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Influence of vascular and transpulmonary pressures on the functional morphology of the pulmonary microcirculation
MICROVASCULARRESEARCH20, 295-306 (1980)
Influence of Vascular and Transpulmonary Pressures on the Functional Morphology of the Pulmonary Microcirculation ROBERT W . MAZZONE
Department of Medicine, M-023, University of Cal~)rnia, San Diego, La Jolla, Califi~rnia 92093 Received August 28, 1979 The effects of airway and vascular pressures on pulmonary capillary morphology were examined by rapidly freezing in situ dog lungs under Zone 1, 2, and 3 conditions at transpulmonary pressures (ew) of 5, 15, and 25 cm HzO. The tissue was preserved using a freeze substitution technique and analyzed by transmission electron microscopy. Capillary and septal wall widths were measured on random sections, and an estimate of the thickness of the blood:gas barrier was made. In addition, the depths of any folds penetrating into the capillary lumen were also measured. Under Zone 2 and 3 conditions, increasing the perfusing pressure increased the mean capillary width, but at higher Ptp, this effect was reduced. Mean fold depth was reduced as etp was increased under both Zone 2 and 3 conditions. At l o w P w (5 cm H20), mean fold depth was unresponsive to changes in perfusing pressure, but decreased significantly as perfusing pressure increased when the Ptp was 15 or 25 cm HzO. Wall thickness was unaffected by change in Pw. Under Zone 2 conditions, wall thickness was independent of perfusing pressure. However, under Zone 3 conditions, at etp of 15 and 25 cm H20, there was a significant decrease in wall thickness as perfusing pressure was increased. Presumably, this decrease in wall thickness is due to distention of pulmonary capillaries at medium and high inflation pressures.
INTRODUCTION The functional morphology of the pulmonary microcirculation is exquisitely sensitive to the relationship between airway and vascular pressures (Glazier et al., 1969). Using vascular perfusion fixation techniques, Gil and Weibel (1972) showed that, in spite of a constant perfusion pressure, the alveolar septum folds and unfolds much like an accordion in response to differing airway pressures. Assimacopoulos et al. (1976) extended these observations in their study of the influence of lung volume on alveolar-capillary configuration and showed that folding could influence capillary morphology. However, since the morphology of the pulmonary capillaries is dependent upon a variety of physiologic conditions, microscopic analysis of pulmonary structure-function relationships requires that the lung be fixed under precisely defined physiologic conditions. With airway and vascular fixation techniques, sone doubt will always exist as to the exact relationship between airway and vascular pressures at the level of the pulmonary capillaries (Mazzone et al., 1978; 1979). This problem can be circumvented to a great degree by stabilizing lung structure under specific physiologic conditions through rapid freezing. By combining the isolated, perfused, in situ lung preparation (West et al., 1964) 295 0026-2862/80/060295-12502.00/0 Copyright © 1980 by Academic Press, Inc. All rightsof reproductionin any form reserved. Printed in U.S.A.
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with the rapid freeze procedure (Staub and Storey, 1962), Glazier et al. (1969) were able to conduct a light microscopic analysis of the relationship between various combinations of vascular and alveolar pressures on pulmonary blood flow. This study, however, was limited by the limited resolution of the light microscope which did not allow visualization of the finer details of pulmonary capillary morphology such as capillary folds. Since the rib cage is removed in this preparation, transpulmonary pressure is constant throughout the lung. The vascular pressure, on the other hand, is affected by the hydrostatic column. If the lung is rapidly frozen in a vertical position, the functional relationship between perfusion pressure and transpulmonary pressure can be preserved under precisely defined conditions. In this study, the methods of Glazier et al. (1969) have been combined with a new procedure described recently by this laboratory (Mazzone et al., 1978). In this manner, the morphology of the pulmonary capillaries fixed under controlled physiologic conditions has been examined by transmission electron microscopy. METHODS The lungs of 21 mongrel dogs (20-30 kg) were ventilated and perfused in situ according to the procedure of West et al. (1964) as modified by Warrell et al. (1971). Briefly, dogs were anesthetized by intravenous injection of phenobarbitol sodium (30 mg/kg) and a tracheostomy was performed. The pleura was exposed by left-side thoracotomy. Heparin (2000 IU/kg) was administered and after 3 min the animal was bled (0.6-1 liters). The pleura was punctured, the heart fibrillated, and the rib cage rapidly removed. The aorta was tied off and a cannula was introduced through the right ventricle into the pulmonary artery. Blood returning from the lungs was collected by a cannula placed in the left atrial appendage. Total surgical time was 8 to 12 min. The perfusion circuit consisted of a Sarns roller pump, a bubble trap and heat exchanger, and a venous reservoir. Pulmonary artery (Part) and venous pressures (Pven) were measured with saline manometers. Pulmonary artery pressure was controlled by regulating the speed of the pump, while pulmonary venous pressure was regulated by raising or lowering the venous reservoir. All experiments were performed with the dogs suspended in the vertical position. The lungs were ventilated with a Harvard respirator supplied with a mixture of 14% 02, 6% CO.,, balance N2 at an end-expiratory pressure of 3-5 cm H20. This gas mixture has been shown to yield normal blood:gas values (Warrell et al., 1971). Airway pressure was measured by a saline manometer connected to the tracheostomy. The lungs were rapidly frozen with liquid Freon 22 cooled to - 155° with liquid Nz. Approximately 2 liters of Freon 22 were poured into a funnel connected to a brass tube which had fine holes approximately 5 mm apart. The Freon was sprayed onto the lung surface for approximately 35 to 45 sec. Following this, the lung was rapidly removed and placed into liquid N2. Perfusion time prior to freezing was never more than 15 min. Physiologic conditions. Lungs were frozen under conditions of Zone 1 (Pa~v > Part > Pven), Zone 2 (Part > Palv > Pven), and Zone 3 (Part > Pven > Pair). Transpulmonary pressures were set at 5, 15, or 25 cm H20. All lungs were frozen
MORPHOLOGY OF P U L M O N A R Y CAPILLARIES
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on the deflation limb of the pressure-volume curve (Gil and Weibel, 1972; Pain and West, 1966). Processing of frozen tissue. Samples of frozen lung approximately 0.5 cm in diameter and 2 mm in thickness were obtained at known vertical distances referenced to either the Zone 1-2 or the Zone 2-3junctions. Each sample of tissue was fractured into quarters while in liquid N2, and these were placed in a solution of 70% ethylene glycol containing 4% glutaraldehyde and 2% paraformaldehyde at - 8 0 ° . The solution and samples were then allowed to warm to - 5 0 ° and were kept at this temperature for 24 hr. The details of the procedure for processing the frozen lung for TEM has been described previously (Mazzone et al., 1978; 1979) and will only be mentioned briefly. After the substitution and fixation period, the samples were thawed to +4 ° and placed in Hanks' salt solution (Grand Island Biological) containing 4% glutaraldehyde and 2% paraformaldehyde (ph 7.4, and 340 mOsm). After 1 hr the tissue was washed with Hanks' and treated with 2% osmium tetroxide for 2 hr in the dark. Samples were then washed with maleate buffer and block-stained with 1.5% uranyl acetate for 90 min in the dark. The tissue was dehydrated in acetone and infiltrated with Spurr low-viscosity embedding media (Polysciences). Samples were then embedded and cured for 48 hr at +60-80 °. Sections, 750-1000/~ in thickness were obtained using a diamond knife (Dupont Ind.) and an LKB ultratome 111. These sections were mounted on 200-mesh copper grids, poststained with aqueous uranyl acetate and Reynolds lead citrate, and examined on a Zeiss EM9 transmission electron microscope. Histological measurements. The sampling procedure used was based on a multi-tier sampling procedure (Weibel and Knight, 1964). Approximately four to six blocks of tissue were prepared for each sample of frozen lung. Two blocks from each group were randomly chosen for analysis. These were sectioned, mounted, and stained as described above. Five microscopic fields from each section were chosen randomly and photographed at a primary magnification of × 1500. In the event lung tissue was not seen in the field, it was discarded and a new area chosen randomly. The dimensions of the capillary width and septal width were measured at 5-~m intervals perpendicular to the alveolar septum. No measurements were made within 10/.~m of septal junctions so that corner vessels were excluded from the analysis. Septa directly attached to the pleura, large blood vessels, or airways were also not analyzed. In addition, measurements of capillary width did not include the thickness of the capillary endothelium. The depth of any folds penetrating into the capillary lumen was also measured in the micrographs. This was done by drawing a line connecting the alveolar epithelium on both sides of the folds and then measuring the distance from this line to the inner edge of the capillary endothelium. Fold depth was measured only if the depth of the fold exceeded the width of the fold at the epithelial surface. If graphical analysis suggested linear relationships for any of the data, a leastsquares linear regression was performed. Several statistical tests were performed on the data. First, a test for coincidental regression was conducted on each set of data (Zar, 1974). Second, the linear regressions were also tested for parallelism (Goldstein, 1964). If this test failed to show significant difference in the slopes, the y-intercepts were then tested for significance (Zar, 1979). These tests were per-
