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Pathophysiological Role of Endothelins in Pulmonary Microcirculatory Disorders Due to Intestinal Ischemia and Reperfusion
Journal of Surgical Research 87, 143–151 (1999) Article ID jsre.1999.5694, available online at http://www.idealibrary.com on
Pathophysiological Role of Endothelins in Pulmonary Microcirculatory Disorders Due to Intestinal Ischemia and Reperfusion 1 Hiroshi Mitsuoka, M.D.,* ,2 Naoki Unno, M.D., Ph.D.,* Takashi Sakurai, Ph.D.,† Hiroshi Kaneko, M.D., Ph.D.,* Shohachi Suzuki, M.D., Ph.D.,* Hiroyuki Konno, M.D., Ph.D.,* Susumu Terakawa, M.D., Ph.D.,† and Satoshi Nakamura, M.D., Ph.D.* *Second Department of Surgery and †Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan Submitted for publication November 2, 1998
Background. This study was conducted to investigate pulmonary microcirculatory disorders caused by intestinal ischemia reperfusion (IIR), and the pathophysiological roles of endothelin (ET) in acute lung injury (ALI). Methods. Male rats were pretreated with normal saline or a nonselective ET receptor antagonist (TAK-044) and subjected to IIR (60 min of intestinal ischemia and 180 min of reperfusion). The right upper lobe of the lung was examined by intravital confocal microscopy. Results. The size of arterioles and venules was not significantly reduced during IIR, but the functional capillary density (FCD) decreased significantly. TAK044 improved the pulmonary microhemodynamics, inhibiting the accumulation of leukocytes, the pulmonary edema, and the decrease of FCD. Conclusions. In the early stage of IIR, pulmonary microhemodynamics seemed more likely to be disturbed by the decrease of FCD, than by arteriolar or venular vasoconstriction. ETs decrease the FCD, promoting the interaction between leukocytes and pulmonary vessels. © 1999 Academic Press Key Words: pulmonary microcirculation; endothelin; ARDS; lung; intestinal ischemia and reperfusion; multiple organ failure; laser confocal microscopy; functional capillary diameter; in vivo; rat. INTRODUCTION
Intestinal ischemia and reperfusion (IIR) have been regarded as primary adaptive responses to shock. Acute lung injury (ALI) after IIR is a well-established model of 1
Supported in part by a research grant from the Ministry of Education, Science and Culture of Japan (06671195). 2 To whom reprint requests should be addressed at Second Department of Surgery, Hamamatsu University School of Medicine, 3600 Handacho, Hamamatsu 431-31 Japan. Fax: 81-53-435-2273.
remote organ injury, related to acute respiratory distress syndrome (ARDS). This experimental lung injury has been commonly studied from the aspect of pulmonary edema. However, the augmentation of vascular permeability is only one facet of ALI. For instance, increased pulmonary vascular resistance (PVR) and pulmonary arterial hypertension (PAH) contribute to the morbidity or mortality of ALI [1]. Nevertheless, these aspects of this experimental ALI have been discussed in only a few studies [2, 3]. Recent studies have demonstrated that ALI after IIR is multifactorial and mediated by various vasoactive substances. Endothelins (ETs) are released from the damaged endothelial cells (ECs) during IIR [4, 5], while the intravenous injection of ET-1 can cause pulmonary edema [6]. Accordingly, ETs are highly anticipated to promote ALI, whereas the pathophysiological roles of ETs under these circumstances have not been elucidated. ETs have both vasoconstrictive and proinflammatory actions [7–9]. Therefore, morphological and functional studies should be combined with the evaluation of edema, when the pathophysiological roles of such a multifunctional substance are investigated. In a previous study, we made a minute observation of subpleural microcirculation, utilizing intravital confocal microscopy [3]. This technique enabled us to observe the functional and morphological changes in pulmonary microcirculation, as well as to evaluate the severity of vascular leakage under an intravital condition. Microcirculatory events, which might be induced by ET-related mechanisms and causative of increased PVR or PAH, could be directly observed by this new technique. This study was conducted to investigate pulmonary microcirculation in the early phase of ALI from various aspects. In addition to the three-dimensional observation of pulmonary edema and the analysis of pulmonary blood distribution [3], the morphological changes
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0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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in subpleural microvessels (the arteriolar or venular diameter and the functional capillary density) were investigated. Furthermore, the pathophysiological roles of ET in the early phase of ALI were indirectly investigated, using a nonselective receptor antagonist. MATERIALS AND METHODS The experiments were approved by the Institutional Animal Use and Care committee of the Hamamatsu University School of Medicine, and were performed in accordance with the National Institute of Health guidelines for the care and handling of animals. Animal preparation. Male Sprague-Dawley rats (290 –340 g) were fasted with free access to water for 12 h before operation. An animal was anesthetized by intraperitoneal injection of 25 mg/kg of sodium pentobarbital and placed on an acrylic plate in the semi-left decubitus position. The abdomen was opened through a midline incision. The superior mesenteric artery (SMA) was looped with a thread of 3-O nylon. A polyethylene tubing was inserted onto the SMA and guided the looping thread out of the body. The SMA was clamped by pulling the thread to cause the intestinal ischemia. The right jugular vein was cannulated with polyethylene tubing (PE-50), through which normal saline was infused continuously at a rate of 1 mL/h. The right carotid artery was also cannulated with another line of polyethylene tubing (PE-50) connected to a blood pressure transducer. The mean arterial pressure (MAP) was recorded with a multichannel monitor (78342A Monitor, Hewlett Packard, Palo Alto, CA). A tracheotomy was made and polyethylene tubing (PE-240) was inserted into the trachea. With a ventilator for small animals (Model SN-480-7, Shimano, Tokyo, Japan), respiration of the rat was maintained mechanically with a tidal volume of 2.0 mL, a breath rate of 70 per minute, and positive end expiratory pressure (PEEP) of 2 cm H 2O. To prevent atelectasis, the lung was overinflated periodically (about once in 1 h) with an exhale port of the ventilator occluded for 2 breaths. The right chest wall was carefully removed not to hurt the lung, and the chest opening was covered with a sheet of plastic wrap to minimize evaporation. The sheet was removed during the microscopic observation, and the exposed lung was moistened by topical application of normal saline. A cover glass (24 3 60 3 0.17 mm) was set horizontally above the chest, so that the surface of the exposed lung would contact with the glass gently in the inspiratory phase. The stage of the microscope (Labphoto, Nikon, Tokyo, Japan) was detached, and an acrylic plate holding the animal was mounted on the microscope instead. Fluorescence microscopy. Bovine serum albumin labeled with fluorescein isothiocynate (FITC-BSA, Sigma, St. Louis, MO) was dissolved in normal saline at a concentration of 10 mg/mL before the initial injection. Vecronium bromide (Masculax, Organon Inc, the Netherlands) was dissolved in normal saline at 1 mg/mL. The animal received 7 mg of FITC-BSA and 0.3 mg of vecronium bromide intravenously soon after the thoracotomy, and an initial observation was performed 20 min after that. FITC-BSA and vecronium were administered at a rate of 2 and 0.1 mg per 1 h, to maintain the fluorescence level of the vasculature and the chemical paralysis. Videomicroscopic data were obtained while the lung was briefly (,40 s) inflated with air pressurized at 10 cm H 2O. A confocal laser microscope utilizing a microlens-attached Nipkow disk as a scanner (CSU-10, Yokogawa, Tokyo, Japan) was connected to the microscope. The right upper lobe of the lung was observed with an 310 objective lens (Type E, NA 5 0.5, Nikon, Tokyo, Japan) or an 320 objective lens (FL, NA 5 0.5, Nikon) under epifluorescence illumination with a 488-nm beam from a multiline argon laser (Model 2213150MLYJ, Cyonics, San Jose, CA). The output level of the laser was kept constant throughout the experiments. Images were detected with an image-intensified charge-coupled device (ICCD) camera (DAS-512, Imagista, Tokyo, Japan). The rate of image detection was 4 frames/s with the 320 objective lens, and it was decreased to 2 frames/s with the
