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Ethyl pyruvate ameliorates ileus induced by bowel manipulation in mice
Ethyl pyruvate ameliorates ileus induced by bowel manipulation in mice Tomoyuki Harada, MD,a Beverley A. Moore, PhD,c Runkuan Yang, MD, PhD,a Ruy J. Cruz Jr, MD, PhD,a Russell L. Delude, PhD,a and Mitchell P. Fink, MD,a,b Pittsburgh, Pa
Background. Ethyl pyruvate (EP) improves survival, decreases proinflammatory cytokine expression, and ameliorates organ dysfunction in mice who have lethal sepsis or were subjected to hemorrhagic shock. Herein, we tested the hypothesis that treatment with EP can prevent the development of ileus after bowel manipulation, a phenomenon that is mediated by an inflammatory response in the bowel wall. Methods. C57Bl/6 mice underwent operative manipulation of the small intestine or were subjected to a sham procedure. Some of the mice subjected to gut manipulation were pre- and post-treated with 4 doses of EP (40 or 80 mg/kg per dose), whereas others received similar volumes of the vehicle for EP. Gastrointestinal transit of a nonabsorbable marker was assessed by gavaging the mice with the tracer 24 hours after operation and assessing its concentration 90 minutes later in bowel contents from the stomach, 10 equally long segments of small intestine, the cecum, and 2 equally long segments of colon. The contractile responses of ileal circular smooth muscle to graded concentrations of bethanechol were assessed by using standard organ bath methodology. Expression of interleukin-6 and inducible nitric oxide synthase transcripts in ileal muscularis propria was assessed by using the semiquantitative reverse transcriptase-polymerase chain reaction. Results. In sham-operated controls, the mean (± SE) geometric center for the transit marker was 10.0 ± 0.5, whereas for vehicle-treated mice subject to bowel manipulation, the value for this parameter was 3.5 ± 0.1 (P < .05). When mice subjected to bowel manipulation were treated with several 40 mg/kg doses of EP, the geometric center was 7.3 ± 1.0 (P < .05 vs sham-operated group). Gut manipulation impaired intestinal smooth muscle contractility in vitro and increased steady-state levels of interleukin-6 and inducible nitric oxide synthase messenger RNA. Treatment with EP ameliorated these effects of gut manipulation. Conclusions. EP warrants further evaluation as a therapeutic agent to ameliorate postoperative ileus. (Surgery 2005;138:530-7.) From the Departments of Critical Care Medicine,a Surgery,b and Medicine,c University of Pittsburgh School of Medicine
A TRANSIENT EPISODE of ileus, defined as impaired propulsive bowel motility, is generally regarded as a normal response to abdominal operation.1 However, the development of ileus can contribute to discomfort during the postoperative period as a result of abdominal distention, nausea, and emesis. In some instances, postoperative ileus can contribute to the development of more serious complications, including acute gastric dilatation, Supported by National Institutes of Health grant GM 1 RO1 GM068481-01. Accepted for publication April 11, 2005. Reprint requests: Mitchell P. Fink, MD, 616A Scaife Hall, 3550 Terrace St, Pittsburgh PA 15261. E-mail: [email protected]. edu. 0039-6060/$ - see front matter Ó 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2005.04.006
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pulmonary aspiration, respiratory compromise, cardiac arrhythmias, anastomotic dehiscence, or intestinal perforation. In rodents, intestinal manipulation (even when very limited in extent) initiates the activation of an inflammatory response in the smooth muscle coats of the bowel that is characterized by increased expression of proinflammatory cytokines, augmented production of nitric oxide and prostaglandins, and infiltration of the tissue by leukocytes.2-7 Collectively, these cellular and humoral inflammatory events suppress intestinal motility (ie, promote the development of ileus). Ethyl pyruvate (EP), a simple aliphatic ester derived from pyruvic acid, has been shown to be an effective anti-inflammatory agent in a variety of in vivo and in vitro model systems.8,9 For example, when mice with hemorrhagic shock are resuscitated with a balanced salt solution containing EP
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instead of a control solution without EP, activation of the proinflammatory transcription factor nuclear factor-jB is inhibited, and the expression of several proinflammatory genes is downregulated.10 Similarly, EP increases survival time and reduces circulating levels of interleukin (IL)-6 and nitrite, a marker of nitric oxide production in rats injected with a lethal dose of lipopolysaccharide (LPS).11 EP also inhibits activation of the nuclear factor-jB in Caco-2 human enterocyte–like cells stimulated with a mixture of tumor necrosis factor (TNF), IL-1b, and interferon-c12 and RAW 264.7 murine macrophage–like cells stimulated with LPS.13 Pretreating mice with EP before administration of a lethal dose of LPS inhibits secretion of the key proinflammatory cytokine TNF.13 In addition, EP blocks secretion of high mobility group B1 (HMGB1) by LPS-stimulated RAW 264.7 cells and blocks the release of this protein into the circulation of mice challenged with LPS.13 HMGB1 is a DNA-binding nuclear protein that was recently shown to function as a proinflammatory cytokine and a late-acting mediator of LPS- and sepsis-induced mortality in mice.14-16 Remarkably, treating mice with EP 12 or 24 hours after the onset of severe infection ameliorates the development of renal dysfunction,17 improves survival,13 and decreases circulating concentrations of both TNF17 and HMGB1.13 Prompted by these findings, we hypothesized that treatment of mice with EP before and after intestinal manipulation would downregulate postoperative inflammatory changes in the gut wall and ameliorate development of postoperative ileus. The experiments described herein were designed to test these hypotheses. METHODS Animals and materials. The research protocol complied with the regulations regarding animal care as published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Pittsburgh Medical School. Male C57BL/6 mice weighing 20 to 25 g (Jackson Laboratories, Bar Harbor, Me) were used in this study. The animals were maintained at the University of Pittsburgh Animal Research Center with a 12-hour light-dark cycle and had free access to standard laboratory feed and water. Animals were not fasted before the experiments. All chemicals were purchased from Sigma Chemical Co (St. Louis, Mo) unless otherwise noted. Animal model of postoperative ileus. Ileus was induced in mice by gentle operative manipulation of the small intestine, as described by Kalff et al.4
