**1. Introduction**

Nociceptive threshold (NT) testing is widely used in studies of pain, analgesia, hyperalgesia and allodynia. Such testing records the intensity of a stimulus that causes pain to the test subject. Ethical, humane and repeatable methods for applying the stimuli and evaluating the responses are essential for producing robust data and maximizing animal welfare. A ramped noxious stimulus, usually thermal, mechanical or electrical, is applied, and the intensity of the stimulus at which a clear aversive response occurs is taken as the nociceptive threshold [1]. The threshold is expected to increase

after analgesic treatment and to decrease with hyperalgesia and allodynia. Nociceptive threshold testing is very widely used for translational studies in rodents, both for drug development as well as for fundamental study of the physiology of pain. This is exemplified in, for example, Gugliandolo and colleagues' investigation into neuropathic pain in mice [2]. They used latency of the response to a thermal stimulus applied to the underside of the foot (Hargreaves test) and the withdrawal threshold for mechanical pressure on the plantar area of the foot (electronic von Frey filament test) to evaluate the hypersensitivity induced by sciatic nerve crush and its prevention by the treatment under investigation. Although rodents are not physically restrained for these tests, they must be contained in a special cage and be in close proximity to the tester, which may a ffect their behaviour [3]. Other tests such as the tail flick [1] and the Randall-Sellito method [4] used in rodents may involve actual physical restraint with even greater impact on behaviour. Larger animals are used less commonly for translational studies but their inclusion is essential for investigation into species-specific physiology and pharmacodynamics. The greater size of these species enables the equipment supplying the stimulus to be attached to the animal, for which rodents are too small. Remote control of this equipment without restraint or close proximity of the tester allows the animal to display natural behaviour.

There are a number of essential criteria for NT testing [5]. The stimulus must be repeatable over a few hours and reproducible over the weeks or months of a longitudinal study. The stimuli must not cause tissue damage, both for animal welfare and also so that the stimulated site is not sensitized to subsequent tests. It is essential that the stimulus is stopped as soon as the animal responds, and that there is a cut-out to prevent tissue damage if no response occurs. The end point of the test that indicates when the subject experiences pain, the NT, is a behavioural escape response and must be clearly identified. For instance, this includes stamping the foot or lifting the leg when a limb site is used. Turning to nose or bite at, and bending or jumping away from a site on the body is also seen. Detection of this response depends on the animal behaving normally throughout testing. Restraint and an unfamiliar environment may prevent the animal from displaying normal behaviour and impede acquisition of robust NTs. A relaxed, undisturbed animal ensures that responses are not suppressed by fear or distraction. It is often a requirement of studies investigating the e ffects of both analgesics (increased NT) and increased sensitivity (decreased NT) that the same animal can be tested at intervals over a period of at least 24 h. Testing over several days may be needed for investigation of, for instance, inflammatory pain and its treatment.

Thermal and mechanical testing has been used for many years in large (non-rodent) animals, particularly for analgesic drug development in the target species [6,7]. A thermal system designed specifically for cats was reported in 2002 [8] and has subsequently been widely used for analgesic drug evaluation in this species [9,10]. The thermal probe applied to the body was connected to the control unit by a cable. The cat was able to move around an individual cage while attached, but the cable was vulnerable to damage from the cat and caused some restriction of movement. The inherent disadvantages of a wired system were underlined by experience of its use in the cat. All the behavioural criteria described above apply to this species, but its unique temperament and desire to chase and bite the cables made a wireless control system highly desirable.

In horses and farm animals, the need for a cable connection generally led to the animal being restrained in stocks. Mechanical NT testing has also commonly been carried out in these larger species by exerting noxious pressure from a blunt ended pin pushed onto the surface of a limb [11–13]. These systems have all been driven by a variously sized and often noisy control box, again necessitating restraint in stocks. The animal is close to potentially fear-inducing or distracting machinery and human contact, which both prevent normal behaviour and limit the duration of a continuous study.

The potential advantages of testing unrestrained animals with a small and silent system were apparent. A wireless, remote controlled thermal NT testing system was first developed for cats and subsequently extended to larger animals, and then followed by a mechanical system for larger animals. Remote control of NT testing presents an additional challenge to purely telemetric recording as the power to drive the noxious stimulus must also be remotely controlled and attached to the animal.

This report describes the development and early validation of remote controlled systems for thermal and mechanical NT testing in a number of large (non-rodent) animal species. The data presented were collected during preparation for subsequent research protocols from animals tested in Australia, Brazil, Germany, Norway, Saudi Arabia, Switzerland, the UK and the USA under the authorization of the appropriate animal use ethical committee for each research establishment. Some of the data were presented at the World Congress of Veterinary Anaesthesia in 2006 [14] and to the Association of Veterinary Anaesthetists in 2008 [15].

### **2. Materials and Methods**
