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J Appl Physiol 104: 88-96, 2008. First published October 25, 2007; doi:10.1152/japplphysiol.00610.2007
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Changes in macrovessel pulmonary blood flow distribution following chronic hypoxia: assessed using synchrotron radiation microangiography

Daryl O. Schwenke,1,2 James T. Pearson,3 Kenji Kangawa,1 Keiji Umetani,4 and Mikiyasu Shirai5

1Department of Biochemistry, National Cardiovascular Center Research Institute, Suita, Osaka, Japan; 2Department of Physiology, University of Otago, Dunedin, New Zealand; 3Department of Physiology and Monash Centre for Synchrotron Science, Monash University, Melbourne, Australia; 4Japan Synchrotron Radiation Research Institute, Hyogo, Japan; and 5Faculty of Health Sciences, Hiroshima International University, Hiroshima, Japan

Submitted 10 June 2007 ; accepted in final form 22 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Structural and functional changes of the pulmonary circulation, particularly during the pathogenesis of pulmonary arterial hypertension (PAH), remain to be fully elucidated. In this study, we utilized monochromatic synchrotron radiation (SR) microangiography to assess changes in pulmonary arteriole blood flow in the intact-chest rat after 4 wk of chronic hypoxia. Sprague-Dawley rats were exposed to normoxia (N-rats) or chronic hypoxia (10% O2; CH-rats) for 28 days. Rats were anesthetized, and microangiography was performed on the left lung to assess 1) the branching distribution of pulmonary arteriole blood flow (internal diameter >80 µm) and 2) dynamic changes in vessel lumen diameter during acute hypoxic (8% O2 for 4 min) pulmonary vasoconstriction (HPV) before and after β-adrenoceptor blockade (2 mg/kg iv propranolol). Using SR angiography, we observed that the number of opaque third- and fourth-generation vessels (100–300 µm) for CH-rats was significantly fewer than the number for N-rats. The magnitude of HPV was not different between CH-rats and N-rats. β-Adrenoceptor blockade accentuated the HPV in 200- to 300-µm vessels for CH-rats, but even more so in N-rats. However, in CH-rats, β-adrenoceptor blockade also accentuated the HPV in 100- to 200-µm vessels. In summary, we utilized SR to assess gross blood flow changes and functional changes (i.e., HPV) of the pulmonary circulation in PAH. These results highlight the benefits of SR for assessing pulmonary circulatory pathology. Of particular importance, future use of SR will provide an effective method for assessing potential therapeutic treatments for PAH.

pulmonary microvessels; hypoxia; intact chest; rat

ALVEOLAR HYPOXIA, A POTENTIAL adverse complication associated with numerous respiratory disorders, causes pulmonary vasoconstriction, which is reversible on reoxygenation. However, when sustained, the elevated shear stress within the pulmonary vasculature causes endothelial cell injury dysfunction (2), ultimately leading to the pathogenesis of pulmonary arterial hypertension (PAH). PAH is characterized by irreversible vascular remodeling and medial thickening of thin muscular-walled vessels (100–300 µm), as well as the formation of new muscle around nonmuscular or partially muscular vessels (50–150 µm) (12, 23, 24, 31). The ensuing decrease in vessel internal caliber increases pulmonary vascular resistance and, consequently, increases the workload of the heart, enhances the risk of heart failure, and is, therefore, closely associated with an increased mortality.

Several studies have indicated that the increase in pulmonary vascular resistance is further exacerbated during chronic hypoxia by a reduction in the number of perfused blood vessels within the pulmonary vascular bed, due to either vessel occlusion (i.e., extensive medial thickening) or the loss of vessels, also known as "rarefaction" or "pruning" of small vessels (8, 11, 25). More recent evidence, however, has cast doubt on this paradigm, with some reports now indicating that angiogenesis is evident within the pulmonary circulation during chronic hypoxia (13, 14, 16).

The underlying mechanisms governing the pathogenesis of PAH remain to be fully elucidated. One of the limitations in understanding the pathology of the lung has been the inability to clearly visualize blood flow within the pulmonary vascular bed. More conventional methods of assessing the vascular anatomy of the diseased lung necessitate removal of the lung from the animal (i.e., in vitro) for angiography (i.e., X-ray) or microsection analysis. Conventional angiography methods have considerable limitations in visualizing the vessels that are most susceptible to pathological changes, i.e., <200 µm (37, 40). Therefore, a technique for visualizing the pulmonary circulation within a closed-chest model, i.e., under intact neurohumoral regulation, is required to better understand the structural and functional changes in vivo and, ultimately, to assess specific treatments for lung disorders more reliably.