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formed on data obtained under each zonal condition. In addition, Zone 2 data were compared to Zone 3 data to determine statistical significance between zonal conditions. Finally, the slopes of the regression lines were tested to determine if they were significantly different from zero, using Student's t test. RESULTS In all figures, the measurements have been plotted as a function of known vertical distance down the lung relative to either the Zone 1-2 or Zone 2-3 junction. In these experiments, the perfusion pressures were always adjusted so that the Zone 1-2 or Zone 2-3 junctions were maintained at the same height relative to the top of the lung for all three transpulmonary pressures. Hence, at a given distance down the lung, the capillary transmural pressure; that is, the difference between alveolar and capillary pressures, will be approximately the same at that point for all three transpulmonary pressures used. Thus, any differences seen in lungs frozen at various transpulmonary pressures will be due primarily to changes in transpulmonary pressure. This will be particularly true for those lungs frozen under Zone 3 conditions. Since pulmonary vascular pressures increase down the lung due to the hydrostatic column, moving down the distance axis is equivalent to increasing perfusing pressure. When lungs were rapidly frozen under Zone 3 conditions, the arterial-venous pressure difference was always 2 to 4 cm H20. Since blood flow under Zone 3 conditions is governed by the arterial-venous pressure difference, capillary pressure at any given distance down the lung may be estimated within 1-2 cm HzO (Glazier et al., 1969). Thus, the distance axis is, under these conditions, essentially equivalent to the capillary perfusing pressure in centimeters of
H20. Under Zone 2 conditions, the distance axis is not equivalent to the exact perfusing pressure. Since changes in the transpulmonary pressure may alter the pulmonary vascular resistance, the exact capillary pressure at a given distance down the lung cannot be known with assurance. However, plotting the measurements as a function of distance down the lung is still a useful technique since the capillary pressure will be increasing with distance down the lung due to hydrostatic effects. Capillary width. Figure 1 shows the mean capillary width (_ 1 SEM) plotted as a function of distance down the lung. Note that the distance axis has been divided to show data obtained under both Zone 1 and Zone 2 conditions. Linear regressions have been fitted using a least-squares technique. The tests for coincidental regression confirm (F (4,44) = 7.49, P < 0.05) that the data are not adequately described by a single regression line. Under Zone 1 conditions (Pa~v > Part), capillary width was not zero. This was due to the presence of red blood cells and plasma presumably trapped in the capillaries. Other investigators have shown that capillary volume never reaches zero, regardless of how high the alveolar pressure is raised above the arterial pressure (Dean et al., 1970; Staub, 1966; Vreim and Staub, 1974). Vreim and Staub (1974) and Staub (1966) have demonstrated histologically that this was because of trapped red cells and plasma. When arterial pressure increased above alveolar pressure, the mean capillary
299
MORPHOLOGY OF PULMONARY CAPILLARIES
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FIG. 1. Mean capillary width (_+ 1 SEM) plotted as a function of distance down the lung for Ptp of 5, 15, and 25 cm H20. Note that the distance axis has been divided so that data obtained above and below the Zone l-2junction can be shown on the same graph. The data have been fitted using a least-squares linear regression. (M = slope, r = correlation coefficient.)
width appeared to increase linearly, regardless of the transpulmonary pressure at which the lungs were frozen. Increasing the transpulmonary pressure served to reduce the slopes of the lines relating mean capillary width and distance down the lung. The slope of the regression line forPtp = 5 cm HzO was significantly different (t = 3.37, df= 31 , P < 0.05) than that forPto = 15 cm HzO orPtp = 25 cm H20 (t = 3.59, df = 29, P < 0.05). However, no significant difference could be found in the slope of the lines f o r P w = 15 cm H20 compared toPto = 25 cm H20 (t = 0.8419), although the y-intercepts were significantly different (t = 2.69; df = 32). In addition, all three regression lines had slopes which were significantly different from zero (Ptp = 5, 15, 25 cm H~O; t = 8.34, 3.3, 9.4, df = 13, 17, 14; P < 0.05). Figure 2 shows mean capillary width (_+ 1 SEM) plotted as a function of distance • 5
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FIG. 2. Mean capillary width (_ 1 SEM) plotted as a function of distance down the lung for Zone 3 preparations at Ptp of 5, 15, and 25 cm HzO. The data have been fitted using a least-squares linear regression over the range of 0 to 30 cm down the lung. Note that past this point the mean capillary width tends toward a plateau at all three Ptp- (M = slope, r = correlation coefficient.)
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ROBERT W. MAZZONE
down the lung for dog lungs frozen under Zone 3 conditions. As distance down the lung increased, the mean capillary width also increased. When transpulmonary pressure was 5 cm H20, mean capillary width reached a maximum of 6 . 5 - 7 / z m approximately 35 cm down the lung. Increases in the transpulmonary pressure caused a decrease in the maximum mean capillary width as well as a decrease in the slopes of the regression lines. The regression lines shown in Fig. 2 were calculated over the range of 0 to 30 cm down the zone. The data were not adequately described by a single regression line (F (4,31) = 21.2, P < 0.05). Analysis of the slopes showed that all three were significantly different from z e r o ( P t p = 5, 15, 25 cm H20; t -- 5.48, 3.19, 2.87; df= 10, 13, 8; P < 0.05). The slope of the regression line for Ptp = 5 c m H20 was significantly different from that for Ptp = 15 cm H20 (t = 3.15; df = 25; P < 0.05) and also from Ptp = 25 c m H 2 0 (t = 2.42; df = 20; P < 0.05). No significant difference could be demonstrated between the slopes of the regression lines for P w = 15 and 25 cm H20 (t = 0.3535). In addition, no significant differences in the elevations of the two lines could be detected. A comparison of mean capillary width under Zone 1-2 and Zone 3 was fitted by separate regression lines ( P t p = 5, 15, 25 cm H20; F = 36.5, 4.27, 44.23; df = 2,33, 2,30, 2,22; P < 0.05). For each Ptp under Zone 2 and 3 conditions, no significant differences in the slopes of the lines could be found (Ptp = 5, 15, 25 cm H20; t = 0.547, 0.4545, 1.296; df = 25, 31,34), although there were significant differences in the elevations (Pro = 5, 15, 25 c m HzO; t = 4.07, 3.1, 3.84; df = 24, 31, 23; P < 0.05). Fold depth. Figure 3 shows the mean fold depth (___1 SEM) plotted as a function of distance down the lung for the Zone 1-2 preparations while the mean fold depth obtained from Zone 3 preparations is shown in Fig. 4. Note the striking similarities between these sets of data. For Zone 2 conditions (Fig. 3) the data have been fitted by separate regression lines (F (4,40) = 76.8, P < 0.05). The slope of the regression line for P w = 5 cm H20 was significantly different from that for P t p = 15 and 25 cm H20 (t = 2.38, 2.69; df = 17, 14; P < 0.05). No significant difference could be found between the
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FIG. 3. Mean fold depth (+- 1 SEM) plotted as a function of distance down the lung for Zone 1-2 preparations. The slope of the linear regression at a Pip of 5 cm n z o was not significant, while the slopes of the regression lines at a Ptp of 15 and 25 cm H20 were significant (P < 0.05). (M = slope, r = correlation coefficient.)
