TRAP-5

Aim:Exposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presumably by removing free radicals which otherwise help activate inflammatory response and damage cells.Main methods:RAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell damage, production of inflammatory mediators, inducible nitric oxide synthase (iNOS) expression, NF-κB p65 activation, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined.Key findings:After cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflammatory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Accordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We found, by assaying cells treated with LPS and/or DMPO for 15-60 min, that DMPO inhibited the phosphorylation of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO inhibited LPS-induced ROS production.Significance:DMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.

Membranous organelles are major endogenous sources of reactive oxygen and nitrogen species. When present at high levels, these species can cause macromolecular damage and disease. To better detect and scavenge free radical forms of the reactive species at their sources, we investigated whether nitrone spin traps could be selectively targeted to intracellular membranes using a bioorthogonal imaging approach. Electron paramagnetic resonance imaging demonstrated that the novel cyclic nitrone 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO) could be used to target the nitrone moiety to liposomes composed of phosphatidyl choline. To test localization with authentic membranes in living cells, fluorophores were introduced via strain-promoted alkyne-nitrone cycloaddition (SPANC). Two fluorophore-conjugated alkynes were investigated: hexynamide-fluoresceine (HYA-FL) and dibenzylcyclooctyne-PEG4-5/6-sulforhodamine B (DBCO-Rhod). Computational and mass spectrometry experiments confirmed the cycloadduct formation of DBCO-Rhod (but not HYA-FL) with diC12PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12PO and DBCO-Rhod demonstrated clear localization of fluorescence with intracellular membranes. These results indicate that targeting of nitrone spin traps to cellular membranes is feasible, and that a bioorthogonal approach can aid the interrogation of their intracellular compartmentalization properties.

Introduction:Pediatric trauma management is a high-stress, high-risk, low-frequency event, and exposure through simulation can help identify and address knowledge gaps. Pediatric residents are likely to provide care for children with traumatic injuries, and it is important they are skilled in performing a rapid trauma assessment.Methods:We developed a simulation-based rapid pediatric trauma assessment curriculum for pediatric residents in the setting of a mass casualty disaster. The patients were 5-year-olds portrayed by mannequins with varying injuries including intracranial hemorrhage, solid organ injury, and open extremity fractures. Critical actions included assigning roles, completing primary assessment within 2 minutes, and giving summary statement and management priorities within 5 minutes using clear communication techniques. We created a badge-sized reference card as well as scenario-specific debriefing tools to facilitate assessment and discussion of learning objectives following the simulation.Results:We conducted two sessions with a total of 49 participants. The case was rated as highly relevant (session 1,m= 4.7; session 2,m= 4.9) and realistic (session 1,m= 4.8; session 2,m= 4.4) by participants on a 5-point Likert scale. During the two sessions participants completed the primary survey in an average of 2.46 and 2.29 minutes, respectively, and the secondary survey with summary statement in an average of 5.08 and 4.27 minutes, respectively.Discussion:This educational resource supports the setup, production, and debriefing of a low-fidelity simulation focused on the pediatric trauma assessment for the novice learner. Also included are educational reference materials and a participant evaluation form.