Andreas D. Niederbichler

An essential role for complement C5a in the pathogenesis of septic cardiac dysfunction

Defective cardiac function during sepsis has been referred to as “cardiomyopathy of sepsis.” It is known that sepsis leads to intensive activation of the complement system. In the current study, cardiac function and cardiomyocyte contractility have been evaluated in rats after cecal ligation and puncture (CLP). Significant reductions in left ventricular pressures occurred in vivo and in cardiomyocyte contractility in vitro. These defects were prevented in CLP rats given blocking antibody to C5a. Both mRNA and protein for the C5a receptor (C5aR) were constitutively expressed on cardiomyocytes; both increased as a function of time after CLP. In vitro addition of recombinant rat C5a induced dramatic contractile dysfunction in both sham and CLP cardiomyocytes, but to a consistently greater degree in cells from CLP animals. These data suggest that CLP induces C5aR on cardiomyocytes and that in vivo generation of C5a causes C5a–C5aR interaction, causing dysfunction of cardiomyocytes, resulting in compromise of cardiac performance.

Inhibition of complement C5a prevents breakdown of the blood-brain barrier and pituitary dysfunction in experimental sepsis.

IntroductionSeptic encephalopathy secondary to a breakdown of the blood-brain barrier (BBB) is a known complication of sepsis. However, its pathophysiology remains unclear. The present study investigated the effect of complement C5a blockade in preventing BBB damage and pituitary dysfunction during experimental sepsis.MethodsUsing the standardised caecal ligation and puncture (CLP) model, Sprague-Dawley rats were treated with either neutralising anti-C5a antibody or pre-immune immunoglobulin (Ig) G as a placebo. Sham-operated animals served as internal controls.ResultsPlacebo-treated septic rats showed severe BBB dysfunction within 24 hours, accompanied by a significant upregulation of pituitary C5a receptor and pro-inflammatory cytokine expression, although gene levels of growth hormone were significantly attenuated. The pathophysiological changes in placebo-treated septic rats were restored by administration of neutralising anti-C5a antibody to the normal levels of BBB and pituitary function seen in the sham-operated group.ConclusionsCollectively, the neutralisation of C5a greatly ameliorated pathophysiological changes associated with septic encephalopathy, implying a further rationale for the concept of pharmacological C5a inhibition in sepsis.

Attenuating Burn Wound Inflammatory Signaling Reduces Systemic Inflammation and Acute Lung Injury

The relationship between local inflammation and the subsequent systemic inflammatory response is poorly described. In a burn injury model, the dermal inflammatory response may act as an ongoing trigger for the systemic inflammatory response syndrome (SIRS) and subsequent systemic complications. We hypothesized that topical attenuation of burn wound inflammatory signaling will control the dermal inflammatory source, attenuate SIRS, and reduce acute lung injury. Mice received a 30% total body surface area burn. Subgroups were treated with specific p38 MAPK inhibitor or vehicle, which was topically applied to wounds. Topical p38 MAPK inhibition significantly reduced burn wound inflammatory signaling and subsequent systemic expression of proinflammatory cytokines and chemokines. In vitro macrophage functional assays demonstrated a significant attenuation in serum inflammatory mediators from animals receiving the topical inhibitor. Topical p38 MAPK inhibition resulted in significantly less pulmonary inflammatory response via reduction of pulmonary neutrophil sequestration, pulmonary cytokine expression, and a significant reduction in pulmonary microvascular injury and edema formation. Although dermal activating transcription factor-2, a downstream p38 MAPK target, was significantly reduced, there was no reduction in pulmonary activating transcription factor-2 expression, arguing against significant systemic absorption of the topical inhibitor. These experiments demonstrate a strong interaction between dermal inflammation and systemic inflammatory response. Attenuating local inflammatory signaling appears effective in reducing SIRS and subsequent systemic complications after burn injury.

Ability of antioxidant liposomes to prevent acute and progressive pulmonary injury.

