Gastric and small bowel ileus after severe burn in rats: The effect of cyclooxygenase-2 inhibitors

burns 35 (2009) 1180–1184

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Gastric and small bowel ileus after severe burn in rats: The effect of cyclooxygenase-2 inhibitors Hermes M. Oliveira b,c,e, Hanaa S. Sallam b,d, Jonathan Espana-Tenorio a,c, David Chinkes a,c, Dai H. Chung a,c, Jiande D.Z. Chen b, David N. Herndon a,c,* a

Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA Department of Internal Medicine/Division of Gastroenterology, University of Texas Medical Branch, Galveston, TX, USA c Shriners Hospitals for Children, Galveston, TX, USA d Department of Physiology, Faculty of Medicine, Suez Canal University, Egypt e Federal University of Minas Gerais, Belo Horizonte, Brazil b

article info

abstract

Article history:

Gastrointestinal (GI) ileus is a common complication after severe burns. Selective cycloox-

Accepted 23 February 2009

ygenase-2 inhibitors (COX-2i) improved post-operative ileus, but its effect on burn-induced GI dysmotility is unknown. Our aim was to test whether a COX-2i improves gastric emptying

Keywords:

(GE) and small bowel transit (SBT) after burn. Experiment on GE: rats were anesthetized and

Burn

randomized into sham/scald burn, treated/untreated with COX-2i. Six hours after burn, rats

COX-2 inhibitors

received a phenol red meal and were sacrificed 30 min later. Gastric emptying was deter-

Gastric emptying

mined based on the percentage of phenol red recovered in harvested stomachs. Experiment

Small bowel ileus

on SBT: rats received a duodenostomy and were scald/sham burned 5 days later. Six hours after burn, rats received a phenol red meal through the duodenostomy catheter and were sacrificed 100 min later. Geometric center (GC) was calculated for SBT. GE was decreased significantly in burned vs. sham animals ( p < 0.001). SBT was significantly impaired in burned vs. sham animals ( p < 0.001). The COX-2i improved GE in the burn rats but not GE in the control rats or SBT in the burn rats. COX-2i improves burn-induced delayed GE, suggesting the mediation of the latter via the prostaglandin pathway. # 2009 Published by Elsevier Ltd and ISBI.

1.

Introduction

The gastrointestinal tract is usually injured during the early phase of severe burns (2nd and 3rd degree burn greater than 40% total body surface area ‘‘TBSA’’) [1–5]. Clinical signs of gastrointestinal injury include feeding intolerance, uncontrollable diarrhea, abdominal distension, gastric ulcers, decreased blood flow to the splanchnic bed, delayed gastric emptying and prolonged ileus, which may induce bacterial translocation, aspiration pneumonia and sepsis [1,3–8].

Prostaglandins have an important role in the maintenance of the gastrointestinal integrity [8]. Cyclooxygenase, a key enzyme required for the synthesis of prostaglandins, which exists as COX-1 and -2 isoforms, appear to be critically involved in the gut mucosal defense after burns [8]. Although the exact mechanism of action of these two COX-isoforms is not fully understood, studies suggest that both COX-1 and -2 are produced constitutively, while COX-2 is also triggered by various inflammatory states [9–11]. Recent studies have shown that prostaglandins, through induction of COX-2, play

* Corresponding author at: Shriners Hospitals for Children, 815 Market Street, 7th Floor, Medical Staff, Galveston, TX 77550, USA. Tel.: +1 409 770 6731; fax: +1 409 770 6919. E-mail addresses: [email protected], [email protected] (D.N. Herndon). 0305-4179/$36.00 # 2009 Published by Elsevier Ltd and ISBI. doi:10.1016/j.burns.2009.02.022

burns 35 (2009) 1180–1184

a major role in causing post-operative ileus [12]. This was not unexpected, as selective COX-2 inhibitors have the ability to improve post-operative ileus after major surgeries [13]. Decreased intestinal and colonic transit following a severe burn is usually accompanied by a delay in gastric emptying [2]. The aim of this study was to test whether a selective COX-2 inhibitor would improve gastric emptying and intestinal transit after severe burns in a rat experimental model.

