Thermal and mechanical nociceptive threshold testing in horses: a review

Veterinary Anaesthesia and Analgesia, 2011, 38, 3–14

doi:10.1111/j.1467-2995.2010.00580.x

REVIEW

Thermal and mechanical nociceptive threshold testing in horses: a review Emma J Love, J Murrell & H R Whay Department of Clinical Veterinary Science, University of Bristol, Langford, UK

Correspondence: Emma J Love, Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK. E-mail: [email protected]

Abstract Objective This review evaluates the thermal and mechanical nociceptive threshold testing techniques that have been used in horses and discusses them with reference to their applications, limitations and the factors which can influence both the testing procedure itself and the animal’s responses. Methods to optimise the reliability and repeatability of the testing procedures are suggested and the potential clinical applications discussed. Databases used Web of Science and Medline. Conclusions Thermal and mechanical nociceptive threshold testing techniques have valuable roles in both the identification of altered nociceptive function and the pre-clinical evaluation of analgesics in horses. Keywords analgesia, analgesiometry, horse, nociceptive threshold testing, pain.

animals with naturally occurring painful conditions or following surgery. The three types of acute stimuli that have been used in preclinical experimental assessment of nociception in the horse are electrical, thermal and mechanical. Each type of noxious stimulation produces a different type of pain sensation in people (Arndt & Klement 1991) and quantitatively different changes in the electroencephalogram in rats (Murrell et al. 2007). This review evaluates the acute thermal and mechanical nociceptive threshold testing techniques which have been used in horses. Electrical stimuli have also been used widely in horses and will be discussed in a future review article. In this article, thermal and mechanical stimuli will be discussed with reference to their limitations, and the factors which can influence both the testing procedure itself, and the animal’s responses. Methods to optimise the reliability and repeatability of the testing procedures will also be suggested and the potential clinical applications discussed. Nociceptive threshold testing

Introduction Objective assessment of pain in animals presents a real challenge to both clinicians and researchers and this impedes the quantitative assessment of the efficacy of analgesics. In horses many analgesic doses are extrapolated from other species as there are limited data on both efficacy and pharmacokinetics. Nociceptive threshold testing, or analgesiometry, can be used experimentally to assess the efficacy of analgesics and clinically to evaluate hyperalgesia in

Nociceptive threshold testing involves the application of a quantifiable stimulus to a body part until a behavioural or physiological response is observed (‘end-point’), at which point application of the stimulus is terminated. The ideal characteristics of a nociceptive stimulus for analgesiometry were described by Beecher (1957). In summary, the stimulus should be repeatable, reliable and easy to apply with a clear end-point. The intensity of the stimulus should be related to the perceived pain intensity; it should be able to detect the effects of weak 3

Nociceptive threshold testing in horses EJ Love et al.

analgesics and a dose–response relationship; and should not result in lasting tissue damage or harm to the animal. Nociceptive threshold testing using heat is widely used in laboratory animals and the ‘tail flick’ reflex response (D’Amour & Smith 1941), ‘tail immersion’ (Luttinger 1985), ‘hind paw thermal withdrawal’ (Hargreaves et al. 1988) and ‘hot plate test’ (Woolfe & MacDonald 1944) are well established pre-clinical models of nociception in rats and mice. Nociceptive threshold testing can be used experimentally to assess the efficacy of analgesics and to determine optimal doses for subsequent clinical evaluation. An increase in nociceptive thresholds following analgesic administration is accepted as an indication of an antinociceptive effect. It enables objective quantification of a drug’s effect by using a defined end-point and can be used to investigate the antinociceptive efficacy of a range of doses. Promising drug dosages can then be evaluated in clinical situations, where pain is a much more complex and dynamic phenomenon, before final dosage recommendations are made (Roughan & Flecknell 2002). Defining an end-point for a nociceptive test is not always straightforward due to species and individual variability. Variations in skin thickness and blood flow (Monteiro-Riviere et al. 1990), hair covering and epidermal pigmentation (Pringle et al. 1999) and nociceptor distribution may influence peripheral perception of the stimulus used. This means that it is important to evaluate and adapt nociceptive threshold testing methods for each species so that optimal results can be obtained (Love et al. 2008). The significance of the end-point should also be considered since these may be reflexes or reflect more complex conscious perception of pain. One potential problem associated with detecting an end-point following all types of stimulation, particularly relevant to horses, is the location of the test site. The limbs have been used for all types of stimulation with limb movement taken as the end-point of the test when stimulation is stopped. Administration of some analgesics, particularly opioids, to horses may produce locomotor stimulation which can interfere with detection of hoof withdrawal or limb movement as an end-point (Kamerling et al. 1985b). The use of a ‘skin twitch’ as the end-point may be difficult in environments where insects may also elicit this response: application of insect repellent should be considered to try to reduce this confounding factor. 4

