The role of gut injury in shock-induced systemic inflammation and multiple organ dysfunction syndrome (MODS) has been known for several decades (1, 2). Although gut-origin sepsis was originally ascribed to bacterial translocation (1), recent studies from our and other laboratories have shown that gut-induced systemic inflammation and MODS are due to nonmicrobial factors released from the injured postshock gut that reach the systemic circulation via the intestinal lymphatics (3). These studies have led to the gut lymph hypothesis of MODS (3). However, the role of mesenteric lymph as a contributory factor to SIRS and MODS has only been studied in nonlethal model of gut I/R. Thus, it is not known whether factors released from the gut into the intestinal lymphatics contribute to mortality in lethal gut I/R models. Consequently, we investigated the role of mesenteric lymph in the pathogenesis of lethal shock using the splanchnic artery occlusion (SAO) gut I/R model. The SAO-induced shock model was used because it is a severe form of shock that is characterized by profound circulatory failure and a high mortality (4). If the notion that factors from the ischemic gut carried in the mesenteric lymph contribute to circulatory failure and shock is true, then preventing these mediators from entering the systemic circulation by ligating the mesenteric lymph duct should limit the degree of shock and improve survival. In addition to testing this hypothesis, we also investigated the effect of mesenteric lymph duct ligation (LDL) on the induction of an augmented NO response because the NO pathway seems to play an important role in SAO-associated vascular dysfunction (5, 6).
MATERIALS AND METHODS
Adult male specific pathogen-free Sprague-Dawley rats weighing 325 to 450 g were used in all the experiments. Animals underwent a 7-day acclimatization period with 12-h light/dark cycles under barrier-sustained conditions, during which time they had free access to water and laboratory chow (Teklad 22/5 Rodent diet [W] 8640; Harlan Teklad, Madison, Wis). The rats were maintained in accordance with the guidelines of the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, and the experiments were approved by the New Jersey Medical School Animal Care and Use Committee. Animals from three different commercial vendors were used (Taconic Farms, Germantown, NY; Charles River Laboratories, Wilmington, Mass; and Harlan Teklad) in these experiments.
The basic hypothesis underlying this study is that gut-derived factors in the mesenteric lymph play a major role in the mortality observed in the SAO shock model. To test this hypothesis, the efficacy of LDL in preventing SAO-induced circulatory failure and death was tested. In this experiment, the SAO rats (Taconic Farms) had their celiac artery and superior mesenteric artery (SMA) temporarily occluded for 20 min, after which the gut was reperfused. This 20-min SAO period was based on pilot studies showing that this length of SAO was associated with a high mortality rate (data not shown). In the SAO-LDL group, the mesenteric lymph duct was ligated immediately before SAO. A control sham-SAO group was included that underwent all the same surgical manipulations as the actual SAO animals except that after the celiac and mesenteric arteries were exposed, they were not occluded. Blood pressure was continuously monitored via the femoral artery catheter until the end of the third hour of gut reperfusion, after which the animals were decannulated and placed back in their cages and observed for 24-h survival. Animals had free access to water and chow during the observation period.
Because the NO pathway has been shown to be involved in SAO-induced shock (5, 6), in a second set of studies, separate groups of animals were killed at 3 h after gut reperfusion, and blood samples (0.5 mL) were obtained for plasma nitrate/nitrite measurements, livers were harvested for iNOS protein measurements, and ileal tissues were processed for morphologic assessment of gut injury. Both of these two sets of studies were performed using Sprague-Dawley rats from Taconic Farms.
Subsequent longer-term 7-day SAO survival studies were carried out with male Sprague-Dawley rats purchased from the following three animal vendors; 1) Taconic Farms, 2) Charles River Laboratories, and 3) Harlan Teklad.
Rats were anesthetized with pentobarbital (50 mg/kg, i.p.), after which they underwent femoral artery cannulation. A midline laparotomy was performed, and the gut was eviscerated and rotated left laterally. The celiac artery and SMA were isolated near their aortic origins. Splanchnic artery occlusion was induced by temporarily occluding both celiac artery and SMA for 20 (24-h survival and NO experiments) or 30 (7-day survival studies) min using 0-silk ligatures. In animals that underwent LDL, the lymph duct was carefully isolated from the SMA and tied separately using a 3-0 silk ligature before SAO. During the procedure, the intestines were kept warm by placing them between gauze pads soaked with warmed 0.9% NaCl solution. After the occlusion period, the gut was reperfused by removing the ligatures around the celiac and SMA. The abdomen was then closed in two layers. Sham-SAO rats underwent the same procedure except that the arteries were not occluded. Heating pads or lamps were used as necessary to maintain the animal's core temperature greater than 36.3°C.
