1Department of General Surgery and 2Institute for Clinical and Experimental Surgery, University of Saarland, 66421 Homburg/Saar, Germany
Submitted 14 May 2002 ; accepted in final form 24 February 2003
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ABSTRACT |
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experimental pancreatitis; intravital microscopy; endothelin
ETs exist in three different isoforms (ET-1, ET-2, ET-3) (22), which are found not only in endothelial cells but also in other cell types of nerval, respiratory, urinary, and gastrointestinal tissue (17, 18, 21, 47). Expression of ETs and the corresponding receptors have also been demonstrated for the exocrine pancreas (19). Moreover, in the pancreatic gland, ETs have been shown to cause a deterioration of nutritive blood flow, an inflammatory response, and acinar cell necrosis (38, 39), which reflect the three distinct characteristics of acute necrotizing pancreatitis.
In acute experimental pancreatitis, both improvement and aggravation of disease have been reported as a result of ET-1 application (28, 31). The administration of a selective ET-1 receptor antagonist was shown to reduce pancreatitis-associated mortality (15), whereas simultaneous inhibition of the ETA and the ETB receptor was not capable of improving survival (13). This clearly demonstrates that the role of ETs in the pathogenesis of acute pancreatits is still controversial and that the individual function of ETA and ETB receptors is not yet fully understood. In particular, little is known regarding the contribution of the individual ET receptor functions on the development of pancreatitis-associated microvascular injury.
Using a rat model of sodium-taurocholate-induced pancreatitis and intravital microscopy, we therefore studied whether selective inhibition of ETA receptor function or combined ETA and ETB receptor blockade affects the development of microcirculatory failure, inflammation, and parenchymal injury.
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MATERIALS AND METHODS |
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After the animal was positioned for microscopy, two different observation areas (450 x 350 µm each) in the corpus and the tail of the gland were selected randomly. Subsequently, animals were treated either with an ET receptor antagonist or with vehicle for control. The ETA+B receptor antagonist was applied in two different concentrations, where the high concentration of 10 mg/kg body wt is thought to mediate a simultaneous inhibition of both receptors, whereas the low concentration of 4 mg/kg body wt predominantly inhibits the ETA receptor.
Baseline conditions were recorded for a 4-min period before acute pancreatitis was induced by retrograde, volume (0.6 ml)-, time (45 min)-, and pressure (<35 mmHg)-controlled infusion of sodium taurocholate (0.4%) by using an infusion pump (Braun Melsungen). The end of the intraductal infusion was defined as the beginning of the experiment (t = 0), and the microcirculatory parameters were recorded for an additional 120 min, choosing the same observation area as for baseline recording. Pancreatitis was confirmed by p-amylase-level analysis, which was performed before and after the experiment, as well as by histological examination.
In vivo microscopy of the pancreas. The pancreatic microcirculation was analyzed by means of a modified Leitz-Orthoplan epifluorescent microscope (100-W mercury lamp, excitation wavelength 450490 nm, emission wavelength >515 nm, final magnification x656 with a Leitz x20/0.32 long-distance objective). Experiments were recorded with a low-light charge-coupled device camera (model FK 6990; Pieper, Schwerte, Germany) and a SVHS video recorder (model AG-7350-E, Panasonic, Tokyo, Japan). For off-line analysis of the experiments, time was continuously superimposed by using a time/date generator (model VTG 33, FOR-A, Tokyo, Japan).
Assessment of microcirculatory parameters. Red blood cell (RBC) flow velocity in pancreatic capillaries was measured by means of syngeneic FITC-labeled erythrocytes, stained basically as described by Zimmerhackl et. al. (50). After a 10-min equilibration period before pancreas microscopy, 0.15 ml FITC-labeled RBCs was injected intravenously. Pancreatic microvessels were visualized by negative contrast due to staining of the pancreatic tissue with sodium fluorescein (50 µg/100 g body wt). The capillaries were differentiated from arterioles and venules by RBCs passing in single-file formation and by the individual microvascular diameter.
Each observation area displayed parts of three to four pancreatic lobules in which nine different capillaries were selected. Thus the selected capillaries were proportionally perfused by different feeding vessels. RBC velocities were determined off-line by using digitized frame-to-frame analysis, employing computer-supported image analysis (Lobulus, Medvis, Homburg, Germany). Because of the non-Newtonian flow properties of blood, the absolute values may not really reflect actual flow, but they should allow the assessment of relative changes compared with baseline conditions and between the groups.
