Pulmonary microvascular and perivascular interstitial geometry during development of mild hydraulic edema

Daniela Negrini1, Anna Candiani2, Federica Boschetti2, Beatrice Crisafulli3, Massimo Del Fabbro1, Dario Bettinelli3, and Giuseppe Miserocchi3

1 Dipartimento di Medicina, Chirurgia e Odontoiatria, Università degli Studi, 20133 Milan; 2 Dipartimento di Bioingegneria, Politecnico di Milano, 20133 Milan; and 3 Dipartimento di Medicina Sperimentale, Ambientale e Biotecnologie Mediche, Università di Milano-Bicocca, 20052 Monza, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To study pulmonary arteriolar vasomotion in control conditions and in the transition to hydraulic edema, changes in subpleural pulmonary arteriolar diameter and perivascular interstitial volume were evaluated in anesthetized spontaneously breathing rabbits. Images of subpleural pulmonary microvessels were recorded in control conditions and for up to 180 min during a 0.5 ml · kg-1 · min-1 intravenous saline infusion through an intact parietal pleural window. Images were digitized and analyzed with a semiautomatic procedure to determine vessel diameter and perivascular interstitial thickness from which interstitial fluid volume was derived. In control vessels, the diameter of ~30-, ~50-, and ~80-µm arterioles and the perivascular interstitial thickness were fairly stable. During infusion, the diameter increased maximally by 20% in ~30-µm vessels, was unchanged in ~50-µm vessels, and decreased by 25% in ~80-µm arterioles; the perivascular interstitial volume increased by 54% only around 30-µm microvessels. In papaverine-treated rabbits, all arterioles dilated and a larger increase in perivascular interstitial thickness was observed. The data suggest that the opposite vasomotor behavior of 30- and 80-µm arterioles during development of mild edema may represent a local specific response of the pulmonary microcirculation to reduce capillary pressure in the face of an increased transendothelial fluid filtration, thus counteracting progression toward severe edema.

pulmonary arteriolar vasomotion; perivascular interstitial volume; mild interstitial edema


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE NORMAL LUNG, at variance with other organs that can withstand a large increase in tissue hydration without showing severe functional impairment, requires a precisely controlled fluid balance to minimize the alveolocapillary membrane thickness and guarantee respiratory gas exchange. Several different mechanisms, including interstitial matrix composition and compliance (7, 10-13), fluid removal through the pulmonary lymphatics (17), and low functional capillary pressure, are involved in maintaining the adequate hydration of the pulmonary interstitial space. Previous studies with the micropuncture technique performed in our and other laboratories allowed us to measure the hydraulic pressure profile in the superficial pulmonary microvascular network in the intact lung in control conditions (2, 3, 9) and during the development of mild hydraulic lung edema (4, 8). The latter condition was shown to cause, together with a decrease in total vascular flow resistance with respect to the control value, an increase in the percent precapillary arteriolar resistance and a simultaneous decrease in postcapillary resistance due to either venous congestion or capillary recruitment (8).

The present study describes the precapillary vasomotor activity in spontaneously breathing rabbits in control conditions and during the development of interstitial lung edema induced through slow-rate intravenous saline infusion (7). We integrated the information attained from the changes in the microvascular pressure profile in edema with the measurement of microvessel size and volume of the perivascular interstitial space. Vessel diameter and perivascular interstitial volume were measured with a previously developed digital-image technique (1, 19) based on the statistical treatment of the gray-level distribution in the region under study. To differentiate between the effect of purely mechanical factors such as vessel transmural pressure and active vasomotion in determining the changes in vessel diameter during edema development, we also examined the superficial pulmonary microvasculature after intravenous administration of the smooth muscle relaxant papaverine both in control conditions and during saline infusion. The data revealed that during development of mild edema, the specific behavior of the pulmonary precapillary microvasculature in the diameter range of ~80 µm may play an active role in controlling the functional capillary pressure, thus limiting fluid filtration into the parenchyma, counteracting further progression toward alveolar edema.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General preparation. Experiments were performed on 14 adult New Zealand White rabbits (weight range 2.6 ± 0.2 kg) anesthetized with a bolus of 2.5 ml/kg of a saline solution containing 0.25 g/ml of urethane plus 10 mg/ml of pentobarbital sodium injected into an ear vein. The absence of an eyelid closure reflex and arterial blood pressure were used as estimates of the proper level of anesthesia during the experiment. When necessary, subsequent doses of 0.5 ml of anesthetic were administered through a venous line. A carotid artery and the homolateral jugular vein were cannulated with a saline-filled catheter connected to a physiological pressure transducer for continuous arterial pressure monitoring and for providing a route for intravenous saline infusion.

