106,456-461
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
(1990)
The Effect of NO2 Exposure on Perfusate Distribution in Isolated Rat Lungs: Pulmonary versus Bronchial Circulation EDWARD M. POSTLETHWAIT,*,~ Pulmonary
AKHIL
BIDANI,*
AND
MICHAEL
J. EvANs*$,§
Research Laboratories, Departments of *Internal Medicine, ~Pharmacology and Toxicology, $Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77550: and §Shriners Burns Institute
Received
June 4, 1990; accepted
August
and
29, 1990
The Effect of NO, Exposure on Perfusate Distribution in Isolated Rat Lungs: Pulmonary versus Bronchial Circulation. POSTLETHWAIT, E. M., BIDANI, A., AND EVANS, M. J. (1990). Toxical. Appl. Pharmacol. 106, 456-461. Isolated rat lung (IPL) studies have suggested that the pulmonary uptake of inhaled nitrogen dioxide (NO,) is governed via a chemical reaction-dependent process which results in NO*-derived reaction products diffusing into the vascular space. Experimental results indicated that substantial proportions of this reactive absorption occur in distal sites. However, gas phase deposition in proximal locations cannot be ruled out due to the lack of information on bronchial perfusion in rat IPL preparations. Consequently, we evaluated the presence of pulmonary-to-bronchial anastomotic perfusate flow in control and NOz-exposed (10.3 ppm) rat IPL. Monastral blue (MB) was used as a vascular marker and was infused into the pulmonary artery catheter either for recirculation at time zero or as an end-experiment (60 min) bolus. In addition, MB was infused into control in situ preparations to observe intact bronchial circulations. Lungs were prepared for routine evaluation by light microscopy. In situ MB was observed in all pulmonary and bronchial vessels. In IPL, MB was observed only in far terminal airway-associated vessels. No differences were observed in MB distribution between bolus (endexperiment) and recirculated (time zero) applications. NO* exposure produced no effect on MB distribution. We conclude that in rat IPL: (1) negligible anastomotic flow occurs from the pulmonary into the bronchial circulation, (2) nonedemagenic NOz exposures do not alter existing perfusate distribution, and (3) the perfusate appearance of inhalation-derived species results from gas phase deposition only in distal sites which have ready accessibility to the pulmonary circulation. Q 1990 Academic
Press. Inc.
Recently, we have employed an isolated rat lung preparation (IPL) to study the interactions between nitrogen dioxide (NOJ and the pulmonary airspace surface (Postlethwait and Bidani, 1989, 1990). This model allows for more reproducible control of gas/tissue interfacial conditions than can be achieved with in vivo preparations. Experimental results have suggested that the absorption of inhaled NO2 from pulmonary airspaces is, in part, rate-limited by irreversible chemical reaction(s) between NO2 and epithelial constituents. This mode of gas absorption differs from those commonly identified as mediators of pulmo0041-008X/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
nary gas uptake, i.e., physical solubility, vascular perfusion, gas phase and tissue diffusion, and reversible chemical reaction. Additional experimental evidence suggests that the reactive absorption and resulting chemical transformation of inhaled NO2 occurs on or near the airspace surface (Postlethwait and Bidani, 1989; Postlethwait et al., 1990). NO2 is a clearly established pulmonary toxin which, as a result of low concentration exposures, generates lesions relatively specific to the distal airways (Evans and Freeman, 1980). Both the site-specific pathology and mathematical models (Overton and Miller, 456
NO2
EFFECTS
ON
IPL
PERFUSATE
1988; Miller et al., 1982) imply that significant NO? deposition occurs in distal locations. In the IPL, substantial proportions of absorbed NOz appear, as transformation products, in the perfusion media (Postlethwait and Bidani, 1989; Postlethwait and Mustafa, 1989). In this preparation, perfusate inflow is limited to a catheter securedwithin the pulmonary artery. Consequently, the vascular spaceappearance (clearance) of NOz-derived reaction products may indicate that their formation occurred in locations perfused by the pulmonary circulation rather than in more proximal areasnormally perfused by the bronchial circulation. However, the degreeto which clearance of reactive absorption products can be used to establish and/or corroborate the anatomic location of gasphasedeposition is not clear since information concerning the cross-perfusion between pulmonary and bronchial circulations in rat IPL preparations is limited. Vascular anastomoses,which connect the bronchial systemic and pulmonary circulations, have been extensively documented (Deffebach et al., 1987). Functionally, anastomotic flow compensates for injury-induced alterations in pulmonary blood flow and may be related to survival of airway tissuesduring lung transplantation (Lakshminarayan et al., 1987; Saunders et al., 1984). The extent and direction of anastomotic blood flow depends on a number of factors which are principally related to the respective hydrostatic pressuresoccurring within the two systems(Deffebach et al., 1987). In our isolated lung preparation, pulmonary vascular pressureswould be expected to exceed bronchial pressuressince the systemic circulation is disrupted during surgical isolation. However, if bronchial flow were to occur, any effluent would collect within the single recirculated perfusate pool and, therefore, would not be uniquely recognized. Accordingly, we wished to establishwhether flow from the pulmonary to the bronchial circulation existed and, if so, to which portions of the conducting airways. Isolated rat lungs were perfused with a vascular indicator (Monastral blue, MB) with and without NO2 ex-
DISTRIBUTION
457