2.

Methods

2.1.

Animals

Adult male Sprague–Dawley1 rats (300–350 g, Harlan Sprague– Dawley, Houston, TX) were housed in wire-bottom cages in a temperature-controlled environment at 22 8C, humidity 40%, and 12 h light–dark cycle. Rats had free access to regular chow pellets and drinking water. There was a minimum of 1 week of acclimatization, prior to the initiation of this experiment. The animals were randomized into four groups: sham burn (n = 12), burn (n = 13), sham burn + a selective COX-2 inhibitor (n = 8) and burn + a selective COX-2 inhibitor (n = 13).

2.2.

Ethics approval

The study was approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX, USA). The experiments were performed in adherence to the guidelines of the National Institutes of Health Guidelines on the use of laboratory animals.

2.3.

Experimental design

2.3.1.

Gastric emptying

Animals, deprived of food for 24 h and of water for 3 h, were anesthetized with isofluorane (Abbott Laboratories, North Chicago, IL) inhalation (2–3%). Intramuscular Buprenorphine (0.1 mg/kg, once just before burn; Reckitt Benckiser Healthcare Ltd., UK) was used as a pain-killer in all animals. Rats underwent burn injury as previously described [14]. Briefly, rats were shaved on the dorsal and ventral surface of the trunk and abdomen, and a 60% total body surface area burn was performed by immersing the dorsum in 96 8C heated water for 10 s and ventral surface for 2 s according to the Walker–Mason burn model. Burned rats were resuscitated immediately with Parkland formula (4 ml/kg/%TBSA/24 h), in which a total of 24 ml of Ringer’s lactate solution (for the first 8 h post-burn) was given intraperitoneally (i.p.). The sham animals received 12 ml of the same solution. Animals in the selective COX-2 inhibitor-treated group received 10 mg/kg of SC-58125 (Cayman Chemical, Ann Arbor, MI) i.p., 30 min after the burn injury. They were returned to the cage after gaining consciousness.

2.3.2.

Meal administration and phenol red (PSP) method

Six hours after burn, all rats received gavage feeding of a prewarmed (35 8C) phenol red meal. Under quick general anesthesia with 2–3% isofluorane inhalation, the rats were

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sacrificed by cervical dislocation 30 min after gavage feed. Four animals without any kind of manipulations were killed immediately after the gavage feeding to serve as an internal control. The entire stomach was carefully isolated, ligated just before the cardia and after the pylorus, and removed. The gastric emptying measurement was done as described by Scarpignato et al. [15]. Shortly: preparation of the meal—50 mg of phenol red (Sigma, St. Louis, MO) were diluted in 100 ml aqueous methylcellulose (1.5%, Fisher Scientific, Fair Lawn, NJ) solution and used as meal. Methylcellulose was dispersed in hot water (80 8C) under continuous stirring. The solution was then allowed to cool to 35 8C and the phenol red was added. Intensity and duration (5 h) of agitation were kept constant in order to obtain solutions of 400 centipoises viscosities. This high viscosity meal was selected to obtain a gastric emptying slower than obtained with simple aqueous solutions. Assay of phenol red: the stomach and its content (meal + possible gastric secretions) was homogenized using a Fischer Scientific PowerGen 700 homogenizer with 100 ml of NaOH 0.1N. The mixture was kept at room temperature for 1 h. 5 ml of the supernatant was added to 0.5 ml of trichloroacetic acid solution (20%, w/v) to precipitate the proteins. After centrifugation (2500  g during 20 min) the supernatant was added to 4 ml of NaOH (0.5N) to develop the maximum intensity of color. The solutions were read using a Beckman DU 650 spectrophotometer (fixed wavelength of 560 nm). Calculations: gastric emptying was determined by the percentage of phenol red meal emptied during the 30-min testing period for each rat and compared with internal controls taken as 100% retention.

