Splanchnic Artery Occlusion Research Articles

Metabolic Tissue Swelling and Local Microcirculation in Splanchnic Artery Occlusion Shock: Implications for Critical Illness.

Trauma is a leading cause of death in the United States. Advancements in shock resuscitation have been disappointing because the correct upstream mechanisms of injury are not being targeted. Recently, significant advancements have been shown using new cell-impermeant molecules that work by transferring metabolic water from swollen ischemic cells to the capillary, which restores tissue perfusion by microcirculatory decompression. The rapid normalization of oxygen transfer improves resuscitation outcomes. Since poor resuscitation and perfusion of trauma patients also causes critical illness and sepsis and can be mimicked by ischemia-reperfusion of splanchnic tissues, we hypothesized that inadequate oxygenation of the gut during trauma drives development of later shock and critical illness. We further hypothesized that this is caused by ischemia-induced water shifts causing compression no-reflow. To test this, the superior mesenteric artery of juvenile anesthetized swine was occluded for 30 minutes followed by 8 hours of reperfusion to induce mild splanchnic artery occlusion (SAO) shock. One group received the impermeant polyethylene glycol 20,000 Da (PEG-20k) that prevents metabolic cell swelling, and the other received a lactated Ringer's vehicle. Survival doubled in PEG-20k-treated swine along with improved macrohemodynamics and intestinal mucosal perfusion. Villus morphometry and plasma inflammatory cytokines normalized with impermeants. Plasma endotoxin rose over time after reperfusion, and impermeants abolished the rise. Inert osmotically active cell impermeants like PEG-20k improve intestinal reperfusion injury, SAO shock, and early signs of sepsis, which may be due to early restoration of mucosal perfusion and preservation of the septic barrier by reversal of ischemic compression no-reflow. SIGNIFICANCE STATEMENT: Significant advancements in treating shock and ischemia have been disappointing because the correct upstream causes have not been targeted. This study supports that poor tissue perfusion after intestinal ischemia from shock is caused by capillary compression no-reflow secondary to metabolic cell and tissue swelling since selectively targeting this issue with novel polyethylene glycol 20,000 Da-based cell-impermeant intravenous solutions reduces splanchnic artery occlusion shock, doubles survival time, restores tissue microperfusion, and preserves gut barrier function.

Expression of concern: Protective role of PI3-kinase-Akt-eNOS signalling pathway in intestinal injury associated with splanchnic artery occlusion shock.

Expression of concern: Protective role of PI3-kinase-Akt-eNOS signalling pathway in intestinal injury associated with splanchnic artery occlusion shock.

Heart period and blood pressure characteristics in splanchnic arterial occlusion shock-induced collapse.

The nature of hemodynamic instability typical of circulatory shock is not well understood, but an improved interpretation of its dynamic features could help in the management of critically ill patients. The objective of this work was to introduce new metrics for the analysis of arterial blood pressure (ABP) in order to characterize the risk of catastrophic outcome in splanchnic arterial occlusion (SAO) shock. Continuous ABP (fs=1kHz) was measured in rats during experimental SAO shock, which induced a fatal pressure drop (FPD) in ABP. The FPD could either be slow (SFPD) or fast (FFPD), with the latter causing cardiovascular collapse. Time series of mean arterial pressure, systolic blood pressure and heart period were derived from ABP. The sample asymmetry-based algorithm Heart Rate Characteristics was adapted to compute the Heart Period Characteristics (HPC) and the Blood Pressure Characteristics (BPC). Baroreflex sensitivity (BRS) was assessed by means of a bivariate model. The approach to FPD of the animals who collapsed (FFPD) was characterized by higher BRS in the low frequency band versus SFPD animals (0.36±0.15 vs. 0.19±0.12ms/mmHg, p value=0.0196), bradycardia as indicated by the HPC (0.76±0.57 vs. 1.94±1.27, p value=0.0179) and higher but unstable blood pressure as indicated by BPC (3.02±2.87 vs. 1.47±1.29, p value=0.0773). The HPC and BPC indices demonstrated promise as potential clinical markers of hemodynamic instability and impending cardiovascular collapse, and this animal study suggests their test in data from intensive care patients.