In a recent study, we (32) demonstrated the validity and accuracy of synchrotron radiation (SR) microangiography for visualizing pulmonary blood flow within vessels in a closed-chest rat model and for assessing dynamic changes in vessel caliber associated with acute hypoxic pulmonary vasoconstriction (HPV). Although resolution limitations of SR only enabled vessels with an internal diameter >80 µm to be visualized, many vessels that are susceptible to pathological changes during chronic hypoxia are well within the resolution capabilities of SR. In contrast to conventional angiography systems, SR is characterized by high brilliance and extreme collimation (36), allowing enhanced sensitivity to contrast material and superior image quality in terms of spatial and density resolution because divergence and scatter of X-ray photons are eliminated.

The primary aim of this study was to utilize SR microangiography to effectively highlight changes in pulmonary blood flow distribution, following the pathogenesis of chronic hypoxia-induced PAH. Because of the resolution limitations of SR, we only assessed those pulmonary arterioles with an internal diameter >80 µm. The structural changes in the pulmonary circulation associated with chronic hypoxia have been shown to alter the functional properties of the pulmonary vasculature, in particular, the vasoconstriction response to acute hypoxia (i.e., HPV) (19, 22). This altered HPV has been attributed, at least in part, to the modulatory effects of the sympathetic nervous system on the pulmonary vasculature (33, 35). Therefore, we also aimed to utilize the high definition of SR to assess the adverse changes in pulmonary reactivity to acute hypoxia and, furthermore, to distinguish the local intrinsic response (i.e., HPV) from that of the extrinsic neural response (i.e., sympathetic modulation) by using propranolol to inhibit β-adrenoceptor activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Experiments were conducted on 10 male Sprague-Dawley rats (10 wk old; ~220–320 g body wt). All rats were on a 12:12-h light-dark cycle at 25 ± 1°C and were provided with food and water ad libitum. Rats were housed in standard normoxic conditions (n = 5) or were continuously housed in a hypoxic chamber (10 ± 0.1% O2) for 4 wk (n = 5), except for a 10-min interval each day when the chamber was cleaned. The hypoxic gas mixture was prepared from N2 (gas cylinders) and compressed air and was continuously delivered to the hypoxic chamber (30 liter capacity) at a flow rate of ~8 l/min. All experiments were approved by the local Animal Ethics Committee and conducted in accordance with the guidelines of the Physiological Society of Japan.

Anesthesia and surgical preparation.   Rats were anesthetized with pentobarbital sodium (60 mg/kg ip). Supplementary doses of anesthetic were periodically administered (~15 mg·kg–1·h–1 ip). Throughout the experimental protocol, body temperature was maintained at 37°C using a rectal thermistor coupled with a thermostatically controlled heating pad.

The trachea was cannulated, and the lungs were ventilated with a rodent ventilator (SN-480-7; Shinano, Tokyo, Japan). The inspirate gas was enriched with O2 (~50% O2), and the ventilator settings were adjusted (tidal volume ~3.5 ml; frequency of ~70 /min). A femoral artery and vein were cannulated for measurement of systemic arterial blood pressure (ABP) and drug administration, respectively. A 20-gauge BD Angiocath catheter (Becton Dickinson), with the tip at a 30-degree angle, was inserted into the jugular vein and advanced into the right ventricle for administering contrast agent and intermittently measuring right ventricular pressure (RVP).

The rat was securely fastened to a clear Perspex surgical plate, which had a single window opening directly beneath the thorax area. The surgical plate was then fixed in a vertical position in front of the beam pathway, so that the synchrotron beam could pass perpendicular to the sagittal plane from anterior to posterior through the rat thorax and ultimately to a SATICON X-ray camera described below.

Microangiographic system.   The pulmonary circulation was visualized with SR microangiography at the SPring-8 BL28B2 beam-line facility (Hyogo, Japan). The use of SR for visualizing the pulmonary microcirculation in the closed-chest rat has previously been described in detail (32).

In brief, SR has a broad and continuous spectrum from the infrared to the X-ray regions. A single crystal monochromator was used to select a single energy of SR, producing X-rays of a very narrow energy bandwidth for imaging. This SR system comprised a monochromatic 33.2-keV X-ray source, just above the iodine K-edge energy for maximal contrast.

X-rays transmitted through the rats were detected by an X-ray detector (Hitachi Denshi Techno-System, Tokyo, Japan) incorporating a SATICON X-ray pickup tube (Hamamatsu Photonics, Shizuoka, Japan). The biomedical imaging SATICON X-ray camera has a resolution of 1,050 scanning lines and can record images at a maximum speed of 30 frames/s for up to 30 s. The shutter open time used in this study was 2.6–3.0 ms/frame. The detector features a 9.5-µm equivalent pixel size projected onto the input area and an input field size of 9.5 x 9.5 mm. High-resolution images were stored in a digital frame memory system with 1,024 x 1,024 pixel format and 10-bit resolution.

