Early structural changes of the heart after experimental polytrauma and hemorrhagic shock

<div><p>Evidence is emerging that systemic inflammation after trauma drives structural and functional impairment of cardiomyocytes and leads to cardiac dysfunction, thus worsening the outcome of polytrauma patients. This study investigates the structural and molecular changes in heart tissue 4 h after multiple injuries with additional hemorrhagic shock using a clinically relevant rodent model of polytrauma. We determined mediators of systemic inflammation (keratinocyte chemoattractant, macrophage chemotactic protein 1), activated complement component C3a and cardiac troponin I in plasma and assessed histological specimen of the mouse heart via standard histomorphology and immunohistochemistry for cellular and subcellular damage and ongoing apoptosis. Further we investigated spatial and quantitative changes of connexin 43 by immunohistochemistry and western blotting. Our results show significantly increased plasma levels of both keratinocyte chemoattractant and cardiac troponin I 4 h after polytrauma and 2 h after induction of hypovolemia. Although we could not detect any morphological changes, immunohistochemical evaluation showed increased level of tissue high-mobility group box 1, which is both a damage-associated molecule and actively released as a danger response signal. Additionally, there was marked lateralization of the cardiac gap-junction protein connexin 43 following combined polytrauma and hemorrhagic shock. These results demonstrate a molecular manifestation of remote injury of cardiac muscle cells in the early phase after polytrauma and hemorrhagic shock with marked disruption of the cardiac gap junction. This disruption of an important component of the electrical conduction system of the heart may lead to arrhythmia and consequently to cardiac dysfunction.</p></div

Histological and humoral markers of cardiac damage.

<p>(A) Plasma levels of cardiac troponin I (cTnI) as a marker of cardiac damage from animals 4 h after the insult of polytrauma and hemorrhagic shock (PTHS; n = 8) and native controls (CTRL, n = 4). Results are presented as amount of protein per total protein in plasma to unmask diluting effects from volume resuscitation. (B) Representative images of immunohistochemical (IHC) assessment of tissue cleaved caspase 3. Magnification: 100x (bar: 50 μm). (C) Representative hematoxylin and eosin stained sections of cardiac tissue, showing no signs of marked histomorphological changes. Magnifications: 100x (upper images, bar: 50 μm) and 200x (lower images, bar: 100 μm) for each group. (D) Representative images and densitometric analysis of IHC preparations of tissue high-mobility group box 1 (HMGB1) showing increased signal in samples from PTHS animals. Magnification: 100x (bar: 50 μm). For histological evaluation: n = 5 (PTHS); n = 5 (CTRL). DSR: density sum red. Experimental means were compared for statistical significance using the unpaired t-test (D) and Mann-Whitney rank sum test (A). *: p<0.05.</p

Plasma levels of inflammatory mediators and hemoglobin.

<p>Levels of (A) haemoglobin (Hb) in blood and (B) macrophage chemotactic protein 1 (MCP-1), (C) keratinocyte chemoattractant (KC) and (D) complement component C3a in plasma of animals 4 h after infliction of polytrauma and hemorrhagic shock (PTHS; n = 7 for Hb, n = 8 for MCP-1, KC and C3a) and native control animals (CTRL; n = 8 for Hb, n = 4 for MCP-1, n = 5 for C3a and KC). Results (B, C, D) are presented as amount of protein per total protein in plasma to unmask diluting effects from volume resuscitation. For statistical comparison of experimental means, unpaired t-tests (A, C, D) and Mann-Whitney rank sum test (B) were performed. *: p<0.05.</p

Experimental blunt chest trauma-induced myocardial inflammation and alteration of gap-junction protein connexin 43

<div><p>Objective</p><p>Severe blunt chest trauma in humans is associated with high mortality rates. Whereas lung tissue damage and lung inflammation after blunt chest trauma have extensively been investigated, the traumatic and posttraumatic effects on the heart remain poorly understood. Therefore, the purpose of this study was to define cardiac injury patterns in an experimental blunt chest trauma model in rats.</p><p>Methods</p><p>Experimental blunt chest trauma was induced by a blast wave in rats, with subsequent analysis of its effects on the heart. The animals were subjected either to a sham or trauma procedure. Systemic markers for cardiac injury were determined after 24 h and 5 days. Postmortem analysis of heart tissue addressed structural injury and inflammation 24 h and 5 days after trauma.</p><p>Results</p><p>Plasma levels of extracellular histones were elevated 24 h and 5 days after blunt chest trauma compared to sham-treated animals. In the heart, up-regulation of interleukin-1β 24 h after trauma and increased myeloperoxidase activity 24 h and 5 days after trauma were accompanied by reduced complement C5a receptor-1 expression 24 h after trauma. Histological analysis revealed extravasation of erythrocytes and immunohistochemical analysis alteration of the pattern of the gap-junction protein connexin 43. Furthermore, a slight reduction of α-actinin and desmin expression in cardiac tissue was found after trauma together with a minor increase in sarcoplasmatic/endoplasmatic reticlulum calcium-ATPase (SERCA) expression.</p><p>Conclusions</p><p>The clinically highly relevant rat model of blast wave-induced blunt chest trauma is associated with cardiac inflammation and structural alterations in cardiac tissue.</p></div

Blood gases after 30, 90 and 120 minutes of incubation.

Blood samples were incubated using the MPC model with either fondaparinux (FPX) or enoxaparin (ENOX) or with the CHC-coating. Measurements via blood gas analysis (BGA) of pO2 and pCO2 were performed before (Baseline) and after 30, 90, and 120 min of incubation. Data was considered exploratory and for further reference, therefore no statistical testing was performed. Values presented as mean (± s.e.m); All samples: n = 5. (EPS)</p

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Local inflammation in cardiac injury after blunt chest trauma.

<p>A. Increased myeloperoxidase (MOP) activity in left ventricular cardiac tissue 24 h and 5 d after blunt chest trauma compared to sham procedure. B. Elevation of proinflammatory cardiodepressive cytokine IL-1β expression in left ventricular homogenates 24 h after blunt chest trauma compared to sham procedure as assessed by qRT-PCR. C. Representative western blot for C5aR1 of left ventricular tissue homogenates. Densitometry revealed diminished C5aR1 protein expression in left ventricular homogenates 24 h after blunt chest trauma compared to sham procedure. D. C5aR expression in left ventricular homogenates 24 h and 5 d after blunt chest trauma and after sham procedure as assessed by qRT-PCR. p<0.05; *differences were significant to sham procedure, For all frames n = 8 for each bar.</p

Systemic and local effects of blunt chest trauma.

<p>Apparence of extracellular histones in plasma 24 h and 5 d after blunt chest trauma or sham, * differences to sham procedure were significant, p<0.05; n = 4 for each bar. B. Representative H.E staining of left ventricles 24 h or 5 d after blunt chest trauma or sham procedure as indicated.</p

Structural alterations in the heart after blunt chest trauma.

<p>Alteration of gap junctional protein connexin 43 (Cx43) after blunt chest trauma in the heart. A. Representative distribution of Cx43 in cardiac tissue after sham procedure or 24 h or 5d after blunt chest trauma as indicated. B. Representative western blot for Cx43 of left ventricular tissue homogenates. Densitometry revealed no significant increase in protein expression in left ventricular homogenates 24 h and 5 d after blunt chest trauma compared to sham procedure. For all frames n = 4 for each bar.</p