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PurposeTrauma and hemorrhagic shock (T/HS) is a major cause of morbidity and mortality. Existing treatment options are largely limited to source control and fluid and blood repletion. Previously, we have shown that enteral protease inhibition improves outcomes in experimental models of T/HS by protecting the gut from malperfusion and ischemia. However, enteral protease inhibition was achieved invasively, by laparotomy and direct injection of tranexamic acid (TXA) into the small intestine. In this study, we tested a minimally invasive method of enteral protease inhibitor infusion in experimental T/HS that can be readily adapted for clinical use.MethodsWistar rats were exsanguinated to a mean arterial blood pressure (MABP) of 40 mmHg, with laparotomy to induce trauma. Hypovolemia was maintained for 120 min and was followed by reperfusion of shed blood. Animals were monitored for an additional 120 min. A modified orogastric multi-lumen tube was developed to enable rapid enteral infusion of a protease inhibitor solution while simultaneously mitigating risk of reflux aspiration into the airways. The catheter was used to deliver TXA (T/HS + TXA) or vehicle (T/HS) continuously into the proximal small intestine, starting 20 min into the ischemic period.ResultsRats treated with enteral protease inhibition (T/HS + TXA) displayed improved outcomes compared to control animals (T/HS), including significantly improved MABP (p = 0.022) and lactate (p = 0.044). Mass spectrometry-based analysis of the plasma peptidome after T/HS indicated mitigation of systemic proteolysis in T/HS + TXA.ConclusionMinimally invasive, continuous enteral protease inhibitor delivery improves outcomes in T/HS and is readily translatable to the clinical arena.

PurposeTrauma and hemorrhagic shock (T/HS) is a major cause of morbidity and mortality. Existing treatment options are largely limited to source control and fluid and blood repletion. Previously, we have shown that enteral protease inhibition improves outcomes in experimental models of T/HS by protecting the gut from malperfusion and ischemia. However, enteral protease inhibition was achieved invasively, by laparotomy and direct injection of tranexamic acid (TXA) into the small intestine. In this study, we tested a minimally invasive method of enteral protease inhibitor infusion in experimental T/HS that can be readily adapted for clinical use.MethodsWistar rats were exsanguinated to a mean arterial blood pressure (MABP) of 40 mmHg, with laparotomy to induce trauma. Hypovolemia was maintained for 120 min and was followed by reperfusion of shed blood. Animals were monitored for an additional 120 min. A modified orogastric multi-lumen tube was developed to enable rapid enteral infusion of a protease inhibitor solution while simultaneously mitigating risk of reflux aspiration into the airways. The catheter was used to deliver TXA (T/HS + TXA) or vehicle (T/HS) continuously into the proximal small intestine, starting 20 min into the ischemic period.ResultsRats treated with enteral protease inhibition (T/HS + TXA) displayed improved outcomes compared to control animals (T/HS), including significantly improved MABP (p = 0.022) and lactate (p = 0.044). Mass spectrometry-based analysis of the plasma peptidome after T/HS indicated mitigation of systemic proteolysis in T/HS + TXA.ConclusionMinimally invasive, continuous enteral protease inhibitor delivery improves outcomes in T/HS and is readily translatable to the clinical arena.

There is a need to develop markers for early detection of organ failure in shock that can be noninvasively measured at point of care. We explore here the use of volatile organic compounds (VOCs) in expired air in a rat peritonitis shock model. Expired breath samples were collected into Tedlar gas bags and analyzed by standardized gas chromatography. The gas chromatograms were digitally analyzed for presence of peak amounts over a range of Kovach indices. Following the induction of peritonitis, selected volatile compounds were detected within about 1 h, which remained at elevated amounts over a 6 h observation period. These VOCs were not present in control animals without peritonitis. Comparisons with know VOCs indicate that they include 1,4-diaminobutane and trimethylamine N-oxide. When pancreatic digestive proteases were blocked with tranexamic acid in the intestine and peritoneum, a procedure that serves to reduce organ failure in shock, the amounts of VOCs in the breath decreased spontaneously to control values without peritonitis. These results indicate that peritonitis shock is accompanied by development of volatile organic compounds that may be generated by digestive enzymes in the small intestine. VOCs may serve as indicators for detection of early forms of autodigestion by digestive proteases.

