Methods in molecular biology
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Traumatic brain injury (TBI) is one of the most common causes of death and disability, and cerebral hypoxia is a frequently occurring harmful secondary event in TBI patients. The hypoxic conditions that occur on the scene of accident, where the airways are often obstructed or breathing is in other ways impaired, could be reproduced using animal TBI models where oxygen delivery is strictly controlled throughout the entire experimental procedure. ⋯ Different models of traumatic brain injury could be used to inflict desired injury type, whereas effects then could be studied using radiological, physiological and functional tests. In order to confirm that the brain has been affected by a hypoxic injury, appropriate substances in the affected cerebral tissue, cerebrospinal fluid, or serum should be analyzed.
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Controlled cortical impact (CCI) is a commonly used and highly regarded model of brain trauma that uses a pneumatically or electromagnetically controlled piston to induce reproducible and well-controlled injury. The CCI model was originally used in ferrets and it has since been scaled for use in many other species. This chapter will describe the historical development of the CCI model, compare and contrast the pneumatic and electromagnetic models, and summarize key short- and long-term consequences of TBI that have been gleaned using this model. In accordance with the recent efforts to promote high-quality evidence through the reporting of common data elements (CDEs), relevant study details-that should be reported in CCI studies-will be noted.
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Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. ⋯ The use of these tools requires careful consideration with regard to the input data, and the interpretation demands a critical approach. This chapter provides a summary of the most appropriate tools for systems analysis of phosphoproteomics datasets, and the considerations that are critical for acquiring meaningful output.
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We describe here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyzes yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-Infrared fluorescent dye. ⋯ Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues.
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Patients with severe traumatic brain injury (TBI) frequently present with concomitant injuries that may cause secondary brain injury and impact outcomes. Animal models have been developed that combine contemporary models of TBI with a secondary neurologic insult such as hypoxia, shock, long bone fracture, and radiation exposure. ⋯ Here, we review these models and their collective contribution to the literature on TBI. In addition, we provide protocols and notes for two well-characterized models of TBI plus hemorrhagic shock.