Experimental protocol.   The rat was positioned in front of the beam line so that the upper segment of the left lobe was positioned in front of the SATICON X-ray camera in alignment with the 9.5 x 9.5-mm imaging field (i.e., between the 2nd and 3rd rib; Fig. 1). Subsequently, baseline heart rate (HR), RVP, and ABP data were collected. Immediately before vessels were imaged, the three-way stopcock on the right ventricle catheter was opened to a clinical autoinjector (Nemoto Kyorindo, Tokyo, Japan), which was used to inject a single bolus of contrast agent (Iomeron 350; Eisai, Tokyo, Japan) at high speed (0.4 ml/s). For each 2-s period of scanning, 100 frames were recorded. Rats were given at least 10 min to recover from each injection of contrast agent. Regular inspection between contrast injections confirmed that the pulmonary vasculature was clear of agent within this period of time.


Figure 1
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  		Fig. 1. A schematic drawing of the arterial circulation of the left lung in a rat. The square box represents the position of the 9.5 x 9.5-mm imaging window used for microangiography of the pulmonary microvessels. [Reprinted from Schwenke et al. (32).]

 
After baseline imaging was completed, rats were exposed to acute hypoxia (8% O2 in N2) for 4 min. During acute hypoxia, ABP, HR, and RVP data were continuously recorded until the 3rd min, after which recording of RVP was stopped, and the catheter was switched from the pressure transducer to the clinical injector for imaging. Lung microangiography was performed on the hypoxic lung after the 4th min of hypoxia.

After recovery from the acute hypoxic test, rats were administered the β-receptor blocker propranolol (2 mg/kg iv). After 10–15 min was allowed for all cardiovascular variables to stabilize, pulmonary microangiography was repeated before and after acute hypoxia.

Data acquisition and analysis.   The RVP and ABP signals were detected by separate Deltran pressure transducers (Utah Medical Products), and the signals were relayed to PowerLab bridge amplifiers (ML117, ADInstruments) and then continuously sampled at 500 Hz with an eight-channel MacLab/8s interface hardware system (ADInstruments) and recorded on a Macintosh Power Book G4 using Chart software (version 5.0.1, ADInstruments). HR was derived from the arterial systolic peaks.

From the 2-s period of image collection, one frame per scan (one scan = 100 frames) was selected for image enhancement and analysis. Furthermore, only those frames recorded at, or near, end systole were used for assessing and comparing pulmonary vessel diameter between baseline and hypoxic conditions.

All imaged vessel branches were counted. Where possible, the widths of two to four vessels of each branching generation (2nd to 4th generation) were measured to ensure that a wide variety of vessel sizes was selected from each frame. Vessels were categorized according to internal diameter: 100–200, 200–300, 300–500, and >500 µm. The internal diameter of individual vessels was measured before and after acute hypoxia, with and without β-receptor blockade (i.e., propranolol treatment).

Image analysis.   The computer-imaging program Image Pro-plus (version 4.1, Media Cybernetics) was used to enhance the contrast and clarity of angiogram images. To enhance images, a temporal subtraction operation was performed for flat-field correction using summation results of 10 consecutive frames acquired before contrast agent injection. The summation image taken before injection was subtracted from a single raw image taken after injection to eliminate the superimposed background structure.

Image Pro-plus was also used to evaluate the vessel internal diameter. A 100-µm-thick tungsten filament, which had been placed directly across the corner of the detector's window, appeared in all of the recorded images and was subsequently used as a reference for calculating vessel diameter (µm). The line-profile function of Image Pro-plus was used to measure changes in pixel intensity (brightness) along manually drawn segments spanning 10–40 pixels on either side in the direction perpendicular to the vessel (see Fig. 2). The first edge of the vessel was determined as the pixel at which intensity decreased by 1.5 standard deviations below that of the preceding 10–40 pixels (depending on space between vessels). The opposite criterion was applied to ascertain the distal edge of the vessel. This boundary segmentation procedure was performed at two different points along the length of the vessel, and the average of the two values was used for data collation. To assess reproducibility, the procedure for measuring vessel width was repeated by a second observer. Of the total number of vessels analyzed (91 vessels from normoxic and chronic hypoxic rats; see RESULTS), 50 vessels from various branching generations were randomly selected from the appropriate angiograph image for analyses by a second observer. The value for each vessel width that was analyzed by both observers was highly reproducible. Regression analysis indicated that the measurements from both observers were highly correlated (y = 6.752 + 0.983x, r2 = 0.975) and had an average difference of 6 ± 3 µm (for 100- to 200-µm vessels) to 9 ± 4 µm (for 300–500 µm).