301
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FIG. 4. Mean fold (_+1 SEM) plotted as a function of distance down the lung for the Zone 3 preparations. As in Fig. 3, the slope of the regression line at aPtp of 5 cm HzO was not significant, while the slopes of the regression lines for/gtp of 15 and 25 cm H~O were significant (P < 0.05). (M = slope, r = correlation coefficient.) slopes for Ptp = 15 and Pto = 25 cm H 2 0 (t = 0.455; df= 30), although there was a significant difference in the elevation of the two lines (t = 2.78; df = 29; P < 0.05). Finally, only the slopes of the regression lines for Pro = 15 and 25 cm H~O were significantly different from zero (t = 2.38, 2.69; df = 17, 14; P < 0.05). The fold depth data obtained under Zone 3 conditions (Fig. 4) have also been fitted by separate regression lines (F (4,41) = 116.3, P < 0.05). The slopes of the regression lines for Ptp = 5 c o m p a r e d to 15 cm H20 and 5 c o m p a r e d to 25 cm H 2 0 were significantly different (t = 3.19, 2.64; df = 23, 18; P < 0.05). H o w e v e r , no significant difference was o b s e r v e d b e t w e e n the slopes of the lines for Ptp = 15 c o m p a r e d to 25 cm H 2 0 (t = 0.538; df = 23). The elevation of these two lines were significantly different (t = 2.056; df = 31; P < 0.05). In addition, the slopes of the regression lines for Ptp = 15 and 25 cm H 2 0 were significantly different from zero (Pw = 15, 25 cm H20; t = 4.62, 4.6; df = 18, 12; P < 0.05). This was not true for etp = 5 cm HzO (t = 1.32; df = 11). A comparison of Zone 1-2 versus Zone 3 data failed to show any significant difference in the slopes of the regression lines for any given Ptp ( P t p = 5, 15, 25 cm HzO; t = 0.116, 1.445, 0.3835; df = 22, 32, 2 I). H o w e v e r , for P t p = 15 and 25 cm H20, the elevations were significantly higher under Zone 3 conditions (Ptp = 15, 25 cm H20; t = 2.38, 2.75; df = 36, 24; P < 0.05). This was not true for Pro = 5 cm HzO (t = 1.678; df = 24). Presumably, the increased fold depth o b s e r v e d in the Zone 3 preparations is related to capillary recruitment and distension. U n d e r Zone 2 conditions, some capillaries were not actively perfused (Warcell et al., 1971) and hence their size is less than under Zone 3 conditions. F r o m the data, it would appear that this results in a slightly smaller fold depth. More importantly, h o w e v e r , under both Zone 2 and Zone 3 conditions, transpulmonary pressure and perfusing pressure have similar effects upon capillary fold depth. The statistical analysis therefore shows that increasing transpulmonary pressure caused a marked reduction in fold depth. F u r t h e r m o r e , fold depth is only responsive to changes in hydrostatic pressure at Ptp = 15 or 25 cm HzO.
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ROBERT W. MAZZONE
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FIG. 5. Wall thickness, defined as the difference between mean septal width and mean capillary width, plotted as a function of distance down Zone 2. Transpulmonary pressure had no significant influence on wall thickness. In addition, the slopes of all these regression lines were not significant. (M = slope, r = correlation coefficient.)
Wall thickness. An indication of the thickness of the barrier to gas exchange was obtained by subtracting the mean capillary width from the mean septal wall width. Figure 5 shows these data, defined as the wall thickness, plotted as a function of distance down the lung for the Zone 2 preparations, while Fig. 6 shows the data for the Zone 3 preparations. As with the fold depth data, there is some similarity between these two sets of results. Under both Zone 2 and Zone 3 conditions, increasing the transpulmonary pressure had no significant effect upon wall thickBess. There is one striking difference between data shown in Figs. 5 and 6. Under Zone 2 conditions (Fig. 5), the slopes of the regression lines for wall thickness versus distance for each transpulmonary pressure were not significantly different from zero (Pw = 5, 15, 25 cm H20; t = 0.12, 1.59; 0.018; df = 10, 13, 11). This implies that increasing the perfusing pressure did not significantly alter the wall thickness, regardless of the transpulmonary pressure, at least under Zone 2
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FIG. 6. Wall thickness plotted as a function of distance down Zone 3. As with the Zone 2 preparations, transpulmonary pressure had no significant influence on wall thickness. The slope of the regression line for Pip of 5 cm H20 was not significant. However, at Ptp of 15 and 25 cm H20, wall thickness decreased significantly (P < 0.05) with increasing distance down the lung, (M = slope, r = correlation coefficient.)
