Sagar S. Damle

Plasma from stored packed red blood cells and MHC class I antibodies causes acute lung injury in a 2-event in vivo rat model

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion death. We hypothesize that TRALI requires 2 events: (1) the clinical condition of the patient and (2) the infusion of antibodies against MHC class I antigens or the plasma from stored blood. A 2-event rat model was developed with saline (NS) or endotoxin (LPS) as the first event and the infusion of plasma from packed red blood cells (PRBCs) or antibodies (OX18 and OX27) against MHC class I antigens as the second event. ALI was determined by Evans blue dye leak from the plasma to the bronchoalveolar lavage fluid (BALF), protein and CINC-1 concentrations in the BALF, and the lung histology. NS-treated rats did not evidence ALI with any second events, and LPS did not cause ALI. LPS-treated animals demonstrated ALI in response to plasma from stored PRBCs, both prestorage leukoreduced and unmodified, and to OX18 and OX27, all in a concentration-dependent fashion. ALI was neutrophil (PMN) dependent, and OX18/OX27 localized to the PMN surface in vivo and primed the oxidase of rat PMNs. We conclude that TRALI is the result of 2 events with the second events consisting of the plasma from stored blood and antibodies that prime PMNs.

HMGB1 is markedly elevated within 6 hours of mechanical trauma in humans.

High-mobility group box 1 (HMGB1) is a late mediator of the systemic inflammation associated with sepsis. Recently, HMGB1 has been shown in animals to be a mediator of hemorrhage-induced organ dysfunction. However, the time course of plasma HMGB1 elevations after trauma in humans remains to be elucidated. Consequently, we hypothesized that mechanical trauma in humans would result in early significant elevations of plasma HMGB1. Trauma patients at risk for multiple organ failure (ISS ≥15) were identified for inclusion (n = 23), and postinjury plasma samples were assayed for HMGB1 by enzyme-linked immunosorbent assay. Comparison of postinjury HMGB1 levels with markers for patient outcome (age, injury severity score, units of red blood cell (RBC) transfused per first 24 h, and base deficit) was performed. To investigate whether postinjury transfusion contributes to elevations of circulating HMGB1, levels were determined in both leuko-reduced and non-leuko-reduced packed RBCs. Plasma HMGB1 was elevated more than 30-fold above healthy controls within 1 h of injury (median, 57.76 vs. 1.77 ng/mL; P < 0.003), peaked from 2 to 6 h postinjury (median, 526.18 ng/mL; P < 0.01 vs. control), and remained elevated above control through 136 h. No clear relationship was evident between postinjury HMGB1 levels and markers for patient outcome. High-mobility group box 1 levels increase with duration of RBC storage, although concentrations did not account for postinjury plasma levels. Leuko-reduced attenuated HMGB1 levels in packed RBCs by approximately 55% (P < 0.01). Plasma HMGB1 is significantly increased within 1 h of trauma in humans with marked elevations occurring from 2 to 6 h postinjury. These results suggest that, in contrast to sepsis, HMGB1 release is an early event after traumatic injury in humans. Thus, HMGB1 may be integral to the early inflammatory response to trauma and is a potential target for future therapeutics.

Arachidonic acid in postshock mesenteric lymph induces pulmonary synthesis of leukotriene B4

Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remains elusive. Previous work has shown that administration of a phospholipase A(2) (PLA(2)) inhibitor attenuated postshock ALI and also identified a non-ionic lipid within the postshock mesenteric lymph (PSML) responsible for polymorphonuclear neutrophil (PMN) priming. Consequently, we hypothesized that gut-derived leukotriene B(4) (LTB(4)) is a key mediator in the pathogenesis of ALI. Trauma/hemorrhagic shock (T/HS) was induced in male Sprague-Dawley rats and the mesenteric duct cannulated for lymph collection/diversion. PSML, arachidonic acid (AA), and a LTB(4) receptor antagonist were added to PMNs in vitro. LC/MS/MS was employed to identify bioactive lipids in PSML and the lungs. T/HS increased AA in PSML and increased LTB(4) and PMNs in the lung. Lymph diversion decreased lung LTB(4) by 75% and PMNs by 40%. PSML stimulated PMN priming (11.56 +/- 1.25 vs. 3.95 +/- 0.29 nmol O(2)(-)/min; 3.75 x 10(5) cells/ml; P < 0.01) that was attenuated by LTB(4) receptor blockade (2.64 +/- 0.58; P < 0.01). AA stimulated PMNs to produce LTB(4), and AA-induced PMN priming was attenuated by LTB(4) receptor antagonism. Collectively, these data indicate that splanchnic ischemia/reperfusion activates gut PLA(2)-mediated release of AA into the lymph where it is delivered to the lungs, provoking LTB(4) production and subsequent PMN-mediated lung injury.

