Serial measurements increase the accuracy of laser Doppler assessment of burn wounds.

Traditional methods of judging burn depth by clinical evaluation of the wound based on appearance and sensation remain in wide use but are subject to individual variation by examiner. In addition to the clinical difficulties with burn wound management, observer dependency of wound assessment complicates clinical trials of burn wound therapy. A laser Doppler flowmeter with a multichannel probe was used to measure burn wound perfusion as a tool to predict wound outcome. Serial measurement with laser Doppler flowmetry had an 88% specificity and a positive predictive value of 81% for identifying nonhealing wounds. These results suggest that laser Doppler flowmetry is a potentially useful tool for burn wound assessment.

Stat proteins play a role in tumor necrosis factor alpha gene expression.

Trauma produces dysfunction in immunity, which appears to be partially related to alterations in the cytokine response. Signal transducer and activator of transcription proteins (STATs) mediate activation of several cytokine genes. However, the effect of STAT proteins on tumor necrosis factor-alpha (TNFalpha) activation is not fully defined. We identified binding sites for STAT 3 and STAT 5/6 within the promoter region of TNFalpha and hypothesize that alterations in these sites would affect TNFalpha expression. The TNFalpha promoter was inserted into the luciferase reporter vector, and binding sites for STAT 3, STAT 5/6, and activator protein-1 (AP-1) were mutated using site-directed mutagenesis. Murine macrophages were transfected with the resultant plasmids, then incubated with and without lipopolysaccharide (LPS) or IFNalpha. Gene expression was measured by dual luciferase assay. Mutation of the STAT 3 binding site was associated with decreased LPS-inducible activity. Mutation of the AP-1 and STAT 5/6 consensus binding sites alone had no effect on TNFalpha expression. However, combined mutation of both STAT 5/6 and AP-1 was associated with increased LPS-inducible activity. Mutations of the STAT binding sites in the promoter region of TNFalpha affect TNFalpha gene expression. These results suggest a regulatory role for STATs in TNF gene transcription.

Inhibition of leukocyte-mediated tissue destruction by synthetic fibronectin peptide (Trp-9-Tyr).

Burns are surrounded by an inflammatory zone of stasis that can progress to ischemia and extension of burn size. Synthetic fibronectin peptides have reduced tissue destruction in several models of inflammation. In this study, we postulate that administration of the peptide Trp-9-Tyr will alter the progression of tissue destruction following thermal injury. Baseline cutaneous blood flow was measured on New Zealand White rabbits with a laser doppler blood-flow meter. While the rabbits were under general anesthesia, 6 full-thickness burns were produced on the rabbits' backs. Blood flow in the zones of stasis was followed daily, and the number of zones that progressed to necrosis was determined at 72 hours. There were 3 experimental groups. Ten control animals received saline. Ten were treated with Trp-9-Tyr for 24 hours postburn. Ten received Trp-9-Tyr for 48 hours. Animals treated with Trp-9-Tyr had higher blood flow and less necrosis in the zones of stasis than did control animals, which was evident at 24 hours but more significant at 48 hours.

Torn ascending aorta missed by transesophageal echocardiography.

Transesophageal echocardiography has become a commonly used screening tool for traumatic tears of the descending aorta. The role of transesophageal echocardiography for ascending aortic tears is not yet well-defined. We report an ascending aortic tear imaged by aortography but missed on transesophageal echocardiography.

The effect of antimicrobial agents on the induction of tumour necrosis factor by alveolar macrophages in vitro in response to endotoxin.

Alveolar macrophages from New Zealand white rabbits were incubated with twice the MIC of amikacin, ciprofloxacin, aztreonam, ceftazidime and imipenem and exposed to either 10(4), 10(5) or 10(6) cfu/mL live Pseudomonas aeruginosa ATCC 27853 or 0.1, 1 or 10 mg/L purified lipopolysaccharide (LPS) derived from P. aeruginosa to determine the effects of different classes of antimicrobial agent on production of tumour necrosis factor (TNF). Incubation of macrophages with ciprofloxacin and amikacin resulted in less TNF activity after exposure to live P. aeruginosa than was found for saline, aztreonam, ceftazidime or imipenem (P < 0.05). However, no significant differences were found between any of the agents after macrophages had been exposed to purified LPS. Different antimicrobial agents therefore appear to exert different effects in vitro on the TNF response of macrophages to bacterial stimulation.

The 1996 Clinical Research award. Use of a pneumatonometer in burn scar assessment.

The evaluation of wound outcome after burn injury is a challenging problem in the performance of clinical trials evaluating potential impact on wound healing and scar formation. The purpose of this study was to determine whether an ocular tonometer could be adapted to provide an objective measurement of scar compliance. A pneumatonometer was used to perform measurements of cutaneous compliance at 8 anatomic areas (14 separate sites) on each of 17 normal volunteers and on 59 burn scars. Comparison of different anatomic sites showed there to be significant differences in the cutaneous compliance of different areas. The aggregate compliance of the burn scars in all sites was less than that of the control sites. These results indicate that the pneumatonometer can discern differences in the compliance of normal skin and differences between normal skin and scar and suggest that it may be a useful tool in the objective assessment of scar formation.

