INTRODUCTION

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REGIONAL DOSIMETRY OF INHALED substances within the respiratory tract is important for characterizing tissue burdens, as well as the pathways for diffusion and clearance. Although numerous investigations have focused on the fractional deposition of aerosols within the conducting airways as a function of particle diameter (7), relatively few studies have examined the fate of soluble substances placed onto the epithelial surface. The rate of diffusion of a substance into submucosal tissue depends on its lipid and water solubilities, size and shape. Lipophilic molecules pass mainly via transcellular routes, and hydrophilic molecules pass via paracellular routes (2). The conducting airways are supplied by systemic arteries. Because the bronchial circulation anastomoses with pulmonary veins (25) and drains into the left heart, the clearance of soluble substances deposited in the lung by the blood perfusing the subcarinal airways is difficult to assess in vivo separately from the pulmonary vascular component (17).

When direct in vivo assessment of clearance into the tracheal vasculature has been evaluated, the uptake of soluble tracers by the tracheal venous outflow was inversely related to the level of airway perfusion (10). Therefore, when tracheal perfusion was increased by vasoactive agonists or mechanical means, the uptake of soluble tracer by the vasculature decreased. Conversely, when tracheal inflow decreased, the venous concentration of tracer increased. This led to the supposition that, if the rate of tracheal perfusion rises, then tissue fluid volume will increase and enhance the interstitial barrier to vascular uptake. Thus, during high inflow, clearance of soluble substances from the tracheal surface into the vasculature would be delayed. For decreased tracheal blood flow, tracer concentration in the venous outflow was increased presumably due to a prolonged dwell time.

The purpose of the present study was to test the hypothesis that clearance of soluble tracers from the surface of subcarinal airways is dependent on bronchial vascular perfusion and that uptake of tracer into bronchial blood would parallel vascular clearance phenomenon reported for the extrathoracic tracheal airway (10). We utilized an instrumented sheep model for our in vivo investigation and the small (492 Da), soluble, hydrophilic radiolabeled tracer ^99mtechnetium-labeled diethylene triamine pentaacetic acid (^99mTc-DTPA) to gauge vascular uptake. In addition, we used two-dimensional imaging of the airways after site-specific delivery of the tracer directly onto the bronchial surface by using a flexible bronchoscope. The two major pathways of clearance, a vascular route and epithelial mucociliary function, were observed and found to be interdependent.

	METHODS

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The study protocol was approved by The Johns Hopkins Animal Care and Use Committee. Anesthesia was induced in sheep (25-35 kg) with intramuscular ketamine (30 mg/kg) and subsequently maintained with intravenous pentobarbital sodium (20 mg · kg¹ · h¹). The sheep were positioned supine on a surgical table, and, after a tracheotomy was performed, the animals were intubated and paralyzed with pancuronium bromide (2 mg iv). The lungs were mechanically ventilated with a tidal volume of 10-12 ml/kg at a rate (12-15 breaths/min) sufficient to maintain normal blood gases. Then, 5 cmH₂O positive end-expiratory pressure were applied. The left thorax was opened at the fifth intercostal space, and heparin (20,000 U iv) was administered. The esophageal and thoracic tracheal branches of the bronchoesophageal artery were ligated as previously described (23). The bronchial branch of the bronchoesophageal artery was isolated, cannulated, and perfused (0.6 ml · min¹ · kg¹) with autologous blood withdrawn from the descending aorta and pumped through a variable-speed roller pump (22, 24)