310 lens. The contrast of the image was enhanced in real time with a digital image processor (ARGUS-20, Hamamatsu photonics, Hamamatsu, Japan), and both the mode and the degree of enhancement were unchanged through the experiments. The processed image was displayed on a video monitor screen (PVM-1454Q, Sony, Tokyo, Japan) and recorded simultaneously with an S-VHS format recorder (AG-7750, Panasonic, Osaka, Japan). A series of confocal images were acquired, while the objective lens was shifted stepwise along the optical axis. Experimental procedure. Rats were divided into three groups. The control and IIR groups received normal saline as a vehicle. The TAK group was pretreated with a nonselective ET receptor antagonist (TAK-044, Takeda Chemical Industries, Osaka, Japan). Either normal saline or TAK-044 was intravenously administered to animals before the operative procedure. TAK-044 was dissolved to a concentration of 1 mg/mL with normal saline and intravenously administered at a dose of 3 mg/kg. The SMA in the IIR (n 5 5) and the TAK group (n 5 5) of rats was clamped for 60 min and then released for 180 min. Rats in the control group (n 5 5) were treated similarly, but without the intestinal ischemia. Hemodynamic data were obtained 20 min after thoracotomy, at 30 and 55 min during the SMA clamp, and at 60, 90, 120, 150, and 180 min during the reperfusion. Videomicroscopic images were obtained and recorded 20 min after thoracotomy (i.e., an initial observation), at 30 and 55 min during the SMA clamp, and at 60, 120, and 180 min during the reperfusion. The observation was started with the 310 objective lens. The observation with the 320 objective lens was made 5 min after that. Three-dimensional images of the subpleural vasculature were reconstructed from the successive images obtained by shifting the focus, using public domain software (NIH-Image 1.61). Vascular diameter. Images were reproduced from the videotape by digitizing frames at a resolution of 640 3 480 pixels with an 8-bit accuracy, using NIH 1.61 on a personal computer (Power Macintosh 7600/120, Apple Computer Inc., Cupertino, CA). Pulmonary arterioles and venules were classified by the direction of the blood flow. They were three-dimensionally observed by shifting the focus stepwise. Diameters of arterioles or venules were not measured at the level of the subpleural capillary network, since the divergence or convergence of capillaries from (into) these microvessels occurred frequently (Fig. 2B). Instead, images acquired at the level of the deepest alveolar cross section were utilized for the measurement of diameters, since divergence (or convergence) occurred less frequently at this level. The diameter was measured with the image analyzing software (NIH 1.61), which measures the length by the pixels. The stage micrometer was observed with this system, and the factor to convert the unit (from the pixels to the mm) was calculated beforehand. Diameters of three or four arterioles (or venules) were averaged to represent the size of the vessels. Functional capillary density in the subpleural capillary network. Images detected at the confocal level of the subpleural capillary network with the 320 objective lens were used for this purpose. Capillaries supplied with the blood flow were illuminated by the fluorescence of FITC-BSA. A square area of 200 3 200 pixels was randomly selected, and the functional capillary density (FCD; the density of perfused capillaries in the pulmonary interstitium) was calculated according to the following equation: Functional capillary density 5
Total number of pixels in capillaries . 40,000
Three-dimensional analysis for pulmonary edema. Images acquired with the 320 objective lens were used for the evaluation of the interstitial edema and the alveolar leakage. The change of fluorescence intensity was considered to be caused by the leakage of FITCBSA. To evaluate the development of interstitial edema, the interstitial fluorescent intensity was measured by placing regions of
MITSUOKA ET AL.: ENDOTHELINS IN MICROCIRCULATION interest (ROIs) on the centers of meshes in the capillary network. The brightness of each pixel, which was expressed in 8-bit accuracy (i.e., the absolute white is 255, while the absolute black is 0), was regarded as the brightness of fluorescence. At least 10 measurements were made in each time point and averaged. Development of the interstitial edema was evaluated from a ratio of the average fluorescence intensity at a given period to the average intensity at the initial observation (i.e., 20 min after the thoracotomy). Development of edema 5
Average fluorescence intensity at a given period . Average fluorescence intensity at the initial observation
At about 50 mm below the capillary network, the fluorescent intensity was measured by placing the ROI at the centers of the alveoli. At least five measurements were made in each time point and the acquired values were averaged. Development of the alveolar leakage was evaluated from a ratio of the average fluorescence intensity at a given period to the average intensity at the initial observation. Histological examination. After detection of the image, animals were sacrificed by bloodletting with their abdominal aorta dissected. Thereafter, the main pulmonary artery was cannulated by a 23gauge needle, and the residual blood in the lung was rinsed out by 40 ml of heparinized normal saline pressurized at 40 cm/H 2O, while the lung was kept inflated with the air pressurized at 10 cm/H 2O. Lung specimens were fixed with 10% buffered formalin for 5 days and then embedded in paraffin. Four-micrometer sections were made and stained with hematoxylin and eosin. In each specimen, the mean numbers of polymorphonuclear cells (PMNs) per 100 alveolar profiles were calculated. Counting was made in the subpleural alveoli, and also in the alveoli which were located approximately 3 mm below the visceral pleura. The number obtained was regarded as the number of PMNs which had been making firm contact with pulmonary ECs or already migrated into the pulmonary interstitium. The other blood cells, which had made loose contact with ECs, were supposed to be rinsed out of the pulmonary vessels. Temporal profile of fluorescent dye distributed in pulmonary microcirculation. Either normal saline or TAK-044 was intravenously administered to animals before the operative procedure as stated above. A rat in the IIR group (n 5 4) or the TAK group (n 5 4) was subjected to IIR (60 min of ischemia followed by 180 min of reperfusion). The rats in the control group (n 5 4) were not subjected to the IIR treatment. After 120 min of reperfusion (or 180 min after the laparotomy for the control group), animals were anesthetized again and then mounted on the acrylic plate. The right jugular vein was cannulated, and the mechanical ventilation was applied as described above. The right chest wall was carefully removed. After 180 min of reperfusion (or 240 min after the laparotomy for the control group), 7 mg of FITC-BSA and 0.3 mg of vecronium bromide were injected intravenously. Twenty minutes after the injection, the lung was inflated with air pressurized at 10 cm H 2O. The subpleural area was observed with the 310 objective lens, and the level of focus was fixed at the depth of the alveolar cross section. Twenty seconds after the respiratory arrest, another 3 mg of FITC-BSA was injected and the optical images were then detected at a rate of 2 frames/s. To make the temporal profile of fluorescence intensity distributed in the pulmonary microcirculation, the average fluorescence intensity in ROI was measured every 0.5 s for 30 s after the incremental injection of FITC-BSA. Since the longer duration of respiratory arrest caused severe arrhythmia or cardiac arrest (especially in the IIR group), this study was limited in duration. The ROI was set larger than 5000 pixels in each image and located over the alveolar septum. The change of fluorescence intensity under ROI was then expressed as a ratio of the average fluorescence intensity at a given period to the average fluorescence intensity just before the additional injection. In each animal, the pattern of the curve indicating the incre-
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ment in fluorescence was obtained. The duration of the first curve was measured and used as an indicator for the pulmonary microcirculatory status. However, the injected bolus should pass the sampling point more than once [3, 10]. To minimize the effect of the recirculation, the duration of the first curve was obtained by extrapolating the curve, after replotting the curve in semilogarithmic form which made the exponential segment linear [3, 10]. Statistics. Data are expressed as means 6 standard errors of the mean (SEM). Statistical comparisons were performed by using oneway ANOVA followed by Tukey’s test or two-way analysis of variance for repeated measures followed by Duncan’s test where appropriate. When a P value is less than 0.05, the difference was considered to be statistically significant.
RESULTS
Hemodynamics In the control group, MAP was over 100 mm Hg throughout the experiments. MAP in the IIR and TAK group of rats gradually decreased during the intestinal ischemia, and a further decrease was observed instantaneously after intestinal reperfusion. The rats in the IIR group did not recover from the hypotension for 180 min after reperfusion. MAP in the TAK group of rats recovered in a small degree at 180 min after intestinal reperfusion, but it was not statistically different from that in the IIR group (Fig. 1). Morphological Changes in Subpleural Microvessels From a series of confocal images detected during the experiments, a three-dimensional image of the alveolar capillaries in an intravital condition was reconstructed (Fig. 2A). Vasoconstriction of arterioles or venules was not recognized during IIR treatment. FCD in the subpleural area was severely reduced (Fig. 2B–2E). Diameter of Microvessels in the Subpleural Area The arterioles with a diameter of 21– 40 mm (30.1 6 1.2), and the venules with a diameter of 24 –36 mm (28.3 6 0.9) were observed in the subpleural area. The size of these vessels did not change significantly during IIR (Fig. 3). Functional Capillary Density in the Subpleural Capillary Network The decrease of FCD was not detected during intestinal ischemia. In the IIR group, FCD was significantly decreased after intestinal reperfusion (P , 0.01 vs control group). The difference became more significant in the later period of reperfusion. The pretreatment with TAK-044 could attenuate the decrease significantly (P , 0.01 vs IIR group) (Fig. 4). Development of Pulmonary Edema The development of interstitial edema and alveolar leakage was separately evaluated (i.e., threedimensionally). Although the interstitial fluores-
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FIG. 1. Mean arterial blood pressure. Time indicates minutes after the induction of intestinal ischemia.
cence tended to increase during intestinal ischemia, no statistical difference was detected between the control and the IIR group. Sixty minutes of intestinal reperfusion increased the interstitial fluorescence in the IIR group (P , 0.05 vs control group). At 180 min of reperfusion, the mean fluorescence intensity in the pulmonary interstitium of the IIR group was about 2.5-fold higher than that of the control group.
