IT IS WELL KNOWN that the development. Gut-Derived Mesenteric Lymph. A Link Between Burn and Lung Injury PAPER

Gut-Derived Mesenteric Lymph A Link Between Burn and Lung Injury PAPER Louis J. Magnotti, MD; Da-Zhong Xu, MD; Qi Lu, MD; Edwin A. Deitch, MD Background: Previously, we showed that mesenteric lymph generated following hemorrhagic shock increases endothelial cell permeability and contributes to lung injury. It has also been shown that lymph produced at the site of burn injury plays a role in altering pulmonary vascular hemodynamics. In addition, previous experimental work has suggested that organs and tissues distant from the injury site may contribute to pulmonary dysfunction. One explanation would be that gutderived inflammatory factors (in addition to those produced locally at the site of injury) are reaching the pulmonary circulation, where they exert their effects via the gut lymphatics. Hypotheses: The 2 hypotheses herein were that (1) gutderived factors carried in the mesenteric lymph of rats generated following thermal injury will contribute to lung injury and (2) intestinal bacterial overgrowth will potentiate the degree of burn-induced lung injury. These hypotheses were tested by examining the effect of mesenteric lymph flow interruption prior to thermal injury on burn-induced lung injury in rats with a normal intestinal bacterial flora and in rats with intestinal Escherichia coli overgrowth. These rats were termed E coli monoassociated rats. Methods: Normal intestinal bacterial flora and monoassociated male Sprague-Dawley rats were subjected to sham burn, 40% total body surface area burn, or lymphatic division plus burn. After 3 hours, 10 mg of Evans blue was injected to measure lung permeability. After the rats were killed, a bronchoalveolar lavage was performed and the fluid analyzed spectrophotometrically. Bronchoalveolar lavage fluid protein content, pulmonary myeloperoxidase activity, and alveolar apoptosis served to further quantitate lung injury. Results: Both normal intestinal bacterial flora and monoassociated-burned rats exhibited significant increases in lung permeability, bronchoalveolar lavage fluid protein content, myeloperoxidase activity, and alveolar apoptosis. The combination of monoassociation and thermal injury resulted in even further increases in lung injury over thermal injury alone. Lymphatic division prior to thermal injury ameliorated burn-induced increases in lung permeability, bronchoalveolar lavage fluid protein content, pulmonary myeloperoxidase accumulation, and alveolar apoptosis in both normal intestinal bacterial flora and monoassociated rats. Conclusions: The results of this study support the hypothesis that gut-derived factors carried in the mesenteric lymph contribute to burn-induced lung injury and may therefore play a role in postburn respiratory failure and suggest that intestinal bacterial overgrowth primes the host such that when animals are exposed to a second stimulus (such as thermal injury) an exaggerated response occurs. Arch Surg. 1999;134:1333-1341 From the Department of Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark. IT IS WELL KNOWN that the development of adult respiratory distress syndrome complicates the recovery of patients with severe thermal injury. 1 Acute lung injury (lung leak) has also been observed in rats subjected to thermal injury, and thermal injury is associated with a variety of responses that may contribute to lung leak. 2,3 For example, damage to the pulmonary vascular endothelium following acute thermal injury has been shown to produce interstitial edema in experimental animals 4,5 and severely burned humans. 6,7 Although the relationship between thermal injury and pulmonary dysfunction has been recognized for many years, the pathogenesis of microvascular damage to the lung following thermal injury, as well as the burninduced local microvascular changes, are not well understood. However, since there is a high incidence of respiratory failure (adult respiratory distress syndrome) in patients with large surface area burns in the absence of direct pulmonary injury, 8,9 it appears that factors produced outside of the lung itself contribute to pulmonary dysfunction. 1333

MATERIALS AND METHODS ANIMALS Specific pathogen-free male Sprague-Dawley rats (Taconic Farms Inc, Germantown, NY) weighing between 250 and 300 g were housed under barrier-sustained conditions at a temperature of 25 C with 12-hour light/dark cycles. The rats had free access to water and rat chow (Harlan Teklad Laboratory Chow, Teklad 22/5 Rodent Diet [W] 8640; Harlan Teklad, Madison, Wis). All rats were maintained in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the experiments were approved by the New Jersey Medical School Animal Care Committee, Newark. EXPERIMENTAL DESIGN The first set of experiments tested whether the division of the mesenteric lymphatics prior to a thermal injury would prevent or reduce burn-induced pulmonary injury in rats with normal intestinal bacterial flora. Rats (N = 32, 8 rats per group), anesthetized with 25 mg/kg of pentobarbital sodium and 0.3 mg/kg of buprenorphine hydrochloride given intraperitoneally, were placed into 1 of the following 4 groups: (1) control (no celiotomy), (2) sham burn (celiotomy), (3) burn (40% total body surface area [TBSA]), or (4) lymphatic division immediately prior to thermal injury. Following a 3-hour recovery period, all animals were injected intravenously with 10 mg of Evans blue (EB) to examine lung