Local tracer delivery. ^99mTc-DTPA was used to assess clearance from the airways and uptake by the bronchial circulation. DTPA was freshly prepared on each experiment day as ^99mTc-labeled DTPA (Medi-Physics, Arlington Heights, IL). Occasionally, ^99mTc-DTPA was sampled predelivery and assayed for unbound ^99mTc with silica gel media and thin-layer chromatography to verify the labeling procedure (3). Local airway delivery was performed to ensure deposition of the tracer exclusively onto surfaces of conducting airways perfused by the bronchial circulation. A fiber-optic bronchoscope (50-mm OD, Olympus, Lake Success, NY) was advanced through the trachea, beyond the carina, and into a fourth-generation bronchus. A polyethylene catheter with a microspray nozzle at the tip was advanced through the working channel of the bronchoscope and visualized beyond the end of the bronchoscope (11). After ventilation was momentarily stopped, 6-10 µl of ^99mTc-DTPA that had been loaded into the catheter tip were sprayed radially onto the airway wall. Absolute activity delivered was determined by measuring the catheter tip after it was loaded and again after deposition of label to the airways. The average activity delivered was 63 µCi (84% delivery efficiency). The catheter was then retracted into the channel of the bronchoscope, and the bronchoscope was removed from the animal. Controlled ventilation resumed, and serial dynamic images of clearance of the ^99mTc-DTPA were acquired every 2 min for 30 min using NucLear MAC (Scientific Imaging, Littleton, CO) and a gamma camera (MaxiCamera, General Electric, Waukesha, WI). Animals were imaged from the ventral aspect, and the camera was set with a 15% window around the peak energy of 140 keV and shielded by a parallel-hole collimator. Radioisotope delivery and clearance data were quantitated with techniques modified from Foster and Stetkiewicz (8). The initial bronchial image acquired immediately after delivery of the ^99mTc-DTPA was stored on a computer screen, and this enabled a region of interest to be selected by cursor manipulation and drawn to cover the site of ^99mTc-DTPA delivery. For the clearance of ^99mTc-DTPA, activity time plots were constructed for the region of interest and the retention of radioactivity during the 30 min washout was corrected for radioactive decay and expressed as a percentage of the ^99mTc-DTPA delivered to the region at time 0 (immediately after the nozzle catheter and bronchoscope were withdrawn from the bronchial airway). Because remaining activity before the second deposition was always such a small amount (<l2% of the original delivered activity), we did not background correct. This level has become a selection criteria for normal tracer clearance. Systemic venous blood samples (0.5 ml) were withdrawn from the inferior vena cava via a catheter inserted into a femoral vein every 6 min for the 30-min time period. Activity in blood was counted in a gamma counter (GammaTrac, TmAnalytic, Tampa, FL). Estimates of total blood ^99mTc-DTPA were made by multiplying counts per minute per milliliter by a nominal blood volume equal to 8.5% body weight (18).

Protocols. Airway clearance and blood uptake of ^99mTc-DTPA were measured in sheep 1) during control bronchial artery perfusion, 2) when bronchial perfusion was stopped by turning off the perfusion pump, and 3) during increased bronchial perfusion to a high level (3 times control = 1.8 ml · min¹ · kg¹). Two groups of sheep were studied. In the "no-flow group" (n = 7), each sheep was studied first during control flow conditions. After an ~60- to 90-min recovery period, blood flow was stopped and, 5 min later, ^99mTc-DTPA was deposited in the same airway that had been studied for the control flow condition. In another group of sheep (n = 5), the effects of high flow were determined. In this "high-flow" group, sheep were first studied during control flow conditions followed by a 60- to 90-min recovery period. Five minutes before the second ^99mTc-DTPA deposition in the same airway, bronchial perfusion was increased. This experimental design was implemented so that paired comparisons could be made within a specific airway so as to 1) eliminate issues of attenuation of radioactivity due to regional differences in airway geometry and the subsequent two-dimensional image acquisition and 2) eliminate error due to left-right lung differences in clearance (9). We did not randomize the order of this sequence because previously we have demonstrated the stability of the preparation over the time period of the measurements (21). Additionally, we were concerned that the experimental flow conditions imposed might alter subsequent control conditions, adding uncertainty to any control clearance measures acquired at a late time point. Bronchoscopy was performed by mapping into a specific fourth-generation bronchus for the control flow deposition. The subsequent deposition during an altered bronchial blood flow condition took place in the same location.

Statistics. All data are presented as means ± SE. The nonparametric Wilcoxon matched-pairs signed-rank test was used to evaluate differences between paired comparisons during control and experimental flow conditions. We compared the percent retained activity at 30 min from the retention-time data and the average retention time(activity × time/activity). A two-tailed P value of 0.05 was accepted as significant.