On the other hand, the pretreatment with a nonselective ET receptor antagonist (TAK-044) prevented the development of interstitial edema due to IIR. Statistical difference between the TAK and the IIR group was also detected at 60, 120, and 180 min after reperfusion. Similarly, pretreatment with TAK-044 could also attenuate the alveolar leakage (Fig. 5).
FIG. 2. Morphological changes in subpleural vessels after IIR. (A) Three-dimentionally reconstructed image of the alveolar capillies under an intravital condition. (B and C) Before IIR, and (D and E) 180 min after reperfusion.
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FIG. 3. Arteriolar and venular diameters. (A) Arteriolar diameter. (V) Venular diameter. Time indicates minutes after the induction of intestinal ischemia.
Accumulation of PMN
Pulmonary Microcirculation in the Subpleural Area
After IIR, the accumulation of PMNs was observed in both subpleural and deeper areas (Fig. 6). The number of PMNs was significantly higher in the IIR group in the subpleural area (P , 0.01 vs control group), and also in the deeper area (P , 0.01 vs control group). Pretreatment with TAK-044 could reduce the accumulation of PMNs (P , 0.05 vs IIR group) (Fig. 7).
The fluorescence intensity of alveolar septum was measured in the series of images detected after the pulsewise injection of the fluorescent dye (Fig. 8). With the statistical difference (P , 0.01), the duration of the first curve in the IIR group (12.8 6 1.4 s) was longer than that in the control group (8.5 6 0.6 s). The duration of the first curve in the TAK group (11.0 6
FIG. 4. ischemia.
Functional capillary density in the subpleural capillary network. Time indicates minutes after the induction of intestinal
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FIG. 5. Development of pulmonary edema. The time indicates minutes after the induction of intestinal ischemia. (Top) Interstitial edema, and (Bottom) alveolar leakage.
1.2 s) was significantly shorter than that in the IIR group (P , 0.01 vs IIR). DISCUSSION
Pulmonary edema caused by IIR has been commonly used as a model of multiple organ failure (MOF) and ARDS. Various methods have been utilized to evaluate the severity of pulmonary edema. Among them, the leak index study of 125I-albumin, which evaluates the severity of augmented vascular permeability, seems to be used most frequently. On the other hand, other aspects of this experimental lung injury have been seldom investigated. In the present study, the ALI in the early stage of IIR was investigated from various aspects, including the size of microvessels, FCD, the interstitial edema, the alveolar leakage, and the temporal profile of the dye injected to the pulmonary microcirculation. Due to technical limitations, our observation by the intravital microscopy was restricted only to the superficial area, approximately as deep as 100 mm from the visceral pleura [3]. New equipment is expected to be
developed, in order to observe the deeper area. In the perfused lung study, however, the blood flow in subpleural vessels (as deep as 2 mm from visceral pleura) was reported to be more sensitive to vasoactive substances than that in the other part of the lung [11], and the microvessels with diameters of 30–40 mm seem to be very sensitive to vasoactive substances [12]. Consequently, microscopic findings in the subpleural area could be at least a key to solve the mechanisms of pulmonary microcirculatory disorders caused by IIR. In the present study, almost the same degree of leukocyte accumulation was observed at both the superficial and the deeper lesions. Vasoconstriction, microvascular plugging, and extrinsic compression due to interstitial edema have been suggested as the potential mechanisms for pulmonary microcirculatory disorders in ALI, which could be manifested as the increased PVR or PAH [13–16]. In the present study, however, the diameter of subpleural vessels did not change significantly. Similar findings were also reported by Carter and colleagues [2]. They reported that these vessels seemed to be fully dilated rather than constricted, reducing sensitivity to the vasoconstrictive stimulus. It is unclear why these subpleural vessels did not constrict during IIR. We cannot deny the possibility that the experimental procedures including anesthesia or exposing the lung to the atmosphere might reduce constriction in the subpleural vessels [2]. It is also possible that the constrictive ability of these vessels might be reduced by NO-driven mechanisms, which play a role in reducing the constrictive ability of microvessels in the cremaster flap after septic shock [17]. In marked contrast to the diameters of microvessels, a significant decrease in FCD took place in the IIR group. Consequently, the decreased FCD could be one of the major factors which impaired pulmonary microhemodynamics in the early stage of IIR. The mechanisms of the decrease are still unclear. In hepatic microcirculation, lipocytes (or Ito cells) are supposed to decrease the FCD, squeezing the sinusoid in response to ET [18]. In pulmonary microcirculation, pericytes around the capillary have the same type of contractile filaments as myofiblasts, and they may act like Ito cells [19, 20]. However, there is no strong evidence for this conjecture. On the other hand, the decrease in FCD might more likely be caused by microvascular plugging or extrinsic compression due to interstitial edema [15, 16]. In fact, the larger number of leukocytes was trapped by the lung in the IIR group, and the interstitial edema was significantly increased at 180 min after IIR. Obviously, the augmented vascular permeability and microvascular plugging are mediated by the interaction between leukocytes and endothelial cells. This interaction is enhanced by multifarious factors. Endothelins are released from the activated or damaged ECs during IIR
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FIG. 6. Histology of the lung. Alveoli located approximately 3 mm below the visceral pleura are shown.
[4, 5]. ETs are potent vasoconstrictors, and the accumulating evidence indicates that they also have proinflammatory actions, such as superoxide production of neutrophils [9] and cytokine release from macrophages or monocytes [22]. In addition, abnormalities in circulating levels of ET-1 have been reported in ARDS [21]. Thus, ETs could be one of the major promoters of ALI. Receptors for ETs are widely distributed in pulmonary microcirculation, including pulmonary ECs and vascular smooth muscle cells [24]. Two types of ET receptors, ET-A and ET-B, have specific vasoactive effects after binding to ETs [23]. Due to mutual interaction, simultaneous inhibition of these receptors is required to prevent the deleterious effects of ETs on lung functions [25]. TAK-
044 is a newly synthesized cyclic hexapeptide which has a nonselective antagonistic effect on both ET-A and ET-B receptors [26]. Once injected intravenously, the concentration of TAK-044 reaches the maximum in several minutes [26]. After that, TAK-044 could stay effective for about 4 h, blocking the pathological effect of injected ET-1, without any major side effects [27]. According to our preliminary experiments (n 5 3), the intravenous injection of TAK-044 alone (i.e., without IIR) made no significant difference in pulmonary microcirculation, compared to that in the control group (e.g., at 4 h after injection: MAP, 85 6 6.0 mm Hg; arteriolar diameters, 28.2 6 1.2 mm; venular diameters, 30.2 6 1.9 mm; FCD, 0.615 6 0.053; interstitial edema, 1.35 6 0.05; and alve-
FIG. 7. Accumulation of PMN.
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olar leakage, 1.18 6 0.05). (Data are not plotted in graphs.) Therefore, it is supposed that TAK-044 prevented the pulmonary microcirculatory disorders by inhibiting the ET-related pathophysiological mechanisms. In the present study, the number of PMNs in the lung was significantly decreased by pretreatment with TAK-044. The expression of adhesion molecules on the neutrophil surface is augmented by ET-1 [22]. Consequently, ETs are highly suspected to promote the interaction between leukocytes and ECs. It was reported that the production of ET was increased in the intact lung in response to unilateral pulmonary ischemia reperfusion [28]. Thus, ETs originated from the pulmonary ECs might be responsible for the pulmonary microcirculatory disorders caused by IIR. In the present study, it is not determined whether the pulmonary microcirculatory disorders were induced by ETs originated from the intestinal ECs or the pulmonary ECs. Further studies are necessary to clarify the origin of ET which promoted ALI during IIR. Pretreatment with a nonselective receptor antagonist successfully attenuated the development of pulmonary edema. The pretreatment might have improved the cardiac performance after shock [29], and thus contributed to this improvement. It is also likely that TAK-044 inhibited the augmentation of vascular permeability which was caused directly by ET. However, controversy exists about whether ETs raise the pulmonary vascular permeability by themselves. Other substances such as PAF may be prerequisite for ETs to promote the pulmonary edema [30]. In our experiments, the intestinal ischemia was induced by complete clamping of the SMA. Intestinal ischemia in this method might be too severe to model the splanchnic ischemia during septic shock, burn, or cardiopulmonary shock. This model might approximate ALI after the ruptured abdominal aneurysm [31], in which the intestinal blood flow is extremely decreased. However, even the short and small decrease in the intestinal blood flow may cause unpredictable severe intestinal ischemia, since intestinal mucosa has a specific susceptibility [32, 33]. This evidence indicates that monitoring intestinal oxygenation is especially important in handling patients with the various types of shock [34]. In conclusion, we demonstrated that pulmonary microhemodynamics could be disturbed by the severe decrease of FCD, rather than by arteriolar or venular vasoconstriction in the early phase of IIR. The interstitial edema and microvascular plugging seemed to be the major causes for this decrease. The basic mechanism involved may be the interaction between ECs and
FIG. 8. Temporal profile of fluorescence in alveolar septum after incremental injections of fluorescent dye. (Top) The control group (n 5 4), (middle) the IIR group (n 5 4), and (bottom) the TAK group (n 5 4).