MORPHOLOGY OF P U L M O N A R Y CAPILLARIES
303
conditions. However, the wall thickness obtained from the Zone 3 preparations shows a different trend. At transpulmonary pressures of either 15 or 25 cm H20, wall thickness decreased significantly (Ptp = 15, 25 cm H20; t = 3.23, 2.87; d f = 17, 12; P < 0.05) as perfusion pressure increased. However, at a transpulmonary pressure of 5 cm H20, wall thickness did not decrease significantly (t = 1.8; d f = 11) as perfusing pressure increased. Thus, it would appear that under Zone 3 conditions at high inflation pressures, increasing the perfusion pressure may reduce the barrier to gas exchange. DISCUSSION Limitation o f Techniques
DeFouw and Berendsen (1978) have performed a stereological analysis of isolated, airway-fixed dog lungs following perfusion for 30, 60, and 120 min. Their results showed progressive morphological changes. However, in this study, the in situ lung preparations were ventilated and perfused for less than 15 min and then rapidly frozen under a defined set of physiologic conditions. It should be pointed out that DeFouw and Berendsen (1978) removed the lungs completely from the animal so that lymphatic drainage might have been altered. This is probably not the case in lungs ventilated and perfused in situ (Warrell et al., 1971). The freezing techniques used in this study provide extremely good preservation to a depth of about 2 mm below the pleural surface (Mazzone et al., 1978; 1979). Glazier et al. (1969) analyzed capillary morphology by light microscopy of frozen lung. Results obtained 1 and 4 mm below the pleural surface agreed within 5%. They concluded that as long as alveolar septa directly attached to the pleura were excluded from the measurements, analysis of lung tissue 1 to 2 mm below the pleura was indicative of events occurring in the more central regions of the lung. Capillary Caliber
The results presented here support the idea that as transpulmonary pressures increase, narrowing of the capillaries occurs. Glazier and colleagues (1969) performed light microscopy on rapidly frozen dog lungs. Their results showed that the number of red cells along the alveolar septum was markedly reduced when the transpulmonary pressure was increased from 10 to 25 cm H20. Assimacopoulos et al. (1976) examined lung fixed by vascular perfusion at several inflation pressures. Their morphological findings showed that the capillary surface to volume ratio, which is an inverse estimate of capillary width, increased slightly from 0 to 15 cm H20 airway pressure, then showed a very abrupt rise as airway pressure was increased to 20 cm H20. Additionally, Forrest (1976; 1978) examined rat lungs fixed by airway instillation at different transpulmonary pressures and found that as inflation pressure was increased from a collapsed state, capillary volume (Ve) initially increased. However, as the transpulmonary pressure increased further, there was a striking decrease in Ve. All these results would imply that capillaries narrow as inflation pressures increase. The results of this study suggest that increasing the transpulmonary pressure will cause a significant increase in pulmonary vascular resistance. For example, under Zone 3 conditions, as transpulmonary pressure is increased to 25 cm H20, the mean capillary width is reduced by about 50%, 30 cm down the zone. Certainly
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ROBERT W . M A Z Z O N E
this would have a significant influence on the vascular resistance of the pulmonary capillaries. Furthermore, increasing the perfusion pressure at these higher transpulmonary pressures probably will not alter the caliber of these vessels significantly. Other investigators have reported a relationship between pulmonary vascular resistance and transpulmonary pressure. For example, Ross et al. (1961) and Thomas et al. (1961) were the first to report that as either transpulmonary pressure or lung volume increased, pulmonary vascular resistance first decreased, then increased. This observation was confirmed by Dawson et al. (1977). West (1969) has proposed that this relationship is due to stretching of the alveolar wall with subsequent narrowing of the pulmonary capillaries. Calculation of the pressure drop across various portions of the pulmonary vasculature supports this idea since experimental results indicate that the larger fraction of the total pressure drop at high lung volumes is due to the pressure drop across the alveolar vessels (Grimm et al., 1977). The statistical analysis suggests that there are differences between the regression upon whether the lungs are frozen under Zone 2 or Zone 3 conditions, but that the effect of increases in perfusing pressure are essentially identical, regardless of whether or not the lungs are perfused under Zone 2 or Zone 3 conditions. The results suggest that perfusing lungs under different zonal conditions changes the intercept of the regression lines, and that the increase in capillary width as perfusion pressure changes is unaffected by whether or not the lungs are in Zone 2 or Zone 3. Fold Depth
Gil and Weibel (1972) demonstrated that alveolar surface changes accompanying lung volume changes were the result of folding and unfolding the septa. Furthermore, Assimacopoulos et al. (1976) found that the number and numerical density of capillary folds decreased as the lungs were inflated. It was argued that the capillary bed became more rectilinear with an increasing degree of alveolar expansion, presumably due to the unfolding of the alveolar wall. It should be mentioned that the folds observed by these authors were really pleating of the alveolar wall and did not necessarily penetrate the capillary lumen. The results shown in Fig. 3 extend the observations of Gil and Weibel (1972), Untersee et al. (1972), and Assimacopoulos et al. (1976). It is apparent from the statistical analysis that as transpulmonary pressure was increased, the depth of the capillary folds decreased markedly. Furthermore, it appeared that this fall was greater for transpulmonary pressure changes of 5 to 15 cm H20 than for 15 to 25 cm H20. This would agree with the observations of Forrest (1979). The exact cause of capillary folds is not known, although a possible factor might be surface-tension forces. That these forces can influence capillary folding is supported by the work of Gil et al. (1979). These investigators fixed air-filled and saline-filled lungs for electron microscopy. In air-filled lungs, numerous pleats and folds were seen and these were usually filled with pools of surfactant. On the other hand, very little pleating and folding were seen in the saline-filled lungs. Capillary folds may play a role in determining pulmonary vascular resistance at low lung volumes. At a transpulmonary pressure of 5 cm HzO, fold depth averaged about 2/~m. Considering that the maximum mean capillary width under Zone 3
MORPHOLOGY OF PULMONARY CAPILLARIES
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conditions was about 6.5 ~m, this would mean a reduction in capillary caliber of approximately 32%. This could significantly increase vascular resistance at the capillary level and, in fact, this may be a contributory factor to the increased pulmonary vascular resistance noted at low lung volumes (Dawson et al., 1977; Grimm et al., 1977; West, 1969). Indeed, Forrest (1976; 1979) has argued that unfolding of the alveolar wall is primarily responsible for the increase in capillary volume determined in rat lungs fixed at a collapsed and slightly expanded state. Wall thickness
It is apparent from Figs. 5 and 6 and the statistical analysis that transpulmonary pressure does not have any significant influence on wall thickness. In addition, under Zone 2 conditions, wall thickness appears to be unaffected by perfusing pressure. However, under Zone 3 conditions at transpulmonary pressures of 15 and 25 cm H20, wall thickness was found to decrease significantly (t = 3.23, 2.87; df = 17, 12; P < 0.05) as perfusing pressure was increased. It has been suggested that increases in pulmonary blood flow are accommodated for predominantly by recruitment under Zone 2 conditions, and by distension, under Zone 3 conditions (Glazier et al., 1969). Thus, it may be that distention of the pulmonary capillaries produces a reduction in the wall thickness. Presumably, this is only seen at high transpulmonary pressures because the majority of unfolding of the alveolar wall has occurred, and therefore the capillaries are narrowed. When capillary pressure is increased under these conditions, the wall thickness is reduced. ACKNOWLEDGMENTS The author gratefully acknowledges the helpful comments and criticisms ofDrs. John West, John Evans, and Frank L. Powell. Carlita Durand, Richard Matthews, and Ernesto Aviles provided expert technical assistance throughout this study. Supported by NIH Grants HL 21943 and 17731.
REFERENCES ASSIMACOPOULOS, A., GUGGENHEIM, R., AND KAPANCI, Y. (1976). Changes in alveolar capillary configuration at different levels of lung inflation in the cat: An ultrastructural and morphometric study. Lab. Invest. 34, 10-22. DAWSON, C. A., GRIMM, D. J., AND LINEHAN, J. H. (1977). Effects of lung inflation on longitudinal distribution of pulmonary vascular resistance. J. Appl, Physiol. 43, 1089-1092. DEAN, J. N., DUTY, V. E., AND JOHNSON, R. L. (1970). Pressure-volume characteristics of the pulmonary capillary bed. The Physiologist 13, 177. DEFOUw, D. O., AND BERENDSEN, P. B. (1978). Isolated perfused dog lungs: A comparative stereological comparison with normal dog lungs. Amer. J. Anat. 151, 337-344. FORRES/, J. B. (1976). Lung tissue plasticity: Morphometric analysis of anisotropic strain in liquid filled lungs. Respir. Physiol. 27, 223-239. FORREST, J. B. (1979). Structural aspects of gas exchange. Fed. Proc. 38, 209-214. GIL, J., AND WEIBEL, E. R. (1972). Morphological study of the pressure-volume hysteresis in rat lungs fixed by vascular perfusion. Respir. Physiol. 15, 190-213. GIL, J., BACHOFEN,H., GEHR, P., AND WEIBEL, E. R. (1979). Alveolar volume to surface area relation in air and saline-filled lungs fixed by vascular perfusion. J. Appl. Physiol. 47, 990-1001. GLAZIER, J. B., HUGHES, J. M. B., MALONEY, J. E., AND WEST, J. B. (1969). Measurements of capillary dimensions and blood volume in rapidly frozen lung. J. Appl. Physiol. 26, 65-76. GOLDSTEIN, A. (1964). In "Biostatistics" p. 144. Macmillan Co., New York.