An "intact pleural window" (surface area ~0.3 cm2) was prepared in the fifth to sixth intercostal space to allow a view of the edge of the lower lobe where subpleural arteriolar branchings are relatively abundant (7). With the animal lying supine, the skin and external intercostal muscles on the right side of the chest were resected, and a surface area of ~0.5 cm2 of internal intercostal muscles was removed down to the endothoracic fascia. Under stereomicroscopic view and with fine forceps, the endothoracic fascia was carefully stripped, exposing the parietal pleura through which the lung surface with its superficial microvascular network could be clearly detected (Fig. 1). This approach allowed us to visualize the lung surface through the intact parietal pleura, leaving the lung chest wall mechanics intact at negative pleural pressure and atmospheric alveolar pressure and allowing the animal to maintain its spontaneous breathing throughout the whole experiment.


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Fig. 1.   Results of the semiautomatic procedure for edge detection of pulmonary arteriolar branching in a control rabbit lung. Blue, inner border of the blood vessel; yellow, edge between perivascular interstitium and lung parenchyma. Bar, 200 µm.

Experimental protocol. Images of the lung surface were obtained in four groups of spontaneously breathing rabbits: 1) control animals (n = 4); 2) animals (n = 5) receiving 0.5 ml · kg-1 · min-1 of a saline infusion into the jugular vein for 180 min according to a protocol widely used in our laboratory to determine the slow development of interstitial edema (7, 8, 10, 12, 13); 3) control animals (n = 2) receiving a single intrajugular dose of 5 mg of papaverine to reach a plasma concentration of ~10-4 M (15, 18); and 4) animals (n = 3) receiving an intravenous infusion of 0.5 ml · kg-1 · min-1 of a saline solution for 180 min after intravenous administration of papaverine. Papaverine is an unspecific cAMP-phosphodiesterase inhibitor that causes a generalized relaxation of the smooth muscle vessel wall.

At the end of the experiment, the animals were euthanized with an anesthetic overdose.

Image acquisition and analysis. Images of the lung surface through the intact parietal pleura were acquired with a stereomicroscope (×3 magnification; Nikon SMZ-2T) connected to a video camera (S-VHS, Panasonic WV-F15E, PAL format), an analog monitor (JVC TM-150 PSN-K), a video recorder (Panasonic AG-7355, PAL format), and a frame grabber (Imaging Technology PCVISIONplus 512, Woburn, MA) with a resolution of 512 × 512 pixels and 256 gray levels (8 bits/pixel) installed on a personal computer (Hewlett-Packard PC Vectra 486/33VL) equipped with image-processing software (OPTIMAS version 4.02, Optimas, Edmonds, WA). Image analysis and calculations were performed with an AST Bravo P133 personal computer. Recordings lasting 15-30 s were taken immediately after identification of the vascular branching to be studied and every 20 min throughout the experiment up to 180 min from the initial acquisition; each image included a view of an arteriolar branching characterized, on average, by three to four main bifurcations. Figure 1 shows an example of a typical black-and-white image of the lung surface obtained through the intact transparent parietal pleura; the arteriolar branching is the darker object surrounded by a continuous brighter layer corresponding to the perivascular interstitial space, where the outer limit consists of the true parenchyma where the alveoli can be clearly distinguished. Arterioles were recognized based on the direction of blood flow. Because venules, unlike arterioles, are not easily found on the lung surface at the edge of the lower lobe, we restricted our analysis to arteriolar branchings.