Functional capillary density (FCD) was defined as the capillary length of
all RBC-perfused capillaries per observation area. According to the method of
Schmidt-Schönbein et al.
(44), a grid system with a
grid width of 50 µm was superimposed on the monitor, and the number of
intersections (Nc) between the grid and the capillaries
was counted. The FCD was calculated according to the equation
Lc = /2 · Nc/L,
where L represents the total length of the grid system. Overall
changes in total nutritive perfusion were assessed from the mean number of
perfused capillaries intersecting with the grid system and the individual
capillary volumetric blood flow. This perfusion index (PI) was calculated
according to the equation
,
where S represents a surface unit of the grid system,
reflects the volumetric blood
flow
(
),
V is velocity; and r is radius
(26). All off-line
measurements were performed by a blinded evaluator.
Pancreas histology and scoring system. For histology of pancreatic corpus and tail, formalin-fixed samples were embedded in paraffin and cut in 4-µm sections. Slides were stained with hematoxylin and eosin. Severity of acute pancreatitis was microscopically assessed by a blinded investigator according to Spormann et. al. (45) [edema: mild = 1, moderate = 2, severe = 3; fat necrosis: <2 sections = 3, 35 sections = 5, >5 sections = 7; parenchymal necrosis: focal (<5%) = 3, and/or sublobular = 5, and/or lobular (>20%) = 7; hemorrhages: mild = 3, moderate = 5, severe = 7]. The onset of an inflammatory response was examined by counting chloracetate esterase (CAE)-stained leukocytes in 50 high-power fields per animal (33).
Experimental protocol. Animals were pretreated intravenously with a combined ETA+B receptor antagonist [Tenzostan (Ro-61-0612); Actelion, Allschwill, Switzerland] (9) either at a high concentration of 10 mg/kg body wt (n = 8) or at a low concentration of 4 mg/kg body wt (n = 8). Additional animals (n = 8) received 1 µg/kg body wt of the ETA receptor antagonist (BSF-3021466/208075; Knoll, Ludwigshafen, Germany) to induce selective ETA receptor inhibition. Nontreated animals that only underwent acute pancreatitis served as controls (n = 8).
To demonstrate, whether the experimental model is stable over time and to exclude any deterioration due to preparation, a group of eight animals underwent no acute pancreatitis. Four of those eight animals underwent only a sham operation, whereas the other four animals were treated with a retrograde, volume (0.6 ml)-, time (45 min)-, and pressure (<35 mmHg)-controlled intraductal infusion of physiological sodium solution into the main pancreatic duct.
Statistical analysis. All results are expressed as means ± SE. Statistical differences within each group were determined by repeated-measures ANOVA, which was, if signifi-cant, followed by the Student-Newman-Keuls test. Different groups were statistically compared by one-way ANOVA followed by post hoc Student-Newman-Keuls test. When criteria for parametric tests were violated, the respective nonparametric test (Friedman repeated-measures ANOVA on ranks, Kruskal-Wallis analysis, and a signed-rank test) was used. A value of P < 0.05 was considered significant.
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RESULTS |
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As shown in Table 1, both sham operation and retrograde intraductal infusion of physiological sodium solution did not cause a significant deterioration of microcirculatory parameters such as mean capillary diameters, RBC velocity, FCD, and PI over the 2-h observation period. Accordingly, no significant tissue damage (Spormann score: sham operation 0.3 ± 0.3 and intraductal infusion physiological sodium solution 0.5 ± 0.3) and no significant recruitment of activated leukocytes (sham operation 6 ± 2 CAE-stained leukocytes/50 high-power fields and intraductal infusion physiological sodium solution 12 ± 6 CAE-stained leukocytes/50 high-power fields) were found in those animals.
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Neither induction of experimental pancreatitis nor pretreatment with the different ET receptor antagonists caused a change in macrocirculatory parameters over the entire observation period (Table 2). This proves that pancreatitis- and treatment-associated effects on microcirculatory parameters were not due to macrocirculatory changes.
