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J Appl Physiol 95: 2047-2054, 2003. First published August 15, 2003; doi:10.1152/japplphysiol.00925.2002
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Gut mucosal damage during endotoxic shock is due to mechanisms other than gut ischemia

Suzana M. Lobo,1 Daniel De Backer,2 Qinghua Sun,2 Zizi Tu,2 George Dimopoulos,2 Jean-Charles Preiser,2 Nathalie Nagy,2 Bernard Vray,2 Vincent Vercruy,2 Renato Giuseppe Giovanni Terzi,3 and Jean-Louis Vincent2

1Department of Intensive Care, Faculdade de Medicina de Sao José do Rio Preto, 15091-450 Sao José; and 3Emergency Department, University of Sao Paulo, 05508-010 Sao Paulo, Brazil; and 2Department of Intensive Care, Erasme Hospital, Free University of Brussels, 1070 Brussels, Belgium

Submitted 10 October 2002 ; accepted in final form 2 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Whether the gut alterations seen during sepsis are caused by microcirculatory hypoxia or disturbances in cellular metabolic pathways associated with mitochondrial respiration remains controversial. We hypothesized that hypoperfusion or hypoxia and local production of nitric oxide might play an important role in the development of gut mucosal injury during endotoxic shock and investigated their roles by using differing levels of fluid resuscitation and occlusion of the superior mesenteric artery (SMA). Anesthetized New Zealand rabbits were allocated to group I (sham, n = 8); group II [low-dose endotoxin (LPS, Escherichia coli-055:B5, 150 µg/kg)/fluid resuscitation (12 ml · kg-1 · h-1); n = 8]; group III [high-dose LPS (1 mg/kg)/fluid resuscitation (12 ml · kg-1 · h-1); n = 8]; group IV [high-dose LPS (1 mg/kg)/hypovolemia (4 ml · kg-1 · h-1 fluids); n = 8]; and group V [SMA ligation/fluid resuscitation (12 ml · kg-1 · h-1); n = 4]. Luminal gut lactate concentrations and PCO2 gap increased in groups IV and V (P < 0.05), reflecting alterations in gut perfusion. Interestingly, significant histological alterations were observed in all LPS groups but not in group V. Blood and luminal gut nitrate/nitrite concentrations increased only in group IV. The mechanism of gut injury in endotoxic shock seems unrelated to hypoxia and release of nitric oxide. Gut dysfunction may occur as a result of so-called "cytopathic hypoxia."

sepsis; lactate; nitric oxide; cytopathic hypoxia

GUT INJURY SEEN DURING SEPSIS has been associated with the development of organ dysfunction and multiple-organ failure (10, 27). However, the pathophysiological mechanisms underlying sepsis-induced gut injury are unclear, and whether it is caused by microcirculatory hypoxia or disturbances in cellular metabolic pathways associated with mitochondrial respiration remains controversial. Indeed, blood flow redistribution may result in regional ischemia in the gastrointestinal tract during sepsis despite a globally increased systemic oxygen delivery (O2). Gut alterations, including ileal mucosal acidosis (1, 12, 37, 45), increase in mucosal permeability (13, 37), and gut mucosal injury (6, 20, 30, 49), have often been reported after administration of lipopolysaccharide (LPS) or live bacteria and in experimental peritonitis. Vallet et al. (44) reported a decrease in ileal mucosal PO2 in endotoxic dogs, and Hasibeder et al. (18) noted a decrease in jejunal mucosal PO2 in endotoxic pigs. Others have made similar observations in various animal models (1, 48). Recently, Theisen et al. (41) reported alterations in microvascular blood flow to the gut mucosa in a porcine model of hyperdynamic endotoxic shock, despite mesenteric blood flow being maintained.

However, other studies have suggested normal or elevated blood flow or tissue PO2 in endotoxemia or experimental sepsis. VanderMeer et al. (45) reported ileal mucosal acidosis despite maintained mucosal perfusion and oxygenation in endotoxic pigs in association with normal or even higher mucosal PO2 levels, as measured with multiwired PO2 electrodes. Using colored microspheres, Revelly et al. (32) showed an increased mucosal flow to the gut in endotoxic pigs, and Crouser et al. (6) demonstrated that mucosal gut flow was maintained despite mucosal acidosis and alterations in gut oxygen metabolism in a feline ileal preparation. After cecal ligation and puncture in rats, there was a 42% increase in microvascular flow to the midjejunum in early sepsis shown by using laser-Doppler flow measurements and colloidal carbon infusion (46).

In addition to the possible effects of hypoperfusion on endotoxin-induced gut iujury, LPS may induce alterations in gastrointestinal oxygen metabolism. Changes in ileal oxygen metabolism have been correlated with the severity of mitochondrial injury in feline ileum (7). Cellular alterations referred to as "cytopathic hypoxia" may be implicated in multiple organ failure, in which case production of ATP may be decreased despite normal PO2 values in the vicinity of mitochondria within cells (11). A number of different biochemical mechanisms or mediators have been suggested to account for cellular dysfunction and cytopathic hypoxia in sepsis, including inactivation of pyruvate dehydrogenase, reversible inhibition of cytochrome oxidase by nitric oxide (NO), inhibition of mitochondrial respiratory complexes by peroxynitrite, and activation of the nuclear enzyme poly(ADP-ribosyl) polymerase (3, 13, 28, 29, 43, 51).