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The operative procedures were performed under aseptic conditions. After inducing general anesthesia with sodium pentobarbital (90 mg/kg im), a midline abdominal incision was performed. Using a rolling motion with 2 cotton-tipped applicators, we gently compressed the small intestine along its length from the duodenaljejunal junction to the ileocecal junction. After completion of the standardized gut manipulation procedure, the intestine was returned to its normal anatomic location within the peritoneal cavity, and the incision was closed in 2 layers with the use of running 4-0 silk suture. Sham-operated animals were subjected to the anesthesia and the laparotomy incision but did not undergo gut manipulation. Experimental design. In the first experiment, we measured intestinal transit of a nonabsorbable tracer, fluorescein isothiocyanate--labeled dextran with an average molecular mass of 70 kDa (FD70), in 5 groups of mice (n = 5-9 each): CONT (no operation and no treatment); SHAM (general anesthesia and celiotomy but no gut manipulation); RLS (gut manipulation plus treatment with Ringer’s lactate solution); EP40 (gut manipulation plus treatment with a relatively low dose of EP); and EP80 (gut manipulation plus treatment with a higher dose of EP). All treatments were administered as ip injections of 0.6 mL of fluid at the following time points relative to the time of operation: 6 hours, 0.5 hours, +6 hours, and +12 hours). Each injection provided mice in the EP40 group with a dose of EP equal to 40 mg/kg body weight. Each injection provided mice in the EP80 group with a dose of EP equal to 80 mg/kg. For mice in the EP40 group, EP was prepared as a 13.8 mmol/L solution in Ringer’s lactate solution (RLS). For mice in the EP80 group, EP was prepared as a 27.6 mmol/L solution in RLS. The second experiment included the same groups as above. In this experiment, segments of small intestine were obtained 24 hours after operative manipulation for estimation of the expression of transcripts of several proinflammatory genes. The sample size was 8 for each condition. The third experiment was designed to assess intestinal smooth muscle contractility in vitro. In this experiment, 4 treatment groups (n = 4 each) were studied: CONT (ie, normal mice not subjected to any treatments); CONT-EP40 (normal mice injected with 40 mg/kg doses EP at 30 h, 24.5 h, 18 hours and 12 hours relative to the measurement of gut mucosal contractility); RLS (same protocol as the RLS group above); and EP40 (same protocol as the EP40 group above). Circular smooth muscle contractility was measured in the
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RLS and EP40 groups 24 hours after operative manipulation). Determination of intestinal motility. FD70 was used as a tracer for motility studies, by using standard methods as previously described by Moore et al.18 Briefly, gastrointestinal transit was measured 24 hours after surgery by evaluating the distribution of an enteral dose of FD70. Mice were gavaged with 200 lL of FD70 dissolved in distilled water (2.5 mg/mL). Ninety minutes later, the animals were killed with an overdose of halothane. The entire gastrointestinal tract, from stomach to distal colon, was excised and divided into 14 segments: stomach, small intestine (divided into 10 segments of equal length), cecum, and colon (2 segments of equal length). The luminal content of each segment was collected into a small tube and suspended in 1 mL of distilled water. The samples were mixed vigorously, then clarified by centrifugation (14,000g for 5 minutes). The supernatants were collected and fluorometrically assayed for FD70 concentration, with the use of an excitation wavelength of 492 nm (slit width, 2.5 nm) and an emission wavelength of 515 nm (slit width, 10 nm) with the use of a Fusion microplate reader (PerkinElmer Boston, Mass). The transit of FD70 along the gastrointestinal (GI) tract was summarized by calculating the geometric center (GC) for the distribution.19 Semiquantitative reverse transcriptase-polymerase chain reaction. Segments of ileum were harvested 24 hours after manipulation or after the sham procedure. The small intestine was opened along the antimesenteric border, and the mucosa was scraped away with the use of a glass microscope slide. Total RNA was extracted from the muscularis propria with chloroform and TRI Reagent (Molecular Research Center, Cincinnati, Ohio) exactly as directed by the manufacturer. The total RNA was treated with DNAFree (Ambion, Houston, Tex) as instructed by the manufacturer by using 10 units of DNase 1/10 lg RNA. Two lg of total RNA was reverse transcribed in a 40 lL reaction volume containing 0.5 lg of oligo(dT)15 (Promega, Madison, Wis), 1 mmol/L of each deoxyribonucleoside triphosphate (dNTP), 15 U avian myeloblastosis virus reverse transcriptase (RT) (Promega) and 1 U/lL of recombinant RNasin ribonuclease inhibitor (Promega) in 5 mmol/L MgCl2, 10 mmol/L TRIS HCl, 50 mmol/L KCL, 0.1% Triton X-100, pH 8.0. The reaction mixtures were preincubated at 21°C for 10 minutes before DNA synthesis. The RT reactions were carried out for 50 minutes at 42°C and were heated to 95°C for 5 minutes to terminate the reaction. Reaction mixtures (50 lL)
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for polymerase chain reaction (PCR) were assembled with the use of 5 lL of complementary DNA template, 10 units AdvanTaq Plus DNA Polymerase (Clontech, Palo Alto, Calif), 200 mmol/L of each dNTP, 1.5 mmol/L MgCl2, and 1.0 mmol/L of each primer in 1 3 AdvanTaq Plus PCR buffer. PCR reactions were performed with the use of a GeneAmp Model 9700 thermocycler (Perkin Elmer, Norwalk, Conn). Amplification was initiated with 5 minutes of denaturation at 94°C. The PCR conditions were denaturation at 94°C for 45 seconds, annealing at 61°C for 45 seconds, and polymerization at 72°C for 45 seconds. To ensure amplification was in the linear range, we identified empirically the optimal number of cycles (33). After the last cycle of amplification, the samples were incubated at 72°C for 10 minutes and then held at 4°C. The 5# and 3# primers for IL-6 were CTG GTG ACA ACC ACG GCC TCC CCT and ATG CTT AGG CAT AAC GCA CTA GGT, respectively; the expected product length was 600 bp. The 5# and 3# primers for inducible nitric oxide synthase (iNOS) were CAC CAC AAG GCC ACA TCG GAT T and CCG ACC TGA TGT TGC CAT TGT T, respectively; the expected product length was 426 bp. 18S Ribosomal RNA was amplified to verify equal loading. For this reaction, the 5# and 3# primers were CCC GGG GAG GTA GTG ACG AAA AAT and CGC CCG CTC CCA AGA TCC AAC TAC, respectively; the expected product length was 200 bp. Ten microliters of each PCR reaction was electrophoresed on a 2% agarose gel, scanned in NucleoVision imaging workstation (NucleoTech, San Mateo, Calif), and quantified with the use of GelExpert release 3.5. Determination of intestinal smooth muscle contractility. In vitro circular muscle mechanical activity was measured as previously described.20 Mice were killed 24 hours after operative manipulation, and segments of the midileum were removed through a midline incision and submerged in preoxygenated Krebs-Ringer bicarbonate buffer (KRB consisting of 137.4 mmol/L Na+, 5.9 mmol/L K+, 2.5 mmol/L Ca2+, 1.2 mmol/L Mg2+, 134 mmol/L Cl, 15.5 mmol/L HCO3, 1.2 mmol/L H2PO4, and 11.5 mmol/L glucose). A segment of intestine was opened along the mesenteric border in a Sylgaard-lined dish. The opened intestine was pinned with the mucosal side facing up, and the mucosa was carefully stripped off the underlying muscularis propria. Circular muscle strips were prepared from the muscle sheet by cutting 2 3 6–mm strips parallel to the circular muscle fibers. Then, the muscle strips were affixed to isometric force transducers (WPI, Sarasota, Fla)
