Intact Glucocorticoid Receptor Dimerization Is Deleterious in Trauma-Induced Impaired Fracture Healing.

Following severe trauma, fracture healing is impaired because of overwhelming systemic and local inflammation. Glucocorticoids (GCs), acting via the glucocorticoid receptor (GR), influence fracture healing by modulating the trauma-induced immune response. GR dimerization-dependent gene regulation is essential for the anti-inflammatory effects of GCs. Therefore, we investigated in a murine trauma model of combined femur fracture and thoracic trauma, whether effective GR dimerization influences the pathomechanisms of trauma-induced compromised fracture healing. To this end, we used mice with decreased GR dimerization ability (GRdim). The healing process was analyzed by cytokine/chemokine multiplex analysis, flow cytometry, gene-expression analysis, histomorphometry, micro-computed tomography, and biomechanical testing. GRdim mice did not display a systemic or local hyper-inflammation upon combined fracture and thorax trauma. Strikingly, we discovered that GRdim mice were protected from fracture healing impairment induced by the additional thorax trauma. Collectively and in contrast to previous studies describing the beneficial effects of intact GR dimerization in inflammatory models, we report here an adverse role of intact GR dimerization in trauma-induced compromised fracture healing.

CD28 Deficiency Ameliorates Thoracic Blast Exposure-Induced Oxidative Stress and Apoptosis in the Brain through the PI3K/Nrf2/Keap1 Signaling Pathway.

Blast exposure is a worldwide public health concern, but most related research has been focused on direct injury. Thoracic blast exposure-induced neurotrauma is a type of indirect injuries where research is lacking. As CD28 stimulates T cell activation and survival and contributes to inflammation initiation, it may play a role in thoracic blast exposure-induced neurotrauma. However, it has not been investigated. To explore the effects of CD28 on thoracic blast exposure-induced brain injury and its potential molecular mechanisms, a mouse model of thoracic blast exposure-induced brain injury was established. Fifty C57BL/6 wild-type (WT) and fifty CD28 knockout (CD28-/-) mice were randomly divided into five groups (one control group and four model groups), with ten mice (from each of the two models) for each group. Lung and brain tissue and serum samples were collected at 12 h, 24 h, 48 h, and 1 week after thoracic blast exposure. Histopathological changes were detected by hematoxylin-eosin staining. The expressions of inflammatory-related factors were detected by ELISA. Oxidative stress in the brain tissue was evaluated by determining the generation of reactive oxygen species (ROS) and the expressions of thioredoxin (TRX), malondialdehyde (MDA), SOD-1, and SOD-2. Apoptosis in the brain tissue was evaluated by TUNEL staining and the levels of Bax, Bcl-xL, Bad, Cytochrome C, and caspase-3. In addition, proteins of related pathways were also studied by western blotting and immunofluorescence. We found that CD28 deficiency significantly reduced thoracic blast exposure-induced histopathological changes and decreased the levels of inflammatory-related factors, including IL-1ß, TNF-α, and S100ß. In the brain tissue, CD28 deficiency also significantly attenuated thoracic blast exposure-induced generation of ROS and expressions of MDA, TRX, SOD-1, and SOD-2; lowered the number of apoptotic cells and the expression of Bax, cleaved caspase-3, Cytochrome C, and Bad; and maintained Bcl-xL expression. Additionally, CD28 deficiency significantly ameliorated thoracic blast exposure-induced increases of p-PI3K and Keap1 and the decrease of Nrf2 expression in the brain. Our results indicate that CD28 deficiency has a protective effect on thoracic blast exposure-induced brain injury that might be associated with the PI3K/Nrf2/Keap1 signaling pathway.

The Role of Cystathionine-γ-Lyase In Blunt Chest Trauma in Cigarette Smoke Exposed Mice.