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Capillary diameters. Induction of acute pancreatitis by retrograde intraductal infusion of sodium taurocholate caused a significant decrease in pancreatic capillary diameters, which was found to be sustained over the entire 2-h observation period (Fig. 1). Pretreatment with the combined ETA and ETB receptor antagonist in a concentration of 10 mg/kg body wt had no effect on the decline of capillary diameters after induction of acute pancreatitis. However, pretreatment with the ETA+B receptor antagonist in a concentration of only 4 mg/kg body wt inhibited the pancreatitisinduced decrease in capillary diameters over the entire observation period. Selective inhibition of the ETA receptor was not only effective in also completely abolishing the pancreatitis-induced decrease in capillary diameters but it even induced a slight increase in diameters when compared with baseline conditions (Fig. 1).
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RBC velocity. Initiation of acute pancreatitis resulted in a steep drop of RBC velocity from 654 ± 24 to 327 ± 26 µm/s, which reached its maximum at 6 min after the end of retrograde intraductal infusion of sodium taurocholate (Fig. 2). This initial decrease in RBC velocity of 47% was followed by a sustained reduction of 3238%, reflecting a significantly decreased mean RBC velocity of 367425 µm/s during the entire 2-h observation period. The decrease in mean RBC velocity was a consequence of an increased number of capillaries with either stasis or low-flow conditions and of a reduced number of capillaries that presented with very high flow velocity, reflecting a substantial increase in heterogeneity of RBC velocities.
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Pretreatment with the combined ETA and ETB receptor antagonist in a concentration of 10 mg/kg body wt showed a transient recovery of RBC velocity after the initial decrease, whereas this receptor antagonist given in the lower concentration of 4 mg/kg body wt caused a generally less pronounced decrease in RBC velocity (Fig. 2). Compared with the capillary RBC velocity distribution after pancreatitis without treatment, both concentrations of the combined ETA+B receptor antagonist increased the number of capillaries with stasis of RBC flow, and, after application of the lower concentration, a higher number of capillaries with normal and high RBC velocities was also observed.
Pretreatment with the selective ETA receptor antagonist caused a nonsignificant initial decrease in RBC velocity and a recovery to almost baseline values (Fig. 2). Thus, compared with pancreatitis without treatment, the selective blockade of the ETA receptor showed a less pronounced decrease of mean RBC velocity on induction of pancreatitis, which was due to only a small number of capillaries with stasis and a high number of capillaries with normal or high RBC velocities.
FCD. After induction of acute pancreatitis, quantitative analysis
of RBC-perfused pancreatic capillaries revealed that the decline of blood flow
velocity was paralleled by significant perfusion failure in 60% of the
initially perfused nutritive capillaries
(Fig. 3). This was indicated by
a reduction of FCD from 530 ± 12 cm1 at
baseline to 183 ± 11 cm1 at 2 h after
induction of pancreatitis.
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Pretreatment with the combined ETA+B receptor antagonist, regardless of the concentration used, had no significant effect on the pancreatitis-related decrease of FCD when compared with pancreatitis without treatment. In contrast, pretreatment with the high concentration (10 mg/kg body wt) of the combined ETA+B receptor antagonist resulted even in a more pronounced decrease of FCD with values of 114 ± 28 cm1 at 2 h after induction of pancreatitis (Fig. 3).
Pretreatment with the selective ETA receptor antagonist,
however, caused a significant attenuation of the pancreatitis-related decrease
in FCD. At 2 h after induction of pancreatitis, only 40% of the initially
perfused nutritive capillaries were found nonperfused, as was also reflected
by a FCD of still 334 ± 8 cm1
(Fig. 3).
PI. Experimental pancreatitis was characterized by a decline of the PI to only 1825% of the initial nutritive perfusion at baseline conditions. Simultaneous blockade of the ETA and ETB receptor (ETA+B receptor antagonist, 10 and 4 mg/kg body wt) showed an overall nutritive perfusion failure, which was slightly less pronounced but not significantly different when compared with the PI measured in animals undergoing acute pancreatitis without treatment (Fig. 4).
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In contrast, selective blockade of the ETA receptor was effective in significantly attenuating the pancreatitis-related decrease in overall nutritive perfusion (Fig. 4). After pretreatment with the selective ETA receptor antagonist, a decrease of the PI to only 6070% was calculated, which indicates improved overall nutritive perfusion, when compared with all other groups studied.