permeability. Twenty minutes later, the rats were killed, the lungs were harvested, a bronchoalveolar lavage was performed, and the fluid analyzed spectrophotometrically. The lungs were then fixed in formalin, processed, and embedded in paraffin. Sections (4 µm) were cut and stained for apoptosis using an in situ apoptosis detection kit. Representative samples from each group were then examined for alveolar cell apoptosis by counting the number of apoptotic nuclei in 20 high-power fields. Levels of pulmonary myeloperoxidase (MPO) activity were used to quantitate neutrophil sequestration in the lung. The second group of experiments was performed to assess the extent, if any, to which intestinal bacteria would contribute to burn-induced lung injury. In these experiments, monoassociated rats (n = 6 per group) were used in place of the normal intestinal bacterial flora rats. All procedures were identical for the 2 groups with the exception of the process of monoassociation. In addition, the mesenteric lymph nodes (MLNs), liver, spleen, and lungs from the monoassociated rats were excised, weighed, homogenized, and examined for bacterial translocation. ESCHERICHIA COLI C25 MONOASSOCIATION Streptomycin sulfate (4 mg/ml) and benzyl penicillin sodium (2 mg/ml) were administered to the rats in their drinking water ad libitum for 4 days. This antibiotic regimen reduces the total aerobic cecal bacterial population levels from 10 6 to less than 10 2 colony-forming units per gram of cecum. 18,19 The antibiotic-decontaminated rats had 5 ml of E coli C25 (a streptomycin-resistant strain) added to each 250 ml of drinking water for 3 days during which time they continued to receive streptomycin sulfate (4 mg/ml). BURN MODEL The procedures used to burn the rats were adapted from those described by Walker and Mason. 20 The rats were anesthetized withpentobarbitalsodium(25mg/kg) andbuprenorphinehydrochloride (0.3 mg/kg) given intraperitoneally. The hair was shavedfromthebackandabdomen, andbothareasweretreated with a depilatory agent. The rat was placed in a plastic holder containing an opening that exposed 20% of the body surface area. 20 The back burn was produced by immersing the back of the animal through the template into boiling water (100 C) for 10 seconds; the abdominal burn was a 5-second immersion scald 19 to produce a third-degree burn. All rats received a 40% TBSA burn and were resuscitated with 5 ml of sterile isotonic sodium chloride solution injected intraperitoneally to prevent shock. Rectal temperature was monitored periodicallythroughouttherecoveryperiodandmaintainedataround 37 C. A heat lamp positioned over the animal was used to prevent hypothermia. The control (no celiotomy) and sham burn (celiotomy) groupswereanesthetized, placedintheplastictemplate, and exposed to room temperature water. DIVISION OF MESENTERIC LYMPHATIC VESSELS The efferent mesenteric lymphatic vessel was identified and divided as described previously 16 and the animal subjected to sham or actual burn. For this reason, it has been hypothesized that inflammatory mediators generated at the site of burn injury play a role in the development of adult respiratory distress syndrome. 10 This concept is supported by clinical and experimental data showing that the fluid generated locally at the site of burn injuries is biologically active and markedly depresses cardiac contractility 11 and cell-mediated immunity, 12-14 as well as altering pulmonary vascular hemodynamics. 15 Thus, acute lung injury following thermal injury may result from an overwhelming systemic inflammatory response driven by the release, activation, and interaction of endogenous immunoregulatory factors, including cytokines. 6 Nevertheless, even though lymph collected locally from the site of a burn can produce distant organ injury and/or dysfunction, this does not exclude other organs distant from the site of initial injury, such as the gut, from contributing to this pulmonary failure. Previously, we have shown that mesenteric lymph generated following hemorrhagic shock contributes to lung injury by increasing lung permeability, pulmonary neutrophil sequestration, and alveolar apoptosis. 16,17 Since thermal injury is associated with intestinal ischemia, it is possible that a major thermal injury can induce the intestine and its gut-associated lymphoid tissue to produce and release immunoinflammatory factors that reach the lung and systemic circulation via the intestinal lymphatics in a manner similar to what is seen following hemorrhagic shock. Since the lung represents the first vascular bed exposed to mesenteric lymph, we tested the hypothesis that mesenteric lymph generated after thermal injury will contribute to lung injury and that bacterial overgrowth will further increase the degree of burn-induced lung injury. 1334

