Ann Thorac Surg 1999;67:323-328
© 1999 The Society of Thoracic Surgeons
Original Articles
Pulmonary artery occlusion and reperfusion causes microvascular constriction in the rabbit lung
Daniel L. Miller, MDa,
Andrew M. Roberts, PhDb
a Department of Cardiothoracic Surgery, University of Louisville School of Medicine, Louisville, Kentucky, USA
b Department of Physiology and Biophysics, and Center for Applied Microcirculatory Research, University of Louisville School of Medicine, Louisville, Kentucky, USA
Address reprint requests to Dr Miller, Division of General Thoracic Surgery, Mayo Clinic, 200 First St, SW, Rochester, MN 55905
e-mail: miller.danielmd{at}mayo.edu
Presented at the Forty-fourth Annual Meeting of the Southern Thoracic Surgical Association, Naples, FL, Nov 6–8, 1997
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Abstract
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Introduction. Reperfusion injury of the lung after ischemia is associated with altered alveolar blood flow and ventilation-perfusion mismatch, which is a significant cause of morbidity and mortality after lung transplantation. We examined the effect of ischemia and reperfusion on the tone of individual subpleural arterioles in the pulmonary circulation by using video microscopy with polarized epiillumination.
Methods. In 11 open-chested rabbits anesthetized with pentobarbital (2.3 to 2.5 kg), we ventilated the lungs through the lower trachea (air or 50% oxygen) and examined the response of subpleural arterioles (diameter 75 ± 13 µm) to ischemia (76 ± 32 min) of the right lung caused by occluding the right main pulmonary artery. Observations were made during baseline, occlusion, and during early (20 to 32 min) and late (48 to 63 min) reperfusion using a long working distance lens (550x) with a dipping cone held at the pleural surface while the lungs were statically inflated (10 cm H2O) with oxygen for brief periods. Data are expressed as mean ± standard deviation.
Results. Arteriolar diameter was decreased 57% ± 19% during early reperfusion. There was a decrease in blood flow and alveolar walls were pale, indicating reduced capillary perfusion. During late reperfusion, arteriolar diameter was diminished (19% ± 26%); flow was still reduced. Overall pulmonary vascular resistance increased during early reperfusion but returned to baseline level during late reperfusion. Arterial partial pressure of oxygen averaged 200 mm Hg during ischemia and reperfusion.
Conclusions. Constriction of small arterioles by ischemia and reperfusion can have a significant effect on the early phase of ventilation-perfusion mismatch and pulmonary dysfunction by altering alveolar perfusion. This response does not appear to be mediated by hypoxia because it was not prevented by ventilation with oxygen.
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Introduction
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Many pulmonary diseases or conditions are characterized by variable periods of ischemia. In acute lung injury, such as adult respiratory distress syndrome, and in pulmonary embolism, vascular obstruction and vasoconstriction together cause ischemia. In addition, acute pulmonary dysfunction after lung transplantation, otherwise known as reimplantation response, can result from interruption in blood supply [1]. As with other organs, the injury that develops after pulmonary ischemia might result from the ischemia or might be related to reperfusion after blood supply is restored. Because of this uncertainty, the injury that develops in this setting is often referred to as ischemia and reperfusion injury.
Acute lung injury damages the pulmonary vasculature at several levels thus impairing gas exchange. Vascular abnormalities include increased pulmonary artery pressure, altered vascular reactivity, vascular obstruction, intrapulmonary shunting, increased permeability, and ventilation and perfusion mismatch [2]. Thus, some form of vascular alteration plays a central role in the mechanism of acute lung injury.
During ischemia and reperfusion lung injury, pulmonary artery pressure could increase as a result of mechanical changes, such as vascular obstruction, edema, and decreased pulmonary compliance [3]. In addition, numerous vasoactive mediators are released as part of an inflammatory response to the tissue injury [4]. These mediators appear to cause an increase in pulmonary vascular resistance. More recent studies have also linked endothelial injury and dysfunction to the altered vascular reactivity [5, 6]. Many questions remain concerning the pathophysiologic mechanisms responsible for abnormal pulmonary vascular reactivity caused by ischemia and reperfusion of the lung and the vascular levels at which they occur.