Pancreas histology. Histomorphology after sodium taurocholate-induced acute pancreatitis was analyzed semiquantitavely by using the Spormann score. Pancreatitis without treatment was characterized by severe interstitial edema formation, considerable necrosis of fatty tissue, distinct sublobular and lobular parenchymal necrosis, as well as hemorrhage, resulting in a Spormann score of 17 ± 1, which reflects severe, acute, necrotizing pancreatitis.
Pretreatment with the combined ETA+B receptor antagonist in a concentration of 10 mg/kg body wt had no beneficial effect on the pancreatitis-associated tissue injury, whereas the lower concentration of 4 mg/kg body wt caused a significant reduction of the tissue damage (Fig. 5). The Spormann score had improved to 11 ± 1, which was mainly due to a decrease in degree and amount of fatty tissue and parenchymal cell necrosis.
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Pretreatment with the selective ETA receptor antagonist caused a further attenuation of tissue damage, which was reflected by a Spormann score of only 9 ± 1. This reduction was caused by a significant decrease in interstitial edema formation as well as by an attenuation of fatty tissue and parenchymal cell necrosis (Fig. 5).
Inflammatory response. The inflammatory response in acute pancreatitis is characterized by accumulation of leukocytes in postcapillary venules and infiltration of these cells into tissue. In nontreated pancreatitis, a total of 207 ± 29 CAE-stained leukocytes were counted in 50 high-power fields, 113 ± 22 CAE-stained leukocytes per 50 high-power fields were found in the pancreatic tissue, whereas 94 ± 9 CAE-stained leukocytes per 50 high-power fields were counted in postcapillary venules (Fig. 6).
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Pretreatment with the combined ETA+B receptor antagonist in a dose of 10 mg/kg body wt caused an increase of the number of CAE-stained leukocytes (total: 340 ± 90, tissue: 188 ± 61, postcapillary venules: 151 ± 31 per 50 high-power fields), whereas pretreatment with a dose of 4 mg/kg body wt resulted in a significant attenuation of CAE-stained leukocyte recruitment (total: 175 ± 33, tissue: 93 ± 22, postcapillary venules: 83 ± 16 per 50 high-power fields) (Fig. 6).
After pretreatment with the selective ETA receptor antagonist, only a total of 121 ± 16 CAE-stained leukocytes were counted in 50 high-power fields, which reflects a significant reduction of pancreatitis-related recruitment of activated leukocytes, when compared with nontreated pancreatitis controls (Fig. 6). More interestingly, pretreatment with the selective ETA receptor antagonist predominantly caused a decreased number of CAE-stained leukocytes (41 ± 8 per 50 high-power fields) that had already emigrated in the pancreatic tissue, whereas the number of CAE-stained leukocytes accumulating in postcapillary venules (80 ± 8 per 50 high-power fields) remained almost unchanged, compared with the nontreated pancreatitis group.
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DISCUSSION |
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Before induction of experimental pancreatitis, the animals were pretreated with either a combined ETA+B receptor antagonist or a highly selective ETA receptor antagonist. The ETA+B receptor antagonist was applied in two different concentrations, where the high concentration of 10 mg/kg body wt is thought to mediate a simultaneous inhibition of both receptors, whereas the low concentration of 4 mg/kg body wt predominantly inhibits the ETA receptor. The control group, which did not undergo acute experimental pancreatitis, proves that the experimental model was stable over time and that no deterioration was caused by the preparation. Retrograde intraductal infusion of physiological sodium solution caused no significant deterioration of microcirculatory or histological parameters, which excludes nonspecific pressure effects as the cause of experimental pancreatitis (40). The difference in mean baseline capillary diameters between the untreated control groups and the groups pretreated reflects inhibition of ET caused vasoconstriction by ET receptor antagonists.
Pretreatment with the high concentration of the ETA+B receptor antagonist showed no significant improvement of pancreatitis-induced microcirculatory injury when compared with animals that underwent pancreatitis without treatment. Evaluation of the magnitude of perfusion failure by determination of the FCD revealed an even higher number of nonperfused capillaries after pretreatment. Accordingly, evaluation of tissue damage and inflammatory response caused by experimental pancreatitis proved an aggravation rather than an improvement as a result of pretreatment with the high dose of the ETA+B receptor antagonist.
