INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

PERFUSION OF THE PULMONARY acinus is generally homogeneous (12). There is, however, a significantly heterogeneous component, the cause of which is unknown. Potential causes of acinar perfusion heterogeneity include 1) dispersion of blood within the arterial tree before it enters the acinus (3), 2) multiple inputs to the acinus from supernumerary arteries, the vessels of the pulmonary arterial tree that branch more frequently than the airway tree (6), 3) stratification of perfusion within the acinus (13, 18, 19), and 4) the complex branching structure of the septal capillary network within each alveolar wall that contains pathways of varying length, diameter, and resistance (2, 4, 5, 7, 10, 11, 16, 17). To investigate the contribution of these septal capillaries to acinar perfusion heterogeneity, the passage of fluorescent dye boluses through the subpleural microcirculation of isolated dog lung lobes was videotaped using fluorescence microscopy. As the videotapes were replayed, dye-dilution curves were recorded from each of the tributary branches of Y-shaped venules that drained single acini. For each Y-shaped venule, the mean appearance time difference between the tributary pairs was calculated from the dye curves. Simultaneous dye passage through these branches posited homogeneous acinar perfusion, whereas different passages suggested heterogeneous acinar perfusion (12). With each venule serving as its own control, we increased airway pressure modestly, which derecruited the complex alveolar septal capillaries and directed flow through the relatively less complex corner vessels [the vessels lying in the junctions where at least 3 alveolar walls intersect (1, 8)]. Under these conditions, we hypothesized that acinar perfusion heterogeneity would decrease because perfusion of the complex septal capillary networks would be reduced.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal preparation. These experiments were approved by the Animal Care Committee of the Indiana University School of Medicine. Healthy adult male mongrel dogs (21-27 kg, n = 9) were anesthetized by pentobarbital sodium (30-40 mg/kg iv), intubated, and mechanically ventilated with room air via a constant-volume respirator. After heparinization (1,000 U/kg), the animals were rapidly exsanguinated through a cannula (3-mm ID) placed in the left common carotid artery. With the lungs inflated to a constant airway pressure of 5 mmHg, a left thoracotomy was performed, and the left upper lobe was excised to provide access to the left lower lobe. The left lower lobar artery was cannulated with a Teflon fluorinated ethylene polypropylene cannula (6-mm ID), and the left lower lobe bronchus was clamped to maintain constant inflation. The left lower lobe was then excised along with a cuff of left atrium and placed on a microscope stand. The left atrial cuff was secured around another Teflon fluorinated ethylene polypropylene cannula (10-mm ID), and the lobe was perfused with autologous heparinized whole blood (hematocrit 32-43%). The time between complete exsanguination and reperfusion of the lobe was <30 min. Blood was pumped through a windkessel to dampen pump vibrations and trap bubbles, a 20-µm-pore filter to remove microaggregates, a heat exchanger to warm the blood to 37-38°C, and finally a dye injection loop before entering the lobe (Fig. 1). Venous blood drained passively from the lobe into a reservoir. The height of the tubing between the vein and the reservoir could be raised or lowered to change venous pressure. The lobe was ventilated with 6% CO₂-17% O₂-77% N₂ at a tidal volume of 100 ml. End-expiratory pressure was set to 5 mmHg by a water overflow on the expiratory limb of the ventilator. Arterial and venous pressures were measured continuously with two transducers (model P23 XL, Statham) zeroed at the level of the microcirculatory observations and connected to polyethylene (PE-200) tubing, the tips of which were located at the ends of the arterial and venous cannulas. Airway pressure was measured intermittently (Statham P23 XL transducer).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Schematic of the experimental setup. PA, alveolar pressure; Ppa and Ppv, pulmonary arterial and venous pressure, respectively; ICCD, intensified charge-coupled device.

Data collection. The lobe was suspended by two small spring-backed paper clips attached to opposite edges of the lobe (18) and raised until the uppermost pleural surface (the diaphragmatic surface in this orientation) came into contact with a transparent window. A 1.3-cm² area on the surface of the lobe was observed through the window, which was surrounded by a vacuum ring to prevent lateral movement (14, 15). The subpleural microcirculation under the window was observed with a modified Olympus BH2 reflectance microscope coupled to a Leitz Ultropak illuminator and a ×11 objective. Bright-field illumination through the Ultropak illuminator was provided by a 200-W mercury arc lamp mounted on an optical bench. This light source was heavily filtered with a combination of dichroic infrared-reflecting filters, broad band-pass ultraviolet-absorbing filters to prevent tissue damage, and a narrow band-pass interference filter to illuminate the field only with the mercury green line (546 nm). This wavelength was absorbed by Hb, thereby increasing the contrast between the erythrocytes and surrounding tissue. Illumination for fluorescence microscopy was provided by a 100-W mercury arc mounted on the sidearm of the Olympus microscope. This light was also filtered by dichroic infrared-reflecting filters and ultraviolet-absorbing filters. The light from this arc passed through a blue band-pass exciter filter (410-480 nm) and a high-pass dichroic mirror (cutoff wavelength 480 nm) that reflected the exciting light down through the objective onto the subpleural microcirculation beneath the window. Emitted light from the lung passed back through the objective, the dichroic mirror, and a yellow high-pass barrier filter (cutoff wavelength 510 nm). Video recordings of the subpleural microcirculation were made with a video recorder (model AU650 MII, Panasonic) and a Cohu (model 5510) intensified charge-coupled device camera that was attached to the microscope with a zoom adapter.