Baroreflex sensitivity during intestinal ischemia (1169.6)

Autonomic control of heart rate (HR) during prolonged intestinal ischemia was investigated by mathematical analysis of cardiac baroreflex. 24 Wistar rats underwent splanchnic arterial occlusion (SAO) under anesthesia and received different treatments, which included enteral saline or glucose, intramuscular xylazine and i.v. glycopyrrolate. High fidelity continuous arterial blood pressure (ABP) was measured by a catheter placed in the femoral artery. Time series of systolic blood pressure (SBP) and heart period (HP) were derived from the continuous ABP. Baroreflex gain (from SBP to HP) was calculated both in the low frequency (LF, 0.25‐0.75 Hz) and high frequency (HF, 0.75‐2.5 Hz) spectral bands. After a variable delay, ABP began to steadily decrease until either sacrifice at 4 hours after SAO or collapse by a sudden, fast fatal drop (FFD) of ABP. FFD was prevented by treatment with glycopyrrolate. Rats who displayed the FFD had a higher LF gain in the period (~ 5 mins) preceding the FFD with respect to the post‐SAO period (0.22 ± 0.11 ms/mmHg vs. 0.36 ± 0.15 ms/mmHg, p‐value < 0.05). These preliminary results suggest a sympathetic hyperactivation in the FFD rats and hint that the sympathetic system reaches saturation before the FFD, with the possibility of a sudden shift of the sympatho‐vagal balance towards a parasympathetic driven control of HR.Grant Funding Source: Supported by the ShockOmics project of the European Union (Grant#602706)

Breakdown of Mucin as Barrier to Digestive Enzymes in the Ischemic Rat Small Intestine

Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO) was used to study the degradation of two mucin isoforms (mucin 2 and 13) and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4). In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell disruption.

What’s New in Shock, March 2012?

What’s New in Shock, March 2012?

Disruption of the Mucosal Barrier During Gut Ischemia Allows Entry of Digestive Enzymes Into the Intestinal Wall

Intestinal ischemia is associated with high morbidity and mortality, but the underlying mechanisms are uncertain. We hypothesize that during ischemia the intestinal mucosal barrier becomes disrupted, allowing digestive enzymes access into the intestinal wall initiating autodigestion. We used a rat model of splanchnic ischemia by occlusion of the superior mesenteric and celiac arteries up to 30 min with and without luminal injection of tranexamic acid as a trypsin inhibitor. We determined the location and activity of digestive proteases on intestinal sections with in situ zymography, and we examined the disruption of two components of the mucosal barrier: mucin isoforms and the extracellular and intracellular domains of E cadherin with immunohistochemistry and Western blot techniques. The results indicate that nonischemic intestine has low levels of protease activity in its wall. After 15-min ischemia, protease activity was visible at the tip of the villi, and after 30 min, enhanced activity was seen across the full thickness of the intestinal wall. This activity was accompanied by disruption of the mucin layer and loss of both intracellular and extracellular domains of E cadherin. Digestive protease inhibition in the intestinal lumen with tranexamic acid reduced morphological damage and entry of digestive enzymes into the intestinal wall. This study demonstrates that disruption of the mucosal epithelial barrier within minutes of intestinal ischemia allows entry of fully activated pancreatic digestive proteases across the intestinal barrier triggering autodigestion.

Severe Intestinal Ischemia Can Trigger Cardiovascular Collapse and Sudden Death Via a Parasympathetic Mechanism

Hemorrhagic shock and splanchnic arterial occlusion (SAO) followed by reperfusion are associated with high mortality. However, rapid cardiovascular failure and death may also occur before reperfusion in hemorrhagic shock and SAO. We show in a rat SAO model that, upon gut ischemia, mean arterial blood pressure transiently elevates and then drops fatally in one of two time courses: (i) gradually over ∼1 to 3 h or (ii) rapidly (often by >80 mmHg) over a period of 1 to 6 min. We hypothesize that fast fatal pressure drops (FFPDs) are due to failure of autonomic nervous system control. To test this, we treated rats with Glucose (10%) in the small intestinal lumen and intramuscularly administered xylazine to activate the parasympathetic nervous system or with a muscarinic anticholinergic (glycopyrrolate) or by total subdiaphragmatic vagotomy to attenuate parasympathetic nervous system activity. We also tested nafamostat mesilate (ANGD [6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate]), a protease inhibitor efficacious in preventing blood pressure loss in SAO with reperfusion, in the intestinal lumen. Fifty percent of animals receiving xylazine and Glucose died by FFPD (vs. 33% with neither, not statistically significant). Total subdiaphragmatic vagotomy or glycopyrrolate treatment significantly reduced the incidence to 0% (P < 0.008), although slow fatal pressure drops still occurred. ANGD did not prevent FFPDs, but delayed onset of slow fatal pressure drops (P < 0.013). These results suggest that gut ischemia can cause sudden death via an autonomic nervous system mechanism and that SAO with Glucose and xylazine may serve as a useful model for the study of neurogenic shock or autonomic dysregulation associated with sudden death.

