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reperfusion
Pentoxifylline protects splanchnic prostacyclin synthesis during mesenteric ischemia/reperfusion S.L Myers, J.W. Horton, R. Hernandez, P. B. Walker, and W. G. Vaughan Dep a r t m e nt s of Surgery, University of Texas Southwestern Medical Center and the Dallas Veterans Administration Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75235
This study examines the hypothesis that pentoxifylline protects splanchnic PGI2 synthesis during severe mesenteric ischemia/ reperfusion injury. Anesthetized Sprague-Dawley rats (300 grams) were subjected to sham or superior mesenteric artery occlusion for 20 minutes followed by 30 minutes of reperfusion. The ischemia/ reperfusion groups received either enteral allopurinol (10 mg/kg) daily for 5 days prior to ischemia, PTX (50 mg/kg) 10 minutes prior to ischemia or carrier. The superior mesenteric artery was cannulated and removed with its intact intestine (SV + SI). The SV + SI was perfused in vitro with oxygenated Krebs buffer. The venous effluent was collected and assayed for release of 6-keto-PGF1~, PGE2 and thromboxane B2 by enzyme immunoassay. Severe mesenteric ischemia/ reperfusion decreased SV + SI 6-keto-PGF~ release by 40% compared to the sham group but did not alter release of PGE2 or thromboxane B2. Pretreatment of the animals with PTX and not allopurinol preserved SV + SI 6-keto-PGFl~ release at all times of perfusion to a level similar to the sham group. These data showed that severe mesenteric ischemia/reperfusion injury abolished release of endogenous splanchnic PGI2. PTX exerted a protective effect against severe mesenteric ischemia/reperfusion injury by maintaining release of splanchnic PGI2, a potent endogenous splanchnic vasodilator.
An abstract of this paper was presented at the 16th Annual Conference on Shock in Santa Fe New Mexico, June, 1993. Address correspondence to: Stuart I. Myers, M.D., Department of Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75235.
© 1994 Butterworth-Heinemann
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Keywords: Mesenteric ischemia; mesenteric PGIz; pentoxifylline; ischemia/ reperfusion injury
Introduction The gastrointestinal tract has been described as the motor organ in the evolution of multiple organ failure following ischemia/reperfusion injury? ,2 The syndrome of multiple organ failure begins to manifest clinically by the onset of enteric sepsis (bacterial translocation) and ileus suggesting that altered splanchnic blood flow is an important initiating factor for this syndrome. 1-5Formation of oxygen-derived free radicals have been shown to be an important factor involved with altered intestinal function following acute ischemia/reperfusion injury.6-s Use of scavengers of oxygen-derived free radicals has been shown to prevent both splanchnic vasoconstriction and intestinal permeability following acute hemorrhage/ reperfusion injury. 9,1°One mechanism for ODFR induced splanchnic vasoconstriction was recently shown to be the inhibition in the synthesis and release of splanchnic PGI2, a potent endogenous vasodilator by ODFR formationY Recently, Pentoxifylline (PTX) has been shown to increase survival in animals treated with hemorrhagic shock. These studies showed that PTX increased tissue oxygenation, increased oxygen consumption, decreased leukocyte adhesiveness and increased intestinal microvascular blood flow. 11-14 PTX has been proposed to improve intestinal microvascular blood flow by the decreased "plugging" of microvascular vessels by increasing deformability of leukocytes. This hypothesis was supported by in vitro studies that showed that PTX increases leukocyte deformability, decreases leukocyte adherence, enhances chemotaxis and blocks the action of inflammatory cytokines on leukocyte function? s-19 The present study examines the hypothesis that PTX maintains splanchnic PGIz release following severe ischemia/reperfusion injury of the intestine. We will utilize both PTX and allopurinol (xanthine oxidase inhibitor which prevents ODFR formation) to more specifically identify a mechanism of action involved with PTX protection of splanchnic PGI2 release following severe ischemia/reperfusion injury.
Methods
Surgical Model Adult male Sprague-Dawley rats (350 grams) were housed and used in compliance with the regulations of the animal care facility of the University of Texas Southwestern Medical Center and the Dallas VAMC. All animals were allowed food and water ad lib 18 hours prior to the experi138
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ment; at this time solid food was removed but animals had free access to water until time of the experiment. Experimental animals received either enteral allopurinol (N = 8)(10), a xanthine oxidase inhibitor, (10 mg/ kg) daily for five days prior to mesenteric ischemia or PTX (N= 10), a phosphodiesterase inhibitor, (50 mg/kg) intravenously, 10 minutes prior to mesenteric ischemia and 1 minute prior to reperfusion. A third group of untreated animals underwent mesenteric I/R and served as ischemic controls. An additional group (N=9) served as untreated, nonischemic (Sham) controls. The animals were anesthetized with inhalational methoxyflurane; ketamine hydrochloride (20 mg/kg IM) was administered to supplement anesthesia. The ventral neck and abdomen were shaved and prepped followed by cannulation of the carotid artery for blood gas analysis and h e m o d y n a m i c monitoring. The internal jugular vein was accessed for the acquisition of blood samples and the administration of fluids and pharmacologic agents. Heparin (500 units/kg) was injected intravenously in all rats after surgical preparation. Arterial blood gas measurements were monitored prior to ischemia/reperfusion and 30 minutes following reperfusion (see below). Mean arterial blood pressure and heart rate were continuously monitored by a Grass recorder (Grass Instrum e n t Co., Quincy, Mass.).
Preparation of the Rat for Mesenteric Ischemia/Reperfusion Injury Body temperature was maintained at 38°C by use of a heating pad and a heating lamp. Animals underwent laparotomy and the superior mesenteric artery (SMA) and collateral vessels were occluded as previously described. 2° In the sham group, rats underwent laparotomy with SMA and collateral vessel isolation without occlusion. All rats were studied at identical time periods regardless of group assignment. After 20 minutes of complete ischemia, the vascular clip was removed from the SMA and the bowel was reperfused for 30 minutes and then prepared for isolated perfusion studies.
Preparation of the ex vivo Isolated Mesenteric Perfusion After 30 minutes of reperfusion, the SMA was rapidly cannulated and removed with its end organ intestine (SV + SI preparation). The SV + SI was perfused ex-vivo by a modification of the techniques described by McGregor and Myers. ~,2~-23The SV + SI was perfused with oxygenated Krebs-Henseleit (O2/CO2 95:5, PO2 460 + 10 m m Hg) buffer at 3 m l / m i n with a Cole-Palmer peristaltic pump (Ph 7.40) at 37°C. The ends of the bowel were vented outside the warming apparatus to avoid distention. Perfusion pressure was monitored via a side arm of the arterial cannula using a Statham pressure transducer. The splanchnic venous effluent was collected after 5, 15, 30, 45 and 60 minutes of perfusion. Each sample
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was placed in siliconized micro-centrifuge tubes and immediately frozen at - 4 0 ° C until assayed by enzyme immunoassay techniques.
