Segmental microvascular permeability in ischemia-reperfusion injury in rat lung

Pavel L. Khimenko and Aubrey E. Taylor

Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Segmental microvascular permeabilities were measured using pre- and postalveolar vessel capillary filtration coefficient (Kfc) values (ml · min-1 · cmH2O-1 · 100 g-1) in isolated rat lungs subjected to ischemia-reperfusion (I/R). Total Kfc values measured in flowing and nonflowing lungs were highly correlated (r = 0.98, P < 0.0001). Kfc values were then measured in another group of lungs under no-flow conditions when airway pressure was increased to 20 cmH2O and either the arterial or venous pressure was elevated to 7-8 cmH2O to measure the prealveolar and postalveolar Kfc values. Control total and postalveolar Kfc values were 0.0225 ± 0.001 and 0.0219 ± 0.001 ml · min-1 · cmH2O-1 · 100 g-1, respectively, and the prealveolar permeability was extremely small (0.00003 ± 0.00005 ml · min-1 · cmH2O-1 · 100 g-1). Kfc values were again made in nonflowing lungs that had been subjected to 45 min of ischemia followed by 30 min of reperfusion. After I/R, the total membrane Kfc increased 10-fold to 0.2597 ± 0.006 ml · min-1 · cmH2O-1 · 100 g-1, the prealveolar Kfc increased to 0.0677 ± 0.003 ml · min-1 · cmH2O-1 · 100 g-1, and the postalveolar Kfc increased to 0.1354 ± 0.008 ml · min-1 · cmH2O-1 · 100 g-1 (P < 0.05 for all I/R values). These data indicate that normal solvent microvascular permeability was predominantly postalveolar, and after I/R damage, the postalveolar (venular) permeability comprised 52% of the total, whereas the prealveolar and alveolar vessels comprised only 27 and 23%, respectively, of the total Kfc.

alveolar; extra-alveolar permeability; filtration coefficient


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY EDEMA has been classically described as resulting from either increases in pulmonary capillary pressure, loss of endothelial barrier function, or a combination of both. Alveolar edema does not occur until the tendency for fluid to be filtered from the capillaries can no longer be opposed by edema safety factors, i.e., increases in tissue fluid pressure, protein osmotic gradient, and lymph flow. Our studies have shown that 45 min of ischemia followed by 30 min of reperfusion results in extensive endothelial damage. However, the relative contribution of alveolar and extra-alveolar microvessels to the total fluid filtration occurring in this form of endothlial injury is not known. It has been thought that the microvascular permeability changes would occur in alveolar capillaries, since they contain such a large membrane surface area. However, published studies indicate that substantial filtration can occur in extra-alveolar vessels in lung and increases to higher levels when lungs are damaged by oleic acid. In fact, after this form of endothelial damage, the capillary filtration coefficient (Kfc) of postalveolar and alveolar vessels comprises 46 and 45% of the total Kfc (Kfc,TOT), respectively (1, 9, 10). Studies by Mitzner and Robotham (11) show that 50% of the total pulmonary filtration in dog lungs is located within the alveolar vessels of the lungs, whereas pre- and postalveolar permeabilities contribute 23 and 27%, respectively. Studies conducted by Iliff (8) and Albert et al. (2) show smaller contributions of alveolar vessels (36 and 38%, respectively). All studies quoted above were conducted in dog lungs subjected to hydrostatic stress or damaged with air bubbles, glass-bead embolization, or oleic acid.

The distribution of microvascular leakage sites between alveolar and extra-alveolar vessels is important to know in order to understand different lung pathologies. When adult respiratory distress syndrome patients are ventilated using high positive end-expiratory pressure values (17), only a small correlation was found between lung water accumulation and the airway pressure, but a better correlation was observed between lung water and the level of pulmonary artery pressure relative to pleural pressure. Because the inflammatory response in the peripheral circulation occurs mainly in the postcapillary segments of the circulation, it is important to know which segments of the lung's circulation are damaged during the inflammatory process. This information will be useful to compare data collected from various pulmonary endothelial monolayers with similar studies conducted using isolated lungs. The present study was designed to determine the relative contributions of alveolar and extra-alveolar vessels to the total microvascular permeability increases occurring in rat lungs subjected to ischemia-reperfusion (I/R) injury. In this study, the classic Kfc measured in nonflowing rat lungs was used to determine the segmental permeability of the lung vascular compartments in lungs subjected to I/R.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated perfused lung. Male CD rats (250-350 g body wt; Charles River Laboratories) were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). A tracheostomy was performed, and lungs were ventilated with 21% O2-5% CO2 (Harvard rodent ventilator, model 683) at a rate of 50 breaths/min, a tidal volume of 15 ml/kg body wt, and a positive end-expiratory pressure of 2 cmH2O. After median sternotomy, heparin (300 IU) was injected into the right ventricle, and cannulas were placed in the pulmonary artery and left ventricle. The heart, lungs, and mediastinal structures were removed en bloc and suspended from a force-displacement transducer (Grass, model FT03) into a humidified chamber to monitor weight changes. The lungs were perfused at a constant flow of 0.03 ml/g body wt using Earle's balanced salt solution (200 mg/l CaCl2, 400 mg/l KCl, 97.9 mg/l MgSO4, 6,800 mg/l NaCl, 140 mg/l NaH2PO4 · H2O, 1,000 mg/l glucose, and 10 mg/l phenol red) containing 0.21% NaHCO3 and 4% BSA. The first 75 ml of perfusate, which contained a large amount of residual blood cells and plasma, were discarded. An additional 50 ml of perfusate were used for recirculation. Pulmonary arterial and pulmonary venous pressures were continuously monitored with pressure transducers (Gould-Statham, model P23 ID) and were recorded on a polygraph recorder (Grass, model 7E).

