Distensibility of the Pulmonary Arteries of Normoxia and Hypoxia Adapted Rats Robert C. Molthen1,2,3, Steve T. Haworth1, Amy E. Heinrich1 , Christopher C. Hanger 4, David L. Roerig 2, and Christopher A. Dawson1,2,3

1 Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 532262 Research Service, Department of Veterans Affairs, Zablocki VAMC, Milwaukee, WI 53295 3 Department of Biomedical Engineering, Marquette University, Milwaukee, WI 53201-18814 Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226

This work supported by HL 19298, the Whitaker Foundation, the W.M. Keck Foundation and the Department of Verterans Affairs.

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Figure 2 : Digital acquisition of images and control of hardware is managed by a Windows NT based workstation. Three dimensional micro-CT reconstructions are obtained by using the Feldkamp cone-beam filtered backprojection algorithm on the multiple 2D planar projection images acquired of the object (lung) from different angles as the specimen stage rotates through 360 degrees. Various computer platforms are used for reconstruction, rendering, and morphometric analysis.

Figure 4 : The lung is suspended by a tracheal and pulmonary artery cannula in a low attenuation plastic cyclinder. Perfluorooctyl bromide, for x-ray contrast, is introduced into the pulmonary arteries and adjusted to maintain an intravascular pressure of 5.4, 12, 21, or 30 mmHg. The airways are maintained at a pressure of 6 mmHg.

Lungs were removed from Sprague-Dawley rats after 21 day exposure to anomoxic or hypoxic (10% O2) environment. Relevant biometric parameters including pulmonary perfusion pressure versus flow were measured, and thedistensibility of small (diameters less than 0.50 mm), medium (diameters 0.5 - 1.4 mm), and large (diameters greater than 1.4 mm) arteries was measured by micro-focal computed tomography. The diameter, D, vs. pressure, P, relation was described by D(P) = Do(1+ αP), where Do is the unstressed vessel diameter and αis a distensibily coefficient. Distensibility, α, of the pulmonary arteries is shown to increase significantly with hypoxic exposure.

This work supported by HL 19298, the W.M. Keck Foundation and the Department of Veterans Affairs.

Program No. 308.10

Abstract (No. 8272)

5.4 mmHg12 mmHg21 mmHg30 mmHg

Hypoxiaadapted

Normoxiaadapted

Chronic hypoxia is often used as an experimental stimulus for invoking pulmonary hypertension and vascular remodeling in the rat. Vascular remodeling has been studied in the past, however, information about the intact morphology and mechanical properties of the remodeled vessels has been limited by previously used experimental techniques and other technical issues. The extant literature on the morphometry of the pulmonary arterial tree comes almost exclusively from histological measurements performed on plastic corrosion casts. This laborious method of histological data collection has discouraged even small scale studies of modified pulmonary arterial architecture resulting from vascular remodeling. As an alternative, x-ray micro-computed tomography increases the throughput enough to make such studies possible. Key advantages of this technique include a three-dimensional digital data set, that the data is comprised of all the resolvable vessels with their correct spatial orientation and connectivity, and that the data can be collected on the same lung under different experimental conditions allowing for evaluation of functional vascular mechanics in addition to anatomical structure. This work quantifies changes in the mechanical properties of the pulmonary arteries after exposure to normobaric hypoxia determined by perfusion studies and morphometric measurements made on isotropic, three-dimensional micro-CT volumes.