During spontaneous breathing, the main disturbance to the image sharpness was provided by continuous tissue movement due to cardiac and respiratory activities. For each recording time frame, we selected the clearest image suitable for analysis, i.e., the image allowing the most precise definition of the object's contour; this was done by always recording the images during the end-expiratory pause when tissue movement was limited to the cardiac stroke. This also guaranteed that all the images were taken at comparable lung volumes corresponding, for all rabbits, to their functional residual capacity.

Principles of the analysis. The transparency of the parietal pleura allowed clear identification of an arteriolar branching; in the typical example in Fig. 1, arterioles of progressively decreasing diameter are recognizable on both the right and left branchings of the main artery (not shown in the lowermost part of the picture). Rectangular areas were chosen along the arteriolar path; each area, defined as a "region of interest" (ROI), included an arteriolar segment, the adjacent perivascular interstitial layer on both sides of the vessel, and, more externally, the pulmonary parenchyma (1). On each branching, several ROIs were located over arteriolar segments of different diameter. According to the image analysis developed in a previous study (19), we distinguished the three adjacent compartments (vessel, perivascular interstitium, and lung parenchyma) by mathematically defining the borders delimiting one from the other with a semiautomatic procedure based on calculation of the moving average and the moving variance of the gray level of the three compartments. Briefly, within each ROI, a line ~450 µm long was traced perpendicular to the vessel main axis, and analysis of variance (ANOVA) of the gray-level distribution along the line was performed automatically through the scanning procedure mentioned in Image acquisition and analysis. ANOVA calculates, along the drawn line, the distribution of the average gray color, the SD of the gray level (sigma  matrix), and the SD of the sigma  matrix element, i.e., the SE of the gray level (tau  matrix). It has been shown that a peak in the sigma  matrix profile allows identification of the border between the vessel and the perivascular interstitium, i.e., of two compartments in which the average gray levels are significantly different. A peak in the tau  matrix profile identifies the edge between the perivascular interstitium and the parenchyma in which the average gray levels are similar but display different scattering in the gray color level (1, 19).

In each ROI, we sampled the vessel diameter and the corresponding perivascular interstitial thickness at ~20 adjacent sites at a distance of ~60 µm along the vessel length. Figure 1 shows the result of the image processing with the semiautomatic procedure in the ROIs (6 on the left main branching and 4 on the main right branching) in an arteriolar tree of a rabbit in control conditions. The analysis allowed definition of the inner border of the blood vessel identified by the blue pixels, whereas the outer yellow pixels mark the edge between the outer perivascular interstitial border and the surrounding lung parenchyma.

Based on this analysis, we also developed a macro in ALI language in OPTIMAS environment that automatically calculates the microvessel diameter and its perivascular interstitial thickness in a region of the digital image defined by the user (1). Results are presented as diameters and perivascular interstitial volumes for each ROI identified on a microvessel. The interstitial volume for unit vessel length was calculated from the interstitial thickness, and the vessel diameter was calculated as the annulus surface area, considering the vessel and the surrounding interstitial space as two concentric straight cylinders. The average vessel diameter and perivascular volume in the ROI were considered representative for the whole vessel segment, the latter being defined as the vessel length between two consecutive branchings.

The method used to evaluate the geometry of the microvessels and perivascular interstitial space was validated by measuring objects of known dimensions and by varying the ratio of gray level among the objects (1, 19). We also validated the optical system (stereomicroscope) by comparing the measurements of calibrated pattern and microvessels to those obtained from a conventional compound microscope at the same magnification (1).

