Postshock mesenteric lymph is a significant source of systemic inflammation

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS and quantitative real-time RT-PCR. Results. Both L-NAME and 1400w blocked the LPS induced TIMP-1 transcription and protein production. While both agents markedly decreased LPS-induced MMP-9 transcription, no MMP-9 protein was detected in the gastric mucosa. Neither agent had any effect on MMP-2 or TIMP-2 transcription nor did they inhibit the LPS-induced increase in MMP-2 protein. L-NAME had no effect, while 1400w induced TIMP-2 protein; however, the fold increase in TIMP-2 was not as great as for MMP-2, favoring continued matrix degradation. Conclusions. Both constitutive and selective NOS inhibitors modulate gastric MMP and TIMP transcription and protein production. However, neither agent appears to significantly affect the predominant active gelatinase, MMP-2. P84. Ketamine Does Not Reverse Lipopolysacharide (LPS) Induced Ileus. J.W. Suliburk, MD, E.A. Gonzalez, MD, S.D. Moore-Olufemi, MD, N.W. Weisbrodt, PhD, D.W. Mercer, MD. University of Texas at Houston Medical School Department of Surgery. Introduction. LPS causes a gastrointestinal ileus and gastric luminal fluid accumulation. Ketamine, an antiinflammatory agent, attenuates accumulation of luminal fluid. However its effects on gastrointestinal transit during endotoxemia are unknown. The purpose of this study was to determine if the antiinflammatory properties of ketamine improve impaired gastrointestinal transit due to LPS. We hypothesized that ketamine would improve impaired GI transit caused by LPS. Methods. Rats were given ketamine (70 mg/kg ip) or saline 1 h prior to LPS (20 mg/kg, ip) or saline injection. Five hours following LPS injection, rats were gavaged with 0.1 ml of 5 mM FITC Dextran followed by 0.9 ml of saline. After 30 min rats were sacrificed; transit was determined as the mean geometric center of the FITC Dextran tracer signal; gastric fluid accumulation was determined, and gastric and ileal mucosa were harvested for analysis of iNOS as an inflammatory marker (Western immunoblot). Results are reported as mean ⫾ SE (n ⱖ 5 per group; ANOVA). Results. As shown in the table above, ketamine did

TABLE—ABSTRACT P84

Sal/Sal Sal/LPS Ket/Sal Ket/LPS

GI transit

Gastric fluid

Gastric iNOS

Ileal iNOS

6.35 ⫾ .16 2.71 ⫾ .34* 6.33 ⫾ .38 2.86 ⫾ .28*

.21 ⫾ .02 2.71 ⫾ .46* .24 ⫾ .08 1.23 ⫾ .4**

.03 ⫾ .001 .31 ⫾ .13* .02 ⫾ .01 .13 ⫾ .02**

.02 ⫾ .001 .4 ⫾ .08* .01 ⫾ .002 .24 ⫾ .02**

* P ⬍ .05 versus saline counterpart; ** P ⬍ .05 versus saline/LPS

not reverse LPS-induced gastrointestinal ileus. However it did decrease LPS-induced gastric luminal fluid accumulation and blunted iNOS expression in both the stomach and the ileum. Conclusion. These data indicate that ketamine’s ability to attenuate gastric fluid accumulation is not due to improved gastric emptying. Moreover, while iNOS may play a role in LPS-induced gastric luminal fluid accumulation, it does not appear to play a significant role in the gastrointestinal ileus caused by LPS. P85. Role of Neutrophils and Kupffer Cells in Hepatic Cells in Hepatic Infection: Do We Have It Wrong? D.R. Jeyarajah, MD, M.L. Kielar, MD, X. Zhou, MD, P. Karimi, MD, N.L. Frantz, BA, C.Y. Lu, MD. Southwestern Medical School. Introduction. Systemic infection is localized and cleared by the liver within minutes. Kupffer cells (KCs) are thought to be the critical cell involved in bacterial clearance. Work in this field has been performed with Listeria Monocytogenes, an intracellular pathogen that is not seen often in the clinical setting. This work examines

the role of KCs and neutrophils in hepatic clearance of E. coli, an organism that frequently causes sepsis. Methods. C57BL/6 mice were rendered KC-deficient with Gadolinium chloride (Gd) or neutropenic with anti-Ly6 monoclonal antibody therapy. KC depletion was confirmed by demonstrating decreased uptake of colloidal carbon, an agent taken up by KCs, or by immunohistochemistry using F4/80 mAb. Control animals received PBS injection. Specific hepatic infection with various doses (2 ⫻ 10 5-10 6) of E. coli was performed by direct injection into the portal vein (PV). Colony counts (CFU) recovered from the liver and peripheral blood, and histology of the liver, were assessed at 10 min and 6 h after infection (n ⫽ 4/grp). Survival was assessed in neutropenic animals (n ⫽ 8/grp). Results. KC depletion did not result in alterations of trapping (at 10 min) or clearance of E. coli in the liver, as measured by bacterial growth. Depletion of neutrophils resulted in similar trapping, but significantly greater bacterial growth by 6 h after infection. Similarly, neutrope-