(1)

(2)

Measurement of capillary recruitment. In 21 alveolar walls (9 lobes), the level of septal capillary recruitment was determined at airway pressures of 5 and 15 mmHg by recording the perfusion pattern of 1-3 alveoli in the field for 1 min. The videotapes from each 1-min observation period were replayed, and the perfused capillary segments were traced onto separate sheets of clear acetate placed over the video monitor. A capillary segment was considered to be perfused if one or more erythrocytes passed through the segment during the 1-min observation period. The length of the perfused capillaries was measured from the tracings with a digitizing pad, planimetry software, and a microcomputer. The area of the observed alveolar walls was measured with the same system. Because subpleural alveolar facets in the isolated lobe at an airway pressure of 5 mmHg can be approximated by flat disks with an average area of 8,000 µm², the alveolar wall area measured at 5 mmHg was divided by 8,000 µm² to obtain the number of average-sized alveolar walls in the observed alveolar facets. Because the alveolar diameters increased at an inflation pressure of 15 mmHg, the measured wall area was divided by the average wall area of 10,000 µm², the average area at this inflation pressure. This normalization permitted us to compare results between individual alveoli, between animals, and between treatments. Dividing the total length of perfused capillaries by the normalized alveolar area indicated how many times perfused capillaries crossed an average alveolar wall at its diameter. Defined mathematically, the capillary perfusion index (CPI) is

(3)

The level of capillary recruitment can be readily estimated from the CPI.

Statistics. Measurements of cardiorespiratory variables, MT, and the CPI at low and high airway pressure conditions were compared with each other by use of paired two-tailed t-tests. Blood gases at the beginning and at the end of the study were also tested for differences with a paired two-tailed t-test. We accepted P < 0.05 as significant for all statistical tests.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

An example of the passage of fluorescein dye through a venular pair is shown in Fig. 2. In Fig. 2, top, the base of the Y-shaped venule is not visible because it flows directly down into the lung. Passage of dye 8.98 s after injection is shown in Fig. 2, middle. At that time the dye bolus was at the peak of its brightness during its passage through the faster branch. No dye, however, had arrived in the slower branch. In Fig. 2, bottom, 12.36 s after injection, the fast branch was nearly depleted of dye, and the slower branch was now filled with dye, at the peak of its brightness. The discrepancy between appearance times through these small venules draining adjoining neighborhoods can be significant. The dye curves from these branches are shown in Fig. 3. MT was 15.2% as calculated from Eq. 2.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Passage of fluorescein dye through a venular pair. Top: direction of flow is indicated by arrows; the base of the Y-shaped venule is not visible, because it flows directly down into the lung. Middle: passage of dye 8.98 s after injection, when the dye bolus was at the peak of its brightness during its passage through the faster branch; no dye had arrived in the slower branch. Bottom: 12.36 s after injection, the faster branch was nearly depleted of dye, and the slower branch was then filled with dye, at the peak of its brightness. Discrepancy between appearance times in these small venules draining adjoining neighborhoods can be significant. To the extent that there was uniform appearance of dye in the arteriolar branches feeding the acinus, the venular appearance time differences reflect acinar capillary transit time differences and, thus, uneven acinar blood flow with respect to acinar blood volume.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Dye curves from branches in venule shown in Fig. 2. Difference in mean appearance times (MT) was 15.2% calculated from Eq. 2.

When airway pressure was increased from 5 to 15 mmHg, two changes were observed in the pulmonary microcirculation of the nine animals studied. Perfusion heterogeneity between the venular tributaries decreased in 14 of 15 pairs and increased in 1 pair. On average, MT decreased by 60% (P < 0.01) when airway pressure was increased (Fig. 4, Table 1). Of the 21 alveoli studied in the same 9 animals, when airway pressure was increased, the CPI decreased in 18 alveoli, did not change in 2 alveoli, and increased in the remaining alveolus. On average, the level of capillary recruitment decreased by 70% (P < 0.01) with higher airway pressure (Fig. 5, Table 1).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   As septal capillaries were derecruited when airway pressure was increased, venular perfusion heterogeneity decreased in 14 of 15 venular branch pairs, as shown by a 60% reduction in MT between branches of the Y-shaped venules.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Level of capillary recruitment, as measured by capillary perfusion index (CPI), decreased when airway pressure was increased from 5 to 15 mmHg.