Statistical analysis. Absolute values of microvessel diameter for the three groups of microvessels in control conditions were compared by one-way ANOVA. In Figs. 2-5, the average diameter and perivascular interstitial volume under control and treatment conditions at progressive times are expressed as the ratio of the value at time t to the initial value at time 0 (Dt/D0 or Vt/V0, respectively). The significance of the effect of treatment (saline infusion against control conditions in Fig. 2, papaverine treatment against control conditions in Fig. 3, and saline infusion after papaverine against saline infusion in Fig. 4) on Dt/D0 and Vt/V0 (Fig. 5) was tested for the three vessel groups by two-way ANOVA. This analysis is suitable to test the effect of two experimental factors that are varied simultaneously for each experimental group; in the present analysis, the two experimental factors considered were time from the beginning of observations and the experimental condition studied, namely control, saline infusion, papaverine administration, and papaverine plus saline infusion. A two-factor design was used to test for the differences between samples grouped according to the value of each factor and for interaction between the factors. Differences between mean values were considered significant at P < 0.05. Whenever two-way ANOVA detected a significant difference, all pairwise multiple comparison procedures were performed (Bonferroni t-test) and the corresponding t and P values are reported in RESULTS. Data are means ± SD.


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Fig. 2.   Average time course of vessel diameter [expressed as a ratio of value at time t to initial value at time 0 (Dt/D0)] in control conditions and during saline infusion in ~30-µm (A), ~50-µm (B), and ~80 µm (C) arterioles. Values are means ± SD of the absolute values expressed as Dt/D0. During saline infusion, Dt/D0 significantly increased in 30-µm arterioles, did not change in 50-µm arterioles, and significantly decreased in 80-µm arterioles compared with that in control arterioles. In both experimental conditions, no significant dependence of Dt/D0 on time was found. See RESULTS for details of 2-way ANOVA and Bonferroni t-test.



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Fig. 3.   Time course of Dt/D0 for ~30-µm (A), ~50-µm (B), and ~80-µm (C) arterioles after papaverine treatment compared with control arterioles. Values are means ± SD of the absolute values expressed as Dt/D0. Papaverine caused a significant increase in Dt/D0 in all types of vessels. See RESULTS for details of 2-way ANOVA and Bonferroni t-test .



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Fig. 4.   Time course of Dt/D0 for ~30-µm (A), ~50-µm (B), and ~80-µm (C) arterioles after administration of papaverine with and without saline infusion. Values are means ± SD of the absolute values expressed as Dt/D0. Saline infusion caused a further significant increase in Dt/D0 in 30-µm and 50-µm vessels but not in the 80-µm arterioles. Note that in all vessels types, there was a significant dependence of Dt/D0 on time. See RESULTS for details of 2-way ANOVA and Bonferroni t-test.



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Fig. 5.   Volume of the perivascular interstitium [expressed as a ratio of value at time t to initial value at time 0 (Vt/V0)] for ~30-µm (), ~50-µm (), and ~80-µm (black-triangle) arterioles in control conditions (A), during saline infusion (B), after administration of papaverine (C), and during infusion in papaverine-treated rabbits (D). Values are means ± SD of the absolute values expressed as Vt/V0. Vt/V0 did not change over time in control conditions but significantly increased around 30-µm vessels during saline infusion. Papaverine caused a significant increase in Vt/V0 around all type of vessels and a further significant fluid accumulation around 50- and 80-µm arterioles after infusion in papaverine-treated rabbits. See RESULTS for details of 2-way ANOVA and Bonferroni t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

On the basis of their average diameter in the first image recorded in control conditions, three microvessel groups of significantly different diameters of ~30 µm (average 38.7 ± 6.4 µm, range 20-39 µm), ~50 µm (average 55.7 ± 7.1 µm, range 40-64 µm), and ~80 µm (average 78.9 ± 8.6 µm, range 65-100 µm) were identified. Table 1 shows the number of animals used and the number of arterioles examined in the different experimental protocols grouped according to the initial vessel caliper.

                              
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Table 1.   No. of animals and observed microvessels grouped according to initial average caliper

During the infusion period, the animals received on average 191 ± 6 ml of saline solution, causing the hematocrit (Hct) value to drop significantly from 41.2 ± 5.7% in control conditions to 25.8 ± 6.0% at the end of the volume expansion period (P < 0.05). Mean systemic arterial pressure was 82 ± 5.7 mmHg in control conditions and remained essentially unchanged during infusion. Papaverine administration caused a transitory drop in systemic arterial pressure, to ~75 mmHg (not significant), with a return to control values within 10 min and remaining steady thereafter. During infusion in papaverine-treated animals, systemic pressure tended to decrease linearly by ~25 mmHg over the 180-min period.