MITSUOKA ET AL.: ENDOTHELINS IN MICROCIRCULATION
leukocytes, which is promoted by ET. Pretreatment with TAK-044, a nonselective ET receptor antagonist, improved the pulmonary microhemodynamics in ALI, inhibiting the accumulation of leukocytes, the pulmonary edema, and the decrease of FCD.
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17.
REFERENCES 18. 1. 2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Zappol, W. M., and Snider, M. T. Pulmonary hypertension in severe acute respiratory failure. N. Engl. J. Med. 296: 476, 1977. Carter, M. B., Wilson, M. A., Wead, W. B., and Garrison, R. N. Pulmonary subpleural arteriolar diameters during intestinal ischemia/reperfusion. J. Surg. Res. 59: 51, 1995. Mitsuoka, H., Sakurai, T., Unno, N., Kaneko, H., Suzuki, S., Nakamura, S., Baba, S., and Terakawa, S. Intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia reperfusion injury in the rat. Crit. Care Med. 1999, in press. Mashima, S., Shirakami, G., Mitsuyoshi, A., Nakagami, M., Morimoto, T., Terasaki, M., Nakao, K., Yamabe, H., Yamaoka, Y., and Ozawa, K. Evaluation of the protective effect of a novel prostacyclin analog on mesenteric circulation following warm ischemia. Eur. Surg. Res. 28: 14, 1996. Yegen, C., Aktan, A. O., Buyukgebiz, O., Haklar, G., Yalcin, A. S., Yalin, R., and Ercan, S. Effect of verapamil and iloprost (ZK 36374) on endothelin release after mesenteric ischemia reperfusion injury. Eur. Surg. Res. 26: 69, 1994. Helset, E., Lindal, S., Olsen, R., Myklebust, R., and Jorgensen, L. Endothelin-1 causes sequential trapping of platelets and neutrophils in pulmonary microcirculation in rats. Am. J. Physiol. 271: L538, 1996. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411, 1988. Lopez, F. A., Riesco, A., Espinosa, G., Digiuni, E., Cernadas, M. R., Alvarez, V., Monton, M., Rivas, F., Gallego, M. J., Egido, J., et al. Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart. Circulation 88: 1166, 1993. Ishida, K., Takeshige, K., and Minakami, S. Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl phenylalanine. Biochem. Biophys. Res. Commun. 173: 496, 1990. Kelman, G. R. In G. R. Kelmans (Ed.), Applied Cardiovascular Physiology. Cardiovascular Measurements. London: Butterworth 1971. Pp. 210 –242. Hakim, T. S. Is flow in subpleural region typical of the rest of the lung? A study using laser Doppler flowmetry. J. Appl. Physiol. 72: 1860, 1992. MacLean, M. R., McCulloch, K. M., and Baird, M. Endothelin ETA- and ETB-receptor-mediated vasoconstriction in rat pulmonary arteries and arterioles. J. Cardiovasc. Pharmacol. 23: 838, 1994. Nagasaka, Y., Ishigaki, M., Okazaki, H., Huang, J., Matsuda, M., Noguchi, T., Toga, H., Fukunaga, T., Nakajima, S., and Ohya, N. Effect of pulmonary blood flow on microvascular pressure profile determined by micropuncture in perfused cat lungs. J. Appl. Physiol. 77: 1834, 1994. Zapol, W. M., and Snider, M. T. Vascular components of ARDS. Clinical pulmonary hemodynamics and morphology. Am. Rev. Respir. Dis. 136: 471, 1987. Vesconi, S., Rossi, G. P., Pesenti, A., Fumagalli, R., and Gattinoni, L. Pulmonary microthrombosis in severe adult respiratory distress syndrome. Crit. Care Med. 16: 111, 1988.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32. 33. 34.
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Tomashefski, J. F., Jr., Davies, P., Boggis, C., Greene, R., Zapol, W. M., and Reid, L. M. The pulmonary vascular lesions of the adult respiratory distress syndrome. Am. J. Pathol. 112: 112, 1983. Hollenberg, S. M., Tangora, J. J., Piotrowski, M. J., Easington, C., and Parrillo, J. E. Impaired microvascular vasoconstrictive responses to vasopressin in septic rats. Crit. Care Med. 25: 869, 1997. Zhang, J. X., Pegoli, W., and Clemens, M. G. Endothelin-1 induces direct constriction of hepatic sinusoids. Am. J. Physiol. 266: G624, 1994. Murphy, D. D., and Wagner, R. C. Differential contractile response of cultured microvascular pericytes to vasoactive agents. Microcirculation 1: 121, 1944. Weibel, E. R. On pericytes, particularly their existence on lung capillaries. Microvasc. Res. 8: 218, 1974. Langleben, D., DeMarchie, M., Laporta, D., Spanier, A. H., Schlesinger, R. D., and Stewart, D. J. Endothelin-1 in acute lung injury and the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 148: 1646, 1993. Helset, E., Sildnes, T., Slejelid, R., and Konopski, Z. S. Endothelin-1 stimulates human monocytes release in vitro to release TNF-a, IL-1b, and IL-6. Mediators Inflamm. 2: 417, 1993. Arai, H., Hori, S., Aramori, I., Ohkubo, H., and Nakanishi, S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348: 730, 1990. Fukuroda, T., Kobayashi, M., Ozaki, S., Yano, M., Miyauchi, T., Onizuka, M., Sugishita, Y., Goto, K., and Nishikibe, M. Endothelin receptor subtypes in human versus rabbit pulmonary arteries. J. Appl. Physiol. 76: 1976, 1994. Stefan, U., and Roland, L. F. The interaction of endothelin receptor responses in the isolated perfused rat lung. NaunynSchmiedeberg’s Arch. Pharmacol. 356: 392, 1977. Ikeda, S., Awane, Y., Kusumoto, K., Wakimasu, M., Watanabe, T., and Fujino, M. A new potent endothelin receptor antagonist, TAK-044, shows long-lasting inhibition of both ETA and ETB mediated blood pressure responses in rats. J. Pharmacol. Exp. Ther. 270: 728, 1994. Haynes, W. G., Ferro, C. J., O’Kane, K. P., Somerville, D., Lomax, C. C., and Webb, D. J. Systemic endothelin receptor blockade decreases peripheral vascular resistance and blood pressure in humans. Circulation 93: 1860, 1996. Okada, M., Yamashita, C., Okada, M., and Okada, K. Contribution of endothelin-1 to warm ischemia/reperfusion injury of the rat lung. Am. J. Respir. Crit. Care Med. 152: 2105, 1995. Weitzberg, E., Hemsen, A., Rudehill, A., Modin, A., Wanecek, M., and Lundberg, J. M. Bosentan improved cardiopulmonary vascular performance and increased plasma levels of endothelin-1 in porcine endotoxin shock. Br. J. Pharmacol. 118: 617, 1996. Filep, J. G., Sirois, M. G., Rousseau, A., Fournier, A., and Sirois, P. Effects of endothelin-1 on vascular permeability in the conscious rat: interactions with platelet-activating factor. Br. J. Pharmacol. 104: 797, 1991. Lindsay, T. F., Walker, P. M., and Romaschin, A. Acute pulmonary injury in a model of ruptured abdominal aortic aneurysm. J. Vasc. Surg. 22: 1, 1995. Schoenberg, M. H., and Beger, H. G. Reperfusion injury after intestinal ischemia. Crit. Care Med. 21: 1376, 1993. Bounous, G. Acute necrosis of the intestinal mucosa. Gastroenterology 82: 1457, 1982. Nordin, A., Makisalo, H., Mildh, L., and Hockerstedt, K. Gut intramucosal pH as an early indicator of effectiveness of therapy for hemorrhagic shock. Crit. Care Med. 26: 1110, 1998.
