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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: J Pharmacol Toxicol Methods. 2011 Aug 7;64(2):180–186. doi: 10.1016/j.vascn.2011.08.001

Phenylephrine-Modulated Cardiopulmonary Blood Flow Measured with Use of X-ray Digital Subtraction Angiography

MingDe Lin a,b, Yi Qi a, Antonia F Chen c,d, Cristian T Badea a, G Allan Johnson a,b,**
PMCID: PMC3200449  NIHMSID: NIHMS322505  PMID: 21846505

Abstract

Introduction

Cardiopulmonary blood flow is an important indicator of organ function. Limitations in measuring blood flow in live rodents suggest that rapid physiological changes may be overlooked. For instance, relative measurements limit imaging to whole organs or large sections without adequately visualizing vasculature. Additionally, current methods use small samples and invasive techniques that often require killing animals, limiting sampling speed, or both. A recently developed high spatial- and temporal-resolution x-ray digital subtraction angiography (DSA) system visualizes vasculature and measures blood flow in rodents. This study was the first to use this system to measure changes in cardiopulmonary blood flow in rats after administering the vasoconstrictor phenylephrine.

Methods

Cardiopulmonary blood flow and vascular anatomy was assessed in 11 rats before, during, and after recovery from phenylephrine. After acquiring DSA images at 12 time points, a calibrated non-parametric deconvolution technique using singular value decomposition (SVD) was applied to calculate quantitative aortic blood flow in absolute metrics (mL/min). Trans-pulmonary transit time was calculated as the time interval between maximum signal enhancement in the pulmonary trunk and aorta. Pulmonary blood volume was calculated based on the central volume principle. Statistical analysis compared differences in trans-pulmonary blood volume and pressure, and aortic diameter using paired t-tests on baseline, peak, and late-recovery time points.

Results

Phenylephrine had dramatic qualitative and quantitative effects on vascular anatomy and blood flow. Major vessels distended significantly (aorta, ~1.2-times baseline) and mean arterial blood pressure increased ~2 times. Pulmonary blood volume, flow, pressure, and aortic diameter were not significantly different between baseline and late recovery, but differences were significant between baseline and peak, as well as peak and recovery time points.

Discussion

The DSA system with calibrated SVD technique acquired blood flow measurements every 30 seconds with a high level of regional specificity, thus providing a new option for in vivo functional assessment in small animals.

Keywords: angiography, blood flow, cardiac output, cardiovascular, imaging, methods, phenylephrine, pulmonary, rat, small animal

1. INTRODUCTION

Visualization of blood vessels and measurement of blood flow in small animals provide immediate anatomical and functional indicators of disease or response to drug administration or other stimulus. Cardiopulmonary blood flow is an indicator of heart and lung function that can be used to derive many other important physiological parameters. Current methods for visualizing cardiopulmonary blood flow, such as labeled microspheres, ultrasound Doppler, thermodilution, magnetic flowmetry, magnetic resonance imaging (MRI), computed tomography (CT), or positron emission tomography (PET), are inadequate because measurements are conducted at slow sampling speeds, do not provide regional specificity, or require killing the animal. To address these shortcomings, an optimized x-ray digital subtraction angiography (DSA) system was developed (Lin et al., 2009) to visualize and measure cardiopulmonary blood flow changes in absolute metrics at heart-beat time resolution with sub-millimeter spatial resolution. Previous work demonstrated that this DSA system could obtain minimally invasive absolute blood flow measurements in a live rat on a pixel-by-pixel basis in the aorta and that relative measurements could be obtained for entire organs, specific blood vessels, and even parenchymal tissue. X-ray DSA is ideal for acute studies because samples can be acquired approximately every 30 seconds.

The current study is the first application of our small-animal x-ray DSA system in measuring real-time physiological changes before, during, and after drug administration. The vasoconstrictor phenylephrine was used to stimulate rat cardiopulmonary vasculature and blood flow as in previous studies (Doggrell et al., 1998; Herlihy et al., 1992; Lin et al., 2006; Segel et al., 1987; Tatchum-Talom et al., 1995). The study’s specific aim was to apply the x-ray DSA system in conjunction with a calibrated deconvolution method in order to measure drug-induced perturbations in cardiopulmonary blood flow (Lin et al., 2009).

