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Title: The use of novel techniques to study the roles of cytokines in joint pain and inflammation
Author: Barton, Nicola J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Rheumatoid arthritis (RA) is a common, chronic, autoimmune, inflammatory disease characterized by persistent synovitis that results in the progressive destruction ofjoints. The cellular and molecular basis of the inflammation is complex and multifactorial. During the progression of the disease many types of cells are activated, which in turn secrete a variety of mediators, including cytokines, which initiate and perpetuate the disease. Rat adjuvant-induced unilateral arthritis is a well established RA disease model and use of this model has facilitated the understanding of the pathology of joint inflammation. The model closely mimics the pathology of human RA, including histopathological changes, cell infiltration, as well as hypersensitivity and swelling of the joint. Measurements of spontaneous pain and hypersensitivity states are assessed in this model. However, no objective measure of joint hypersensitivity is used to assess experimental arthritic joint pain in laboratory rodents. To that end, the pressure application device (PAD) was developed to align pre-clinical measures to those used clinically and help the translation of animal studies to human conditions. PAD was able to detect FCA-induced hypersensitivity in mice and rats, observed as a decrease in limb withdrawal thresholds (LWTs) of around 60% and 40% respectively, compared with basal levels in normal joints. PAD subsequently detected prednisolone analgesia in both species, which was abolished after dosing ceased. PAD also showed significant reversal of evoked mechanical hypersensitivity in arthritic animals treated with morphine or celecoxib, which was comparable to that measured by the weight distribution readout. PAD provides a novel, accurate behavioural tool for detecting localised primary mechanical hypersensitivity in two animal models of chronic inflammatory joint pain. The infiltration of cells and release of inflammatory proteins in the synovial tissue and joint space is a key characteristic of synovitis. Measuring the levels of these in the synovial fluid can provide information about the underlying pathophysiology of joint disease. Furthermore changes occurring in the synovial fluid can be used as biomarkers of disease; therefore the joint perfusion method was developed to evaluate the inflammatory protein and cell content of rat knee joints, to further validate the adjuvant-induced arthritis model, as well as to determine the effects of inflammatory insults or the effect of antiinflammatory, analgesic or anti-rheumatic drugs. This technique proved to be reliable and consistent when perfusing the joint cavity, and regular volumes of sample were easily collected. This technique is therefore a valuable addition to protocols which use homogenates of entire joints to assess inflammatory mediator content. The temporal expression patterns of cytokines and inflammatory cells in the knee joints of rats following induction of arthritis were determined using the novel perfusion technique. Cytokine expression altered over time as arthritis progressed from the acute to the more "chronic" phase. The proportion of inflamed joints that contained detectable levels of each mediator measured was significantly increased during the study. This suggests that it may be the presence of the protein, even at low levels, that is important for the development and maintenance of joint inflammation and hypersensitivity. In addition, significant correlations between measures of joint swelling or mechanical hypersensitivity and levels of cytokines in inflamed joints were seen. Prednisolone did not affect the absolute levels of cytokines in inflamed joints, although it reduced the percentage of inflamed joints that contained detectable levels of ILla and IL6. This suggests that the steroid appears to have an all-or-none effect in terms of cytokine expression levels in this study. The roles of ILip and IL6 in joint pain and inflammation were assessed. The contribution of the activity of primary afferent fibres to joint pain and hypersensitivity after administration of intra-articular ILip or IL6 was investigated by recording action potentials from primary afferent nerves innervating the knee joint. IL1 p caused a transient increase in the frequency of basal neural discharge by 88% within three hours. It also decreased the threshold of mechanical stimulation required to evoke neural activity by 50% between one and four hours after injection. In contrast, IL6 did not affect the frequency of basal neural discharge or the mechanical threshold. Neither ILip nor IL6 affected the neural discharge frequency to mechanical stimulation above the threshold. The induction of basal neural activity resembles the occurrence of spontaneous pain during inflammation, such as that measured by the incapacitance tester as a result of intra-articular IL1 p or IL6. A reduction in the LWT, measured by PAD, following ILip or IL6 occurred within a few hours, similar to the decrease in the mechanical threshold to von Frey hairs in primary afferents after ILip, as a result of neuronal sensitization. Although ILip or IL6 did not cause swelling of the joint, they did induce mechanical hypersensitivity within a couple of hours, which lasted for up to four days. Intra-articular IL1 p or IL6 had no effect on joint structure, bone or cartilage. ILip and IL6 evoked increases in the expression of ILip, IL6 and TNFa within the first eight hours, and additionally elevated levels of ILla, IL2, IL4 (IL6-treated only) and IL10 (ILlp-treated only) from day one post administration. ILip also resulted in recruitment of inflammatory cells into the synovial cavity one day after administration. In conclusion, this study has developed and validated two novel techniques to study experimental joint pain and inflammation in rodents; the behavioural measure of joint mechanical hypersensitivity, PAD; and the joint perfusion technique to assess inflammatory mediator and cell content of synovial fluid. These methods have been used alongside other techniques to show the temporal cytokine expression patterns during adjuvant-induced arthritis and the relationship of these to swelling and hypersensitivity of the joint. The roles of ILip and IL6 in evoking joint pain and hypersensitivity were also investigated. This data supports the hypothesis that ILip and IL6 are directly involved in the development of joint pain, but cannot alone elicit swelling or joint damage at doses sufficient to evoke hypersensitivity. Furthermore, similarities between this animal model of joint disease and human RA have been demonstrated that further validate the model as a valuable pre-clinical tool to study the inflammatory process of human RA. Moreover, consolidation of these similarities helps improve the confidence of novel drug screening using this model prior to use in the clinic.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available