Pathophysiological Role of Endothelins in Pulmonary Microcirculatory Disorders Due to Intestinal Ischemia and Reperfusion 1 Hiroshi Mitsuoka, M.D.,* ,2 Naoki Unno, M.D., Ph.D.,* Takashi Sakurai, Ph.D.,† Hiroshi Kaneko, M.D., Ph.D.,* Shohachi Suzuki, M.D., Ph.D.,* Hiroyuki Konno, M.D., Ph.D.,* Susumu Terakawa, M.D., Ph.D.,† and Satoshi Nakamura, M.D., Ph.D.* *Second Department of Surgery and †Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan Submitted for publication November 2, 1998
Background. This study was conducted to investigate pulmonary microcirculatory disorders caused by intestinal ischemia reperfusion (IIR), and the pathophysiological roles of endothelin (ET) in acute lung injury (ALI). Methods. Male rats were pretreated with normal saline or a nonselective ET receptor antagonist (TAK-044) and subjected to IIR (60 min of intestinal ischemia and 180 min of reperfusion). The right upper lobe of the lung was examined by intravital confocal microscopy. Results. The size of arterioles and venules was not significantly reduced during IIR, but the functional capillary density (FCD) decreased significantly. TAK044 improved the pulmonary microhemodynamics, inhibiting the accumulation of leukocytes, the pulmonary edema, and the decrease of FCD. Conclusions. In the early stage of IIR, pulmonary microhemodynamics seemed more likely to be disturbed by the decrease of FCD, than by arteriolar or venular vasoconstriction. ETs decrease the FCD, promoting the interaction between leukocytes and pulmonary vessels. © 1999 Academic Press Key Words: pulmonary microcirculation; endothelin; ARDS; lung; intestinal ischemia and reperfusion; multiple organ failure; laser confocal microscopy; functional capillary diameter; in vivo; rat. INTRODUCTION
Intestinal ischemia and reperfusion (IIR) have been regarded as primary adaptive responses to shock. Acute lung injury (ALI) after IIR is a well-established model of 1
Supported in part by a research grant from the Ministry of Education, Science and Culture of Japan (06671195). 2 To whom reprint requests should be addressed at Second Department of Surgery, Hamamatsu University School of Medicine, 3600 Handacho, Hamamatsu 431-31 Japan. Fax: 81-53-435-2273.
remote organ injury, related to acute respiratory distress syndrome (ARDS). This experimental lung injury has been commonly studied from the aspect of pulmonary edema. However, the augmentation of vascular permeability is only one facet of ALI. For instance, increased pulmonary vascular resistance (PVR) and pulmonary arterial hypertension (PAH) contribute to the morbidity or mortality of ALI [1]. Nevertheless, these aspects of this experimental ALI have been discussed in only a few studies [2, 3]. Recent studies have demonstrated that ALI after IIR is multifactorial and mediated by various vasoactive substances. Endothelins (ETs) are released from the damaged endothelial cells (ECs) during IIR [4, 5], while the intravenous injection of ET-1 can cause pulmonary edema [6]. Accordingly, ETs are highly anticipated to promote ALI, whereas the pathophysiological roles of ETs under these circumstances have not been elucidated. ETs have both vasoconstrictive and proinflammatory actions [7–9]. Therefore, morphological and functional studies should be combined with the evaluation of edema, when the pathophysiological roles of such a multifunctional substance are investigated. In a previous study, we made a minute observation of subpleural microcirculation, utilizing intravital confocal microscopy [3]. This technique enabled us to observe the functional and morphological changes in pulmonary microcirculation, as well as to evaluate the severity of vascular leakage under an intravital condition. Microcirculatory events, which might be induced by ET-related mechanisms and causative of increased PVR or PAH, could be directly observed by this new technique. This study was conducted to investigate pulmonary microcirculation in the early phase of ALI from various aspects. In addition to the three-dimensional observation of pulmonary edema and the analysis of pulmonary blood distribution [3], the morphological changes
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in subpleural microvessels (the arteriolar or venular diameter and the functional capillary density) were investigated. Furthermore, the pathophysiological roles of ET in the early phase of ALI were indirectly investigated, using a nonselective receptor antagonist. MATERIALS AND METHODS The experiments were approved by the Institutional Animal Use and Care committee of the Hamamatsu University School of Medicine, and were performed in accordance with the National Institute of Health guidelines for the care and handling of animals. Animal preparation. Male Sprague-Dawley rats (290 –340 g) were fasted with free access to water for 12 h before operation. An animal was anesthetized by intraperitoneal injection of 25 mg/kg of sodium pentobarbital and placed on an acrylic plate in the semi-left decubitus position. The abdomen was opened through a midline incision. The superior mesenteric artery (SMA) was looped with a thread of 3-O nylon. A polyethylene tubing was inserted onto the SMA and guided the looping thread out of the body. The SMA was clamped by pulling the thread to cause the intestinal ischemia. The right jugular vein was cannulated with polyethylene tubing (PE-50), through which normal saline was infused continuously at a rate of 1 mL/h. The right carotid artery was also cannulated with another line of polyethylene tubing (PE-50) connected to a blood pressure transducer. The mean arterial pressure (MAP) was recorded with a multichannel monitor (78342A Monitor, Hewlett Packard, Palo Alto, CA). A tracheotomy was made and polyethylene tubing (PE-240) was inserted into the trachea. With a ventilator for small animals (Model SN-480-7, Shimano, Tokyo, Japan), respiration of the rat was maintained mechanically with a tidal volume of 2.0 mL, a breath rate of 70 per minute, and positive end expiratory pressure (PEEP) of 2 cm H 2O. To prevent atelectasis, the lung was overinflated periodically (about once in 1 h) with an exhale port of the ventilator occluded for 2 breaths. The right chest wall was carefully removed not to hurt the lung, and the chest opening was covered with a sheet of plastic wrap to minimize evaporation. The sheet was removed during the microscopic observation, and the exposed lung was moistened by topical application of normal saline. A cover glass (24 3 60 3 0.17 mm) was set horizontally above the chest, so that the surface of the exposed lung would contact with the glass gently in the inspiratory phase. The stage of the microscope (Labphoto, Nikon, Tokyo, Japan) was detached, and an acrylic plate holding the animal was mounted on the microscope instead. Fluorescence microscopy. Bovine serum albumin labeled with fluorescein isothiocynate (FITC-BSA, Sigma, St. Louis, MO) was dissolved in normal saline at a concentration of 10 mg/mL before the initial injection. Vecronium bromide (Masculax, Organon Inc, the Netherlands) was dissolved in normal saline at 1 mg/mL. The animal received 7 mg of FITC-BSA and 0.3 mg of vecronium bromide intravenously soon after the thoracotomy, and an initial observation was performed 20 min after that. FITC-BSA and vecronium were administered at a rate of 2 and 0.1 mg per 1 h, to maintain the fluorescence level of the vasculature and the chemical paralysis. Videomicroscopic data were obtained while the lung was briefly (,40 s) inflated with air pressurized at 10 cm H 2O. A confocal laser microscope utilizing a microlens-attached Nipkow disk as a scanner (CSU-10, Yokogawa, Tokyo, Japan) was connected to the microscope. The right upper lobe of the lung was observed with an 310 objective lens (Type E, NA 5 0.5, Nikon, Tokyo, Japan) or an 320 objective lens (FL, NA 5 0.5, Nikon) under epifluorescence illumination with a 488-nm beam from a multiline argon laser (Model 2213150MLYJ, Cyonics, San Jose, CA). The output level of the laser was kept constant throughout the experiments. Images were detected with an image-intensified charge-coupled device (ICCD) camera (DAS-512, Imagista, Tokyo, Japan). The rate of image detection was 4 frames/s with the 320 objective lens, and it was decreased to 2 frames/s with the
310 lens. The contrast of the image was enhanced in real time with a digital image processor (ARGUS-20, Hamamatsu photonics, Hamamatsu, Japan), and both the mode and the degree of enhancement were unchanged through the experiments. The processed image was displayed on a video monitor screen (PVM-1454Q, Sony, Tokyo, Japan) and recorded simultaneously with an S-VHS format recorder (AG-7750, Panasonic, Osaka, Japan). A series of confocal images were acquired, while the objective lens was shifted stepwise along the optical axis. Experimental procedure. Rats were divided into three groups. The control and IIR groups received normal saline as a vehicle. The TAK group was pretreated with a nonselective ET receptor antagonist (TAK-044, Takeda Chemical Industries, Osaka, Japan). Either normal saline or TAK-044 was intravenously administered to animals before the operative procedure. TAK-044 was dissolved to a concentration of 1 mg/mL with normal saline and intravenously administered at a dose of 3 mg/kg. The SMA in the IIR (n 5 5) and the TAK group (n 5 5) of rats was clamped for 60 min and then released for 180 min. Rats in the control group (n 5 5) were treated similarly, but without the intestinal ischemia. Hemodynamic data were obtained 20 min after thoracotomy, at 30 and 55 min during the SMA clamp, and at 60, 90, 120, 150, and 180 min during the reperfusion. Videomicroscopic images were obtained and recorded 20 min after thoracotomy (i.e., an initial observation), at 30 and 55 min during the SMA clamp, and at 60, 120, and 180 min during the