2. MATERIALS AND METHODS

2.1 Animals

All animal studies were conducted with approval of the Duke Institutional Animal Care and Use Committee and complied with welfare-related assessments and interventions before, during, and after study experiments. The 11 male rats (Fischer-344, Charles River, Wilmington, MA) were of similar body weight and age (235±20 g, 79±4 days); they were housed in a pathogen-free animal facility located in the building where experiments were conducted to minimize stress from moving between locations. The semi-opaque polycarbonate cages (19 × 10.5 × 8 inches) with filter tops had individual ventilation and bedding material; rats had access ad libitum to food and water. Cages were kept at a constant temperature of 70°F and cleaned every 2-3 days. No more than three rats were housed per cage. The rats had 12-hour light/dark cycles. Prior to experimentation, three catheters were placed in each rat in the following locations: 1) right jugular vein at the root of the superior vena cava ([polyethylene-50, 22.2-cm length] for injection of x-ray contrast agent), 2) femoral artery (for blood pressure measurements), and 3) tail vein (for drug infusion).

2.2 Experimental System

The x-ray contrast injection system has been described in (Lin et al., 2006 IEEE). Briefly, the computer-controlled system delivered repeatable (<12% variation) microliter (μL) volumes of x-ray contrast agent at specific time points in the cardiac and ventilatory cycles. Previous work (Lin et al., 2008) had indicated that these small injection volumes (~100μL) have little effect on blood flow physiology. In the current study, blood pressure was measured with use of a direct blood pressure kit (DBP1001, Kent Scientific Corporation, Torrington, CT) simultaneously with acquisition of DSA images. Blood pressure measurements were normalized to pre-drug state for all rats. Animals were anesthetized using 1-3% Isoflurane, ventilated (60 breaths/min, 2-cc tidal volume), kept warm with a heat lamp controlled via feedback from a rectal thermocouple (37±0.1°C), and monitored for parameters such as heart rate and blood pressure, all as reported previously (Lin et al., 2009). At the end of the study, rats were killed with an overdose of anesthetic agent (250 mg/kg, Nembutal [Ovation Pharmaceuticals, Deerfield, IL]) delivered by intraperitoneal (IP) injection.

In DSA, a sequence of x-ray images is acquired before and after vascular injection of x-ray contrast agent. The pre-injection images are averaged to create a mask from which the post-injection images are subtracted. Subtraction yields a series of dynamic x-ray images that show changes in contrast enhancement of blood vessels. The subtracted images provide both anatomical vessel imaging and a time sequence of changes in vessel size, which can then be used to derive blood flow metrics.

The radiographic system had been developed previously for high spatial (100 μm × 100 μm) and temporal resolution (every rat heartbeat) (Lin et al., 2006), with techniques optimized for small-animal x-ray DSA (Lin et al., 2006; Lin et al., 2008). The exposure parameters were 80 kVp, 160 mA, and 10 ms. Each DSA dataset was composed of 30 x-ray images per contrast injection. Images were acquired during systole after induction of apnea. For each dataset, a micro-injector delivered one bolus (100±11μL) of contrast agent (Isovue 370, 370mg iodine/mL, Bracco Diagnostics, Princeton, NJ) into the right atrium via the right jugular vein.

2.3 Study Design

DSA datasets were acquired before, during, and after administration of vasoconstrictor (phenylephrine hydrochloride 1%, 10 mg/mL, [American Regent Laboratories, Inc., Shirley, NY]). Phenylephrine was chosen because it lacks chronotropic and inotropic activity (Segel et al., 1987). Figure 1 shows the timeline for acquiring images and infusing drug. First, a pre-drug DSA scan was performed. Then, phenylephrine was infused at 0.125 mL/min for two minutes. During infusion, scans were performed every 30 seconds; the last scan in the infusion time series was obtained two minutes after discontinuing infusion. This timeline ensured capture of rapid changes in blood flow (i.e., demonstration of physiologically adequate temporal resolution). Recovery-phase DSA scans were performed at 5, 8, and 22 minutes after start of drug infusion to capture the return of blood flow to baseline state. For each rat, a total of 12 DSA datasets (30 images per set) were acquired. As each dataset was acquired, blood pressure measurements were recorded from the indwelling catheter in the femoral artery.