posure, and the anatomic location of the indicator was evaluated. We found no evidence of Monastral blue in any location except for the terminal airways and, therefore, concluded that perfusion of the more proximal conducting airways is negligible in the rat IPL. The perfusate appearance of agents inspired by isolated rat lungs (or their reaction products) must result from deposition only in peripheral siteswhich have direct accessibility to the pulmonary circulation. METHODS Animals. Viral antigen-free, male, Sprague-Dawley rats (Harlan Sprague-Dawley, Houston, TX), weighing between 250 and 275 gl were used for all studies. Animals were allowed free access to food and water until induction of anesthesia. Immediately prior to lung isolation, animals were briefly anesthetized with 4% halothane in air, followed by 70 mg/kg intraperitoneal Na pentobarbital. Foot pinch was used to determine depth of anesthesia. Isolated lung preparation. Details of the lung surgical isolation and IPL and NO* exposure systems have been previously described (Postlethwait and Bidani, 1990). Briefly, after tracheal cannulation, ventilation was maintained by positive pressure support while the chest was opened and the pulmonary artery cannulated via a trocar inserted into the right ventricle. The pulmonary vascular bed was flushed (50 ml, perfusion media) while the heart was resected and the lungs removed en bloc. Ventilation and perfusion were briefly interrupted while the lungs were transferred to the temperature-controlled (37°C) IPL apparatus. Lungs were placed in an artificial thorax and the respective cannulae connected for either perfusate or ventilation gas delivery. The perfusate was continuously recirculated and equilibrated with 95% sir/5% COZ. Subatmospheric ventilation (50 breaths/min) was accomplished by cyclically altering pressures (pleural) within the lung chamber. In order to limit rebreathing, humidified ventilation gas (95% sir/5% CO& maintained as a bias flow across the tracheal cannula, was delivered in excess of peak inspiratory flow. Ventilation gas was collected via a sample port located downstream from the tracheal cannula connection. The lungs were monitored for transpulmonary pressures (1 O/3 cm HZO), ventilation gas system pressure and flow, and breath-by-breath tidal volume (2.2 + 0.2 ml). Perfusate flow to the lung (gravity driven) was adjusted to 12- 15 ml/min which correspondingly produced pulmonary artery pressures ranging from 8 to 10 cm H20. Perfusate consisted of Krebs’ Ringer bicarbonate buffer (pH 7.4) containing 8.33 mM glucose and 6 g% bovine serum albumin (fraction V).
458
POSTLETHWAITE,
BIDANI,
For NOZ exposures, NO2 (105 ppm in Nz; Liquid Carbonic, Houston, TX) was diluted into the ventilation gas and rapidly delivered to the tracheal cannula. Lungs were exposed to 10.3 f 0.1 ppm NOz. The system for control of gas flow, mixing, and delivery consisted of materials resistant to chemical interaction. Gas samples were collected into Teflon bags from which three separate aliquots of known volume were withdrawn into glass syringes containing Saltzman reagent (Intersociety Committee, 1977). NOz concentrations were determined colotimetrically against daily standard curves. Monascral blue. Monastral blue B (3% suspension; Sigma Chemical Co., St. Louis. MO), an insoluble copper-based phthalocyanine, was used as the vascular marker (Joris et al., 1982). MB was applied under three experimental conditions. ( 1) At the end of the 60 min ventilation/perfusion period, in both control and NO,-exposed IPL, 1.0 ml Monastral blue was manually infused directly into the flowing perfusate within the pulmonary artery cannula. After infusion, the cannula was immediately clamped and the lungs,were removed for fixation. (2) Monastral blue (1.5 ml) was added to the perfusate shortly after installation of the lungs$nto the IPL apparatus and allowed to recirculate for the duration of the experimental period (both control and NO,-exposed). (3) In order to visualize Monastral blue distribution in a model with an intact bronchial circulation, an open-chested in sitzl preparation was utilized. Animals were anesthetized as above and placed on positive-pressure support ventilation concomitant with a midline thoracotomy, and a loose suture was threaded around the pulmonary artery and aorta. One milliliter of Monastral blue was slowly infused into the ascending vena cava after which the arterial suture was pulled tight. The lungs and heart were quickly removed en bloc for fixation. Lung jixation and histology. Lungs were fixed by tracheal instillation of 2% glutaraldehyde (in 0.08 M cacodylic buffer, pH 7.4) to a final pressure of 25 cm HzO. Instillation pressures were maintained for 30 min, after which the trachea was tied and the lungs were placed in a specimen vial containing the fixative. The left lung was embedded in paraffin, sectioned at 6 Km, and counterstained with 1% eosin. Six different sections from each lung were studied. Each section contained the main axial bronchus and several lateral bronchi (large airways) in addition to bronchioles and terminal bronchioles (small airways). The lamina propria associated with each airway and the alveoli were examined for the presence or absence of Monastral blue by two observers. The tracheas were not prepared for microscopic analysis.
RESULTS
In situ controls. Infusion of Monastral blue into the ascending vena cava resulted in rapid color distribution throughout the entire ani-
AND
EVANS
ma1body. Histologically, Monastral blue was observed in all pulmonary and bronchial vesselswhich included the terminal airspace vascular beds as well as the bronchial vesselslocated within the lamina propria of both large and small conducting airways. Isolated perfused lungs. During the surgical isolation procedure, lungs blanched a uniform white when the vascular bed was initially flushed with perfusate. By grossexamination, after 60 min of ventilation/perfusion, the bolus injections of Monastral blue produced a homogeneous blue color over the entire pleural surface, indicating that perfusion was still active to all original sites. We observed no MBassociatedcoloration of IPL tracheas. NO2 exposure did not alter color distribution of the bolus injection. When Monastral blue was allowed to recirculate, the initial color intensity of the lung surfaces diminished with time, most likely as a result of particles adhering to the inner walls of the perfusate tubing. However, color distribution on the pleural surfaces remained uniform and was not altered by NO2 exposure. Neither control perfusions nor NO2 exposure of isolated rat lungs (60 min) produced overt alterations in either lung fluid balance or histology. Relative to the histology samples obtained from in situ preparations, light microscopic examination of isolated perfused lungs revealed no evidence of fluid accumulation in perivascular, peribronchial, or alveolar spaces.No extravascular Monastral blue particles were observed in experimental preparations. Table 1 summarizes our observations of pulsed and recirculated Monastral blue distribution within both control and N02-exposed isolated perfused lungs. We observed Monastral blue in alveolar-associated vesselsunder all experimental conditions. However, regardless of the isolated lung experimental condition, we could not detect the vascular marker in any airway-associated vessels, suggesting that negligible anastomotic perfusate flow occurred from the pulmonary into the bronchial circulation.