LUNG PERMEABILITY Following a 3-hour recovery period, the rats were reanesthetized with pentobarbital sodium (25 mg/kg), and the femoral artery was isolated by aseptic techniques and cannulated with polyethylene-50 tubing (0.023 in [inner diameter] 0.038 in [outer diameter]) containing 0.1 ml of 10 U/mL of heparinized saline. 21 Next, aseptic cannulation of the internal jugular vein was performed using a 50-gauge silicone catheter. At this point, all animals were injected with 10 mg of EB via the internal jugular catheter. After 5 minutes (allowing for complete circulation of the EB), a blood sample (1.5 ml) was withdrawn from the femoral artery catheter. This blood sample was centrifuged at 1500 rpm at 4 C for 20 minutes, and the resultant plasma was serially diluted to form a standard curve. Twenty minutes after injection of the EB, all rats were killed and the lungs harvested. Bronchoalveolar lavage was performed on the excised lungs using isotonic sodium chloride solution. Five milliliters of isotonic sodium chloride solution was instilled, rinsed in and out 3 times, and collected. The lavage was repeated 3 times. The combined recovered bronchoalveolar lavage fluid (BALF) was consistently 12.5 ml or more. The BALF was then centrifuged at 1500 rpm at 4 C for 20 minutes to remove any cells, and the supernatant fluid was assayed spectrophotometrically at 620 nm for dye concentration. 22 The concentration of EB in the BALF was then plotted on the standard curve, and the percentage relative to that present in the plasma was determined. QUANTITATION OF ALVEOLAR APOPTOSIS Alveolar apoptosis was quantitated as previously described. 16 The morphologic characteristics of the apoptotic cells included chromatin condensation, separation of the cell from surrounding cells, cell shrinkage, and the formation of apoptotic bodies. 23 MPO ASSAY The previously frozen lung tissue was homogenized for 30 seconds in 4 ml of 20-mmol/L potassium phosphate buffer, ph 7.4, and centrifuged for 30 minutes at 40 000g at 4 C. The pellet was resuspended in 4 ml of 50-mmol/L potassium phosphate buffer, ph 6.0, containing 0.5 g/dl of hexadecyltrimethyl ammonium bromide. Samples were sonicated for 90 seconds at full power, incubated in a 60 C water bath for 2 hours, and centrifuged. The supernatant (0.1 ml) was added to 2.9 ml of 50-mmol/L potassium phosphate buffer, ph 6.0, containing 0.167 mg/ml o-dianisidine and 0.0005% hydrogen peroxide. Absorbance of 460 nm of visible light (A 460 ) was measured for 3 minutes. The MPO activity per gram of lung tissue was calculated as follows: MPO activity (units per gram of tissue) = ( A 460 13.5)/lung weight (grams), where A 460 = rate of change in absorbance at 460 nm between 1 and 3 minutes. The coefficient 13.5 was empirically determined such that 1 U of MPO activity is the amount of enzyme that will reduce 1-µmol/L peroxide per minute. 24 BALF PROTEIN ASSAY The total protein content (grams per deciliter) of the BALF (for all 4 groups) was determined using a hand refractometer (Milton Roy Co, Rochester, NY). TESTING FOR BACTERIAL TRANSLOCATION Following determination of lung permeability, the MLNs, liver, and spleen were excised, weighed, and homogenized. Organ homogenates were quantitatively cultured on blood and MacConkey agar plates. The cecum was also excised, treated in the same way as the other tissues, and serial dilutions were plated onto blood and MacConkey agar plates for culturing total aerobic bacteria and gramnegative enteric bacteria, respectively. The plates were then examined after 24 hours of aerobic incubation at 37 C. 18,19 STATISTICAL ANALYSIS All data were analyzed by analysis of variance with the Tukey-Kramer multiple comparisons procedures unless otherwise specified. All data are expressed as the mean ± SD. Statistical significance was considered to be reached when P.05. RESULTS Lung permeability was examined using EB as a permeability probe and was found to be greatest in those rats subjected to 40% TBSA burn. This was true of both rats with a normal gut bacterial flora and those that had been monoassociated with E coli prior to thermal injury (Table 1 and Table 2). Thermal injury resulted in a nearly 3-fold increase in lung permeability over nonburned animals. Division of the mesenteric lymphatics prior to thermal injury reduced this observed increase in pulmonary permeability to about half of that observed in the burned animals. However, there was still a small but significant difference in pulmonary permeability between the burned rats whose lymphatics were divided prior to thermal injury and the nonburned animals (both with and without celiotomy). The changes in pulmonary permeability were consistent with another sign of lung permeability, BALF protein content. Thermal injury produced a significant increase in the total protein content of the BALF over all groups (Tables 1 and 2). Division of the mesenteric lymphatics prior to thermal injury reduced the BALF protein content but did not fully prevent the burn-induced increase in BALF protein content. Since MPO levels are a reliable and quantitative marker for neutrophil accumulation in tissues, 25 pulmonary MPO levels were used to assess the degree of pulmonary neutrophil infiltration. Lung MPO levels were significantly higher in the burned rats compared with nonburned rats (Tables 1 and 2). As was true of changes in lung permeability, lymphatic division prior to thermal injury only partially prevented the increase in pulmonary MPO accumulation observed following a 40% TBSA burn. 1335

