Patricia Sikes

Effect of a bradykinin antagonist on the local inflammatory response following thermal injury

Partial and full thickness burns with intervening zones of stasis were created on the backs on New Zealand White rabbits (n = 23). Either saline or the bradykinin receptor antagonist, NPC 17731, was administered. Skin blood flow was measured hourly using a laser Doppler blood flowmeter. After 4 h skin samples were harvested for assessment of tissue oedema (wet/dry weights) and leucocyte accumulation (immunohistochemistry). Statistical analysis was performed using Analysis of variance (ANOVA) and Mann-Whitney U test with a level of significance at P < 0.05. It was found that blood flow was decreased postburn in all groups. Bradykinin antagonist resulted in increased blood flow in partial thickness burns and zones of stasis compared to saline-treated animals (P < 0.05). Pretreatment with bradykinin antagonist showed reduced tissue oedema in full thickness burns (P < 0.05). No significant difference was observed in leucocyte accumulation between both groups. These data suggest a role for bradykinin in the pathogenesis of postburn microvascular changes which is independent of leucocyte-mediated injury.

Activation of Stress-responsive Pathways by the Sympathetic Nervous System in Burn Trauma

We have shown previously that burn trauma activates the stress responsive proteins, p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK), and NF-&kgr;B, and we have shown further that p38 MAPK is an important mediator of cardiomyocyte TNF-&agr; secretion and cardiac dysfunction in burn trauma. Since burn trauma causes a rise in circulating catecholamine levels, we hypothesized that this increased sympathetic activity may function as an upstream activator of the p38 MARK pathway in burn trauma. This study determined whether the &agr;1-adrenergic receptor ligand phenylephrine could mimic burn trauma activation of p38 MAPK, JNK, and NF-&kgr;B nuclear translocation; and the effect of the &agr;1-adrenergic receptor antagonist prazosin on either phenylephrine or burn-mediated activation of the stress response pathway was examined. Sprague Dawley rats were divided into seven groups: Group 1, controls; Group 2, phenylephrine-treated (2 &mgr;g/kg, i.v.) control rats; Group 3, phenylephrine-treated plus prazosin-treated (1 mg/kg, i.v.) control rats; additional rats were given burn over 40% total body surface area (TBSA) and received vehicle (1 mL of 2% sucrose, PO) plus fluid resuscitation (Group 4), while in Group 5, burn rats were given prazosin (1 mg/kg, PO) plus fluid resuscitation. In Groups 6 and 7, sham-burned rats were given either vehicle (1 mL of 2% sucrose, PO) or prazosin (1 mg/kg, PO) to provide appropriate controls. Administration of phenylephrine to rats caused a significant activation of cardiac p38 MAPK/JNK activities (Western blot) and cardiac NF-&kgr;B nuclear translocation (electrophoretic mobility shift assay, EMSA). Prazosin blocked phenylephrine mediated changes in p38 MAPK/JNK activities. Burn trauma activated cardiac p38 MAPK/JNK and NF-&kgr;B, increased TNF-&agr; secretion by cardiomyocytes, and impaired cardiac function. Prazosin treatment in burns interrupted the burn-mediated signaling cascade, decreasing TNF-&agr; secretion by cardiomyocytes and preventing post-burn cardiac contractile dysfunction. Thus, burn trauma-related sympathetic activity likely activates the stress-responsive cascade, which regulates myocardial TNF-&agr; transcription/translation and culminates in cardiac contraction and relaxation defects.

Sodium/hydrogen exchange activity in sepsis and in sepsis complicated by previous injury: 31P and 23Na NMR study

Objective:Sepsis or septic shock occurs frequently in sick and injured patients and is associated with a significant mortality. Myocardial contractile dysfunction has been proposed to be a major determinant of sepsis-related mortality. This study was directed to examine the role of Na+/H+ exchange activity in myocardial defects after sepsis or after sepsis complicated by a previous burn injury. Design:Laboratory study. Setting:University research laboratory. Subjects:Sprague-Dawley rats (300–350 g, males). Interventions:Cardiac function, cellular Na+ and Ca2+, myocardial pH, and high-energy phosphates were examined in perfused hearts harvested after sepsis alone (intratracheal Streptococcus pneumoniae, 0.4 mL of 1 × 107 CFU/mL), after sepsis complicated by previous burn injury (40% total body surface area), and after amiloride (a selective inhibitor of Na+/H+ exchange) treatment of either sepsis alone or sepsis plus burn. Measurements and Results:The ratio of Na+ signal from the intracellular compartment (Na+i) compared with an external standard (monitored by 23Na-NMR spectroscopy, TmDOTP−4 shift reagent) increased by 70% in sepsis alone and by 41% in sepsis complicated by previous burn injury compared with shams. Cardiac adenosine triphosphate and intracellular pH (31P nuclear magnetic resonance spectroscopy) were unchanged by sepsis or sepsis plus burn. Left ventricular pressure and maximal change in pressure over time were reduced after sepsis or after sepsis plus burn injury. Amiloride treatment in either sepsis or sepsis complicated by a previous burn injury prevented myocardial Na+ and Ca2+ accumulation, attenuated sepsis-related lactic acidosis, and improved left ventricular function. Conclusion:Our results suggest that sepsis-related cardiac dysfunction is mediated, in part, by Na+/H+ exchange activity, and inhibition of Na+/H+ exchange activity improves cardiac function after sepsis alone or sepsis complicated by a previous injury.