Figure 2 shows the time course of vessel diameter (expressed as Dt/D0) in control conditions and during saline infusion for each microvessel group (30 µm, Fig. 2A; 50 µm, Fig. 2B; and 80 µm, Fig. 2C). One may appreciate that in all vessels, both in control conditions and with saline infusion, Dt/D0 was not constant but oscillated, although not significantly (by no more than 12% of the initial control value), over time. The oscillatory behavior of the individual data is witnessed by the variability of the average data and by the not significant dependence of the data with time (two-way ANOVA). Saline infusion caused different specific responses in the three vessel groups. Indeed, two-way ANOVA performed on the average data and the pairwise multiple comparison procedure (Bonferroni t-test) indicated that over time, infusion caused a significant vasodilation in 30-µm vessels (t = 3.684; P = 0.005), a not-significant change in 50-µm vessels, and a significant vasoconstriction in 80-µm arterioles (t = 4.519; P = 0.001). The interaction between time dependence and experimental condition (control or infusion) was not significant and accounted for 6.1, 8.7, and 2.7% of the total variance in the 30-µm, 50-µm, and 80-µm vessels, respectively.

Figure 3 presents the comparison between the time course of the Dt/D0 for the three microvessel groups in rabbits treated with papaverine relative to that observed in control animals. Two-way ANOVA showed that papaverine caused a significant progressive increase in the microvascular diameter of 30-µm (t = 5.319; P < 0.001), 50-µm (t = 3.369; P = 0.008), and 80-µm (t = 4.597; P = 0.001) arterioles, attaining a maximum vasodilation of 30, 15, and 23% of initial diameter in 30-, 50-, and 80-µm arterioles, respectively.

Saline infusion in papaverine-treated rabbits caused, compared with papaverine alone-treated rabbits, a further small but significant vasodilation in 30-µm (t = 3.074; P = 0.013; Fig. 4A) and 50-µm (t = 4.279; P = 0.002; Fig. 4B) arterioles but not in the 80-µm arterioles (Fig. 4C). Interestingly, after papaverine, Dt/D0 data displayed a smaller variability compared with data from no-papaverine-treated animals, and, in fact, there was a significant time dependence of the Dt/D0 values in 30-µm (two-way ANOVA: F = 6.69; P < 0.0001), 50-µm (F = 5.27; P < 0.0001), and 80-µm (F = 4.87; P < 0.0001) arterioles.

Under control conditions at time 0, the average interstitial thickness around arterioles of 30-, 50-, and 80-µm diameter was 25.1 ± 7.1, 26.3 ± 7.5, and 25.2 ± 6.9 µm, respectively, yielding a perivascular volume for unit vessel length of 5.3 × 10-3 ± 1.2 × 10-3, 6.8 × 10-3 ± 1.8 × 10-3, and 8.2 × 10-3 ± 2.9 × 10-3 mm3, respectively.

