Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo through intra-articular injections of lubricin in a rat OA model where the inhibition of systemic inflammatory signaling and reduction in pain were observed. Lubricin plays an important role in regulating the inflammatory environment under both homeostatic and tissue injury states.

Our limited ability to assess spontaneous pain in rodent models of painful human conditions may be associated with a translational failure of promising analgesic compounds in to clinical use. If measurement of spontaneous pain behaviours can be used to generate an analgesic intervention score their use could expand to guide the use of analgesics, as mandated by regulatory bodies and ethical and welfare obligations. One such measure of spontaneous pain, the Rat Grimace Scale (RGS), has recently been described and shown to exhibit reliability. However, reliability of measurement scores is context and content specific, and further testing required to assess translation to a heterogenous setting (different model, raters, environment). The objectives of this study were to perform reliability testing with the Rat Grimace Scale in a heterogenous setting and generate an analgesic intervention score for its use. In a randomised, blinded study, sixteen adult female rats received one of three analgesia treatments (0.05 mg/kg buprenorphine subcutaneously, 1 mg/kg meloxicam subcutaneously, 0.2 mg/kg oral buprenorphine in jelly) peri-operatively (telemetry unit implantation surgery). Rats were video-recorded (before, 1–6 and 12 hours post-operatively) and images collected for independent scoring by three blinded raters using the RGS, and five experts based on “pain/no pain” assessment. Scores were used to calculate inter- and intra-rater reliability with an intraclass correlation coefficient and generate an analgesic intervention score with receiver operating characteristic curve analysis. The RGS scores showed very good inter- and intra-rater reliability (0.85 [0.78–0.90 95% CI] and 0.83 [0.76–0.89], respectively). An analgesic intervention threshold of greater than 0.67 was determined. These data demonstrate that the RGS is a useful tool which can be successfully employed in a heterogenous setting, and has the potential to guide analgesic intervention.

This study confirms that the three pain models induce pain in rodents and showed that peak pain coincided with peak mechanical hypersensitivity. However, mechanical hypersensitivity remained once pain subsided, mimicking the human experience of CFA injection. These findings further our understanding of the roles of, and relationship between, these assays in the assessment of nociception and pain.

Gradual filling of a chamber with carbon dioxide is currently listed by the Canadian Council on Animal Care guidelines as a conditionally acceptable method of euthanasia for rats. Behavioural evidence suggests, however, that exposure to carbon dioxide gas is aversive. Isoflurane is less aversive than carbon dioxide and may be a viable alternative, though objective data are lacking for the period leading up to loss of consciousness. It has been shown that during negative states, such as pain and distress, rats produce ultrasonic vocalizations. The objective of this study was to detect ultrasonic vocalizations during exposure to carbon dioxide gas or isoflurane as an indicator of a negative state. Specialized recording equipment, with a frequency detection range of 10 to 200 kHz, was used to register these calls during administration of each agent. Nine female Sprague-Dawley rats were exposed to either carbon dioxide or isoflurane on two different occasions. All rats vocalized in the ultrasonic range (30 to 70 kHz) during exposure to carbon dioxide. When exposed to isoflurane, no calls were detected from any of the animals. The frequent occurrence of ultrasonic vocalizations during carbon dioxide exposure suggests that the common practice of carbon dioxide euthanasia is aversive to rats and that isoflurane may be a preferable alternative.