In contrast, pretreatment with the low concentration of the ETA+B receptor antagonist revealed a signifi-cantly less pronounced reduction of capillary diameters, whereas deterioration of RBC velocity, FCD, and PI were not affected. However, determination of leukocyte recruitment and parenchymal tissue injury still displayed a significant attenuation when compared with nontreated pancreatitis controls. Furthermore, the selective inhibition of the ETA receptor was effective to almost abrogate the pancreatitis-induced microcirculatory disorders, parenchymal injury, and inflammatory response. The decline in overall leukocyte recruitment was caused by preventing leukocytes to emigrate into the pancreatic tissue, whereas leukocyte accumulation in postcapillary venules remained almost unchanged, compared with the nontreated pancreatitis group.
This might suggest that selective inhibition of the ETA receptor prevents migration of leukocytes in acute experimental pancreatitis, which is in accordance with findings demonstrating that ET-1 ETA receptor interaction plays a key role in leukocyte adhesion and migration (8, 10, 23, 43, 51). Furthermore, there is an increasing body of evidence that ETA receptor antagonists inhibit ET-1-mediated adhesion and migration of leukocytes, whereas selective ETB receptor antagonists are ineffective (10, 43, 51).
The fact that simultaneous inhibition of ETA and ETB receptors caused no improvement, whereas more pronounced or selective inhibition of the ETA receptor did improve the time course of experimental pancreatitis, might be explained by the physiological function of the ETB receptor. Stimulation of the ETB receptor mediates the clearance of ETs and the liberation of nitric oxide, which counteracts the ET-mediated vasoconstriction (16, 32, 36, 42). Therefore, simultaneous inhibition of ETA and ETB receptors results in both sustained high local ET concentrations and deficiency of nitric oxide-mediated vasodilation, which may mask the beneficial effect of inhibition of the ETA receptor. This might also explain that simultaneous, maximal inhibition of ETA and ETB receptors caused some aggravation of microcirculatory disorders, and it extends the knowledge based on results of others who could not demonstrate a survival benefit in acute experimental pancreatitis after treatment with the combined ETA+B receptor antagonist bosentan (13). This is also in compliance with the current opinion that selective ETA receptor antagonists have a number of still hypothetical advantages over nonselective ET receptor antagonists (1). Moreover, the role of the ETB receptor in circulatory homeostasis is known to be ambiguous as reflected in either vasodilation or vasoconstriction ascribed to the receptor, whereas the clearing function for ET-1 of the ETB receptor seems to be of pivotal importance (42). In addition, a more recent study (11) could not demonstrate significant benefits for selective ETB receptor inhibition in acute experimental pancreatitis.
Our results showing that selective inhibition of the ETA receptor attenuates pancreatitis-induced microcirculatory failure, inflammation, and parenchymal injury is in good concordance with the finding that transgenic rats with ET-1 receptor overexpression develop a more severe pancreatitis and that the administration of a selective ET-1 receptor antagonist in acute pancreatitis reduces fluid sequestration, improves microcirculation, and ameliorates mortality (14, 15).
Overall, these findings implicate the pivotal contribution of the ETA receptor in the pathogenesis of the pancreatitis-associated microcirculatory failure, and they underline the key function of ETs in acute necrotizing pancreatitis.
In normal pancreas, ETs and especially ET-1 mediate microcirculatory deteriorations, which are associated with local inflammation and intrapancreatic tissue damage (38, 39). These recent studies have also shown that the magnitude of microcirculatory failure is strictly related to the amount of final tissue damage, indicating that ischemia may act as a major cause of acinar cell necrosis. In accordance, it has been shown that temporary short-term pancreatic ischemia causes reversible functional and morphological changes (4, 25), whereas long-lasting ischemia results in necrotizing pancreatitis (30) and that therapeutic approaches, which aim at preventing microcirculatory stasis, limit the extent of pancreatic necrosis (24). In line with this, the present study now demonstrates that selective inhibition of the ETA receptor causes a significant attenuation of the pancreatitis-associated nutritive perfusion failure, which is paralleled by a significant reduction of pancreatits-associated inflammation and tissue injury. Thus we conclude that microcirculatory failure with subsequent ischemia caused by ETs coupling onto the ETA receptor is a determining factor in the pathogenesis of acute necrotizing pancreatits and that inhibition of the ETA receptor function may represent an interesting, novel treatment approach.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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