We developed a rabbit model to investigate the role of blood flow alterations, tissue hypoxia, and NO release in the development of gut mucosal injury during endotoxic shock.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal model. Thirty-six specific pathogen-free New Zealand rabbits (2.7-3.4 kg body wt) were handled according to the rules of the local Animal Care Committee after institutional approval for animal investigations was obtained. Induction doses of ketamine (20 mg/kg) and xylazine (4 mg/kg) were given intramuscularly for sedation and anesthesia, and were followed by a continuous intravenous infusion of ketamine (15-35 mg · kg-1 · h-1) started 3 h after induction. An intravenous catheter was inserted into an ear vein for venous access (Surflo IV catheter, 18 gauge x 2 in.), and 20 ml of saline solution were given as a bolus. Cefazoline (50 mg/kg) and chloramphenicol (12.5 mg/kg) were administered intravenously. Tracheotomy was performed, and the animals were ventilated (Servo ventilator, Siemens, Solna, Sweden) with 40-60% of inspired oxygen fraction, tidal volume of 7-10 ml/kg, and a respiratory rate of 40 breaths/min further adjusted to maintain an arterial PO2 of >80 Torr and an arterial PCO2 (PaCO2) between 35 and 45 Torr.

Surgical procedure. A 16-gauge polyethylene catheter was inserted into the right carotid artery and connected to a pressure transducer to enable continuous recording of arterial pressure. Another catheter (Surflo IV catheter, 22 gauge x 2 in.) was placed in the jugular vein for recording of right atrial pressure and venous access. A midline laparotomy was performed, and ultrasonic flow probes (Transonic System, Ithaca, NY) were placed around the superior mesenteric artery (SMA) and the abdominal aorta (aorta) just above the origin of the celiac trunk to continuously measure flow. A bowel segment was delineated in the midileum. An antimesenteric enterotomy was performed, and inflow (Foley catheter 8) and outflow (Foley catheter 10) catheters were placed to delimit a 5-cm segment. After the catheters were positioned in the ileum, the balloons were gently inflated with water. Once inflated, the balloons permitted the perfusion of a closed area of the gut lumen with gut luminal perfusate (GLP) solution. A larger catheter size was used for the outflow to facilitate perfusate withdrawal. In a second gut segment, a tonometric catheter (TRIP tonometry catheter, Datex) was placed through a minimal antimesenteric wall incision and secured with a purse-string suture. Body temperature was maintained with a heating lamp. The animals were allowed to recover for 60 min before the experimental protocol was started. Hemodynamic measurements and blood samples were obtained at baseline and every hour up to 4 h after LPS administration.

GLP. The bowel segment was carefully rinsed with heated (37°C) physiological saline before a perfusate solution (RPMI 1640 medium, Sigma Chemical, St. Louis, MO) was infused at a rate of 6 ml/h. In addition, 10,000 KIU/ml of aprotinin (Trasylol, Bayer) were added per 10 ml of perfusate solution to inhibit proteolytic activity. The lumen of the proximal catheter was used for continuous infusion, and the lumen of the distal catheter was used to recover the perfusate every hour. GLP samples were taken hourly, centrifuged at 3,000 rpm, filtered through a 0.22-µm filter (Millex, Bedford, OR) to eliminate bacteria, and stored at -80°C.

Experimental protocol. The animals were randomized to four groups, and a fifth group (group V) was added after preliminary analysis of the results of the original four groups (Table 1). Group I (sham) received placebo and 12 ml · kg-1 · h-1 fluids (Ringer lactate). Group II (low-dose LPS/fluid resuscitation) received intravenous 150 µg/kg LPS (Escherichia coli-055:B5, DIFCO Laboratories, Detroit, MI) and 12 ml · kg-1 · h-1 fluids. Group III (high-dose LPS/fluid resuscitation) received 1 mg/kg LPS + 12 ml · kg-1 · h-1 fluids. Group IV (high-dose LPS/hypovolemia) received 1 mg/kg LPS + 4 ml · kg-1 · h-1 fluids. Group V (gut ischemia/fluid resuscitation) received ligation of superior mesenteric artery (SMA) and 12 ml · kg-1 · h-1 fluids. All animals received intravenous lactated Ringer solution as fluid resuscitation throughout the experimental protocol and a 20-ml intravenous bolus of hydroxyethyl starch (6%; molecular weight 200,000; D/0.5, Haes-Steril) at the end of the surgical instrumentation. A continuous intravenous infusion of hydroxyethyl starch was started after the bolus in all animals except group IV, to provide a total volume of 20 ml/kg over 5 h. Immediately after baseline measurements, LPS was diluted in normal saline (1 mg/ml) and administered intravenously over 3 min to groups II-IV. Group V had the peritoneum reopened for ligation of the SMA at this time point.


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  		Table 1. Characteristics of rabbits

 

Analytical methods. Blood-gas analyses were performed on arterial samples (ABL-30, Radiometer, Copenhagen, Denmark). Samples for lactate analysis were stored on ice and analyzed (ABL-30, Radiometer) within 10-30 min. Blood samples were centrifuged immediately (3,000 g for 20 min), and plasma was stored at -80°C.