We recently showed that acute oxidant-related lung injury (ALI) in rats after application of 2-chloroethyl ethyl sulfide (CEES) is attenuated by the airway instillation of antioxidants. We investigated whether intratracheal administration of antioxidant-containing liposomes immediately after instillation of CEES would attenuate short-term as well as long-term (fibrotic) effects of CEES-induced lung injury. In the acute injury model (4 h after injury), N-acetylcysteine (NAC)-containing liposomes were protective and reduced to baseline levels both the lung permeability index and the appearance of proinflammatory mediators in bronchoalveolar lavage fluids from CEES-exposed lungs. Similar results were obtained when rat alveolar macrophages were incubated in vitro with either CEES or lipopolysaccharide in the presence of NAC-liposomes. When lung fibrosis 3 weeks after CEES was quantitated by using hydroxyproline content, liposomes containing NAC or NAC + glutathione had no effects, but liposomes containing alpha/gamma-tocopherol alone or with NAC significantly suppressed the increase in lung hydroxyproline. The data demonstrate that delivery of antioxidants via liposomes to CEES-injured lungs is, depending on liposomal content, protective against ALI, prevents the appearance of proinflammatory mediators in bronchoalveolar fluids, and suppresses progressive fibrosis. Accordingly, the liposomal strategy may be therapeutically useful in CEES-induced lung injury in humans.

Topical p38MAPK inhibition reduces dermal inflammation and epithelial apoptosis in burn wounds.

ABSTRACT Thermal injury induces dermal inflammatory and proapoptotic signaling. These phenomena extend burn wound size and trigger a systemic inflammatory response, factors known to adversely affect outcomes. p38MAPK is known to trigger inflammatory responses and induce epithelial proapoptotic genes. We hypothesize that topical p38MAPK inhibition will attenuate excessive inflammatory and apoptotic signaling and reduce dermal tissue loss. Rats were given a 30% total body surface area partial thickness burn or sham injury. Some of the animals were treated with a p38MAPK inhibitor or vehicle, which was applied directly to the wound. Dermal inflammation was investigated with enzyme-linked immunosorbent assay, reverse transcriptase polymerase chain reaction, myeloperoxidase assay, and Evans blue extravasation. Apoptotic changes were detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and Caspase-3 in situ staining. Burn injury activated dermal p38MAPK and induced a significant rise in dermal IL-6, TNF-&agr;, and IL-1&bgr; expression. Neutrophil sequestration, microvascular damage, and hair follicle apoptosis were significantly elevated after injury. Topical p38MAPK inhibition significantly attenuated downstream dermal p38MAPK targets, proinflammatory cytokine expression, neutrophil sequestration, and microvascular injury. A significant reduction in hair follicle apoptosis was seen. This study demonstrates the attenuation of burn-induced cellular stress by topical application of p38MAPK inhibitors. Blunting early excessive inflammatory signaling may be an efficient strategy to improve patient outcomes after burn injury.

Cardiomyocyte function after burn injury and lipopolysaccharide exposure : Single-cell contraction analysis and cytokine secretion profile

ABSTRACT A component of multiorgan dysfunction in burned patients is heart failure. Burn trauma induces cytokine synthesis of interleukin (IL) 1&bgr;, IL-6, and tumor necrosis factor &agr; (TNF-&agr;) which can negatively impact cardiac function. Infectious complications are common following severe burn injury. We hypothesized that burn injury and lipopolysaccharide (LPS) exposure independently influence peak cardiomyocyte contraction and cytokine secretion. Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 1, 6, 12, and 24 h after burn, cardiomyocytes were isolated and incubated with increasing LPS doses. Peak sarcomere shortening and contractile velocity parameters were recorded using a variable-rate video camera with sarcomere length detection software. Supernatants were assayed for IL-1&bgr;, IL-6, and TNF-&agr; by ELISA. Peak sarcomere shortening was decreased in the burn group at 1, 6, 12, and 24 h after burn. IL-1&bgr;, IL-6, and TNF-&agr; levels were increased in cardiomyocytes isolated 1 h after burn compared with sham controls, but returned to sham levels at 6, 12, and 24 h after burn. LPS exposure caused dose-dependent decreases in sarcomere shortening in sham and burn animals. LPS exposure did not produce increased cardiomyocyte cytokine expression. Burn injury diminished peak sarcomere shortening. Whereas exposure to LPS did not have an effect on cardiomyocyte cytokine expression, LPS significantly inhibited sarcomere shortening in a dose-dependent fashion. Combined burn and LPS exposure inhibited sarcomere shortening more than each alone. These results demonstrate that LPS exposure and burn injury independently decrease peak cardiac shortening. These decreases did not directly correlate with the levels of cytokines released in response to each stressor.

