INTRODUCTION

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THE IMPETUS FOR DEVELOPING a technique to determine how rapidly pulmonary capillaries recruit came from an experiment designed to determine the effect of gravity on the distribution of pulmonary blood flow. In that study, pigs were flown on the National Aeronautics and Space Administration (NASA) KC-135 aircraft (3). The flight pattern was a series of parabolic arcs that produced alternating 25-s periods of zero gravity and 1.8 G. During each new gravitational state, a 6- to 8-s period was allotted for the pulmonary circulation of the pig to reach equilibrium. Fluorescently labeled microspheres were then injected over an ~5-s period at the junction of the caudal vena cava and the right atrium. A 10- to 14-s interval remained for distribution of the microspheres. The 15-µm microspheres lodged at the entrance to the pulmonary capillary bed and thus reflected flow at that level. One potential problem with these brief exposures to microgravity was that there might not have been sufficient time for the pulmonary microcirculation to reach steady state. As a result, the distribution of microspheres could have been affected by a transitional state in the pulmonary circulation, rather than by the intended gravitational state alone. Although the KC-135 experiment prompted this study, the response time of the pulmonary microcirculation is of interest in a number of areas. For example, how quickly gas-exchange surface area is recruited is important in exercise physiology, and the speed with which the microcirculation fills and empties is essential information for vascular occlusion studies.

The aim of the current study was to determine the length of time necessary for the pulmonary capillaries to reach steady state after a step change in blood flow. We reasoned that, if we knew how rapidly capillaries recruited after a sudden change in flow, we would then have an approximation of the response time of the microcirculation. We used isolated canine lung lobes perfused by two pumps in parallel. To produce step changes in flow, one of the pumps was turned off to halve flow or turned on to double flow. Changes in subpleural pulmonary capillary recruitment were recorded by videomicroscopy to determine how rapidly capillary recruitment reached steady state. These changes, which occur at a maximal distance from the hilum, represent an upper limit approximation of the response for the rest of the pulmonary capillaries and their feeding vessels.

	METHODS

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Animal preparation. In accordance with our institutional guidelines, healthy adult male dogs (20-25 kg, n = 6) were anesthetized with pentobarbital sodium (30-40 mg/kg iv), intubated, and mechanically ventilated with room air with the use of a Harvard 607D animal respirator. After the dogs were given heparin (1,000 U/kg iv), they were rapidly exsanguinated via the left common carotid artery. The first 120 ml of blood lost from each animal was replaced with 120 ml of 10% Dextran 40 (molecular mass of 40 kDa) in saline solution (1). After a left thoracotomy, the left lower lobar pulmonary artery was cannulated with a 6-mm-ID Teflon fluorinated ethylene polypropylene cannula, and the left lower lobe was excised, along with a cuff of left atrium, and placed on a microscope stand. The left atrial cuff was secured around another cannula (9 mm ID), and the lobe was perfused with autologous heparinized whole blood (hematocrit 34-44%). Care was taken to exclude all air bubbles from the circuit before perfusion was initiated. The time interval to reperfusion was <30 min.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Schematic of apparatus used to perfuse left lower lung lobe under conditions of steady blood flow, microvascular pressure, and alveolar inflation pressure. CCD, charge-coupled device. PA, alveolar pressure; Ppa, pulmonary arterial pressure; Ppv, pulmonary venous pressure.

Microcirculatory observations. The subpleural microcirculation under the window was observed with a Leitz Ultropak surface-illuminating microscope (×11 objective) coupled to a 200-W mercury arc lamp. The light was heavily filtered to prevent tissue damage by infrared and ultraviolet light. A narrow band-pass interference filter was used to illuminate the field with the mercury green line (546 nm). This wavelength was absorbed by hemoglobin, thereby increasing the contrast between the erythrocytes and surrounding tissue (12).