NO. Stable end products of NO metabolism, nitrate (NO3-), and nitrite (NO2)-) concentrations were determined spectro-photometrically from serum and GLP by using the Griess reaction. Briefly, for NO2- measurements, the absorbance was measured at 540 nm in a microplate ELISA reader (Titertek multiscan MCC/340, MKII, Eflab, Finland). NO3- level was determined after stoichiometrical reduction to NO2-.

Tonometry. For tonometry measurements, 1 ml of 0.9% saline was placed in the silicone balloon of the tonometer and allowed to equilibrate for 30 min. The first 0.7 ml aspirated was discharged, and analysis was immediately performed in the remaining 0.3 ml in a blood-gas analyzer (ABL 500 radiometer). Ileum PCO2 was corrected for incomplete equilibration time during the 30-min sampling periods by multiplying PCO2 by 1.24 (5). Ileal mucosal-PaCO2 gradient (PaCO2 gap) was calculated as the difference between ileal PCO2 and PaCO2.

Grading of mucosal damage. At the end of the experiment, each animal received a lethal injection of pentobarbital sodium, and the ileum was immediately removed. A segment was taken for optical microscopy and fixed in 10% formaldehyde saline followed by sectioning and staining with hematoxilin and eosin. Mucosal histology was graded as previously described (4) by using the following scale: grade 0, normal mucosa; grade 1, subepithelial space formation; grade 2, extension of the subepithelial space with moderate lifting of the epithelial layer from the lamina propria; grade 3, massive epithelial lifting down the sides of the villi; grade 4, denuded villi with lamina propria and dilated capillaries exposed; and grade 5, digestion and disintegration of the lamina propria, hemorrhage, and ulceration.

Data analysis. Results are presented as means ± SD. Significance was tested by repeated-measures ANOVA. Bonferroni adjustment was used for multiple comparisons. Linear regression was used to test the relation between gut lactate release and PaCO2-gap and SMA. The grade of histological damage was evaluated by using a Mann-Whitney rank sum test. A P value of <0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
All groups received a similar amount of fluids, except the hypovolemic group (group IV) (Table 1). Body temperatures were maintained between means of 37.8 and 38.5°C.

Hemodynamics. After LPS administration, mean arterial pressure decreased significantly in group II (low LPS, high fluid; from 76 ± 11 to 52 ± 12 mmHg at 3 h; P < 0.05), group III (high LPS, high fluid; from 69 ± 6 to 52 ± 7 mmHg at 3 h; P < 0.05), and especially in group IV (from 68 ± 8 to 38 ± 8 mmHg at 3 h; P < 0.05) (Fig. 1). At 1, 2, and 3 h, group III had significantly lower mean arterial pressure than group I (56 ± 9 vs. 72 ± 7 mmHg at 1 h; 51 ± 9 vs. 80 ± 14 mmHg at 2 h; and 52 ± 7 vs. 77 ± 18 mmHg at 3 h; P < 0.05 for all). aorta was significantly lower at 1 h in group III (49 ± 9 ml/min) and group IV (45 ± 10 ml/min) than in group I (82 ± 37 ml/min) (P < 0.05 for all). After this transient decline, aorta returned to baseline in all groups (P = not significant). SMA increased significantly above baseline in group III (from 52 ± 9 to 84 ± 12 ml/min at 2 h, 88 ± 17 ml/min at 3 h, and 81 ± 17 ml/min at 4 h; P < 0.05 for all) (Fig. 1).



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  		Fig. 1. Mean arterial pressure (MAP; A), aortic blood flow (aorta; B), mesenteric blood flow (SMA; C), and ileal mucosal PCO2 gradient (PCO2-gap; D) at baseline (time 0) and hourly. Values are means ± SD. {blacksquare}, Group I; {triangleup}, group II; {blacktriangledown}, group III; {bullet}, group IV; {circ}, group V. *P < 0.05 vs. baseline.

 

Tonometry-derived measurements. PaCO2 gap increased significantly in group IV (from 1.7 ± 6.6 to 17.2 ± 11.0 Torr at 3 h and to 19 ± 9 Torr at 4 h) and group V (from 6.2 ± 4.0 to 62 ± 38 Torr at 1 h, 64 ± 35 Torr at 2 h, and 69 ± 22 Torr at 4 h) (P < 0.05 for all) (Fig. 1). PaCO2 gap was significantly higher in group V compared with all other groups at 1, 2, 3, and 4 h (P < 0.05 for all).

Lactate measurements. An increase in arterial lactate concentrations occurred in group III (from 2.2 ± 0.6 to 4.3 ± 2.0 meq/l at 4 h; P < 0.05) and group IV (from 2.0 ± 0.7 to 6.7 ± 1.3 meq/l at 4 h; P < 0.05) (Fig. 2). Lactate concentrations were considerably higher in group IV than in the other groups at all time points (Fig. 2). Luminal gut lactate concentrations increased significantly in group IV (from 0.2 ± 0.1 to 1.1 ± 1.3 meq/l at 4 h; P < 0.05) and, especially, in group V (from 0.3 ± 0.3 to 5.6 ± 0.7 meq/l at 4 h; P < 0.05) (Fig. 2). In group V, but not in groups II-IV, there was a significant relation between gut lactate release and SMA (r = 0.47, P = 0.0008) and between gut lactate and PaCO2 gap (r = 0.62, P = 0.0001) (Fig. 3).