Because in vivo responses of the pulmonary vasculature often have been based on the resistance calculated across the entire vascular bed, actual sites at which resistance changes occur remain unknown. There is limited information concerning effects of ischemia and reperfusion on specific vascular segments or on the microcirculation of the intact circulation [7]. Although the microcirculation is a target of the injury, few studies have characterized microvascular changes in the intact lung during ischemia and reperfusion. One purpose of the present study was to observe directly the effects of acute ischemia and reperfusion on basal tone of small pulmonary arterioles at the alveolar level in the intact lung. Whereas previous studies focused on vascular changes that developed in response to prolonged periods of pulmonary artery occlusion, our aim was to measure microvascular responses during short periods of ischemia and reperfusion. We tested the hypothesis that short periods of pulmonary ischemia followed by reperfusion, leads to arteriolar constriction and reduced alveolar perfusion. Using a method of intravital video microscopy, we observed individual subpleural pulmonary arterioles when the lungs were statically inflated to a constant pressure. This approach allowed us to evaluate regulatory changes in microvessels that normally have low tone and are an important distribution site of blood flow to the alveolar wall.
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Materials and methods
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Surgical preparation
Experiments used 11 male rabbits (2.3 ± 0.12 kg) anesthetized intramuscularly with a ketamine-xylazine mixture (1 mL/kg containing 37.5 mg ketamine and 5 mg xylazine per mL) followed by sodium pentobarbital injected into an ear vein or femoral vein slowly, to reach a loading dose of approximately 40 mg/kg. Supplemental doses of sodium pentobarbital (approximately 6 to 8 mg/kg) were given intravenously as needed to maintain an adequate level of anesthesia as defined by an absence of active corneal reflex, whisker twitching, and a pedal reflex to toe pinching. The trachea was cannulated low in the neck with a 4-mm noncuffed endotracheal tube while the rabbits breathed room air spontaneously. Systemic arterial blood pressure and heart rate were measured through a catheter placed in the left femoral artery that was connected to a pressure transducer (Gould-Statham P23ID; Gould Electronics, Oxnard, CA). A second catheter was placed in the left femoral vein for subsequent administration of anesthetics, drugs, and maintenance balanced saline solutions.
The rabbits were placed on the movable stage of an intravital microscope. Each animal was ventilated with air (supplemental oxygen when needed) by a cycle-triggered ventilator (model CTP-930; CWE, Inc, Ardmore, PA). Tracheal pressure was recorded from a port on the tracheal catheter, which was connected to a volumetric pressure transducer (PT-5; Grass Instruments Co, Quincy, MA). Tidal volume and frequency were set according to a standard nomogram and adjusted depending on arterial blood gases and pH, which were measured periodically. The chest was opened by a sternotomy, and a pericardial cradle was fashioned. To minimize damage to the lungs, the pleural cavities were opened but not entered. An ultrasonic flow probe was placed around the main proximal pulmonary artery to measure pulmonary blood flow (model T-206; Transonic Systems, Inc, Ithaca, NY). Pulmonary artery pressure was recorded through a catheter (3 Fr, Cook) placed into the main pulmonary artery through the right ventricle and secured with a silk ligature. Left atrial pressure was recorded through a catheter placed in the left atrium via the left atrial appendage. These catheters were connected to pressure transducers (Grass, P23ID). Catheters were flushed with heparinized saline (2.5 IU/mL) to maintain catheter patency. The signals representing systemic arterial blood pressure, heart rate, tracheal pressure, pulmonary artery flow, pulmonary artery pressure, and left atrial pressure were amplified and recorded by a polygraph system (Grass, Model 7D). A microscopic flow loop ligature or removable hemostatic clip was placed carefully around the right pulmonary artery transpericardially and medial to the superior vena cava so that pulmonary blood flow to the right lung could be occluded temporarily. A schematic drawing of the surgical preparation is shown in Figure 1.
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Fig 1. SURGICAL PREPARATION. Schematic drawing of instrumentation using a sternotomy approach. (P.A. = pulmonary artery; L.A. = left atrium).
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After placing the rabbit in the left decubitus position, the right chest was opened via the fifth intercostal space. The microscope objective was positioned above the right lung. Pulmonary arterioles at the pleural surface were observed using closed-circuit video microscopy as described previously [8], while lung inflation was maintained by switching from the ventilator to a system that delivered oxygen at a constant pressure of approximately 10 cm H2O.