Plasma nitrite/nitrate assay
Blood samples (0.5 mL) were collected in heparinized syringes from the animals at the end of the third hour of gut reperfusion. Plasma nitrite and nitrate (NO2 −/NO3 −) measurements were performed spectrophotometrically using the Griess reagent as previously described (7).
The mucosal structure of the terminal ileum harvested at 3 h after gut reperfusion was analyzed by light microscopy in a blinded fashion. After harvesting, the ileal samples were fixed by luminal perfusion and subsequent immersion in 10% buffered formalin overnight at room temperature. The tissue was then dehydrated and embedded in paraffin. Five random fields (magnification ×100) were evaluated per animal. The incidence of ileal villous damage was determined by dividing the number of injured villi by the total number of villi examined.
Western blot analysis
Whole cell extracts were prepared by homogenizing liver tissue samples in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCL, pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol, 0.5 mM 4,2-aminoethyl-benzenesulfonylfluoride, 1 mM NaVO3, and 1 µg/mL of aprotinin, leupeptin, and pepstatin). After 30 min in ice, the samples were centrifuged, and the supernatant was collected. Protein concentrations were determined using Bradford protein assay reagent (BioRad, Hercules, Calif). On a 3% to 8% Tris-acetate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, Calif), 50 µg of protein was separated, transferred to nitrocellulose membrane, and blocked with 5% nonfat dried milk in TBS-T (50 mM Tris-HCL, pH 7.5, 140 mM NaCl, 0.1% Tween) for 1 h. The membrane was incubated with iNOS or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Assays Designs, Ann Arbor, Mich) at a 1:1,000 dilution in blocking buffer overnight at 4°C. After washing several times with TBS-T, the membrane was incubated for 1 h with horseradish peroxidase-conjugated antirabbit secondary antibody (1:2,000; Santa Cruz Biotechnology, Santa Cruz, Calif) and developed with enhanced chemiluminescence reagents (Amersham, Arlington Heights, Ill). Densitometry was performed using the AlphaImager 3400 imaging system and AlphaEase FC software (Alpha Innotech, San Leandro, Calif). Densitometric values for iNOS expression were expressed relative to GAPDH loading control.
ANOVA with the post hoc Tukey-Kramer multiple comparison test was used to analyze the differences between groups. Results are expressed as mean± SD. P values less than 0.05 were considered statistically significant.
In the first set of experiments, the ability of LDL to affect survival in rats subjected to 20 min of SAO was tested during the summer months (July to September). All of the control rats subjected to either sham-SAO or sham-SAO-LDL survived the 24-h observation period. In contrast, none of the Taconic rats subjected to 20 min of SAO survived past 6h, and 40% of the rats had died within 3 h of gut reperfusion, with the mean survival time of the SAO rats being 3.75 h. In contrast, none of the SAO-LDL rats had died at 6 h, and 60% of the SAO-LDL survived 24 h, at which point the surviving rats were killed (Fig. 1A). The increase in survival in the lymph duct-ligated-SAO rats was associated with a better preservation of their MAP (Fig. 1B). This blunting of the SAO-induced hypotensive response in the LDL group was apparent both immediately after gut reperfusion (0 time period) and during the 180-min observation period. None of the sham-SAO rats died, and their MAP remained stable throughout the 3-h observation period (data not shown). Thus, LDL both abrogated the magnitude of SAO-induced shock and increased survival.
Because increased iNOS-induced NO levels have been implicated in SAO-induced shock (5, 6), we measured plasma nitrite/nitrate and hepatic iNOS protein levels in rats subjected to 20 min of SAO (studies carried out in September). As expected, plasma nitrite/nitrate levels were significantly increased in the rats 3 h after 20 min of SAO, and this increase in plasma nitrite/nitrate was significantly abrogated in the SAO rats undergoing LDL (Fig. 2A). In fact, the plasma nitrate/nitrite levels in the SAO group were 3-fold higher than those of the SAO + LDL group. Consistent with the decrease in plasma nitrite/nitrate levels, LDL also abrogated SAO-induced hepatic iNOS protein induction (Fig. 2B). Assessment of intestinal injury using gut histology showed that both SAO and SAO + LDL animals had an equal magnitude of villous injury (Fig. 2C). This indicates that the protection offered by lymph duct ligation was not due to a reduction in gut injury and is consistent with the hypothesis that factors exiting the gut via the mesenteric lymph contribute to SAO-induced shock and mortality as well as an augmented NO response.