Table 1. Effect of Preburn Lymphatic Division (LD) on Lung Injury in Rats With a Normal Gut Intestinal Bacteria Flora* n Lung Permeability, % Evans Blue Dye in BALF BALF Protein, g/dl MPO Activity, U/g Tissue Alveolar Apoptosis, No. of Apoptotic Nuclei per HPF Control 8 4.1 ± 0.6 1.3 ± 0.1 8.2 ± 1.2 1.0 ± 0.2 Sham burn 8 4.1 ± 1.0 1.3 ± 0.1 7.9 ± 0.4 0.8 ± 0.1 Burn 8 13.2 ± 0.9 2.0 ± 0.1 14.5 ± 1.2 3.3 ± 0.2 LD + burn 8 6.1 ± 0.8 1.6 ± 0.1 10.0 ± 0.5 1.7 ± 0.3 *BALF indicates bronchoalveolar lavage fluid; MPO, myeloperoxidase; and HPF, high-power field. Data are expressed as mean ± SD. Statistical analysis was via analysis of variance with Tukey-Kramer multiple comparisons procedures. P.001 vs all groups. P.01 vs control and sham burn group. P.05 vs control and sham burn group. Table 2. Effect of Preburn Lymphatic Division (LD) Plus Monoassociation (MA) on Lung Injury* n Lung Permeability, % Evans Blue Dye in BALF BALF Protein, g/dl MPO Activity, U/g Tissue Alveolar Apoptosis, No. of Apoptotic Nuclei per HPF MA + control 6 4.6 ± 0.7 1.2 ± 0.1 11.1 ± 0.7 0.8 ± 0.1 MA + sham burn 6 5.8 ± 0.3 1.4 ± 0.1 13.7 ± 0.6 1.1 ± 0.1 MA + burn 6 15.9 ± 1.9 2.3 ± 0.1 26.6 ± 2.8 4.0 ± 0.6 MA + LD + burn 6 8.1 ± 0.9 1.8 ± 0.2 18.9 ± 1.3 2.1 ± 0.2 *BALF indicates bronchoalveolar lavage fluid; MPO, myeloperoxidase; and HPF, high-power field. Data are expressed as mean ± SD. Statistical analysis was via analysis of variance with Tukey-Kramer multiple comparisons procedure. P.001 vs all groups. P.01 vs MA + control group. P.01 vs MA + control and sham burn groups. P.01 vs MA + control and.05 vs MA + and sham burn groups. Last, there was a nearly 3-fold increase in the incidence of alveolar apoptosis in those rats subjected to a 40% TBSA burn compared with both nonburned animals and those whose mesenteric lymphatics had been divided prior to thermal injury (Tables 1 and 2). Lymphatic division prior to thermal injury reduced alveolar apoptosis to about half of that observed in the burned animals, paralleling those changes seen with extravasation of EB into the lung. The Figure illustrates the differential pattern of lung injury in rats after manipulation of their gut microflora. Monoassociation coupled with thermal injury resulted in even further increases in lung injury over thermal injury alone for all the parameters examined. Significant increases in lung permeability and pulmonary MPO accumulation were observed in all rats whose intestine had been colonized with streptomycin-resistant E coli C25 (Figure, A and C). In addition, monoassociation resulted in small but significant increases in BALF protein content (P =.001 vs normal intestinal bacterial flora undergoing 40% TBSA burn) and alveolar apoptosis (P =.02 and P =.049 vs normal intestinal bacterial flora undergoing sham and actual burn, respectively) (Figure 1, B and D). To further evaluate the host response to both bacterial overgrowth and thermal injury, enteric bacterial translocation to the MLNs and distant organs (the liver, spleen, and lungs) was measured. All E coli C25 monoassociated rats had viable intestinal bacteria present in their MLNs following sham or actual burn, except for one of the control animals (Table 3). There was no statistical difference in the magnitude of bacterial translocation to the MLNs between the groups, although the burned animals exhibited the greatest degree of translocation. There was no evidence of distant bacterial spread to the liver, spleen, or lungs in any of the groups. Cecal population levels of gram-negative enteric bacteria and total aerobes were similar among the groups (Table 4). COMMENT Considerable clinical and experimental evidence documenting the connection between severe thermal injury, respiratory failure, and the subsequent development of adult respiratory distress syndrome exists. 1,9 The cause of acute lung injury, without a preceding history of inhalation injury, has been variously ascribed to the production of mediators produced both at the site of injury 10 or by other injured organs. 26 Previously, we showed that mesenteric lymph generated following hemorrhagic shock increases endothelial cell permeability and contributes to lung injury, supporting the possibility that gut-derived cell-injurious factors are reaching the lung and systemic circulation via the intestinal lymphatics. 16,17 Not only does the gut-associated lymphoid tissue compose the body s largest lymphoid organ, but the intestinal immune system is continually exposed to potential proinflammatory stimuli. 27 Since thermal injury is associated with intestinal ischemia, loss of gut barrier function, and subsequent bacterial translocation, 28-30 it appears logical that the level of gutassociated lymphoid tissue activation should also be increased and that the gut may then be involved in the pathogenesis of pulmonary injury following thermal injury. Consequently, we tested the hypothesis that after 1336

18 A Normal Flora (n = 8 per ) Monoassociated (n = 6 per ) P =.01 3.0 B P =.001 % Evans Blue in BALF 10 P =.007 P =.003 BALF Protein, g/dl 1.5 2 0 30 C P<.001 5.0 D P =.049 MPO Activity, U/g Tissue 20 10 P<.001 P<.001 No. of Apoptotic Nuclei per HPF 2.5 P =.02 0 Control Sham Burn Lymphatic Division + Burn Burn 0 Control Sham Burn Lymphatic Division + Burn Burn Comparison of lung injury between rats with normal intestinal bacterial flora and monoassociated rats following either sham burn or actual thermal injury with and without lymphatic division. A, Lung permeability (mean ± SD in the percentage of Evans blue found in the bronchoalveolar lavage fluid [BALF]) in rats with normal intestinal bacterial flora (gray bars) and Escherichia coli C25 monoassociation (black bars). Statistical analysis was via a 2-tailed t test. B, Bronchoalveolar lavage fluid protein content (mean ± SD in grams per deciliter) in rats with normal intestinal bacterial flora (gray bars) and E coli C25 monoassociation (black bars). Statistical analysis was via a 2-tailed t test. C, Pulmonary myeloperoxidase (MPO) activity (mean ± SD in units of activity per gram of lung tissue) in rats with normal intestinal bacterial flora (gray bars) and E coli C25 monoassociation (black bars). Statistical analysis was via Mann-Whitney U test. D, Alveolar apoptosis (mean ± SD in number of apoptotic nuclei per high-power field [HPF]) in rats with normal intestinal bacterial flora (gray bars) and E coli C25 monoassociation (black bars). Statistical analysis was via a 2-tailed t test. Table 3. Effect of Monoassociation (MA) Plus Preburn Lymphatic Division (LD) on Incidence and Magnitude of Bacterial Translocation (BT) to Mesenteric Lymph Nodes (MLNs) n %of Bacteria in MLNs* Total Aerobes, 10 3 Gram-negative Enteric Bacteria 10 3 MA + control 6 83 3.8 ± 2.0 3.2 ± 1.7 MA + sham burn 6 100 7.7 ± 0.8 6.4 ± 0.6 MA + burn 6 100 9.1 ± 1.9 8.6 ± 0.7 MA + LD + burn 6 100 7.6 ± 1.2 5.9 ± 1.1 *Statistical analysis was via the Fisher exact test. Data are expressed as mean ± SEM of 10 3 colony-forming units per gram of MLNs except where indicated. Statistical analysis was via analysis of variance. a nonlethal but severe burn, gut-derived factors are present in the mesenteric lymph and contribute to burninduced lung injury. This hypothesis appears valid based on the following experimental observations. First, rats subjected to a 40% TBSA burn demonstrated significant increases in lung permeability, BALF protein content, MPO accumulation, and alveolar apoptosis. Second, lymphatic division prior to thermal injury reduced the burninduced increases observed in lung permeability, BALF Table 4. Effect of Monoassociation (MA) Plus Preburn Lymphatic Division (LD) on Cecal Bacterial Population Levels* n Total Aerobes Gram-negative Enteric Bacteria MA + control 6 9.7 ± 0.2 9.7 ± 0.2 MA + sham burn 6 9.7 ± 0.2 9.7 ± 0.2 MA + burn 6 9.6 ± 0.2 9.6 ± 0.2 MA + LD + burn 6 10.0 ± 0.2 9.9 ± 0.2 *Data are expressed as mean ± SD log 10 colony-forming units per gram. Statistical analysis was via analysis of variance with Tukey-Kramer multiple comparisons procedure. P.05 vs MA + burn group. protein content, MPO accumulation, and alveolar apoptosis. Thus, thermal injury resulted in a significant and reproducible increase in lung permeability, as measured by EB extravasation into the lung, which was approximately 3-fold higher than that observed in the nonburned animals. This increase in lung permeability was partially prevented by lymphatic division prior to the induction of the thermal injury. Second, we tested the hypothesis that bacterial overgrowth would contribute to burn-induced lung injury. This hypothesis was based on experimental observa- 1337

tions that alterations in the gut flora play a role in modulating the cytokine response to tissue injury 18 and that overgrowth of potentially pathogenic bacteria in the gastrointestinal tract can alter the anatomic and physical barrier function of the intestinal mucosa, eventually resulting in enteric bacteria translocating to extraintestinal sites or the systemic circulation. 31 Additionally, there is evidence that selective antibiotic decontamination of the gastrointestinal tract reduces systemic cytokine and endotoxin levels following cardiac surgery 32,33 and in patients in the intensive care unit. 34 Therefore, to further evaluate the role of the gut flora in modulating the extent of distant organ injury, rats were subjected to bacterial overgrowth (monoassociation) prior to thermal injury. The results obtained in the monoassociated rats paralleled those found in rats with a normal gut flora. That is, rats subjected to a 40% TBSA burn demonstrated significant increases in lung permeability, BALF protein content, MPO accumulation, and alveolar apoptosis, while lymphatic division prior to thermal injury reduced these burninduced increases. Since bacterial overgrowth plus thermal injury led to an even further increase in lung permeability, BALF protein content, MPO accumulation, and alveolar apoptosis than observed with thermal injury alone, it appears that bacterial overgrowth accentuates the magnitude of burn-induced lung injury. One explanation for these observations may be that the ischemic gut 28-30 produces or contains factors that, because of the loss of gut barrier function, are preferentially transported via the lymphatic circulation. These factors are then carried via the lymphatic route to the pulmonary circulation before passing through any other organ. Thus, the lung will receive the first volley of cytotoxic gut-derived products postinjury. Consequently, lymphatic division prior to thermal injury prevents the subsequent transport of injurious factors that are produced or released during the period of gut ischemia/ reperfusion. However, since the division of the mesenteric lymphatics failed to completely prevent burninduced changes in pulmonary function, it appears that other lung-injurious factors besides those present in the mesenteric lymph are produced after thermal injury. That is, although most factors that contribute to lung injury appear to be derived from the injured gut via the intestinal lymphatic route, it is likely that toxic factors are also being produced and/or released locally at the site of injury (burn lymph). It appears likely that these burn site generated toxins are transported via the lymphatic system into the venous circulation, where they produce their pulmonary hemodynamic and toxic effects. 35 The observation that pulmonary MPO activity increased following thermal injury is consistent with the work of others using similar models of thermal injury. 