reperfusion. The observation was started with the 310 objective lens. The observation with the 320 objective lens was made 5 min after that. Three-dimensional images of the subpleural vasculature were reconstructed from the successive images obtained by shifting the focus, using public domain software (NIH-Image 1.61). Vascular diameter. Images were reproduced from the videotape by digitizing frames at a resolution of 640 3 480 pixels with an 8-bit accuracy, using NIH 1.61 on a personal computer (Power Macintosh 7600/120, Apple Computer Inc., Cupertino, CA). Pulmonary arterioles and venules were classified by the direction of the blood flow. They were three-dimensionally observed by shifting the focus stepwise. Diameters of arterioles or venules were not measured at the level of the subpleural capillary network, since the divergence or convergence of capillaries from (into) these microvessels occurred frequently (Fig. 2B). Instead, images acquired at the level of the deepest alveolar cross section were utilized for the measurement of diameters, since divergence (or convergence) occurred less frequently at this level. The diameter was measured with the image analyzing software (NIH 1.61), which measures the length by the pixels. The stage micrometer was observed with this system, and the factor to convert the unit (from the pixels to the mm) was calculated beforehand. Diameters of three or four arterioles (or venules) were averaged to represent the size of the vessels. Functional capillary density in the subpleural capillary network. Images detected at the confocal level of the subpleural capillary network with the 320 objective lens were used for this purpose. Capillaries supplied with the blood flow were illuminated by the fluorescence of FITC-BSA. A square area of 200 3 200 pixels was randomly selected, and the functional capillary density (FCD; the density of perfused capillaries in the pulmonary interstitium) was calculated according to the following equation: Functional capillary density 5
Total number of pixels in capillaries . 40,000
Three-dimensional analysis for pulmonary edema. Images acquired with the 320 objective lens were used for the evaluation of the interstitial edema and the alveolar leakage. The change of fluorescence intensity was considered to be caused by the leakage of FITCBSA. To evaluate the development of interstitial edema, the interstitial fluorescent intensity was measured by placing regions of
MITSUOKA ET AL.: ENDOTHELINS IN MICROCIRCULATION interest (ROIs) on the centers of meshes in the capillary network. The brightness of each pixel, which was expressed in 8-bit accuracy (i.e., the absolute white is 255, while the absolute black is 0), was regarded as the brightness of fluorescence. At least 10 measurements were made in each time point and averaged. Development of the interstitial edema was evaluated from a ratio of the average fluorescence intensity at a given period to the average intensity at the initial observation (i.e., 20 min after the thoracotomy). Development of edema 5
Average fluorescence intensity at a given period . Average fluorescence intensity at the initial observation
At about 50 mm below the capillary network, the fluorescent intensity was measured by placing the ROI at the centers of the alveoli. At least five measurements were made in each time point and the acquired values were averaged. Development of the alveolar leakage was evaluated from a ratio of the average fluorescence intensity at a given period to the average intensity at the initial observation. Histological examination. After detection of the image, animals were sacrificed by bloodletting with their abdominal aorta dissected. Thereafter, the main pulmonary artery was cannulated by a 23gauge needle, and the residual blood in the lung was rinsed out by 40 ml of heparinized normal saline pressurized at 40 cm/H 2O, while the lung was kept inflated with the air pressurized at 10 cm/H 2O. Lung specimens were fixed with 10% buffered formalin for 5 days and then embedded in paraffin. Four-micrometer sections were made and stained with hematoxylin and eosin. In each specimen, the mean numbers of polymorphonuclear cells (PMNs) per 100 alveolar profiles were calculated. Counting was made in the subpleural alveoli, and also in the alveoli which were located approximately 3 mm below the visceral pleura. The number obtained was regarded as the number of PMNs which had been making firm contact with pulmonary ECs or already migrated into the pulmonary interstitium. The other blood cells, which had made loose contact with ECs, were supposed to be rinsed out of the pulmonary vessels. Temporal profile of fluorescent dye distributed in pulmonary microcirculation. Either normal saline or TAK-044 was intravenously administered to animals before the operative procedure as stated above. A rat in the IIR group (n 5 4) or the TAK group (n 5 4) was subjected to IIR (60 min of ischemia followed by 180 min of reperfusion). The rats in the control group (n 5 4) were not subjected to the IIR treatment. After 120 min of reperfusion (or 180 min after the laparotomy for the control group), animals were anesthetized again and then mounted on the acrylic plate. The right jugular vein was cannulated, and the mechanical ventilation was applied as described above. The right chest wall was carefully removed. After 180 min of reperfusion (or 240 min after the laparotomy for the control group), 7 mg of FITC-BSA and 0.3 mg of vecronium bromide were injected intravenously. Twenty minutes after the injection, the lung was inflated with air pressurized at 10 cm H 2O. The subpleural area was observed with the 310 objective lens, and the level of focus was fixed at the depth of the alveolar cross section. Twenty seconds after the respiratory arrest, another 3 mg of FITC-BSA was injected and the optical images were then detected at a rate of 2 frames/s. To make the temporal profile of fluorescence intensity distributed in the pulmonary microcirculation, the average fluorescence intensity in ROI was measured every 0.5 s for 30 s after the incremental injection of FITC-BSA. Since the longer duration of respiratory arrest caused severe arrhythmia or cardiac arrest (especially in the IIR group), this study was limited in duration. The ROI was set larger than 5000 pixels in each image and located over the alveolar septum. The change of fluorescence intensity under ROI was then expressed as a ratio of the average fluorescence intensity at a given period to the average fluorescence intensity just before the additional injection. In each animal, the pattern of the curve indicating the incre-
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ment in fluorescence was obtained. The duration of the first curve was measured and used as an indicator for the pulmonary microcirculatory status. However, the injected bolus should pass the sampling point more than once [3, 10]. To minimize the effect of the recirculation, the duration of the first curve was obtained by extrapolating the curve, after replotting the curve in semilogarithmic form which made the exponential segment linear [3, 10]. Statistics. Data are expressed as means 6 standard errors of the mean (SEM). Statistical comparisons were performed by using oneway ANOVA followed by Tukey’s test or two-way analysis of variance for repeated measures followed by Duncan’s test where appropriate. When a P value is less than 0.05, the difference was considered to be statistically significant.
RESULTS
Hemodynamics In the control group, MAP was over 100 mm Hg throughout the experiments. MAP in the IIR and TAK group of rats gradually decreased during the intestinal ischemia, and a further decrease was observed instantaneously after intestinal reperfusion. The rats in the IIR group did not recover from the hypotension for 180 min after reperfusion. MAP in the TAK group of rats recovered in a small degree at 180 min after intestinal reperfusion, but it was not statistically different from that in the IIR group (Fig. 1). Morphological Changes in Subpleural Microvessels From a series of confocal images detected during the experiments, a three-dimensional image of the alveolar capillaries in an intravital condition was reconstructed (Fig. 2A). Vasoconstriction of arterioles or venules was not recognized during IIR treatment. FCD in the subpleural area was severely reduced (Fig. 2B–2E). Diameter of Microvessels in the Subpleural Area The arterioles with a diameter of 21– 40 mm (30.1 6 1.2), and the venules with a diameter of 24 –36 mm (28.3 6 0.9) were observed in the subpleural area. The size of these vessels did not change significantly during IIR (Fig. 3). Functional Capillary Density in the Subpleural Capillary Network The decrease of FCD was not detected during intestinal ischemia. In the IIR group, FCD was significantly decreased after intestinal reperfusion (P , 0.01 vs control group). The difference became more significant in the later period of reperfusion. The pretreatment with TAK-044 could attenuate the decrease significantly (P , 0.01 vs IIR group) (Fig. 4). Development of Pulmonary Edema The development of interstitial edema and alveolar leakage was separately evaluated (i.e., threedimensionally). Although the interstitial fluores-
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FIG. 1. Mean arterial blood pressure. Time indicates minutes after the induction of intestinal ischemia.
cence tended to increase during intestinal ischemia, no statistical difference was detected between the control and the IIR group. Sixty minutes of intestinal reperfusion increased the interstitial fluorescence in the IIR group (P , 0.05 vs control group). At 180 min of reperfusion, the mean fluorescence intensity in the pulmonary interstitium of the IIR group was about 2.5-fold higher than that of the control group.