Video microscopy system
Microscopic images were obtained using a modified triocular microscope (Zeiss, Thornwood, NY) designed for incident illumination. A color videocamera attached to the microscope transmitted images to a color monitor, which were stored on videotape for offline analysis using a videocassette recorder. The objective lens (Leitz L10 iris, numerical aperture 0.22) was attached to a glass dipping cone (5 mm diameter at the tip) that could be positioned just above the focal plane of the lens (Fig 2). Final combined optical and video magnification of the intravital microscopy system was approximately 550x. When the dipping cone was lowered gently to the pleural surface, movements of the lung were restricted and focus was easily maintained. Fine adjustment of focus was obtained by adjusting the position of the dipping cone with respect to the lens. With this method, vessels could be visualized for several minutes before raising the lens and switching back to the ventilator. Between observations the surface of the lung was kept moist with warm saline (0.9%) solution. We found that it was possible to make repeated observations at the same time without altering vessel diameter or alveoli size [8].
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Fig 2. INTRAVITAL VIDEO MICROSCOPE. Drawing of the triocular microscope using incident illumination. The objective lens (L10 iris) was attached to a glass dipping cone. A color video camera was attached to the microscope to transmit images to a color monitor and then stored on videotape. Final magnification of the microscope was 550x.
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Arterioles were identified by observing the direction of blood flow in relation to the bifurcation pattern. Internal vessel diameters were measured offline directly from the screen of the video monitor using a vernier caliper and a calibration factor (100 µm/45 mm on the monitor screen) determined from the image of a graticule that was recorded. Images of microvessels were recorded continuously during the observation periods and measured later at intervals using the stop-action feature of the video recorder.
Protocol
After instrumentation, rabbits were allowed to stabilize for approximately 30 minutes. Baseline data were obtained, which included systemic arterial blood pressure, heart rate, tracheal pressure, pulmonary artery blood flow, pulmonary artery pressure, and left atrial pressure. Pulmonary vascular resistance was calculated by dividing the pressure gradient between the pulmonary artery and the left atrium by the pulmonary blood flow. Throughout each experiment, arterial blood samples were taken periodically for blood-gas analysis.
A subpleural arteriole was identified and its video image was recorded for future measurement. The same arteriole was observed at least twice during the control period to insure that its diameter was stable. The right pulmonary artery was then occluded, and inspection of the pleural surface was done to insure ischemia within the lung. A video image was recorded of the ischemic arteriole that had been identified during baseline measurements. The right lung was ventilated throughout the entire period of ischemia along with the left lung. After 76 ± 32 minutes of ischemia, the right pulmonary artery occluder was released. Hemodynamic data and video images were obtained during reperfusion in the same manner as during baseline and occlusion. Measurements obtained during early reperfusion were taken between 20 and 33 minutes and during late reperfusion between 48 and 63 minutes after releasing the occluder. Times varied in each experiment because it was not always possible to locate the particular arteriole when desired. Hemodynamic data were recorded continuously, but arteriolar diameters were observed only when the lungs were held statically inflated for brief periods.
Data analysis
Arteriolar images were recorded continuously during observation periods, and diameters were measured at 10-second intervals and averaged over 30 seconds (each period usually included three to four measurements). Hemodynamic data were averaged over 15- to 30-second periods immediately before static lung inflation (during mechanical ventilation) and during static lung inflation when arterial measurements were taken. In each experiment, changes from baseline were calculated individually and averaged to give group means. Baseline values were taken as the average of two separate measurement periods during control. Data are reported as means ± standard deviation. Baseline values were compared with those obtained during occlusion and reperfusion by a one-way repeated measures analysis of variance. Differences were identified with a Tukey post-hoc comparison test [9]. A t test was used to compare values between the two different groups. For all of those comparisons, a p value of less than 0.05 was considered statistically significant.
Animal care
All rabbits received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1987).
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Results
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In the 11 rabbits, we observed subpleural pulmonary arterioles in the right lung while simultaneously recording systemic and pulmonary hemodynamic measurements, as described above. During baseline, the resting arteriolar diameter averaged 75 ± 13 µm (Fig 3). During occlusion of the right pulmonary artery, the subpleural arteriolar diameter averaged 43 ± 8 µm (Fig 4). The lung was pale and the vessels were patent, but without blood flow. When the occluder was removed, blood flow slowly returned to the right lung, and observations of the microvasculature showed vasoconstriction during early and late reperfusion. Reperfusion caused a significant (p < 0.014) decrease of 57% ± 19% in arteriolar diameter at 20 to 33 minutes of reperfusion (Fig 5). During late reperfusion (48 to 63 minutes) arteriolar diameter was decreased from baseline by 19% ± 26%, which was not significantly different from baseline values (Fig 6). Although arteriolar diameter recovered with time, the surrounding lung tissue appeared to be injured and edematous, and the alveolar walls were not as well perfused as at baseline. Oxygenation of the animals remained stable throughout the experimental protocol with arterial partial pressure of oxygen averaging above 200 mm Hg during ischemia and reperfusion.