Enzyme Immunoassays (EIA) Venous effluent was analyzed for 6-keto-PGG~ (metabolite of PGI2), thromboxane B2 (TxB2) and PGE2 on Krebs-Henseleit buffer by enzymatic immunoassay as previously described. 2~The EIA reagents were purchased from Cayman Chemical Co. (Ann Arbor, MI). Inter-assay and intra-assay variation was 7-10% and were similar to those reported by our laboratory for RIAs. 23
Histology At the time of cannulation of the superior mesenteric artery and removal of the Sv + SI preparation (just prior to initiation of the ex-vivo perfusion), a 2 cm segment of distal jejunum was excised from nonischemic Sham controls, the ischemic controls and the ischemic groups treated with allopurinol and pentoxifylline. The specimens were placed in 10% formalin and submitted for histopathologic examination in a blinded fashion. Ischemic lesions were graded on a scale of 0 to 5 as described by Megison et al. 24 Grade 0 represents normal villi. Grade 1 represents the development of subepithelial space at the apex of the villus; capillary congestion. Grade 2 represents extension of subepithelial space with moderate separation of mucosa from lamina propria. Grade 3 represents extensive epithelial separation from lamina propria down the sides of the villi, with ulceration at villus tips. Grade 4 represents areas of denuded villi, dilated capillaries and increased cellularity of lamina propria. Grade 5 represents disintegration of lamina propria and hemorrhagic ulceration.
Statistical Analysis Prostanoid levels were assessed by a repeated measures analysis of variance. This analysis allows for comparisons among groups and across times, as well as evaluating differential change among groups over time (interaction). Results were considered significant when p < 0.05. Post hoc multiple comparisons were made using a Bonferroni strategy at the 0.05 level. This data analysis was performed after prostanoid levels were log-transformed (base 10) since it was observed that variability was correlated to mean level. Analysis of histologic injury score, arterial pH, PCO2 content, PO~ content, mean arterial blood pressure and heart rate was performed by analysis of variance and use of the Bonferroni strategy at the 0.05 level. All data is reported as mean + S.E.M. Analysis was performed using SAS software.
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Pentoxifylline protects splanchnic during eicosanoid ischemia: Myers et al. Results The mean arterial pressures and heart rates were similar at the basal time of measurement in all groups (Table 1 ). Mean arterial pressures measured in all ischemia/reperfusion groups after 30 minutes of reperfusion and at the 50 minute time period in the sham group decreased to a similar level. Heart rate was unchanged in the 50 minute time period of measurement in the Sham group (Table 1). The heart rate significantly decreased following 30 minutes of reperfusion in the untreated ischemia/reperfusion group (p < 0.02 compared to basal level) whereas heart rate significantly increased in the PTX group (p < 0.009 compared to basal measurement). Basal measurement of arterial pH was similar in the sham and all ischemia/reperfusion groups and was unchanged at the 50 minute measurement in the sham group and after 30 minutes of reperfusion in all ischemia/reperfusion groups (Table 2). Basal measurements of arterial PO2 and PCO~ content were similar in all groups (Table 2). Arterial PO~ and PCO~ content were not changed at the 50 minute time period of measurement in the sham group. Arterial PCO2 content in all ischemia/reperfusion groups decreased to similar levels after 30 minutes of reperfusion (Table 2). Arterial PO2 content increased in the PTX group after 30 minutes of reperfusion but was unchanged in the other experimental groups (Table 2). The ex-vivo perfused sham rat SV + SI preparation released 6-ketoPGFI~, PGE2 and TxB2 at all times of isolated perfusion (Figures 1-3). The major eicosanoid released from the SV + SI preparation was 6-keto-PGFl~ which was released 4 fold or greater than the other eicosanoids assayed TABLE 1. injury'
Effect of pentoxifylline and allopurinol on systemic pressure and heart rate during ischemia/reperfusion
Sham MAPS-03 Tin. (mmHg) MAPS-504 min. (mmHg) HRS-03 Tin. HR6-504 Tin.
92.0 64.2 207.7 197.5
_+6.6 _+4.0 x _+9.8 _+ 12.0
Carrier + I/R 2 100 61.1 219 180
~+4.0 ___4.8 × --- 9.4 ___8.2 x
Pentoxifylline + I/R 2 88.2 64.2 233 271
_+6.0 _+3.5 x _+7.2 xx _+ 12.0 x
AIIpurinol + I/R 2 103.3 70.6 201 185
_+4.5 _+8.3 x _+ 10.1 _+7.8 x
1Data expressed as Mean _+S . E . M (N = 8 or more). 2Superior mesenteric ischemia for 20 T i n followed by 30 T i n of reperfusion. 3Basal measurement (prior to ischemia). 450 minutes in Sham group or just following 30 minutes of reperfusion. 5Mean arterial pressure. 8Heart Rate. Xp < .05 level compared to same group at 0 time level by ANOVA. xxp < .05 level compared to Sham, AIIopurinol and Carrier I/R groups by ANOVA.
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Pentoxifylline protects splanchnic during eicosanoid ischemia: TABLE 2. injury 1
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Effect of pentoxifylline and allopurinol an arterial blood gas measurements during ischemia/reperfusion
Sham pHS-03 pHS-504 PO26-03 PO28-504 PCO27-03 PCO~7-504
7.38 _+0.001 7.41 + 0.006 91.2 _+3.1 97.9 _+5.0 27.0 _+0.9 34.7 _+1.5
Carrier
Pentoxifylline
AIIopurinol
+ I/R2
+ I/R 2
+ I/R 2
7.39 _+0.001 7.40 _+0.001 94.1 + 7.0 103.0 _+7.4 35.0 _+2.3 29.8 _+1.8 X8
7.40 _+0.001 7.43 _+0.007 85.2 _+3.2 102.0 +_4.8 x 35.3 +_1.4 22.7 _+1.1 x8
7.38 _+0.001 7.39 _+0.001 80.0 +_5.2 88.6 +_6.2 36.6 +_0.9 29.9 _+1.7x8
1Data expressed as Mean_+S.E.M. (N = 8 or more). 2Superior mesenteric ischemia for 20 rain followed by 30 min of reperfusion. 3Basal measurement prior to ischemia. 450 minute measurement in Sham group or just following 30 minutes of reperfusion. SArterial pH. 6Arterial oxygen content. 7Arterial PC02 content. 8p < .05 compared to Sham by ANOVA. xp < .05 compared to same group at 0 time measurement.