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and mounted in standard horizontal mechanical organ chambers that were continuously superfused with KRB equilibrated with 95% O2-5% CO2 and maintained at 37 ± 0.5°C. Contractions were recorded, measured, and stored in a computer with the use of analog-to-digital hardware and software package (Acknowledge; Biopac Systems, Santa Barbara, Calif). Tissues were equilibrated for 1 hour and then incrementally stretched to an optimum length that produced the maximum spontaneous contractile amplitude. After a baseline recording period of 20 minutes, contractilityresponse curves were generated by exposing the tissues to increasing concentrations of the muscarinic agonist bethanechol (0.3-300 lmol/L) for 10 minutes, followed by intervening 10-minute washout periods. The magnitude of the contractile response was determined by integrating the area under the tracing. This response was then normalized to the tissue cross-sectional area by converting the length and weight (1.03 mg/mm2) of the strip to square millimeters of tissue, and reporting the results as gmm2s1. Statistical methods. Results are presented as means ± SE. Intestinal transit and smooth muscle contractility data were analyzed by analysis of variance followed by Fisher’s least significant difference test. The specific statistical approach used is indicated in the legend for the relevant figure. P values < .05 were considered significant. Data obtained from RT-PCR were not analyzed statistically in view of the semiquantitative nature of the assay employed.10 RESULTS Gastrointestinal transit. In the CONT group (ie, completely normal mice not subjected to general anesthesia and laparotomy or operative manipulation of the gut) and the SHAM group (ie, mice subjected to general anesthesia and celiotomy, but not gut manipulation), the enterally administered fluorescent tracer was rapidly transported in an aboral direction within the intestine such that the peak signal 90 minutes after administration was in the distal ileum (Figs 1, A and 1, B). In contrast, in the RLS group (ie, mice subjected to gut manipulation but treated only with RLS), the distribution of the fluorescent tracer was shifted toward the more proximal segments of the GI tract (Fig 1, C). The FD70 distribution, as assessed by calculating the GC, was significantly different in the RLS group from that observed in the CONT or SHAM groups (Fig 2). Pre- and posttreatment of mice with 4 doses of EP (either 40 or 80 mg/kg per
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Fig 1. Distribution along the GI tract of FD70 90 minutes after enteral administration in normal animals and mice subjected to bowel manipulation. Mice in the CONT group (A; n = 9) were not subjected to operation and did not receive any treatments. Mice in the SHAM group (B; n = 5) were subjected to general anesthesia and celiotomy, but did not undergo manipulation of the intestines. Mice in the RLS group (C; n = 9) were subjected to bowel manipulation 24 hours before administration of the oral tracer and were treated with RLS. Mice in the EP40 group (D; n = 9) were subjected to gut manipulation and treated with 4 doses of EP (40 mg/kg each). Mice in the EP80 group (n = 7) were subjected to manipulation and treated with 4 doses of EP (E; 80 mg/kg each). Segments are as described in Methods. Results are presented as means ± SE.
dose) partially ameliorated the derangement in intestinal motility induced by operative manipulation (Figs 1, D and 1, E). The beneficial effect of treatment with EP was statistically significant (Fig 2). The treatment effects for the 2 doses were statistically indistinguishable. Expression of IL-6 and iNOS. Estimates regarding changes in the expression of several proinflammatory gene products in muscularis were determined with the use of semiquantitative RTPCR. Twenty-four hours after operation, manipulation of the gut was associated with increased expression of IL-6 and iNOS messenger RNA (mRNA) in the muscularis propria of the ileum (Figs 3 and 4, respectively). Circular smooth muscle contractility. Tonic contraction response curves of intestines were generated by exposing the tissues to increasing concentrations of the muscarinic agonist bethanechol. The addition of bethanechol (0.3 to 300 lmol/L) to the superfusate-elicited tonic and phasic contractions with magnitudes that were concentration dependent. The mean circular muscle contractile responses generated in response to
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Fig 2. Mean ± SE GC for the distribution of FD70 90 minutes after enteral administration of the tracer. The groups are the same as those shown in Fig 1. Higher numbers indicate better progression of the FD70 tracer into the distal GI tract. *P < .05 vs CONT; yP < .05 vs RLS.
bethanechol for 3 groups of mice (CONT, RLS, and EP40) are shown in Figure 5. Operative manipulation led to a ;50% reduction in the maximal capacity of the muscle to contract in response to bethanechol although the concentration needed to elicit a half-maximal response (ie, the EC50) was not appreciably different. Maximal contractile responses were restored to control levels in mice treated with EP (Fig 6). Responses from unoperated animals treated with EP were not different from CONT (data not shown). Figure 7, A, depicts a typical response to 100 lmol/L when circular muscle contractility was assessed with the use of a smooth muscle preparation from a mouse in the RLS group. Figure 7, B depicts a typical response to 100 lmol/L when circular muscle contractility was assessed with the use of a smooth muscle preparation from a mouse in the EP40 group. The peak contractile responses to 100 lmol/L bethanechol was significantly lower in the RLS group than in the CONT group, and the peak contractile responses to 100 lmol/L bethanechol was significantly greater in the EP40 group than in the RLS group (Fig 6). DISCUSSION Postoperative ileus is, at least in part, caused by the initiation of an inflammatory cascade within the intestinal muscularis propria that consists of the induction of proinflammatory cytokines, the massive recruitment and extravasation of inflammatory leukocytes into the muscularis, and the release of mediators---notably nitric oxide and prostaglandins---that directly inhibit smooth muscle contractility.3,4,21-23 The pluripotent cytokine IL-6 appears to be another important mediator of inflammation-induced alterations in gut motility.25-27 Since we have shown that EP has beneficial pharmacologic effects in other pathologic
Fig 3. Expression of IL-6 mRNA in intestinal smooth muscle. Groups are the same as in the legend for Fig 1. Results were obtained with the use of semiquantitative RT-PCR. Bands were scanned in NucleoVision imaging workstation (NucleoTech) and quantified with the use of GelExpert release 3.5. Data in bar graphs are means ± SE (n = 7 per condition).
Fig 4. Expression of iNOS mRNA in intestinal smooth muscle. Groups are the same as in the legend for Fig 1. Results were obtained by using the methods described in the legend for Fig 3. Data in bar graphs are means ± SE (n = 7 per condition).
conditions that are associated with increased expression of proinflammatory proteins,10,12,13,17,25-27 we sought to evaluate the effects of this simple compound in a murine model of postoperative
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Fig 5. Contraction of ileal circular smooth muscle in vitro in response to graded concentrations of the muscarinic agonist bethanechol. Smooth muscle was obtained from normal animals (CONT), mice subjected to bowel manipulation 24 hours earlier and treated with RLS (RLS), and mice subjected to bowel manipulation and treated with 4 doses of EP (40 mg/kg per dose; EP40). Data are means ± SE (n = 4 animals per group). Fig 7. Representative tracings from smooth muscle preparations from mice subjected to bowel manipulation 24 hours earlier and treated with RLS (A) or 4 doses of EP (40 mg/kg per dose; B) and incubated with 100 lmol/L bethanechol. Abbreviations are the same as those in Fig 5.
