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Introduction: Deregulation of polymorphonuclear neutrophils (PMNs) is an essential step in the development of inflammatory complications upon trauma. Different neutrophil subtypes have been identified recently, however, the role of neutrophil subtypes in immunoregulation upon trauma is unclear. We hypothesize that extensive trauma surgery causes instant progressive heterogeneity of the blood neutrophil pool, and increased appearance of young (CD16(dim)/CD62L(bright)) neutrophils in peripheral blood.Material and methods: A standardized extensive thoraco-abdominal porcine trauma surgery model was utilized, and 12 animals were included. Blood was collected at defined timepoints and neutrophil numbers and subtypes were studied by flowcytometry. Neutrophil subtypes were identified by differences in cell surface expression levels of CD16 (Fc gamma RIII) and CD62L (L-selectin). Porcine neutrophil subtypes were further characterized after flow sorting.Results: Eleven animals survived the 3-hour surgical protocol. Neutrophil numbers dropped significantly from a mean of 8,6 +/- 3,5 x 10(6) to 2,4 +/- 1,8 x 10(6) cells/ml during 180 min, (p

Deregulation of polymorphonuclear neutrophils (PMNs) is an essential step in the development of inflammatory complications upon trauma. Different neutrophil subtypes have been identified recently, however, the role of neutrophil subtypes in immunoregulation upon trauma is unclear. We hypothesize that extensive trauma surgery causes instant progressive heterogeneity of the blood neutrophil pool, and increased appearance of young (CD16A standardized extensive thoraco-abdominal porcine trauma surgery model was utilized, and 12 animals were included. Blood was collected at defined timepoints and neutrophil numbers and subtypes were studied by flowcytometry. Neutrophil subtypes were identified by differences in cell surface expression levels of CD16 (FcγRIII) and CD62L (L-selectin). Porcine neutrophil subtypes were further characterized after flow sorting.Eleven animals survived the 3-hour surgical protocol. Neutrophil numbers dropped significantly from a mean of 8,6 ± 3,5 × 10Standardized extensive trauma surgery was associated with instant progressive neutropenia and increased heterogeneity of the blood neutrophil pool. Furthermore, three different neutrophil subsets in peripheral porcine blood were identified over the course of surgery. Further studies should clarify their precise role in the development of early organ failure upon extensive trauma surgery. This for the first time exemplifies experimentally the time constraints and impact of damage control surgery after severe trauma.

Background Hemorrhagic shock (HS) is known to induce an inflammatory response by activating the immune system. This response is mainly caused by primed polymorphonuclear granulocytes (PMNs). Trauma patients often require mechanical ventilation (MV), which can cause additional pulmonary and systemic inflammation. The aim of this study was to evaluate the role of MV in the development of systemic and pulmonary inflammation in a HS model in rats. Materials and methods In male Sprague–Dawley rats, the effect of MV and HS on the systemic and pulmonary inflammatory responses was measured and compared. In five groups (control, sham, MV, HS, and MV + HS), the inflammation was measured at time point 300 min after the start of the experiment. Results The systemic inflammatory response, expressed in absolute numbers of PMNs in blood and blood growth related oncogene (GRO-KC) levels, was significantly higher in MV rats compared with that in other groups. The pulmonary inflammatory response, expressed by PMNs in bronchoalveolar lavage fluid (BALF), BALF interleukin 6, BALF GRO-KC, and myeloperoxidase activity, was significantly higher in all ventilated rats compared with that in the controls or HS rats. There was, however, no additional effect of HS in MV as the inflammatory indices were similar in both groups. Conclusions Our data show that HS alone has minimal effect on the development of inflammation. MV (alone or in combination with HS) is the determining factor in inducing an inflammatory response. These results emphasize the importance of local (pulmonary) ventilation-induced damage in the development of systemic inflammation.

During acute inflammation, 3 neutrophil subsets are found in the blood: neutrophils with a conventional segmented nucleus, neutrophils with a banded nucleus, and T-cell-suppressing CD62L

Background and purpose—Nonunion is common in femoral fractures. Previous studies suggested that the systemic immune response after trauma can interfere with fracture healing. Therefore, we investigated whether there is a relation between peripheral blood cell counts and healing of femur fractures. Patients and methods—62 multi-trauma patients with a femoral fracture presenting at the University Medical Centre Utrecht between 2007 and 2013 were retrospectively included. Peripheral blood cell counts from hematological analyzers were recorded from the 1st through the 14th day of the hospital stay. Generalized estimating equations were used to compare outcome groups. Results—12 of the 62 patients developed nonunion of the femoral fracture. The peripheral blood-count curves of total leukocytes, neutrophils, monocytes, lymphocytes, and platelets were all statistically significantly lower in patients with nonunion, coinciding with significantly higher CRP levels during the first 2 weeks after trauma in these patients. Interpretation—Patients who developed femoral nonunion after major trauma demonstrated lower numbers of myeloid cells in the peripheral blood than patients with normal fracture healing. This absent rise of myeloid cells seems to be related to a more severe post-traumatic immune response.