It has been previously shown that intestinal proteases translocate into the circulation during hemorrhagic shock and contribute to proteolysis in distal organs. However, consequences of this phenomenon have not previously been investigated using high-throughput approaches. Here, a shotgun label-free quantitative proteomic approach was utilized to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats to verify the possible role of uncontrolled proteolytic activity in shock. Plasma was collected from rats after hemorrhagic shock (HS) consisting of 2-h hypovolemia followed by 2-h reperfusion, and from healthy control (CTRL) rats. A new two-step enrichment method was applied to selectively extract peptides and low molecular weight proteins from plasma, and directly analyze these samples by tandem mass spectrometry. One hundred twenty-six circulating peptides were identified in CTRL and 295 in HS animals. Ninety-six peptides were present in both conditions; of these, 57 increased and 30 decreased in shock. In total, 256 peptides were increased or present only in HS confirming a general increase in proteolytic activity in shock. Analysis of the proteases that potentially generated the identified peptides suggests that the larger relative contribution to the proteolytic activity in shock is due to chymotryptic-like proteases. These results provide quantitative confirmation that extensive, system-wide proteolysis is part of the complex pathologic phenomena occurring in hemorrhagic shock.

Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance, but the mechanisms are unknown. We recently demonstrated that reduction of the extracellular domain of the insulin receptor by degrading proteases may lead to a reduced ability to maintain normal plasma glucose values. In shock, transfer of digestive enzymes from the lumen of the intestine into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors is effective in reducing the level of the inflammatory reactions. To determine the degree to which blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg for 2 h) at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid in polyethylene glycol and physiological electrolyte solution), and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared with control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine led to a lesser decrease in insulin receptor density compared with controls without protease blockade. Glucose tolerance test indicates a significant increase in plasma glucose levels 2 h after hemorrhagic shock, which are reduced to control values in the presence of protease inhibition in the lumen of the intestine. The transient reduction of the plasma glucose levels after an insulin bolus is significantly attenuated after shock but is restored when digestive enzymes in the lumen of the intestine are blocked. These results suggest that in hemorrhagic shock elevated microvascular extracellular digestive enzyme activity causes insulin receptor dysfunction, hyperglycemia, and reduced ability to regulate blood glucose values.

Physiological fluid shear stress evokes pseudopod retraction in normal leukocytes by a mechanism that involves the formyl peptide receptor (FPR) as mechanosensor. In hypertensives, such as the spontaneously hypertensive rat (SHR), leukocytes lack the normal fluid shear response. The increased activity of matrix metalloproteinases (MMPs, including MMP-9) in SHR plasma is associated with cleavage of several cell membrane receptors. We hypothesize that the attenuated fluid shear response in leukocytes (neutrophils) of the SHR is due to extracellular proteolytic cleavage of the FPR. We show that suspended SHR neutrophils in whole blood sheared in a cone-and-plate device or individual neutrophils adherent to a glass surface and subject to fluid shear exhibited reduced pseudopod retractions compared with neutrophils of control Wistar-Kyoto (WKY) rats. SHR neutrophils and naive Wistar rat neutrophils exposed to SHR plasma also exhibited impaired fluid shear responses as shown by their inability to project pseudopods with fluid shear. Labeling of extracellular FPR revealed that the FPR density in SHR neutrophils is on average 27% reduced compared with those of the WKY rats. Exposure of Wistar rat neutrophils to the gelatinase MMP-9 (final concentration 5 nM) led to attenuation of fluid shear response and decrease in extracellular FPR density. Chronic treatment of the SHR with a broad-acting MMP inhibitor, doxycycline, significantly improved the fluid shear response and increased the FPR extracellular density of SHR neutrophils. These results suggest that proteolytic cleavage of the FPR may interfere with normal fluid shear-induced pseudopod retractions in SHR neutrophils. formyl peptide receptor; pseudopod projection; cone-and-plate device; N-formyl-methionine-leucine-phenylalanine; immunohistochemistry doi: 10.1152/ajpcell.00157.2010.