Fig 6. Peak contractile responses of ileal circular smooth muscle in response to incubation with 100 lmol/L bethanechol. Groups are the same as those described in Fig 5. Data are means ± SE (n = 4 animals per group).
ileus. As the data presented herein shows, treatment with EP beginning before the operative procedure and continuing for 12 hours afterwards significantly ameliorated ileus induced by a standardized brief duration of intestinal manipulation. In addition, treatment with EP normalized ileal smooth muscle function as assessed ex vivo by measuring contractile responses of circular muscle strips to graded concentrations of the parasympathomimetic bethanachol. Consistent with data published in numerous previous studies by the Bauer laboratory at the University of Pittsburgh,3,18,28,29 intestinal manipulation in the present study was associated with marked upregulation of the expression of IL-6 and iNOS transcripts in the muscularis propria of the ileum. In previously reported studies, immunohis-
tochemical methods have documented that intestinal manipulation increases the expression of the proteins encoded by the IL-6, iNOS, and cyclooxygenase-2 (COX-2) genes.3,28 Furthermore, ex vivo studies have verified that compared with normal tissue, musularis tissue from rodents subjected to bowel manipulation 24 hours earlier produces increased amounts of nitric oxide.3,29 Both iNOS and COX-2 are enzymes that enhance the production of autocoids that have been implicated as being important in the development of ileus due to bowel manipulation3,22,30 or other proinflammatory stimuli.31,32 IL-6 may impair intestinal motility directly24 or indirectly by enhancing the overall magnitude of the inflammatory response. In many previous human studies, treatment with various prokinetic agents, such as metoclopramide or erythromycin (a motilin agonist), failed to ameliorate postoperative ileus.33-39 Thus, using prokinetic therapy to ameliorate postoperative ileus is not likely to be a fruitful approach. In contrast, it is noteworthy that various pharmacologic anti-inflammatory strategies have been shown to ameliorate postoperative ileus in experimental animals. Some of the strategies that have been employed include treatment with the gaseous small molecule, carbon monoxide,29 various
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isoform-selective COX-2 inhibitors,22,30 and the protein tyrosine kinase inhibitor AG 126.18 The data presented herein showing decreased steadystate levels of IL-6 and iNOS mRNA in EP-treated compared with vehicle-treated mice are consistent with the view that treatment with this agent downregulated the inflammatory response in the smooth muscle coats of the bowel after operative manipulation. Although it is impossible to be certain that the anti-inflammatory effects of EP were responsible for the salutary effects of this agent with respect to gut motility and smooth muscle contractility, this explanation seems to us to be highly plausible. EP is chemically closely related to the endogenous metabolite pyruvic acid, a compound that is well-known to be an effective scavenger of the reactive oxygen species (ROS), hydrogen peroxide.40-42 Therefore, it is not too surprising that in addition to its properties as an anti-inflammatory agent, EP also has been shown in several studies to inhibit lipid peroxidation induced by hemorrhagic shock in vivo or by exposure of murine macrophage–like cells to LPS in vitro.43,44 It is conceivable, therefore, that the salutary effects of EP observed in the present study were related to scavenging of hydrogen peroxide or other ROS. Few if any previous studies, however, have examined the effects of ROS scavengers in models of inflammation-induced ileus, so making reference to the published literature provides little guidance as to whether this hypothesis is reasonable and worthy of further testing. Only limited data are available, regarding in vivo dose-response effects for EP in various animal models of human disease. In a study evaluating the effects of EP on a variety of parameters in mice subjected to mesenteric ischemia and reperfusion (I/R), EP was administered as a single bolus dose immediately before restoring perfusion to the small intestine.45 Three doses of EP, ranging from 17 to 150 mg/kg, were evaluated. In this study, there was a clear dose-response effect for some parameters (eg, inhibition of post-I/R hepatic TNF mRNA expression), whereas for other parameters (eg, amelioration of post-IR ileal mucosal hyperpermeability), the 2 highest doses tested (50 and 150 mg/kg) provided similar benefits. In a study evaluating the effects of delayed treatment with EP on survival of septic mice, repetitive doses of 4 mg/kg provided slight benefit, whereas clear protection against mortality was afforded by repetitive doses of 40 mg/kg (the lower dose used in the present study).13 In the present series of experiments, both dosing regi-
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mens for EP (40 mg/kg per dose and 80 mg/kg per dose) were similarly efficacious, presumably because the lower dose was already greater than the dose required for maximal achievable efficacy. CONCLUSION We showed herein that pre- and posttreatment with EP ameliorated the development of gut dysmotility in a murine model of postoperative ileus. We believe these findings support the view that EP warrants further evaluation, ultimately leading to clinical trials for the prevention of ileus in patients undergoing major abdominal operations.
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14. Yang H, Wang H, Li J, et al. Reversing established sepsis with antagonists of endogenous HMGB1. Proc Natl Acad Sci USA 2004;101:296-301. 15. Wang H, Bloom O, Zhang M, et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science 1999;285:248-51. 16. Andersson U, Wang H, Palmblad K, et al. High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 2001; 192:565-70. 17. Miyaji T, Hu X, Yuen PST, et al. Ethyl pyruvate decreases sepsis-induced acute renal failure and multiple organ damage in aged mice. Kidney Int 2003;64:1620-31. 18. Moore BA, Turler A, Pezzone MA, Dyer K, Grandis J, Bauer AJ. Tyrphostin AG 126 inhibits development of postoperative ileus induced by surgical manipulation of murine colon. Am J Physiol Gastrointest Liver Physiol 2004;286: G214-24. 19. Miller MS, Galligan JJ, Burks TF. Accurate measurement of intestinal transit in the rat. J Pharmacol Methods 1981;6: 211-7. 20. Eskandari MK, Kalff JC, Billiar TR, Lee KK, Bauer AJ. Lipopolysaccharide activates the muscularis macrophage network and suppresses circular smooth muscle activity. Am J Physiol Gastrointest Liver Physiol 1997;273:G727-34. 21. Kalff JC, Carlos TM, Schraut WH, Billiar TR, Simmons RL, Bauer AJ. Surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus. Gastroenterology 1999;117:378-87. 22. Schwarz NT, Kalff JC, Turler A, et al. Prostanoid production via COX-2 as a causative mechanism of rodent postoperative ileus. Gastroenterology 2001;121:1354-71. 23. De Winter BY, Boeckxstaens GE, De Man JG, Moreels TG, Herman AG, Pelckmans PA. Differential effect of indomethacin and ketorolac on postoperative ileus in rats. Eur J Pharmacol 1998;344:71-6. 24. Natale L, Piepoli AL, De Salvia MA, et al. Interleukins 1 beta and 6 induce functional alteration of rat colonic motility: an in vitro study. Eur J Clin Invest 2003;33:704-12. 25. Yang R, Uchiyama T, Alber SM, et al. Ethyl pyruvate ameliorates distant organ injury in a murine model of acute necrotizing pancreatitis. Crit Care Med 2004;32:1453-9. 26. Yang R, Han X, Delude RL, Fink MP. Ethyl pyruvate ameliorates acute alcohol-induced liver injury and inflammation in mice. J Lab Clin Med 2003;142:322-31. 27. Sappington PL, Fink ME, Yang R, Delude RL, Fink MP. Ethyl pyruvate provides durable protection against inflammation-induced intestinal epithelial barrier dysfunction. Shock 2003;20:521-8. 28. Kalff JC, Turler A, Schwarz NT, et al. Intra-abdominal activation of a local inflammatory response within the human muscularis externa during laparotomy. Ann Surg 2003;237:301-15. 29. Moore BA, Otterbein LE, Turler A, Choi AM, Bauer AJ. Inhaled carbon monoxide suppresses the development of postoperative ileus in the murine small intestine. Gastoenterology 2003;124:377-91.
