Methods in molecular biology
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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.
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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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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.
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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.
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Protocols for high-resolution respirometry (HRR) of intact cells, permeabilized cells, and permeabilized muscle fibers offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques and small needle biopsies of muscle. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling and substrate control. ⋯ Substrate control with electron entry separately through Complex I (pyruvate + malate or glutamate + malate) or Complex II (succinate + rotenone) restricts ETS capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific branches of the ETS. Oxygen levels are maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental oxygen limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in HRR.