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  		Fig. 2. Serum lactate (A) and luminal gut lactate (B) measurements at baseline (time 0) and hourly. Values are means ± SD. {blacksquare}, Group I; {triangleup}, group II; {blacktriangledown}, group III; {bullet}, group IV; {circ}, group V. *P < 0.05 vs. baseline.

 


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  		Fig. 3. Comparison between SMA (A) and PCO2-gap (B) with luminal gut lactate release. {blacksquare}, Group I; {triangleup}, group II; {blacktriangledown}, group III; {bullet}, group IV; {circ}, group V.

 

Serum and gut concentrations of NO2-/NO3-. After LPS administration, NO2-/NO3- (NOx) concentrations significantly increased in serum and decreased in the GLP in group IV (from 149 ± 32 to 223 ± 55 µmol/l, and from 675 ± 161 to 422 ± 224 µmol/l, respectively; both P < 0.05) (Fig. 4). These levels were unaltered in all other groups.



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  		Fig. 4. Levels of serum (A) and gut luminal perfusate (B) nitrate/nitrite (NOx) at baseline and 4 h afer LPS. Values are mean ± SD. {blacksquare}, Group I; {triangleup}, group II; {blacktriangledown}, group III; {bullet}, group IV; {circ}, group V.*P < 0.05 vs. baseline.

 

Histological damage. All ileum specimens obtained in control animals were normal (degree 0). Mild to severe alterations were observed in all endotoxin groups. No histological alterations were detected after prolonged occlusion of the SMA (group V; Fig. 5).



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  		Fig. 5. Column scatterplots represent grades of histological injury in groups. Within-group comparisons: aP < 0.05 vs. group I. bP < 0.05 vs. group II.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cellular dysfunction and injury secondary to different types of shock are explained usually by a diminished cellular oxygen availability resulting from decreased arterial oxygen tension (hypoxic hypoxia), hemoglobin concentration (anemic hypoxia), or blood flow (stagnant hypoxia). In sepsis, however, a number of studies have suggested that tissue injury can occur despite adequate tissue oxygenation (11), and our findings show convincingly that endotoxin can induce histological lesions by mechanisms other than tissue hypoxia. Our observations are consistent with the development of so-called "cytopathic hypoxia," i.e., altered oxygen utilization despite adequate oxygen availability in the vicinity of the cell mitochondria. Hypoxia is unlikely to have played a major contribution to the histological lesions observed in our study for several reasons. First, hypoxemia and anemia were absent, and mesenteric blood flow was even increased after generous fluid resuscitation. Even though LPS administration induced an early hypotensive state, mesenteric oxygen delivery was well preserved. Second, mucosal PCO2 did not increase, and this can reasonably exclude a reduction in regional blood flow (5). Finally, the absence of luminal lactate release in fluid-resuscitated animals suggests the absence of gut ischemia. It was only when hypovolemia was purposely maintained (group IV) or when ischemia was induced by ligation of SMA (group V) that increases in PaCO2 gap and luminal gut release of lactate occurred.

Our observations are in accord with results from other studies. Fink et al. (13) showed that gut permeability alterations occurred after endotoxin administration but not after a similar mechanically induced reduction in mesenteric flow, suggesting that hypoperfusion by itself is not responsible for increased permeability. In a feline ileum preparation, epithelial necrosis occurred early after LPS administration, despite unaltered ileal tissue oxygen content, blood volume, and blood flow (8). Revelly et al. (33) reported that marked decreases in mucosal ATP content related to permeability and translocation alterations induced by LPS were not improved by massive resuscitation. In an isolated pig hindlimb preparation, the LPS-induced decrease in the skeletal muscle cytochrome aa3 redox status did not depend on oxygen delivery (14). Finally, in vitro studies indicate that cell dysfunction occurs within minutes of exposure to endotoxin and that a major functional derangement involves the inhibition of pyruvate dehydrogenase activity, pyruvate and lactate accumulation in the cell, and deranged oxidative phosphorylation (11).

Our observations indicate that these changes can occur as early as 4 h after LPS administration. Other studies have also reported that LPS or bacteria can rapidly induce tissue injury. Hersch et al. (20) reported that tissue injury, including the gut, antedated multiple-organ dysfunction in a normotensive sheep model with cecal ligation and perforation. Yu and Martin (49), using the same scoring system for mucosal damage (4) that we used, demonstrated gut mucosal injury in normotensive septic rats 24 h after tracheal instillation of Pseudomonas aeruginosa. Apoptosis is likely to be involved in histological lesions in association with necrosis as a result of cytotoxic mediators. Crouser et al. (8) reported that ileal necrosis and apoptosis could occur as early as 2 and 4 h, respectively, after LPS administration. Hotchkiss et al. (21) found extensive focal crypt epithelial and lymphocyte apoptosis in intestinal tissues obtained intraoperatively from patients with acute traumatic injuries.