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30. Josephs MD, Cheng G, Ksontini R, Moldawer LL, Hocking MP. Products of cyclooxygenase-2 catalysis regulate postoperative bowel motility. J Surg Res 1999;86:50-4. 31. De Winter BY, Bredenoord AJ, De Man JG, Moreels TG, Herman AG, Pelckmans PA. Effect of inhibition of inducible nitric oxide synthase and guanylyl cyclase on endotoxin-induced delay in gastric emptying and intestinal transit in mice. Shock 2002;18:125-31. 32. Eskandari MK, Kalff JC, Billiar TR, Lee KK, Bauer AJ. LPSinduced muscularis macrophage nitric oxide suppresses rat jejunal circular muscle activity. Am J Physio 1999;277: G478-86. 33. Jepsen S, Klaerke A, Nielsen PH, Simonsen O. Negative effect of Metoclopramide in postoperative adynamic ileus. A prospective, randomized, double blind study. Br J Surg 1986;73:290-1. 34. Davidson ED, Hersh T, Brinner RA, Barnett SM, Boyle LP. The effects of metoclopramide on postoperative ileus. A randomized double-blind study. Ann Surg 1979;190:27-30. 35. Seta ML, Kale-Pradhan PB. Efficacy of metoclopramide in postoperative ileus after exploratory laparotomy. Pharmacotherapy 2001;21:1181-6. 36. Cheape JD, Wexner SD, James K, Jagelman DG. Does metoclopramide reduce the length of ileus after colorectal surgery? A prospective randomized trial. Dis Colon Rectum 1991;34:437-41. 37. Tolleson PO, Cassuto J, Faxen A, Rimback G, Mattsson E, Rosen S. Lack of effect of metoclopramide on colonic motility after cholecystectomy. Eur J Surg 1991;157:355-8. 38. Bonacini M, Quiason S, Reynolds M, Gaddis M, Pemberton B, Smith O. Effect of intravenous erythromycin on postoperative ileus. Am J Gastroenterol 1993;88:208-11. 39. Smith AJ, Nissan A, Lanouette NM, et al. Prokinetic effect of erythromycin after colorectal surgery: randomized, placebocontrolled, double-blind study. Dis Colon Rectum 2000;43: 333-7. 40. Bunton CA. Oxidation of a-diketones and a-keto-acids by hydrogen peroxide. Nature 1949;163:144. 41. Constantopoulos G, Barranger JA. Non-enzymatic decarboxylation of pyruvate. Anal Biochem 1984;139:353-8. 42. Andrae U, Singh J, Ziegler-Skylakakis K. Pyruvate and related a-ketoacids protect mammalian cells in culture against hydrogen peroxide-induced cytotoxicity. Toxicol Lett 1985;28:93-8. 43. Song M, Kellum JA, Kaldas H, Fink MP. Evidence that glutathione depletion is a mechanism responsible for the anti-inflammatory effects of ethyl pyruvate in cultured LPSstimulated RAW 264.7 cells. J Pharmacol Exp Ther 2004; 308:307-16. 44. Tawadrous ZS, Delude RL, Fink MP. Resuscitation from hemorrhagic shock with Ringer’s ethyl pyruvate solution improves survival and ameliorates intestinal mucosal hyperpermeability in rats. Shock 2002;17:473-7. 45. Uchiyama T, Delude RL, Fink MP. Dose-dependent effects of ethyl pyruvate in mice subjected to mesenteric ischemia and reperfusion. Intensive Care Med 2003;29:2050-8.
Background. Ethyl pyruvate (EP) improves survival, decreases proinflammatory cytokine expression, and ameliorates organ dysfunction in mice who have lethal sepsis or were subjected to hemorrhagic shock. Herein, we tested the hypothesis that treatment with EP can prevent the development of ileus after bowel manipulation, a phenomenon that is mediated by an inflammatory response in the bowel wall. Methods. C57Bl/6 mice underwent operative manipulation of the small intestine or were subjected to a sham procedure. Some of the mice subjected to gut manipulation were pre- and post-treated with 4 doses of EP (40 or 80 mg/kg per dose), whereas others received similar volumes of the vehicle for EP. Gastrointestinal transit of a nonabsorbable marker was assessed by gavaging the mice with the tracer 24 hours after operation and assessing its concentration 90 minutes later in bowel contents from the stomach, 10 equally long segments of small intestine, the cecum, and 2 equally long segments of colon. The contractile responses of ileal circular smooth muscle to graded concentrations of bethanechol were assessed by using standard organ bath methodology. Expression of interleukin-6 and inducible nitric oxide synthase transcripts in ileal muscularis propria was assessed by using the semiquantitative reverse transcriptase-polymerase chain reaction. Results. In sham-operated controls, the mean (± SE) geometric center for the transit marker was 10.0 ± 0.5, whereas for vehicle-treated mice subject to bowel manipulation, the value for this parameter was 3.5 ± 0.1 (P < .05). When mice subjected to bowel manipulation were treated with several 40 mg/kg doses of EP, the geometric center was 7.3 ± 1.0 (P < .05 vs sham-operated group). Gut manipulation impaired intestinal smooth muscle contractility in vitro and increased steady-state levels of interleukin-6 and inducible nitric oxide synthase messenger RNA. Treatment with EP ameliorated these effects of gut manipulation. Conclusions. EP warrants further evaluation as a therapeutic agent to ameliorate postoperative ileus. (Surgery 2005;138:530-7.) From the Departments of Critical Care Medicine,a Surgery,b and Medicine,c University of Pittsburgh School of Medicine
A TRANSIENT EPISODE of ileus, defined as impaired propulsive bowel motility, is generally regarded as a normal response to abdominal operation.1 However, the development of ileus can contribute to discomfort during the postoperative period as a result of abdominal distention, nausea, and emesis. In some instances, postoperative ileus can contribute to the development of more serious complications, including acute gastric dilatation, Supported by National Institutes of Health grant GM 1 RO1 GM068481-01. Accepted for publication April 11, 2005. Reprint requests: Mitchell P. Fink, MD, 616A Scaife Hall, 3550 Terrace St, Pittsburgh PA 15261. E-mail: [email protected]. edu. 0039-6060/$ - see front matter Ó 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2005.04.006
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pulmonary aspiration, respiratory compromise, cardiac arrhythmias, anastomotic dehiscence, or intestinal perforation. In rodents, intestinal manipulation (even when very limited in extent) initiates the activation of an inflammatory response in the smooth muscle coats of the bowel that is characterized by increased expression of proinflammatory cytokines, augmented production of nitric oxide and prostaglandins, and infiltration of the tissue by leukocytes.2-7 Collectively, these cellular and humoral inflammatory events suppress intestinal motility (ie, promote the development of ileus). Ethyl pyruvate (EP), a simple aliphatic ester derived from pyruvic acid, has been shown to be an effective anti-inflammatory agent in a variety of in vivo and in vitro model systems.8,9 For example, when mice with hemorrhagic shock are resuscitated with a balanced salt solution containing EP