Effect of a sea snake (Enhydrina schistosa) venom on the ganglionic nicotinic actions of acetylcholine.

Abstract The effects of venom from a common sea snake, Enhydrina schistosa, on the actions of acetylcholine in the superior cervical ganglion, the adrenal medulla and in the atropinized cat were investigated. The crude venom reduced nictitating membrane responses to preganglionic nerve stimulation and intra-arterial injection of acetylcholine, without lowering the responses to postganglionic nerve stimulation or intra-arterial adrenaline. In the eviscerated cat, the venom also antagonized rises in heart rate and blood pressure induced by in-injections into the coeliac artery of acetylcholine but not those of histamine. Pressor effects due to splanchnic nerve stimulation and carotid artery occlusion were blocked. In the atropinized cat, the venom depressed heart rate and pressor responses to intravenous acetylcholine, but not the responses to adrenaline. It was concluded that the venom antagonized the actions of acetylcholine at autonomic ganglia and the adrenal medulla.

Protective effects of apocynin, an inhibitor of NADPH oxidase activity, in splanchnic artery occlusion and reperfusion

SAO shock is a severe form of circulatory shock produced by I/R of the splanchnic organs. SAO causes an enhanced formation of ROS, which contributes to the pathophysiology of shock. Apocynin is a naturally occurring, methoxy-substituted catechol, experimentally used as an inhibitor of NOX. It can decrease the production of ROS from activated neutrophils and macrophages, inhibiting the assembly of NADPH-oxidase activity. In this study, we wanted to evaluate the pharmacological action of apocynin in rats subjected to SAO shock, which was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 45 min. After this period of occlusion, the clamps were removed. Administration of apocynin i.p. (5 mg/kg i.p. 10% DMSO) 5 min before reperfusion significantly reduced the (1) histological evidence of tissue injury, (2) proinflammatory cytokine production (TNF-α, IL-1β), (3) adhesion molecules (ICAM-1, P-selectin), (4) nitrotyrosine formation, (5) NF-κB expression, and (6) apoptosis (Bax, Bcl-2, Fas-L, and TUNEL). These results could imply a future use of apocynin in the treatment of I/R shock.

Rapid Death in Severe Gut Ischemia is Prevented By Parasympathetic Blockade

Splanchnic arterial occlusion raises blood pressure (BP) in rats initially but within hours, even without reperfusion, BP falls irreversibly. Fatal pressure drops occur either over hours (slow) or in just minutes (fast). The fast BP drops occur without significant reduction of pulse pressure or pulse rate suggesting death is from neurogenic shock (i.e. dilation of peripheral blood vessels resulting in BP loss despite maintenance of cardiac contractility). Fast fatal pressure drops (FFPDs) occur more frequently and earlier if the rat is treated with intramuscular xylazine and glucose in the intestinal lumen, both stimulators of the parasympathetic nervous system (PNS). Thus we hypothesized parasympathetic inhibition by glycopyrrolate, a muscarinic anticholinergic, in intestinal ischemia could reduce incidence of rapid death. The results show that glycopyrrolate (1 mg/kg) significantly delayed onset of the fatal pressure drop (115±31 vs. 71±34 min post‐occlusion) and completely prevented FFPDs (0 of 6 vs. 5 of 8 animals with a fast death without glycopyrrolate), but not the slow form of pressure reduction. These results suggest that in severe intestinal ischemia, parasympathetic blockade prevents rapid pressure reductions, an observation that is consistent with a similar protection seen after vagotomy. Support: HL 67825, 76180, GM85072 and an unrestricted educational gift from Leading Ventures.