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Fig 3. Subpleural arteriole during baseline conditions. Normal caliber of vessel is visible as well as normal alveolar perfusion.
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Fig 4. Subpleural arteriole during ischemia. There is no evidence of arteriolar blood flow, and scattered alveoli are pale secondary to ischemia.
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Fig 5. Subpleural arteriole during early (30-minute) reperfusion. Significant vasoconstriction is evident as well as minimal perfusion of the associated alveolar walls.
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Fig 6. Subpleural arteriole during late (60-minute) reperfusion. Arteriole diameters are near baseline dimensions; however, significant edema is present in arteriolar walls. The lung parenchyma appears to be injured, inflamed, and edematous.
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During early reperfusion, pulmonary artery blood flow was 28% ± 16% lower (p < 0.05). Pulmonary artery and left atrial pressures were not significantly different from baseline, but the calculated pulmonary vascular resistance across both lungs increased during occlusion. Mean arterial pressure decreased during early reperfusion, whereas pulmonary arterial flow slowed during ischemia and early reperfusion but improved during late reperfusion. The hemodynamic effects of ischemia and reperfusion of the right lung in conjunction with microvascular measurements are summarized in Table 1. A representative hemodynamic tracing is shown in Figure 7. During early reperfusion, arteriolar constriction and pulmonary vascular resistance were more closely related to changes in pulmonary vascular tone than to systemic blood pressure, cardiac output, and oxygenation. Arteriolar constriction continued during reperfusion when systemic blood pressure, heart rate, and oxygenation were not different from baseline (Table 2).
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Fig 7. Hemodynamic tracing of a representative animal taken at (A) baseline, (B) 10 minutes of reperfusion, (C) 30 minutes of reperfusion, and (D) 60 minutes of reperfusion. Microvascular observations were made during the periods when tracheal pressure was held constant (black dots). (PT = tracheal pressure; PPA = pulmonary artery pressure; QPA = pulmonary blood flow; PLA = left atrial pressure; ABP = systemic arterial blood pressure; HR = heart rate).
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Comment
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The idea that reperfusion of previously ischemic tissue can cause injury has been developed primarily from studies involving skeletal muscle, small intestine, and the heart [7, 10, 11]. In these organs, interruption of the arterial blood supply leads to tissue hypoxia. The lung, however, has been a more difficult organ to evaluate for ischemia and reperfusion injury, because lung parenchyma has access to three oxygen sources: a dual blood supply and the airways. The current study found that altered vascular reactivity occurs in the lung after 1 to 2 hours of ischemia followed by reperfusion. These findings are not likely artifacts of surgical technique or deterioration in the experimental protocol because they were related to the time course of the reperfusion protocol.
Our results show that in the intact lung, small pulmonary arterioles undergo a period of constriction in response to reperfusion, which indicates that these vessels play an important role in regulation of alveolar blood flow and are not passive conduits. Individual pulmonary arterioles smaller than 100 µm in diameter are capable of a no-reflow response to a relatively short period of ischemia and reperfusion. This behavior is similar to that which has been described for microvessels in skeletal muscle [7] and coronary vascular beds [11]. Thus, our finding indicates that arterioles close to the alveolar level have the ability to participate actively in the vascular response resulting in pulmonary hypertension and reduced gas exchange associated with pulmonary embolism or other causes of obstruction of the pulmonary vascular bed.
Although our data indicate that small arterioles several orders upstream from the pulmonary capillaries contribute to the increase in pulmonary vascular resistance during the earlier phase of reperfusion, additional studies are needed to determine the mechanism for the vasoconstriction. In other types of ischemic injury, the overall increase in pulmonary vascular resistance could result from mechanical factors or release of humoral mediators [12]. The vasoconstriction that we observed in the lung does not appear to be caused by hypoxia, which was prevented by ventilation of the lungs with a high oxygen mixture and occurred while the lungs were statically inflated with oxygen during microvascular observation periods. Arteriolar constriction and pulmonary vascular resistance returned to near-baseline values during late reperfusion.
In summary, this model seems to be useful for evaluating the physiologic consequences of pulmonary ischemia and reperfusion injury. This model could enable us to determine the mechanism of reperfusion injury in the lung and could help to determine which pharmaceutical interventions, if any, will minimize or eliminate this potentially fatal phenomenon from occurring after lung transplantation.
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Abstract
Introduction