Neutrophils comprise a heterogeneous population of cells essential for bacterial eradication, and defects in neutrophil function are associated with increased susceptibility to infection. In this study, neutrophils from healthy controls were shown to prevent bacterial proliferation for at least 48 hours when cocultured with methicillin-resistant Staphylococcus aureus (MRSA) in tissue-like scaffolds by establishing a bacteriostatic environment inside their phagolysosome. This intracellular bacterial containment is independent of reactive oxygen species because neutrophils that lack a functional nicotinamide adenine dinucleotide phosphate-oxidase complex displayed no defect in intracellular bacterial containment, whereas killing of the pathogen was impaired. During acute inflammation, a subset of CD16bright/CD62Ldim hypersegmented neutrophils displayed normal phagocytosis associated with a remarkably poor capacity to contain bacteria intracellularly. Conversely, CD16dim-banded neutrophils were the only neutrophil subset that adequately contained MRSA. These findings demonstrate a clear neutrophil heterogeneity in their antimicrobial capacity and the appearance of neutrophil subsets with a clear differentiation in functionality during acute inflammation. Furthermore, this study provides an evolutionary basis for the rapid release of banded neutrophils into the circulation during acute inflammation.

Myocardial infarction (MI) induces an inflammatory response in which neutrophils fulfill a prominent role. Mean neutrophil volume (MNV) represents the average size of the circulating neutrophil population. Our goal was to determine the effect of MI on MNV and investigate the mechanisms behind MNV elevation. MNV of 84 MI patients was compared with the MNV of 209 stable angina patients and correlated to simultaneously measured CK levels. Fourteen pigs were subjected to temporary coronary balloon occlusion and blood was sampled at multiple time points to measure MNV. Echocardiography was performed followed by ex vivo infarct size assessment after 72 h. MNV was higher in MI patients compared to stable angina patients (602 SD26 AU vs. 580 SD20 AU, p

Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting translational failure. Experimentally, MI is induced artificially and the effect of the experimental procedures may influence outcome and thus clinical applicability. The aim of this study was to investigate if invasive surgery, as in the common open chest MI model affects IS and cardiac function. Twenty female landrace pigs were subjected to MI by transluminal balloon occlusion. In 10 of 20 pigs, balloon occlusion was preceded by invasive surgery (medial sternotomy). After 72 hrs, pigs were subjected to echocardiography and Evans blue/triphenyl tetrazoliumchloride double staining to determine IS and area at risk. Quantification of IS showed a significant IS reduction in the open chest group compared to the closed chest group (IS versus area at risk: 50.9 ± 5.4% versus 69.9 ± 3.4%, P = 0.007). End systolic LV volume and LV ejection fraction measured by echocardiography at follow-up differed significantly between both groups (51 ± 5 ml versus 65 ± 3 ml, P = 0.033; 47.5 ± 2.6% versus 38.8 ± 1.2%, P = 0.005). The inflammatory response in the damaged myocardium did not differ between groups. This study indicates that invasive surgery reduces IS and preserves cardiac function in a porcine MI model. Future studies need to elucidate the effect of infarct induction technique on the efficacy of pharmacological therapies in large animal cardioprotection studies.

Introduction.Flow cytometry markers have been proposed as useful predictors for the occurrence of posttraumatic inflammatory complications. However, currently the need for a dedicated laboratory and the labour-intensive analytical procedures make these markers less suitable for clinical practice. We tested an approach to overcome these limitations.Material and Methods.Neutrophils of healthy donors were incubated with antibodies commonly used in trauma research: CD11b (MAC-1), L-selectin (CD62L), FcγRIII (CD16), and FcγRII (CD32) in active form (MoPhab A27). Flow cytometric analysis was performed both on a FACSCalibur, a standard flow cytometer, and on a Cell-Dyn Sapphire, a routine haematology analyser.Results.There was a high level of agreement between the two types of analysers, with 41% for FcγRIII, 80% for L-selectin, 98% for CD11b, and even a 100% agreement for active FcγRII. Moreover, analysis on the routine haematology analyser was possible in less than a quarter of the time in comparison to the flow cytometer.Conclusion.Analysis of neutrophil phenotype on the Cell-Dyn Sapphire leads to the same conclusion compared to a standard flow cytometer. The markedly reduced time necessary for analysis and reduced labour intensity constitutes a step forward in implementation of this type of analysis in clinical diagnostics in trauma research.