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instead of a control solution without EP, activation of the proinflammatory transcription factor nuclear factor-jB is inhibited, and the expression of several proinflammatory genes is downregulated.10 Similarly, EP increases survival time and reduces circulating levels of interleukin (IL)-6 and nitrite, a marker of nitric oxide production in rats injected with a lethal dose of lipopolysaccharide (LPS).11 EP also inhibits activation of the nuclear factor-jB in Caco-2 human enterocyte–like cells stimulated with a mixture of tumor necrosis factor (TNF), IL-1b, and interferon-c12 and RAW 264.7 murine macrophage–like cells stimulated with LPS.13 Pretreating mice with EP before administration of a lethal dose of LPS inhibits secretion of the key proinflammatory cytokine TNF.13 In addition, EP blocks secretion of high mobility group B1 (HMGB1) by LPS-stimulated RAW 264.7 cells and blocks the release of this protein into the circulation of mice challenged with LPS.13 HMGB1 is a DNA-binding nuclear protein that was recently shown to function as a proinflammatory cytokine and a late-acting mediator of LPS- and sepsis-induced mortality in mice.14-16 Remarkably, treating mice with EP 12 or 24 hours after the onset of severe infection ameliorates the development of renal dysfunction,17 improves survival,13 and decreases circulating concentrations of both TNF17 and HMGB1.13 Prompted by these findings, we hypothesized that treatment of mice with EP before and after intestinal manipulation would downregulate postoperative inflammatory changes in the gut wall and ameliorate development of postoperative ileus. The experiments described herein were designed to test these hypotheses. METHODS Animals and materials. The research protocol complied with the regulations regarding animal care as published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Pittsburgh Medical School. Male C57BL/6 mice weighing 20 to 25 g (Jackson Laboratories, Bar Harbor, Me) were used in this study. The animals were maintained at the University of Pittsburgh Animal Research Center with a 12-hour light-dark cycle and had free access to standard laboratory feed and water. Animals were not fasted before the experiments. All chemicals were purchased from Sigma Chemical Co (St. Louis, Mo) unless otherwise noted. Animal model of postoperative ileus. Ileus was induced in mice by gentle operative manipulation of the small intestine, as described by Kalff et al.4
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The operative procedures were performed under aseptic conditions. After inducing general anesthesia with sodium pentobarbital (90 mg/kg im), a midline abdominal incision was performed. Using a rolling motion with 2 cotton-tipped applicators, we gently compressed the small intestine along its length from the duodenaljejunal junction to the ileocecal junction. After completion of the standardized gut manipulation procedure, the intestine was returned to its normal anatomic location within the peritoneal cavity, and the incision was closed in 2 layers with the use of running 4-0 silk suture. Sham-operated animals were subjected to the anesthesia and the laparotomy incision but did not undergo gut manipulation. Experimental design. In the first experiment, we measured intestinal transit of a nonabsorbable tracer, fluorescein isothiocyanate--labeled dextran with an average molecular mass of 70 kDa (FD70), in 5 groups of mice (n = 5-9 each): CONT (no operation and no treatment); SHAM (general anesthesia and celiotomy but no gut manipulation); RLS (gut manipulation plus treatment with Ringer’s lactate solution); EP40 (gut manipulation plus treatment with a relatively low dose of EP); and EP80 (gut manipulation plus treatment with a higher dose of EP). All treatments were administered as ip injections of 0.6 mL of fluid at the following time points relative to the time of operation: 6 hours, 0.5 hours, +6 hours, and +12 hours). Each injection provided mice in the EP40 group with a dose of EP equal to 40 mg/kg body weight. Each injection provided mice in the EP80 group with a dose of EP equal to 80 mg/kg. For mice in the EP40 group, EP was prepared as a 13.8 mmol/L solution in Ringer’s lactate solution (RLS). For mice in the EP80 group, EP was prepared as a 27.6 mmol/L solution in RLS. The second experiment included the same groups as above. In this experiment, segments of small intestine were obtained 24 hours after operative manipulation for estimation of the expression of transcripts of several proinflammatory genes. The sample size was 8 for each condition. The third experiment was designed to assess intestinal smooth muscle contractility in vitro. In this experiment, 4 treatment groups (n = 4 each) were studied: CONT (ie, normal mice not subjected to any treatments); CONT-EP40 (normal mice injected with 40 mg/kg doses EP at 30 h, 24.5 h, 18 hours and 12 hours relative to the measurement of gut mucosal contractility); RLS (same protocol as the RLS group above); and EP40 (same protocol as the EP40 group above). Circular smooth muscle contractility was measured in the
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RLS and EP40 groups 24 hours after operative manipulation). Determination of intestinal motility. FD70 was used as a tracer for motility studies, by using standard methods as previously described by Moore et al.18 Briefly, gastrointestinal transit was measured 24 hours after surgery by evaluating the distribution of an enteral dose of FD70. Mice were gavaged with 200 lL of FD70 dissolved in distilled water (2.5 mg/mL). Ninety minutes later, the animals were killed with an overdose of halothane. The entire gastrointestinal tract, from stomach to distal colon, was excised and divided into 14 segments: stomach, small intestine (divided into 10 segments of equal length), cecum, and colon (2 segments of equal length). The luminal content of each segment was collected into a small tube and suspended in 1 mL of distilled water. The samples were mixed vigorously, then clarified by centrifugation (14,000g for 5 minutes). The supernatants were collected and fluorometrically assayed for FD70 concentration, with the use of an excitation wavelength of 492 nm (slit width, 2.5 nm) and an emission wavelength of 515 nm (slit width, 10 nm) with the use of a Fusion microplate reader (PerkinElmer Boston, Mass). The transit of FD70 along the gastrointestinal (GI) tract was summarized by calculating the geometric center (GC) for the distribution.19 Semiquantitative reverse transcriptase-polymerase chain reaction. Segments of ileum were harvested 24 hours after manipulation or after the sham procedure. The small intestine was opened along the antimesenteric border, and the mucosa was scraped away with the use of a glass microscope slide. Total RNA was extracted from the muscularis propria with chloroform and TRI Reagent (Molecular Research Center, Cincinnati, Ohio) exactly as directed by the manufacturer. The total RNA was treated with DNAFree (Ambion, Houston, Tex) as instructed by the manufacturer by using 10 units of DNase 1/10 lg RNA. Two lg of total RNA was reverse transcribed in a 40 lL reaction volume containing 0.5 lg of oligo(dT)15 (Promega, Madison, Wis), 1 mmol/L of each deoxyribonucleoside triphosphate (dNTP), 15 U avian myeloblastosis virus reverse transcriptase (RT) (Promega) and 1 U/lL of recombinant RNasin ribonuclease inhibitor (Promega) in 5 mmol/L MgCl2, 10 mmol/L TRIS HCl, 50 mmol/L KCL, 0.1% Triton X-100, pH 8.0. The reaction mixtures were preincubated at 21°C for 10 minutes before DNA synthesis. The RT reactions were carried out for 50 minutes at 42°C and were heated to 95°C for 5 minutes to terminate the reaction. Reaction mixtures (50 lL)