On the other hand, the pretreatment with a nonselective ET receptor antagonist (TAK-044) prevented the development of interstitial edema due to IIR. Statistical difference between the TAK and the IIR group was also detected at 60, 120, and 180 min after reperfusion. Similarly, pretreatment with TAK-044 could also attenuate the alveolar leakage (Fig. 5).
FIG. 2. Morphological changes in subpleural vessels after IIR. (A) Three-dimentionally reconstructed image of the alveolar capillies under an intravital condition. (B and C) Before IIR, and (D and E) 180 min after reperfusion.
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FIG. 3. Arteriolar and venular diameters. (A) Arteriolar diameter. (V) Venular diameter. Time indicates minutes after the induction of intestinal ischemia.
Accumulation of PMN
Pulmonary Microcirculation in the Subpleural Area
After IIR, the accumulation of PMNs was observed in both subpleural and deeper areas (Fig. 6). The number of PMNs was significantly higher in the IIR group in the subpleural area (P , 0.01 vs control group), and also in the deeper area (P , 0.01 vs control group). Pretreatment with TAK-044 could reduce the accumulation of PMNs (P , 0.05 vs IIR group) (Fig. 7).
The fluorescence intensity of alveolar septum was measured in the series of images detected after the pulsewise injection of the fluorescent dye (Fig. 8). With the statistical difference (P , 0.01), the duration of the first curve in the IIR group (12.8 6 1.4 s) was longer than that in the control group (8.5 6 0.6 s). The duration of the first curve in the TAK group (11.0 6
FIG. 4. ischemia.
Functional capillary density in the subpleural capillary network. Time indicates minutes after the induction of intestinal
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FIG. 5. Development of pulmonary edema. The time indicates minutes after the induction of intestinal ischemia. (Top) Interstitial edema, and (Bottom) alveolar leakage.
1.2 s) was significantly shorter than that in the IIR group (P , 0.01 vs IIR). DISCUSSION
Pulmonary edema caused by IIR has been commonly used as a model of multiple organ failure (MOF) and ARDS. Various methods have been utilized to evaluate the severity of pulmonary edema. Among them, the leak index study of 125I-albumin, which evaluates the severity of augmented vascular permeability, seems to be used most frequently. On the other hand, other aspects of this experimental lung injury have been seldom investigated. In the present study, the ALI in the early stage of IIR was investigated from various aspects, including the size of microvessels, FCD, the interstitial edema, the alveolar leakage, and the temporal profile of the dye injected to the pulmonary microcirculation. Due to technical limitations, our observation by the intravital microscopy was restricted only to the superficial area, approximately as deep as 100 mm from the visceral pleura [3]. New equipment is expected to be
developed, in order to observe the deeper area. In the perfused lung study, however, the blood flow in subpleural vessels (as deep as 2 mm from visceral pleura) was reported to be more sensitive to vasoactive substances than that in the other part of the lung [11], and the microvessels with diameters of 30–40 mm seem to be very sensitive to vasoactive substances [12]. Consequently, microscopic findings in the subpleural area could be at least a key to solve the mechanisms of pulmonary microcirculatory disorders caused by IIR. In the present study, almost the same degree of leukocyte accumulation was observed at both the superficial and the deeper lesions. Vasoconstriction, microvascular plugging, and extrinsic compression due to interstitial edema have been suggested as the potential mechanisms for pulmonary microcirculatory disorders in ALI, which could be manifested as the increased PVR or PAH [13–16]. In the present study, however, the diameter of subpleural vessels did not change significantly. Similar findings were also reported by Carter and colleagues [2]. They reported that these vessels seemed to be fully dilated rather than constricted, reducing sensitivity to the vasoconstrictive stimulus. It is unclear why these subpleural vessels did not constrict during IIR. We cannot deny the possibility that the experimental procedures including anesthesia or exposing the lung to the atmosphere might reduce constriction in the subpleural vessels [2]. It is also possible that the constrictive ability of these vessels might be reduced by NO-driven mechanisms, which play a role in reducing the constrictive ability of microvessels in the cremaster flap after septic shock [17]. In marked contrast to the diameters of microvessels, a significant decrease in FCD took place in the IIR group. Consequently, the decreased FCD could be one of the major factors which impaired pulmonary microhemodynamics in the early stage of IIR. The mechanisms of the decrease are still unclear. In hepatic microcirculation, lipocytes (or Ito cells) are supposed to decrease the FCD, squeezing the sinusoid in response to ET [18]. In pulmonary microcirculation, pericytes around the capillary have the same type of contractile filaments as myofiblasts, and they may act like Ito cells [19, 20]. However, there is no strong evidence for this conjecture. On the other hand, the decrease in FCD might more likely be caused by microvascular plugging or extrinsic compression due to interstitial edema [15, 16]. In fact, the larger number of leukocytes was trapped by the lung in the IIR group, and the interstitial edema was significantly increased at 180 min after IIR. Obviously, the augmented vascular permeability and microvascular plugging are mediated by the interaction between leukocytes and endothelial cells. This interaction is enhanced by multifarious factors. Endothelins are released from the activated or damaged ECs during IIR
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FIG. 6. Histology of the lung. Alveoli located approximately 3 mm below the visceral pleura are shown.
[4, 5]. ETs are potent vasoconstrictors, and the accumulating evidence indicates that they also have proinflammatory actions, such as superoxide production of neutrophils [9] and cytokine release from macrophages or monocytes [22]. In addition, abnormalities in circulating levels of ET-1 have been reported in ARDS [21]. Thus, ETs could be one of the major promoters of ALI. Receptors for ETs are widely distributed in pulmonary microcirculation, including pulmonary ECs and vascular smooth muscle cells [24]. Two types of ET receptors, ET-A and ET-B, have specific vasoactive effects after binding to ETs [23]. Due to mutual interaction, simultaneous inhibition of these receptors is required to prevent the deleterious effects of ETs on lung functions [25]. TAK-
044 is a newly synthesized cyclic hexapeptide which has a nonselective antagonistic effect on both ET-A and ET-B receptors [26]. Once injected intravenously, the concentration of TAK-044 reaches the maximum in several minutes [26]. After that, TAK-044 could stay effective for about 4 h, blocking the pathological effect of injected ET-1, without any major side effects [27]. According to our preliminary experiments (n 5 3), the intravenous injection of TAK-044 alone (i.e., without IIR) made no significant difference in pulmonary microcirculation, compared to that in the control group (e.g., at 4 h after injection: MAP, 85 6 6.0 mm Hg; arteriolar diameters, 28.2 6 1.2 mm; venular diameters, 30.2 6 1.9 mm; FCD, 0.615 6 0.053; interstitial edema, 1.35 6 0.05; and alve-
FIG. 7. Accumulation of PMN.
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olar leakage, 1.18 6 0.05). (Data are not plotted in graphs.) Therefore, it is supposed that TAK-044 prevented the pulmonary microcirculatory disorders by inhibiting the ET-related pathophysiological mechanisms. In the present study, the number of PMNs in the lung was significantly decreased by pretreatment with TAK-044. The expression of adhesion molecules on the neutrophil surface is augmented by ET-1 [22]. Consequently, ETs are highly suspected to promote the interaction between leukocytes and ECs. It was reported that the production of ET was increased in the intact lung in response to unilateral pulmonary ischemia reperfusion [28]. Thus, ETs originated from the pulmonary ECs might be responsible for the pulmonary microcirculatory disorders caused by IIR. In the present study, it is not determined whether the pulmonary microcirculatory disorders were induced by ETs originated from the intestinal ECs or the pulmonary ECs. Further studies are necessary to clarify the origin of ET which promoted ALI during IIR. Pretreatment with a nonselective receptor antagonist successfully attenuated the development of pulmonary edema. The pretreatment might have improved the cardiac performance after shock [29], and thus contributed to this improvement. It is also likely that TAK-044 inhibited the augmentation of vascular permeability which was caused directly by ET. However, controversy exists about whether ETs raise the pulmonary vascular permeability by themselves. Other substances such as PAF may be prerequisite for ETs to promote the pulmonary edema [30]. In our experiments, the intestinal ischemia was induced by complete clamping of the SMA. Intestinal ischemia in this method might be too severe to model the splanchnic ischemia during septic shock, burn, or cardiopulmonary shock. This model might approximate ALI after the ruptured abdominal aneurysm [31], in which the intestinal blood flow is extremely decreased. However, even the short and small decrease in the intestinal blood flow may cause unpredictable severe intestinal ischemia, since intestinal mucosa has a specific susceptibility [32, 33]. This evidence indicates that monitoring intestinal oxygenation is especially important in handling patients with the various types of shock [34]. In conclusion, we demonstrated that pulmonary microhemodynamics could be disturbed by the severe decrease of FCD, rather than by arteriolar or venular vasoconstriction in the early phase of IIR. The interstitial edema and microvascular plugging seemed to be the major causes for this decrease. The basic mechanism involved may be the interaction between ECs and
FIG. 8. Temporal profile of fluorescence in alveolar septum after incremental injections of fluorescent dye. (Top) The control group (n 5 4), (middle) the IIR group (n 5 4), and (bottom) the TAK group (n 5 4).