36,37 Consistent with changes in lung permeability and alveolar apoptosis, lymphatic division prior to thermal injury failed to fully prevent the observed increase in pulmonary MPO accumulation seen with a 40% TBSA burn. One possible explanation for this observation may be that following an ischemic insult and mesenteric reperfusion, circulating neutrophils are primed either by direct interaction with the endothelium of the injured gut or by inflammatory mediators released from the gut into the mesenteric lymphatics or the portal circulation. 38 A second possibility is that factors produced locally at the site of thermal injury are responsible for, or at least partially contribute to, neutrophil priming in addition to the mesenteric lymph. Alternatively, local and distal factors may be acting synergistically to produce their pulmonary effects. Under these circumstances, the mesenteric lymph would only be partially responsible for the activation and recruitment of pulmonary neutrophils. Others have suggested that thermal injury promotes lung injury through systemic complement and/or neutrophil activation and accumulation in the lung with the subsequent production of reactive oxygen species since lung injury following thermal injury is prevented by treatment with antioxidants, neutrophil depletion, and xanthine oxidase inhibition. 2,39,40 Important questions arising from our studies include the following: What toxic factors might be present in the mesenteric lymph, and how do they contribute to burn-induced lung injury? The answer to these questions is not known, although certain mediators have been implicated in burn-induced lung injury. For example, several studies 4,41 have implicated complement activation in the pathogenesis of acute lung injury following thermal skin injury (burn). Specifically, increases in lung permeability developed progressively over a 6-hour period and paralleled changes in complement levels. Leff et al 3 and Burton et al 37 found that rats subjected to a 30% second-degree burn had increased serum catalase activity and xanthine oxidase activity, respectively. In a rat intestinal model of ischemia/reperfusion injury, Koike et al 42 demonstrated that following 45 minutes of mesenteric ischemia, intestinal phospholipase A 2 was activated, resulting in neutrophil priming and subsequent lung albumin leak. Finally, mesenteric lymph, collected from rats subjected to various periods of hemorrhagic shock was found to contain elevated levels of both interleukin 6 and tumor necrosis factor. 43 Thus, based on the results of the present study and a review of all of the literature, we propose the following mechanistic sequence by which gut injury (ischemia/ reperfusion) following thermal injury contributes to lung injury: (1) there is an elaboration of proinflammatory mediators both from the gut as well as from the site of injury; (2) these gut-derived mediators are subsequently transported to the lung via the intestinal lymphatics, where they contribute to (3) endothelial cell activation and neutrophil sequestration, (4) increased protein (albumin) leak, and (5) lung injury. CONCLUSIONS In summary, the concept that the intestinal lymphatics are the primary route by which gut-derived cellinjurious factors are reaching the systemic circulation and distant organs is supported by several lines of evidence. First, our laboratory has demonstrated that mesenteric lymph generated following hemorrhagic shock increases endothelial cell permeability to a greater extent than portal vein plasma as well as contributing to lung injury. Second, we know from many animal studies that the mesenteric lymphatics are the primary route 1338

Statement of Clinical Relevance Recent clinical studies in victims of major trauma have questioned the clinical relevance of gut-origin sepsis and the role of the gut as an organ responsible for the development of multiple organ failure. In these studies, researchers failed to document the presence of detectable bacteria in either the portal blood or mesenteric lymph nodes of trauma victims, including several patients who ultimately went on to develop systemic infection and/or multiple organ failure. One possible explanation for this apparent controversy is that gut ischemia can induce gutassociated lymphoid tissue to produce and release cytokines or other proinflammatory factors even in the absence of detectable portal bacteremia. Previously, we documented that the mesenteric lymph generated after hemorrhagic shock contains toxic factors and contributes to lung injury. The results of the present study showing that burn-induced lung injury can be ameliorated by ligation of the mesenteric lymph duct suggest that gut ischemia, following a significant thermal injury, can also induce the intestine and the gutassociated lymphoid tissue to produce and release immunoinflammatory factors that reach the lung and the systemic circulation via the intestinal lymphatics. These results thus further validate the role of the intestinal lymph as a carrier of factors that predispose to distant-organ injury. overgrowth of intestinal bacteria causes an increase in the production and subsequent release of gut-derived factors into the mesenteric lymph and primes the host such that when the animal is subsequently exposed to a second stimulus (such as thermal injury), an exaggerated response occurs as evidenced by increased lung injury. In conclusion, the present study was carried out to test whether thermal injury can generate mesenteric lymph that results in distant organ (lung) injury