C5a-Blockade Improves Burn-Induced Cardiac Dysfunction

We previously reported that generation of the anaphylatoxin C5a is linked to the development of cardiac dysfunction in sepsis due to C5a interaction with its receptor (C5aR) on cardiomyocytes. Burn injury involves inflammatory mechanisms that can lead to C5a generation as well. In this study, we investigated the effects of C5a blockade on burn-induced cardiac dysfunction. Using a standardized rat model of full thickness scald injury, left ventricular pressures were recorded in vivo followed by in vitro assessment of sarcomere contraction of single cardiomyocytes. Left ventricular pressures in vivo and cardiomyocyte sarcomere contractility in vitro were significantly reduced following burn injury. In the presence of anti-C5a Ab, these defects were greatly attenuated 1, 6, and 12 h after burn injury and completely abolished 24 h after burn. In vitro incubation of cardiomyocytes with bacterial LPS accentuated the impaired contractility, which was partially prevented in cardiomyocytes from burned rats that had received an anti-C5a Ab. Based on Western blot analyses, real-time PCR, and immunostaining of left ventricular heart tissue, there was a significant increase in cardiomyocyte expression of C5aR after burn injury. In conclusion, an in vivo blockade of C5a attenuates burn-induced cardiac dysfunction. Further deterioration of contractility due to the exposure of cardiomyocytes to LPS was partially prevented by C5a-blockade. These results suggest a linkage between C5a and burn-induced cardiac dysfunction and a possible contribution of LPS to these events.

Attenuating burn wound inflammation improves pulmonary function and survival in a burn-pneumonia model.

Objective:We previously showed that topical inhibition of inflammatory signaling in burn wounds reduced systemic inflammatory response and burn-induced pulmonary inflammation. We hypothesized that this topical intervention would attenuate burn-induced lung injury, improve pulmonary function, protect lungs from bacterial invasion, and reduce mortality. Design:Controlled, in vivo, laboratory study. Setting:University laboratory. Subjects:Female mice, 8–10 wks old. Interventions:Animals received 30% total body surface area burn followed by topical application of a specific inhibitor of p38 mitogen-activated protein kinase, a key inflammatory signaling pathway, or vehicle to the wound. Twenty-four hours after injury, pulmonary collagen deposition and pulmonary function were assessed. One day postburn, some of the animals received intratracheal instillation of Klebsiella pneumoniae and were subsequently monitored for 7 days. Measurements and Main Results:Topical inhibition of p38 mitogen-activated protein kinase significantly decreased pulmonary collagen deposition and prevented a decline in pulmonary function at 1 day after burn injury. Compared with sham controls, animals with burn injury had a significantly higher mortality in response to intratracheal bacterial challenge. Application of p38 mitogen-activated protein kinase inhibitor to the burn wound attenuated pulmonary neutrophil infiltration and reduced the mortality rate to a level experienced by sham controls. Conclusions:Inflammatory source control in burn wounds with topical p38 mitogen-activated protein kinase inhibition attenuates acute lung injury, avoids pulmonary dysfunction, protects lungs from bacterial challenge, and improves survival.