Monoclonal antibody to intercellular adhesion molecule-1 reduces cardiac contractile dysfunction after burn injury in rabbits.

Cardiac dysfunction occurs after thermal injury but the pathogenesis is nuclear; leukocytes have been implicated in the pathogenesis of multiorgan dysfunction after burn injury. White blood cell activation and entry into tissue involve the use of a number of adhesion molecules, including intercellular adhesion molecule (ICAM)-1, CD54. We asked the question: will administration of the monoclonal antibody (R6.5) directed against ICAM-1 alter the cardiac dysfunction which we have previously shown to occur after thermal injury? Previously instrumented New Zealand White rabbits were anesthetized and given a full-thickness burn over 30% of the total body surface area by applying brass probes heated to 100 degrees C to the animals' backs for 15 sec. Animals were monitored for 24 hr and given lactated Ringer's (LR) solution (4 cc/kg/% burn, Parkland formula) with additional LR given to maintain cardiac output and urine output. Three experimental groups were studied: sham burn controls had catheters placed and were monitored for 24 hr (N = 8); burn rabbits were divided into vehicle treated (saline, 1 ml/kg, N = 6) or R6.5 treated (2 mg/kg, N = 6). Vehicle or antibody was administered 30 min postburn and every 8 hr until 24 hr postburn; at this time, rabbits were sacrificed and hearts were harvested for in vitro assessment of contractile performance (Langendorff). Compared to values measured in sham burn controls, burn injury caused cardiac contractile depression as indicated by a fall in left ventricular pressure (LVP) (77 +/- 2 vs 56 +/- 3 mm Hg, P = 0.01), +dP/dt (1223 +/- 64 vs 842 +/- 64 mm Hg/sec, P = 0.001), and -dP/dt (973 +/- 63 vs 666 +/- 42 mm Hg/sec, P = 0.01). Administration of R6.5 significantly improved cardiac contractile function compared to the vehicle-treated burns as indicated by higher LVP (67 +/- 2 mm Hg, P > 0.05), +dP/dt (1017 +/- 33 mm Hg/sec, P > 0.05), and -dP/dt (858 +/- 40 mm Hg/ sec, P > 0.05) than values measured in vehicle-treated burns. These results suggest that ICAM-1-mediated WBC activation and/or tissue entry are involved in the pathogenesis of cardiac dysfunction following thermal injury.

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.

Inhibition of selectin- and integrin-mediated inflammatory response after burn injury.

Inflammation and microvascular injury in the areas adjacent to burn wounds produces extension of postburn tissue necrosis. Leukocytes are potent mediators of the local inflammatory response preceding tissue necrosis, and the selectin and integrin adhesion molecules have been implicated in leukocyte-mediated tissue destruction. We sought to examine the role of L-selectin (CD62-L) and CD18 in leukocyte accumulation and tissue necrosis following burn injury. New Zealand White rabbits (n = 36) were subjected to burn injury and were randomized to treatment with saline (control) or monoclonal antibodies to L-selectin or CD18. Animals given the anti-L-selectin antibody demonstrated reduced immunohistochemical evidence of leukocyte accumulation at 24 hr postinjury but did not show improved wound perfusion or reduced tissue necrosis. Animals in the anti-CD18 group showed significantly improved tissue survival and improved tissue perfusion but had grades of leukocyte accumulation similar to those in the control group. These observations suggest that leukocyte accumulation is partially L-selectin dependent and that leukocyte accumulation alone is not sufficient to cause changes in blood flow and tissue destruction, both of which appear to be largely CD18 mediated.

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.

The role of neutrophils in peritoneal adhesion formation.

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.

Is octreotide beneficial following pancreatic injury?

BACKGROUND: Pancreatic injury is often associated with multiple complications related to uncontrolled pancreatic exocrine secretion, including pancreatic fistula, pseudocyst, and intra-abdominal abscesses. Somatostatin analogues such as octreotide have been shown to decrease pancreas-related morbidity following major pancreatic resection in patients with pancreatic neoplasms and acute severe pancreatitis. This study was conducted to determine whether or not the administration of octreotide influences the incidence and severity of abdominal complications following pancreatic injury. PATIENTS AND METHODS: Patients with intraoperative diagnosis of pancreatic injury over a 6-year period were studied retrospectively. Specific complications assessed include abdominal abscesses, pseudocyst, pancreatitis, and pancreatic fistula. Statistical analysis of qualitative variables was by chi-square analysis, and analysis of quantitative variables by Student's t-test (P < 0.05). RESULTS: Injury to the pancreas was identified in 96 patients. Sixteen early deaths (< 48 hours) and one late death occurred, for a mortality of 18%, leaving 80 patients as the study population; 21 patients received octreotide and 55 patients did not. Pancreatic fistula occurred in 32 patients (40%). When stratified by pancreatic injury severity, there was no significant difference in complication rates, although patients treated with octreotide had a higher rate of fistula formation (48% versus 40%), longer duration of fistula drainage, and longer hospital stay compared with untreated patients. CONCLUSION: Although adverse patient selection may be a factor in this retrospective survey, the magnitude of observed differences raises concerns regarding the empiric administration of octreotide to such patients pending prospective study.