Kfc,TOT. Kfc was used as a measurement of capillary permeability with methods previously described (6, 9) and modified for use in nonflowing rat lungs (3). Briefly, the pulmonary arterial and venous lines were connected by side branches through three-way stopcocks to a reservoir containing the perfusion solution. The reservoir was adjusted to produce an initial no-flow pressure of 7.5 cmH2O. After an isogravimetric state was achieved, the reservoir was rapidly elevated by an additional 7-8 cmH2O for 15 min. The increase in lung weight was recorded, and the rate of weight change (Delta W/Delta t) calculated during the 6- to 15-min interval was analyzed using a linear regression of the log10-transformed rates of weight changes per minute. The initial rate of weight gain was then calculated by extrapolating Delta W/Delta t to time 0. Kfc was calculated by dividing Delta W/Delta t at time 0 by the change measured in no-flow pressure that occurred on perfusion reservoir elevation. The filtration coefficient was normalized using the baseline lung wet weight and is expressed as milliliters per minute per centimeter water per 100 g lung tissue and is referred to as Kfc,TOT.

Measurements of Kfc under no-flow conditions. To measure Kfc under no-flow conditions, a technique modified from Becker et al. (3) was used. The pulmonary arterial and venous lines were connected by side branches through three-way stopcocks to a reservoir containing the perfusion solution, and the reservoir was adjusted to produce an initial no-flow pressure of 7.5 cmH2O. Next, the perfusion pump was turned off, and the reservoir was connected to the lungs (n = 7). After 2-3 min of equilibration, the reservoir was rapidly elevated and adjusted to 15 cmH2O for 15 min, and the lung weight gain was recorded. Kfc was calculated by the method used for measurements made during flow conditions.

Segmental Kfc values. After Kfc,TOT was evaluated, the an alveolar pressure was raised to 20 cmH2O, and the lung (n = 5) was allowed to again attain an isogravimetric pressure at 7.5 cmH2O, which required ~2-3 min. It has been shown that an alveolar inflation pressure of 20 cmH2O does not cause changes in either pulmonary vascular resistance or Kfc values in rat lungs (14), and values as high as 25 cmH2O in dog lungs produced no damage (2, 10). After equilibration, only the prealveolar pressure was elevated by 7-8 cmH2O for an additional 15 min, and the lung weight was recorded. Next, the prealveolar pressure was decreased to 7.5 cmH2O, and the lung was allowed to attain a new isogravimetric state. Only the postalveolar pressure was increased by 7-8 cmH2O, and the lung weight gain again was recorded for 15 min. The pre-Kfc and post-Kfc values were then calculated from the tracings as previously described. After this baseline measurement, segmental Kfc and Kfc,TOT values were again measured in nonflowing lungs after 45 min of ischemia, followed by 30 min of reperfusion. Kfc,TOT was measured at 30 min, and the pre- and post-Kfc values were measured after 50 and 70 min after reperfusion, respectively, under the same nonflowing conditions and pressure elevations as used in the control measurements.


    RESULTS
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INTRODUCTION
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DISCUSSION
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As shown in Fig. 1, the no-flow Kfc [Kfc(NO)] was significantly correlated with the Kfc measured during flow. The correlation between Kfc measurements made in these two different conditions is very high (r = 0.98, P < 0.0001) and is described by the following regression equation
<IT>K</IT><SUB>fc(NO)</SUB> = 0.64 × [<IT>K</IT><SUB>fc</SUB>] − 0.03
where [Kfc] is Kfc concentration.


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Fig. 1.   Measurement of no-flow (NO) capillary filtration coefficient (Kfc) and flow Kfc in the same lungs (n = 7).