Although pulmonary arterial pressure increased significantly when airway pressure was raised, the gradient between pulmonary arterial pressure and airway pressure was unchanged (Table 2). Pulmonary venous pressure was also held constant (Table 2). Blood gases did not change over the course of the study (Table 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We investigated the contribution of alveolar septal capillaries to intra-acinar perfusion heterogeneity by measuring appearance time differences in the tributary arms of small Y-shaped venules that drained a single acinus. When the complex septal capillary networks were derecruited by high positive airway pressure, venular perfusion became proportionally more homogeneous.

These results show a strong correlation, but the correlation does not establish cause and effect. Of the 15 venular pairs observed, 14 became more homogeneous when airway pressure was increased. In the odd case that became more heterogeneous with high airway pressure, one of the alveoli in the neighborhood of that venule had more septal capillary recruitment. Of course, each venule drains many alveolar walls, making the association limited. Nevertheless, it is intriguing that the only venular pair that behaved in the opposite manner from the others was associated with an alveolar recruitment pattern that was also opposite from other alveoli in this study. These associations are consistent and suggest that septal capillary pathlength and resistance differences are important contributors to intra-acinar perfusion heterogeneity.

We have considered several issues in reaching these conclusions. First, we assumed that the tributary branches of each Y-shaped venule drained the same acinus. Previously, we tested this assumption by comparing MT of the largest venules with MT the smallest venules on the surface of the lung (12), reasoning that the largest venules would be more likely to drain more than one acinus and would therefore be more likely to have dissimilar transit times. However, MT of the largest branches was not different from that of the smallest branches. This result implied that all venular branches in this size range drain a single acinus. Even if the branches did drain more than one acinus, the decreased heterogeneity of perfusion still resulted from derecruitment of septal capillaries.

We also assumed that the results obtained from the isolated lobes are similar to results from intact animals. In pilot studies of intact dogs, MT between venular branches was 0-5% in ~40% of venular branch pairs and 5-10% in ~35% of pairs (n = 20 pairs), results similar to the isolated lobe data previously reported (12). Although MT values reported here were greater, because the venular branch pairs were selected for their heterogeneity to provide a clear test of the hypothesis, it still seems reasonable that these results would apply to intact animals.

Lamm et al. (8) previously showed that septal capillary blood flow is diverted through corner vessels when airway pressure is increased. Our results indicate that the pathways of perfusion through corner vessels are more spatially homogeneous than the pathways through septal capillaries. Increasing airway pressure, however, may have distended corner vessels. Permutt et al. (9) showed that extra-alveolar vessels (which likely include the corner vessels in our study) increase in volume when the lung is expanded by increased airway pressure. If increasing airway pressure did distend corner vessels, that effect may have contributed to the increased homogeneity of acinar perfusion; however, we have no evidence from this study bearing either way on that possibility.

Even when septal capillaries were significantly derecruited by high airway pressure, there remained some heterogeneous venular appearance times. The remaining heterogeneity could have resulted from transit time differences in arteriolar inlet flow (3), which would have been unaltered by septal capillary derecruitment. Another possibility comes from the incomplete septal derecruitment at an airway pressure of 15 mmHg. Although a CPI of 89 µm at high airway pressure is a low value, i.e., a single perfused capillary pathway would not cross an average alveolar wall at its 100-µm diameter, some septal capillary perfusion remained. If we assume a linear relationship between septal capillary recruitment and perfusion heterogeneity, then we can speculate on the effect of complete capillary derecruitment. To estimate the effect of complete septal capillary derecruitment, we plotted CPI against perfusion heterogeneity (Fig. 6). When the relationship was extrapolated to zero CPI (no septal capillary perfusion), the y-axis was intercepted at 1-2%. If this extrapolation is valid, it suggests a number of interesting possibilities. First, it implies that septal capillaries account for nearly all acinar perfusion heterogeneity. Furthermore, it suggests that corner vessels are homogeneously perfused. Finally, the extrapolation suggests that whatever heterogeneity exists in arteriolar flow has little effect on acinar perfusion homogeneity, even under these conditions where observations are made on the surface of the lung, a location providing a maximal arterial length for dispersion to occur.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   When capillary recruitment was plotted against perfusion heterogeneity and the line was extrapolated to the y-axis, the intercept occurs between 1 and 2%, suggesting that septal capillaries accounted for nearly all acinar perfusion heterogeneity.