Methods in molecular biology
-
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.
-
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.
-
Normal and tumor stem cells are present in rare quantities in tissues and this has historically represented a major hurdle to in-depth investigations of their biology. In the case of the mammary gland, the relative promiscuity of the immunophenotypical markers described in several studies for the isolation of human and mouse mammary stem cells limits their usefulness, in particular when highly purified mammary stem cell fractions are required for an in-depth molecular and functional characterization (Stingl et al. Nature 439:993-997, 2006; Shackleton et al. ⋯ Cell 138:1083-1095, 2009). Following mammosphere dissociation, the differential degree of PKH26 epifluorescence displayed by stem cells compared to precursor cells is exploited for their purification by FACS sorting. As a result, the scarcely represented PKH26-labeled mammary stem cells are purified to near homogeneity and can be used for further molecular and biological studies.
-
Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. ⋯ However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene.
-
Developments in psychoneuroimmunology (PNI) need to be translated into personalized medicine to achieve better clinical outcomes. One of the most critical steps in this translational process is to identify systemic biomarkers for better diagnosis and treatment. Applications of systems biology approaches in PNI would enable the insights into the correlations among various systems and different levels for the identification of the basic elements of the psychophysiological framework. ⋯ The understanding of the general systemic pathways among different disorders may contribute to the transition from the disease-centered medicine to patient-centered medicine. Integrative strategies targeting these factors and pathways would be useful for the prevention and treatment of a spectrum of diseases that share the common links. Examples of the translational implications of potential PNI biomarkers and networks in diseases including depression, Alzheimer's disease, obesity, cardiovascular disease, stroke, and HIV are discussed in details.