Olprinone attenuates the development of ischemia/reperfusion injury of the gut

Splanchnic artery occlusion (SAO) shock is a severe form of circulatory shock produced by ischemia and reperfusion of the splanchnic organs. The occlusion and reperfusion of the splanchnic arteries causes activation and adhesion of polymorphonuclear neutrophils (PMNs), release of proinflammatory substances and the formation of both species of oxygen and nitrogen derivatives free radicals. Olprinone is a specific phosphodiesterase-III inhibitor that has many properties; one of which is anti-inflammatory actions at therapeutic concentrations clinically used for heart failure. In this study, we wanted to evaluate the pharmacological action of olprinone (a PDEIII inhibitor) on SAO shock in mice. SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 30 min. After this period of occlusion, the clamps were removed. Olprinone was given at a dose of 0.2 mg/kg i.p. 15 min before reperfusion. Our results indicated that olprinone up-regulated cAMP in injured ileum tissue, and decreased the ileum tissue damage after 1 h of reperfusion in SAO shock mice. Moreover, olprinone decreased NF-kappaB expression; the nitration of tyrosine residues; the phosphorylation of p38 MAPK and JNK; cytokine production (TNF-alpha and IL-1beta); ICAM-1 and P-selectin expression and apoptosis in the injured ileum. These results could imply a future use of olprinone in the therapy of ischemia and reperfusion shock.

Liver X receptor agonist treatment reduced splanchnic ischemia and reperfusion injury

LXR is another member of the superfamily of nuclear hormone receptors that heterodimerizes with RXR and regulates the intracellular levels of cholesterol through gene induction of enzymes and proteins involved in the cholesterol metabolism and transport. LXR ligands inhibit the gene expression of proinflammatory mediators in immunostimulated macrophages; in vivo studies have shown that activation of LXR reduces the inflammatory response in a murine model of contact dermatitis and atherosclerosis. No reports have addressed a role for LXRs in pathophysiology of intestinal ischemia. The aim of this study was to investigate the effects of T0901317, a potent LXR ligand, in a mouse model of SAO shock, which was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 30 min. After this period of occlusion, the clamps were removed. Mice were killed at 60 min after reperfusion. This study provides the evidence that T0901317, LXR agonist, modulates: the development of SAO shock; the infiltration of the tissue with PMNs; the expression of TNF-alpha and IL-1beta; the nitration of tyrosine residues; NF-kappaB expression; the MAPK phosphorylation (ERK, JNK, and p38); FasL; apoptosis; Bax and Bcl-2 expression; and the degree of tissue injury caused by SAO shock. Our results imply that LXR agonists may be useful in the therapy of inflammation.

RECOMBINANT HUMAN ERYTHROPOIETIN PREVENTS LIPOPOLYSACCHARIDE-INDUCED VASCULAR HYPOREACTIVITY IN THE RAT

Erythropoietin (EPO) is a hypoxia-inducible hormone that is essential for normal erythropoiesis in the bone marrow. Administration of recombinant human-EPO is currently being used for the therapy of anemia associated with chronic renal failure and cancer. Moreover, EPO reduces organ injury in experimental hemorrhagic as well as in splanchnic artery occlusion shock and preserves cardiac function after experimental cardiac I/R. Erythropoietin receptors are widely distributed in the cardiovascular system, including endothelial, smooth muscle, cardiac, and other cell types, and nonhematopoietic effects of EPO are increasingly recognized. Thus, the vasculature may be a biological target of EPO. Therefore, the aim of our study was to investigate whether EPO exerts a protective effect in septic shock by modulating vascular dysfunction and hyporeactivity. Rats received EPO (300 U/kg, i.v.) or vehicle 30 min before and 1 and 3 h after LPS (8 x 10 U/kg, i.v.). In vivo and ex vivo (aortic rings) experiments were performed to evaluate the vascular response to contracting and vasodilating agents. The expression of iNOS, intercellular adhesion molecule 1, poly(ADP)ribose polymerase, Bcl-xl, and Bcl-2 was evaluated by Western blot analysis in the rat aorta. We demonstrate that EPO significantly prevents LPS-induced vascular hyporeactivity and endothelial dysfunction. Interestingly, EPO inhibits the increase in iNOS, poly(ADP)ribose polymerase, and intercellular adhesion molecule 1 expression in the aorta of endotoxemic rats and attenuated the decline in the expression of both Bcl-xl and Bcl-2 caused by LPS. In conclusion, our data support the view that EPO has important nonerythropoietic effects protecting organ and tissue against injury and indicate that EPO may be useful in the therapy of patients with septic shock.