in the sham group (Table 1, Figures 1-3). Release of 6-keto-PGFl~, TxB2 and PGE2 in all groups was highest at 5 or perfusion and fell slightly after 15 and 30 minutes perfusion (Figures 1-3). Subjecting the untreated rats to mesenteric ischemia for 20 minutes followed by reperfusion for 30 minutes decreased release of vasodilator 6-keto-PGG~ from the in vitro perfused splanchnic bed when compared to the sham group (p < 0.05) (Table 1, Figure 1). After 30 minutes of perfusion,mesenteric ischemia for 20 minutes followed by 30 minutes of reperfusion decreased splanchnic release of 6-keto-PGF~ by approximately forty percent when compared to the sham group. In contrast, rats pretreated with PTX and subjected to mesenteric ischemia followed by reperfusion synthesized and released quantities of 6-keto-PGFl~ similar to those from sham animals (Table 1, Figure 1). Pretreatment of the rats with allopurinol did not protect splanchnic release of 6-keto-PGG~ following severe mesenteric ischemia followed by reperfusion (Table 1). In vitro perfused splanchnic release of PGE2 was not significantly altered by mesenteric ischemia, treatment with carrier, allopurinol or PTX (Table 1, Figure 2). Release of TxB2 was minimal in all groups of animals and was not altered by mesenteric ischemia followed by reperfusion (Table 1, Figure 3). Use of allopurinol pretreatment had virtually no detectable effect on the production and release of splanchnic prostanoids in our model of severe mesenteric ischemia (Table 3). 142
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Minutes of Perfusion FIGURE 1. The effect of pentoxifylline on superior mesenteric artery (SMA) 6-keto-PGFl~ release. Rats received pentoxifylline (PTX, open box) or carrier (filled box) and were subjected to SMA occlusion and reperfusion as described under Methods and compared to Sham controls (hatched box). SV + SI preparations were perfused in vitro and the venous effluent collected after 5, 15, and 30 minutes of perfusion and assayed for 6-keto-PGFl~ (PGI2 metabolite). Data is calculated as ng 6-keto-PGF,, released/minute and a logarithmic (base 10) transformation (LN) is used prior to analysis of the data. Data is reported as mean _+S.E.M. (N = 10, * indicates significance vs. PTX and sham groups at p < 0.05 by ANOVA).
Histologic examination showed a significant increase in injury score from 0.6 _+ 0.2 in the nonischemic Sham group to 4.1 + 0.1 (p < 0.0001) in the untreated ischemia/reperfusion group. Treatment with PTX or allopurinol significantly decreased the injury score to 3.4 +_ 0.1 (p < 0.05) and 2.6 + 0.8 (p < 0.05) respectively when compared to the untreated ischemia/reperfusion group. However the injury score for both the PTX and allopurinol groups remained significantly higher (at p < 0.0002) when compared to the Sham group.
Discussion Hemorrhagic shock is a c o m m o n l y encountered clinical entity which contributes to the mortality of critically ill patients.~-5 For decades, the treatment of these disorders of ischemia/reperfusion has been the logical replacement of volume losses with red cells, crystalloid and colloid solu-
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1.25 [
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0.75
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Minutes of Perfusion FIGURE 2. The effect of pentoxifylline on superior mesenteric artery (SMA) TxB~ release. Rats received pentoxifylline (PTX, open box) or carrier (filled box) and were subjected to SMA occlusion and reperfusion as described under Methods and compared to Sham controls (hatched box). SV + SI preparations were perfused in vitro and the venous effluent collected after 5, 15, and 30 minutes of perfusion and assayed for TxB2 (thromboxane A2 metabolite). Data is calculated as ng TxB2 released/minute and a logarithmic (base 10) transformation (LN) is used prior to analysis of the data. Data is reported as mean _+S.E.M. (N= 10). Untreated ischemic control group (carrier) was not significantly different than the PTX and sham groups (at p < 0.05 by ANOVA).
tions; however the adverse sequelae of shock are often progressive and may lead to multiple organ failure despite seemingly adequate initial resuscitation. 1-4 The gastrointestinal tract has been purported to play a contributing role in the pathogenesis of multiple organ failure. 1,,~ This hypothesis encompasses the notion that clinical shock and resuscitation lead to a decrease in splanchnic microcirculatory blood flow. This concept is not new since, during periods of shock, the sympathetic nervous system mediates responses that preserve blood flow to more vital structures (i.e., brain, heart, lung), thus, depriving other organs of adequate blood flow. When this vasoconstrictive response becomes prolonged, severe reductions in microcirculatory flow of less central organs leads to initiation and enhancement of an inflammatory process. This phenomenon, characterized by the production and release of inflammatory mediators (TNF, eicosanoids, complement and cytokines), aggregation of red cells, neutro144
Prostaglandins 1994:47, February
Pentoxifylline protects splanchnic during eicosanoid ischemia: Myers et al. 0.75
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FIGURE 3. The effect of pentoxifylline on superior mesenteric artery (SMA) PGE2 release. Rats received pentoxifylline (PTX, open box) or carrier (filled box) and were subjected to SMA occlusion and reperfusion as described under Methods and compared to Sham controls (hatched box). SV + SI preparations were perfused in vitro and the venous effluent collected after 5, 15, and 30 minutes of perfusion and assayed for PGE2. Data is calculated as ng PGE2 released/minute and a logarithmic (base 10) transformation (LN) is used prior to analysis of the data. Data is reported as mean+-S.E.M. ( N - 1 0 ) . Untreated ischemic control group (carrier) was not significantly different from the PTX and sham groups (at p < 0.05 by ANOVA).
TABLE 3.
PGI23 PGE2 TxB24
Effect of allopurinol on splanchnic eicosanoid release during ischemia/reperfusion injury,
Sham
Carrier + I/R 2
3.4 +- 0.2 0.4 +- 0.2 0.9-+0.1
2.08 + 0.3 X 0.2 _+0.2 0.7+_0.1
Pentoxifylline + I/R 2 3.14 +_0.1 0.65 _+0.1 0.8+_0.1
Allopurinol + I/R 2 1.7 +- 0.09 X 0.3_+ 0.1 0.9_+0.07
'Data expressed as ng/min (Mean +_S.E.M., N = 8 or more) at 5 Tin of perfusion. 2Superior mesenteric ischemia for 20 Tin followed by 30 Tin of reperfusion. 36-keto-PGG,,. 4Thromboxane B2. ×p < .05 level by repeated measure AOVA compared to Sham and pentoxifylline groups.