Early assessment of pediatric burn wounds by laser Doppler flowmetry.

Evaluation of burn wound depth in pediatric patients is often difficult. A Laser Doppler Flowmeter with a temperature-controlled multichannel probe was used to measure burn wound perfusion as a tool to predict wound outcome. The average perfusion levels for wounds that healed spontaneously in fewer than 21 days was significantly higher than the average perfusion levels for wounds that required excision and grafting or were not healed by day 21 after burn injury. Laser Doppler Flowmetry showed high positive predictive values for "nonhealing" wounds on postburn days 1, 2, and 3. These results suggest that Laser Doppler Flowmetry is a useful tool for burn wound assessment in pediatric patients.

Differential effects of monoclonal antibody blockade of adhesion molecules on in vivo susceptibility to soft tissue infection.

Leukocyte adherence to endothelial cells has been implicated in the pathogenesis of microvascular injury as well as in host defense against various infectious microorganisms. Administration of monoclonal antibodies directed against the beta chain of the leukocyte integrins inhibits leukocyte-endothelial-cell adherence and has been reported to modulate ischemia-reperfusion and inflammatory injury. However, such inhibition of adhesion molecule function adversely affects resistance to infection. The following studies were carried out to determine whether monoclonal antibodies to other adhesion molecules, including L-selectin (CD62L), and CD11a (the alpha chain of LFA-1), also increase susceptibility to infection. New Zealand White rabbits were shaved and given subcutaneous injections on their dorsa with 10(9) CFU of Staphylococcus aureus ATCC 25923 at two sites and with 10(8) CFU at two sites. A second set of rabbits were given subcutaneous injections with 10(8) CFU of P. aeruginosa ATCC 27853 at two sites and with 10(7) CFUs at two sites. The animals were monitored for 1 week. There were three blinded experimental groups: controls given saline and two groups given blocking monoclonal antibodies to either L-selectin (Dreg-200) or CD11a (R7.1). In contrast to monoclonal antibodies to CD18, none of the monoclonal antibodies significantly increased the risk of abscess formation by S. aureus, although inhibition of CD11a increased the rate of abscess formation by P. aeruginosa.

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 (M phi) 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 M phis 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 M phis 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 M phi populations.

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.

Laser Doppler flowmetry in burn wounds.

Traditional methods for judgment of burn depth by clinical evaluation of the wound based on appearance and sensation remain in wide use but are subject to individual variation by examiner. In addition to the clinical difficulties with burn wound management, observer dependency of wound assessment complicates clinical trials of burn wound therapy. A laser Doppler flowmeter with a newly designed multichannel probe was used to measure burn wound perfusion as a tool to predict wound outcome. Wounds that healed spontaneously in fewer than 21 days showed higher average perfusion levels than those that required excision and grafting or that were not healed by day 21 after the burn. Laser Doppler flowmetry had a positive predictive value of 100% for nonhealing wounds on postburn days 1 and 3. These data suggest that laser Doppler flowmetry is a potentially useful tool for burn wound assessment.

Enhancement of peritoneal macrophages reduces postoperative peritoneal adhesion formation.

Postoperative adhesion formation results from a fibroproliferative inflammatory reaction. Macrophages are critical in the final resolution of the inflammatory process and tissue repair, including modulation of proliferation and differentiation of fibroblasts and secretion of neutral proteases like plasminogen activator. We, therefore, studied the influence of peritoneal macrophage enhancement on postoperative adhesion formation in five groups of rabbits. Group 1 was a control with normal peritoneum. Animals in group 2 had increased macrophage population in their peritoneum by intraperitoneal injection of protease peptone 3 days before adhesion induction. In group 3, animals were treated by protease peptone as in group 2 and then depleted of the increased macrophage population by peritoneal lavage before adhesion induction. In group 4 macrophages were transplanted from animals enriched as in group 2 into a nonenriched peritoneum at the time of adhesion induction. Group 5 had a normal peritoneum with peritoneal lavage before adhesion induction. Peritoneal adhesions were induced at laparotomy by repairing a peritoneal defect in two different models. It was found that enhancement of peritoneal macrophages by protease peptone reduced markedly the degree of postoperative adhesion formation. After depletion of the enhanced peritoneal macrophages by peritoneal lavage the degree of adhesion formation was equivalent to that of controls. Finally, macrophage transplantation into a nonenhanced macrophage peritoneum also reduced the degree of postoperative adhesion formation. It is concluded that enhancement of peritoneal macrophages reduces postoperative peritoneal adhesion formation.

Interference with the function of leukocyte adhesion molecules by monoclonal antibodies: a new approach to burn injury.

Monoclonal antibody to intercellular adherence molecule-1 (ICAM-1) was used to inhibit leukocyte adherence in two models of burn injury. The antibodies to ICAM-1 had significant effects in preserving microvascular blood flow in burn wounds, and in modulating the systemic response to major burn injury. These results suggest a central role for leukocytes in the pathophysiologic response to burn injury.