Insight in microcirculation and histomorphology during burn shock treatment using in vivo confocal-laser-scanning microscopy ☆,☆☆

PURPOSE Microcirculatory disturbances are well known during shock; however, the accompanied histomorphological alterations are widely unknown. We used high resolution confocal-laser-scanning microscopy for the evaluation of microcirculation and histomorphology during Burn Shock treatment. METHODS Confocal-laser-scanning microscopy was performed in 10 burn shock patients (4 women, 6 men; aged 40.6 +/- 11.4 years, burn extent >20% body surface area) initially and 24 hours after shock resuscitation. Ten matched hemodynamic stable burn intensive care unit patients served as controls. The following parameters were evaluated: quantitative blood cell flow, cell size of the granular layer, basal layer thickness, and epidermal thickness. RESULTS Quantitative blood cell flow in controls was 62.45 +/- 3.39 cells per minute. Burn shock significantly reduced blood cell flow to 37.27 +/- 3.64 cells per minute; fluid resuscitation effectively restored baseline blood flow (65.18 +/- 3.76 cells per minute) after 24 hours. Granular cell size was 793.61 +/- 41.58 microm(2) in controls vs 644.27 +/- 42.96 microm(2) during burn shock. Post resuscitation granular cell size measured 932.74 +/- 38.83 microm(2). Basal layer thickness was 14.84 +/- 0.59 microm in controls, 13.26 +/- 0.54 microm in burn patients at admission and before resuscitation, and 17.50 +/- 0.46 microm after resuscitation. Epidermal thickness in control patients was 49.60 +/- 2.36 microm, 37.83 +/- 2.47 microm in burn patients at admission and 69.50 +/- 3.18 microm after resuscitation. CONCLUSIONS Confocal-laser-scanning microscopy provides a noninvasive tool for simultaneous evaluation of microcirculation and tissue histomorphology. It may help to assess the adequacy of and response to resuscitation of burn patients early after trauma.

Burn-induced Heart Failure: Lipopolysaccharide Binding Protein Improves Burn and Endotoxin-induced Cardiac Contractility Deficits

BACKGROUND Burn injury is frequently complicated by bacterial infection. Following burn injury, exposure to endotoxin produces a measurable decrease in cardiomyocyte sarcomere contractile function. Lipopolysaccharide-binding protein (LBP) is an acute phase protein that potentiates the recognition of lipopolysaccharide (LPS) by binding to the lipid A moiety of LPS. In this study, we sought to determine the effect of recombinant rat LBP (rLBP) on cardiomyocyte sarcomere function after burn or sham injury in the presence or absence of bacterial endotoxin. METHODS Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 24 h post-injury, cardiomyocytes were isolated, plated at 50,000 cells/well, and incubated with 50 μg/mL LPS and rLBP or chloramphenicol acetyltransferase (BVCat, an irrelevant control protein produced using the same expression system as rLBP) at concentrations by volume of 1%, 5%, 10%, and 30%. Subsets of cardiomyocytes were incubated with 5% rat serum or 30% rLBP and blocking experiments were conducted using an LBP-like synthetic peptide (LBPK95A). In vitro sarcomere function was measured using a variable rate video camera system with length detection software. RESULTS Co-culture of burn and sham injury derived cardiomyocytes with high-dose rLBP in the presence of LPS resulted in a significant reduction to the functional impairment observed in peak sarcomere shortening following exposure to LPS alone. LBP-like peptide LBPK95A at a concentration of 20 μg/mL, in the presence of LPS, abolished the ability of 30% rLBP and 5% rat serum to restore peak sarcomere shortening of cardiomyocytes isolated following burn injury to levels of function exhibited in the absence of endotoxin exposure. CONCLUSIONS In the setting of LPS challenge following burn injury, rLBP at high concentrations restores cardiomyocyte sarcomere contractile function in vitro. Rather than potentiating the recognition of LPS by the cellular LPS receptor complex, rLBP at high concentrations likely results in an inhibitory binding effect that minimizes the impact of endotoxin exposure on cardiomyocyte function following thermal injury.