Figure 5A shows that in control conditions, the perivascular interstitial volume expressed as Vt/V0 did not change significantly over time for the three groups of microvessels. During saline infusion (Fig. 5B), Vt/V0 around 30-µm microvessels significantly increased (Bonferroni t-test: t = 4.065; P = 0.003) up to a maximum of 54% at 140 min, whereas around 50-µm (F = 2.38; P = 0.12) and 80-µm (F = 3.32; P = 0.072) microvessels, it remained essentially unchanged with respect to the initial value. In the papaverine-treated animals (Fig. 5C), Vt/V0 significantly increased with respect to control values around 30-µm (t = 3.37; P = 0.008), 50-µm (t = 6.051; P < 0.001), and 80-µm (t = 3.037; P = 0.014) arterioles, suggesting that papaverine caused a generalized increase in endothelial permeability, causing an increased fluid accumulation into the perivascular interstitium. Saline infusion in papaverine-treated animals (Fig. 5D) caused, with respect to papaverine alone, a further small but significant increase in perivascular interstitial volume surrounding 50-µm (t = 4.407; P = 0.002) but not surrounding 30-µm (F = 1.82; P = 0.1832) and 80-µm (F = 2.25; P = 0.1378) arterioles. On average, the maximum increase in interstitial fluid volume, attained at 140 min from the onset of infusion, was 40.3 ± 5.7% for all the three groups of vessels.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The analytic procedure utilized in the present study to determine the size of the superficial pulmonary microvasculature and adjacent perivascular interstitial thickness has been developed in the past on spontaneously breathing rabbits in control conditions. Image analysis allows the recognition of the delimiting edge between the vessel, perivascular interstitium, and parenchyma based on the gray-image level or, more specifically (see METHODS), on the calculation of the moving average of the gray level for detection of the vessel edge and of the moving variance of the gray level for determination of the interstitial to parenchymal tissue edge (1, 19). On the basis of the decrease in the Hct value, assuming that no erythrocytes escape through the vessel walls and an initial plasma volume of 90 ml (~3.5% of body weight), one can estimate that, at the end of infusion, plasma volume increased by ~60%. Such a plasma expansion, by diluting the red cells, might, in principle, have modified the gray-level distribution of the initial control image, reducing the difference between vessel and perivascular interstitial gray level and thus affecting the edge detection procedure. However, as clearly addressed in previous studies by Crisafulli et al. (1) and Venturoli et al. (19), the sensitivity of the present methods was validated with variable differences in the gray level between objects; hence the precision of the semiautomatic procedure used was high enough to guarantee that the gray-level difference between the three compartments under study was sharp enough even in case of marked hemodilution.

As addressed in METHODS, semiautomatic edge detection recognizes the passage between the inner vessel border and the surrounding interstitial tissue and between the outer border of the latter and the tissue parenchyma. Hence the measured perivascular interstitial thickness might be systematically overestimated by including the thickness of the endothelial vessel layer. However, considering that the endothelial plus the basement membrane layer is thinner than 0.1 µm, this error would not exceed 1% of the observed perivascular interstitial tissue thickness (1, 19). The present study describes the changes in diameter occurring in the superficial pulmonary arterioles with a diameter smaller than 100 µm in control conditions and during development of interstitial pulmonary edema induced by a slow intravenous saline infusion. From the data shown in Fig. 2, it appears that the vessel diameter undergoes phases of moderate contraction and dilation, likely due to the cyclic contraction of the smooth muscles of the arteriolar wall; as observed in RESULTS, this behavior causes a large data variability. Two-way ANOVA allowed distinguishing between the data variability attributable to the time dependence of the phenomenon under study and the specific experimental treatment. From the data shown in Fig. 2, it may be difficult to attribute a clear pattern and a level of significance to cyclic phenomena. Indeed, by assuming, in principle, a sinusoid-like pattern of the contraction-relaxation phase, the observation of such a phenomenon in our in vivo experimental approach would allow a clear recognition of the sinusoidal pattern only in cases where 1) the vessel diameter started simultaneously at the same phase of the cycle and 2) all the observed vessels displayed the same contractile pace. These two conditions are very unlikely to be simultaneously met in an in vivo approach, where one can actually expect to observe the effect of a complex superimposition of different periods and phases in the contraction pattern of the vessels. In this respect, the cyclic behavior seems to account for the great variability in the data points in Fig. 2; indeed, it is evident from the SD of the mean values that both the cyclic changes in diameter and the variability in the diameter values are greatly reduced in papaverine-treated animals in which the contractile capability of the smooth muscle cells is greatly reduced or abolished.