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phils and platelets causes further sludging of flow and contributes to the local ischemic process. 2s,~6 PTX is a methylxanthine, phosphodiesterase inhibitor that increases intracellular cAMP levelsY Its potential role in lessening the effects of the "no reflow" phenomenon makes PTX a promising agent for improving microcirculatory abnormalities that develop as a result of ischemia/reperfusion. Several studies have shown that PTX increases survival of animals subjected to acute ischemia/reperfusion injury.ll-14 These studies showed that use of PTX during ischemia/reperfusion injury increased oxygen consumption, tissue oxygen uptake, and increased hepatic and intestinal microvascular blood flow. The exact mechanisms of action of PTX in increasing survival following ischemia/reperfusion injury are not known. However, several in vitro studies provide insight into the possible mechanisms of action of PTX. PTX has been shown to decrease viscosity and increase red blood cell and neutrophil deformability,2s-3° improving flow through the microcirculation. PTX has been shown to decrease superoxide release during neutrophil and macrophage activation and to decrease adhesion by the neutrophil26,29,3~ Several studies have demonstrated that PTX inhibits cytokine (tumor necrosis factor, IL-1) induced neutrophil activation thus preventing lysosomal degranulation and superoxide production and neutrophil adhesion to endothelium. ~9,~3° Locally synthesized eicosanoids have been shown to have a role in the regulation of splanchnic blood flow during normal and pathologic states.F, 3234 PGI~ has been shown to be the predominant eicosanoid released by the splanchnic bed, is a known potent inhibitor of platelet aggregation and is a splanchnic arterial vasodilator. 34Both of these physiologic actions of PGI2 combine to contribute to maintenance of splanchnic vascular tone and in particular to antagonize sympathetic arterial vasoconstriction, g~,3,~ We have previously shown that PGI2 is the predominant eicosanoid released by the splanchnic vascular bed. These studies showed that the splanchnic bed can dramatically increase endogenous synthesis and release of PGI2 in response to acute hemorrhage.3s However this endogenous mechanism of the bowel to attempt to maintain splanchnic blood flow during acute hemorrhage is overwhelmed by both prolonged hemorrhage and reperfusion of shed blood, a6,37Subsequent studies showed that production of locally produced ODFRs inhibited both splanchnic PGI2 release and superior mesenteric blood flow during reperfusion following acute hemorrhage? These studies did not examine the effects of PTX on protection of splanchnic PGI2 release during severe ischemia/reperfusion injury. The experiments presented in this study were designed to test the hypothesis that PTX maintains splanchnic PGI2 release during severe intestinal ischemia/reperfusion injury. The experimental model used to examine this hypothesis was the selective clipping of the superior mesen-
146
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teric artery for 20 minutes followed by reperfusion for 30 minutes which produced a relatively severe localized intestinal ischemia/reperfusion injury. The hemodynamic data showed that the SMA clip model did not alter systemic hemodynamics as mean arterial blood pressure, arterial pH, PCO2 or PO2 were not significantly altered by intestinal ischemia/ reperfusion or PTX or allopurinol treatment (Tables 1, 2). The splanchnic eicosanoid release data demonstrated that pentoxifylline protects splanchnic prostacyclin production and release in a model of severe ischemia/ reperfusion injury. This would theoretically promote continued microcirculatory flow to the bowel mucosa and prevent or lessen the ischemic insult brought about by hypovolemic or low-flow shock states. In contrast to PTX treatment, allopurinol, a xanthine oxidase inhibitor 38 had no demonstrable effect on the release of prostacyclin as evidenced by reduced levels of 6-keto-PGFl~ similar to those of the untreated ischemic animals. It is entirely possible that the severity of ischemia in our model overwhelmed any effect that allopurinol may have had in preventing the formation of these highly reactive oxygen radicals. This concept is corroborated by findings from our previous studies that showed an inability of the splanchnic bed to compensate for episodes of prolonged hemorrhagic shock, g6 One must also consider the possibility that the allopurinol pretreatment period was too short or that the dosage was inadequate, in this study, to cause inhibition of xanthine oxidase. The histologic studies support the eicosanoid release data described above. PTX pretreatment significantly decreased the intestinal injury score when compared to the untreated ischemia/reperfusion group. However, despite normalizing release of endogenous splanchnic PGI2 release, PTX pretreatment did not completely reverse the intestinal injury. These findings suggest that splanchnic PGI2 synthesis is only one of the factors which contributes to maintaining normal intestinal viability during ischemia/reperfusion injury. Although the exact mechanisms of action exerted by PTX in our study are unknown we propose that PTX contributes to maintaining splanchnic PGI2 release during severe mesenteric ischemia/reperfusion injury by several mechanisms of action which have been previously documented in several in vitro and in vivo studies. These mechanisms of action include decreasing viscosity, increasing deformability of red blood cells and neutrophils, and by contributing to prevention of the activation of neutrophils by cytokines or local tissue factors. Prevention of the activation of neutrophils would decrease neutrophil adhesion and superoxide production in the microvasculature.lS-18,29 Although extrapolation of data collected from this model to the human can be made only with extreme caution, our data may have clinical implications. Maintaining splanchnic microcirculatory flow during period of ischemic or hemorrhagic shock is a worthy goal that, if achieved, may prevent intestinal barrier failure and its concomitant
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sequelae. PTX has a variety of pharmacologic properties that may be beneficial in helping achieve the aforementioned goal, yet, the relative importance of these properties of pentoxifylline awaits definition in future laboratory and clinical trials.
Acknowledgments These studies were supported by the National Institutes of Health grants GM-38529 (S.I.M), GM-21681 (J.W.H.) and VA Merit Grant (S.I.M).
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30.
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expression, degranulation, and superoxide production. Journal of Leukocyte Biology 47:244. 1990. Bessler, H., Gilgal, R., Djaldetti, M., and Zahavi, I. Effect of pentoxifylline on the phagocytic activity, CAMP levels, and superoxide anion production by monocytes and polymorphonuclear cells. Journal of Leukocyte Biology 40:747. 1986. Hammerschmidt, D.E., Kotasek, D., McCarthy, T., Huh, P.W., Freyburger, G., and Vercellotti, G.M. Pentoxifylline inhibits granulocyte and platelet function, including granulocyte priming by platelet activating factor. J. Lab. Clin. Med. 112:254. 1988. Armstrong, M., Jr., Needham, D., Hatchell, D.L., and Nunn, R.S. Effect of pentoxifylline on the flow of polymorphonuclear leukocytes through a model capillary. Angiology 41:253. 1990. Sullivan, G.W., Carper, H.T., Novick, W.J., Jr., and Mandell, G.L. Inhibition of the inflammatory action of inteleukin-1 and tumor necrosis factor (alpha) on neutrophil function by pentoxifylline. Infection and Immunity 56:1722. 1988. Megison, S.M, Horton, J.W., Chao, H., and Walker, P.B. A new model for intestinal ischemia in the rat. J. Surg. Res. 49:168. 1990. McGregor, D.D. The effect of sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels of the rat. J Physiol 177:21. 1965. Myers, S.I., Hernandez, R., White, D.J., and Horton, J.W. Burn injury decreased splanchnic PGI~ release is restored by treatment with lazaroid. Prostaglandins 45:535. 1993. Reed, M.K., Taylor, B., and Myers, S.I. The effect of hypoxia on rat splanchnic prostanoid output. Prostaglandins 38:599. 1989. Megison, S.M., Horton, J.W., Chao, H., and Walker, P.B. Prolonged survival and decreased mucosal injury after low-dose enteral allopurinol prophylaxis in mesenteric ischemia. J. Ped. Surg. 25:917. 1990. Barroso-Aranda, J., Schmid-Schonbein, G.W., Zweifach, B.W., and Engler, R.L. Granulocytes and the no-reflow phenomenon in irreversible hemorrhagic shock. Circ. Res. 63:437. 1988. Vedder, N.B., Winn, R.K., Rice, C.L., Chi, E.Y., Arfors, K.E., and Harlan, J.M. A monoclonal antibody to the adherence-promoting glycoprotein, CD18, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. J. Clin. Invest. 8•:939. 1988. Muller, R. Pentoxifylline: A biomedical profile. J Med •0:307. 1979. Bagge, U., Amundson, B., and Lauritzen, C. White blood cell deformability and plugging of skeletal muscle in hemorrhagic shock. Acta Physiol Scand 108:159. 1980. Bertocchi, F., Proserpio, P., Lampugnani, M.G., and Dejana, E. The effect of pentoxifylline on polymorphonuclear cell adhesion to cultured endothelial cells. In: Mandel, G.L., Novick, W.Z. (Jr.), Eds: Pentoxifylline and Leukocyte Function. Heochst-Roussel Pharm., Inc., Somerville, 1988. p.68. Sullivan, G.W., Carper, H.T., Novick, W.J., Jr., and Mandell, G.L. Inhibition of the inflammatory action of interleukin-1 and tumor necrosis factor (Alpha) on neutrophil function by pentoxifylline. Infect. Immun. 56:1722. 1988.
