Methods in molecular biology
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The cold pressor test is a reliable pain model in which subjects submerge their hands and forearms into ice water while onset to pain, pain intensity, and tolerance are assessed. Although originally developed as a model for hypertension, the paradigm leads to development of reproducible pain responses allowing assessment to analgesic medications. ⋯ A recent study suggests that methodological discrepancies may contribute to such inconsistencies. The model may be more reproducible by utilizing consistent protocols.
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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.
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In the last decade, radiological neuroimaging techniques have enhanced the study of mechanisms involved in the development and maintenance of neuropathic pain. Recent findings suggest that neuropathic pain in certain pain syndromes (e.g., complex regional pain syndrome/reflex sympathic dystrophy, phantom-limb pain) is associated with a functional reorganization and hyperexitability of the somatosensory and motor cortex. Studies showing that the reversal of cortical reorganization in patients with spontaneous or provoked pain is accompanied by pain relief stimulated the search for novel alternatives how to modulate the cortical excitability as a strategy to relieve pain. ⋯ Both techniques (TMS and tDCS) have been clinically investigated in healthy volunteers as well as in patients with various clinical pathologies and variety of pain syndromes. Although there is less evidence on tDCS as compared with TMS, the findings on tDCS in patients with pain are promising, showing an analgesic effect of tDCS, and observations up to date justify the use of tDCS for the treatment of pain in selected patient populations. tDCS has been shown to be very safe if utilized within the current protocols. In addition, tDCS has been proven to be easy to apply, portable and not expensive, which further enhances great clinical potential of this technique.
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The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. ⋯ The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.
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Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Recently, our group demonstrated that three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, are associated with experimental pain sensitivity and onset of temporomandibular joint disorder. In order to determine the functional mechanisms whereby these haplotypes contribute to pain processing, a series of in vitro experiments were performed. ⋯ Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data provide the first demonstration that combinations of commonly observed alleles in the coding region of the human COMT gene can significantly affect the secondary structure of corresponding mRNA transcripts, which in turn leads to dramatic alterations in the translation efficiency of enzyme crucial for a variety of essential functions. The protocols applied to the study of these molecular genetic mechanisms are detailed herein.