	RESULTS

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Baseline bronchial artery pressure was 84 ± 5 mmHg for the 12 sheep studied. This pressure was obtained during perfusion at the control flow (16 ± 1 ml/min), which had been set based on sheep body weight (26 ± 2 kg). Mean systemic arterial pressure for the group of sheep studied was 92 ± 2 mmHg. Peak inspiratory pressure was 17 ± 1 cmH₂O. The time course of ^99mTc-DTPA clearance during control bronchial perfusion is presented in Fig. 1. The retention at 30 min after deposition averaged 20 ± 6% of the initial activity as measured by gamma camera imaging. The average retention time was 10.9 ± 0.9 min. This time course of clearance can be compared with clearance that was observed during no-flow conditions when the perfusion pump was stopped. The bronchial retention of ^99mTc-DTPA was significantly greater during the no-flow condition, with retention at 30 min averaging 32 ± 8% of the initial deposited amount, and the average retention time was 12.5 ± 0.6 min (both P = 0.028 from control).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Bronchial retention of soluble radiotracer vs. time curves. Values are average retentions (± SE) for a bronchial region observed in 7 sheep at indicated times for each condition: control bronchial flow () and no bronchial blood flow (). Retention is expressed as percentage of technetium-99m-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA) activity initially delivered to the airway surface; time = 0 min immediately follows delivery of radioactivity. Radiolabeled tracer retention is significantly greater during the no-flow condition relative to control perfusion (P < 0.05).

In the second series of sheep, the effects of high blood flow on ^99mTc-DTPA retention was determined. In these animals, bronchial blood flow was increased to 300% control flow (43 ± 4 ml/min), which resulted in a bronchial artery perfusion pressure of 183 ± 11 mmHg. The effects of high blood flow are compared with control flow ^99mTc-DTPA kinetics in the same animals in Fig. 2. Although the initial time points are essentially superimposable, by 30 min a difference in retention was observed with the high-flow responses being prolonged (control: 13 ± 4% vs. high flow: 25 ± 4%; P = 0.043). The average retention time for this group during high flow was 12.0 ± 0.8 min compared with the control average retention time of 9.9 ± 0.8 min (P = 0.043).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Bronchial retention-time data comparing control bronchial flow () to high-flow conditions (). Values are average retentions (± SE) for a bronchial region observed in 5 sheep. See Fig. 1 legend for details. Radiolabeled tracer retention is significantly greater during the high-flow condition relative to control perfusion (P < 0.05).

To evaluate the uptake of ^99mTc-DTPA into the blood for the three different flow conditions, we measured counts per minute per one-half milliliter needed to be normalized for the total delivered ^99mTc-DTPA. Blood activity was converted to microcuries per milliliter and the total circulating blood volume was estimated as 8.5% body weight (18). Thus the total blood activity could be estimated and the time course of changes in blood activity as a percentage of the total delivered activity could be calculated for the three blood flow conditions. Blood ^99mTc-DTPA concentrations were obtained for four of seven sheep in the no-flow series and five of five sheep in the high-flow series. All control flow data from both groups studied were averaged and are presented in Fig. 3. The control flow condition resulted in an average group maximum systemic venous ^99mTc-DTPA concentration by 18 min after delivery. Blood activity averaged 16% for all the control flow experiments. The area under the activity-time curve for the control flow condition was significantly greater than during the high-flow condition (P = 0.043) but was not statistically different from the stopped-flow condition (P = 0.273).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Normalized 99mTc-DTPA activity in blood as a percentage of the delivered dose over the course of 30 min. Values are average blood activities (± SE); control flow: n = 9 sheep, no flow: n = 4 sheep, high flow: n = 5 sheep.

Because at any time point the activity in the blood and the amount retained at the site of deposition did not account for all the delivered tracer, another clearance route in addition to renal excretion appeared to be operative. Figure 4 shows the gamma camera image series from one representative experiment where radioactivity can be visualized moving up the airway by mucociliary activity. To determine the contribution of this clearance pathway, we first estimated the amount of renal excretion and then used mass balance to determine the overall distribution of ^99mTc-DTPA. We used mean values to determine estimates of each component. The total delivered (100%) = percent in blood + percent cleared by kidneys + percent retained at airway site + percent mucociliary clearance. We estimated renal clearance as 4.5% of the blood volume per minute (6, 20) and used the mean blood activity values in microcuries that were used to calculate the results presented in Fig. 3. The percent retained values are taken from Figs. 1 and 2. Figure 5 shows the distribution of ^99mTc-DTPA activity as measured in the blood, excreted by the kidney, retained in the airway, and attributed to mucociliary clearance at the two time points where the maximum differences in blood activity (18 min; A) or retained activity (30 min; B) were observed. Estimates for the renal and mucociliary components demonstrate the magnitude of these pathways. Additionally, mucociliary clearance tended to be decreased only during the no-flow experiments.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Dynamic series of gamma camera images observed in 1 sheep over the course of 30 min demonstrating mucociliary pathway for clearance. Each frame represents 2-min acquisition. Activity (bright spots) can be visualized in transit from initial delivery site into the tracheal airway.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Distribution of 99mTc-DTPA activity in blood, excreted by the kidneys (calculated estimate), retained in the airway, and attributed to mucociliary clearance (calculated estimate) at 18 min (A) and 30 min (B).