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Babior, B.M., Kipnes, R.S., and Curnutte, J.T. The production by leukocytes of superoxide. A potential bactericidal agent. J. Clin. Invest. 52:741. 1973. Coupar, I.M., and McLennan, P.L. The influence of prostaglandins on noradrenaline-induced vasoconstriction in isolated perfused mesenteric blood vessels of the rat. Br. J. Pharmacol. 62:651. 1978. Lipton, H.L., Chapnick, B.M., Hyman, A.L., and Kadowitz, P.G. Inhibition of vasoconstrictor responses by prostacyclin (PGI~) in the feline mesenteric vascular bed. Arch Int Pharmacodyn 241:214. 1979. Dusting, G.J., Moncada, S., and Vane, J.R. Vascular actions of arachidonic acid and metabolites in perfused mesenteric and femoral beds of the dog. Eur. J. Pharmacol. 49:65. 1978. Myers, S.I., Reed, M.K., Taylor, B., Smith, G., and Phan, T. Splanchnic prostanoid production: effect of hemorrhagic shock. J. Surg. Res. 48:579. 1990. Myers, S.I. and Small, J. Prolonged hemorrhagic shock decreased splanchnic prostacyclin synthesis. J. Surg. Res. 50:417. 1991. Myers, S.I. and Hernandez, R. Role of oxygen-derived free radicals on rat splanchnic eicosanoid production during acute hemorrhage. Prostaglandins 44:25. 1992. Itoh, M. and Guth, P.H. Role of oxygen-derived free radicals in hemorrhagic shock-induced gastric lesions in the rat. Gastroenterology 88:1162. 1985.
Editor: J.R. Fletcher
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Received: 8-17-93
Accepted: 12-22-93
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This study examines the hypothesis that pentoxifylline protects splanchnic PGI2 synthesis during severe mesenteric ischemia/ reperfusion injury. Anesthetized Sprague-Dawley rats (300 grams) were subjected to sham or superior mesenteric artery occlusion for 20 minutes followed by 30 minutes of reperfusion. The ischemia/ reperfusion groups received either enteral allopurinol (10 mg/kg) daily for 5 days prior to ischemia, PTX (50 mg/kg) 10 minutes prior to ischemia or carrier. The superior mesenteric artery was cannulated and removed with its intact intestine (SV + SI). The SV + SI was perfused in vitro with oxygenated Krebs buffer. The venous effluent was collected and assayed for release of 6-keto-PGF1~, PGE2 and thromboxane B2 by enzyme immunoassay. Severe mesenteric ischemia/ reperfusion decreased SV + SI 6-keto-PGF~ release by 40% compared to the sham group but did not alter release of PGE2 or thromboxane B2. Pretreatment of the animals with PTX and not allopurinol preserved SV + SI 6-keto-PGFl~ release at all times of perfusion to a level similar to the sham group. These data showed that severe mesenteric ischemia/reperfusion injury abolished release of endogenous splanchnic PGI2. PTX exerted a protective effect against severe mesenteric ischemia/reperfusion injury by maintaining release of splanchnic PGI2, a potent endogenous splanchnic vasodilator.
An abstract of this paper was presented at the 16th Annual Conference on Shock in Santa Fe New Mexico, June, 1993. Address correspondence to: Stuart I. Myers, M.D., Department of Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75235.
© 1994 Butterworth-Heinemann
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Keywords: Mesenteric ischemia; mesenteric PGIz; pentoxifylline; ischemia/ reperfusion injury
Introduction The gastrointestinal tract has been described as the motor organ in the evolution of multiple organ failure following ischemia/reperfusion injury? ,2 The syndrome of multiple organ failure begins to manifest clinically by the onset of enteric sepsis (bacterial translocation) and ileus suggesting that altered splanchnic blood flow is an important initiating factor for this syndrome. 1-5Formation of oxygen-derived free radicals have been shown to be an important factor involved with altered intestinal function following acute ischemia/reperfusion injury.6-s Use of scavengers of oxygen-derived free radicals has been shown to prevent both splanchnic vasoconstriction and intestinal permeability following acute hemorrhage/ reperfusion injury. 9,1°One mechanism for ODFR induced splanchnic vasoconstriction was recently shown to be the inhibition in the synthesis and release of splanchnic PGI2, a potent endogenous vasodilator by ODFR formationY Recently, Pentoxifylline (PTX) has been shown to increase survival in animals treated with hemorrhagic shock. These studies showed that PTX increased tissue oxygenation, increased oxygen consumption, decreased leukocyte adhesiveness and increased intestinal microvascular blood flow. 11-14 PTX has been proposed to improve intestinal microvascular blood flow by the decreased "plugging" of microvascular vessels by increasing deformability of leukocytes. This hypothesis was supported by in vitro studies that showed that PTX increases leukocyte deformability, decreases leukocyte adherence, enhances chemotaxis and blocks the action of inflammatory cytokines on leukocyte function? s-19 The present study examines the hypothesis that PTX maintains splanchnic PGIz release following severe ischemia/reperfusion injury of the intestine. We will utilize both PTX and allopurinol (xanthine oxidase inhibitor which prevents ODFR formation) to more specifically identify a mechanism of action involved with PTX protection of splanchnic PGI2 release following severe ischemia/reperfusion injury.
Methods
Surgical Model Adult male Sprague-Dawley rats (350 grams) were housed and used in compliance with the regulations of the animal care facility of the University of Texas Southwestern Medical Center and the Dallas VAMC. All animals were allowed food and water ad lib 18 hours prior to the experi138
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ment; at this time solid food was removed but animals had free access to water until time of the experiment. Experimental animals received either enteral allopurinol (N = 8)(10), a xanthine oxidase inhibitor, (10 mg/ kg) daily for five days prior to mesenteric ischemia or PTX (N= 10), a phosphodiesterase inhibitor, (50 mg/kg) intravenously, 10 minutes prior to mesenteric ischemia and 1 minute prior to reperfusion. A third group of untreated animals underwent mesenteric I/R and served as ischemic controls. An additional group (N=9) served as untreated, nonischemic (Sham) controls. The animals were anesthetized with inhalational methoxyflurane; ketamine hydrochloride (20 mg/kg IM) was administered to supplement anesthesia. The ventral neck and abdomen were shaved and prepped followed by cannulation of the carotid artery for blood gas analysis and h e m o d y n a m i c monitoring. The internal jugular vein was accessed for the acquisition of blood samples and the administration of fluids and pharmacologic agents. Heparin (500 units/kg) was injected intravenously in all rats after surgical preparation. Arterial blood gas measurements were monitored prior to ischemia/reperfusion and 30 minutes following reperfusion (see below). Mean arterial blood pressure and heart rate were continuously monitored by a Grass recorder (Grass Instrum e n t Co., Quincy, Mass.).