2.4.

Small bowel transit

Rats received a duodenostomy under 2% isofluorane anesthesia, using a polyethylene tubing (0.86 inner diameter), with the tip of the tube placed 1.5 cm beyond the pylorus and it was exteriorized on the back neck of the rats. Five days later, the rats were submitted to a sham or 60% TBSA burn in the same way as mentioned above. Six hours after burn they received 1 ml of PSP meal (1 mg of PSP in 1.5% methylcellulose solution made in the way of the meal for GE experiment). One-hundred minutes later, rats were sacrificed under anesthesia and the entire small bowel was carefully harvested and divided into 10 equal pieces. The stomach and colon were harvested at the same time as a control for the method. The PSP contents of each segment were measured. Calculations: small bowel transit was determined by geometric center (GC) of the meal inside the intestine using the following formula: GC = Sum of n  Pn for n = 1, 2, . . ., 10 where, n was the number of the intestinal segment and Pn was the percentage of phenol red recovered from the corresponding segment.

2.5.

Statistic analysis

Data was analyzed using one-way analysis of variance (ANOVA) with Tukey’s correction for multiple comparisons between groups and t-test within the paired groups. Data were expressed as mean  standard error of mean (S.E.M.). Significance was accepted at p < 0.05.

1182 3.

burns 35 (2009) 1180–1184

Results

Gastric emptying was significantly decreased 6 h after burn in the burned group, when compared to sham. At this point, 41.7% (1.8) of the phenol red meal was emptied in burned animals, compared to 77.6% (3) in sham animals ( p < 0.001— Fig. 1). Small bowel transit was significantly decreased 6 h after burn in the burned group, when compared to sham. At this point, the GC of the distribution of phenol red meal was 8.5 (0.2) in the sham animals and 3.3 (0.4) in the burned animals ( p < 0.001—Fig. 2). SC-58125 did not improve the percentage of GE in sham burned rats. The percentage of gastric emptying was 77.9 (2.7) vs. 77.6  3; p > 0.05 in SC treated vs. untreated sham burned animals. SC-58125 improved the percentage of gastric emptying in burned rats from 41.69 (1.8) to 59.9 (3.8; p < 0.001) burned vs. burned + SC animals (Fig. 1). SC-58125 was not able to ameliorate the small bowel ileus 6 h after burn in rats. At this time point the GC of the phenol red meal in the burned rats was 3.25 (0.38) and in the burned rats treated with SC-58125 was 3.77 (0.216; p = 0.097) (Fig. 2).

4.

Fig. 1 – Gastric emptying after a severe burn injury, treated with a COX-2 inhibitor, in a rat experimental model. Gastric emptying percentage is significantly decreased 6 h after a severe burn injury, when 41.69% of the phenol red emptied, vs. 77.62% in the sham group ( p < 0.001). SC58125, the selective COX-2 inhibitor was able to ameliorate the delayed gastric emptying after the burn to 59.9% ( p < 0.001 burned vs. burned + SC animals).

Discussion

In this study, we observed the following: (1) severe burn resulted in a marked delay in both gastric emptying and small bowel transit in rats. (2) Selective COX-2i improved burn-induced delayed gastric emptying but not intestinal transit, and showed no effects on gastric emptying in the control rats. Methods for improving burn-induced GI dysmotility have usually included the use of prokinetic drugs [16]. Recently, COX-2i was proven effective in improving burn-induced intestinal dysmotility in an experimental setting [17]. Although subjective of extensive investigation, little is known about the specific roles of COX-1 and COX-2 in gastrointestinal motility. Cyclooxygenase-1 was initially found to be constitutively expressed, while COX-2 was induced by cytokines, hormones and growth factors [18,19]. Unlike non-steroidal inflammatory drugs (NSAIDs), COX-2i does not interfere with platelet aggregation, and have little GI toxicity. Like NSAIDs, COX-2i does have renal impairment effects that require monitoring, especially in patients with renal disease. Long term use of certain COX-2i has been associated with an increased risk of cardiovascular risks and potentially life-threatening skin reactions; which prompted the withdrawal of Rofecoxib (Vioxx1) and Valdecoxib (Bextra1) from the market. In spite of its side effects and potential risks, selective COX2i remained attractive for short term use. COX-2i has been widely used and has shown beneficial results in both animals and humans after trauma. In rats with traumatic brain injury, selective COX-2i provided neuroprotection and improved the brain cognitive function [20,21]. Following femur fracture, hemorrhage and intraperitoneal septic challenge in mice, selective COX-2i normalized proinflammatory cytokines and improved survival [22]. In patients with acute musculoskeletal

