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 ofjoint 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
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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 anti¬
inflammatory, 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 ofjoint 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 ILiß 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 IL1ß or IL6. A reduction in
the LWT, measured by PAD, following ILiß or IL6 occurred within a few hours, similar to
the decrease in the mechanical threshold to von Frey hairs in primary afferents after ILiß, as
a result of neuronal sensitization. Although ILiß 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 IL1ß or IL6 had no effect on joint structure, bone or cartilage.
ILiß 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
(ILiß-treated only) from day one post administration. ILiß 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
