Methods in molecular biology
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Many genetic mutations result in the disruption of (alternative) splicing. Prime examples are the SMN1 and SMN2 genes: a silent mutation in SMN2 leads to the skipping of the constitutive exon 7 in the majority of SMN2 transcripts, while this exon is generally included in SMN1 transcripts. ⋯ There are proteins and drugs that can chance alternative splicing events, e.g. increase the inclusion of exon 7 in SMN2. This chapter describes mini-genes and methods that can be employed to screen for candidate proteins and drugs.
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Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex biological samples. Improved instrumentation, quantification strategies and data analysis tools now enable protein analysis on a genome-wide scale. ⋯ The spectrum of applications ranges from bacteria and eukaryotic cell culture systems to multicellular organisms. Here, we provide a step-by-step protocol on how to plan and perform large-scale quantitative proteome analysis using SILAC, from sample preparation to final data analysis.
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The spinal nerve ligation model of neuropathic pain in rats, as originally described by Kim and Chung (Pain 50:355-363, 1992), provides an excellent venue to study the antinociception and modulation effects of pulsed radiofrequency (PRF) current in pain processing. We describe the procedure of application of PRF current near the exposed L5 dorsal root ganglion (DRG) in rats with L5 spinal nerve ligation injury-induced behavioral hypersensitivity. This method employs the direct visualization of the L5 DRG, allowing for confirmation of the location of the PRF probe adjacent to the DRG.
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Astrocytes produce numerous mediators under conditions of inflammation in the central nervous system. One such mediator is nitric oxide (NO) derived from nitric oxide synthase-2 (NOS-2), the high output, inducible NOS isoform. Expression of NOS-2 and production of NO can be stimulated in astrocyte cultures by combinations of cytokines and lipopolysaccharide, a gram-negative bacterial endotoxin. This chapter details methods to induce and analyze NOS-2 expression and NO production in astrocyte cultures.
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Orofacial pain remains an understudied area in pain research given that most attention has been focused on the spinal system. In this chapter, animal models of neuropathic and inflammatory orofacial pain are presented. Four different types of pain behavior tests are then described for assessing evoked and spontaneous pain behavior in addition to conditional reward behavior. The use of a combination of different pain models and behavior assessments is needed to aid in understanding the mechanisms contributing to orofacial pain in humans for developing effective therapy.