P263

336

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

P262. ENDOTHELIAL CELL DYSFUNCTION AND NOT BLOOD RHEOLOGY DETERMINES TISSUE MYELOPEROXIDASE ACTIVITY AFTER RESUSCITATION FROM HEMORRHAGIC SHOCK. J. E. Campbell 1, R. N. Garrison 2, J. C. Peyton 3, E. R. Zakaria 1; 1University of Louisville, Louisville, KY, 2University of Louisville and VAMC, Louisville, KY, 3VAMC, Louisville, KY Introduction: Conventional resuscitation (CR) of Hemorrhagic Shock (HS) primes neutrophils and damages vascular endothelium thereby modulating leukocyte (WBC)-endothelial interaction. We hypothesize that adjunct direct peritoneal resuscitation (DPR) with clinical peritoneal dialysis solution further modulates the WBC-endothelial interaction. Methods: Anesthetized rats were hemorrhaged to 40% mean arterial pressure for 60 min. Animals were randomized for CR (return of the shed blood plus two volumes of saline), or for CR with adjunctive DPR (25 ml of intraperitoneal clinical peritoneal dialysis solution). Tissue myeloperoxidase (MPO) activity and total water content were assessed in the gut, lung, and liver at time-points 1, 2, and 4 hours post-resuscitation. Results: Adjunctive DPR resulted in no significant change in MPO concentration amongst tissues at corresponding time-points (p⬎0.05, n⫽7 CR, n⫽7 CR ⫹DPR) in all tissues except the lung (p⬍0.05). However, a chronological trend of increasing MPO activity exists from baseline to one hour, and one hour to two hours (p⬍0.05). This MPO activity significantly decreased to baseline at 4h post-resuscitation regardless of the resuscitation regimen as shown in the figure. Conclusion: These data suggest that the gut is an important site for neutrophil priming in shock/resuscitation. DPR-mediated splanchnic vasodilation and hyperperfusion did not translate into changes in tissue MPO activity. These data suggest that endothelial cell dysfunction and not blood rheology is the detrimental factor in WBC-endothelial interaction in hemorrhagic shock with resuscitation.

P263. FLUID RESUSCITATION FOLLOWING HEMORRHAGE DOES NOT INCREASE LATE EDEMA OR VIABILITY LOSS IN RAT SKELETAL MUSCLE AFTER ISCHEMIA-REPERFUSION. D. S. Kauvar, D. G. Baer, T. J. Walters; U.S. Army Institute of Surgical Research, Fort Sam Houston, TX Introduction: Tourniquets are frequently employed for hemorrhage control in military extremity injuries. The complications of tourniquet use in trauma are not well studied. We investigated the combined, late (2 days) effect of hemorrhage and fluid resuscitation with Hextend (HX) or Lactated Ringers’ (LR) on skeletal muscle subjected to tourniquet-induced ischemia-reperfusion in-

jury (I-R). Methods: 26 male Sprague-Dawley rats underwent 33% arterial hemorrhage followed by 3 hour hind limb tourniquet application. Prior to reperfusion, animals were resuscitated with LR (3 times shed volume, n⫽9) or HX (shed volume, n⫽8). Nine controls received no resuscitation (NR). Rats were euthanized after 2 days of reperfusion and the tibialis anterior (TA) and medial gastrocnemius (MG) muscles were examined for edema (wet/dry weight ratio) and viability (nitroblue tetrazolium-NBT reduction). Results: The TA and MG in all groups experienced edema, with all mean wet/dry muscle weight ratios significantly (p⬍0.05) greater than those of the corresponding contralateral limb muscles. No differences in edema were seen between similar tourniquet muscles in the NR, LR, and HX groups. In the TA, all groups experienced significant reductions in viability compared with the contralateral. In the MG, however, this was true only in the NR group. In the LR and HX groups, viability was similar between tourniquet and contralateral muscles. No difference in viability loss between similar muscles in the NR, LR, and HX groups was present. When directly compared, the TA experienced significantly greater edema and viability loss than the MG, regardless of resuscitation group. Conclusions: Resuscitation with HX or LR did not increase edema or viability loss in rat skeletal muscle subjected to hemorrhage, tourniquet-induced ischemia, fluid resuscitation, and two days of reperfusion. Muscles of similar fiber-type composition but different anatomic compartment location respond differently to the combined effect of hemorrhage, resuscitation, and tourniquet-induced I-R.

P264. HYPERTONIC SALINE REDUCES TNF ALPHA INDUCED IKB PHOSPHORYLATION. T. L. Nydam 1 , E. E. Moore 2 , R. C. McIntyre 1 , F. Gamboni-Roberts 1 , P. C. Eckels 2 , A. Banerjee 1 ; 1 University of Colorado Health Science Center, Denver, CO, 2 Denver Health Medical Center, Denver, CO Renewed interest in post-injury resuscitation with hypertonic saline is due to its immunomodulatory effects. Hypertonicity reduces proinflammatory chemokine production, cell surface adhesion molecule expression, and markers of lung injury. The Tumor Necrosis Factor alpha (TN␣) induced Nuclear Factor kappa B (NF-␬B) pathway is a primary mediator of these proinflammatory processes. We therefore hypothesized that hypertonicity inhibits TNF␣-induced phosphorylation of Inhibitor of kappa B (I␬B) and activation of NF-␬B. Methods: Human pulmonary epithelial cells were pretreated with clinically relevant hypertonic media (180 mM NaCl) for 30 minutes, followed by TNF␣ stimulation (10ng/mL). ICAM-1, phosphorylated I␬B␣, and Tumory Necrosis Factor receptor 1 (TNFR1) were measured with western blot analysis of whole cell lysate. Digital microscopy was used to quantify NF-␬B p65 subunit fluorescence and nuclear translocation was calculated from the mean fluorescence intensity (MFI) within the nuclei. Cell surface TNFR1 expression was measured with flow cytometry. Statistical analysis was done using ANOVA (p⬍0.01). Results: TNF␣-induced NF-␬B activation increases ICAM-1 expression in HPECs. Hypertonicity (HT) reduced total ICAM-1 levels as compared to TNF␣ only controls. Hypertonicity reduced TNF␣-induced NF-␬B nuclear translocation by 40% which was due to decreased phosphorylation of I␬B. TNFR1 cell surface expression and total protein levels were unchanged. Conclusion: Hypertonicity reduces TNF␣-induced phosphorylation of I␬B resulting in decreased NF-␬B nuclear translocation and proinflammatory gene expression. The alteration is proximal to phosphorylation of I␬B, yet not due to decreased total TNFR1 levels or cell surface expression.