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GRIMM, D. J., LINEHAN, J. H., AND DAWSON, C. A. (1977). Longitudinal distribution of vascular resistance in the lung. J. Appl. Physiol. 43, 1093-1101. MAZZONE,R. W., DURAND,C., AND WEST,J. B. (1978). Electron microscopy of lung rapidly frozen under controlled physiological conditions. J. Appl. Physiol. 45, 325-333. MAZZONE,R. W., E)URAND,C., AND WEST, J. B. (1979). Electron microscopic appearances of rapidly frozen lung. J. Microscopy. 117, 269-284. PAIN, M. C. F., AND WEST, J. B. (1966). Effect of the volume history of the isolated lung on distribution of bloodflow. J. Appl. Physiol. 21, 1545-1550. Roos, A., THOMAS,L. J., NAGEL,E. L., AND PROMMAS,D. C. (1961). Pulmonary vascular resistance as determined by lung inflation and vascular pressures. J. Appl. Physiol. 16, 77-84. STAUR,N. C. (1966). Effect of alveolar surface tension on the pulmonary vascular bed. Japan Heart J. 7, 386-399. STAUB,N. C., ANDSTOREY,W. F. (1962). Relation between morphological and physiological events in lung studied by rapid freezing. J. Appl. Physiol. 17, 381-390. STAUB, N. C., GEE, M., AND VREIM, C. (1976). Mechanism of alveolar flooding in acute pulmonary edema. In "Lung Liquids" (CIBA Symposium 38), pp. 225-272. North-Holland, New York. THOMAS,L. J., Gm FFO, Z. J., AND ROOS, A. ( 1961). Effect of negative-pressure inflation of the lung on pulmonary vascular resistance. J. Appl. Physiol. 16, 451-456. UNTERSEE, P., GIL, J., AND WEIBEL, E. R. (1972). Morphometric characteristics of the air-blood barrier at different degrees of inflation. Bull. Physiopathol. Respir. 8, 139. VREIM, C. E., AND STAUB, N. C. (1974). Pulmonary vascular pressures and capillary blood volume changes in anesthetized cats. J. Appl. Physiol. 36, 275-279. WARRELL,D. A., EVANS,J. W., CLARKE,R. O., KINGABY,G. P., ANDWEST, J. B. (1971). Patterns of filling in the pulmonary capillary bed. J. Appl. Physiol. 32, 346-356. WEIBEL, E. R., AND BACHOFEN, H. (1979). Structural design of the alveolar septum and fluid exchange. In "Pulmonary Edema" (A. P. Fishman and E. M. Renkin, eds.), pp. 1-20. American Physiol. Soc., Bethesda, Md. WEIBEL, E. R., AND KNIGHT, B. W. (1964). A morphometric study of the thickness of the pulmonary air-blood barrier. J. Cell. Biol. 21, 367-384. WEST, J. B. (1969). Effects of interstitial pressure. In "The Pulmonary Circulation and Interstitial Space" (A. P. Fishman and H. H. Hecht, eds.), pp. 43-63. Univ. of Chicago, Chicago, Illinois. WEST, J. B., DOLLERY,C. T., AND NAIMARK,H. (1964). Distribution of blood flow in isolated lung: relation to vascular and alveolar pressures. J. Appl. Physiol. 19, 713-724. ZAR, J. H. (19774). "'Biostatistical Analysis," p. 234. Prentice-Hall.
Influence of Vascular and Transpulmonary Pressures on the Functional Morphology of the Pulmonary Microcirculation ROBERT W . MAZZONE
Department of Medicine, M-023, University of Cal~)rnia, San Diego, La Jolla, Califi~rnia 92093 Received August 28, 1979 The effects of airway and vascular pressures on pulmonary capillary morphology were examined by rapidly freezing in situ dog lungs under Zone 1, 2, and 3 conditions at transpulmonary pressures (ew) of 5, 15, and 25 cm HzO. The tissue was preserved using a freeze substitution technique and analyzed by transmission electron microscopy. Capillary and septal wall widths were measured on random sections, and an estimate of the thickness of the blood:gas barrier was made. In addition, the depths of any folds penetrating into the capillary lumen were also measured. Under Zone 2 and 3 conditions, increasing the perfusing pressure increased the mean capillary width, but at higher Ptp, this effect was reduced. Mean fold depth was reduced as etp was increased under both Zone 2 and 3 conditions. At l o w P w (5 cm H20), mean fold depth was unresponsive to changes in perfusing pressure, but decreased significantly as perfusing pressure increased when the Ptp was 15 or 25 cm HzO. Wall thickness was unaffected by change in Pw. Under Zone 2 conditions, wall thickness was independent of perfusing pressure. However, under Zone 3 conditions, at etp of 15 and 25 cm H20, there was a significant decrease in wall thickness as perfusing pressure was increased. Presumably, this decrease in wall thickness is due to distention of pulmonary capillaries at medium and high inflation pressures.
INTRODUCTION The functional morphology of the pulmonary microcirculation is exquisitely sensitive to the relationship between airway and vascular pressures (Glazier et al., 1969). Using vascular perfusion fixation techniques, Gil and Weibel (1972) showed that, in spite of a constant perfusion pressure, the alveolar septum folds and unfolds much like an accordion in response to differing airway pressures. Assimacopoulos et al. (1976) extended these observations in their study of the influence of lung volume on alveolar-capillary configuration and showed that folding could influence capillary morphology. However, since the morphology of the pulmonary capillaries is dependent upon a variety of physiologic conditions, microscopic analysis of pulmonary structure-function relationships requires that the lung be fixed under precisely defined physiologic conditions. With airway and vascular fixation techniques, sone doubt will always exist as to the exact relationship between airway and vascular pressures at the level of the pulmonary capillaries (Mazzone et al., 1978; 1979). This problem can be circumvented to a great degree by stabilizing lung structure under specific physiologic conditions through rapid freezing. By combining the isolated, perfused, in situ lung preparation (West et al., 1964) 295 0026-2862/80/060295-12502.00/0 Copyright © 1980 by Academic Press, Inc. All rightsof reproductionin any form reserved. Printed in U.S.A.