Despite the large data variability, two-way ANOVA allowed recognition during saline infusion of a significant trend toward vasodilation in the ~30-µm arterioles and an opposite behavior in the ~80-µm vessels. Although, in the 80-µm diameter vessels, the smooth muscle cell layer is not complete (6), these vessels seem to be well suited to constrict during the development of mild interstitial edema induced with the present experimental protocol. Indeed, it has been previously observed (8) that, using the same protocol, the percent segmental vascular flow resistance in the 30- to 80-µm-diameter arterioles increased by 24.5% with respect to control values, with a reduction in the total pulmonary vascular resistance by ~20%. A significant vasoactive response has also been observed in canine arterioles with a diameter < 70 µm after exposure to hypoxia (3). In addition, it has been shown (3) that the mechanical behavior of <100-µm arterioles is not uniform, the largest fraction of lobar vascular compliance being confined to vessels of diameter smaller than 40 µm (14). Taking into consideration these observations, we focused our attention on the <100-µm arterioles, distinguishing them in three different groups based mainly on the average caliper of the microvessel branching observed on the pleural surface: 1) the smallest arterioles with an average diameter of ~30 µm, emptying into the true capillary bed; 2) larger arterioles of ~80 µm in diameter, surrounded by a likely almost complete smooth muscle layer; and 3) arterioles of 50 µm in diameter, representing a functional intermediate condition. This grouping was supported by the experimental finding that an arteriolar response to saline loading was different according to the initial diameter; indeed, although a significant vasodilation was observed in the smallest vessels (Fig. 2A), the largest ones tended to constrict (Fig. 2C).

The regulation of vessel caliper is a complex phenomenon depending, on one hand, on an active process consisting of vasomotion of the vessel wall; in this respect, pulmonary precapillary vessels are provided, down to a diameter < 100 µm (5), by vasoconstrictor and vasodilator nerve fibers to smooth muscle cells. On the other hand, vessel caliper also depends on transmural pressure, defined as the difference between local intravascular pressure and the pressure acting at the outer surface of the vessel wall (tissue surface pressure). The intravascular pressure along the pulmonary superficial microcirculation has been measured through micropuncture in intact rabbit lungs both in control conditions (9) and during development of hydraulic edema (8). With the same technique, hydraulic pressure has been measured in the perivascular interstitial tissue layer (7, 10), but no recordings of local tissue surface pressure are available. Tissue surface pressure cannot be simply equated to the perivascular fluid interstitial pressure due to the force developing at the attachment of interstitial matrix macromolecules to the outer vessel wall. Therefore, a quantitative estimate of the transmural pressure governing the passive mechanical behavior of the in situ vessel surrounded by an organized matrix is at present not possible.

During saline infusion, a specific trend toward vasodilation was observed in the 30-µm vessels in which the diameters increased by ~20%; this effect is in line with a slight increase in intravascular pressure as previously documented (8) and/or with a vasodilation as suggested by the behavior of these vessels after papaverine treatment. Plasma volume expansion in papaverine-treated rabbits caused a moderate but significant further increase in the diameter of 30- and 50-µm arterioles, a result that could be accounted for by a moderate increase in intravascular pressure and/or an increase in mechanical vascular compliance. Plasma infusion caused, instead, vasoconstriction of the 80-µm vessels in which the diameter decreased by ~25%; this can be ascribed to an increase in vasomotor tone because the effect is reversed after papaverine treatment.