Data analysis. While perfusion was recorded, flow was doubled by turning the second pump on for 24 s and then halved by turning the second pump off for 24 s. This 48-s cycle was repeated an average of five times per alveolus. A stopwatch in a time-date generator (WJ-810, Panasonic) was triggered by turning the second pump on or off to record elapsed time to the millisecond on the videotape. The video recordings were replayed, and all perfused capillary segments were traced onto a sheet of transparency film placed over the video monitor. A capillary segment was defined as any capillary between junctions with other vessels or between a junction and the alveolar boundary. Each capillary segment was numbered. The length of the capillaries was measured using a Summagraphics Summasketch III digitizing pad, Sigmascan planimetry software, and an IBM-compatible computer. The areas of the alveolar walls were measured with the same system. The video recordings were analyzed by dividing them into consecutive 4-s observation periods. For each observation period, the perfusion state (perfused or not perfused) was determined for each capillary segment. If one red blood cell passed through that segment during the 4-s period, it was considered to be perfused. The lengths of perfused capillaries were then summed for each 4-s period to give the total length of capillaries perfused during that period. The capillary perfusion index was subsequently calculated, using the following equation

(1)

Because the subpleural alveolar facets in the upper lung can be approximated by flat disks with an average diameter of 110 µm and an area of 10,000 µm², the alveolar wall area was divided by 10,000 µm² to obtain the number of average walls in the observed alveolar facet. This normalization permitted us to compare results between individual alveoli and between animals.

Ensemble averaging. There is variation in capillary perfusion in each alveolus over time, even when pressure and flow are held steady (13). The large capillary recruitment response caused by a large step change in flow was somewhat masked by this variation. The technique of ensemble averaging isolated the true signal from these moment-to-moment variations.

	RESULTS

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Data were obtained from 360 capillary segments in 11 alveoli from 6 canine lung lobes. An example videomicrograph of one flow change in a single alveolus is shown in Fig. 2. The top panel is one frame from the videotape, with a large alveolus centered in the frame. The contrast between the red blood cells and the background is poor, causing the cells to be invisible (12). For purposes of demonstration, we used a computer enhancement technique to show the path of the red blood cells through the pulmonary capillary bed (6). By overlaying the computer-enhanced images of the moving red blood cells collected over 4 s, the path of the red blood cells through the capillary bed is visible (time = 4 to 0 s in Fig. 2B). At time 0, the second pump was turned on and data were collected over the next 4 s. The pathways that the red blood cells followed through the capillary network during that time (0 to +4 s) are shown in Fig. 2C. Capillary recruitment reached steady state during that interval, as demonstrated in Fig. 2D, which shows that perfusion patterns that resulted during +16 to +20 s are essentially the same as those during 0 to +4 s.

View larger version (69K): [in this window] [in a new window]   Fig. 2.   A: 1-frame videomicrograph from 1 video recording. One alveolus, outlined in white, is centered in the frame. B: computer-enhanced image of alveolus in A showing the path red blood cells took across the alveolus with 1 pump running (flow = 284 ml/min). C: computer-enhanced image of alveolus in A showing the path red blood cells took across the alveolus during the 4-s interval immediately after the second pump was turned on (flow = 694 ml/min). D: computer-enhanced image of alveolus in A showing the path red blood cells took across the alveolus during the 4-s interval 16 s after turning on the second pump (flow = 694 ml/min).

Figure 3 shows an example of the data collected from two 48-s on-off pump cycles in the alveolus shown in Fig. 2. The number of perfused capillaries is expressed as a percentage of maximum recruitment. Although it is obvious in Fig. 3 that there is a significant change in recruitment with flow doubling and halving, there is also variation in the pattern of recruitment and derecruitment from one cycle to the next. To compensate for the variation, we used ensemble averaging (Fig. 4), which shows the effect of step changes in blood flow on capillary recruitment in these subpleural alveoli. The response to increased flow is remarkably rapid, having reached steady state in 4 s (Fig. 4). This agrees with observation of the videotapes, which shows that the response is clearly complete in <4 s and nearly complete in 1.5-2 s (a video demonstration can be viewed at http://jap.physiology.org/cgi/content/full/89/3/1233). However, as discussed in detail elsewhere (13), errors are introduced if the measurements are made over periods <4 s. When the second pump was turned off, slightly over 8 s were required for the derecruitment process to reach steady state (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Percent of maximum capillary recruitment for 1 alveolus over two 48-s on-off pump cycles.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Ensemble average of all data showing the effect of step changes in blood flow on capillary recruitment in subpleural alveoli.

Pulmonary arterial and venous pressures and arterial blood-gas values (Table 1) were normal for this kind of preparation. Baseline pulmonary arterial pressures (one pump on) were stable throughout each experiment.