	DISCUSSION

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The overall goal of this study was to determine how changes in bronchial vascular perfusion would affect the clearance of a soluble substance deposited in a subcarinal airway. Our results showed increased retention of ^99mTc-DTPA at the airway delivery site when bronchial perfusion was interrupted. The average retention of ^99mTc-DTPA was significantly greater during the no-flow condition than that observed during control flow conditions. Although blood ^99mTc-DTPA concentrations were not statistically different from control, blood levels tended to be lower early in the time course but increased over time. Additionally, the estimates of mucociliary clearance demonstrated a decrease during the no-flow condition (Fig. 5). We have previously reported the dependence of mucociliary clearance on bronchial perfusion where decreased bronchial blood flow decreased mucociliary transport of an insoluble tracer (21). However, our results are inconsistent with a previously published report on soluble tracer kinetics in the trachea in which Hanafi and colleagues (10) demonstrated an inverse relationship between blood flow and venous uptake. Using an in vivo sheep preparation, these investigators filled an isolated tracheal segment with ^99mTc-DTPA in a physiological salt solution and then measured tracheal artery and venous blood flow. Thus the experimental preparation eliminated the mucociliary component as a potential pathway of clearance. These investigators showed that decreasing perfusion resulted in an increase in the venous uptake and clearance of the tracer. They suggested that decreased perfusion resulted in a decrease in interstitial fluid volume of the airway wall that might decrease the barrier to tracer uptake into the vasculature. Another potential explanation provided was related to the fact that, in their experimental setup, arterial inflow was never equal to the venous outflow of the isolated segment. Thus perfusion redistribution through collateral vascular channels might have been altered with changes in inflow, obscuring the true changes in tracer concentrations.

In our model, we found that blood uptake only showed trends toward a decrease in uptake and was not totally absent when blood flow was interrupted. This result is likely related to two factors. First, cessation of perfusion does not prevent collateral flow at downstream sites or backflow from the pulmonary circulation. Baile et al. (1), using aggressive methods to obliterate the bronchial vasculature, demonstrated a persistent, albeit greatly decreased, perfusion to the precapillary vessels even after occluding the main bronchial arterial inflow in sheep. Additionally, because mucociliary activity was present during all three flow conditions, delivered ^99mTc-DTPA did not remain exclusively at the site of deposition. At later time points, when tracer was in transit through the tracheal airway, uptake by blood vessels in the trachea where blood flow was not controlled likely contributed to the total systemic venous ^99mTc-DTPA activity. Thus examination of the blood activity during the earliest time points more accurately reflects blood uptake in the subcarinal airways before the mucociliary transport of surface activity into the tracheal airway.

The estimates of total tracer distribution provide further support of our previous results describing the effects of decreased bronchial perfusion on mucociliary clearance of an insoluble tracer (21). In that study, clearance of ^99mTc-labeled sulfur colloid by mucociliary function was delayed when bronchial perfusion was stopped. Although the precise mechanism responsible for delayed mucociliary clearance is not known, some level of bronchial vascular perfusion is likely required to maintain normal ciliary function. Whether the bronchial circulation provides an essential heating and/or humidification function, delivery of metabolic substrate, or endothelial shear-related factors important for normal ciliary function is not clear.