Figure 2
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  		Fig. 2. Top: an angiograph image obtained from a phantom study that assessed X-ray absorption of 9 tubes containing 1) Iopamiron 370 (370 mg/cm3 iodine; Nihon Schering), 2) 127 mg/cm3 iodine (diluted contrast material with distilled water), 3) 64 mg/cm3 iodine, 4) 32 mg/cm3 iodine, 5) 12.8 mg/cm3 iodine, 6) 6.4 mg/cm3 iodine, 7) 1.28 mg/cm3 iodine, 8) distilled water, and 9) air. A 5-mm-thick aluminum mask was overlaid to simulate attenuation by the animal body. Concentrations ranging from 32 to 127 mg/cm3 of iodine simulate typical in vivo conditions for intra-arterial injection techniques. Bottom: computerized line profile (representing the solid line transecting all 9 tubes in top) was performed to show that changes in pixel intensity (i.e., brightness), which reflect the magnitude of absorption, are dependent on iodine concentration.

 
Evaluation of accuracy of measurement.   We (32) have previously described in detail the method of evaluating the accuracy of measurement. In brief, we estimated a margin of error for detecting the edge of a vessel by assessing pixel variability of the reference wire (known diameter of 100 µm). Before commencing the study, we measured wire width at 40 random points along its length. The mean width of this reference wire was 12.99 pixels (SD ± 1.12). This equates to an average pixel size of 7.69 µm (95% confidence interval of 7.496–7.908 µm). Consequently, the 100-µm tungsten wire could accurately be measured to within ~5 µm (range of 97.4–102.7 µm).

In this study, the iodine contrast agent was injected into the right ventricle so that the iodine concentration within the pulmonary circulation would have been diluted, which is likely to have attenuated X-ray absorption and potentially the accuracy of the vessel width measurement. We previously performed preliminary phantom measurements to assess the relationship between X-ray absorption and iodine concentration. The phantom measurements consisted of filling nine tubes, with an internal diameter of 200 µm, with various concentrations of iodinated contrast material ranging from pure agent (370 mg/cm3) to distilled water (i.e., 0 mg/cm3). The line-profile function of Image Pro-plus was used to assess the magnitude of absorption (i.e., brightness) for each concentration of iodinated contrast material (Fig. 2).

The widths of tubes containing an iodine concentration between 32 and 370 mg/cm3 could be accurately measured with a small margin of error, e.g., the width of the tube containing 32 mg/cm3 iodine was measured at 40 points and had a 95% confidence interval of 196.84–203.26 µm. This is a margin of error similar to that obtained for multiple measurements of the tungsten reference wire.

These results show that measurements of 200 µm in diameter can be made precisely for those vessels with an iodine concentration of >32 mg/cm3. Moreover, it seems reasonable to suppose that measurements of ~100 µm in diameter can be made with the required precision for those vessels with an iodine concentration of 64 mg/cm3 or greater.

Statistical analysis.   All statistical analyses were conducted with Statview (version 5.01; SAS Institute). All results are presented as means ± SE. Two-way ANOVA (repeated measures) was used to test whether propranolol significantly altered the dynamic pulmonary vasoconstriction response to acute hypoxia. One-way ANOVA (factorial) was used to test for differences in 1) vessel caliber during normoxia and acute hypoxia and 2) baseline values for normoxic rats (N-rats) compared with chronic hypoxic rats (CH-rats). Where statistical significance was reached, post hoc analyses were incorporated using the paired or unpaired t-test with the Dunnett's correction for multiple comparisons. A P value ≤0.05 was predetermined as the level of significance for all statistical analysis.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Baseline.   Rats had an initial body weight of ~285 g before being placed into a normoxic (N-rats) or hypoxic chamber (CH-rats) for 4 wk. The gain in body weight of CH-rats (~20% increase) was significantly lower (P < 0.01) than that for N-rats (~74% increase) over the 4-wk period. Chronic hypoxia induced PAH (see Table 1), as demonstrated by our observation that systolic RVP of CH-rats was ~120% higher than that of N-rats (P < 0.01). Chronic hypoxia did not significantly alter mean ABP (MABP) or HR.