Previous data (7) have shown that perivascular interstitial pressure increases from approximately -10 to ~4 cmH2O when interstitial edema develops with the same infusion protocol adopted in this study, causing an ~10% increase in total extravascular lung water. Accordingly, in mild interstitial pulmonary edema, a marked increase in interstitial pressure does not seem to interfere with microvascular vasomotion. The different behavior of the 30- and 80-µm-diameter groups leads to the hypothesis of the existence of different mechanisms specifically controlling vasomotion in the smallest precapillary arterioles compared with the larger ones. The vasoactive responses could be related to the changes in shear stress (16). In a laminar flow regime, shear stress at the vessel wall (tau ) may be calculated as tau  = (8 · V · eta b)/D, where V is the average flow velocity, eta b is the blood viscosity, and D is the average vessel diameter. Saline infusion determined an ~37% drop in the initial Hct value. On the basis of the relationship (Einstein law) relating eta b to plasma viscosity (eta p) and Hct as eta b = eta p · (1+ 2.5Hct), one can calculate that eta b would decrease by ~19% over the 180-min period. Cardiac output was not recorded in the present study to avoid any possible disturbance to the image acquisition; however, in a series of experiments with the same infusion rate, directly measured cardiac output increased by ~40% of the control value over the 180-min period (7). For an increase in velocity in the arteriolar segment similar to the increase in cardiac output, these two factors would tend to increase tau  by 13%. In 80-µm vessels, the diameter decreased by ~20%, thereby causing a 40% increase in tau . Therefore, as mild interstitial edema is developing, the increase in shear stress in the 80-µm arterioles is associated with local vasoconstriction, suggesting that the augmented mechanical stimuli may shift the balance between vasoconstrictor and vasodilator mediators in favor of the former. In 30-µm arterioles, a similar calculation led to an estimate of a decrease of ~6% in tau  after saline infusion. This would represent an overestimate because recruitment in this vascular segment was shown to occur, and, therefore, the increase in velocity cannot be equated to the increase in cardiac output. In these vessels, the vasomotor balance is shifted toward vasodilation. Given the complexity and multiplicity of the biochemical pathways triggered by endothelial and smooth muscle cell levels by mechanical and chemical stimuli, a thorough analysis of the cellular mechanisms involved in smooth muscle contraction and/or relaxation is beyond the target of the present study and deserves to be fully examined.

The changes in diameter for 30- and 80-µm vessels during saline infusion are in keeping with the observation made by Negrini (8) based on the microvascular pressure profile observed in intact rabbit lung during hydraulic edema development. In fact, pressure data suggested, in the face of a drop in total flow resistance, an increase in the segmental resistance in the precapillary arterioles larger than ~50 µm and a simultaneous reduction in resistance at the precapillary level. The present study provides direct evidence that the specific site of arteriolar vasoconstriction during edema development in in situ lungs is at the level of the 80-µm arterioles. The opposite vasomotor behavior observed during saline infusion for 30- and 80-µm vessels might reflect a functional response of the pulmonary circulation aimed at maintaining the capillary pressure within narrow limits, thus offsetting the increase in microvascular filtration.

Slow infusion of a saline solution caused interstitial edema through the progressive decrease in plasma colloid osmotic pressure (causing an increase in Starling pressure gradient) and/or to a capillary recruitment, leading to an increase in exchange surface area (7, 8). The perivascular interstitium surrounding the smallest vessels is in continuity with the interstitial space surrounding capillaries. Hence, during saline infusion, an increase in periadventitial interstitial volume has to be expected close to the site of fluid filtration, i.e., around the smaller pre- and/or postcapillary vessels. In the present study, an increased perivascular fluid volume was observed during infusion only around the smallest 30-µm arterioles, whereas no perivascular cuff was observed around larger microvessels where vasoconstriction occurs. Administration of papaverine, causing an increased microvascular permeability (20), determined a greater and more uniform fluid accumulation around all types of vessels. The relatively small increase in perivascular volume, despite a marked increase in interstitial pressure observed in similar experimental conditions (7, 10), reflects the low compliance of the pulmonary interstitial matrix that provides a strong "tissue safety factor" preventing progression toward more severe stages of alveolar edema. This mechanical property is critically dependent on the proteoglycans of the matrix and the basement membrane in which integrity is to some extent affected by increased tissue hydration (10-13). Indeed, interstitial fluid accumulation during the transition toward the development of pulmonary edema was shown to be associated with fragmentation of heparan sulfate proteoglycans (perlecan) of the basal membranes and chondroitin sulfate proteoglycans (versican) of the fiber matrix; the loss of native architecture of these macromolecules determined an increase in microvascular permeability and tissue compliance, favoring fluid accumulation into the tissue (10-13).


    FOOTNOTES

Address for reprint requests and other correspondence: D. Negrini, Dipartimento di Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano, Via Mangiagalli 32, 20133 Milano, Italy (E-mail: Daniela.Negrini{at}unimi.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 January 2001; accepted in final form 22 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 281(6):L1464-L1471
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