In the present study, when bronchial blood flow was increased to three times the normal control value, the clearance of a soluble tracer became delayed. Simultaneous with this greater activity level of the tracer at the intrathoracic deposition site was a decrease in tracer uptake by the blood. This observation strongly supports in vivo the results of Hanafi et al. (10) and their demonstration of an inverse relation in situ between perfusion and vascular absorption from the trachea. A mechanism to explain this paradox, a higher local concentration of soluble marker in the presence of greater tissue perfusion, relates to our laboratory's earlier finding that identical increases in bronchial perfusion (3 times the normal control flow) in this same sheep model induces a significant increase (18%) in the airway wall area, 11% of which was attributed to edema fluid accumulation (4). This leads to our current speculation that an immediate increase in bronchial perfusion enhances the tissue-fluid barrier and delays clearance of soluble tracers from the airway interstitium into the bronchial vasculature. Because mucociliary clearance was not substantially different during high-flow compared with during control perfusion, it appears that the bronchial vasculature during control flow conditions is fully recruited and distended and that increasing flow has little effect on this variable (15). Furthermore, these observations are consistent with our laboratory's methacholine clearance study in which high bronchial flow (2 times control flow) did not reverse agonist-induced bronchospasm more rapidly than control perfusion and decreased bronchial blood flow (50% of control) prolonged agonist-induced bronchoconstriction (23). It is also possible that, with alterations in perfusion, changes in blood flow distribution within the airway wall take place. Increases in blood flow might result in blood flow diversion to the adventitia such that soluble tracer uptake in the mucosa becomes less effective. Whether the changes in pump flow applied in this study preferentially affect submucosal or adventitial perfusion is not known. Finally, increased perfusion might result in net fluid flux into the lumen, thereby limiting the access and uptake of deposited tracer into the blood vessels of the airway mucosa (16). Although the mechanism responsible for the decrease in soluble-tracer uptake into blood during conditions of increased airway perfusion is not known, it may have implications for self-limiting the uptake of potentially injurious soluble particles. For example, respirable irritants such as cotton smoke (19), cigarette smoke (14), and antigen (13) all increase airway blood flow, which may then limit further uptake of soluble particles.

The time course of measurements made in this study warrants further discussion. Although airway retention-time data and blood activity levels were monitored over the course of 30 min, the impact of altering perfusion on clearance mechanisms can be seen most clearly early in the time course. Blood activity levels of Tc-DTPA represent a dynamic process of both uptake by the airway circulation and clearance by the kidney (5). However, tracer clearance from the blood by the kidney was an ongoing process that we did not measure directly and only estimated. As seen in Fig. 3, blood uptake mechanisms during the control flow condition had not reached a maximum until ~18 min. Before this time, uptake mechanisms predominate, whereas, after this maximum is reached, it is likely that clearance by the kidney predominates. The amount attributed to mucociliary clearance is likely a slight overestimate of that clearance route if there is any additional distribution of tracer in extracellular fluid space. We believe our estimates are only slightly in excess because of the visually impressive contribution of this component seen in Fig. 4 and the fact that mucociliary clearance tended to be decreased in the no-flow condition, as we have shown previously (21).

An additional reason for focusing on the distribution of activity at the earlier time points can be realized in the experiments in which bronchial perfusion was stopped. Although mucociliary clearance tended to be decreased relative to the control flow situation (Fig. 5A), a substantial amount of the removal of tracer activity still could be attributed to this component. When labeled tracer reached the trachea, blood uptake mechanisms by the tracheal vasculature were operative, which likely accounts for the slowed but steady increase in blood activity level observed during this no-flow condition (Fig. 3).

Although we used ^99mTc-DTPA to assess soluble-tracer kinetics in the airway, local bronchoscopic deposition of ^99mTc-DTPA has been used previously to assess regional differences in airway epithelial permeability. Wolff and colleagues (26), delivering a 50-µl instillate in a fifth-generation bronchus in dogs, demonstrated significantly prolonged clearance half times (140 min) relative to what was observed in the present study. Furthermore, they saw no evidence of mucociliary clearance of the radiolabeled tracer. These investigators suggested that the halothane anesthesia used in their study depressed mucociliary clearance and that consequently the predominant clearance pathway was through blood uptake. The larger delivered volume relative to what was used in the present study might also have contributed to increased retention. We modeled our delivery system after that used by Lay and colleagues (12). These investigators used the same microspray-nozzle delivery system and 6-µl volume of both insoluble tracer and the radiolabel ^99mTc-pertechnetate. Relative to the insoluble tracer, in their study the highly permeable ^99mTc-pertechnetate showed only slight clearance via the mucociliary route. Interestingly, these investigators also used halothane anesthesia in dogs.

In conclusion, we have demonstrated that the level of bronchial vascular perfusion significantly alters clearance kinetics of the soluble tracer ^99mTc-DTPA deposited locally onto a subcarinal airway. This study and our laboratory's prior investigation (4) suggest that bronchial vascular perfusion can modify the airway interstitial barrier to absorption. Inhaled substances that alter bronchial vascular perfusion are likely to limit further uptake. Although increased blood flow alters the blood uptake of tracer, decreased perfusion also tends to limit mucociliary activity. Thus there is a clear interdependence of bronchial vascular perfusion with the clearance mechanisms of the airway.