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ROBERT W. MAZZONE
with the rapid freeze procedure (Staub and Storey, 1962), Glazier et al. (1969) were able to conduct a light microscopic analysis of the relationship between various combinations of vascular and alveolar pressures on pulmonary blood flow. This study, however, was limited by the limited resolution of the light microscope which did not allow visualization of the finer details of pulmonary capillary morphology such as capillary folds. Since the rib cage is removed in this preparation, transpulmonary pressure is constant throughout the lung. The vascular pressure, on the other hand, is affected by the hydrostatic column. If the lung is rapidly frozen in a vertical position, the functional relationship between perfusion pressure and transpulmonary pressure can be preserved under precisely defined conditions. In this study, the methods of Glazier et al. (1969) have been combined with a new procedure described recently by this laboratory (Mazzone et al., 1978). In this manner, the morphology of the pulmonary capillaries fixed under controlled physiologic conditions has been examined by transmission electron microscopy. METHODS The lungs of 21 mongrel dogs (20-30 kg) were ventilated and perfused in situ according to the procedure of West et al. (1964) as modified by Warrell et al. (1971). Briefly, dogs were anesthetized by intravenous injection of phenobarbitol sodium (30 mg/kg) and a tracheostomy was performed. The pleura was exposed by left-side thoracotomy. Heparin (2000 IU/kg) was administered and after 3 min the animal was bled (0.6-1 liters). The pleura was punctured, the heart fibrillated, and the rib cage rapidly removed. The aorta was tied off and a cannula was introduced through the right ventricle into the pulmonary artery. Blood returning from the lungs was collected by a cannula placed in the left atrial appendage. Total surgical time was 8 to 12 min. The perfusion circuit consisted of a Sarns roller pump, a bubble trap and heat exchanger, and a venous reservoir. Pulmonary artery (Part) and venous pressures (Pven) were measured with saline manometers. Pulmonary artery pressure was controlled by regulating the speed of the pump, while pulmonary venous pressure was regulated by raising or lowering the venous reservoir. All experiments were performed with the dogs suspended in the vertical position. The lungs were ventilated with a Harvard respirator supplied with a mixture of 14% 02, 6% CO.,, balance N2 at an end-expiratory pressure of 3-5 cm H20. This gas mixture has been shown to yield normal blood:gas values (Warrell et al., 1971). Airway pressure was measured by a saline manometer connected to the tracheostomy. The lungs were rapidly frozen with liquid Freon 22 cooled to - 155° with liquid Nz. Approximately 2 liters of Freon 22 were poured into a funnel connected to a brass tube which had fine holes approximately 5 mm apart. The Freon was sprayed onto the lung surface for approximately 35 to 45 sec. Following this, the lung was rapidly removed and placed into liquid N2. Perfusion time prior to freezing was never more than 15 min. Physiologic conditions. Lungs were frozen under conditions of Zone 1 (Pa~v > Part > Pven), Zone 2 (Part > Palv > Pven), and Zone 3 (Part > Pven > Pair). Transpulmonary pressures were set at 5, 15, or 25 cm H20. All lungs were frozen
MORPHOLOGY OF P U L M O N A R Y CAPILLARIES
297
on the deflation limb of the pressure-volume curve (Gil and Weibel, 1972; Pain and West, 1966). Processing of frozen tissue. Samples of frozen lung approximately 0.5 cm in diameter and 2 mm in thickness were obtained at known vertical distances referenced to either the Zone 1-2 or the Zone 2-3junctions. Each sample of tissue was fractured into quarters while in liquid N2, and these were placed in a solution of 70% ethylene glycol containing 4% glutaraldehyde and 2% paraformaldehyde at - 8 0 ° . The solution and samples were then allowed to warm to - 5 0 ° and were kept at this temperature for 24 hr. The details of the procedure for processing the frozen lung for TEM has been described previously (Mazzone et al., 1978; 1979) and will only be mentioned briefly. After the substitution and fixation period, the samples were thawed to +4 ° and placed in Hanks' salt solution (Grand Island Biological) containing 4% glutaraldehyde and 2% paraformaldehyde (ph 7.4, and 340 mOsm). After 1 hr the tissue was washed with Hanks' and treated with 2% osmium tetroxide for 2 hr in the dark. Samples were then washed with maleate buffer and block-stained with 1.5% uranyl acetate for 90 min in the dark. The tissue was dehydrated in acetone and infiltrated with Spurr low-viscosity embedding media (Polysciences). Samples were then embedded and cured for 48 hr at +60-80 °. Sections, 750-1000/~ in thickness were obtained using a diamond knife (Dupont Ind.) and an LKB ultratome 111. These sections were mounted on 200-mesh copper grids, poststained with aqueous uranyl acetate and Reynolds lead citrate, and examined on a Zeiss EM9 transmission electron microscope. Histological measurements. The sampling procedure used was based on a multi-tier sampling procedure (Weibel and Knight, 1964). Approximately four to six blocks of tissue were prepared for each sample of frozen lung. Two blocks from each group were randomly chosen for analysis. These were sectioned, mounted, and stained as described above. Five microscopic fields from each section were chosen randomly and photographed at a primary magnification of × 1500. In the event lung tissue was not seen in the field, it was discarded and a new area chosen randomly. The dimensions of the capillary width and septal width were measured at 5-~m intervals perpendicular to the alveolar septum. No measurements were made within 10/.~m of septal junctions so that corner vessels were excluded from the analysis. Septa directly attached to the pleura, large blood vessels, or airways were also not analyzed. In addition, measurements of capillary width did not include the thickness of the capillary endothelium. The depth of any folds penetrating into the capillary lumen was also measured in the micrographs. This was done by drawing a line connecting the alveolar epithelium on both sides of the folds and then measuring the distance from this line to the inner edge of the capillary endothelium. Fold depth was measured only if the depth of the fold exceeded the width of the fold at the epithelial surface. If graphical analysis suggested linear relationships for any of the data, a leastsquares linear regression was performed. Several statistical tests were performed on the data. First, a test for coincidental regression was conducted on each set of data (Zar, 1974). Second, the linear regressions were also tested for parallelism (Goldstein, 1964). If this test failed to show significant difference in the slopes, the y-intercepts were then tested for significance (Zar, 1979). These tests were per-
298
ROBERT W . M A Z Z O N E
formed on data obtained under each zonal condition. In addition, Zone 2 data were compared to Zone 3 data to determine statistical significance between zonal conditions. Finally, the slopes of the regression lines were tested to determine if they were significantly different from zero, using Student's t test. RESULTS In all figures, the measurements have been plotted as a function of known vertical distance down the lung relative to either the Zone 1-2 or Zone 2-3 junction. In these experiments, the perfusion pressures were always adjusted so that the Zone 1-2 or Zone 2-3 junctions were maintained at the same height relative to the top of the lung for all three transpulmonary pressures. Hence, at a given distance down the lung, the capillary transmural pressure; that is, the difference between alveolar and capillary pressures, will be approximately the same at that point for all three transpulmonary pressures used. Thus, any differences seen in lungs frozen at various transpulmonary pressures will be due primarily to changes in transpulmonary pressure. This will be particularly true for those lungs frozen under Zone 3 conditions. Since pulmonary vascular pressures increase down the lung due to the hydrostatic column, moving down the distance axis is equivalent to increasing perfusing pressure. When lungs were rapidly frozen under Zone 3 conditions, the arterial-venous pressure difference was always 2 to 4 cm H20. Since blood flow under Zone 3 conditions is governed by the arterial-venous pressure difference, capillary pressure at any given distance down the lung may be estimated within 1-2 cm HzO (Glazier et al., 1969). Thus, the distance axis is, under these conditions, essentially equivalent to the capillary perfusing pressure in centimeters of
H20. Under Zone 2 conditions, the distance axis is not equivalent to the exact perfusing pressure. Since changes in the transpulmonary pressure may alter the pulmonary vascular resistance, the exact capillary pressure at a given distance down the lung cannot be known with assurance. However, plotting the measurements as a function of distance down the lung is still a useful technique since the capillary pressure will be increasing with distance down the lung due to hydrostatic effects. Capillary width. Figure 1 shows the mean capillary width (_ 1 SEM) plotted as a function of distance down the lung. Note that the distance axis has been divided to show data obtained under both Zone 1 and Zone 2 conditions. Linear regressions have been fitted using a least-squares technique. The tests for coincidental regression confirm (F (4,44) = 7.49, P < 0.05) that the data are not adequately described by a single regression line. Under Zone 1 conditions (Pa~v > Part), capillary width was not zero. This was due to the presence of red blood cells and plasma presumably trapped in the capillaries. Other investigators have shown that capillary volume never reaches zero, regardless of how high the alveolar pressure is raised above the arterial pressure (Dean et al., 1970; Staub, 1966; Vreim and Staub, 1974). Vreim and Staub (1974) and Staub (1966) have demonstrated histologically that this was because of trapped red cells and plasma. When arterial pressure increased above alveolar pressure, the mean capillary
299
MORPHOLOGY OF PULMONARY CAPILLARIES
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FIG. 1. Mean capillary width (_+ 1 SEM) plotted as a function of distance down the lung for Ptp of 5, 15, and 25 cm H20. Note that the distance axis has been divided so that data obtained above and below the Zone l-2junction can be shown on the same graph. The data have been fitted using a least-squares linear regression. (M = slope, r = correlation coefficient.)