The Kfc,TOT and postalveolar average Kfc values (means ± SD) were 0.0225 ± 0.001 and 0.0219 ± 0.001 ml · min-1 · cmH2O-1 · 100 g-1, respectively, for baseline conditions and are not statistically different, as shown in Fig. 2. The prealveolar permeability was very small, averaging only 0.00003 ± 0.00005 ml · min-1 · cmH2O-1 · 100 g-1. After I/R, Kfc,TOT increased (a 10-fold increase) to 0.2597 ± 0.006 ml · min-1 · cmH2O-1 · 100 g-1. Prealveolar Kfc increased to 0.0677 ± 0.003 ml · min-1 · cmH2O-1 · 100 g-1 (P < 0.05 compared with baseline). Postalveolar Kfc accounted for 50% of the Kfc change (0.1354 ± 0.008 ml · min-1 · cmH2O-1 · 100 g-1; P < 0.05 compared with baseline) associated with I/R injury.


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Fig. 2.   Effect of ischemia-reperfusion (I/R) on segmental microvascular permeability in isolated rat lungs (n = 5). For baseline conditions, postalveolar permeability accounts for 97% of the total endothelial permeability. After I/R injury, the segmental microvascular permeabilities were 26, 22, and 52% for prealveolar, alveolar, and postalveolar segments, respectively. * P < 0.05 compared with baseline.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study clearly shows significant damage of the extra-alveolar microvessels in the total microcirculation of isolated rat lungs subjected to ischemia and reperfusion. The postalveolar (venular) segment of the microvasculature was damaged to the greatest extent, since it comprises ~50% of the Kfc,TOT after I/R challenge. However, it is important to emphasize that both prealveolar and alveolar Kfc values were extremely low in control lungs and both increased significantly after I/R. We have no direct measurements of permeability in the alveolar segment of the microcirculation and used a calculated value. This calculated value may represent an underestimation of permeability of alveolar capillaries, since it was measured later in the experimental protocol. Also, Lamm and Albert (9) studied lung weight gain when pressures in the arterial and venous extra-alveolar vessels were increased either individually or simultaneously. Results of their study indicate that transvascular fluid flux is greater when the hydrostatic stress is applied to each segment separately rather than when applied to both segments simultaneously. Lamm et al. (10) showed that oleic acid challenge increased both postalveolar and alveolar Kfc values significantly in isolated dog lungs. Our studies are in agreement with these segmental permeabilities measured in isolated dog lungs damaged by oleic acid, since both alveolar and postalveolar permeabilities were also significantly increased above control values in our present study. In addition, the prealveolar Kfc was also increased by I/R. A recent study by Chetham et al. (4) showed that thapsigargin causes extensive postalveolar damage but much less damage in the alveolar and prealveolar vessels. Rather than defining alveolar vessels in the classic fashion, Chetham et al.'s study used ultrastructural studies to determine leak sites. However, the postalveolar vessels were the primary filtration sites in control lungs of this study and also had the largest filtration after I/R damage compared with prealveolar and alveolar vessels. It is well known that leukocytes are found in all segments of the pulmonary circulation (5, 7) and are required for I/R damage (12, 13, 16). However, Doerschuk (5) showed that 97% of the marginated cells are in the alveolar vessels and the pools in arterioles and venous segments are extremely small. Also, leukocytes roll in the post- and prealveolar vessels and communicate with endothelial cells and could certainly produce endothelial permeability changes. Although our study is the first done to evaluate pre- and postalveolar sites of damage in I/R, it indicates that postalveolar vessels are more permeable than either prealveolar or alveolar vessels in normal lungs. However, the permeability of all segments increased after the endothelial barrier damage associated with I/R, but the greatest damage occurred within the postalveolar segments of the lung's circulation.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-22549.


    FOOTNOTES

A portion of this work was presented in abstract form at the Experimental Biology 1998 meeting (FASEB J. 45: A777, 1998).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. E. Taylor, Dept. of Physiology, College of Medicine, MSB 3024, Univ. of South Alabama, Mobile, AL 36688-0002 (E-mail: ataylor{at}jaguar1.usouthal.edu).

Received 3 September 1998; accepted in final form 24 February 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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2.   Albert, R. K., S. Lakshminarayan, N. B. Charan, W. Kirk, and J. Butler. Extra-alveolar vessel contribution to hydrostatic pulmonary edema in in situ dog lung. J. Appl. Physiol. 54: 1010-1017, 1983[Abstract/Free Full Text].

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6.   Drake, R., K. A. Gaar, and A. E. Taylor. Estimation of the filtration coefficient of pulmonary exchange vessels. Am. J. Physiol. 234 (Heart Circ. Physiol. 3): H266-H274, 1978[Abstract/Free Full Text].

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Am J Physiol Lung Cell Mol Physiol 276(6):L958-L960
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