Introduction

One group of rats (N = 6) was exposed to normobaric hypoxia (10% O2) for 21 days using an environmental chamber. During conditioning the rats were allowed to eat and drink ad libitum. The second group (N = 6) was housed under similar but normoxic conditions. Each rat was anesthetized with sodium pentobarbital (50 mg/kg ip), heparinzed (200 IU/kg) by right ventricular injection and a blood sample taken for hematocrit determination. The trachea and pulmonary artery were cannulated, the heart and lungs removed en bloc and the heart excised and dissected for right ventricular free wall versus left ventricle plus septum weight determination. The lungs were suspended by the cannulae and perfused with a physiological salt solution (pss) containing 5% serum albumin, and the arterial pressure versus flow relationship measured over a range of 0 to 40 ml/min. The lungs were then placed in an imaging chamber and ventilated with a 15% O2, 6 % CO2 in N2 gas mixture. The pss in the arteries was replaced by perfluorooctyl bromide, to provide x-ray contrast to the arterial vasculatureonly. Ventilation was then halted at a tracheal pressure of 6 mmHg. The arterial pressure was set to 30 mmHg and the lungs rotated in an x-ray beam at 1o increments to obtain 360 planar images. The pressure was lowered successively to 21, 12, and 5.4 mmHg and then returned to 30 mmHg and full CT scans obtained at each pressure. The projection data was transferred to a Linux workstation, preprocessed to compensate for distortions introduced by the imaging chain, and then isotropic reconstructions were obtained through an implementation of the Feldkamp cone-beam algorithm. Mophometricmeasurements were made at each intravascular pressure in identical locations throughout the pulmonary trunk on the isotropic CT volumes in arteries ranging in size from approximately 0.2 to 2.4 mm. Using a linear diameter, D, vs. pressure, P, model and the relation D(P) = Do(1+ αP), where Do is the unstressed vessel diameter and α is a distensibily coefficient, α was determined for all of the measured vessels under each condition.

Methods

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Significant pulmonary hypertension, as evident in the perfusion studies, was present in all of the hypoxia exposed rats. Hypoxic exposure also resulted in significant decrease in body weight, increase in hematocrit, and increase in right ventricular hypertrophy as measured by the ratio of right ventricular free wall versus left ventricle plus septum weight ratio. Measurements made from micro-CT of the intact arterial trees revealed that vascular remodeling and pulmonary hypertension caused by hypoxic exposure resulted in a significant decrease in arterial distensibility when compared to a normoxic control group.

Conclusion

Do (intercept)

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Figure 3 : Perfusion data, flow of physiological salt solution vs. pulmonary artery pressure, in isolated normoxia adapted (N = 6) and hypoxia adapted (N = 6) rat lungs.

Figure 1 : Keck micro-angiographic and micro-computed tomographic imaging suite. Shown in the picture are the micro-focal x-ray source (right), micro-manipulator stage (center), image intensifier/detector (left), and surrounding the scanner, there is space for various experiment specific and physiological support equipment, such as circulation pumps, temperature exchangers, pressure transducers, tubing, wires, etc., positioning and instrument settings are controlled remotely from outside the chamber.

Figure 8 : Intravascular pressure vs. arterial diameter (linear fit to average± SE) plotted for three size ranges of vessels {(Large, greater than 1.4 mm [a&d]), (Medium, between 1.4 and 0.5 mm [b&e]), and (small, less than 0.5 mm [c&f])}in hypoxia (top row) and normoxia (bottom row) adapted rat lungs. Also presented are values of pulmonary arterial distensibility, α, as defined in caption of figure 7, for each group of vessel segments.

Figure 6 : Surface-shaded renderings of micro-CT data acquired from hypoxia (top row) and normoxia (bottom row) adapted rat lungs at intravscularpressure of 30 mmHg (first column), 21 mmHg (second column), 12 mmHg (third column), 5.4 mmHg (fourth column).

Figure 5 : Intravascular pressure vs. arterial diameter plotted for 5 vessel segments, randomly chosen, measured in a normoxia adapted, control rat lung. Data for each vessel segment was fit to a linear pressure vs. diameter relationship proposed by Yen et. al., D(P) = D(0)+βP.

Figure 7 : Pulmonary arterial distensibility, α, from D(P) = Do(1+ αP) relationship of Fung et. al., where α = β/D(0), measured in the pulmonary arteries of normoxia adapted (N=6) and hypoxia adapted (N=6) rat lungs. A global α value for the best linear fit to the data is shown at the top left for the normoxic group (blue) and the hypoxic group (red).

Figure 9 : Results for individual hypoxia (N = 6) and normixia (N = 6) adapted rats : a) Pulmonary arterial distensibility, ¶p < 0.0006, b) Hematocrit, § p < 0.00000002, Body weight, ‡ p < 0.0001, and Right ventricular hypertrophy, p < 0.0008.

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