Methods in molecular biology
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Limited understanding of the cell biology of the breast and breast cancer hampers our ability to develop new therapeutic approaches. Mouse models of mammary gland development and tumourigenesis are key to developing new insights into the biology of both the normal and diseased tissues. Recent advances have enabled the isolation, molecular characterisation and functional analysis of mouse mammary epithelial cell subpopulations from the normal gland, including subpopulations enriched for stem cell behaviour. Application of these techniques both to the normal mammary gland and to tumour models will promote a better understanding of the nature of the different epithelial cell types in the mammary gland, the origins of mammary tumours and the role of tumour stem cells.
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The UV-B pain model utilizes ultraviolet light to induce a small area of inflammation allowing assessment of mechanical and thermal thresholds. Pharmacologic testing has mainly focused on reduction of primary hyperalgesia, although the effect of analgesics on secondary hyperalgesia has also been investigated. The model requires an instrument to precisely generate controlled UV-B tissue hyperalgesia. ⋯ Tissue is then assessed for inflammation using color Doppler imaging or flare measurements. Heat pain thresholds and pain tolerance are often evaluated using a commercially available thermal sensory testing device. Analgesics can be administered to determine the influence on these clinical endpoints.
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RNA interference (RNAi) has become a powerful tool for modulating gene expression. While delivery of small interfering RNAs (siRNAs) has achieved silencing of pain-related genes in various animal models of nociception, delivery of short-hairpin RNA (shRNA) or artificial miRNA (miRNA) to dorsal root ganglia (DRG) has proven particularly challenging. This chapter describes a highly efficient method for in vivo gene silencing in sensory neurons using replication-defective vectors based on herpes simplex virus (HSV). This method can be utilised to obtain a better understanding of gene function, validate novel gene targets in drug discovery and potentially develop new RNAi-mediated approaches to achieve analgesia.
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Migraine is a high prevalence disorder which affects a significant proportion of the general population, especially women during their central and more productive time of the life, thus causing severe disability. The genetic basis of the disease is unknown and the mechanism is poorly understood. The possibility that following a perturbation in the central nervous system, and particularly in the brainstem, trigeminal neurons become hyperexcitable and produce an uncontrolled release of sensory neuropeptides which eventually results in arterial vasodilatation and neuronal sensitization, has been gaining credit from studies in experimental animals and humans. In particular, experimental and clinical data with antagonists of the calcitonin gene-related peptide (CGRP) propose this molecule and its receptor as a major target for migraine treatment.
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The results of clinical studies on the value of preemptive analgesia are far from being unanimous. There are a number of potential problems related to preemptive analgesia that could lead to controversy regarding its clinical significance. The following potential problems are analyzed: (1) terminology, (2) approach to reveal the effect of preemptive analgesia, (3) verification of the direct pharmacological effect of a treatment, (4) partial preemptive effect in control, (5) intensity of noxious stimuli, (6) difference in a drug concentration between study groups during postoperative period, and (7) outcome measures.