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for polymerase chain reaction (PCR) were assembled with the use of 5 lL of complementary DNA template, 10 units AdvanTaq Plus DNA Polymerase (Clontech, Palo Alto, Calif), 200 mmol/L of each dNTP, 1.5 mmol/L MgCl2, and 1.0 mmol/L of each primer in 1 3 AdvanTaq Plus PCR buffer. PCR reactions were performed with the use of a GeneAmp Model 9700 thermocycler (Perkin Elmer, Norwalk, Conn). Amplification was initiated with 5 minutes of denaturation at 94°C. The PCR conditions were denaturation at 94°C for 45 seconds, annealing at 61°C for 45 seconds, and polymerization at 72°C for 45 seconds. To ensure amplification was in the linear range, we identified empirically the optimal number of cycles (33). After the last cycle of amplification, the samples were incubated at 72°C for 10 minutes and then held at 4°C. The 5# and 3# primers for IL-6 were CTG GTG ACA ACC ACG GCC TCC CCT and ATG CTT AGG CAT AAC GCA CTA GGT, respectively; the expected product length was 600 bp. The 5# and 3# primers for inducible nitric oxide synthase (iNOS) were CAC CAC AAG GCC ACA TCG GAT T and CCG ACC TGA TGT TGC CAT TGT T, respectively; the expected product length was 426 bp. 18S Ribosomal RNA was amplified to verify equal loading. For this reaction, the 5# and 3# primers were CCC GGG GAG GTA GTG ACG AAA AAT and CGC CCG CTC CCA AGA TCC AAC TAC, respectively; the expected product length was 200 bp. Ten microliters of each PCR reaction was electrophoresed on a 2% agarose gel, scanned in NucleoVision imaging workstation (NucleoTech, San Mateo, Calif), and quantified with the use of GelExpert release 3.5. Determination of intestinal smooth muscle contractility. In vitro circular muscle mechanical activity was measured as previously described.20 Mice were killed 24 hours after operative manipulation, and segments of the midileum were removed through a midline incision and submerged in preoxygenated Krebs-Ringer bicarbonate buffer (KRB consisting of 137.4 mmol/L Na+, 5.9 mmol/L K+, 2.5 mmol/L Ca2+, 1.2 mmol/L Mg2+, 134 mmol/L Cl, 15.5 mmol/L HCO3, 1.2 mmol/L H2PO4, and 11.5 mmol/L glucose). A segment of intestine was opened along the mesenteric border in a Sylgaard-lined dish. The opened intestine was pinned with the mucosal side facing up, and the mucosa was carefully stripped off the underlying muscularis propria. Circular muscle strips were prepared from the muscle sheet by cutting 2 3 6–mm strips parallel to the circular muscle fibers. Then, the muscle strips were affixed to isometric force transducers (WPI, Sarasota, Fla)
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and mounted in standard horizontal mechanical organ chambers that were continuously superfused with KRB equilibrated with 95% O2-5% CO2 and maintained at 37 ± 0.5°C. Contractions were recorded, measured, and stored in a computer with the use of analog-to-digital hardware and software package (Acknowledge; Biopac Systems, Santa Barbara, Calif). Tissues were equilibrated for 1 hour and then incrementally stretched to an optimum length that produced the maximum spontaneous contractile amplitude. After a baseline recording period of 20 minutes, contractilityresponse curves were generated by exposing the tissues to increasing concentrations of the muscarinic agonist bethanechol (0.3-300 lmol/L) for 10 minutes, followed by intervening 10-minute washout periods. The magnitude of the contractile response was determined by integrating the area under the tracing. This response was then normalized to the tissue cross-sectional area by converting the length and weight (1.03 mg/mm2) of the strip to square millimeters of tissue, and reporting the results as gmm2s1. Statistical methods. Results are presented as means ± SE. Intestinal transit and smooth muscle contractility data were analyzed by analysis of variance followed by Fisher’s least significant difference test. The specific statistical approach used is indicated in the legend for the relevant figure. P values < .05 were considered significant. Data obtained from RT-PCR were not analyzed statistically in view of the semiquantitative nature of the assay employed.10 RESULTS Gastrointestinal transit. In the CONT group (ie, completely normal mice not subjected to general anesthesia and laparotomy or operative manipulation of the gut) and the SHAM group (ie, mice subjected to general anesthesia and celiotomy, but not gut manipulation), the enterally administered fluorescent tracer was rapidly transported in an aboral direction within the intestine such that the peak signal 90 minutes after administration was in the distal ileum (Figs 1, A and 1, B). In contrast, in the RLS group (ie, mice subjected to gut manipulation but treated only with RLS), the distribution of the fluorescent tracer was shifted toward the more proximal segments of the GI tract (Fig 1, C). The FD70 distribution, as assessed by calculating the GC, was significantly different in the RLS group from that observed in the CONT or SHAM groups (Fig 2). Pre- and posttreatment of mice with 4 doses of EP (either 40 or 80 mg/kg per
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Fig 1. Distribution along the GI tract of FD70 90 minutes after enteral administration in normal animals and mice subjected to bowel manipulation. Mice in the CONT group (A; n = 9) were not subjected to operation and did not receive any treatments. Mice in the SHAM group (B; n = 5) were subjected to general anesthesia and celiotomy, but did not undergo manipulation of the intestines. Mice in the RLS group (C; n = 9) were subjected to bowel manipulation 24 hours before administration of the oral tracer and were treated with RLS. Mice in the EP40 group (D; n = 9) were subjected to gut manipulation and treated with 4 doses of EP (40 mg/kg each). Mice in the EP80 group (n = 7) were subjected to manipulation and treated with 4 doses of EP (E; 80 mg/kg each). Segments are as described in Methods. Results are presented as means ± SE.
dose) partially ameliorated the derangement in intestinal motility induced by operative manipulation (Figs 1, D and 1, E). The beneficial effect of treatment with EP was statistically significant (Fig 2). The treatment effects for the 2 doses were statistically indistinguishable. Expression of IL-6 and iNOS. Estimates regarding changes in the expression of several proinflammatory gene products in muscularis were determined with the use of semiquantitative RTPCR. Twenty-four hours after operation, manipulation of the gut was associated with increased expression of IL-6 and iNOS messenger RNA (mRNA) in the muscularis propria of the ileum (Figs 3 and 4, respectively). Circular smooth muscle contractility. Tonic contraction response curves of intestines were generated by exposing the tissues to increasing concentrations of the muscarinic agonist bethanechol. The addition of bethanechol (0.3 to 300 lmol/L) to the superfusate-elicited tonic and phasic contractions with magnitudes that were concentration dependent. The mean circular muscle contractile responses generated in response to
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Fig 2. Mean ± SE GC for the distribution of FD70 90 minutes after enteral administration of the tracer. The groups are the same as those shown in Fig 1. Higher numbers indicate better progression of the FD70 tracer into the distal GI tract. *P < .05 vs CONT; yP < .05 vs RLS.