Preparation of the Rat for Mesenteric Ischemia/Reperfusion Injury Body temperature was maintained at 38°C by use of a heating pad and a heating lamp. Animals underwent laparotomy and the superior mesenteric artery (SMA) and collateral vessels were occluded as previously described. 2° In the sham group, rats underwent laparotomy with SMA and collateral vessel isolation without occlusion. All rats were studied at identical time periods regardless of group assignment. After 20 minutes of complete ischemia, the vascular clip was removed from the SMA and the bowel was reperfused for 30 minutes and then prepared for isolated perfusion studies.
Preparation of the ex vivo Isolated Mesenteric Perfusion After 30 minutes of reperfusion, the SMA was rapidly cannulated and removed with its end organ intestine (SV + SI preparation). The SV + SI was perfused ex-vivo by a modification of the techniques described by McGregor and Myers. ~,2~-23The SV + SI was perfused with oxygenated Krebs-Henseleit (O2/CO2 95:5, PO2 460 + 10 m m Hg) buffer at 3 m l / m i n with a Cole-Palmer peristaltic pump (Ph 7.40) at 37°C. The ends of the bowel were vented outside the warming apparatus to avoid distention. Perfusion pressure was monitored via a side arm of the arterial cannula using a Statham pressure transducer. The splanchnic venous effluent was collected after 5, 15, 30, 45 and 60 minutes of perfusion. Each sample
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was placed in siliconized micro-centrifuge tubes and immediately frozen at - 4 0 ° C until assayed by enzyme immunoassay techniques.
Enzyme Immunoassays (EIA) Venous effluent was analyzed for 6-keto-PGG~ (metabolite of PGI2), thromboxane B2 (TxB2) and PGE2 on Krebs-Henseleit buffer by enzymatic immunoassay as previously described. 2~The EIA reagents were purchased from Cayman Chemical Co. (Ann Arbor, MI). Inter-assay and intra-assay variation was 7-10% and were similar to those reported by our laboratory for RIAs. 23
Histology At the time of cannulation of the superior mesenteric artery and removal of the Sv + SI preparation (just prior to initiation of the ex-vivo perfusion), a 2 cm segment of distal jejunum was excised from nonischemic Sham controls, the ischemic controls and the ischemic groups treated with allopurinol and pentoxifylline. The specimens were placed in 10% formalin and submitted for histopathologic examination in a blinded fashion. Ischemic lesions were graded on a scale of 0 to 5 as described by Megison et al. 24 Grade 0 represents normal villi. Grade 1 represents the development of subepithelial space at the apex of the villus; capillary congestion. Grade 2 represents extension of subepithelial space with moderate separation of mucosa from lamina propria. Grade 3 represents extensive epithelial separation from lamina propria down the sides of the villi, with ulceration at villus tips. Grade 4 represents areas of denuded villi, dilated capillaries and increased cellularity of lamina propria. Grade 5 represents disintegration of lamina propria and hemorrhagic ulceration.
Statistical Analysis Prostanoid levels were assessed by a repeated measures analysis of variance. This analysis allows for comparisons among groups and across times, as well as evaluating differential change among groups over time (interaction). Results were considered significant when p < 0.05. Post hoc multiple comparisons were made using a Bonferroni strategy at the 0.05 level. This data analysis was performed after prostanoid levels were log-transformed (base 10) since it was observed that variability was correlated to mean level. Analysis of histologic injury score, arterial pH, PCO2 content, PO~ content, mean arterial blood pressure and heart rate was performed by analysis of variance and use of the Bonferroni strategy at the 0.05 level. All data is reported as mean + S.E.M. Analysis was performed using SAS software.
140
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Pentoxifylline protects splanchnic during eicosanoid ischemia: Myers et al. Results The mean arterial pressures and heart rates were similar at the basal time of measurement in all groups (Table 1 ). Mean arterial pressures measured in all ischemia/reperfusion groups after 30 minutes of reperfusion and at the 50 minute time period in the sham group decreased to a similar level. Heart rate was unchanged in the 50 minute time period of measurement in the Sham group (Table 1). The heart rate significantly decreased following 30 minutes of reperfusion in the untreated ischemia/reperfusion group (p < 0.02 compared to basal level) whereas heart rate significantly increased in the PTX group (p < 0.009 compared to basal measurement). Basal measurement of arterial pH was similar in the sham and all ischemia/reperfusion groups and was unchanged at the 50 minute measurement in the sham group and after 30 minutes of reperfusion in all ischemia/reperfusion groups (Table 2). Basal measurements of arterial PO2 and PCO~ content were similar in all groups (Table 2). Arterial PO~ and PCO~ content were not changed at the 50 minute time period of measurement in the sham group. Arterial PCO2 content in all ischemia/reperfusion groups decreased to similar levels after 30 minutes of reperfusion (Table 2). Arterial PO2 content increased in the PTX group after 30 minutes of reperfusion but was unchanged in the other experimental groups (Table 2). The ex-vivo perfused sham rat SV + SI preparation released 6-ketoPGFI~, PGE2 and TxB2 at all times of isolated perfusion (Figures 1-3). The major eicosanoid released from the SV + SI preparation was 6-keto-PGFl~ which was released 4 fold or greater than the other eicosanoids assayed TABLE 1. injury'
Effect of pentoxifylline and allopurinol on systemic pressure and heart rate during ischemia/reperfusion
Sham MAPS-03 Tin. (mmHg) MAPS-504 min. (mmHg) HRS-03 Tin. HR6-504 Tin.
92.0 64.2 207.7 197.5
_+6.6 _+4.0 x _+9.8 _+ 12.0
Carrier + I/R 2 100 61.1 219 180
~+4.0 ___4.8 × --- 9.4 ___8.2 x
Pentoxifylline + I/R 2 88.2 64.2 233 271
_+6.0 _+3.5 x _+7.2 xx _+ 12.0 x
AIIpurinol + I/R 2 103.3 70.6 201 185
_+4.5 _+8.3 x _+ 10.1 _+7.8 x
1Data expressed as Mean _+S . E . M (N = 8 or more). 2Superior mesenteric ischemia for 20 T i n followed by 30 T i n of reperfusion. 3Basal measurement (prior to ischemia). 450 minutes in Sham group or just following 30 minutes of reperfusion. 5Mean arterial pressure. 8Heart Rate. Xp < .05 level compared to same group at 0 time level by ANOVA. xxp < .05 level compared to Sham, AIIopurinol and Carrier I/R groups by ANOVA.
