It is well recognised that nociceptive threshold testing does not provide the same stimulus as clinical pain, but it can be used to assess the efficacy of analgesics with cutaneous antinociceptive activity in the preclinical phase for elaboration of adequate doses and dosing intervals.
In the last few decades many different techniques have been developed to perform thermal threshold testing in dogs. Both fixed stimulus, where latency to response is measured, and a threshold stimulus that elicits an effect have been used as end points in analgesiometry. In 1941 Andrews and Workman
] used a “Hardy-Wolff” apparatus
] for thermal threshold testing in dogs. They blackened the dogs` thoracolumbar area of the skin and stimulated with radiant heat. In another study, a filament lamp was attached to the hindlimb of dogs for thermal threshold testing
]. In a recent study, latency response was measured following application of a relatively constant temperature of 60°C with a thermal probe
In the present study, nociceptive thermal threshold was measured by adapting equipment well established for use in cats
]. The testing device has previously been used for nociceptive thermal threshold measurements in horses
], encouraging the adaption to another species.
The equipment was well tolerated by the beagles who wore the band for up to 10 hours without attempting to remove or play with it. In contrast to the first study of this device in cats
] none of the dogs chewed the cable between the probe and the control unit. Thus there was no necessity to unplug the cable between tests which reduced direct manipulation on the dogs. The flexibility of the ribbon cable minimized the restriction on the dogs, keeping a possible influence of restraint induced stress low.
It is important to evaluate and adapt nociceptive threshold testing methods for each species due to species and individual variability. It is known that variations in skin thickness and blood flow
], hair covering and epidermal pigmentation
] as well as nociceptor distribution may influence the thermal threshold.
Previous studies in cats investigated reproducibility by 6–12 repeated tests at 5-15- minute intervals in eight untreated cats and further tests at 24 hours and repeated this on only two occasions 3–6 months later. Reproducibility was improved in cats during development of the equipment and testing procedure by careful attention to detail such as clipping, band placement and precise bladder pressure
] and this accuracy was considered in the present study.
In the first study of this device in cats, a safety cut-out at 60°C led to minor skin lesions
]. The safety cut-out was adapted to 55°C to prevent thermal burns in the present study.
Since there are many factors possibly influencing the nociceptive thermal threshold, baseline reproducibility is required prior to any testing of analgesic agents. In the present study, three of the four saline placebo treatments were assigned prior to drug testing. There was at least six weeks between saline placebo treatment periods to provide reproducible data over several months.
The effect of learning leading to recognition and subsequent avoidance of the stimulus can be another factor influencing the test results
]. Sham testing showed that the dogs did not anticipate the experiment. The testing device used in this study, had the great advantage that direct manipulation of the dogs could be reduced to a minimum and no learned avoidance of the testing device occurred. In addition it is presumed that environmental factors influence the response to painful stimuli in animals
]. Therefore the dogs were habituated to wearing the testing device, the room in which the testing was performed and the person performing the tests.
Comparable to the studies in cats and horses using the same testing device as in the current paper
] thermal threshold did not change significantly after placebo saline injection in the present study.
Measurement of the plasma concentration of levomethadone gave us the opportunity to elucidate the relationship between blood concentration and effect (Figure
); however, it is important to take into consideration that the analgesic effect correlates with the drug concentration at the site of action- effect site (opioid receptors in the central nervous system) and not necessarily directly with the plasma concentration. Additionally, the sampling schedule may not allow very accurate estimates of pharmacokinetic parameters. Nevertheless, a close relationship between serum concentration and nociceptive thermal threshold was demonstrated for levomethadone in this study (Figure
To our knowledge there is little information about the correlation between levomethadone plasma concentration and an analgesic effect in dogs. In this study, serum concentrations of 22.6 to 46.3 ng/mL provided analgesia in dogs. These concentrations were observed during significant elevation of TT and not at steady-state, and the relationship between concentration and effect is unlikely to be direct. It is therefore likely that the changes in effect lagged behind the changes in concentration, and the actual analgesic concentrations may be therefore have been somewhat different. The duration of effect was 15 to 120 minutes after IM injection, with a maximum threshold 45 minutes after injection.
Time to maximal plasma concentration in the present study was reached after 1.25 hours, with a maximal plasma concentration of 46.3
14.0 ng/ml. In a recent study, time to maximal plasma concentration was reached after 1.26 hour, with maximal plasma concentrations of 23.9
14.4 ng/ml after subcutanoues administration of 0.4 mg/kg methadone hydrochloride in dogs
]. Racemic methadone and levomethadone boths provide a comparable analgesic effect. Another study compared analgesia following administration of either 0.3 mg/kg levomethadone or 0.6 mg/kg racemic methadone intramusculary (IM) to cats undergoing ovariohysterectomy. Post operative pain was scored at a number of time points for up to four hours after surgery using a scoring system comprised of physical and behavioural measures, including wound palpation. The authors concluded that both levomethadone and racemic methadone, at the doses tested, provided adequate analgesia after ovariohysterectomy
Opioids commonly produce sedation
] due to their interaction with μ and κ receptors
]. Acepromazine was used to determine the influence of sedation on the testing results. It is the most widely used phenothiazine sedative in veterinary medicine, and is considered not to have a significant analgesic effect
], although there is controversy if phenothiazines produce analgesia
]. In a recent study on cats, acepromazine alone and in combination with tramadol increased the pressure threshold whereas tramadol alone did not
]. In contrast, no increase in thermal threshold occurred after administration of acepromazine in horses, suggesting that acepromazine does not have somatic antinociceptive effects in the horse
In the present study, degree of sedation was compared between treatment groups. Acepromazine produced sedation comparable to levomethadone at the dosage used, but did not increase the nociceptive thermal threshold, indicating that the testing system seems to measure only the analgesic effect and is not influenced by sedation. However, the study design and the small number of dogs tested do not seem appropriate to demonstrate the lack of an effect. Therefore, we cannot confirm with acceptable certainty that saline placebo and acepromazine do not have an effect on nociceptive thermal threshold, even though the data itself shows relatively convincingly that the thermal threshold likely does not change following saline placebo or acepromazine administration.