In this thesis, I have investigated several aspects of the use of intraarticular morphine for the provision of analgesia in dogs, as well as the quantitation of lameness in dogs. Morphine has been used as an intraarticular (i.a.) analgesic in human patients since 1991. Provided aseptic techniques are observed, i.a. injection of opioid drugs is a low-risk procedure with the benefit of avoiding the systemic side effects of morphine administered by more conventional routes. However, at present, there is little scientific evidence for the efficacy of i.a. morphine as an analgesic treatment in canine patients.
In Chapter 2 of my thesis, experiments carried out to validate methods of producing canine models of joint inflammation for use in subsequent studies, are described. Leukaemia Inhibitory Factor was trialed unsuccessfully as an arthritogen in the canine radiocarpal joint. Oleyl alcohol injected into the radiocarpal joints of dogs repeatably produced inflammation and lameness by 12 hours after injection. However, this was an acute and rapidly resolving lameness and so was unsuitable for studies where a more chronic condition was required for the evaluation of efficacy of i.a. opioids. Freund's Complete Adjuvant (FCA) was injected into the radiocarpal joint in a further group of dogs. Although all of these dogs developed an acute lameness, this too resolved within days. Importantly, however, half of the dogs subsequently developed a chronic, mild lameness which was suitable for the development and validation of methods for objective quantification of lameness.
The assessment of a new analgesic technique relies on the quantitation of a parameter that is related to the conscious perception of pain. In my work, two assessment procedures were used. Firstly, a pain scoring method relying on a lameness score in conjunction with a score based on the behavioural response elicited by joint flexion was used. Secondly, a force platform was used to objectively quantify various parameters derived from the downward force applied by a limb whilst weight bearing. Changes due to joint inflammation were considered to be, at least in part, due to pain.
In Chapter 3, radioligand binding studies were carried out on tissue taken fi*om dogs with inflamed joints following the injection of OA. The development of a process by which joint capsule and surrounding tissues could be homogenised, preceded these studies. This process differed from the homogenisation techniques applied to brain tissue, in that joint tissue required an initial incubation period with elastase and collagenase followed by filtration to remove particulate matter. Additionally, there was truncation of the usual multiple wash steps to preserve specific opioid binding in the joint tissue homogenates. Whilst non-inflamed joint tissue showed little specific morphine binding, inflamed joint tissue had a very high density of low affinity morphine binding sites (Bmax = 1032 (± 265) fmol/mg protein, KD = 79 (± 17) nM). These binding sites were clearly different from conventional µt - and µ2 -opioid binding sites, which have comparatively high affinity for morphine (K ≈ 1 nM) and approximately 100-fold lower density. Further indication that these binding sites were not conventional opioid-binding sites was the finding that they were opioid alkaloid sensitive (morphine) but insensitive to the µ- and [sigma]-opioid peptides, DAMGO and DPDPE respectively, and insensitive to the K-opioid ligand U69,593. Morphine, morphine-6-glucuronide and naloxone displaced [3H]-morphine, but oxycodone and pethidine did not. Intriguingly, the binding of [3H]-morphine was enhanced in the presence of the µ-opioid peptide, DAMGO. This enhancement was characterised by an increase in the density of opioid binding sites, but the affinity of the binding site for morphine remained unchanged. Whether this DAMGO enhancement of morphine binding is mediated by µ-opioid receptor homodimers, requires further investigation.
My studies in Chapter 4 were designed to determine the analgesic efficacy of i.a. morphine in dogs with oleyl alcohol induced inflammatory joint pain. As joint pain was evident by 6 h and the presence of specific opioid binding sites had been demonstrated by 12 h after oleyl alcohol administration, i.a. injections were given at 6 h and assessment continued until 12 h. Using this study design, intraarticular morphine (1 mg) was no better than saline as an analgesic treatment. However, as, bupivacaine (1.25 mg) (positive control group) and morphine (1 mg) plus naltrexone (1 mg) were also not significantly different from saline (negative control), in terms of extent and duration of analgesia, the apparent lack of analgesic effect of i.a. morphine (1 mg) may have been due to deficiencies in my trial design, which will need to be addressed in future studies.
Force plate studies were carried out on groups of normal dogs (mixed breeds and greyhounds) to quantify the normal distribution of the downward forces generated by the four limbs as they bear weight at the trot and to determine the repeatability of parameters derived from these forces (Chapter 5). All groups of dogs studied had near perfect left to right symmetry. The values of the derived gait parameters that quantified left to right weight bearing symmetry, and fore to hind limb weight distribution, were highly reproducible for an individual dog over repeated measurements. However, while the left to right gait symmetry parameters were predictable and repeatable for a naive dog, the fore to hind limb gait parameters could not be predicted for an individual dog but were repeatable once an initial reading was known. Clearly, the fore to hind limb weight bearing ratio is an individual dog characteristic.
In Chapter 6, force plate gait evaluation was carried out using three greyhounds with FCA-induced left fore-limb lameness. Loss of lateral symmetry was more marked in response to induced left fore-limb lameness than changes in the fore to hind limb distribution of weight bearing forces. Subsequent force plate gait analysis carried out using five dogs with naturally acquired lameness, confirmed the findings of the study undertaken in greyhounds with induced lameness. Thus, lateral symmetry was used in further studies of lame dogs to monitor changes in the extent of lameness. Finally, preliminary evaluation ofthe efficacy ofmorphine as an i.a. analgesic in dogs using force plate quantification of lameness, showed a non-significant trend for i.a. morphine to restore normal gait. However, detailed studies using a range of doses of i.a. morphine, remain for the future.