Background: Successful translation of pre-clinical treatment strategies into clinical therapies depends on reproducible, conclusive models. To date, two distinctive procedures for the induction of myocardial ischemia-reperfusion to test novel therapies for myocardial infarction (MI) are commonly used. The first method applies coronary artery ligation via an open chest procedure, requiring invasive surgery. Alternatively, the coronary artery is occluded by balloon inflation via a transluminal, minimally invasive procedure. In the current study we hypothesized that invasive surgery as for open chest procedures reduces myocardial infarct size and preserves cardiac function post-MI. Methods: Pigs (n=14) were subjected to 75-minute balloon occlusion followed by 3 days of reperfusion. In 7 pigs, the balloon occlusion was preceded by medial sternotomy (MS) that is normally performed for open chest coronary artery ligation. In the remaining 7 pigs the same procedure was performed without sternotomy (NS). Cardiac damage was determined by histological infarct size, 3D-echocardiography and troponin I measurements. The area at risk (AAR) was assessed through Evans blue injection. Circulating leukocyte numbers were measured at baseline, ischemia and reperfusion. Results: Heart rate and blood pressure did not differ significantly between the two groups during the procedure. Also the area at risk (AAR) was comparable between groups (19.8±1.6% (NS) vs. 19.7±3.4% (MS), p=ns). We observed a significantly smaller infarct size as a percentage of the AAR in pigs subjected to sternotomy (67.6±10.2% (NS) vs. 49.3±14.8% (MS), p=0.024), culminating in a significantly higher left ventricular ejection fraction (39.5±3.8% (NS) vs. 47.9±7.1% (MS), p=0.022). Furthermore, troponin I levels 1 hour after reperfusion were significantly different between the two groups (127.6±100.4 ng/mL (NS) vs. 24.5±13.1 (MS) ng/mL, p=0.030). Leukocyte count after 30 minutes of reperfusion was higher in the MS group than the NS group (14.5±3.1*106 cell/mL (NS) vs. 19.0±3.0*106 cell/mL (MS); p=0.023). Conclusion: Invasive surgery required for an open chest temporary coronary artery ligation reduces infarct size compared to minimally invasive surgery in a porcine model of myocardial infarction, despite increased leukocyte numbers. This confounding effect should be taken into account when assessing therapeutic efficacy for novel treatment strategies to treat myocardial infarction.

Background Systemic inflammation in response to a femur fracture and the additional fixation is associated with inflammatory complications, such as acute respiratory distress syndrome and multiple organ dysfunction syndrome. The injury itself, but also the additional procedure of femoral fixation induces a release of pro-inflammatory cytokines such as interleukin-6. This results in an aggravation of the initial systemic inflammatory response, and can cause an increased risk for the development of inflammatory complications. Recent studies have shown that administration of the serum protein C1-esterase inhibitor can significantly reduce the release of circulating pro-inflammatory cytokines in response to acute systemic inflammation. Objective Attenuation of the surgery-induced additional systemic inflammatory response by perioperative treatment with C1-esterase inhibitor of trauma patients with a femur fracture. Methods The study is designed as a double-blind randomized placebo-controlled trial. Trauma patients with a femur fracture, Injury Severity Score ≥ 18 and age 18-80 years are included after obtaining informed consent. They are randomized for administration of 200 U/kg C1-esterase inhibitor intravenously or placebo (saline 0.9%) just before the start of the procedure of femoral fixation. The primary endpoint of the study is Δ interleukin-6, measured at t = 0, just before start of the femur fixation surgery and administration of C1-esterase inhibitor, and t = 6, 6 hours after administration of C1-esterase inhibitor and the femur fixation. Conclusion This study intents to identify C1-esterase inhibitor as a safe and potent anti-inflammatory agent, that is capable of suppressing systemic inflammation in trauma patients. This might facilitate early total care procedures by lowering the risk of inflammation in response to the surgical intervention. This could result in increased functional outcomes and reduced health care related costs. Trial registration clinicaltrials.gov NCT01275976 (January 12th 2011)