bethanechol for 3 groups of mice (CONT, RLS, and EP40) are shown in Figure 5. Operative manipulation led to a ;50% reduction in the maximal capacity of the muscle to contract in response to bethanechol although the concentration needed to elicit a half-maximal response (ie, the EC50) was not appreciably different. Maximal contractile responses were restored to control levels in mice treated with EP (Fig 6). Responses from unoperated animals treated with EP were not different from CONT (data not shown). Figure 7, A, depicts a typical response to 100 lmol/L when circular muscle contractility was assessed with the use of a smooth muscle preparation from a mouse in the RLS group. Figure 7, B depicts a typical response to 100 lmol/L when circular muscle contractility was assessed with the use of a smooth muscle preparation from a mouse in the EP40 group. The peak contractile responses to 100 lmol/L bethanechol was significantly lower in the RLS group than in the CONT group, and the peak contractile responses to 100 lmol/L bethanechol was significantly greater in the EP40 group than in the RLS group (Fig 6). DISCUSSION Postoperative ileus is, at least in part, caused by the initiation of an inflammatory cascade within the intestinal muscularis propria that consists of the induction of proinflammatory cytokines, the massive recruitment and extravasation of inflammatory leukocytes into the muscularis, and the release of mediators---notably nitric oxide and prostaglandins---that directly inhibit smooth muscle contractility.3,4,21-23 The pluripotent cytokine IL-6 appears to be another important mediator of inflammation-induced alterations in gut motility.25-27 Since we have shown that EP has beneficial pharmacologic effects in other pathologic
Fig 3. Expression of IL-6 mRNA in intestinal smooth muscle. Groups are the same as in the legend for Fig 1. Results were obtained with the use of semiquantitative RT-PCR. Bands were scanned in NucleoVision imaging workstation (NucleoTech) and quantified with the use of GelExpert release 3.5. Data in bar graphs are means ± SE (n = 7 per condition).
Fig 4. Expression of iNOS mRNA in intestinal smooth muscle. Groups are the same as in the legend for Fig 1. Results were obtained by using the methods described in the legend for Fig 3. Data in bar graphs are means ± SE (n = 7 per condition).
conditions that are associated with increased expression of proinflammatory proteins,10,12,13,17,25-27 we sought to evaluate the effects of this simple compound in a murine model of postoperative
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Fig 5. Contraction of ileal circular smooth muscle in vitro in response to graded concentrations of the muscarinic agonist bethanechol. Smooth muscle was obtained from normal animals (CONT), mice subjected to bowel manipulation 24 hours earlier and treated with RLS (RLS), and mice subjected to bowel manipulation and treated with 4 doses of EP (40 mg/kg per dose; EP40). Data are means ± SE (n = 4 animals per group). Fig 7. Representative tracings from smooth muscle preparations from mice subjected to bowel manipulation 24 hours earlier and treated with RLS (A) or 4 doses of EP (40 mg/kg per dose; B) and incubated with 100 lmol/L bethanechol. Abbreviations are the same as those in Fig 5.
Fig 6. Peak contractile responses of ileal circular smooth muscle in response to incubation with 100 lmol/L bethanechol. Groups are the same as those described in Fig 5. Data are means ± SE (n = 4 animals per group).
ileus. As the data presented herein shows, treatment with EP beginning before the operative procedure and continuing for 12 hours afterwards significantly ameliorated ileus induced by a standardized brief duration of intestinal manipulation. In addition, treatment with EP normalized ileal smooth muscle function as assessed ex vivo by measuring contractile responses of circular muscle strips to graded concentrations of the parasympathomimetic bethanachol. Consistent with data published in numerous previous studies by the Bauer laboratory at the University of Pittsburgh,3,18,28,29 intestinal manipulation in the present study was associated with marked upregulation of the expression of IL-6 and iNOS transcripts in the muscularis propria of the ileum. In previously reported studies, immunohis-
tochemical methods have documented that intestinal manipulation increases the expression of the proteins encoded by the IL-6, iNOS, and cyclooxygenase-2 (COX-2) genes.3,28 Furthermore, ex vivo studies have verified that compared with normal tissue, musularis tissue from rodents subjected to bowel manipulation 24 hours earlier produces increased amounts of nitric oxide.3,29 Both iNOS and COX-2 are enzymes that enhance the production of autocoids that have been implicated as being important in the development of ileus due to bowel manipulation3,22,30 or other proinflammatory stimuli.31,32 IL-6 may impair intestinal motility directly24 or indirectly by enhancing the overall magnitude of the inflammatory response. In many previous human studies, treatment with various prokinetic agents, such as metoclopramide or erythromycin (a motilin agonist), failed to ameliorate postoperative ileus.33-39 Thus, using prokinetic therapy to ameliorate postoperative ileus is not likely to be a fruitful approach. In contrast, it is noteworthy that various pharmacologic anti-inflammatory strategies have been shown to ameliorate postoperative ileus in experimental animals. Some of the strategies that have been employed include treatment with the gaseous small molecule, carbon monoxide,29 various
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isoform-selective COX-2 inhibitors,22,30 and the protein tyrosine kinase inhibitor AG 126.18 The data presented herein showing decreased steadystate levels of IL-6 and iNOS mRNA in EP-treated compared with vehicle-treated mice are consistent with the view that treatment with this agent downregulated the inflammatory response in the smooth muscle coats of the bowel after operative manipulation. Although it is impossible to be certain that the anti-inflammatory effects of EP were responsible for the salutary effects of this agent with respect to gut motility and smooth muscle contractility, this explanation seems to us to be highly plausible. EP is chemically closely related to the endogenous metabolite pyruvic acid, a compound that is well-known to be an effective scavenger of the reactive oxygen species (ROS), hydrogen peroxide.40-42 Therefore, it is not too surprising that in addition to its properties as an anti-inflammatory agent, EP also has been shown in several studies to inhibit lipid peroxidation induced by hemorrhagic shock in vivo or by exposure of murine macrophage–like cells to LPS in vitro.43,44 It is conceivable, therefore, that the salutary effects of EP observed in the present study were related to scavenging of hydrogen peroxide or other ROS. Few if any previous studies, however, have examined the effects of ROS scavengers in models of inflammation-induced ileus, so making reference to the published literature provides little guidance as to whether this hypothesis is reasonable and worthy of further testing. Only limited data are available, regarding in vivo dose-response effects for EP in various animal models of human disease. In a study evaluating the effects of EP on a variety of parameters in mice subjected to mesenteric ischemia and reperfusion (I/R), EP was administered as a single bolus dose immediately before restoring perfusion to the small intestine.45 Three doses of EP, ranging from 17 to 150 mg/kg, were evaluated. In this study, there was a clear dose-response effect for some parameters (eg, inhibition of post-I/R hepatic TNF mRNA expression), whereas for other parameters (eg, amelioration of post-IR ileal mucosal hyperpermeability), the 2 highest doses tested (50 and 150 mg/kg) provided similar benefits. In a study evaluating the effects of delayed treatment with EP on survival of septic mice, repetitive doses of 4 mg/kg provided slight benefit, whereas clear protection against mortality was afforded by repetitive doses of 40 mg/kg (the lower dose used in the present study).13 In the present series of experiments, both dosing regi-
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mens for EP (40 mg/kg per dose and 80 mg/kg per dose) were similarly efficacious, presumably because the lower dose was already greater than the dose required for maximal achievable efficacy. CONCLUSION We showed herein that pre- and posttreatment with EP ameliorated the development of gut dysmotility in a murine model of postoperative ileus. We believe these findings support the view that EP warrants further evaluation, ultimately leading to clinical trials for the prevention of ileus in patients undergoing major abdominal operations.
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