Sudden death in severe gut ischemia: a possible role for the autonomic nervous system

Occlusion of blood vessels in the gut is a life‐threatening event. For example, splanchnic arterial occlusion (SAO) followed by reperfusion is an established model for physiologic shock. In addition SAO is highly lethal on its own, either associated with gradual drops in mean arterial blood pressure (MABP) over &lt;1 to 3 hours or with a severe rapid drop in MABP (often &gt; 80 mmHg) over 1 to 6 minutes, leading to left ventricular arrest. The rapid drop occurs after ~ 75‐150 min of ischemia. We observed that high levels of glucose in the lumen of the small intestine decrease the ischemic duration before which a rapid drop may be observed (as early as 30 min) and increase its rate of occurrence (from 29% to 63%). As afferent neurons in the gut are capable of sensing ischemia and/or hyperglycemia, our objective was to determine whether a total subdiaphragmatic vagatomy (TSV), which prevents parasympathetic sensing in the gut, could prevent the rapid drop in MABP. We found that survival time was increased by TSV (P&lt;0.05). While not preventing the gradual drops in MABP, TSV significantly reduced the instances of rapid drops in MABP (P&lt;0.008). These results suggest that meals high in glucose and parasympathetic sensing in the gut may greatly affect the amount of time available to treat gut ischemia before catastrophic cardiovascular failure. Supported in part by HL 67825, 76180 and an unrestricted educational gift from Leading Ventures.

MESENTERIC LYMPH DUCT LIGATION IMPROVES SURVIVAL IN A LETHAL SHOCK MODEL

The goal of this study was to test the hypothesis that factors released from the gut and carried in the mesenteric lymph contribute to mortality in a lethal gut I/R model. To test this hypothesis, a lethal splanchnic artery occlusion (SAO) shock model was used in male Sprague-Dawley rats. In the first set of experiments, ligation of the mesenteric lymph duct (LDL), which prevents gut-derived factors carried in the intestinal lymphatics from reaching the systemic circulation, significantly improved 24-h survival after a 20-min SAO insult (0% vs. 60% survival; P < 0.05). This increase in survival in the LDL-treated rats was associated with a blunted hypotensive response. Because increased iNOS-induced NO levels have been implicated in SAO-induced shock, we measured plasma nitrite/nitrate levels and liver iNOS protein levels in a second group of animals. Ligation of the mesenteric lymph duct significantly abrogated the SAO-induced increase in plasma nitrite/nitrate levels and the induction of hepatic iNOS (P < 0.05). In an additional series of studies, we documented that LDL increased not only 24-h but also long-term 7-day survival. During the course of these studies, we made the unexpected finding that Sprague-Dawley rats from different animal vendors had differential resistance to SAO, and that the time of the year that the experiments were carried out also influenced the results. Nonetheless, in conclusion, these studies support the hypothesis that factors carried in the mesenteric lymph significantly contribute to the development of irreversible shock after SAO.

Plant histaminase as an investigational drug in splanchnic artery occlusion and reperfusion

Background: Amine oxidases are ubiquitous enzymes involved in the metabolism of biogenic amines. Copper amine oxidases catalyze the oxidative deamination of primary amine groups of several biogenic amines, such as putrescine, cadaverine and histamine. Objective: In the present review the effects of a plant amine oxidase (histaminase, EC1.4.3.6), purified from pea seedlings, in the prevention of splanchnic postischemic reperfusion damage are reported. Conclusion: Various studies have clearly indicated that the use of histaminase will offer a good perspective for a novel therapeutic approach in the medical treatment of intestinal ischemia.