TABLE—ABSTRACT P85

anti-Ly6 PBS P-value

10 minutes

6 hours

114,800 ⫾ 22,220 78,320 ⫾ 20,436 0.27

137,440 ⫾ 43,507 22,080 ⫾ 4,817 0.03

nia resulted in decreased survival after infection (25% survival neutropenic/100% survival in neutrophil sufficient). Histologic examination revealed the presence of large clusters of neutrophils in KC-depleted animals, but not KC-sufficient animals, at 6 h after infection, suggesting a role for KCs in control of activated neutrophils that are involved in clearance of infection. Conclusions. Surprisingly, KCs appear not to be critical to initial trapping or control of E. coli; by comparison, neutrophils are critical to the elimination of E. coli. KCs are likely important in controlling neutrophils that have emigrated to the liver to control infection. P86. Postshock Mesenteric Lymph Is a Significant Source of Systemic Inflammation. T. Masuno, MD, A. Cheng, MD, E. Sarin, MD, E. Moore, MD. Denver Health Medical Center and UCHSC. Mesenteric hypoperfusion of circulatory shock is a key event in the pathogenesis of subsequent distant organ injury. Previous studies have shown that postshock mesenteric lymph (PSML) contains cytotoxic mediators elaborated from the ischemic gut and that lymph diversion abrogates postshock acute lung injury. In this study we observed that maximal PSML cytotoxicity occurred during the period of greatest PSML volume contribution into the systemic circulation. Method. Sprague-Dawley rats were subjected to hemorrhagic shock (30 mmHg ⫻ 45 min) and then resuscitated over 2 h in a clinically relevant manner: 2⫻ volume of shed blood using normal saline (NS) in the first 30 min; 50% volume shed blood in the next 30 min; another 2⫻ volume shed blood using NS over the remaining 60 min. Mesenteric lymph was collected hourly and flow rates were recorded up to 6 h postshock. Superoxide was measured from isolated human neutrophils incubated with 5% (v/v) lymph taken from different time points. Results. Shocked rats recovered to baseline hemodynamics within 60 min of resuscitation. Despite blood pressure recovery, mesenteric lymph flow at 2– 4 h postshock increased to 400% above the preshock level (Fig. 1). Mesenteric lymph fractions collected during the peak flow had the greatest cytotoxicity (Fig. 2). Conclusion. With clinically relevant resuscitation of shock, maximal PSML cytotoxicity and flow into the systemic circulation occurs 2– 4h after shock. This finding suggests that the systemic contribution of gut-derived cytotoxic lymph after hemorrhagic shock may be more significant than previously realized.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P87 Model

Volume (ml)

pH (pH)

HO-1 (AU)

iNOS (AU)

Sham SMAO SMAO/LL SMAO/LD SMAO/NS

0.30 ⫾ 0.05a 2.22 ⫾ 0.57b 1.28 ⫾ 0.31 c 0.80 ⫾ 0.23 c 1.03 ⫾ 0.12 c

1.67 ⫾ 0.16a 6.58 ⫾ 0.20b 5.58 ⫾ 0.62b 6.38 ⫾ 0.38b 5.83 ⫾ 0.60b

0.36 ⫾ 0.02a 0.43 ⫾ 0.05a 0.56 ⫾ 0.04b 0.57 ⫾ 0.04b 0.59 ⫾ 0.05b

0.28 ⫾ 0.03a 0.29 ⫾ 0.07a 0.50 ⫾ 0.06b 0.44 ⫾ 0.03b 0.49 ⫾ 0.10b

P88. Inhibition of Intestinal Transit by ResuscitationInduced Gut Edema is Reversed by Administration of L-NIL. S.D. Moore-Olufemi, MD, H. Xue, MD, S.J. Allen, MD, D.H. Oliver, MD, F.A. Moore, MD, N. Weisbrodt, PhD, R. Stewart, DVM, PhD, G. Laine, PhD, C.S. Cox, Jr., MD. The UT-Houston Medical School, Houston, TX and the Texas A&M University, College Station, TX.