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  		Table 1. Systolic RVP, MABP, and HR data of anesthetized N-rats and CH-rats before and after propranolol administration

 
Using SR, we were able to clearly visualize the blood flow of pulmonary microvessels (>80 µm) in the left lung of both N-rats and CH-rats. The typical branching pattern of the pulmonary circulation from the main axial artery of the left lobe to the fourth generation of branching (within the 9.5 x 9.5 mm imaging window) of a N-rat and a CH-rat is presented below (see Fig. 4).


Figure 4
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  		Fig. 4. Typical microangiogram images showing the branching pattern of small pulmonary arteries in N-rats (n = 5) and CH-rats (n = 5). Pulmonary branches to the 4th generation from the left main axial artery (not in the image) were visible. The tungsten wire in the top left of each image is a reference of 100 µm diameter. Images were recorded during air breathing (A) and after 4 min of acute hypoxia (8% O2; B). Images for A and B were obtained from the same respective N-rat or CH-rat. The black arrows point to branches of the pulmonary vessels that have constricted in response to hypoxia.

 
The internal diameter of 52 vessels was measured in five N-rats: 24 vessels with a diameter between 100 and 200 µm, 15 vessels with a diameter between 200 and 300 µm, 7 vessels with a diameter between 300 and 500 µm, and 6 vessels with a diameter >500 µm. In comparison, CH-rats had comparatively fewer vessels (Fig. 3A); thus the internal diameter of 39 vessels was measured in five CH-rats: 15 vessels with a diameter between 100 and 200 µm, 10 vessels with a diameter between 200 and 300 µm, 7 vessels with a diameter between 300 and 500 µm, and 7 vessels with a diameter >500 µm.


Figure 3
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  		Fig. 3. Changes in the number of opaque vessels (means ± SE) (A) and range of vessel sizes (box and whisker graph; B) at each of the first 4 branching generations of the pulmonary circulation in normoxic rats (N-rats; n = 5) and chronic hypoxic rats (CH-rats; n = 5). {dagger}Significant difference between N-rats and CH-rats (P < 0.05).

 
The total number of vessel branches visible within each baseline image (i.e., 9.5 x 9.5-mm imaging window) was counted. As illustrated in Fig. 3A, the number of opaque third- and fourth-generation vessels for CH-rats (9 ± 1 and 16 ± 2 vessels, respectively) was significantly fewer than the number for N-rats (14 ± 1 and 30 ± 2 vessels, respectively; P < 0.05). The numbers of first- and second-generation branches were not significantly different between N-rats and CH-rats (2 and 4 or 5, respectively).

Vessel caliber tended to decrease according to each generation of branching, as well as the distance away from the main axial artery toward the periphery (Fig. 3B). However, often more than one size category could be found within one branching generation. For example, the third generation of branching (135–290 µm) comprised vessels of the 100- to 200-µm and 200- to 300-µm categories (Fig. 3B). The internal diameter of the first-generation branch in CH-rats (740 ± 53 µm) was significantly larger than that shown in N-rats (511 ± 57 µm; P < 0.05), possibly because of the higher distending pressure (i.e., pulmonary arterial pressure) for CH-rats. Vessel caliber was not significantly different between N-rats and CH-rats for the second generation (210–570 µm), third generation (135–290 µm), and fourth generation of branching (90–180 µm) (Fig. 3B).

Responses to acute hypoxia.   N-rats and CH-rats were exposed to 8% O2 for 4 min. Acute hypoxia caused a significant decrease in the internal diameter of all vessels with an internal diameter <500 µm (for N-rats) or <300 µm (for CH-rats) (Figs. 4 and 5). In both groups of rats, the magnitude of constriction tended to increase as vessel caliber decreased, with the greatest degree of vasoconstriction occurring in those vessels with a diameter between 100 and 300 µm. These vessels were generally of the third to fourth generation of branching. There was no significant difference between N-rats and CH-rats regarding the magnitude of HPV for all vessel sizes.


Figure 5
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  		Fig. 5. Relationship between vessel size and the magnitude of pulmonary vasoconstriction (%decrease in vessel diameter) in response to acute hypoxia (8% O2 for 4 min) in N-rats (n = 5) and CH-rats (n = 5) before (left) and after (right) intravenous administration of propranolol (2 mg/kg). *Significant vasoconstriction response to acute hypoxia (P < 0.05). {dagger}Significant difference between N-rats and CH-rats (P < 0.05). {ddagger}Significant change in the magnitude of vasoconstriction after propranolol administration (P < 0.05).