width appeared to increase linearly, regardless of the transpulmonary pressure at which the lungs were frozen. Increasing the transpulmonary pressure served to reduce the slopes of the lines relating mean capillary width and distance down the lung. The slope of the regression line forPtp = 5 cm HzO was significantly different (t = 3.37, df= 31 , P < 0.05) than that forPto = 15 cm HzO orPtp = 25 cm H20 (t = 3.59, df = 29, P < 0.05). However, no significant difference could be found in the slope of the lines f o r P w = 15 cm H20 compared toPto = 25 cm H20 (t = 0.8419), although the y-intercepts were significantly different (t = 2.69; df = 32). In addition, all three regression lines had slopes which were significantly different from zero (Ptp = 5, 15, 25 cm H~O; t = 8.34, 3.3, 9.4, df = 13, 17, 14; P < 0.05). Figure 2 shows mean capillary width (_+ 1 SEM) plotted as a function of distance • 5
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300
ROBERT W. MAZZONE
down the lung for dog lungs frozen under Zone 3 conditions. As distance down the lung increased, the mean capillary width also increased. When transpulmonary pressure was 5 cm H20, mean capillary width reached a maximum of 6 . 5 - 7 / z m approximately 35 cm down the lung. Increases in the transpulmonary pressure caused a decrease in the maximum mean capillary width as well as a decrease in the slopes of the regression lines. The regression lines shown in Fig. 2 were calculated over the range of 0 to 30 cm down the zone. The data were not adequately described by a single regression line (F (4,31) = 21.2, P < 0.05). Analysis of the slopes showed that all three were significantly different from z e r o ( P t p = 5, 15, 25 cm H20; t -- 5.48, 3.19, 2.87; df= 10, 13, 8; P < 0.05). The slope of the regression line for Ptp = 5 c m H20 was significantly different from that for Ptp = 15 cm H20 (t = 3.15; df = 25; P < 0.05) and also from Ptp = 25 c m H 2 0 (t = 2.42; df = 20; P < 0.05). No significant difference could be demonstrated between the slopes of the regression lines for P w = 15 and 25 cm H20 (t = 0.3535). In addition, no significant differences in the elevations of the two lines could be detected. A comparison of mean capillary width under Zone 1-2 and Zone 3 was fitted by separate regression lines ( P t p = 5, 15, 25 cm H20; F = 36.5, 4.27, 44.23; df = 2,33, 2,30, 2,22; P < 0.05). For each Ptp under Zone 2 and 3 conditions, no significant differences in the slopes of the lines could be found (Ptp = 5, 15, 25 cm H20; t = 0.547, 0.4545, 1.296; df = 25, 31,34), although there were significant differences in the elevations (Pro = 5, 15, 25 c m HzO; t = 4.07, 3.1, 3.84; df = 24, 31, 23; P < 0.05). Fold depth. Figure 3 shows the mean fold depth (___1 SEM) plotted as a function of distance down the lung for the Zone 1-2 preparations while the mean fold depth obtained from Zone 3 preparations is shown in Fig. 4. Note the striking similarities between these sets of data. For Zone 2 conditions (Fig. 3) the data have been fitted by separate regression lines (F (4,40) = 76.8, P < 0.05). The slope of the regression line for P w = 5 cm H20 was significantly different from that for P t p = 15 and 25 cm H20 (t = 2.38, 2.69; df = 17, 14; P < 0.05). No significant difference could be found between the
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301
MORPHOLOGY OF P U L M O N A R Y CAPILLARIES
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FIG. 4. Mean fold (_+1 SEM) plotted as a function of distance down the lung for the Zone 3 preparations. As in Fig. 3, the slope of the regression line at aPtp of 5 cm HzO was not significant, while the slopes of the regression lines for/gtp of 15 and 25 cm H~O were significant (P < 0.05). (M = slope, r = correlation coefficient.) slopes for Ptp = 15 and Pto = 25 cm H 2 0 (t = 0.455; df= 30), although there was a significant difference in the elevation of the two lines (t = 2.78; df = 29; P < 0.05). Finally, only the slopes of the regression lines for Pro = 15 and 25 cm H~O were significantly different from zero (t = 2.38, 2.69; df = 17, 14; P < 0.05). The fold depth data obtained under Zone 3 conditions (Fig. 4) have also been fitted by separate regression lines (F (4,41) = 116.3, P < 0.05). The slopes of the regression lines for Ptp = 5 c o m p a r e d to 15 cm H20 and 5 c o m p a r e d to 25 cm H 2 0 were significantly different (t = 3.19, 2.64; df = 23, 18; P < 0.05). H o w e v e r , no significant difference was o b s e r v e d b e t w e e n the slopes of the lines for Ptp = 15 c o m p a r e d to 25 cm H 2 0 (t = 0.538; df = 23). The elevation of these two lines were significantly different (t = 2.056; df = 31; P < 0.05). In addition, the slopes of the regression lines for Ptp = 15 and 25 cm H 2 0 were significantly different from zero (Pw = 15, 25 cm H20; t = 4.62, 4.6; df = 18, 12; P < 0.05). This was not true for etp = 5 cm HzO (t = 1.32; df = 11). A comparison of Zone 1-2 versus Zone 3 data failed to show any significant difference in the slopes of the regression lines for any given Ptp ( P t p = 5, 15, 25 cm HzO; t = 0.116, 1.445, 0.3835; df = 22, 32, 2 I). H o w e v e r , for P t p = 15 and 25 cm H20, the elevations were significantly higher under Zone 3 conditions (Ptp = 15, 25 cm H20; t = 2.38, 2.75; df = 36, 24; P < 0.05). This was not true for Pro = 5 cm HzO (t = 1.678; df = 24). Presumably, the increased fold depth o b s e r v e d in the Zone 3 preparations is related to capillary recruitment and distension. U n d e r Zone 2 conditions, some capillaries were not actively perfused (Warcell et al., 1971) and hence their size is less than under Zone 3 conditions. F r o m the data, it would appear that this results in a slightly smaller fold depth. More importantly, h o w e v e r , under both Zone 2 and Zone 3 conditions, transpulmonary pressure and perfusing pressure have similar effects upon capillary fold depth. The statistical analysis therefore shows that increasing transpulmonary pressure caused a marked reduction in fold depth. F u r t h e r m o r e , fold depth is only responsive to changes in hydrostatic pressure at Ptp = 15 or 25 cm HzO.
302
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FIG. 5. Wall thickness, defined as the difference between mean septal width and mean capillary width, plotted as a function of distance down Zone 2. Transpulmonary pressure had no significant influence on wall thickness. In addition, the slopes of all these regression lines were not significant. (M = slope, r = correlation coefficient.)