Alterations in leukocyte adhesion molecule expression after burn injury

OBJECTIVE To determine if thermal injury alters the expression of leukocyte adhesion molecules. DESIGN This is a controlled experimental animal study. MATERIALS AND METHODS Partial thickness burns were created on the backs of New Zealand White rabbits. At 30 minutes, 2 hours, 4 hours, and 24 hours after burn, skin was harvested for immunohistochemistry. Monoclonal antibodies were used to study changes in intercellular adhesion molecule 1 (ICAM-1), E-selectin, and leukocyte CD11a. Staining was graded on a scale of 0 to 4. MEASUREMENTS AND MAIN RESULTS ICAM-1 was significantly decreased at 24 hours after burn (p < 0.007, Wilcoxon signed rank test). CD11a was increased at 30 minutes (p < 0.02), 2 hours (p < 0.02), and 24 hours (p < 0.006). E-selectin was increased at 2 hours (p < 0.03). CONCLUSION Thermal injury alters the expression of leukocyte adhesion molecules.

Effect of inhibiting leukocyte integrin (CD18) and selectin (L-selectin) on susceptibility to infection with Pseudomonas aeruginosa.

Leukocyte (WBC) adherence to endothelial cells has been implicated in the pathogenesis of microvascular injury. The process of leukocyte adherence is mediated by both the integrin and selectin families of molecules, and their interaction with specific endothelial ligands. Antibodies directed against the leukocyte integrin CD18 and L-selectin have been developed and functionally inhibit leukocyte adherence in models of inflammatory injury. We asked the question: Does inhibition of leukocyte adherence by administration of monoclonal antibody directed against either CD18, integrins (R15.7, R7.1) or against L-selectin (DREG 200) increase susceptibility to infection? New Zealand white rabbits were shaved and injected subcutaneously on their dorsum with Pseudomonas aeruginosa (ATCC#27853) at two sites each of 10(8) and 10(7) colony forming units. Animals were monitored with daily determination of weight, temperature, WBC counts, hematocrit, and killed at 1 week for determination of abscess formation. There were four blinded experimental groups: (1) Saline (2 mL/kg); (2) DREG 200 (2 mg/kg); (3) R7.1 (2 mg/kg); or (4) R15.7 (2 mg/kg). At the 10(7) and 10(8) injection sites the R15.7 group had an increased rate and size of abscess formation compared with controls. The R7.1 group had an increased rate at the 10(8) injection site. There was no significant difference in the percentage of the abscess formation or mean area between the controls and DREG 200-treated groups. We conclude that giving antibody to CD18 increased susceptibility to infection while giving antibody to L-selectin does not.

Alterations In Alveolar Macrophage Tumor Necrosis Factor (tnf) Response Following Hemorrhagic Shock

The purpose of this study was to determine if hemorrhagic shock alters the alveolar macrophage (MØ) tumor necrosis factor (TNF) response to lipopolysaccharide (LPS) stimulation. New Zealand White rabbits underwent hemorrhage and resuscitation. At 1, 2, 3, 5, and 7 days post-shock, both MØs and peripheral whole blood monocytes were incubated in vitro with saline or Escherichia coli LPS. The supernatants were assayed for TNF activity using the L929 bioassay. Alveolar MØs from hemorrhaged animals showed reduced TNF activity during the first 5 days post-hemorrhage. Maximal depression of TNF activity was observed on days 3 and 5 post-hemorrhage (p < .05). In comparison, peripheral whole blood monocytes showed an increased TNF response on post-shock days 2 and 3. These results suggest that hemorrhagic shock and resuscitation differentially affect TNF response in alveolar and peripheral blood MØ populations.

Role of CD14 in hemorrhagic shock-induced alterations of the monocyte tumor necrosis factor response to endotoxin

OBJECTIVE To determine if the shock-induced alterations in whole blood monocyte tumor necrosis factor (TNF) response are mediated by the CD14 receptor. DESIGN Prospective controlled animals experiments. MATERIALS AND METHODS New Zealand White rabbits (n = 15) were subjected to hemorrhage and resuscitation. Blood samples obtained before shock and 24, 72, and 120 hours after shock were stimulated with lipopolysaccharide in the presence or absence of the anti-CD14 monoclonal antibody, 63D3. Tumor necrosis factor was assayed using L929 cells. MEASUREMENTS AND MAIN RESULTS There are no detectable TNF activity in unstimulated blood. The CD14 inhibition resulted in a 55% reduction in baseline TNF activity. After shock, there was a marked increase in TNF activity with lipopolysaccharide stimulation. Addition of 63D3 resulted in a dose-dependent 95% reduction in TNF activity at 24 and 72 hours after shock, (p < 0.05). CONCLUSION The enhanced whole blood monocyte TNF response after hemorrhage is CD14 dependent.