Proteomic profiling of the mesenteric lymph after hemorrhagic shock: Differential gel electrophoresis and mass spectrometry analysis

Experiments show that upon traumatic injury the composition of mesenteric lymph changes such that it initiates an immune response that can ultimately result in multiple organ dysfunction syndrome (MODS). To identify candidate protein mediators of this process we carried out a quantitative proteomic study on mesenteric lymph from a well characterized rat shock model. We analyzed three animals using analytical 2D differential gel electrophoresis. Intra-animal variation for the majority of protein spots was minor. Functional clustering of proteins revealed changes arising from several global classes that give novel insight into fundamental mechanisms of MODS. Mass spectrometry based proteomic analysis of proteins in mesenteric lymph can effectively be used to identify candidate mediators and loss of protective agents in shock models.

Heme oxygenase-1 induction in macrophages by a hemoglobin-based oxygen carrier reduces endotoxin-stimulated cytokine secretion.

The inflammatory response after an insult may provoke further tissue damage, and the macrophage is central in this pathophysiology. Induction of heme oxygenase-1 (HO-1) attenuates postshock organ dysfunction, although the mechanism remains unclear. We hypothesized that HO-1 induction modifies the cytokine profile of LPS-stimulated macrophages. Heme oxygenase-1 was induced in murine and human macrophages with varying concentrations of a hemoglobin-based oxygen carrier (HBOC). Heme oxygenase-1 expression was analyzed by Western blotting of whole cell lysates. Macrophages were pretreated with HBOC for 4 h, then media with LPS were added for up to 24 h. The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) was used to inhibit the effects of HO-1. Supernatants were analyzed for IL-6, IL-10, TNF-&agr;, and monocyte chemotactic protein 1 (MCP-1) by enzyme-linked immunosorbent assay. Incubation of cells with HBOC produced a dose-dependent expression of HO-1. Heme oxygenase-1 expression decreased LPS-stimulated secretion of MCP-1, IL-6, IL-10, and TNF-&agr; at both 4 and 24 h in murine and human macrophages. The addition of ZnPP to inhibit HO-1 partially restored MCP-1 and IL-6 secretion in murine macrophages. Furthermore, immunofluorescent microscopy revealed HBOC-induced HO-1 inhibited LPS-stimulated nuclear translocation of the p65 subunit of nuclear factor-&kgr;B. In summary, HBOC incubation of macrophages induced HO-1 expression, which reduced LPS-mediated cytokine release, and that MCP-1 and IL-6 secretion could be partially restored with ZnPP. These data encourage continued investigation into the role of HO-1 in protecting against posttraumatic organ dysfunction and the clinical potential of HBOC for HO-1 induction.

Secretory phospholipase A2 is required to produce histologic changes associated with gastroduodenal reflux in a murine model