NO2 TABLE HISTOLOGIC PRESENCE (+) CULAR SPACES
EFFECTS
ON
I% shu--controP IPL control--pulse’ IPL N02-pulse IPL control-recirculated IPL NO*--recirculate
PERFUSATE
1
EVALUATION
OF
MONASTRAL
OR
(-)
WITHIN
ABSENCE
AlV~OlaJ CapillXieS
+ + + + +
BLUE INTRAVAS-
Bronchial Experimental preparation
IPL
Small airways
vessels” Large airways
+ -
y Vessels located within the lamina propria of small ways (bronchioles and terminal bronchioles) and large ways (main axial bronchi and lateral bronchi).
f airair-
b Monastral blue injected (vena cava) in situ. ’ At termination of ventilation/perfusion. Monastral blue injected into pulmonary artery cannula and flow immediately stopped. dAfter initial equilibration, Monastral blue added to perfusate and allowed to recirculate throughout experimental period (60 min).
DISCUSSION In this study we determined the regional distribution of an insoluble intravascular marker infused into control and NO?-exposed isolated perfused lungs. In addition, the marker (Monastral blue) was infused into an in situ preparation in which the systemic bronchial circulation remained intact. NO2 exposures were included in order to establish whether NO? inhalation altered perfusate distribution in isolated rat lungs. The results suggest that, under conditions of isolated ventilation/perfusion of pulmonary artery-only cannulated rat lungs, pulmonary-to-bronchial anastomotic perfusate flow does not occur. This generates little or no perfusion of conducting airways. However, the existing distribution of perfusate throughout the pulmonary vascular bed remains essentially unaffected by either the duration of isolated ventilation/perfusion (~60 min) or NO2 exposure. Studies in other isolated lung models (dogs and guinea pigs) had previously demonstrated active pul-
DISTRJBUTION
459
monary-to-bronchial anastomotic flow (Barman et al., 1988; Kroll et al., 1987). This contrast to our current results is, most likely, attributable either to species-related anatomical differences or, potentially, to methodologicalinduced influences (i.e., increased pulmonary venous pressures due to left atria1 cannulation). The maintenance of normal lung fluid homeostasisduring IPL studiesis of fundamental interest since formation of pulmonary edema will eventually limit the usefulnessof this experimental model. Fluid redistribution may generate significant complications when attempting to study the chemical fate, translocation, and distribution of inhaled substances. Based on gravimetric evaluation, previous acute exposuresof our preparation to lessthan 20 ppm NOz (~90 min) have not been demonstrably edemagenic(Postlethwait and Bidani, 1989; Postlethwait and Mustafa, 1989). Our histologic evaluations in this study revealed that control and NO,?-exposedIPL were indistinguishable and that in both, no evident fluid accumulations occurred in alveolar, peribronchial, or perivascular spaces. During the initial IPL surgical isolation procedure, flushing of the pulmonary vasculature with cell-free perfusate resulted in the pleural surface blanching to a uniform white (O’Neil and Tierney, 1974). Areas which were not adequately perfused remained conspicuously pink due to the underlying red blood cells. In a similar fashion, we evaluated perfusate distribution during the experimental period by observing the uniformity of pleural surface coloration induced by Monastral blue infusion. This simple technique does not determine absolute flow rates but rather is a visual assessmentof the persistenceof localized flow at the time of infusion. When the Monastral blue was added to the perfusate early in the experimental period and allowed to recirculate, the depth of the pleural surface coloration tended to decreasewith time, most likely due to MB particles adhering to the walls of the IPL tubing and glassware.This phenomenon inadvertently facilitated continuous
460
POSTLETHWAITE.
evaluation of flow distribution since anatomic areas with impaired flow would have remained darker blue as the MB particle concentration in the circulating perfusate was reduced by tubing adherence. On the basis of these subjective criteria, we suggest that acute exposure of isolated lungs to nonedemagenic NO2 concentrations does not alter perfusate delivery or flow continuity in peripheral lung tissue. We have previously employed isolated lung techniques to investigate the reactive uptake mechanisms, chemical fate, and reaction product partitioning of inhaled NOz (Postlethwait and Bidani, 1989, 1990). Perfusate analysis formed an integral element of those studies. However, interpretations of the resulting data were restricted since the anatomic profile of actively perfused regions was unknown. Our ability to differentiate whether the perfusion-related clearance of reaction products represented proximal versus distal toxicant/lung interactions was limited. The results from this study suggest that only the far distal portions of the airspaces are perfused (Table 1). Therefore, translocation of deposition, transformation, and/or metabolic products into the perfusate is limited to regions which are within ready diffusional distance from the vascular bed directly perfused (nonanastomotic flow) by the pulmonary artery. While we could detect no MB particles in the subsurface vasculature of the IPL terminal bronchiolar lamina propria, these airways have suitably thin walls and are in sufficiently close proximity to the alveolar septal vasculature that diffusible reaction products could translocate into the IPL perfusate. Perfusion through the bronchial circulation may not be required for product removal from these locations. Our current results, in combination with results from earlier studies (Postlethwait and Bidani, 1989) suggest that significant NOz deposition and associated chemical interaction occur in distal airspaces resulting in substantial amounts of NO*-derived products translocating to intravascular spaces perfused directly by the pulmonary artery.
