Methods in molecular biology
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The study of procedural sedation and analgesia has experienced significant development recently. As specific procedural sedation and analgesia agents have been developed and introduced into clinical practice, safety and efficacy studies have been conducted. ⋯ As procedural sedation and analgesia research has expanded, measurement techniques have been refined to allow for precise comparisons between smaller groups of subjects to improve the capacity to compare these procedures. We have used capnography, bispectral EEG analysis, and subject perceptions of pain and recall as surrogate predictors of adverse events in order to compare agents and procedural techniques in procedural sedation and analgesia.
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Animal models of tissue injury have been used to investigate the mechanisms of pain. Here, we describe a variety of animal models that have been used to mimic acute surgical pain in human subjects, which include the plantar, tail, and gastrocnemius incision models. We also provide discussion on animal models of laparotomy, thoracotomy, visceral pain, and bone injury. Preclinical studies using these models have provided insights into the mechanisms and causes of acute surgical pain as well as the treatment options to control postsurgical pain.
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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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Protein kinases (PKs) are widely recognized as valuable targets for disease diagnosis and drug discovery. For this reason, we have developed a sensitive peptide microarray for detecting intracellular PK activity. Peptides are immobilized on a glutaraldehyde-premodified high-amino terminal glass slide, by spotting 2 nL volumes of substrate peptide solutions with an automated microarray spotter. ⋯ The peptide microarray system involves simple peptide immobilization, requires low sample volumes and provides a high density array. Importantly, it provides high sensitivity for detecting PK activities in cell lysates. Thus, the peptide microarray system is expected to be useful for a high-throughput kinase assay to investigate intracellular kinase activity and has potential applications in disease diagnosis and drug discovery.
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Creating a robust and unbiased assay for the study of current and novel analgesics has been a daunting task. Traditional rodent models of pain and inflammation typically rely on a negative reaction to various forms of evoked stimuli to elicit a pain response and are subject to rater interpretation. ⋯ Rats, following prior administration of an activity-decreasing inflammatory insult, will positively increase spontaneous locomotor exploration when given single doses of known analgesics. The RSAA model capitalizes on a rat's spontaneous exploratory behavior in a novel environment with the aid of computer tracking software to quantify movement and eliminate rater bias.