Figure 1.

Figure 1

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Timeline of events showing the relationship between DSA imaging and administering the phenylephrine drug. The drug was infused for 2 minutes, during which DSA image sets were acquired every 30 seconds. Images were acquired up to 4 minutes after the start of the first (pre-drug) DSA dataset. The remaining image sets were acquired at the 5, 8, and 22-minute time points in the recovery phase. A total of 12 DSA datasets (30 images per set) were acquired for each of the 11 rats following this timeline. The pre-drug, post-drug, and recovery time points correspond to the DSA and blood flow maps in the other figures and in the text.

2.4 Cardiopulmonary Blood Flow Calculations

Aortic blood flow was measured using a nonparametric deconvolution technique with singular value decomposition (SVD). The SVD technique, based on work done by Ostergaard et al. (1996), was chosen because it is independent of mathematical tissue models and can measure flow rate on a pixel-by-pixel basis. Briefly, the rate of blood flow (BF) in a vessel can be estimated by deconvolving the effects of the vasculature from contrast concentration curves. In our case, the curves are pixel-value changes of x-ray intensity (time density curve, or TDC) as the contrast agent moves in and out of a region of interest (ROI) over time. Typically, an arterial input function (AIF) in the shape of a square impulse function at a proximal vessel location will become a delayed and dispersed curve at the distal vessel location. Effects of delay in contrast bolus are corrected by realigning the distal TDCs and AIF to a common signal maximum (Calamante et al., 2000; Calamante et al., 2003; Calamante et al., 2006). The relative measurements produced by SVD calculations are corrected for vessel thickness by modulation of blood flow values and calibrated to absolute metrics (Lin et al., 2009). This calibration specifically links relative DSA-based blood flow measurements to absolute metrics based on thermodilution and Fick methods. Although the relevant data were not presented in our previous paper (Lin et. al., 2009), Bland-Altman plots were constructed for this study that showed agreement between DSA, thermodilution, and Fick blood flow measurement methods.

The trans-pulmonary mean transit time (MTT) from the pulmonary trunk to the aorta was calculated as described by Ugander et al. (2010). Figure 2 shows the ROIs in the current study as black squares with white borders. Trans-pulmonary blood volume (BV) was calculated based on the central volume principle (BV = BF × MTT). Each DSA dataset included calibrated aortic BF (mL/min), trans-pulmonary MTT (sec), and trans-pulmonary BV (mL). In addition, we corrected vessel thickness using measurements of aortic inner diameter for all 11 rats and at all time points.

Figure 2.

Figure 2

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The DSA images shown here are from the same rat and are representative of the study group. The columns show DSA image sets acquired pre-drug infusion (0 min), immediately post-drug infusion (2 min), and during recovery (5 min) from the phenylephrine infusion. The images were acquired at the same point in the cardiac cycle with a steady heart rate (370 ± 40 beats per minute) between the three DSA image sets. The y-axis time scale represents the time from injection of the contrast agent (from the 2nd to the 15th heart-beat) for each DSA set. The black squares with white borders in the pre-drug column are the regions of interest chosen for the quantitative blood flow metrics. At all time points, the constriction of the peripheral blood vessels induced by the vasoconstrictor resulted in an increase in central blood volume, and thus distended the major blood vessels. This is clearly seen after the drug has been infused for 2 minutes (post-drug column)—the pulmonary arteries (black arrows), left ventricle (hatched arrow), and aorta (white arrow) are distended compared to the pre-drug and the recovery time points. In addition, almost a full second elapses before the contrast moves into the same systemic phase after the drug injection (2268 ms) when compared to pre-drug and recovery (1296 ms). This is significant considering the 300+ BPM heart rate of the rat. The cardiopulmonary system returns to a pre-drug state soon after the drug infusion was stopped. This can be seen in the likeness of the DSA images (pre-drug and recovery columns) and in the quantitative metrics (SVD transit time maps, mean arterial pressure, and aorta inner diameter) described in the following figures and in the text.