BIDANI.
AND
EVANS
Insoluble oxidant gases such as NO-, and O3 produce pathologic lesions specific to the terminal conducting airways and proximal alveolar regions (Evans and Freeman, 1980). The site-specificity of the injury may relate to regional deposition and/or localized hypersusceptibility. Mathematical models have predicted that NOz uptake rates are greatest in the same distal locations where the NO*induced pathology develops (Overton and Miller, 1988; Miller et al., 1982). The cumulative results from these and our prior isolated lung investigations support the mathematical model-based predictions. A significant amount of the total gas phase removal of inhaled NO2 occurs in distal locations since a large proportion of the resulting NOz-related reaction products appear in the IPL perfusate (Postlethwait and Bidani, 1989; Postlethwait and Mustafa, 1989). Due to the lack of an active bronchial circulation, if notable deposition had occurred in proximal airways, only limited amounts of NOz-derived products could have diffused into the IPL perfusate. Therefore, it is reasonable to assume that the site-specific distal injuries which result from NOz inhalation are, in part, a regional uptake phenomenon and not solely attributable to localized tissue hypersensitivity. Because in the isolated rat lung preparation only the far distal airspaces communicate with actively perfused vascular spaces, events which occur in the conducting airways are not reflected in the perfusate. As a consequence, the applicability of the IPL for studying upper airway metabolism or tissue diffusion of gas phase deposition products may be limited. While short-term observation of the gas phase (only) phenomenon may be undertaken, the IPL would not be the model of choice for investigating the tissue disposition of airborne contaminants which are deposited primarily in the upper airways. The highly heterogeneous distribution of tissue perfusion may limit the experimental applications and certainly necessitates attention to the potential absence of regional flow in isolated lung preparations.
NO*
EFFECTS
ON
IPL
ACKNOWLEDGMENTS This work was supported in part by a grant from the National Institute of Environmental Health Sciences (ES049520 1) and Shriners Crippled Children Hospital Grant 15813. We thank Mr. Shannon Langford, Mr. Robert Cox, and Ms. Ann Burke for their technical assistance.
REFERENCES BARMAN, S. A., ARDELL, J. L., PARKER, J. C., PERRY, M. L.. AND TAYLOR, A. E. (1988). Pulmonary and systemic blood flow contributions to upper airways in canine lung. Amer. J. Physiol. 255, H 1130-H 1135. DEFFEBACH, M. E.: CHARAN, N. B., LAKSHMINARAYAN, S., AND BUTLER, J. (1987). The bronchial circulation: Small, but a vital attribute ofthe lung. Amer. Rev. Respir. Dis. 135, 463-48 1. EVANS, M. J., AND FREEMAN, G. (1980). Morphological and pathological effects of NOz on the rat lung. In Nitrogen Oxides und Their Effecl on Health (S. D. Lee, Ed.), pp. 243-265. Ann Arbor Science, Ann Arbor. Intersociety Committee for Air Sampling and Analysis (1977). Tentative method of analysis for nitrogen dioxide content of the atmosphere (Greiss-Saltzman reaction). In Methods ofAir Sampling andAnalysis, pp. 524-534. American Public Health Association, Washington, DC. JORIS, I., DEGIROLAMI, U., WORTHAM, K.. AND MAJNO, G. (1982). Vascular labelling with Monastral blue B. Stain Technol. 75, 177-183. KROLL, F.. KARLSSON, J.-A., AND PERSSON, C. G. A. (1987). Tracheobronchial microvessels perfused via the
PERFUSATE pulmonary
461
DISTRIBUTION artery
in guinea-pig
isolated
lungs.
Acta
Physiol. Stand. 129, 445-446. LAKSHMINARAYAN, S., JINDAL, S. K., KIRK, W., AND BUTLER, J. (1987). Acute increases in anastomotic bronchial systemic to pulmonary blood flow due to generalized lung injury. f. Appl. Physiol. 62(6), 235% 2361. MILLER, F. J., OVERTON, J. H., MYERS, E. T., AND GRAM HAM, J. A. (1982). Pulmonary dosimetry of nitrogen dioxide in animals and man. In Air Pollution by Nitrogen o,uides, pp. 377-386. Elsevier Scientific, Amsterdam. O’NEIL, J. J.. AND TIERNEY, D. F. (1974). Rat lung metabolism: Glucose utilization by isolated perfused lung and tissue slices. Amer. J. Physiol. 226, 867-873. OVERTON, J. H., AND MILLER, F. J. (1988). Absorption of inhaled reaction gases. In Toxicology of the Lung. pp. 477-507. Raven Press, New York. POSTLETHWAIT. E. M., AND BIDANI, A. (1989). Pulmonary disposition of inhaled NO,-nitrogen in isolated rat lungs. Tox-icol. Appl. Pharmacol. 98, 303-3 12. POSTLETHWAIT, E. M., AND BIDANI, A. (1990). Reactive uptake governs the pulmonary airspace removal of inhaled nitrogen dioxide. J. Appl. P&siol. 68(2), 594603. POSTLETHWAIT, E. M., LANGFORD, S. D., AND BIDANI, A. (1990). Reactive absorption of nitrogen dioxide by pulmonary epithelial lining fluid. J. Appl. Phvsiol. 69(2), 523-531. POSTLETHWAIT, E. M., AND MUSTAFA, M. G. (1989). The effect of altered dose on NO, uptake and transformation in isolated lungs. J. Toxicol. Environ. Health 26, 497507. SAUNDERS, N. R., EGAN, T. M., CHAMBERLAIN, D., AND COOPER, J. D. (1984). Cyclosporine and bronchial healing in canine lung transplantation. J. Thoruc. Cur-
diovasc. Surg. 88, 993-999.
