rate dependent. J. Appl. Physiol. 80:1716–1723. Fedde, M. R. 1986. Respiration. Pages 191–220 in Avian Physiology. 4th ed. P. D. Sturkie, ed. Springer-Verlag, New York. 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. Poult. 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. Poult. Sci. 78:1773–1785.

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

the onset of pulmonary hypertension as the lungs perform their normal function of entrapping particulates. First, physical occlusion of vascular channels reduces the overall cross-sectional area of the pulmonary vasculature, thereby directly increasing the resistance to blood flow. Second, the immune response to entrapped particulates may amplify the pulmonary hypertensive responses through the local release of vasoactive substances such as endothelin1, thromboxane, and serotonin (Heffner and Repine, 1997). Indeed, various particulates and bacterial endotoxins stimulate endothelin-mediated thromboxane-dependent increases in pulmonary vascular resistance and pulmonary arterial pressure in mammals and birds (Morel et al., 1989; Longworth et al., 1994; Staub, 1994; Faltin et al., 1996; Wideman et al., 2001). Thromboxane and serotonin are potent pulmonary vasoconstrictors in broilers, and both of these vasoactive compounds are stored in or produced by the thrombocytes or endothelial cells of domestic fowl (Lacoste-Eleaume et al., 1994; Wideman et al., 1999a, 2001; Chapman and Wideman, 2002). Broilers have a cardiac output averaging ≥150 mL/min per kg BW and a total blood volume averaging ≥8% of BW, consequently broiler lungs routinely process the entire blood volume approximately every 30 s, providing a highly dynamic opportunity for circulating components of the immune system to respond to trapped particulates (Sturkie, 1986; Wideman, 2000). In the present study, micro-particles trapped within the lung parenchyma were quickly surrounded by aggregates of thrombocytes and mononuclear leukocytes, raising the likelihood that regional vasoconstriction in response to focally released thromboxane or serotonin could substantially amplify the overall increase in pulmonary vascular resistance. Accordingly, numerous factors must be taken into account when attempting to explain the observed variability in pulmonary hypertensive responses induced by micro-particle types having different sizes and compositions. Such variability may reflect interactions dependent on the immunostimulatory properties of the micro-particles, particle density as related to the absolute numbers of micro-particles per unit of dry weight, and the size or location of the vascular channels in which the micro-particles become trapped. For example, the polystyrene microspheres are small enough (15-µm diameter) to pass through the terminal arterioles and become trapped within individual capillaries. In that case, only one capillary would be occluded per microsphere injected, and if polystyrene is not inert antigenically, then the subsequent immune reaction would occur within the parabronchial gas exchange capillary bed where arteriole (primary resistance vessel) smooth muscle is virtually absent (Abdalla and King, 1975; King et al., 1978). In contrast, larger micro-particles such as cellulose (average particle length 30 µm) are trapped in the terminal arterial branches. Consequently, each occlusive cellulose micro-particle blocks or reduces blood flow through a multitude of “downstream” capillaries, and the very rapid and marked immune response to trapped cellulose micro-particles occurs within the inter-parabronchial vasculature where abundant arterial smooth muscle is ca-

885

886

WIDEMAN AND ERF West, J. B. 1993. Respiratory Physiology—The Essentials. 5th ed. Williams and Wilkins, Philadelphia. Wideman, R. F. 2000. Cardio-pulmonary hemodynamics and ascites in broiler chickens. Avian Poult. Biol. Rev. 11:21–43. Wideman, R. F. 2001. Pathophysiology of heart/lung disorders: Pulmonary hypertension syndrome in broiler chickens. World’s Poult. Sci. J. 57:289–301. Wideman, R. F., and W. G. Bottje. 1993. Current understanding of the ascites syndrome and future research directions. Pages 1–20 in Nutrition and Technical Symposium Proceedings. Novus International, Inc., St. Louis. Wideman, R. F., G. F. Erf, and M. E. Chapman. 2001. Intravenous endotoxin triggers pulmonary vasoconstriction and pulmonary hypertension in broiler chickens. Poult. Sci. 80:647–655. 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, 1998b. 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 breeder 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., and H. French. 2000. Ascites resistance of progeny from broiler breeders selected for two generations using chronic unilateral pulmonary artery occlusion. Poult. Sci. 79:396–401. Wideman, R. F. and Y. K. Kirby. 1995. Evidence of a ventilationperfusion mismatch during acute unilateral pulmonary artery occlusion in broilers. Poult. Sci. 74:1209–1217. Wideman, R. F., Y. K. Kirby, M. F. Forman, N. Marson, R. W. McNew, and R. L. Owen. 1998a. The infusion rate dependent influence of acute metabolic acidosis on pulmonary vascular resistance in broilers. Poult. Sci. 77:309–321. Wideman, R. F., Y. K. Kirby, C. D. Tackett, N. E. Marson, and R. W. McNew. 1996a. 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., Y. K. Kirby, C. D. Tackett, N. E. Marson, C. J. Tressler, and R. W. McNew, 1996b. Independent and simultaneous unilateral occlusion of the pulmonary artery and extrapulmonary primary bronchus in broilers. Poult. Sci. 75:1417–1427. Wideman, R. F., P. Maynard, and W. G. Bottje. 1999a. Thromboxane mimics the pulmonary but not systemic vascular responses to bolus HCl injections in broiler chickens. Poult. Sci. 78:714–721. Wideman, R. F., P. Maynard, and W. G. Bottje. 1999b. Venous blood pressure in broilers during acute inhalation of 5% carbon dioxide or unilateral pulmonary artery occlusion. Poult. Sci. 78:1443–1451. Wideman, R. F., and C. D. Tackett. 2000. Cardio-pulmonary function in broilers reared at warm or cool temperatures: Effect of acute inhalation of 100% oxygen. Poult. Sci. 79:257–264. Wolfenson, D. 1983. Blood flow through arteriovenous anastomoses and its thermal function in the laying hen. J. Physiol. 334:395–407. Wolfenson, D., A. Berman, Y. F. Frei, and N. Snapir. 1978. Measurement of blood flow distribution by radioactive microspheres in the laying hen (Gallus domesticus). Comp. Biochem. Physiol. 61A:549–554.