OBJECTIVE The earliest response of esophageal mucosa to gastric reflux is the development of oxidative damage and inflammation. These processes contribute to the development of metaplasia known as Barretts esophagus, as well as the progression to malignancy. Secretory phospholipase A(2) is a mediator of inflammation with levels that are increased in Barretts metaplasia and carcinoma when compared with levels in normal samples. Our goal is to determine the role of secretory phospholipase A(2) in the development of reflux-associated changes in the esophageal mucosa. METHODS Secretory phospholipase A(2)-deficient mice (C57BL/6, n = 5) and mice known to express high levels of secretory phospholipase A(2) (BALB/c, n = 5) underwent side-to-side surgical anastomosis of the first portion of the duodenum and gastroesophageal junction, allowing exposure of esophageal mucosa to duodenal and gastric contents duodeno-gastroesophageal anastomosis. Control animals (n = 5) of each strain underwent laparotomy with esophagotomy and repair. Tissue was frozen in embedding medium. Hematoxylin and eosin staining and Ki67 and secretory phospholipase A(2) immunohistochemistry were used to evaluate esophageal tissue and its response to duodeno-gastroesophageal anastomosis. RESULTS Immunofluorescent staining confirmed the absence of secretory phospholipase A(2) in C57BL/6 mice and its presence in BALB/c mice. Hematoxylin and eosin staining demonstrated significant thickening of the esophageal mucosa in response to gastroesophageal reflux in the presence of secretory phospholipase A(2). Mice known to express high levels of secretory phospholipase A(2) also demonstrated increased numbers of proliferating cells. Secretory phospholipase A(2)-deficient mice were immune to the early changes induced by mixed reflux. CONCLUSIONS The presence of secretory phospholipase A(2) appears necessary for early histologic changes produced by exposure of the esophagus to gastroduodenal contents. This enzyme is identified as a promising target for evaluation of mechanisms of carcinogenesis and chemoprevention of esophageal carcinoma.

Hemoglobin-Based Oxygen Carrier Induces Heme Oxygenase-1 in the Heart and Lung but Not Brain

BACKGROUND The clinical sequel of ischemia and reperfusion remains a challenge in several clinical areas. Overexpression of heme oxygenase-1 (HO-1), using viral vectors, endotoxemia, and hypoxia, provides protection against ischemia and reperfusion injury. To date, however, no clinically viable therapy exists to safely induce HO-1. We have recently observed that administration of a hemoglobin-based oxygen carrier (HBOC) attenuates postinjury systemic inflammation. We have further demonstrated that an HBOC can induce HO-1 in vitro. We now explore the tissue-specific induction of heme oxygenase-1 after administration of an HBOC. STUDY DESIGN Rats were infused with doses of HBOC or saline through femoral vein injection (n=5 per group). Animals were sacrificed and organs were flushed. Heart, lung, and brain samples were taken for evaluation of total organ levels of HO-1 induction and for histologic localization of the cellular expression of the HO-1. Heat shock protein 72 levels were also analyzed to determine whether HO-1 induction was a generalized stress response. RESULTS Both the heart and lung demonstrated a dose-dependent induction of total organ HO-1. Interestingly, brain tissue did not have any significant amount of HO-1, either at baseline or after HBOC therapy. The cellular localization of HO-1 between organs was also specific, predominantly occurring in the cardiac myocyte and alveolar macrophages. Heat shock protein 72 levels were not significantly changed in any group examined, suggesting the induction of HO-1 is specific. CONCLUSIONS This study demonstrates that a clinically accessible product, HBOC, can specifically and selectively induce the expression of the protective enzyme HO-1 in vivo. These findings begin to characterize which organ systems may benefit by preischemic treatments with HBOC and further expand potential clinical applications of HBOCs.

Surgery for Tracheobronchomalacia

Tracheobronchomalacia (TBM) refers to a weakening of the anterior tracheal rings leading to splaying and collapse of the central airways. In this report, we review the treatment of TBM, including preoperative workup, intraoperative anesthesia management, and surgical technique for posterior splinting tracheobronchoplasty. Imperative in the preoperative preparation is a stent trial in which an airway stent is placed to temporarily relieve the TBM and reassess for improvement in symptoms. Definitive therapy is then carried out with posterior splinting tracheoplasty or tracheobronchoplasty. Surgical results are generally excellent with the majority of patients having significant improvements in breathing.

Assessment and Management of Postintubation Airway Injuries.

Tracheal laceration is a known complication of endotracheal intubation. This rare complication remains a diagnostic and management challenge for today’s practitioners. This clinical challenge report highlights current surgical and anesthetic management strategies.