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
(1990)
The Effect of NO2 Exposure on Perfusate Distribution in Isolated Rat Lungs: Pulmonary versus Bronchial Circulation EDWARD M. POSTLETHWAIT,*,~ Pulmonary
AKHIL
BIDANI,*
AND
MICHAEL
J. EvANs*$,§
Research Laboratories, Departments of *Internal Medicine, ~Pharmacology and Toxicology, $Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77550: and §Shriners Burns Institute
Received
June 4, 1990; accepted
August
and
29, 1990
The Effect of NO, Exposure on Perfusate Distribution in Isolated Rat Lungs: Pulmonary versus Bronchial Circulation. POSTLETHWAIT, E. M., BIDANI, A., AND EVANS, M. J. (1990). Toxical. Appl. Pharmacol. 106, 456-461. Isolated rat lung (IPL) studies have suggested that the pulmonary uptake of inhaled nitrogen dioxide (NO,) is governed via a chemical reaction-dependent process which results in NO*-derived reaction products diffusing into the vascular space. Experimental results indicated that substantial proportions of this reactive absorption occur in distal sites. However, gas phase deposition in proximal locations cannot be ruled out due to the lack of information on bronchial perfusion in rat IPL preparations. Consequently, we evaluated the presence of pulmonary-to-bronchial anastomotic perfusate flow in control and NOz-exposed (10.3 ppm) rat IPL. Monastral blue (MB) was used as a vascular marker and was infused into the pulmonary artery catheter either for recirculation at time zero or as an end-experiment (60 min) bolus. In addition, MB was infused into control in situ preparations to observe intact bronchial circulations. Lungs were prepared for routine evaluation by light microscopy. In situ MB was observed in all pulmonary and bronchial vessels. In IPL, MB was observed only in far terminal airway-associated vessels. No differences were observed in MB distribution between bolus (endexperiment) and recirculated (time zero) applications. NO* exposure produced no effect on MB distribution. We conclude that in rat IPL: (1) negligible anastomotic flow occurs from the pulmonary into the bronchial circulation, (2) nonedemagenic NOz exposures do not alter existing perfusate distribution, and (3) the perfusate appearance of inhalation-derived species results from gas phase deposition only in distal sites which have ready accessibility to the pulmonary circulation. Q 1990 Academic
Press. Inc.
Recently, we have employed an isolated rat lung preparation (IPL) to study the interactions between nitrogen dioxide (NOJ and the pulmonary airspace surface (Postlethwait and Bidani, 1989, 1990). This model allows for more reproducible control of gas/tissue interfacial conditions than can be achieved with in vivo preparations. Experimental results have suggested that the absorption of inhaled NO2 from pulmonary airspaces is, in part, rate-limited by irreversible chemical reaction(s) between NO2 and epithelial constituents. This mode of gas absorption differs from those commonly identified as mediators of pulmo0041-008X/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
nary gas uptake, i.e., physical solubility, vascular perfusion, gas phase and tissue diffusion, and reversible chemical reaction. Additional experimental evidence suggests that the reactive absorption and resulting chemical transformation of inhaled NO2 occurs on or near the airspace surface (Postlethwait and Bidani, 1989; Postlethwait et al., 1990). NO2 is a clearly established pulmonary toxin which, as a result of low concentration exposures, generates lesions relatively specific to the distal airways (Evans and Freeman, 1980). Both the site-specific pathology and mathematical models (Overton and Miller, 456
NO2
EFFECTS
ON
IPL
PERFUSATE
1988; Miller et al., 1982) imply that significant NO? deposition occurs in distal locations. In the IPL, substantial proportions of absorbed NOz appear, as transformation products, in the perfusion media (Postlethwait and Bidani, 1989; Postlethwait and Mustafa, 1989). In this preparation, perfusate inflow is limited to a catheter securedwithin the pulmonary artery. Consequently, the vascular spaceappearance (clearance) of NOz-derived reaction products may indicate that their formation occurred in locations perfused by the pulmonary circulation rather than in more proximal areasnormally perfused by the bronchial circulation. However, the degreeto which clearance of reactive absorption products can be used to establish and/or corroborate the anatomic location of gasphasedeposition is not clear since information concerning the cross-perfusion between pulmonary and bronchial circulations in rat IPL preparations is limited. Vascular anastomoses,which connect the bronchial systemic and pulmonary circulations, have been extensively documented (Deffebach et al., 1987). Functionally, anastomotic flow compensates for injury-induced alterations in pulmonary blood flow and may be related to survival of airway tissuesduring lung transplantation (Lakshminarayan et al., 1987; Saunders et al., 1984). The extent and direction of anastomotic blood flow depends on a number of factors which are principally related to the respective hydrostatic pressuresoccurring within the two systems(Deffebach et al., 1987). In our isolated lung preparation, pulmonary vascular pressureswould be expected to exceed bronchial pressuressince the systemic circulation is disrupted during surgical isolation. However, if bronchial flow were to occur, any effluent would collect within the single recirculated perfusate pool and, therefore, would not be uniquely recognized. Accordingly, we wished to establishwhether flow from the pulmonary to the bronchial circulation existed and, if so, to which portions of the conducting airways. Isolated rat lungs were perfused with a vascular indicator (Monastral blue, MB) with and without NO2 ex-