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Pentoxifylline protects splanchnic during eicosanoid ischemia: TABLE 2. injury 1
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Effect of pentoxifylline and allopurinol an arterial blood gas measurements during ischemia/reperfusion
Sham pHS-03 pHS-504 PO26-03 PO28-504 PCO27-03 PCO~7-504
7.38 _+0.001 7.41 + 0.006 91.2 _+3.1 97.9 _+5.0 27.0 _+0.9 34.7 _+1.5
Carrier
Pentoxifylline
AIIopurinol
+ I/R2
+ I/R 2
+ I/R 2
7.39 _+0.001 7.40 _+0.001 94.1 + 7.0 103.0 _+7.4 35.0 _+2.3 29.8 _+1.8 X8
7.40 _+0.001 7.43 _+0.007 85.2 _+3.2 102.0 +_4.8 x 35.3 +_1.4 22.7 _+1.1 x8
7.38 _+0.001 7.39 _+0.001 80.0 +_5.2 88.6 +_6.2 36.6 +_0.9 29.9 _+1.7x8
1Data expressed as Mean_+S.E.M. (N = 8 or more). 2Superior mesenteric ischemia for 20 rain followed by 30 min of reperfusion. 3Basal measurement prior to ischemia. 450 minute measurement in Sham group or just following 30 minutes of reperfusion. SArterial pH. 6Arterial oxygen content. 7Arterial PC02 content. 8p < .05 compared to Sham by ANOVA. xp < .05 compared to same group at 0 time measurement.
in the sham group (Table 1, Figures 1-3). Release of 6-keto-PGFl~, TxB2 and PGE2 in all groups was highest at 5 or perfusion and fell slightly after 15 and 30 minutes perfusion (Figures 1-3). Subjecting the untreated rats to mesenteric ischemia for 20 minutes followed by reperfusion for 30 minutes decreased release of vasodilator 6-keto-PGG~ from the in vitro perfused splanchnic bed when compared to the sham group (p < 0.05) (Table 1, Figure 1). After 30 minutes of perfusion,mesenteric ischemia for 20 minutes followed by 30 minutes of reperfusion decreased splanchnic release of 6-keto-PGF~ by approximately forty percent when compared to the sham group. In contrast, rats pretreated with PTX and subjected to mesenteric ischemia followed by reperfusion synthesized and released quantities of 6-keto-PGFl~ similar to those from sham animals (Table 1, Figure 1). Pretreatment of the rats with allopurinol did not protect splanchnic release of 6-keto-PGG~ following severe mesenteric ischemia followed by reperfusion (Table 1). In vitro perfused splanchnic release of PGE2 was not significantly altered by mesenteric ischemia, treatment with carrier, allopurinol or PTX (Table 1, Figure 2). Release of TxB2 was minimal in all groups of animals and was not altered by mesenteric ischemia followed by reperfusion (Table 1, Figure 3). Use of allopurinol pretreatment had virtually no detectable effect on the production and release of splanchnic prostanoids in our model of severe mesenteric ischemia (Table 3). 142
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Pentoxifylline protects splanchnic during eicosanoid ischemia: Myers et al. ~-~ PTX
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Minutes of Perfusion FIGURE 1. The effect of pentoxifylline on superior mesenteric artery (SMA) 6-keto-PGFl~ release. Rats received pentoxifylline (PTX, open box) or carrier (filled box) and were subjected to SMA occlusion and reperfusion as described under Methods and compared to Sham controls (hatched box). SV + SI preparations were perfused in vitro and the venous effluent collected after 5, 15, and 30 minutes of perfusion and assayed for 6-keto-PGFl~ (PGI2 metabolite). Data is calculated as ng 6-keto-PGF,, released/minute and a logarithmic (base 10) transformation (LN) is used prior to analysis of the data. Data is reported as mean _+S.E.M. (N = 10, * indicates significance vs. PTX and sham groups at p < 0.05 by ANOVA).
Histologic examination showed a significant increase in injury score from 0.6 _+ 0.2 in the nonischemic Sham group to 4.1 + 0.1 (p < 0.0001) in the untreated ischemia/reperfusion group. Treatment with PTX or allopurinol significantly decreased the injury score to 3.4 +_ 0.1 (p < 0.05) and 2.6 + 0.8 (p < 0.05) respectively when compared to the untreated ischemia/reperfusion group. However the injury score for both the PTX and allopurinol groups remained significantly higher (at p < 0.0002) when compared to the Sham group.
Discussion Hemorrhagic shock is a c o m m o n l y encountered clinical entity which contributes to the mortality of critically ill patients.~-5 For decades, the treatment of these disorders of ischemia/reperfusion has been the logical replacement of volume losses with red cells, crystalloid and colloid solu-
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Pentoxifylline protects splanchnic during eicosanoid ischemia:
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1.25 [
~ PTX
m 1.00
Cartier Sham
. ~
0.75
[~0
0.50
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5
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Minutes of Perfusion FIGURE 2. The effect of pentoxifylline on superior mesenteric artery (SMA) TxB~ release. Rats received pentoxifylline (PTX, open box) or carrier (filled box) and were subjected to SMA occlusion and reperfusion as described under Methods and compared to Sham controls (hatched box). SV + SI preparations were perfused in vitro and the venous effluent collected after 5, 15, and 30 minutes of perfusion and assayed for TxB2 (thromboxane A2 metabolite). Data is calculated as ng TxB2 released/minute and a logarithmic (base 10) transformation (LN) is used prior to analysis of the data. Data is reported as mean _+S.E.M. (N= 10). Untreated ischemic control group (carrier) was not significantly different than the PTX and sham groups (at p < 0.05 by ANOVA).
tions; however the adverse sequelae of shock are often progressive and may lead to multiple organ failure despite seemingly adequate initial resuscitation. 1-4 The gastrointestinal tract has been purported to play a contributing role in the pathogenesis of multiple organ failure. 1,,~ This hypothesis encompasses the notion that clinical shock and resuscitation lead to a decrease in splanchnic microcirculatory blood flow. This concept is not new since, during periods of shock, the sympathetic nervous system mediates responses that preserve blood flow to more vital structures (i.e., brain, heart, lung), thus, depriving other organs of adequate blood flow. When this vasoconstrictive response becomes prolonged, severe reductions in microcirculatory flow of less central organs leads to initiation and enhancement of an inflammatory process. This phenomenon, characterized by the production and release of inflammatory mediators (TNF, eicosanoids, complement and cytokines), aggregation of red cells, neutro144
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Pentoxifylline protects splanchnic during eicosanoid ischemia: Myers et al. 0.75
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FIGURE 3. The effect of pentoxifylline on superior mesenteric artery (SMA) PGE2 release. Rats received pentoxifylline (PTX, open box) or carrier (filled box) and were subjected to SMA occlusion and reperfusion as described under Methods and compared to Sham controls (hatched box). SV + SI preparations were perfused in vitro and the venous effluent collected after 5, 15, and 30 minutes of perfusion and assayed for PGE2. Data is calculated as ng PGE2 released/minute and a logarithmic (base 10) transformation (LN) is used prior to analysis of the data. Data is reported as mean+-S.E.M. ( N - 1 0 ) . Untreated ischemic control group (carrier) was not significantly different from the PTX and sham groups (at p < 0.05 by ANOVA).