Pretraumatic cigarette smoke (CS) exposure aggravates posttraumatic acute lung injury (ALI). Cystathionine-γ-lyase (CSE) protects against ALI and CS exposure-induced chronic obstructive lung disease (COPD). Therefore, we tested the hypothesis whether genetic CSE knockout (CSE) would aggravate posttraumatic ALI after CS exposure. After 3 to 4 weeks of CS exposure, anesthetized wild-type (WT) and CSE mice underwent blunt chest trauma, surgical instrumentation and 4 h of lung-protective mechanical ventilation. We measured hemodynamics, lung mechanics, gas exchange, metabolism, and acid-base status together with blood and tissue cytokine and chemokine levels, tissue expression of mediator proteins, parameters of oxidative and nitrosative stress, and histology. CSE mice without CS exposure showed higher cytokine and chemokine levels, and this was further enhanced by CS exposure, particularly in males. CS exposure in WT mice aggravated posttraumatic alveolar membrane thickening, dystelectasis, and inflammatory cell accumulation, which was associated with higher thoracopulmonary compliance. Pretraumatic CS exposure in CSE mice produced a similar response, except for less alveolar membrane thickening, most likely due to lung hyperinflation. CS-exposed WT mice showed the most pronounced metabolic acidosis, while CS exposure in CSE mice resulted in the lowest blood glucose levels. Urinary output and anesthesia rate were highest in male CS-exposed CSE animals. In conclusion, in murine acute-on-chronic pulmonary disease, CSE knockout aggravated posttraumatic inflammation, which was further worsened upon pretraumatic CS exposure, and this effect was particularly pronounced in males. Hence, maintaining CSE expression is critically important for stress adaptation during ALI and CS-induced COPD, most likely in a gender-dependent manner.

Role of Complement C5 in Experimental Blunt Chest Trauma-Induced Septic Acute Lung Injury (ALI).

BACKGROUND: Severe blunt chest trauma is associated with high mortality. Sepsis represents a serious risk factor for mortality in acute respiratory distress syndrome (ARDS). In septic patients with ARDS complement activation products were found to be elevated in the plasma. In single models like LPS or trauma complement has been studied to some degree, however in clinically highly relevant double hit models such as the one used here little data is available. Here, we hypothesized that absence of C5 is correlated with a decreased inflammatory response in trauma induced septic acute lung injury. METHODS: 12 hrs after DH in mice the local and systemic cytokines and chemokines were quantified by multiplex bead array or ELISA, activated caspase-3 by western blot. Data were analyzed using one-way ANOVA followed by post-hoc Sidak's multiple comparison test (significance, p≤ 0.05). RESULTS: In lung tissue interleukin (IL)-6, monocyte chemo attractant protein-1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF) was elevated in both C5-/- mice and wildtype littermates (wt), whereas caspase-3 was reduced in lungs after DH in C5-/- mice. Systemically, reduced keratinocyte-derived chemokine (KC) levels were observed after DH in C5-/- compared to wt mice. Locally, lung myeloperoxidase (MPO), protein, IL-6, MCP-1 and G-CSF in brochoalveolar lavage fluid (BALF) were elevated after DH in C5-/- compared to wt. CONCLUSIONS: In the complex but clinically relevant DH model the local and systemic inflammatory immune response features both, C5-dependent and C5-independent characteristics. Activation of caspase-3 in lung tissue after DH was C5-dependent whereas local inflammation in lung tissue was C5-independent.

In vivo high-resolution magic angle spinning magnetic and electron paramagnetic resonance spectroscopic analysis of mitochondria-targeted peptide in Drosophila melanogaster with trauma-induced thoracic injury.

Trauma is the most common cause of mortality among individuals aged between 1 and 44 years and the third leading cause of mortality overall in the US. In this study, we examined the effects of trauma on the expression of genes in Drosophila melanogaster, a useful model for investigating genetics and physiology. After trauma was induced by a non-lethal needle puncture of the thorax, we observed the differential expression of genes encoding for mitochondrial uncoupling proteins, as well as those encoding for apoptosis-related and insulin signaling-related proteins, thus indicating muscle functional dysregulation. These results prompted us to examine the link between insulin signaling and mitochondrial dysfunction using in vivo nuclear magnetic resonance (NMR) with complementary electron paramagnetic resonance (EPR) spectroscopy. Trauma significantly increased insulin resistance biomarkers, and the NMR spectral profile of the aged flies with trauma-induced thoracic injury resembled that of insulin-resistant chico mutant flies. In addition, the mitochondrial redox status, as measured by EPR, was significantly altered following trauma, indicating mitochondrial uncoupling. A mitochondria-targeted compound, Szeto-Schiller (SS)-31 that promotes adenosine triphosphate (ATP) synthesis normalized the NMR spectral profile, as well as the mitochondrial redox status of the flies with trauma-induced thoracic injury, as assessed by EPR. Based on these findings, we propose a molecular mechanism responsible for trauma-related mortality and also propose that trauma sequelae in aging are linked to insulin signaling and mitochondrial dysfunction. Our findings further suggest that SS-31 attenuates trauma-associated pathological changes.