A key finding in our study was the absence of histological lesions after prolonged mechanical occlusion of the SMA. In this group, gut hypoxia must have been present and was reflected by the marked rise in PaCO2 gap in gut lactate production, which was known to be secondary to gut ischemia (40). The well-maintained integrity of the gut mucosal morphology in this group after prolonged ligation of the SMA may be explained by several factors. Initially, the collateral vessels from the caudal mesenteric and celiac axis were not ligated so that some flow could be maintained. Indeed, we confirmed this finding by infusing 5 ml of methylene blue into the right atrium 2 and 3 h after SMA ligation in another rabbit; there was a small amount of methylene blue distributed in a heterogeneous manner in the jejunum and ileum, showing that the collateral vessels maintained some flow. The vascular bed can also compensate for the flow reduction by increasing the oxygen extraction rate. Indeed, blood flow can be decreased to <50% without significantly altering gut oxygen consumption (25). In this range, ileal oxygen consumption could be maintained independent of blood flow. Finally, there was no reperfusion possible, and much histological damage actually occurs during reperfusion. In addition, other cell protective mechanisms, such as induction of heat shock proteins, may have taken place (23).

The involvement of cytopathic hypoxia in the pathogenesis of multiple organ failure may account for the poor results of studies on optimization of oxygen delivery in patients with septic shock (16, 19), except for when the intervention takes place very early to correct other factors such as hypovolemia and cardiac dysfunction (34). This is in contrast with high-risk surgical patients in whom these therapeutic goals are more likely to improve survival (26, 38, 47). Indeed, all efforts to increase cellular oxygen availability may be in vain in the presence of derangements in mitochondrial oxygen metabolism (31).

Even though luminal gut lactate release was not observed, serum lactate concentrations significantly increased after LPS administration, suggesting that blood lactate comes primarily from other sources. Bellomo et al. (2) reported that lactate is even taken up by the gut in early endotoxemia in dogs. Other important sources of lactate in sepsis may be the lung (2, 9) or leukocytes (17). Lactate production in sepsis may arise from sites of inflammation rather than areas of poor perfusion and is related to augmented glycolytic metabolism by inflammatory cells and mitochondrial dysfunction. As in other studies in endotoxic shock, we showed that gut ischemia was present only when fluid replacement was insufficient (13, 50). Adequate fluid administration was essential to maintain splanchnic perfusion but was unable to prevent endotoxin-induced mucosal injury. In the group with fluid restriction, there was more severe hyperlactatemia compared with the other groups, indicating that in this circumstance, due to hypovolemia, the liver clears lactate less efficiently. In contrast, despite severe mesenteric hypoperfusion, animals in group V had significantly lower lactate concentrations due to better resuscitation.

The pathophysiological mechanisms underlying mucosal gut injury during septic shock remain poorly understood. LPS either by itself or via the release of various cytokines is able to alter cellular function by a NO-dependent pathway (3, 28, 29, 42). Studies with well-controlled conditions of oxygenation and perfusion such as in vitro enterocyte monolayers or Ussing chamber models have shown that NO mimics the action of LPS on permeability to both probe and bacteria (15, 28, 36). Salzman et al. (36) have documented that NO donors dilate tight junctions, cause cytoskeletal disruption, and deplete cellular ATP. Changes in the dynamics of NO deplete intracellular levels of ATP, cause marked derangements in the structure of the actin-based cytoskeleton, and increase intracellular ionized calcium concentration (28). In the present study, plasma or gut luminal NO2- and NO3- concentrations did not correlate with LPS-induced ileal mucosal injury, and there was a significant decrease in ileal luminal NOx concentrations compared with baseline during hypovolemia. Crouser et al. (8) also reported that inducible NO synthase induction was not involved in the early ileal epithelial necrosis induced by LPS in a feline model. In pigs with septic shock induced by live bacteria, Snygg et al. (39) noted that jejunal mucosal NO production decreased markedly during severe hypovolemia, whereas stable production was observed after E. coli sepsis. The mechanisms behind the inhibition of ileal mucosal NOx production are not known. Possible mechanisms include the presence of diminished flow and tissue dysoxia secondary to hypovolemia leading to a state of low availability of the substrate L-arginine and/or oxygen, or direct inhibition of cellular enzymes in the intestinal mucosa (39). Thus a very complex relationship between NO and hypoxia or flow may exist, and it is possible that constitutively formed NO plays a role in these changes.

Much of the oxidative injury associated with NO production is mediated by peroxynitrite, a toxic oxidant derived from the reaction of NO and superoxide (29). NO-induced increases in intestinal monolayer permeability can be prevented by peroxynitrite scavengers (28). Agents that interfere with the formation of peroxynitrite, such as superoxide dismutase mimetics and peroxynitrite decomposition catalysts, can also reduce endotoxin-induced cellular injury. In rats, Salvemini et al. (35) found that superoxide dismutase mimetics were able to reduce the LPS-induced increase in microvascular leakage, lipid peroxidation, and epithelial cell injury seen in duodenum and jejunum, suggesting that superoxide and peroxynitrite play a significant role in the pathogenesis of gut injury during endotoxemia. In sepsis or shock, production of peroxynitrite is increased, and it acts as a potent cytotoxic agent producing breaks in cellular DNA. DNA strand breaks activate the repair enzyme poly(ADP-ribose) synthase (PARS), which is able to induce cell energy depletion and increase gut mucosal permeability. There is now increasing evidence that exaggerated PARS activation contributes to cellular injury in sepsis. Jagtap et al. (22) reported that a phenanthridinone inhibitor of PARS was able to ameliorate LPS-induced gut injury in rats. There are few data on other mechanisms involved in LPS-induced gut injury. The naturally occurring protein lactoferrin, by its binding activity to the lipid A portion of LPS, prevented villus atrophy, edema, and vacuolation induced by LPS in the gut of mice; in this model, lactoferrin attenuated the lethal effects of LPS (24).