 
The magnitudes of responses to hypoxia for systolic RVP, MABP, and HR are presented in Fig. 6. Acute hypoxia induced a significant 21% increase in systolic RVP (P < 0.01) in N-rats, which was not significantly different from that observed in CH-rats (27% increase above baseline). This increase in systolic RVP reflects the HPV seen with microangiography. Hypoxia decreased MABP in CH-rats, but significantly more so in N-rats (40% and 54% decrease, respectively). Hypoxia did not alter HR in either N-rats or CH-rats.


Figure 6
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  		Fig. 6. Hemodynamic responses (% change) of N-rats (n = 5) and CH-rats (n = 5) to acute hypoxia (8% O2 for 4 min) before and after intravenous administration of propranolol (2 mg/kg). *Significant difference in the magnitude of response between N-rats and CH-rats (P < 0.05). {dagger}Significant response to propranolol (P < 0.01).

 
Responses to propranolol.   N-rats and CH-rats were administered the β-receptor blocker propranolol (2 mg/kg iv). Propranolol did not significantly alter baseline pulmonary vessel caliber for any of the vessel size groups analyzed, in both N-rats and CH-rats. The lack of effect of propranolol on pulmonary vessel caliber for N-rats and CH-rats was reflected in the insignificant response of systolic RVP to propranolol (Table 1). In N-rats, propranolol did not significantly alter MABP or HR. In CH-rats, propranolol significantly increased MABP (15% increase) and caused a 17% decrease in HR (P < 0.01).

In N-rats, β-receptor blockade significantly exacerbated the magnitude of acute hypoxic vasoconstriction in 200- to 300-µm-sized pulmonary vessels (Fig. 5). However, the systolic RVP, MABP, and HR responses to acute hypoxia were unaltered (Fig. 6). In comparison, β-receptor blockade in CH-rats significantly accentuated the systolic RVP response to acute hypoxia (P < 0.05), reflecting extensive pulmonary vasoconstriction in 200- to 300-µm-sized vessels and, unlike that observed for N-rats, even greater constriction in 100- to 200-µm-sized vessels (Fig. 5). The HPV response of vessels with a caliber >300 µm was not altered by β-receptor blockade in N-rats or CH-rats.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This study has demonstrated the effectiveness of SR for visualizing the adverse anatomic and functional changes of the pulmonary circulation associated with the pathogenesis of pulmonary hypertension in a closed-chest rat model.

PAH.   Although significant advances in the treatment of pulmonary disorders have been made in recent decades, the underlying mechanisms governing the pathogenesis of PAH remain to be fully elucidated. Specifically, anatomic and structural changes within the pulmonary vascular bed during chronic hypoxia remain unclear.

On the basis of the early work of Reid and colleagues (8, 11, 12, 25), it had become accepted that a reduction in the number of perfused vessels within the pulmonary circulation was an important structural pathology that contributed to the sustained increase in vascular resistance during the pathogenesis of PAH. The reduction in perfused vessels was attributed to either the encroachment of smooth muscle into the vessel lumen, thereby occluding blood flow, or by a reduction in the total number of vessels (i.e., pruning).

One limitation of these studies was the inability to clearly identify the region of susceptibility and the degree of pruning or reduced perfusion. Indeed, because of the limited resolution of conventional X-ray systems, the lung had to first be excised for angiography and then pruning was nonquantitatively described from crude angiographs as a decrease in "background haze" or "background filling" (representing small peripheral vessels) (11, 21, 30).

We (32) have previously described the high definition achieved with SR microangiography for visualizing pulmonary microvessels, with an internal diameter >80 µm. In this study, using SR, we were able to confirm that exposure to chronic hypoxia for 4 wk reduced the number of opaque arterioles with internal diameter >80 µm within the pulmonary circulation. Unlike previous studies, this study was also able to identify the region of susceptibility (i.e., third to fourth generation of branching, 100–300 µm) and to quantify the degree of change (e.g., CH-rats had 47% fewer opaque vessels of the third generation than N-rats) within a closed-chest model, i.e., under intact neurohumoral regulation. One significant limitation with SR, however, is that it is not possible to determine whether the reduction in the number of opaque vessels is due to 1) pruning of vessels, 2) occlusion of existing vessels (i.e., complete encroachment of smooth muscle within the vessel lumen), or 3) partial occlusion, reducing the internal diameter below the resolution limit of SR (i.e., 80 µm).

In the latter case, vessels would still be perfused but would not be visualized by SR. Consequently, the change in the number of perfused microvessels could potentially be overestimated, since we were unable to distinguish between completely occluded and partially occluded vessels. Without further improvements in the signal-to-noise ratio of pixel areas containing fifth-generation branches, it is presently not possible to quantify to what extent the microvessels are perfused in the CH-rat model.