DISTRIBUTION
457
posure, and the anatomic location of the indicator was evaluated. We found no evidence of Monastral blue in any location except for the terminal airways and, therefore, concluded that perfusion of the more proximal conducting airways is negligible in the rat IPL. The perfusate appearance of agents inspired by isolated rat lungs (or their reaction products) must result from deposition only in peripheral siteswhich have direct accessibility to the pulmonary circulation. METHODS Animals. Viral antigen-free, male, Sprague-Dawley rats (Harlan Sprague-Dawley, Houston, TX), weighing between 250 and 275 gl were used for all studies. Animals were allowed free access to food and water until induction of anesthesia. Immediately prior to lung isolation, animals were briefly anesthetized with 4% halothane in air, followed by 70 mg/kg intraperitoneal Na pentobarbital. Foot pinch was used to determine depth of anesthesia. Isolated lung preparation. Details of the lung surgical isolation and IPL and NO* exposure systems have been previously described (Postlethwait and Bidani, 1990). Briefly, after tracheal cannulation, ventilation was maintained by positive pressure support while the chest was opened and the pulmonary artery cannulated via a trocar inserted into the right ventricle. The pulmonary vascular bed was flushed (50 ml, perfusion media) while the heart was resected and the lungs removed en bloc. Ventilation and perfusion were briefly interrupted while the lungs were transferred to the temperature-controlled (37°C) IPL apparatus. Lungs were placed in an artificial thorax and the respective cannulae connected for either perfusate or ventilation gas delivery. The perfusate was continuously recirculated and equilibrated with 95% sir/5% COZ. Subatmospheric ventilation (50 breaths/min) was accomplished by cyclically altering pressures (pleural) within the lung chamber. In order to limit rebreathing, humidified ventilation gas (95% sir/5% CO& maintained as a bias flow across the tracheal cannula, was delivered in excess of peak inspiratory flow. Ventilation gas was collected via a sample port located downstream from the tracheal cannula connection. The lungs were monitored for transpulmonary pressures (1 O/3 cm HZO), ventilation gas system pressure and flow, and breath-by-breath tidal volume (2.2 + 0.2 ml). Perfusate flow to the lung (gravity driven) was adjusted to 12- 15 ml/min which correspondingly produced pulmonary artery pressures ranging from 8 to 10 cm H20. Perfusate consisted of Krebs’ Ringer bicarbonate buffer (pH 7.4) containing 8.33 mM glucose and 6 g% bovine serum albumin (fraction V).
458
POSTLETHWAITE,
BIDANI,
For NOZ exposures, NO2 (105 ppm in Nz; Liquid Carbonic, Houston, TX) was diluted into the ventilation gas and rapidly delivered to the tracheal cannula. Lungs were exposed to 10.3 f 0.1 ppm NOz. The system for control of gas flow, mixing, and delivery consisted of materials resistant to chemical interaction. Gas samples were collected into Teflon bags from which three separate aliquots of known volume were withdrawn into glass syringes containing Saltzman reagent (Intersociety Committee, 1977). NOz concentrations were determined colotimetrically against daily standard curves. Monascral blue. Monastral blue B (3% suspension; Sigma Chemical Co., St. Louis. MO), an insoluble copper-based phthalocyanine, was used as the vascular marker (Joris et al., 1982). MB was applied under three experimental conditions. ( 1) At the end of the 60 min ventilation/perfusion period, in both control and NO,-exposed IPL, 1.0 ml Monastral blue was manually infused directly into the flowing perfusate within the pulmonary artery cannula. After infusion, the cannula was immediately clamped and the lungs,were removed for fixation. (2) Monastral blue (1.5 ml) was added to the perfusate shortly after installation of the lungs$nto the IPL apparatus and allowed to recirculate for the duration of the experimental period (both control and NO,-exposed). (3) In order to visualize Monastral blue distribution in a model with an intact bronchial circulation, an open-chested in sitzl preparation was utilized. Animals were anesthetized as above and placed on positive-pressure support ventilation concomitant with a midline thoracotomy, and a loose suture was threaded around the pulmonary artery and aorta. One milliliter of Monastral blue was slowly infused into the ascending vena cava after which the arterial suture was pulled tight. The lungs and heart were quickly removed en bloc for fixation. Lung jixation and histology. Lungs were fixed by tracheal instillation of 2% glutaraldehyde (in 0.08 M cacodylic buffer, pH 7.4) to a final pressure of 25 cm HzO. Instillation pressures were maintained for 30 min, after which the trachea was tied and the lungs were placed in a specimen vial containing the fixative. The left lung was embedded in paraffin, sectioned at 6 Km, and counterstained with 1% eosin. Six different sections from each lung were studied. Each section contained the main axial bronchus and several lateral bronchi (large airways) in addition to bronchioles and terminal bronchioles (small airways). The lamina propria associated with each airway and the alveoli were examined for the presence or absence of Monastral blue by two observers. The tracheas were not prepared for microscopic analysis.
