ABSTRACT
Local delivery of the small (492 daltons), soluble, hydrophilic tracer 99mTc-DTPA onto the bronchial epithelium was employed to gauge absorption of soluble substances from the airway surface. DTP A was freshly prepared as 99mTc-DTPA (Medi-Physics, Arlington Heights, IL). 99mTc-DTPA was randomly sampled predelivery and assayed for unbound 99mTc with silica gel media and thin-layer chromatography to verify the labeling procedures (10). Local airway delivery was performed to ensure deposition of the DTPA exclusively onto bronchial airway surfaces. A fiberoptic bronchoscope (OlympusBF type P10, New Hyde Park, NY) was advanced into the trachea and passed beyond the carina and mapped into a fourth-generation bronchus. A polyethylene catheter with a microspray nozzle (11) at its tip that delivers spray radially onto the epithelial surface was advanced through a channel of the bronchoscope and visualized beyond the end of the bronchoscope within the center of the bronchial lumen. Ventilation was withheld and 6-10 |nL of 99mTc-DTPA was deposited through the nozzle and sprayed directly onto the bronchial wall using 0.7 mL of air to clear the catheter tip (an additional 0.7 mL of air cleared any residual activity from the catheter). Controlled ventilation was resumed and serial images of 99mTc-DTPA retention were recorded every 120 seconds over a 30-minute period using a gamma scintillation camera (MaxiCamera, General Electric Medical Systems, Pittsburgh, PA) set with a 15% window around the peak energy of 140 keV and shielded with a parallel hole collimator. Images were acquired from the ventral aspect of the thorax and the fraction of sprayed droplets initially delivered within the lung was normalized to 100%. All images were background subtracted and decay corrected to time zero (time immediately after droplet delivery) for radioisotopic decay of the 99mTc. Systemic venous blood was sampled (0.5 mL) every 6 minutes during the 30-minute imaging period in order to assess take-up of the 99mTc-DTPA into the blood. Once 99mTc-DTPA is absorbed into the circulation, it is cleared from the blood by the kidneys (12). Blood samples were analyzed by a gamma scintillation counter (GammaTrac, Tm Analytic, Tampa, FL). Total absorption of the 99mTc-DTPA into blood was estimated by the calculation of multiplying the activity in blood (counts/mL) by a nominal blood volume equal to 8.5% of body weight (13). Experimental Protocol
The design of the protocol was to use each animal preparation as its own control and thus to perform measures of 99mTc-DTPA clearance with bronchial perfusion
intact (control condition), followed by redelivery of 99mTc-DTPA and measure clearance with bronchial perfusion interrupted (experimental condition). We did not randomize the order of this sequence, as previously we have demonstrated the stability of the preparation over the time period of the measurements (14). Our concern was that if the experimental condition was evaluated first, then the time course of recovery of the preparation from the no flow condition might extend the measurement period beyond the period during which the preparation was physiologically stable, adding uncertainty to any control clearance measures acquired at a late time point. To eliminate issues of nonhomogeneous tissue attenuation of radioactivity due to regional differences in airway geometry, all sprayed deliveries of 99mTc-DTPA were performed as paired comparisons (control vs. experimental) in the same airways. Residual activity from the initial delivery (in our experience usually <2%) that was not cleared by the delivery time of the second administration of 99mTc-DTPA was considered as nonclearable and added to the room and animal background corrections that were subtracted from acquired images. With the present sheep model and other models, we have had good success in using the bronchoscopic method to map into a specific bronchial airway and deliver radiolabeled tracers directly onto the airway surface and at a later time point in the protocol remap into the airway and redeliver isotopic tracer for a second measurement period of tracer clearance (15).Additional sheep were evaluated with protocols to investigate blood uptake of unbound label, 99mTc04, and clearance of 99mTc-DTPA after vagotomy. Isotopic tracer was delivered in these additional protocols in a manner identical to the methods used above in control and interrupted bronchial blood flow protocols. For the vagotomy protocol the vagus nerves were isolated bilaterally and sectioned as previously described (9). Data Management and Statistics