The lung is one of the most densely vascularized organs; therefore, it is extremely difficult to accurately quantify the number of vessels within the "whole" lung. Some studies have claimed that casting techniques, which apply a high-perfusion pressure to distend and fill all arteries to the level of the capillaries, provides evidence that chronic hypoxic reduces vessel number, supporting the concept of pruning (11, 30). However, the reduction of the number of vessels filled with medium within a cast may simply be the result of occlusion, rather than the loss of vessels.

In the past 5 yr, the paradigm of vessel pruning during chronic hypoxia has been challenged, with some reports indicating that the pulmonary circulation appears to undergo angiogenesis during chronic hypoxia (13, 14, 16). However, the vascular region of angiogenesis is uncertain but is likely to occur (if at all) within the capillary bed and/or venules (not the arterioles) because these are the regions of angiogenesis within the systemic circulation (6, 28, 29). Unfortunately, visualizing the capillary network of the lung is presently beyond the resolution capabilities of SR with iodinized contrast agents.

Limitations of this study.   Our group has previously described the limitations of SR for visualizing pulmonary vessels <80 µm but also reported that, despite this limitation, a majority of vessels that are susceptible to pathological disorders, and that significantly contribute to an increase in vascular resistance, generally have an internal diameter between 50 and <300 µm (11, 32, 37, 40), i.e., resistance vessels that are at least partially muscular (12). However, resistance vessels <70 µm and lacking complete muscular media also constrict in response to hypoxia (10, 27) and therefore are also likely to be susceptible to pathological changes.

When analyzing the vasoconstriction response to acute hypoxia, we could only analyze vessels with an internal caliber >100 µm because vasoconstriction of vessels <100 µm reduced their caliber below the resolution capabilities of SR (i.e., <80 µm).

Another significant limitation of this study is the inability of SR to assess the integrity of the pulmonary arterial vascular wall. Numerous studies have reported that pulmonary vascular remodeling and medial thickening during chronic exposure are significant structural pathologies responsible for the increase in vascular resistance (12, 23, 24, 31). Assessment of vascular remodeling often necessitates histochemical analysis. On the other hand, SR is only able to measure the internal diameter of perfused vessels (assuming the vessel contains sufficient contrast medium) and is therefore a simple, albeit useful, method for assessing gross anatomic changes in the pulmonary circulation of the hypertensive lung. Important information concerning vascular wall thickness and medial thickening cannot be assessed with SR.

In this study, we could not view the circulation of the whole lung; rather, we were restricted to a relatively small field of view of 9.5 x 9.5 mm. We therefore make the assumption that the image captured within the 9 x 9 window is representative of the whole lung circulation. However, changes in vascular resistance (during either acute of chronic hypoxia) are the consequence of global pulmonary vasoconstriction and/or remodeling.

HPV in the rat.   In this study, we used SR to assess the dynamic changes in vessel caliber during acute hypoxia (i.e., HPV). The results of this study concur with our previous report (32) that showed, in N-rats, that all vessels with a diameter <500 µm, especially between 200 and 300 µm, constricted in response to acute hypoxia (8% O2). The unique result of this study is that chronic hypoxia did not significantly alter HPV, as assessed by hemodynamic and microangiography analysis.

HPV has been reported to preferentially occur in vessels with an internal diameter of ~150–300 µm in cats and rabbits (17, 34, 35) and rats (32) and up to 600 µm in dogs (1). Chronic hypoxia has been reported to alter acute HPV due to structural changes in the pulmonary vasculature (22). According to the literature, chronic hypoxia can potentially attenuate (9, 15, 38, 41), enhance, or have no effect on the acute HPV (5, 7, 18). Despite decades of research concerning HPV, the exact mechanism(s) that governs acute HPV is yet to be fully elucidated, although various humoral (e.g., nitric oxide) and neural pathways are likely to be involved [see Moudgil et al. (26) for a review]. In this study, we aimed to specifically assess sympathetic modulation of the HPV by SR microangiography. Although sympathetic fibers innervate the pulmonary vasculature, neural control of "tonic" pulmonary vascular tone is less prominent than that of the systemic vasculature. However, modulation of the pulmonary vasculature by the sympathetic nervous system becomes critically important under stressful conditions, such as hypoxia (33, 35).