2.5 Statistical Analysis

Statistical analysis was performed using paired t-tests comparing baseline, peak, and late-recovery time points. We used paired t-tests to compare differences in pulmonary BV and BP and aortic diameter for the following pairs of time-point values: 1) baseline to peak values (maximum), 2) peak to late-recovery values (late recovery, 20 minutes after discontinuation of drug infusion), and 3) baseline to late-recovery values. Paired t-test results for BF were compared as described above except that the minimum rather than peak values were used because the BF decreased (all other measures increased) with drug administration. Statistical significance was defined as a p-value <0.05. The purpose of these calculations was to show that changes in blood flow metrics before, during, and after drug infusion were primarily due to vasoconstrictor effects rather than the imaging system itself or variability among rats.

3. RESULTS

Bland-Altman plots (Figure 3) created from previous data (Lin et. al., 2009) demonstrate that the same blood flow measurements obtained by x-ray DSA, thermodilution, and Fick methodology show only minimal differences. The coefficient of variance was 15.4% for thermodilution versus Fick, 7.3 % for thermodilution versus DSA, and 15.8% for DSA versus Fick.

Figure 3.

Figure 3

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Bland-Altman plots comparing DSA, thermodilution, and Fick blood flow measurements. These plots graphically show the agreement between the three measurement methods based on the work from (Lin et al., 2009). The x-ray DSA method was previously calibrated to the thermodilution and Fick methods. The heavy line is the mean difference and the dashed line is ± 2 standard deviations. As shown by the plots, different modalities show excellent agreement, and the lowest variability is seen when comparing DSA to thermodilution.

Figure 2 shows representative DSA images for the current rat study group. Images were acquired at baseline (pre-phenylephrine infusion, 0 min), immediately post-drug infusion (2 min), and during recovery (5 min). Time-series images for each DSA set (three columns) were acquired at the same point in the cardiac cycle with a steady heart rate of 370 ± 40 beats per minute between DSA sets. One 100 ± 11μL contrast injection was used for each DSA set. Movement of contrast agent can be followed chronologically from injection into the right heart (162 ms after contrast agent injection), to the lungs, and finally to the left heart and aorta (2268 ms).

Phenylephrine infusion markedly altered both blood flow and vessel anatomy. This is clearly seen at the 2-minute time point (post-drug, discontinuation of infusion): The pulmonary arteries (black arrows), left ventricle (hatched arrow), and aorta (white arrow) were distended compared with both pre-drug and recovery time points. At the same time point, almost a full second elapsed before contrast agent moved into the systemic vasculature (2268 ms) compared with the much faster rate at pre-drug and recovery (1296 ms). This time lapse is important because the rat’s heart rate is quite high, more than 300 beats per minute (BPM).

Statistical analysis confirms that the pre-drug and recovery images in Figure 2 are very similar. Pulmonary blood volume (PBV), BF and BP, as well as aortic diameter, were not significantly different between baseline and late recovery (PBV: p = 0.709, BF: p = 0.699, BP: p = 0.879, aortic diameter: p = 0.05).

BF changed dramatically between the pre-drug and 2-minute time points. BF in the aorta dropped from 33.7 to 28.8 mL/min (15% decrease) (Fig. 4b). Phenylephrine also pronouncedly increased trans-pulmonary MTT (Fig. 4c) and BV (Fig. 4d). These results were expected: Phenylephrine constricts tissue capillaries, which increases vascular resistance and impedes blood flow. In addition, the constricted capillaries increased central blood volume (CBV). There was a statistically significant difference between PBV (p = 0.001), PBF (p = 0.002), BP (p < 0.001), and aortic diameter (p < 0.001) between baseline and peak values and between peak and late-recovery values (all, p < 0.001).

Figure 4.