After completing these studies of rats subjected to 20 min of SAO in the summer, we initiated a long-term 7-day survival study in the winter (December to March). In doing this study, we found that none of the rats subjected to SAO for 20 min were dead at 24 h, nor were a second group subjected to 30 min of SAO. During this period, our University had changed animal vendors from Taconic Farms to Harlan Laboratories. Because our University uses three different animal vendors, we tested whether male Sprague-Dawley rats obtained from different animal vendors would respond differently to SAO before assessing the effect of LDL on long-term survival. In this experiment, performed in April, rats obtained from Taconic Farms, Charles River Laboratories, and Harlan Teklad were subjected to 30 min of SAO. To limit other confounding factors, all animals were received at the same time, from the same litters, housed in the same animal rooms, and the experiments were performed during the same time of the day. As shown in Figure 3A, the Harlan rats were relatively resistant to SAO-induced mortality, with just one of nine rats dying within 24 h and 67% of the rats surviving 7 days. In contrast, both the Taconic and Charles River rats had significantly higher 24-h and 7-day mortality rates after SAO than the Harlan rats, indicating that the vendor source of the rats was an important and underappreciated variable (Fig. 3A). Based on these results, in May, using two additional groups of rats, we next tested the ability of LDL to improve long-term survival in Taconic rats subjected to 30 min of SAO. Consistent with our initial set of studies using Taconic rats subjected to 20 min of SAO, the LDL Taconic rats subjected to 30 min of SAO had improved short- and long-term survival, indicating that the protective effect of LDL on SAO-induced shock is not transient and results in long-term survivors (Fig. 3B).
Irreversible shock is an important contributor to mortality in multiply transfused trauma patients and occurs in up to 36% of patients who die after major trauma (8, 9). For this reason, and because therapy is limited, a number of studies have been performed to clarify the mechanisms and mediators involved in the development of irreversible shock. Although much remains to be learned, a key concept in irreversible shock is that the normal vasoconstrictor response is lost, and that a vasodilated state develops where the vasculature smooth muscle does not adequately respond to normal endogenous vasoconstrictors (10). Several factors have been implicated in this functional failure of the vasculature to respond adequately to vasoconstrictive stimuli, which include increased concentrations of vasodilators, especially NO (5, 6, 11), increased production of proinflammatory factors such as TNF (12, 13), and a relative deficiency of the vasoconstrictor vasopressin (10, 14). However, despite the advances made in clarifying the cellular and molecular mechanisms involved in the pathogenesis of irreversible shock, little is known concerning the source or nature of the factors that initiate the process of irreversible shock. Because irreversible shock is associated with a severe and prolonged gut ischemic insult (1), we investigated the hypothesis that gut factors released into the systemic circulation via the lymphatic, rather than the portal venous pathway, is involved in transducing a reversible to an irreversible shock state. We chose to use the SAO model as a proof-of -principle system to test this hypothesis, rather than hemorrhagic shock, because the SAO model avoids the potentially confounding systemic factors associated with hemorrhagic shock. Additionally, the cellular mechanisms involved in the transition from a reversible to an irreversible shock state seem to be similar between SAO and hemorrhagic shock (5, 6, 10-14). In fact, although septic shock is more physiologically complex than hemorrhagic or SAO-induced shock, the key cellular and molecular events in septic shock seem to be similar to irreversible SAO and hemorrhagic shock in that the smooth muscle vasoconstrictive failure observed during septic shock also seems to involve NO, cytokines, vasopressin, K channels, and the cytoskeleton (15). Thus, we believe that insights gained from the SAO-shock studies might also apply, at least in part, to other irreversible shock states.
The key observation from the current study is that LDL abrogates the SAO-induced shock state and increases long-term survival. Thus, this study shows for the first time that factors released from the ischemic gut into the lymphatic system, but not the portal venous blood, are involved in initiating an irreversible shock state. An important corollary of this observation is that studies investigating only the portal venous blood are unlikely to identify the key mediators that are involved in converting gut-associated reversible to irreversible shock. This notion that gut-derived factors can lead to an irreversible shock state was shown as early as 1957 by Lillehei (16) in dogs subjected to hemorrhagic shock. More recently, Chang (17) documented that enterectomy improved survival in an irreversible rodent hemorrhagic shock model. Although the exact factors in mesenteric lymph that led to the SAO-induced irreversible shock state are not known, we did observe that LDL abrogated both plasma nitrite/nitrate levels and the induction of hepatic iNOS protein levels. Based on this observation and the fact that NO has been implicated as an important factor in the pathogenesis of SAO-induced shock (5) as well as hemorrhagic (11) and septic (15) shock, it seems that the pathogenesis of SAO-induced irreversible shock by mesenteric lymph occurs at least in part via an iNOS-dependent mechanism. This notion is supported by work showing that iNOS inhibition significantly improves survival in rats subjected to an otherwise lethal SAO insult (6, 18) and is consistent with recent work from our laboratory showing that the injection of trauma-hemorrhagic shock (T/HS) mesenteric lymph into naive rats leads to lung injury via an iNOS-dependent mechanism (19) and studies showing that plasma nitrate/nitrite levels correlate with T/HS-induced lung injury.