P87. The Isomeric Composition of Lactated Ringer’s Has No Differential Effect on Gastric Dysfunction After Mesenteric Ischemia/Reperfusion. E.A. Gonzalez, MD, R.A. Kozar, MD, PhD, J.W. Suliburk, MD, F.A. Moore, MD, D.W. Mercer, MD. University of Texas Health Science Center at Houston, Houston, TX. Lactated Ringer’s is the standard of care trauma resuscitation, but the D-isomer of lactate has been shown to activate proinflammation. We have shown that superior mesenteric artery occlusion (SMAO) without resuscitation causes gastric dysfunction characterized by luminal fluid accumulation and alkalization. In the same model, LR resuscitation protected the ileum, but the presence of the D-isomer abrogated this effect. We, therefore, hypothesized that the isomeric composition of LR resuscitation would have a differential effect on gastric dysfunction and inflammation in this model. Rats underwent sham or SMAO for 60 min. Five minutes prior to clamp removal, SMAO animals received no resuscitation, 20 ml/kg IV (our standard) of either LL or LD isomer LR, or 17 ml/kg IV normal saline (NS) as equal salt load control. Animals were sacrificed at 6 h of reperfusion and gastric mucosa was harvested for analysis. Gastric dysfunction was quantitated by gastric volume and luminal pH. HemeOxygenase-1 (HO-1; index of anti-inflammation) and inducible Nitric Oxide Synthase (iNOS; index of proinflammation) protein expression were analyzed by Western immunoblot. Data are reported as mean ⫾ SEM (n ⬎ 6/group; ANOVA). Means with different letters are significantly different. SMAO (no resuscitation) increased gastric volume and pH. All three resuscitative regimens decreased gastric volume, had no effect on pH, and equally induced HO-1 and iNOS. In conclusion, isomeric composition of LR had no differential effect on gastric dysfunction and inflammation after SMAO in contradistinction to its previously observed effects in the ileum. The isomeric immunomodulatory effects of LR appear to be organ specific.

Background. Postresuscitation gut edema and associated gut dysfunction is a common and significant clinical problem seen after traumatic injury and shock. We have previously shown that gut edema without ischemia/reperfusion injury delays intestinal transit. We hypothesized that up regulation of iNOS protein plays a role in delayed intestinal transit associated with gut edema and is reversible by L-NIL. Methods. 1 hour prior to laparotomy, rats were pretreated with 10 mg/kg of L-NIL or vehicle and underwent 80 mL/kg of 0.9% saline ⫹ superior mesenteric vein partial occlusion (Edema) or sham surgery. A duodenal catheter was placed to evaluate intestinal transit using a fluorescent dye. At 6 h, the small bowel was divided and the mean geometric center (MGC) was calculated to express transit. Ileum was harvested for evaluation of iNOS protein expression by Western blotting and MPO activity. Tissue water was determined using the % wet-to-dry weight ratio to assess gut edema. Data are expressed as mean ⫾ SEM, n ⫽ 3– 6 and *P ⬍ 0.05 using ANOVA. Results. Gut edema, expressed as increased

TABLE—ABSTRACT P88

Sham Edema Edema ⫹ L-Nil

Tissue water (%Tissue Water)

Intestinal transit (MGC)

75 ⫾ 1 82 ⫾ 2* 80 ⫾ 1*

4.6 ⫾ 0.3 2.7 ⫾ 0.3* 4.2 ⫾ 0.6

iNOS MPO densitometry activity (arbitrary (milliUnits) units) 6⫾2 2 ⫾ 0.4 4 ⫾ 0.7

0.6 ⫾ 0.03 1.2 ⫾ 0.2 0.8 ⫾ 0.03

tissue water, was associated with a decreased intestinal transit, elevated iNOS protein expression. Pretreatment with L-NIL improved intestinal transit and decreased expression of iNOS protein without decreasing intestinal tissue water compared to edema animals. There was no difference in the MPO activity among groups. Conclusion. Gut edema, in the absence of leukocyte infiltration, delays intestinal transit via an iNOS-mediated mechanism. P89. Heat Stress Modulates and Protects Hepatocyte Membrane Proteins During Sepsis. R. Przkora MD, PhD, M. Jeschke, MD, M. Haslbeck, MD, C. Heyde, MD, W. Ertel, MD, D. Herndon, MD, U. Bolder, MD, D. Herndon, MD. Shriners Burns Hospital for Children, Galveston, TX. Introduction. Hepatic failure is one of the important features of septic multiorgan dysfunction. Previous studies have shown that, in septic rats, heat stress (HS) has a protective effect on bile acid