Figure 4

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These graphs show the hemodynamic changes for 11 rats due to phenylephrine. The drug was infused for 2 minutes and had a dramatic effect on the blood pressure, aorta inner diameter, and blood flow metrics. Once the drug infusion was stopped at 2 minutes, the perturbed physiology quickly reverted to baseline values. By the 5-minute time point (recovery), 3 minutes post-drug infusion, most values returned to pre-drug levels. For example, the aorta inner diameter (a) began at 2.28mm, steadily increased to its peak at 2.64mm, then returned to 2.07mm during recovery. The x-ray based measurement of aortic blood flow (b) showed a decrease with drug infusion and then a return to baseline values during recovery. The trans-pulmonary mean transit time (c) and blood volume (d), like aorta diameter and blood pressure, rose steadily with drug administration and then return to baseline value during recovery.

Variation between measurements was calculated using the coefficient of variation (CV). CV for repeated measurements in the same rat at the same physiologic state had been determined previously to be 8.7% (x-ray DSA), 20.8% (thermodilution), and 13.9% (Fick blood flow measurement methods) (Lin et al., 2009).

We were able to measure inner aortic diameter in the current study because of inherent vessel enhancement from DSA and rapid, synchronized, physiologically driven image acquisition. In addition, we directly measured aortic BP. Average BP normalized to pre-drug values (0 min) during recovery from drug infusion (Fig. 4a). The two-minute-long infusion had dramatic effects on both aortic BP and diameter as shown by SVD-derived metrics. By the 5-minute time point (recovery, 3 minutes after infusion ended), most values had returned to pre-drug conditions. Note in Figure 4a that aortic BP and inner diameter followed the same trend, with both values steadily increasing during infusion and then steadily declining during recovery to pre-drug values. A 104% increase was noted in average normalized BP between baseline (0 min) and end of drug infusion (2 min). Inner diameter showed a simultaneous increase over baseline, from an average inner diameter of 2.3 mm to 2.6 mm (16% increase).

4. DISCUSSION

In this study, we established that rapid changes in the rat cardiopulmonary system due to phenylephrine infusion could be visualized, quantitatively measured, and tracked using our x-ray DSA system; we were secondarily able to calculate blood flow. Furthermore, blood flow changes calculated from x-ray DSA images were compatible with changes observed in pulmonary BP and inner aortic diameter.

We chose phenylephrine as have previous investigators working with rats (Doggrell et al., 1998; Herlihy et al., 1992; Lin et al., 2006; Segel et al., 1987; Tatchum-Talom et al., 1995) because it is a vasoconstrictor without chronotropic or inotropic activity (American Regent Laboratories, 2001; Segel et al., 1987). Phenylephrine acts primarily on pulmonary vessels and capillary beds (American Regent Laboratories, 2001) by inducing constriction of peripheral blood vessels and increasing peripheral vascular resistance (PVR), which increases central blood volume (CBV) and causes distention of major vessels.

In our study, increases in CBV and PVR caused mean arterial pressure to rise almost two-fold from baseline, while average aortic inner diameter increased almost 1.2-fold. These drug-induced changes were clearly visualized and measured using the DSA imaging system. Representative images (Fig. 2) show distention of the pulmonary arteries (solid arrows), aorta (white arrow), and left ventricle (dashed arrow). Blood flow in the aorta decreased. In the setting of small-animal research, these measurements have often been difficult to obtain because a rat’s heart beats more than 300 times per minute. Figure 2 also shows that areas of enhancement after infusion mimic those seen prior to infusion, but are delayed in time.

X-ray DSA images were acquired under the condition of apnea and at the same time point during every cardiac cycle. Thus, there were no subtraction artifacts that could compromise imaging-based measurements and physiological state could be compared between imaging sets. The imaging-based measurements of blood flow and transit time agree with drug-induced morphological changes. Phenylephrine significantly increased blood transit time through the lungs from baseline to end of drug infusion. The observed increase in transpulmonary mean transit time (MTT) is the result of arteriolar constriction. After arterioles constricted, the pulmonary arteries and aorta became distended. The ability to visualize and measure these effects of phenylephrine on lung parenchyma highlights the value of x-ray DSA and points to its utility in future in vivo experiments.

