Methods in molecular biology
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Physical trauma in the central nervous system (CNS) is usually the result of a number of forces in different directions and dimensions. A large number of experimental models have been developed to improve the possibilities to understand the outcome of CNS trauma. ⋯ Models can serve different needs, such as: to test new treatments for injuries, to reveal thresholds for injuries, to provide a better understanding of injury mechanisms, or to test tools and methods for translation between experiments and clinical data. In this chapter, we will discuss on the validation of models and translation between experimental and clinical studies.
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Luminescence exerts an ideal optical readout for imaging living subjects including no external light source, whereas the dim luminescence and poor color pallet should be addressed for the better utilities. To address the demerits and to prevail the advantages, we developed a bright luminescent protein, named yellow Nano-lantern, exhibiting about 10-20 times brighter than wild-type RLuc. In this chapter, we demonstrate two luminescence-based protocols in detail: i.e., (a) multicolor visualization of Ca(2+) dynamics in different cellular compartments in a single cell using Ca(2+) indicators based on cyan- and orange-Nano-lanterns and (b) video-rate tumor detection in a freely moving mouse using yellow Nano-lantern.
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Traumatic brain injury (TBI) is the leading cause of death in young adults in industrialized nations and in the developing world the WHO considers TBI a silent epidemic caused by an increasing number of traffic accidents. Despite the major improvement of TBI outcome in the acute setting in the past 20 years, the assessment, therapeutic interventions, and prevention of long-term complications remain a challenge. ⋯ In addition, limitations and advantages of each TBI model are mentioned. This will hopefully give an insight into the possibilities of each model and be of value in choosing one when designing a study.
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The development and screening of pharmacological modulators of histone deacetylases (HDACs), and particularly sirtuins, is a promising field for the identification of new drugs susceptible to be used for treatment strategies in a large array of welfare-associated, autoimmune and oncologic diseases. Here we describe a comprehensive protocol to evaluate the impact of sirtuin-targeting drugs on inflammatory and innate immune responses in vitro and in a preclinical mouse model of endotoxemia. We first provide an overview on strategies to design in vitro experiments, then focus on the analysis of cytokine production by primary macrophages and RAW 267.7 macrophages at the mRNA and protein levels, and finally describe the setup and follow-up of a mouse model of inflammation-driven endotoxic shock.
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Traumatic brain injury (TBI) is the leading cause of death and disability for people under 45 years of age. Clinical TBI is often the result of disparate forces resulting in heterogeneous injuries. Preclinical modeling of TBI is a vital tool for studying the complex cascade of metabolic, cellular, and molecular post-TBI events collectively termed secondary injury. ⋯ This chapter details the most widely used models of preclinical TBI, including the controlled cortical impact, fluid percussion, blast, and closed head models. Each of these models replicates particular critical aspects of clinical TBI. Prior to selecting a preclinical TBI model, it is important to address what aspect of human TBI is being sought to evaluate.