Anti-Inflammatory and Anti-Apoptotic Effects of Fumonisin B1, an Inhibitor of Ceramide Synthase, in a Rodent Model of Splanchnic Ischemia and Reperfusion Injury

Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study sought to determine whether pharmacological inhibition of ceramide biosynthesis in the intestine attenuates pathophysiological sequelae of shock induced by splanchnic artery occlusion and reperfusion. Ischemia and reperfusion injury was induced in anesthetized rats by clamping both the superior mesenteric artery and the celiac artery for 45 min followed by reperfusion. Within 6 min after reperfusion, animals developed significant systemic hypotension with 100% of the animals dying during the 4-h period of reperfusion. In parallel experiments, animals were necropsied after 60 min of reperfusion, and the ileum was harvested for histological examination and assessment of biochemical changes. Administration of fumonisin B1 (FB1), a competitive and reversible inhibitor of ceramide synthase (3 mg/kg, 15 min before reperfusion), significantly reduced i) the increased ceramide expression as detected by immunohistochemistry; ii) peroxynitrite-mediated protein nitration; iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils following a decrease in intercellular adhesion molecule-1 expression; iv) production of the proinflammatory cytokine tumor necrosis factor-alpha; and v) apoptosis in the ileum. Overall, tissue-protective effects were clearly observed upon histological examination of the ileum. These beneficial events were ultimately linked to decreases in both the development of hypotension and overall mortality. These results implicate ceramide as a key signaling molecule in splanchnic arterial ischemia and reperfusion-induced shock. The broader implications of our results provide a pharmacological rationale for the development of inhibitors of ceramide biosynthesis as novel therapeutics for ischemia and reperfusion-induced shock of several etiologies.

Shock survival improved by combined protease and lipase inhibition in the intestinal lumen

Shock and multiple‐organ failure remain one of the leading causes of death. Our evidence shows it is triggered by a failure of the mucosal barrier in the intestine that is followed by cell death in the intestine and elsewhere. We have recently shown that homogenates of ischemic, but not non‐ischemic, intestines or intestinal wall homogenates digested with proteases from the intestinal lumen contain cytotoxic mediators. We have identified these mediators as free fatty acids (FFAs), already present or created by the action of a lipolytic enzyme present in the intestinal wall. The FFAs are bound to FFA‐binding proteins, such as albumin, unless those proteins are digested, as may occur in shock when the mucosal barrier fails and proteases enter the walls. To test this hypothesis in vivo, we injected saline, aprotinin (protease inhibitor), orlistat (lipase inhibitor), or a combination of the two into the lumen of the small intestine of Wistar rats and subjected them to 120 min of splanchnic arterial occlusion followed by up to 180 min of observed reperfusion. Saline treated animals all died after 86±43 min of reperfusion. Only the combination aprotinin and orlistat treatment significantly improved survival time to 164±11, suggesting that in severe models of shock both luminal protease and luminal lipase inhibition should be explored.

The intestine as source of cytotoxic mediators in shock: free fatty acids and degradation of lipid-binding proteins

Shock and multiple organ failure remain primary causes of late-stage morbidity and mortality in victims of trauma. During shock, the intestine is subject to extensive cell death and is the source of inflammatory factors that cause multiorgan failure. We (34) showed previously that ischemic, but not nonischemic, small intestines and pancreatic protease digested homogenates of normal small intestine can generate cytotoxic factors capable of killing naive cells within minutes. Using chloroform/methanol separation of rat small intestine homogenates into lipid fractions and aqueous and sedimented protein fractions and measuring cell death caused by those fractions, we found that the cytotoxic factors are lipid in nature. Recombining the lipid fraction with protein fractions prevented cell death, except when homogenates were protease digested. Using a fluorescent substrate, we found high levels of lipase activity in intestinal homogenates and cytotoxic levels of free fatty acids. Addition of albumin, a lipid binding protein, prevented cell death, unless the albumin was previously digested with protease. Homogenization of intestinal wall in the presence of the lipase inhibitor orlistat prevented cell death after protease digestion. In vivo, orlistat plus the protease inhibitor aprotinin, administered to the intestinal lumen, significantly improved survival time compared with saline in a splanchnic arterial occlusion model of shock. These results indicate that major cytotoxic mediators derived from an intestine under in vitro conditions are free fatty acids. Breakdown of free fatty acid binding proteins by proteases causes release of free fatty acids to act as powerful cytotoxic mediators.