Molecular and cellular changes in the lumbar spinal cord following thoracic injury: regulation by treadmill locomotor training.

Traumatic spinal cord injury (SCI) often leads to debilitating loss of locomotor function. Neuroplasticity of spinal circuitry underlies some functional recovery and therefore represents a therapeutic target to improve locomotor function following SCI. However, the cellular and molecular mechanisms mediating neuroplasticity below the lesion level are not fully understood. The present study performed a gene expression profiling in the rat lumbar spinal cord at 1 and 3 weeks after contusive SCI at T9. Another group of rats received treadmill locomotor training (TMT) until 3 weeks, and gene expression profiles were compared between animals with and without TMT. Microarray analysis showed that many inflammation-related genes were robustly upregulated in the lumbar spinal cord at both 1 and 3 weeks after thoracic injury. Notably, several components involved in an early complement activation pathway were concurrently upregulated. In line with the microarray finding, the number of microglia substantially increased not only in the white matter but also in the gray matter. C3 and complement receptor 3 were intensely expressed in the ventral horn after injury. Furthermore, synaptic puncta near ventral motor neurons were frequently colocalized with microglia after injury, implicating complement activation and microglial cells in synaptic remodeling in the lumbar locomotor circuitry after SCI. Interestingly, TMT did not influence the injury-induced upregulation of inflammation-related genes. Instead, TMT restored pre-injury expression patterns of several genes that were downregulated by injury. Notably, TMT increased the expression of genes involved in neuroplasticity (Arc, Nrcam) and angiogenesis (Adam8, Tie1), suggesting that TMT may improve locomotor function in part by promoting neurovascular remodeling in the lumbar motor circuitry.

Association between polymorphisms in the fibrinogen alpha- and beta-genes on the post-trauma fibrinogen increase.

Fibrinogen is an acute phase reactant, and therefore its plasma levels increase after severe injury. Polymorphisms in the fibrinogen alpha and beta genes have been found to be associated with plasma levels of fibrinogen, and it has also been suggested that they are associated with the fibrinogen increase in acute phase situations. In forty-five consecutive patients admitted to the Intensive Care Unit after acute cranial or thoracic trauma, we investigated the influence of four polymorphisms at the fibrinogen loci (-455G/A and BclI (beta gene), TaqI and T/A312 (alpha gene)) on the post-trauma increase of the fibrinogen levels. At admission, fibrinogen levels were comparable in the patients with the different genotypes for the four polymorphisms studied. However, patients carrying the -455A allele of the -455G/A polymorphism had a significantly wider variation and higher peak levels of fibrinogen, during their stay at the intensive care unit, than did the -455GA homozygotes (5.1 g/l (SD 1.3) and 5.9 g/l (SD 1.0), respectively, p<0.05). Such difference was not found for the other studied polymorphisms. The present study suggests that the increase of fibrinogen level in acute phase situations like severe trauma is associated with the beta-gene -455G/A polymorphism.

[Human genotyposcopy: the identification of the species, sex and individual by DNA genetic imprints in a case connected with a murder attempt]. / Genotiposkopiia cheloveka: identifikatsiia vida, pola i lichnosti po geneticheskim otpechatkam DNK v sluchae, sviazannom s pokusheniem na ubiistvo.

Blood stains on a knife were identified by DNA genotyposcopy. The statistical validation method has confirmed that the blood stains on material evidence belonged to the victim, the probability of random coincidence being less than 10(-11). The efficacy of using hypervariable locus-specific DNA probes and the possibility of detecting DNA impressions in blood stains stored for more than 3 months have been demonstrated.