and to what extent, if any, intestinal bacteria contribute to that injury. The results of this study, documenting that the division of mesenteric lymph flow prevents burn-induced lung injury and that bacterial overgrowth plus thermal injury increases lung injury over thermal injury alone, support this hypothesis. Further studies will be required to elucidate the causative factor(s) carried in the mesenteric lymph. This study was supported in part by grant GM 36376 from the National Institutes of Health, Bethesda, Md (Dr Deitch). Presented at the 19th Annual Meeting of the Surgical Infection Society, Seattle, Wash, April 30, 1999. Corresponding author: Edwin A. Deitch, MD, Department of Surgery, MSB G506, UMDNJ New Jersey Medical School, 185 S Orange Ave, Newark, NJ 07103 (e-mail: edeitch@umdnj.edu). of bacterial translocation. 44 Third, the MLN complex represents the first and frequently the only site of translocated bacteria. 45 Finally, multiple different experimental models, 46-48 including a model of partial gut ischemia, 46 reveal significantly elevated levels of gut-derived endotoxin in the thoracic duct prior to its subsequent detection in the portal circulation. The development of systemic endotoxemia paralleled the appearance of endotoxin in thoracic duct lymph, and endotoxin was identified in the lymph before significant portal endotoxemia. 46 Thus, the loss of gut barrier function to bacteria and/or endotoxin, 49 especially if coupled with gut injury, could produce a local intestinal inflammatory response. Since the biological effects of both bacteria and endotoxin are mediated primarily by host-derived responses, intestinal bacteria do not need to reach the portal circulation to induce a systemic inflammatory state. Bacterial translocation may be involved in the pathogenesis of the systemic inflammatory state via the activation and subsequent release of cytokines or other inflammatory mediators from the gut-associated lymphoid tissue even in the absence of detectable portal bacteremia. Consequently, it would not be necessary to recover bacteria from either the portal or systemic circulation for gut injury to contribute to a systemic inflammatory state and distant organ injury. Additionally, there is extensive evidence that intestinal bacteria and their products play a major role in the development and maintenance of the host s immune system and that bacterial translocation (in the absence of tissue injury) alters systemic immune responsiveness. 50 Furthermore, based on the results of the present study, it appears that REFERENCES 1. Pruitt BA, Erickson DR, Morris A. Progressive pulmonary insufficiency and other pulmonary complications of thermal injury. J Trauma. 1975;15:369-379. 2. Oldham KT, Guice KS, Till GO, Ward PA. Activation of complement by hydroxyl radical in thermal injury. Surgery. 1988;104:272-279. 3. Leff JA, Burton LK, Berger EM, Anderson BO, Wilke CP, Repine JE. Increased serum catalase activity in rats subjected to thermal skin injury. Inflammation. 1993;17:199-204. 4. Till GO, Beauchamp C, Menapace D, et al. Oxygen radical dependent lung damage following thermal injury of rat skin. J Trauma. 1983;23:269-277. 5. 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Surgery. 1991;109:269-276. DISCUSSION Per-Olof Hasselgren, MD, Cincinnati, Ohio: Last year, Dr Deitch and his group reported similar results in a hemorrhagic shock model, and I think the work that we are seeing today is an important extension of those observations because they indicate that the same mechanisms may not be specific to a certain model or a certain situation but may indeed be a generalized phenomena in shock, injury, and critical illness. I have a couple of questions. One technical, minor question. The burn group that you reported on, did that group also undergo laparotomy and placement of the sponge in the abdomen? That wasn t quite clear from your presentation or from the manuscript. My second question is related to the effectiveness of the division of the lymphatic vessel. How well documented is it that it completely prevents gut lymph from reaching the lung? Is lymph that is drained into the abdominal cavity absorbed and transported further, or does it really stay in the abdomen? My third question is whether you have tried to give the lymph back to the lungs. What you have shown is that by dividing the lymphatic vessels, you prevent or blunt the response in the lung. Have you tried the other side of the coin, that is, giving lymph back to the burn animals? My fourth and final question is what is the active factor in the lymph? Dr Magnotti: Thank you, Dr Hasselgren, for your comments and for reading the paper. I will try to answer your questions as best I can. I will start off with the first question regarding the burn group. The burn group did undergo laparotomy incision and placement of a sponge. The main difference in the groups was the only animals not undergoing the celiotomy incision were the control animals. They were shaved, they were dunked into room temperature water, but did not undergo laparotomy. Lymphatic division, the burn group and the sham burn group all underwent laparotomy incision with placement of the sponge. In regard to your second question, the effectiveness of lymphatic division, we know that in these animals, in the rats, there is a single lymphatic channel that drains the gut. It drains the distal two thirds of the small bowel, the right colon, and approximately two thirds of the transverse colon. It is a single lymphatic channel that comes off. It is located adjacent to the superior mesenteric artery. Division was done visually by cutting it and confirming adequate flow of lymph into the peritoneal cavity. The sponge was placed in order to try to prevent any effects of absorption of the lymph by the peritoneum and taken back in that way, and in that regards, that is why the sponge was placed in order to hopefully prevent any of that. The experiments were again performed with ligation of the lymph channel, and we obtained similar results with ligation of the channel rather than just division of it to hopefully avoid any of the complications of the lymph fluid being reabsorbed. 1340