Fig. 2 – Small bowel motility after a severe burn injury, treated with a COX-2 inhibitor, in a rat experimental model. The geometric center (GC) of the distribution of phenol red meal was 8.5 (W0.2) in the sham animals and 3.25 (W0.4) in burned animals ( p < 0.001). The GC of phenol red meal in the burned rats treated with SC-58125 was 3.77 (W0.2; p = 0.097).

burns 35 (2009) 1180–1184

pain, following surgery or injury, selective COX-2i proved to be as effective as NSAIDs [23–26]. In several animal models of intestinal trauma including ischemia reperfusion and post-operative ileus, COX-2i has remarkable results. In a dog model of intestinal ischemiareperfusion, selective COX-2i reduced the intestinal histologic injury and the elevated serum prostanoids [27]. In postoperative ileus rodent models, both selective and nonselective inhibition of COX-2 have proved effective in improving intestinal transit and/or myoelectrical activity [13,28–31]. Selective COX-2i diminished the inflammatory component of the post-operative ileus by reducing the level of prostanoids and pro-inflammatory cytokines [28,29]. Selective COX-2i had the advantage to accelerate recovery from post-surgical ileus without the undesirable effects of nonselective inhibitors [13]. In a 30% TBSA scald burn animal model, we have recently reported that selective COX-2i improved burn-induced delayed intestinal transit [17]. Treatment of mononuclear cells, obtained from humans with 15% TBSA burn injury, with selective COX-2i normalized COX-2 expression and restored the down-regulated prostaglandin receptors expression [32]. The delay in gastric emptying and small bowel transit observed in this study was in agreement with our previously published data [14] and others [1,2]. Interestingly, in this study, we showed that the use of a COX-2i was able to ameliorate the delayed gastric emptying caused by an extensive burn. However, we could not demonstrate that COX-2i would improve small intestinal ileus after burn. This unique effect of COX-2i on burn-induced delayed gastric emptying has been confirmed, as COX-2i had no effect on gastric emptying in sham burned rats. The improvement in burn-induced delay in gastric emptying with the COX-2 inhibitor suggested a mechanism involving the COX-2 pathway. Both COX isoforms were shown to be required for protection against inflammatory changes in the liver and the small intestine following burn injury [8]. Possible mechanisms for the effect of COX-2i amelioration of burn-induced gastric dysmotility are probably due to inhibition of prostaglandin I2 (PGI2) and prostaglandin E2 (PGE2) synthesis [13,33]. PGI2 and PGE2 are eicosanoids that exert its effect on GI motility both directly and indirectly; directly on GI smooth muscle cells or the interstitial cells of Cajal [11,34], and indirectly by decreasing the threshold of afferent nerves to painful stimuli, thus inducing a hyperalgesic effect that leads to a decreased intestinal motility [13]. Cyclooxygenase inhibitor may also act by interrupting adrenergic reflex pathways, leading to improved intestinal motility [33]. In conclusion, as selective COX-2 inhibition has lead to an improvement of post-burn gastric emptying, we provide evidences that the prostaglandin pathway may play a key role in the delayed gastric emptying induced by major burns.

Acknowledgments This study was supported by Shriners Hospitals for Children Grant # 8556 (Herndon).

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