RESULTS
In situ controls. Infusion of Monastral blue into the ascending vena cava resulted in rapid color distribution throughout the entire ani-
AND
EVANS
ma1body. Histologically, Monastral blue was observed in all pulmonary and bronchial vesselswhich included the terminal airspace vascular beds as well as the bronchial vesselslocated within the lamina propria of both large and small conducting airways. Isolated perfused lungs. During the surgical isolation procedure, lungs blanched a uniform white when the vascular bed was initially flushed with perfusate. By grossexamination, after 60 min of ventilation/perfusion, the bolus injections of Monastral blue produced a homogeneous blue color over the entire pleural surface, indicating that perfusion was still active to all original sites. We observed no MBassociatedcoloration of IPL tracheas. NO2 exposure did not alter color distribution of the bolus injection. When Monastral blue was allowed to recirculate, the initial color intensity of the lung surfaces diminished with time, most likely as a result of particles adhering to the inner walls of the perfusate tubing. However, color distribution on the pleural surfaces remained uniform and was not altered by NO2 exposure. Neither control perfusions nor NO2 exposure of isolated rat lungs (60 min) produced overt alterations in either lung fluid balance or histology. Relative to the histology samples obtained from in situ preparations, light microscopic examination of isolated perfused lungs revealed no evidence of fluid accumulation in perivascular, peribronchial, or alveolar spaces.No extravascular Monastral blue particles were observed in experimental preparations. Table 1 summarizes our observations of pulsed and recirculated Monastral blue distribution within both control and N02-exposed isolated perfused lungs. We observed Monastral blue in alveolar-associated vesselsunder all experimental conditions. However, regardless of the isolated lung experimental condition, we could not detect the vascular marker in any airway-associated vessels, suggesting that negligible anastomotic perfusate flow occurred from the pulmonary into the bronchial circulation.
NO2 TABLE HISTOLOGIC PRESENCE (+) CULAR SPACES
EFFECTS
ON
I% shu--controP IPL control--pulse’ IPL N02-pulse IPL control-recirculated IPL NO*--recirculate
PERFUSATE
1
EVALUATION
OF
MONASTRAL
OR
(-)
WITHIN
ABSENCE
AlV~OlaJ CapillXieS
+ + + + +
BLUE INTRAVAS-
Bronchial Experimental preparation
IPL
Small airways
vessels” Large airways
+ -
y Vessels located within the lamina propria of small ways (bronchioles and terminal bronchioles) and large ways (main axial bronchi and lateral bronchi).
f airair-
b Monastral blue injected (vena cava) in situ. ’ At termination of ventilation/perfusion. Monastral blue injected into pulmonary artery cannula and flow immediately stopped. dAfter initial equilibration, Monastral blue added to perfusate and allowed to recirculate throughout experimental period (60 min).
DISCUSSION In this study we determined the regional distribution of an insoluble intravascular marker infused into control and NO?-exposed isolated perfused lungs. In addition, the marker (Monastral blue) was infused into an in situ preparation in which the systemic bronchial circulation remained intact. NO2 exposures were included in order to establish whether NO? inhalation altered perfusate distribution in isolated rat lungs. The results suggest that, under conditions of isolated ventilation/perfusion of pulmonary artery-only cannulated rat lungs, pulmonary-to-bronchial anastomotic perfusate flow does not occur. This generates little or no perfusion of conducting airways. However, the existing distribution of perfusate throughout the pulmonary vascular bed remains essentially unaffected by either the duration of isolated ventilation/perfusion (~60 min) or NO2 exposure. Studies in other isolated lung models (dogs and guinea pigs) had previously demonstrated active pul-
DISTRJBUTION
459
monary-to-bronchial anastomotic flow (Barman et al., 1988; Kroll et al., 1987). This contrast to our current results is, most likely, attributable either to species-related anatomical differences or, potentially, to methodologicalinduced influences (i.e., increased pulmonary venous pressures due to left atria1 cannulation). The maintenance of normal lung fluid homeostasisduring IPL studiesis of fundamental interest since formation of pulmonary edema will eventually limit the usefulnessof this experimental model. Fluid redistribution may generate significant complications when attempting to study the chemical fate, translocation, and distribution of inhaled substances. Based on gravimetric evaluation, previous acute exposuresof our preparation to lessthan 20 ppm NOz (~90 min) have not been demonstrably edemagenic(Postlethwait and Bidani, 1989; Postlethwait and Mustafa, 1989). Our histologic evaluations in this study revealed that control and NO,?-exposedIPL were indistinguishable and that in both, no evident fluid accumulations occurred in alveolar, peribronchial, or perivascular spaces. During the initial IPL surgical isolation procedure, flushing of the pulmonary vasculature with cell-free perfusate resulted in the pleural surface blanching to a uniform white (O’Neil and Tierney, 1974). Areas which were not adequately perfused remained conspicuously pink due to the underlying red blood cells. In a similar fashion, we evaluated perfusate distribution during the experimental period by observing the uniformity of pleural surface coloration induced by Monastral blue infusion. This simple technique does not determine absolute flow rates but rather is a visual assessmentof the persistenceof localized flow at the time of infusion. When the Monastral blue was added to the perfusate early in the experimental period and allowed to recirculate, the depth of the pleural surface coloration tended to decreasewith time, most likely due to MB particles adhering to the walls of the IPL tubing and glassware.This phenomenon inadvertently facilitated continuous
460
POSTLETHWAITE.