We acknowledge that the technique used to isolate a closed segment of the gut has some limitations. First, the inflation of the balloons inside the bowel may induce local ischemia and pressure damage. However, histological analysis of normal bowel segment and isolated bowel segment did not show major differences. Second, the gut mucosa could have been affected by the continuous washout by the gut perfusate. We do not believe that luminal perfusion affected the gut mucosa because no histological lesions were detected in control animals and the preparation was able to detect an increase in lactate release and mucosal CO2 in the presence of a decreased mesenteric blood flow.

In summary, this study indicates that LPS can induce early ileal mucosal injury by mechanisms other than tissue hypoxia and NO release. Fluid resuscitation is fundamental to maintain intestinal blood flow but does not prevent mucosal damage.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-L. Vincent, Dept. of Intensive Care Medicine, Erasme Univ. Hospital, Route de Lennik 808, 1070 Brussels, Belgium (E-mail: jlvincen{at}ulb.ac.be).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Antonsson JB, Engstrom L, Rasmussen I, Wollert S, and Haglund UH. Changes in gut intramucosal pH and gut oxygen extraction ratio in a porcine model of peritonitis and hemorrhage. Crit Care Med 23: 1872-1881, 1995.[ISI][Medline]
  2. Bellomo R, Kellum JA, and Pinsky MR. Transvisceral lactate fluxes during early endotoxemia. Chest 110: 198-204, 1996.[Medline]
  3. Chavez AM, Menconi MJ, Hodin RA, and Fink MP. Cytokine-induced intestinal epithelial hyperpermeability: role of nitric oxide. Crit Care Med 27: 2246-2251, 1999.[ISI][Medline]
  4. Chiu CJ, McArdle AH, Brown R, Scott HJ, and Gurd FN. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic and metabolic reappraisal. Arch Surg 101: 478-483, 1970.[ISI][Medline]
  5. Creteur J, De Backer D, and Vincent JL. Monitoring of gastric mucosal PCO2 by gas tonometry: in vitro and in vivo validation studies. Anesthesiology 87: 504-510, 1997.[ISI][Medline]
  6. Crouser ED, Julian MW, and Dorinsky PM. Ileal O2-O2 alterations induced by endotoxin correlate with severity of mitochondrial injury. Am J Respir Crit Care Med 160: 1347-1353, 1999.[Abstract/Free Full Text]
  7. Crouser ED, Julian MW, Weinstein DM, Fahy RJ, and Bauer JA. Endotoxin-induced ileal mucosal injury and nitric oxide dysregulation are temporally dissociated. Am J Respir Crit Care Med 161: 1705-1712, 2000.[Abstract/Free Full Text]
  8. Crouser ED, Julian MW, Weisbrode SE, and Dorinsky PM. Ischemia/reperfusion injury to the ileum does not account for the ileal O2-O2 alterations induced by endotoxin. J Crit Care 12: 200-207, 1997.[ISI][Medline]
  9. De Backer D, Creteur J, Zhang H, Norrenberg M, and Vincent JL. Lactate production by the lungs in acute lung injury. Am J Respir Crit Care Med 156: 1099-1104, 1997.[Abstract/Free Full Text]
  10. Deitch EA, Berg R, and Specian R. Endotoxin promotes the translocation of bacteria from the gut. Arch Surg 122: 185-192, 1987.[Abstract]
  11. Fink M. Cytopathic hypoxia in sepsis. Acta Anaesthesiol Scand Suppl 110: 87-95, 1997.[Medline]
  12. Fink MP, Cohn SM, Lee PC, Rothschild HR, Deniz YF, Wang H, and Fiddian-Green RG. Effect of lipopolysaccharide on intestinal intramucosal hydrogen ion concentration in pigs: evidence of gut ischemia in a normodynamic model of septic shock. Crit Care Med 17: 641-646, 1989.[ISI][Medline]
  13. Fink MP, Kaups KL, Wang H, and Rothschild HR. Maintenance of superior mesenteric arterial perfusion prevents increased intestinal mucosal permeability in endotoxic pigs. Surgery 110: 154-161, 1991.[ISI][Medline]
  14. Forget AP, Mangalaboyi J, Mordon S, Guery B, Vallet B, Fourrier F, and Chopin C. Escherichia coli endotoxin reduces cytochrome aa3 redox status in pig skeletal muscle. Crit Care Med 28: 3491-3497, 2000.[ISI][Medline]
  15. Forsythe RM, Xu DZ, Lu Q, and Deitch EA. Lipopolysaccharide-induced enterocyte-derived nitric oxide induces intestinal monolayer permeability in an autocrine fashion. Shock 17: 180-184, 2002.[ISI][Medline]