Others have shown that iNOS and NO are involved in SAO-induced shock and mortality (5, 6, 18). However, because inhibition of iNOS protects the gut from I/R insults (7, 20), it was unclear whether the protective effect of inhibiting iNOS was due to its ability to limit gut injury or the systemic consequences of gut injury. The current study, by showing that gut injury after SAO was not limited by lymph duct ligation, whereas survival was improved, allowed these two variables to be separated. Additionally, based on recent studies, it seems likely that another splanchnic organ, the pancreas, and the small intestine may be involved in the production of the shock-inducing factors carried in the mesenteric lymph. This notion is based on experimental studies showing that both the small intestine and the pancreas can be sources of factors leading to myocardial depression and refractory vasodilatory shock states in a number of conditions, including hemorrhage, sepsis, and pancreatitis (21). In this regard, work from the laboratory of Schmid-Schonbein (22, 23) has documented an important role for intraluminal pancreatic proteases acting on the ischemic small intestine in the pathogenesis of systemic leukocyte activation, shock, and MODS in rodent gut I/R models. In these studies, they found that intraluminal neutralization of pancreatic enzymes with serine protease inhibitors improves survival after SAO-induced shock (24). Since then, our studies in a T/HS model with a mortality of approximately 25% showed that pharmacologic inhibition of pancreatic protease activity and pancreatic duct ligation abrogates gut injury, the production of biologically active mesenteric lymph, and lung injury and neutrophil activation (25-27). Thus, although the exact source of the factors present in the mesenteric lymph from the SAO rats are not known, it is likely that their generation involves the activation of pancreatic proteases, with the lumen of the gut acting on the injured intestine.
An unexpected major observation made while performing these studies was that rats tested from different vendors responded differently to SAO. This observation of vendor variability was somewhat surprising, although the fact that environmental variables as well as the sex and age of the animals can modulate the response of an animal to an insult or stimulus is well known (28-30). Common examples of environmental factors that have been shown to potentially influence experimental results include the conditions under which the animals are housed, acclimatizing cycle, preoperative management, access to food and age (28), and the time of the day the studies are performed (31). The physiologic explanation for the confounding effects of these variables such as the effect of the circadian rhythm on experimental results seems to be related to the fact that certain hormonal levels and immune responses vary with the circadian rhythm (32). In this context, our observation that the susceptibility of the rats to SAO-induced mortality varied based on the time of the year, with the greatest susceptibility to death being seen in the summer, followed by spring and then winter, is consistent with studies showing that the response to experimental infectious challenges and the susceptibility to cancer metastasis can be influenced by the seasons (33-35). Just as with the circadian rhythm, there are emerging data indicating that the immunoinflammatory and hormonal responses vary in normal animals, with an increased immune response in the autumn and winter (36). This seasonal effect on the response to an insult seems to help explain why the animals tested in the spring required a greater insult and had a longer length of survival than animals tested in the summer (i.e. 30 min of SAO vs. 20 min of SAO) to create a lethal model and 24-h survival of 0% vs. 40% to 50%. However, it does not explain the fact that Sprague-Dawley male rats received from one vendor were resistant to SAO-induced mortality, whereas rats of the same strain from two other vendors were susceptible. Although this vendor-related difference in susceptibility to SAO-induced mortality cannot be explained, this observation plus the seasonal effect on outcomes may help explain, at least in part, divergent results from different groups under what seems to be equivalent experimental conditions.
One limitation of this study is that we do not know what the factors are in the mesenteric lymph from the SAO animals that contributed to SAO-induced hypotension, mortality, or iNOS induction. However, we do know from our previous studies in T/HS and burn models, in which gut lymph leads to acute injuries of the lungs and other organs as well as activation of neutrophils, that these effects do not seem to be related to endotoxin or bacterial products in the lymph because these specimens were sterile and did not contain measurable levels of endotoxin (37). Likewise, cytokines within the mesenteric lymph do not seem to be mediating lymph's tissue-injurious or proinflammatory activities (38). However, recently, T/HS lymph has been shown to contain several incompletely identified biologically active protein and lipid factors (37, 39, 40), including a modified albumin species (40). Consequently, it is likely that certain lymph-induced organ and cellular responses may be mediated by different factors acting through different mechanisms. On the other hand, because these lymph studies were performed in models with a modest mortality rate, the degree to which they apply to the highly lethal SAO model remains to be determined. A second limitation of this study is that the changes in iNOS in the rats dying with SAO-induced shock are only associative and should not be overinterpreted. This is especially true in light of the complex balance between the beneficial and deleterious roles played by NO in other forms of shock (41) and the failure of clinical trials of NO inhibition to improve survival in patients with septic (42) or cardiogenic shock (43).
In summary, to our knowledge, this is the first study to show that gut-derived factors carried in the mesenteric lymph are involved in converting a reversible to an irreversible shock state and thus provide proof-of-principle that factors in mesenteric lymph can cause lethality.
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