Downloaded from http://ps.oxfordjournals.org/ at University of Oklahoma on March 28, 2015

Forman, M. F., and R. F. Wideman. 2000. Measurements of pulmonary arterial pressure in anesthetized male broilers at two and seven weeks of age. Poult. Sci. 79:1645–1649. Heffner, J. E., and J. E. Repine. 1997. Platelets. Pages 947–959 in The Lung: Scientific Foundations. 2nd ed. R. G. Crystal, J. B. West, P. J. Barnes, E. R. Weibel, ed. Lippincott-Raven, Philadelphia. Henry, J. D., and M. R. Fedde. 1970. Pulmonary circulation time in the chicken. Poult. Sci. 49:1286–1290. Jandel Scientific. 1994. SigmaStat Statistical Software User’s Manual. Jandel Scientific Software, San Rafael, CA. King, A. S., D. Z. King, and M. A. Abdalla. 1978. The structure of the intrapulmonary vasculature of the domestic fowl. Pages 112–124 in Respiratory Function in Birds, Adult and Embryonic. J. Piiper, ed. Springer-Verlag, New York. Lacoste-Eleaume, A.-S., C. Bleux, P. Que´re´, F. Coudert, C. Corbel, and C. Kanellopoulos-Langevin. 1994. Biochemical and functional characterization of an avian homolog of the integrin GPIIb-IIIa present on chicken thrombocytes. Exp. Cell Res. 213:198–209. Longworth, K. E., K. A. Jarvis, W. S. Tyler, E. P. Steffey, and N. C. Staub. 1994. Pulmonary intravascular macrophages in horses and ponies. Am. J. Vet. Res. 55:382–388. Malik, A. B. 1983. Pulmonary microembolism. Physiol. Rev. 63:1114–1207. Morel, D. R., J. S. Lacroix, A. Hemsen, D. A. Steinig, J.-F. Pittet, and J. M. Lundberg. 1989. Increased plasma and pulmonary lymph levels of endothelin during endotoxin shock. Eur. J. Pharmacol. 167:427–428. National Research Council. 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Owen, R. L., R. F. Wideman, and B. S. Cowen. 1995. Changes in pulmonary arterial and femoral arterial blood pressure upon acute exposure to hypobaric hypoxia in broiler chickens. Poult. Sci. 74:708–715. Peacock, A. J., C. Pickett, K. Morris, and J. T. Reeves. 1989. The relationship between rapid growth and pulmonary hemodynamics in the fast-growing broiler chicken. Am. Rev. Respir. Dis. 139:1524–1530. Peacock, A. J., C. Pickett, K. Morris, and J. T. Reeves. 1990. Spontaneous hypoxaemia and right ventricular hypertrophy in fast growing broiler chickens reared at sea level. Comp. Biochem. Physiol. 97A:537–541. Powell, F. L., R. H. Hastings, and R. W. Mazzone. 1985. Pulmonary vascular resistance during unilateral pulmonary artery occlusion in ducks. Am. J. Physiol. 249:R39–R43. Reeves, J. T., G. Ballam, S. Hofmeister, C. Pickett, K. Morris, and A. Peacock. 1991. Improved arterial oxygenation with feed restriction in rapidly growing broiler chickens. Comp. Biochem. Physiol. 99A:481–485. Scheid, P., and J. P. Holle. 1978. Adjustment of the regional pulmonary circulation to the profile of oxygen pressure along the parabronchus of the duck. Pages 105–109 in Respiratory Function in Birds Adult and Embryonic. J. Piiper, ed. SpringerVerlag, New York. Staub, N. C. 1994. Pulmonary intravascular macrophages. Annu. Rev. Physiol. 56:47–67. Sturkie, P. D. 1986. Heart and circulation: anatomy, hemodynamics, blood pressure, blood flow. Pages 130–166 in Avian Physiology. 4th ed. P. J. Sturkie, ed. Springer-Verlag, New York. Vidyadaran, M. K., A. S. King, and H. Kassim. 1990. Quantitative comparisons of lung structure of adult domestic fowl and red jungle fowl, with reference to broiler ascites. Avian Pathol. 19:51–58.