As far as the question regarding giving the lymph back, have we tried this? No. Has it been done in other settings? Yes. There is some work where burn lymph has been taken from animals and injected back in into normal, healthy animals, and they have been able to reproduce changes in the pulmonary vascular hemodynamics, that was recently shown, but we haven t done it ourselves. It is a good idea now that we have shown that we have established that there is something, we prevented its effects by blocking it, now we just need to reproduce its effects by giving it back. And finally in terms of what factor is present, well, right now there is ongoing research in our lab as to trying to identify the factor, but it appears at this point in time that it is more likely factors that are present. Initial studies have been done separating the lymph that has been collected based on molecular weight and looking at endothelial cell viability tested against different molecular weights, and what we found is that there seems to be biological activity at different molecular weights, not at one cutoff point. We have also done some heat stability studies where the lymph has been exposed to heat, and at certain time points you are able to block its effects, and at other time points you are not. This has been an ongoing process looking at not only the lymph but the lymph time course to see what happens over time. Kenneth Kudsk, MD, Memphis, Tenn: I want to congratulate you on a very nice presentation. The experiment certainly seems to be very consistent with the articles that I have read from Dr Deitch s lab. I have a question about the bacterial overgrowth part of the experiment and the monoassociation. Is this because of having a single organism, and have you documented that there are really increased numbers of bacteria per milliliter of stool, a bacterial overgrowth sort of phenomena? Is it just the number of bacteria there or is there something about the E coli that you are using, that it is more toxic or its products are more toxic, that are accounting for the change? Can you do the same thing, for example, with Pseudomonas or some other gram-negative organism? And then, do you have any lung cultures from these animals to determine whether there are whole bacteria that are being translocated to the pulmonary circulation? Dr Magnotti: Thank you. To answer your questions, I will answer the second question first. When we did the antibiotic decontamination in monoassociation models, we did harvest the lungs and looked for bacterial translocation in the lungs of all the animals, and we found no translocated bacteria. In fact, we looked at the animals at 3 hours, which was kind of early to get distant bacterial translocation. It usually occurs after about 24 hours. But we did have positive bacterial translocation in all animals to the mesenteric lymph nodes except one of the control animals. So we did know that it was occurring. Whether they would have been propagated onward after 24 hours is something that would need to be looked at, but 3 hours is relatively early to see distant bacterial translocation. In terms of the bacterial overgrowth model, we use an E coli C25 strain, which is useful because it is streptomycin resistant, so that is why after giving the penicillin and streptomycin we continue with the E coli and the streptomycin to prevent other bacteria from growing and to keep the animal s gut flora pure at the E coli C25. It is E coli, so it is a normal constituent of the gut, and it is nonpathogenic, and those are the 3 characteristics of the E coli C25 that makes it useful in terms of this model. We do have documentation that antibiotic decontamination alone will reduce the typical cecal bacterial populations from about 10 6 to less than 10 2, and that the monoassociation significantly increases it; by just doing fecal smears on these animals, we can see how much is increased. Whether you can do it with Pseudomonas, I don t know if it would be as easy and whether you would be sure that you just have a pure single culture of one organism there. Cora Ogle, MD, Cincinnati, Ohio: I have a question about your burn model. You said you give 20% of the burn to the abdomen. Do you think you could induce heat shock proteins in your intestine and maybe compromise your results because of all that has been said now about the protective effect of heat shock proteins? Dr Magnotti: Whether or not heat shock proteins can be induced, it is difficult for me to say. The burn, it is not a long burn, it is a 5-second immersion in terms of the abdominal burn and the back burn, the back burn being 10 seconds. Could they be induced? Yes. But is it likely given that it is such a short period of exposure? I don t think so. So I don t think that is playing a factor. 1341