	DISCUSSION

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In this study, we made direct measurements of the temporal response of pulmonary capillary recruitment to step changes in flow. When flow was suddenly doubled, subpleural capillary recruitment reached steady state in <4 s. Observation of the videotape showed that steady state was reached in ~2 s (see video demonstration at http://jap.physiology.org/cgi/content/full/89/3/1233). When flow was halved, capillary recruitment reached steady state in ~8 s.

Several issues need to be considered in interpreting these data. First, the distance from the hilum to the subpleural capillaries in the left lower lobe of these dogs is similar to the distance from the hilum to the periphery of the pigs flown on the NASA aircraft (3). Second, we assume that the subpleural capillaries are an upper limit approximation of how quickly the pulmonary capillary bed as a whole responds to changes in flow. This assumption is based on the fact that the subpleural capillaries are the most distant from the hilum and therefore would be the last to respond to flow changes in the lobar pulmonary artery. The third assumption is that the subpleural capillaries will recruit and derecruit in the same way as capillaries in the rest of the lung. The subpleural capillary network is less dense than interior networks (5, 7, 11) and therefore might not recruit in the same way as interior capillaries. In a study by Short et al. (10), subpleural capillaries were shown to accurately reflect recruitment in interior capillaries, thus giving us assurance that our observations of recruitment patterns are both a reasonable and conservative representation of the response time of capillary recruitment in the lung as a whole.

Steady state was reached rapidly after flow was increased. Considering the time required to stretch the vessel walls in the arterial vascular tree, and the very large number of capillaries available for recruitment, it is remarkable that the response was so rapid. When the second pump was turned off, slightly over 8 s were required for the derecruitment process to reach steady state. Possibly, the slower response to reduced flow came from energy stored in the elastic tissue of the arterial tree, functioning in a similar manner to the maintenance of arterial pressure during diastole.

We considered several different methods for measuring the response time of the microcirculation to flow changes. A first possibility would be to measure the rate of lobar weight gain after a step change in flow. Drake et al. (2) showed that a step change in perfusion pressure produced a rapid increase in lobar weight due to filling of vessels, followed by a slow, steady increase due to filtration of fluid from the vascular space. Another possibility was to place flow probes on the arterial and venous cannulas. The probes would show when blood flow out of the lobe had reached equilibrium with blood flow into the lobe after a step change in flow. We rejected these methods, however, because both measured the response time of the entire lobar circulation, including the veins distal to the microcirculation, rather than the response of the gas-exchanging vessels, which was the measurement directly relevant to the microsphere experiment.

We also considered using labeled red blood cells to make these measurements, a method our laboratory has used in the past to study flow changes after vessel occlusion maneuvers (8). Although, with enough measurements, this approach could have produced the needed data, it presented a number of technical difficulties. First, to produce labeled cells that circulated freely in the appropriate concentration is not trivial. Second, to obtain the needed flow measurements, frame-by-frame red cell velocity measurements were required. The red blood cell velocities at the highest flow rates used in the present study would cause the cells to appear as blurred streaks on each frame of the videotape, thereby making it difficult to track single cells from frame to frame and introducing potential errors into the measurements. High-speed videomicroscopy, which would eliminate the blurring, was impractical due to the low level of light emitted by fluorescently labeled red blood cells. For this reason, an intensified charge-coupled-device camera run at maximum gain was needed to record the images at normal speed (8). Because of these problems with other methods, we used the measurement of capillary recruitment, for it provided the needed time resolution, isolated the response of the gas-exchange vessels, and increased our success rate relative to previous experiments in which labeled red cells were used.

The impetus for this study was to determine whether the 25-s-long exposure to microgravity in the NASA KC-135 aircraft was long enough for the pulmonary microcirculation to reach steady state. Our data show that it is highly probable. We never imagined, however, that a capillary bed, well known for its low resistance and high degree of compliance, would respond in such a way that, when pulmonary blood flow was suddenly doubled, recruitment of the capillaries would reach steady state in <4 s. This finding is additional evidence of the impressive design of the lung: the large reserve of gas-exchange surface area (14), the thin and delicate alveolar-capillary membrane that is also strong (16), the efficient fractal character of the airways and vessels (4, 15), the robust design of the system of capillary perfusion (13), and, as demonstrated in this study, the rapidity of the response of the gas-exchange vessels to increased flow.