evaluation of flow distribution since anatomic areas with impaired flow would have remained darker blue as the MB particle concentration in the circulating perfusate was reduced by tubing adherence. On the basis of these subjective criteria, we suggest that acute exposure of isolated lungs to nonedemagenic NO2 concentrations does not alter perfusate delivery or flow continuity in peripheral lung tissue. We have previously employed isolated lung techniques to investigate the reactive uptake mechanisms, chemical fate, and reaction product partitioning of inhaled NOz (Postlethwait and Bidani, 1989, 1990). Perfusate analysis formed an integral element of those studies. However, interpretations of the resulting data were restricted since the anatomic profile of actively perfused regions was unknown. Our ability to differentiate whether the perfusion-related clearance of reaction products represented proximal versus distal toxicant/lung interactions was limited. The results from this study suggest that only the far distal portions of the airspaces are perfused (Table 1). Therefore, translocation of deposition, transformation, and/or metabolic products into the perfusate is limited to regions which are within ready diffusional distance from the vascular bed directly perfused (nonanastomotic flow) by the pulmonary artery. While we could detect no MB particles in the subsurface vasculature of the IPL terminal bronchiolar lamina propria, these airways have suitably thin walls and are in sufficiently close proximity to the alveolar septal vasculature that diffusible reaction products could translocate into the IPL perfusate. Perfusion through the bronchial circulation may not be required for product removal from these locations. Our current results, in combination with results from earlier studies (Postlethwait and Bidani, 1989) suggest that significant NOz deposition and associated chemical interaction occur in distal airspaces resulting in substantial amounts of NO*-derived products translocating to intravascular spaces perfused directly by the pulmonary artery.
BIDANI.
AND
EVANS
Insoluble oxidant gases such as NO-, and O3 produce pathologic lesions specific to the terminal conducting airways and proximal alveolar regions (Evans and Freeman, 1980). The site-specificity of the injury may relate to regional deposition and/or localized hypersusceptibility. Mathematical models have predicted that NOz uptake rates are greatest in the same distal locations where the NO*induced pathology develops (Overton and Miller, 1988; Miller et al., 1982). The cumulative results from these and our prior isolated lung investigations support the mathematical model-based predictions. A significant amount of the total gas phase removal of inhaled NO2 occurs in distal locations since a large proportion of the resulting NOz-related reaction products appear in the IPL perfusate (Postlethwait and Bidani, 1989; Postlethwait and Mustafa, 1989). Due to the lack of an active bronchial circulation, if notable deposition had occurred in proximal airways, only limited amounts of NOz-derived products could have diffused into the IPL perfusate. Therefore, it is reasonable to assume that the site-specific distal injuries which result from NOz inhalation are, in part, a regional uptake phenomenon and not solely attributable to localized tissue hypersensitivity. Because in the isolated rat lung preparation only the far distal airspaces communicate with actively perfused vascular spaces, events which occur in the conducting airways are not reflected in the perfusate. As a consequence, the applicability of the IPL for studying upper airway metabolism or tissue diffusion of gas phase deposition products may be limited. While short-term observation of the gas phase (only) phenomenon may be undertaken, the IPL would not be the model of choice for investigating the tissue disposition of airborne contaminants which are deposited primarily in the upper airways. The highly heterogeneous distribution of tissue perfusion may limit the experimental applications and certainly necessitates attention to the potential absence of regional flow in isolated lung preparations.
NO*
EFFECTS
ON
IPL
ACKNOWLEDGMENTS This work was supported in part by a grant from the National Institute of Environmental Health Sciences (ES049520 1) and Shriners Crippled Children Hospital Grant 15813. We thank Mr. Shannon Langford, Mr. Robert Cox, and Ms. Ann Burke for their technical assistance.
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PERFUSATE pulmonary
461
DISTRIBUTION artery
in guinea-pig
isolated
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Acta
Physiol. Stand. 129, 445-446. LAKSHMINARAYAN, S., JINDAL, S. K., KIRK, W., AND BUTLER, J. (1987). Acute increases in anastomotic bronchial systemic to pulmonary blood flow due to generalized lung injury. f. Appl. Physiol. 62(6), 235% 2361. MILLER, F. J., OVERTON, J. H., MYERS, E. T., AND GRAM HAM, J. A. (1982). Pulmonary dosimetry of nitrogen dioxide in animals and man. In Air Pollution by Nitrogen o,uides, pp. 377-386. Elsevier Scientific, Amsterdam. O’NEIL, J. J.. AND TIERNEY, D. F. (1974). Rat lung metabolism: Glucose utilization by isolated perfused lung and tissue slices. Amer. J. Physiol. 226, 867-873. OVERTON, J. H., AND MILLER, F. J. (1988). Absorption of inhaled reaction gases. In Toxicology of the Lung. pp. 477-507. Raven Press, New York. POSTLETHWAIT. E. M., AND BIDANI, A. (1989). Pulmonary disposition of inhaled NO,-nitrogen in isolated rat lungs. Tox-icol. Appl. Pharmacol. 98, 303-3 12. POSTLETHWAIT, E. M., AND BIDANI, A. (1990). Reactive uptake governs the pulmonary airspace removal of inhaled nitrogen dioxide. J. Appl. P&siol. 68(2), 594603. POSTLETHWAIT, E. M., LANGFORD, S. D., AND BIDANI, A. (1990). Reactive absorption of nitrogen dioxide by pulmonary epithelial lining fluid. J. Appl. Phvsiol. 69(2), 523-531. POSTLETHWAIT, E. M., AND MUSTAFA, M. G. (1989). The effect of altered dose on NO, uptake and transformation in isolated lungs. J. Toxicol. Environ. Health 26, 497507. SAUNDERS, N. R., EGAN, T. M., CHAMBERLAIN, D., AND COOPER, J. D. (1984). Cyclosporine and bronchial healing in canine lung transplantation. J. Thoruc. Cur-
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