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leukocytes, which is promoted by ET. Pretreatment with TAK-044, a nonselective ET receptor antagonist, improved the pulmonary microhemodynamics in ALI, inhibiting the accumulation of leukocytes, the pulmonary edema, and the decrease of FCD.
16.
17.
REFERENCES 18. 1. 2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Zappol, W. M., and Snider, M. T. Pulmonary hypertension in severe acute respiratory failure. N. Engl. J. Med. 296: 476, 1977. Carter, M. B., Wilson, M. A., Wead, W. B., and Garrison, R. N. Pulmonary subpleural arteriolar diameters during intestinal ischemia/reperfusion. J. Surg. Res. 59: 51, 1995. Mitsuoka, H., Sakurai, T., Unno, N., Kaneko, H., Suzuki, S., Nakamura, S., Baba, S., and Terakawa, S. Intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia reperfusion injury in the rat. Crit. Care Med. 1999, in press. Mashima, S., Shirakami, G., Mitsuyoshi, A., Nakagami, M., Morimoto, T., Terasaki, M., Nakao, K., Yamabe, H., Yamaoka, Y., and Ozawa, K. Evaluation of the protective effect of a novel prostacyclin analog on mesenteric circulation following warm ischemia. Eur. Surg. Res. 28: 14, 1996. Yegen, C., Aktan, A. O., Buyukgebiz, O., Haklar, G., Yalcin, A. S., Yalin, R., and Ercan, S. Effect of verapamil and iloprost (ZK 36374) on endothelin release after mesenteric ischemia reperfusion injury. Eur. Surg. Res. 26: 69, 1994. Helset, E., Lindal, S., Olsen, R., Myklebust, R., and Jorgensen, L. Endothelin-1 causes sequential trapping of platelets and neutrophils in pulmonary microcirculation in rats. Am. J. Physiol. 271: L538, 1996. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411, 1988. Lopez, F. A., Riesco, A., Espinosa, G., Digiuni, E., Cernadas, M. R., Alvarez, V., Monton, M., Rivas, F., Gallego, M. J., Egido, J., et al. Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart. Circulation 88: 1166, 1993. Ishida, K., Takeshige, K., and Minakami, S. Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl phenylalanine. Biochem. Biophys. Res. Commun. 173: 496, 1990. Kelman, G. R. In G. R. Kelmans (Ed.), Applied Cardiovascular Physiology. Cardiovascular Measurements. London: Butterworth 1971. Pp. 210 –242. Hakim, T. S. Is flow in subpleural region typical of the rest of the lung? A study using laser Doppler flowmetry. J. Appl. Physiol. 72: 1860, 1992. MacLean, M. R., McCulloch, K. M., and Baird, M. Endothelin ETA- and ETB-receptor-mediated vasoconstriction in rat pulmonary arteries and arterioles. J. Cardiovasc. Pharmacol. 23: 838, 1994. Nagasaka, Y., Ishigaki, M., Okazaki, H., Huang, J., Matsuda, M., Noguchi, T., Toga, H., Fukunaga, T., Nakajima, S., and Ohya, N. Effect of pulmonary blood flow on microvascular pressure profile determined by micropuncture in perfused cat lungs. J. Appl. Physiol. 77: 1834, 1994. Zapol, W. M., and Snider, M. T. Vascular components of ARDS. Clinical pulmonary hemodynamics and morphology. Am. Rev. Respir. Dis. 136: 471, 1987. Vesconi, S., Rossi, G. P., Pesenti, A., Fumagalli, R., and Gattinoni, L. Pulmonary microthrombosis in severe adult respiratory distress syndrome. Crit. Care Med. 16: 111, 1988.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32. 33. 34.
151
Tomashefski, J. F., Jr., Davies, P., Boggis, C., Greene, R., Zapol, W. M., and Reid, L. M. The pulmonary vascular lesions of the adult respiratory distress syndrome. Am. J. Pathol. 112: 112, 1983. Hollenberg, S. M., Tangora, J. J., Piotrowski, M. J., Easington, C., and Parrillo, J. E. Impaired microvascular vasoconstrictive responses to vasopressin in septic rats. Crit. Care Med. 25: 869, 1997. Zhang, J. X., Pegoli, W., and Clemens, M. G. Endothelin-1 induces direct constriction of hepatic sinusoids. Am. J. Physiol. 266: G624, 1994. Murphy, D. D., and Wagner, R. C. Differential contractile response of cultured microvascular pericytes to vasoactive agents. Microcirculation 1: 121, 1944. Weibel, E. R. On pericytes, particularly their existence on lung capillaries. Microvasc. Res. 8: 218, 1974. Langleben, D., DeMarchie, M., Laporta, D., Spanier, A. H., Schlesinger, R. D., and Stewart, D. J. Endothelin-1 in acute lung injury and the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 148: 1646, 1993. Helset, E., Sildnes, T., Slejelid, R., and Konopski, Z. S. Endothelin-1 stimulates human monocytes release in vitro to release TNF-a, IL-1b, and IL-6. Mediators Inflamm. 2: 417, 1993. Arai, H., Hori, S., Aramori, I., Ohkubo, H., and Nakanishi, S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348: 730, 1990. Fukuroda, T., Kobayashi, M., Ozaki, S., Yano, M., Miyauchi, T., Onizuka, M., Sugishita, Y., Goto, K., and Nishikibe, M. Endothelin receptor subtypes in human versus rabbit pulmonary arteries. J. Appl. Physiol. 76: 1976, 1994. Stefan, U., and Roland, L. F. The interaction of endothelin receptor responses in the isolated perfused rat lung. NaunynSchmiedeberg’s Arch. Pharmacol. 356: 392, 1977. Ikeda, S., Awane, Y., Kusumoto, K., Wakimasu, M., Watanabe, T., and Fujino, M. A new potent endothelin receptor antagonist, TAK-044, shows long-lasting inhibition of both ETA and ETB mediated blood pressure responses in rats. J. Pharmacol. Exp. Ther. 270: 728, 1994. Haynes, W. G., Ferro, C. J., O’Kane, K. P., Somerville, D., Lomax, C. C., and Webb, D. J. Systemic endothelin receptor blockade decreases peripheral vascular resistance and blood pressure in humans. Circulation 93: 1860, 1996. Okada, M., Yamashita, C., Okada, M., and Okada, K. Contribution of endothelin-1 to warm ischemia/reperfusion injury of the rat lung. Am. J. Respir. Crit. Care Med. 152: 2105, 1995. Weitzberg, E., Hemsen, A., Rudehill, A., Modin, A., Wanecek, M., and Lundberg, J. M. Bosentan improved cardiopulmonary vascular performance and increased plasma levels of endothelin-1 in porcine endotoxin shock. Br. J. Pharmacol. 118: 617, 1996. Filep, J. G., Sirois, M. G., Rousseau, A., Fournier, A., and Sirois, P. Effects of endothelin-1 on vascular permeability in the conscious rat: interactions with platelet-activating factor. Br. J. Pharmacol. 104: 797, 1991. Lindsay, T. F., Walker, P. M., and Romaschin, A. Acute pulmonary injury in a model of ruptured abdominal aortic aneurysm. J. Vasc. Surg. 22: 1, 1995. Schoenberg, M. H., and Beger, H. G. Reperfusion injury after intestinal ischemia. Crit. Care Med. 21: 1376, 1993. Bounous, G. Acute necrosis of the intestinal mucosa. Gastroenterology 82: 1457, 1982. Nordin, A., Makisalo, H., Mildh, L., and Hockerstedt, K. Gut intramucosal pH as an early indicator of effectiveness of therapy for hemorrhagic shock. Crit. Care Med. 26: 1110, 1998.