  16. Gattinoni L, Brazzi L, Pelosi P, Latini R, Tognoni G, Pesenti A, and Fumagalli R. A trial of goal-oriented hemodynamic therapy in critically ill patients. N Engl J Med 333: 1025-1032, 1995.[Abstract/Free Full Text]
  17. Haji-Michael PG, Ladriere L, Sener A, Vincent JL, and Malaisse WJ. Leukocyte glycolysis and lactate output in animal sepsis and ex vivo human blood. Metabolism 48: 779-785, 1999.[ISI][Medline]
  18. Hasibeder W, Germann R, Wolf HJ, Haisjackl M, Hausdorfer H, Riedmann B, Bonatti J, Pfaller K, Gruber E, Schwarz B, Waldenberger P, and Furtner B. The effects of short-term endotoxemia and dopamine on mucosal oxygenation in the porcine jejunum. Am J Physiol Gastrointest Liver Physiol 270: G667-G675, 1996.[Abstract/Free Full Text]
  19. Hayes MA, Timmins AC, Yau EH, Palazzo M, Hinds CJ, and Watson D. Elevation of systemic oxygen delivery in the treatment of critically ill patients. N Engl J Med 330: 1717-1722, 1994.[Abstract/Free Full Text]
  20. Hersch M, Gnidec AA, Bersten AD, Troster M, Rutledge FS, and Sibbald WJ. Histologic and ultrastructural changes in nonpulmonary organs during early hyperdynamic sepsis. Surgery 107: 397-410, 1990.[ISI][Medline]
  21. Hotchkiss RS, Schmieg RE Jr, Swanson PE, Freeman BD, Tinsley KW, Cobb JP, Karl IE and Buchman TG. Rapid onset of intestinal epithelial and lymphocyte apoptotic cell death in patients with trauma and shock. Crit Care Med 28: 3207-3217, 2000.[ISI][Medline]
  22. Jagtap P, Soriano FG, Virag L, Liaudet L, Mabley J, Szabo E, Hasko G, Marton A, Lorigados CB, Gallyas F Jr, Sumegi B, Hoyt DG, Baloglu E, VanDuzer J, Salzman AL, Southan GJ and Szabo C. Novel phenanthridinone inhibitors of poly-(adenosine 5'-diphosphate-ribose) synthetase: potent cytoprotective and antishock agents. Crit Care Med 30: 1071-1082, 2002.[ISI][Medline]
  23. Kawana K, Miyamoto Y, Tanonaka K, Han-no Y, Yoshida H, Takahashi M, and Takeo S. Cytoprotective mechanism of heat shock protein 70 against hypoxia/reoxygenation injury. J Mol Cell Cardiol 32: 2229-2237, 2000.[ISI][Medline]
  24. Kruzel ML, Harari Y, Chen CY, and Castro GA. Lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice. Inflammation 24: 33-44, 2000.[ISI][Medline]
  25. Kvietys PR and Granger DN. Relationship between intestinal blood flow and oxygen uptake. Am J Physiol Gastrointest Liver Physiol 242: G202-G209, 1982.[Abstract/Free Full Text]
  26. Lobo SM, Salgado PF, Castillo VG, Borim AA, Polachini CA, Palchetti JC, Brienzi SL, and de Oliveira GG. Effects of maximizing oxygen delivery on morbidity and mortality in high-risk surgical patients. Crit Care Med 28: 3396-3404, 2000.[ISI][Medline]
  27. Marshall JC, Christou NV, Horn R, and Meakins JL. The microbiology of multiple organ failure. Arch Surg 123: 309-315, 1988.[Abstract]
  28. Menconi MJ, Unno N, Smith M, Aguirre DE, and Fink MP. Nitric oxide donor-induced hyperpermeability of cultured intestinal epithelial monolayers: role of superoxide radical, hydroxyl radical, and peroxynitrite. Biochim Biophys Acta 1425: 189-203, 1998.[Medline]
  29. Mercer DW, Smith GS, Cross JM, Russell DH, Chang L, and Cacioppo J. Effects of lipopolysaccharide on intestinal injury: potential role of nitric oxide and lipid peroxidation. J Surg Res 63: 185-192, 1996.[ISI][Medline]
  30. Neviere RR, Pitt-Hyde ML, Piper RD, Sibbald WJ, and Potter RF. Microvascular perfusion deficits are not a prerequisite for mucosal injury in septic rats. Am J Physiol Gastrointest Liver Physiol 276: G933-G940, 1999.[Abstract/Free Full Text]
  31. Petrucci O Jr, Didomizio GSAD, Gorios ST, Araujo S, Saraiva JFK, and Terzi RGG. Regulation of tissue oxygen extraction in septic and cardiogenic shock. Rev Bras Terap Intens 3: 185, 1991.
  32. Revelly JP, Ayuse T, Brienza N, Fessler HE, and Robotham JL. Endotoxic shock alters distribution of blood flow within the intestinal wall. Crit Care Med 24: 1345-1351, 1996.[ISI][Medline]
  33. Revelly JP, Liaudet L, Frascarolo P, Joseph JM, Martinet O, and Markert M. Effects of norepinephrine on the distribution of intestinal blood flow and tissue adenosine triphosphate content in endotoxic shock. Crit Care Med 28: 2500-2506, 2000.[ISI][Medline]