A limitation of x-ray DSA is that it enables only planar imaging. We took steps to reduce the impact of superposition by carefully synchronizing contrast injection with image acquisition; in addition, regions of interest for calculating MTT were drawn with attention to minimizing overlap of vessels/parenchyma. X-ray phantom-based measurements had been conducted in previous work (Lin et. al., 2009) to correct for modulation in vessel thickness and contrast bolus delay effects, as well as to show the linearity of DSA signal with various vessel thicknesses and with contrast injection volume. The same approach can be extended to tomographic DSA (Badea et al., 2007) to mitigate superposition effects.

DSA- and SVD-based blood flow calculations provided regional measurements in live rats on a temporal scale (~ every 30 seconds with imaging at every heartbeat sample) that captures rapid changes in physiologic state. This technology is an exciting solution for future in vivo functional phenotyping of dynamic changes due to disease, drug challenge, and/or drug therapy.

The high temporal resolution of the current x-ray DSA system allowed us to reduce the number of animals needed for experiments in comparison to work done with use of imaging techniques that operate at similar fields of view and spatial resolutions but have temporal resolutions on the order of minutes. Thus, the x-ray DSA system has another advantage compared with other imaging techniques. A range of measurement resolutions were established in previous studies—global (whole lung average, Cander et al., 1959; Gedeon et al., 1980; Rabinovitch et al., 1983; Vincent et al., 1993), regional sections (lung lobe, image reconstructed slab, Doctor et al., 1998; Gauger et al., 1997; Glenny et al., 1991; Martin et al., 1982; Presson Jr. et al., 1997; Reed et al., 1970; Schoepf et al., 2000; West et al., 1960; West et al., 1964; Zhang et al., 1997), and pulmonary arteries (Milnor et al. 1966; Reddy et al., 1995; Redington et al., 1991; Sanders et al., 1983; Williams et al., 1954). The technique most similar to the methods reported here used positron emission tomography (PET) with dogs (Rabinovitch et al., 1983), with results mapping regional (axial lung slab) pulmonary blood flow. However, these earlier measurements in dogs were relative and the regions were large (~1 cm2-resolution slabs), making this method too coarse to observe a rat’s aorta.

In summary, we infused phenylephrine and acquired X-ray DSA images before, during, and after administration, with a total of 12 DSA sets per rat (11 animals total) over 22 minutes. The vasoconstrictor caused major vessel distention, retardation of blood flow, increased blood pressure, and increased aortic inner diameter. After completion of drug infusion, physiologic metrics returned to pre-drug values. DSA imaging also captured drug-induced morphological changes. The study was the first application of our small-animal x-ray DSA system in measuring cardiopulmonary changes due to drug infusion. This ability to measure absolute aortic blood flow (cardiac output), transpulmonary transit time, and mathematically coupled pulmonary blood volume with high spatial and temporal resolution in living animals can advance in vivo functional phenotyping. Applications include blood flow modulation by drugs (e.g., nitroprusside), diseases (e.g., lung perfusion induced by hypoxia), and/or genetic models (e.g., spontaneous hypertension). The technique can also be extended to measurement of blood flow in the liver, kidney, and solid tumors, as well as visualization of relevant vessels (Badea et al., 2006; Lin et al., 2008).

6. ACKNOWLEDGMENTS

The authors wish to thank Jim Pollaro, M.S., for the ventilator software-control interface and monitoring system, Laurence Hedlund, Ph.D., for animal support and surgery, and Sally Zimney, M.Ed., and Elizabeth Coolidge-Stolz, M.D., for editorial assistance. All work was performed at the Duke Center for In Vivo Microscopy, an NIH/NCRR National Biomedical Technology Research Center (P41 RR005959) and NCI Small Animal Imaging Resource Program (U24 CA092656).

SPONSORS OF FUNDING: NIH/NCRR P41 RR005959, NCI U24 CA092656

Footnotes

5. CONFLICT OF INTEREST STATEMENT: The authors have no conflicts of interest associated with this work.

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