Radioisotope delivery and clearance data were quantitated with techniques modified from Foster and Freed (15). The initial bronchial image acquired immediately after delivery of the 99mTc-DTPA was stored on a video screen, and this enabled a region of interest to be selected by cursor manipulation and drawn to cover the airway 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 within the region during the 30-minute washout was corrected for background and radioactive decay and expressed as a percentage of the 99mTc-DTPA delivered to the region at time zero (immediately after the nozzle catheter and bronchoscope were withdrawn from the bronchial airway). The natural logarithm of the proportion of radioactivity remaining within an airway region was plotted as a function of time. The semilogarithmic regression line for the interval from peak radioactivity after delivery to the end of the observation time point was
determined by a least-squares fit. The slope of the regression line was determined according to the equation, A = Aoe~kt, where Ao is the y intercept and A is the count rate at any time t. The slope of the line is the rate constant (k) for the clearance of 99mTc-DTPA from the bronchus, and it can be converted to a clearance half-time (t50, clearance index) by t50 = 0.693Ik (15). Corrections to the clearance analysis of 99mTc-DTPA were not made for nonairway epithelial radioactivity, because it has been demonstrated that such corrections over the 30-minute imaging period do not significantly affect the measured clearance rate (6,16,17).Comparisons of right to left bronchial clearance, and normal bronchial blood flow to interrupted bronchial blood flow treatment on the 99mTc-DTPA clearance half-times were accomplished by a paired t-test analysis. Effects of interrupted bronchial blood flow on the bronchial retention levels of 99mTc-DTPA were analyzed with analysis of variance for repeated measures and a Newman-Keuls post hoc test for significance of the differences. A p-value of <0.05 was considered significant. RESULTS
A total of 12 sheep were investigated and the baseline bronchial artery pressure was 87 ± 5 mmHg. The mean pressures were obtained during perfusion at the control flow (17 ± 1 mL/min), which had been set based on sheep body weight (27.5 ±1 .7 kg). Mean systemic arterial pressure for the group of sheep evaluated was 95 ± 2 mmHg. Peak inspiratory pressure was 17 ± 2 cm H20.Table 1 shows 99mTc-DTPA clearance half-times for right and left lobar bronchi that were measured in 10 sheep during the 30-minute imaging period of control bronchial perfusion. The mean half-times for the right and left lobar bronchi were 14.8 (SEM ± 2.7) minutes and 8.6 (SEM ± 1.7) minutes, respectively. The difference in mean clearance half-times between right and left bronchial regions was significant. In eight of the sheep during control bronchial perfusion the corresponding absorption of 99mTc-DTPA into the systemic blood was assessed, and the time course of this event is demonstrated in Figure 1. The activity present in systemic venous blood peaked on average at 18.5 minutes after delivery of the 99mTc-DTPA and decreased thereafter to plateau levels that ranged between 60 and 80% of the maximum level. To further characterize the clearance pathways, an estimate of the total 99mTc-DTPA present in blood was extrapolated from the amount in a 0.5 mL sample and blood volume of the animal (estimated as 8.5% of the mass in kg) (13). Thus, the blood sample with the maximum amount of radioactivity during the sampling period (0-36 min postdelivery) was used to calculate the fraction of 99mTc-DTPA delivered to the airway that cleared to blood; on average this occurred at the 18.5-minute time point postdelivery. Knowing the initial amount of 99mTc-DTPA delivered to each bronchial segment,
the amount retained, and the fraction cleared to the blood enabled an estimate of the amount cleared by a second pathway for clearance, i.e., mucociliary function, from the start (delivery of isotope onto the bronchial surface) to the point at which concentrations of the tracer were maximal in blood. Using this mass balance approach, the fraction of delivered activity absorbed into the vasculature, cleared by mucociliary function, or retained at the delivery site was on average0.179, 0.432, and 0.389, respectively. For comparison the mean (± SEM) of each component as a fraction of the amount sprayed onto the airway surface are presented in Figure 2. Thus, at the time point when 99mTc-DTPA levels in blood were maximal, approximately 18% of the 99mTc-DTPA delivered to the airway had cleared into the circulation and 43% followed a mucociliary pathway, and this difference was significant.In additional control sheep we investigated with a separate protocol the absorption into blood of the unbound label, 99mTc-pertechnetate (99rnTc04_), delivered directly to the bronchial surface in the same manner as the 99mTc-DTPA.