  34. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, Peterson E, and Tomlanovich M. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 345: 1368-1377, 2001.[Abstract/Free Full Text]
  35. Salvemini D, Riley DP, Lennon PJ, Wang ZQ, Currie MG, MacArthur H, and Misko TP. Protective effects of a superoxide dismutase mimetic and peroxynitrite decomposition catalysts in endotoxin-induced intestinal damage. Br J Pharmacol 127: 685-692, 1999.[ISI][Medline]
  36. Salzman AL, Menconi MJ, Unno N, Ezzel RM, Casey DM, Gonzalez PK, and Fink MP. Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BB intestinal epithelial monolayers. Am J Physiol Gastrointest Liver Physiol 268: G361-G373, 1995.[Abstract/Free Full Text]
  37. Salzman AL, Wang H, Wollert PS, VanderMeer TJ, Compton CC, Denenberg AG, and Fink MP. Endotoxin-induced ileal mucosal hyperpermeability in pigs: role of tissue acidosis. Am J Physiol Gastrointest Liver Physiol 266: G633-G646, 1994.[Abstract/Free Full Text]
  38. Shoemaker WC, Appel PL, Kram HB, Waxman K, and Lee TS. Prospective trial of supranormal values of survivors as therapeutic goals in high-risk surgical patients. Chest 94: 1176-1186, 1988.[ISI][Medline]
  39. Snygg J, Fandriks L, Bengtsson J, Holm M, Pettersson A, and Aneman A. Jejunal luminal nitric oxide during severe hypovolemia and sepsis in anesthetized pigs. Intensive Care Med 27: 1807-1813, 2001.[ISI][Medline]
  40. Tenhunen JJ, Kosunen H, Alhava E, Tuomisto L, and Takala JA. Intestinal luminal microdialysis: a new approach to assess gut mucosal ischemia. Anesthesiology 91: 1807-1815, 1999.[ISI][Medline]
  41. Theisen M, Trager K, Tugtekin I, Stehr A, Ploner F, Georgieff M, Radermacher P, and Matejovic M. Effects of nicotinamide, an inhibitor of PARS activity, on gut and liver O2 exchange and energy metabolism during hyperdynamic porcine endotoxemia. Intensive Care Med 27: 586-592, 2001.[ISI][Medline]
  42. Unno N, Menconi MJ, Salzman AL, Smith M, Hagen S, Ge Y, Ezzell RM, and Fink MP. Hyperpermeability and ATP depletion induced by chronic hypoxia or glycolytic inhibition in Caco-2BBe monolayers. Am J Physiol Gastrointest Liver Physiol 270: G1010-G1021, 1996.[Abstract/Free Full Text]
  43. Unno N, Wang H, Menconi MJ, Tytgat SH, Larkin V, Smith M, Morin MJ, Chavez A, Hodin RA, and Fink MP. Inhibition of inducible nitric oxide synthase ameliorates endotoxin-induced gut mucosal barrier dysfunction in rats. Gastroenterology 113: 1246-1257, 1997.[ISI][Medline]
  44. Vallet B, Lund N, Curtis SE, Kelly D, and Cain SM. Gut and muscle tissue PO2 in endotoxemic dogs during shock and resuscitation. J Appl Physiol 76: 793-800, 1994.[Abstract/Free Full Text]
  45. VanderMeer TJ, Wang H, and Fink MP. Endotoxemia causes ileal mucosal acidosis in the absence of mucosal hypoxia in a normodynamic porcine model of septic shock. Crit Care Med 23: 1217-1226, 1995.[ISI][Medline]
  46. Wang P, Zhou M, Rana MW, Ba ZF, and Chaudry IH. Differential alterations in microvascular perfusion in various organs during early and late sepsis. Am J Physiol Gastrointest Liver Physiol 263: G38-G43, 1992.[Abstract/Free Full Text]
  47. Wilson J, Woods I, Fawcett J, Whall R, Dibb W, Morris C, and McManus E. Reducing the risk of major elective surgery: randomised controlled trial of preoperative optimisation of oxygen delivery. Br Med J 318: 1099-1103, 1999.[Abstract/Free Full Text]
  48. Xu D, Qi L, Guillory D, Cruz N, Berg R, and Deitch EA. Mechanisms of endotoxin-induced intestinal injury in a hyperdynamic model of sepsis. J Trauma 34: 676-682, 1993.[ISI][Medline]
  49. Yu P and Martin CM. Increased gut permeability and bacterial translocation in Pseudomonas pneumonia-induced sepsis. Crit Care Med 28: 2573-2577, 2000.[ISI][Medline]
  50. Zhang H, Smail N, Cabral A, Cherkaoui S, Peny MO, and Vincent JL. Hepato-splanchnic blood flow and oxygen extraction capabilities during experimental tamponade: effects of endotoxin. J Surg Res 81: 129-138, 1999.[ISI][Medline]
  51. Zingarelli B, Day BJ, Crapo JD, Salzman AL, and Szabo C. The potential role of peroxynitrite in the vascular contractile and cellular energetic failure in endotoxic shock. Br J Pharmacol 120: 259-267, 1997.[ISI][Medline]



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