Research was conducted to examine the enhanced DNA fragmentation in the thymus of spontaneously hypertensive rats. The in situ deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end-labeling approach was applied to the thymus tissue of spontaneously hypertensive rat (SHR) as well as Wistar-Kyoto rats with or without adrenalectomy. Results indicate that increased DNA fragmentation is one of the mechanisms associated with thymic atrophy, a feature reflecting immune suppression in SHR.

Microsurgical methods and imaging techniques were developed for the detailed study of normal murine embryos' left ventricular (LV) chamber and the LV ejection fraction at embryo day (ED) 10.5 to 11 and ED13.5 to 14.5. The methods used intravital color and fluorescence videomicroscopy. The methods led to the study of systolic and diastolic LV chamber volumes and function. These methods could be important tools in the in vivo studies of size, form and capacity of the ventricles in mutant murine embryos.

We have recently characterized a cardiac model of ventricular chamber defects in retinoid X receptor [alpha] (RXR[alpha]) homozygous mutant ( - / - ) gene-targeted mice. These-mice display generalized edema, ventricular chamber hypoplasia, and muscular septal defects, and they die at embryonic day 15. To substantiate our hypothesis that the embryos are dying of cardiac pump failure, we have used digital bright-field and fluorescent video microscopy and in vivo microinjection of fluorescein-labeled albumin to analyze cardiac function. The affected embryos showed depressed ventricular function (average left ventricular area ejection fraction, 14%), ventricular septal defects, and various degrees of atrioventricular block not seen in the RXR[alpha] wild-type (+/+) and heterozygous (+/-) littermates (average left ventricular area eJection fraction, 50%). The molecular mechanisms involved in these ventricular defects were studied by evaluating expression of cardiac-specific genes known to be developmentally regulated. By in situ hybridization, aberrant, persistent expression of the atrial isoform of myosin light chain 2 was identified in the ventricles. We hypothesize that retinoic acid provides a critical signal mediated through the RXRa pathway that is required to allow progression of development of the ventricular region of the heart from its early atrial-like form to the thick-walled adult ventricle. The conduction system disturbances found in the RXR[alpha] -/- embryos may reflect a requirement of the developing conduction system for the RXRa signaling pathway, or it may be secondary to the failure of septal development.

Endogenous nitric oxide regulates oxidative changes in cells and arteriolar and venular microvascular endothelium and also prevents leukocyte margination in venules. An iron-catalyzed oxidative process catalyzes intracellular hyperoxide generation in microvascular endothelium. This is enhanced by endogenous NO suppression and the hyperoxide release regulates leukocyte recruitment.

A study was conducted to define temporal alterations of oxidative metabolism in microvascular endothelium in vivo connected with neutrophil adhesion and diapedesis. In postcapillary venules where neutrophils display adhesion and transendothelial transport, the endothelial oxidative events in microvasculature treated with platelet-activating factor (PAF) take place. An endothelial cell-dependent oxyradical propagation function is involved in venular oxidative alterations during neutrophil adhesion by a reaction which is iron-catalyzed, and through which the limited number of adherent neutrophils can cause considerable oxidative stress on venular endothelium.

Effective training requires a professional approach and one that fits the style of the facilitator and the environment. Collaboration and relationships are central concepts to such an approach. This article describes the foundation and core values of a 'professionally packaged' training and four strategies for trainers to set a professional tone. Also included are eight types of participants that can be challenging and ways to handle them with dignity and respect. [ABSTRACT FROM AUTHOR]

The transitional process that takes place when a Child and Youth Practitioner moves into their first supervisory position is one that presents many challenges and a number of exciting opportunities to increase their impact on quality care for children and families. It also presents an opportunity to help others grow and develop their practice. The new supervisor should keep in mind that the process of becoming an excellent supervisor is a developmental one which will take some time and parallel much of the developmental process they experienced as a direct practitioner. The new supervisor should be aware that excellent supervision is rooted in relationship and the same foundational practices and values of the Child and Youth Care Profession in general. This article will present a framework to help the newly appointed supervisor better understand and make this challenging and exciting transition in their career. [ABSTRACT FROM AUTHOR]