TABLE 3.
PGI23 PGE2 TxB24
Effect of allopurinol on splanchnic eicosanoid release during ischemia/reperfusion injury,
Sham
Carrier + I/R 2
3.4 +- 0.2 0.4 +- 0.2 0.9-+0.1
2.08 + 0.3 X 0.2 _+0.2 0.7+_0.1
Pentoxifylline + I/R 2 3.14 +_0.1 0.65 _+0.1 0.8+_0.1
Allopurinol + I/R 2 1.7 +- 0.09 X 0.3_+ 0.1 0.9_+0.07
'Data expressed as ng/min (Mean +_S.E.M., N = 8 or more) at 5 Tin of perfusion. 2Superior mesenteric ischemia for 20 Tin followed by 30 Tin of reperfusion. 36-keto-PGG,,. 4Thromboxane B2. ×p < .05 level by repeated measure AOVA compared to Sham and pentoxifylline groups.
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phils and platelets causes further sludging of flow and contributes to the local ischemic process. 2s,~6 PTX is a methylxanthine, phosphodiesterase inhibitor that increases intracellular cAMP levelsY Its potential role in lessening the effects of the "no reflow" phenomenon makes PTX a promising agent for improving microcirculatory abnormalities that develop as a result of ischemia/reperfusion. Several studies have shown that PTX increases survival of animals subjected to acute ischemia/reperfusion injury.ll-14 These studies showed that use of PTX during ischemia/reperfusion injury increased oxygen consumption, tissue oxygen uptake, and increased hepatic and intestinal microvascular blood flow. The exact mechanisms of action of PTX in increasing survival following ischemia/reperfusion injury are not known. However, several in vitro studies provide insight into the possible mechanisms of action of PTX. PTX has been shown to decrease viscosity and increase red blood cell and neutrophil deformability,2s-3° improving flow through the microcirculation. PTX has been shown to decrease superoxide release during neutrophil and macrophage activation and to decrease adhesion by the neutrophil26,29,3~ Several studies have demonstrated that PTX inhibits cytokine (tumor necrosis factor, IL-1) induced neutrophil activation thus preventing lysosomal degranulation and superoxide production and neutrophil adhesion to endothelium. ~9,~3° Locally synthesized eicosanoids have been shown to have a role in the regulation of splanchnic blood flow during normal and pathologic states.F, 3234 PGI~ has been shown to be the predominant eicosanoid released by the splanchnic bed, is a known potent inhibitor of platelet aggregation and is a splanchnic arterial vasodilator. 34Both of these physiologic actions of PGI2 combine to contribute to maintenance of splanchnic vascular tone and in particular to antagonize sympathetic arterial vasoconstriction, g~,3,~ We have previously shown that PGI2 is the predominant eicosanoid released by the splanchnic vascular bed. These studies showed that the splanchnic bed can dramatically increase endogenous synthesis and release of PGI2 in response to acute hemorrhage.3s However this endogenous mechanism of the bowel to attempt to maintain splanchnic blood flow during acute hemorrhage is overwhelmed by both prolonged hemorrhage and reperfusion of shed blood, a6,37Subsequent studies showed that production of locally produced ODFRs inhibited both splanchnic PGI2 release and superior mesenteric blood flow during reperfusion following acute hemorrhage? These studies did not examine the effects of PTX on protection of splanchnic PGI2 release during severe ischemia/reperfusion injury. The experiments presented in this study were designed to test the hypothesis that PTX maintains splanchnic PGI2 release during severe intestinal ischemia/reperfusion injury. The experimental model used to examine this hypothesis was the selective clipping of the superior mesen-
146
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teric artery for 20 minutes followed by reperfusion for 30 minutes which produced a relatively severe localized intestinal ischemia/reperfusion injury. The hemodynamic data showed that the SMA clip model did not alter systemic hemodynamics as mean arterial blood pressure, arterial pH, PCO2 or PO2 were not significantly altered by intestinal ischemia/ reperfusion or PTX or allopurinol treatment (Tables 1, 2). The splanchnic eicosanoid release data demonstrated that pentoxifylline protects splanchnic prostacyclin production and release in a model of severe ischemia/ reperfusion injury. This would theoretically promote continued microcirculatory flow to the bowel mucosa and prevent or lessen the ischemic insult brought about by hypovolemic or low-flow shock states. In contrast to PTX treatment, allopurinol, a xanthine oxidase inhibitor 38 had no demonstrable effect on the release of prostacyclin as evidenced by reduced levels of 6-keto-PGFl~ similar to those of the untreated ischemic animals. It is entirely possible that the severity of ischemia in our model overwhelmed any effect that allopurinol may have had in preventing the formation of these highly reactive oxygen radicals. This concept is corroborated by findings from our previous studies that showed an inability of the splanchnic bed to compensate for episodes of prolonged hemorrhagic shock, g6 One must also consider the possibility that the allopurinol pretreatment period was too short or that the dosage was inadequate, in this study, to cause inhibition of xanthine oxidase. The histologic studies support the eicosanoid release data described above. PTX pretreatment significantly decreased the intestinal injury score when compared to the untreated ischemia/reperfusion group. However, despite normalizing release of endogenous splanchnic PGI2 release, PTX pretreatment did not completely reverse the intestinal injury. These findings suggest that splanchnic PGI2 synthesis is only one of the factors which contributes to maintaining normal intestinal viability during ischemia/reperfusion injury. Although the exact mechanisms of action exerted by PTX in our study are unknown we propose that PTX contributes to maintaining splanchnic PGI2 release during severe mesenteric ischemia/reperfusion injury by several mechanisms of action which have been previously documented in several in vitro and in vivo studies. These mechanisms of action include decreasing viscosity, increasing deformability of red blood cells and neutrophils, and by contributing to prevention of the activation of neutrophils by cytokines or local tissue factors. Prevention of the activation of neutrophils would decrease neutrophil adhesion and superoxide production in the microvasculature.lS-18,29 Although extrapolation of data collected from this model to the human can be made only with extreme caution, our data may have clinical implications. Maintaining splanchnic microcirculatory flow during period of ischemic or hemorrhagic shock is a worthy goal that, if achieved, may prevent intestinal barrier failure and its concomitant
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sequelae. PTX has a variety of pharmacologic properties that may be beneficial in helping achieve the aforementioned goal, yet, the relative importance of these properties of pentoxifylline awaits definition in future laboratory and clinical trials.
Acknowledgments These studies were supported by the National Institutes of Health grants GM-38529 (S.I.M), GM-21681 (J.W.H.) and VA Merit Grant (S.I.M).
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Editor: J.R. Fletcher
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Received: 8-17-93
Accepted: 12-22-93
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