Hypoxia is a potent activator of pulmonary sympathetic nerve activity. The increase in sympathetic nerve activity has been reported to attenuate the local vasoconstrictor effects of hypoxia via a β-adrenoceptor-mediated vasodilator mechanism, especially when the inspired level of O2 is ≤8% O2 (33, 35). For example, Shirai et al. (35) reported that HPV was greater for moderate (10% O2) than for severe hypoxia (5% O2), but only if the hypoxia was global (i.e., whole body). If the hypoxic stimulus was restricted to just the lung (i.e., regional hypoxia), the magnitude of vasoconstriction was proportional to the degree of hypoxia (i.e., greatest for 5% O2). Shirai et al. (35) subsequently demonstrated that the vasoconstrictor response to severe global hypoxia is offset by a sympathetic β-adrenoceptor-mediated vasodilatory mechanism.

In our study, we observed that sympathetic β-adrenoceptor blockade (using propranolol) in N-rats did not modify baseline vascular tone, but it did accentuate HPV of those vessels 200–300 µm in diameter. These results support the concept that modulation of the pulmonary vasculature by the sympathetic nervous system appears to be an important homeostatic response for limiting the magnitude of vasoconstriction under hypoxic conditions.

Interestingly, although β-adrenoceptor blockade accentuated HPV in this study, it did not significantly alter the magnitude of the systolic RVP to acute hypoxia in N-rats. This difference may be attributed to a decrease in cardiac output (not measured in this study), since β-receptor blockade has been reported to significantly reduce cardiac output (4, 20). Alternatively, the magnitude of vasoconstriction may not have been sufficient to elicit a significant change in the systolic RVP response, since 1) propranolol accentuated the HPV of only the 200- to 300-µm vessels and 2) the vasoconstriction observed within the 9.5 x 9.5-mm field of view may not be representative of the whole lung (as discussed above).

The mechanism(s) responsible for alterations of the HPV after chronic hypoxia remains poorly understood. In this study, we observed that β-receptor blockade in CH-rats significantly accentuated the HPV, not only in the 200- to 300-µm vessels as observed in N-rats but also in the 100- to 200-µm vessels. Consequently, the systolic RVP response to acute hypoxia was also enhanced by propranolol. These results indicate that sympathetic modulation of the HPV becomes critically enhanced after chronic hypoxia.

Although the mechanisms for these observations need to be further researched, we speculate that the enhanced HPV may be attributable to 1) the formation of new muscle around nonmuscular or partially muscular vessels (50–150 µm) and 2) an increase in sympathetic innervation of the pulmonary vasculature. HPV is intrinsic to the lung, and, although modulated by the endothelium, the core mechanism is in the smooth muscle cell (26). Peripheral arterioles undergo medial thickening during chronic hypoxia. Therefore, in this study, it may be possible that the potential of the 100- to 200-µm vessels to constrict was enhanced after chronic hypoxia. However, the difference in the vasoconstrictive response of 100- to 200-µm vessels between N-rats and CH-rats was only apparent after β-receptor blockade. Studies have shown that chronic hypoxia significantly increases β-receptor number within the lung (3, 39), so that modulation of HPV by the sympathetic nervous system is enhanced after chronic hypoxia. These reports are in agreement with the observations of this study.

Future directions.   The primary aim of this study was to demonstrate the effectiveness of SR for "visualizing" the pathological changes in pulmonary microcirculation in a closed-chest rat model after the development of pulmonary hypertension. As a result, this study has provided a foundation with which future investigative studies can build to further elucidate the adverse changes in the pulmonary circulation during hypertension. Specifically, we used only one stimulant (acute hypoxia) to test the reactivity of the pulmonary circulation. However, the question still remains as to whether reactivity to other specific vasoactive agents is altered in hypertension (e.g., reactive oxygen species, nitric oxide, endothelin-1, etc.) and what region of the microcirculation is most susceptible to pathological changes (i.e., vessel branching generation and vessel size). Ultimately, it is anticipated that SR will provide an effective means of assessing potential therapeutic or prophylactic treatments for PAH.

In summary, we have demonstrated the effectiveness of SR for assessing changes in pulmonary blood flow distribution and functional changes (i.e., HPV) associated with the pathogenesis of PAH. Despite some limitations, the observations from this study can provide future direction for investigating the potential mechanisms responsible for these pathological changes. Of particular importance, future use of SR will provide an effective method for assessing the potential treatments for PAH.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported in part by the "Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO)" and also in part by a Grant-in-Aid for Scientific Research (16659210) and a Monash Synchrotron Fellowship (J. T. Pearson). We also acknowledge financial support from the access to Major Research Facilities Programme, which is a component of the International Science Linkages Programme (Australian Government).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The SR experiments were performed at the BL28B2 in the SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (Proposal 2006BO464-NL2-NP).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. O. Schwenke, Dept. of Physiology, Univ. of Otago, PO Box 56, Dunedin, New Zealand (e-mail: daryl.schwenke{at}stonebow.otago.ac.nz)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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