Wall thickness. An indication of the thickness of the barrier to gas exchange was obtained by subtracting the mean capillary width from the mean septal wall width. Figure 5 shows these data, defined as the wall thickness, plotted as a function of distance down the lung for the Zone 2 preparations, while Fig. 6 shows the data for the Zone 3 preparations. As with the fold depth data, there is some similarity between these two sets of results. Under both Zone 2 and Zone 3 conditions, increasing the transpulmonary pressure had no significant effect upon wall thickBess. There is one striking difference between data shown in Figs. 5 and 6. Under Zone 2 conditions (Fig. 5), the slopes of the regression lines for wall thickness versus distance for each transpulmonary pressure were not significantly different from zero (Pw = 5, 15, 25 cm H20; t = 0.12, 1.59; 0.018; df = 10, 13, 11). This implies that increasing the perfusing pressure did not significantly alter the wall thickness, regardless of the transpulmonary pressure, at least under Zone 2
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MORPHOLOGY OF P U L M O N A R Y CAPILLARIES
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conditions. However, the wall thickness obtained from the Zone 3 preparations shows a different trend. At transpulmonary pressures of either 15 or 25 cm H20, wall thickness decreased significantly (Ptp = 15, 25 cm H20; t = 3.23, 2.87; d f = 17, 12; P < 0.05) as perfusion pressure increased. However, at a transpulmonary pressure of 5 cm H20, wall thickness did not decrease significantly (t = 1.8; d f = 11) as perfusing pressure increased. Thus, it would appear that under Zone 3 conditions at high inflation pressures, increasing the perfusion pressure may reduce the barrier to gas exchange. DISCUSSION Limitation o f Techniques
DeFouw and Berendsen (1978) have performed a stereological analysis of isolated, airway-fixed dog lungs following perfusion for 30, 60, and 120 min. Their results showed progressive morphological changes. However, in this study, the in situ lung preparations were ventilated and perfused for less than 15 min and then rapidly frozen under a defined set of physiologic conditions. It should be pointed out that DeFouw and Berendsen (1978) removed the lungs completely from the animal so that lymphatic drainage might have been altered. This is probably not the case in lungs ventilated and perfused in situ (Warrell et al., 1971). The freezing techniques used in this study provide extremely good preservation to a depth of about 2 mm below the pleural surface (Mazzone et al., 1978; 1979). Glazier et al. (1969) analyzed capillary morphology by light microscopy of frozen lung. Results obtained 1 and 4 mm below the pleural surface agreed within 5%. They concluded that as long as alveolar septa directly attached to the pleura were excluded from the measurements, analysis of lung tissue 1 to 2 mm below the pleura was indicative of events occurring in the more central regions of the lung. Capillary Caliber
The results presented here support the idea that as transpulmonary pressures increase, narrowing of the capillaries occurs. Glazier and colleagues (1969) performed light microscopy on rapidly frozen dog lungs. Their results showed that the number of red cells along the alveolar septum was markedly reduced when the transpulmonary pressure was increased from 10 to 25 cm H20. Assimacopoulos et al. (1976) examined lung fixed by vascular perfusion at several inflation pressures. Their morphological findings showed that the capillary surface to volume ratio, which is an inverse estimate of capillary width, increased slightly from 0 to 15 cm H20 airway pressure, then showed a very abrupt rise as airway pressure was increased to 20 cm H20. Additionally, Forrest (1976; 1978) examined rat lungs fixed by airway instillation at different transpulmonary pressures and found that as inflation pressure was increased from a collapsed state, capillary volume (Ve) initially increased. However, as the transpulmonary pressure increased further, there was a striking decrease in Ve. All these results would imply that capillaries narrow as inflation pressures increase. The results of this study suggest that increasing the transpulmonary pressure will cause a significant increase in pulmonary vascular resistance. For example, under Zone 3 conditions, as transpulmonary pressure is increased to 25 cm H20, the mean capillary width is reduced by about 50%, 30 cm down the zone. Certainly
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this would have a significant influence on the vascular resistance of the pulmonary capillaries. Furthermore, increasing the perfusion pressure at these higher transpulmonary pressures probably will not alter the caliber of these vessels significantly. Other investigators have reported a relationship between pulmonary vascular resistance and transpulmonary pressure. For example, Ross et al. (1961) and Thomas et al. (1961) were the first to report that as either transpulmonary pressure or lung volume increased, pulmonary vascular resistance first decreased, then increased. This observation was confirmed by Dawson et al. (1977). West (1969) has proposed that this relationship is due to stretching of the alveolar wall with subsequent narrowing of the pulmonary capillaries. Calculation of the pressure drop across various portions of the pulmonary vasculature supports this idea since experimental results indicate that the larger fraction of the total pressure drop at high lung volumes is due to the pressure drop across the alveolar vessels (Grimm et al., 1977). The statistical analysis suggests that there are differences between the regression upon whether the lungs are frozen under Zone 2 or Zone 3 conditions, but that the effect of increases in perfusing pressure are essentially identical, regardless of whether or not the lungs are perfused under Zone 2 or Zone 3 conditions. The results suggest that perfusing lungs under different zonal conditions changes the intercept of the regression lines, and that the increase in capillary width as perfusion pressure changes is unaffected by whether or not the lungs are in Zone 2 or Zone 3. Fold Depth
Gil and Weibel (1972) demonstrated that alveolar surface changes accompanying lung volume changes were the result of folding and unfolding the septa. Furthermore, Assimacopoulos et al. (1976) found that the number and numerical density of capillary folds decreased as the lungs were inflated. It was argued that the capillary bed became more rectilinear with an increasing degree of alveolar expansion, presumably due to the unfolding of the alveolar wall. It should be mentioned that the folds observed by these authors were really pleating of the alveolar wall and did not necessarily penetrate the capillary lumen. The results shown in Fig. 3 extend the observations of Gil and Weibel (1972), Untersee et al. (1972), and Assimacopoulos et al. (1976). It is apparent from the statistical analysis that as transpulmonary pressure was increased, the depth of the capillary folds decreased markedly. Furthermore, it appeared that this fall was greater for transpulmonary pressure changes of 5 to 15 cm H20 than for 15 to 25 cm H20. This would agree with the observations of Forrest (1979). The exact cause of capillary folds is not known, although a possible factor might be surface-tension forces. That these forces can influence capillary folding is supported by the work of Gil et al. (1979). These investigators fixed air-filled and saline-filled lungs for electron microscopy. In air-filled lungs, numerous pleats and folds were seen and these were usually filled with pools of surfactant. On the other hand, very little pleating and folding were seen in the saline-filled lungs. Capillary folds may play a role in determining pulmonary vascular resistance at low lung volumes. At a transpulmonary pressure of 5 cm HzO, fold depth averaged about 2/~m. Considering that the maximum mean capillary width under Zone 3
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conditions was about 6.5 ~m, this would mean a reduction in capillary caliber of approximately 32%. This could significantly increase vascular resistance at the capillary level and, in fact, this may be a contributory factor to the increased pulmonary vascular resistance noted at low lung volumes (Dawson et al., 1977; Grimm et al., 1977; West, 1969). Indeed, Forrest (1976; 1979) has argued that unfolding of the alveolar wall is primarily responsible for the increase in capillary volume determined in rat lungs fixed at a collapsed and slightly expanded state. Wall thickness
It is apparent from Figs. 5 and 6 and the statistical analysis that transpulmonary pressure does not have any significant influence on wall thickness. In addition, under Zone 2 conditions, wall thickness appears to be unaffected by perfusing pressure. However, under Zone 3 conditions at transpulmonary pressures of 15 and 25 cm H20, wall thickness was found to decrease significantly (t = 3.23, 2.87; df = 17, 12; P < 0.05) as perfusing pressure was increased. It has been suggested that increases in pulmonary blood flow are accommodated for predominantly by recruitment under Zone 2 conditions, and by distension, under Zone 3 conditions (Glazier et al., 1969). Thus, it may be that distention of the pulmonary capillaries produces a reduction in the wall thickness. Presumably, this is only seen at high transpulmonary pressures because the majority of unfolding of the alveolar wall has occurred, and therefore the capillaries are narrowed. When capillary pressure is increased under these conditions, the wall thickness is reduced. ACKNOWLEDGMENTS The author gratefully acknowledges the helpful comments and criticisms ofDrs. John West, John Evans, and Frank L. Powell. Carlita Durand, Richard Matthews, and Ernesto Aviles provided expert technical assistance throughout this study. Supported by NIH Grants HL 21943 and 17731.
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