A major limitation of nociceptive threshold testing is that it clearly does not provide the same stimulus as clinical pain, and therefore, may not detect the analgesic effects of all classes of analgesic drugs under clinical conditions. For example, sole application of the acute stimuli described in this review may not be appropriate to detect the antinociceptive effects of non steroidal anti-inflammatory drugs (NSAIDS) (Steagall et al. 2007). To investigate NSAID analgesia or antinociception it is necessary to induce an inflammatory lesion with an irritant substance such as kaolin and this has been done successfully in cats (Giraudel et al. 2005; Taylor et al. 2007). In addition, the complexity of clinical pain means that it is unrealistic to expect a single threshold testing modality to reproduce the complete pain experience (Nielsen et al. 2009) although a more comprehensive evaluation can be accomplished by using more than one type of stimulus (Dixon et al. 2002). Many nociceptive neurones respond to more than one type of stimulus. C and A fibre nociceptors can respond to both mechanical and thermal noxious stimuli. A fibre nociceptors can be sub-divided into three groups: high threshold mechanoreceptive units that are activated by noxious mechanical stimuli only; mechano-heat units that are activated by noxious mechanical stimuli and noxious heat and; mechanocold units that are activated by noxious mechanical stimuli and noxious cold stimuli (Djouhri & Lawson 2004). Despite the limitations of nociceptive threshold testing the increases in thermal and mechanical thresholds in cats after opioid (meperidine) administration (Millette et al. 2008) seem to mirror the clinical analgesic effects in the post-operative period following ovariohysterectomy in cats (Slingsby & Waterman-Pearson 1998). Nociceptive threshold testing can also be used to identify altered nociceptive function in animals with naturally occurring or experimentally induced inflammatory conditions such as ‘footrot’ in sheep (Nolan 2002) and mastitis in cattle (Kemp et al. 2008). An affected animal would be expected to have lower nociceptive thresholds than a normal animal due to hyperalgesia. Thermal stimulation Thermal stimulation of the skin is used to simulate superficial (cutaneous) pain. Two different methods of thermal stimulation are often used: latency to a response following application of a relatively

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constant temperature; and measurement of the temperature at which a response occurs (threshold temperature) when there is a ramped increase in temperature. In rats it has been demonstrated that when the rate of heating applied to the paw is fast (6.5 °C second)1) Ad fibres are activated, whereas the response is mediated by C fibres when heating times are slow (0.9 °C second)1) (Yeomans & Proudfit 1996). A reliable method for the preferential activation of each fibre type using contact heat has been described in rats which allows the role of each type of fibre in pain processing to be determined and also the differential sensitivity of each type of fibre to analgesics to be studied (McMullan et al. 2004). In horses responses to thermal stimulation of the skin have been measured using slow heating rates, therefore, they are likely to be mediated by C fibres. Techniques using measurement of latency to a response (Pippi & Lumb 1979; Kalpravidh et al. 1984; Kamerling et al. 1985b; Olbrich & Mosing 2003; Carregaro et al. 2007; Dhanjal et al. 2009) and measurement of threshold temperatures (Robertson et al. 2005; Sanchez et al. 2007, 2008; Love et al. 2008; Elfenbein et al. 2009) are used. Radiant heat Latency to response to a thermal stimulus in horses has been measured by focusing a radiant heat source (lamp) on a clipped and blackened area of skin above the coronary band of a forelimb. The elapsed time was recorded electronically and limb movement (hoof withdrawal) taken as the end-point at which heating was stopped (Pippi & Lumb 1979; Kalpravidh et al. 1984; Queiroz-Neto et al. 1998; Carregaro et al. 2007). Following administration of analgesics, an increase in the time elapsed between application of the stimulus and the response compared to pre-treatment values was interpreted as an antinociceptive effect. In order to overcome the confounding effect of opioid induced locomotor stimulation on the testing procedure, alternative sites to the coronary band have been used. Placing the stimulus on the skin over the withers (Kamerling et al. 1985b) and the skin at the dorsal point of the shoulder (Carregaro et al. 2007) have been evaluated, with a skin twitch taken as the end point. The skin twitch latency was found to be a more sensitive method of determining antinociception to a thermal stimulus after opioid administration in horses than the latency to hoof withdrawal. This

was because it was not possible to differentiate the hoof withdrawal reflex from opioid induced locomotor activity, whereas interpretation of the skin twitch end-point was still possible (Kamerling et al. 1985b). Difficulties with methods that measure latency to a response include inaccuracies associated with timing. These become more significant if time between exposure and response to the stimulus is short since both the reaction time of the animal and of the observer (unless an automated system is used for detection of the end-point) will constitute a proportionally greater period of the measured time. The use of a lamp as a heat source has the advantage that it is not directly in contact with the skin so does not produce a tactile stimulus. However, it does have several inherent problems. Skin will both reflect and absorb the radiation, though this can be partially overcome by blackening the skin with ink. The amount of radiation required to increase the temperature of the skin to the threshold temperature depends on the conduction properties of the skin, which will vary between different sites on the body, and also with the initial temperature of the skin. An assumption is made that the rise in skin temperature is related to the time of exposure to the heat lamp although skin temperature is rarely measured. The rate of increase in skin temperature is not linear and varies with the square root of time (Le Bars et al. 2001). A time-limit for skin exposure to the heat lamp usually is set to prevent burns (Carregaro et al. 2007; Dhanjal et al. 2009). Some horses may fail to respond to the stimulus within the set time-limit resulting in exclusion from a study population (Dhanjal et al. 2009). Alternatively, the intensity of the lamp may be altered for individual horses to achieve a pre-defined latency in all animals. These measures could potentially bias the results of the study since the included horses may not be representative of the population (Kamerling et al. 1985a). This is particularly important since the high costs of scientific investigations involving horses mean that low numbers of animals are used. It is vital that the current applied to the lamp remains constant, since changes in current will produce changes in both the intensity and the electromagnetic spectrum of the radiation that is produced. A change in the current applied to the bulb can alter the volume of skin that is stimulated which introduces a systematic and unquantifiable error in the measurement (Le Bars et al. 2001).

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The effect of learning leading to recognition and subsequent avoidance of the stimulus may occur in up to 50% of horses though this can be eliminated by including ‘sham tests’ in the protocols (Kamerling et al. 1985b). This involves regularly shining a non-heat producing lamp on the skin test site (Kamerling et al. 1985b). Thermal stimulation of the skin at 20-minute intervals for up to three and a half hours produced consistent latencies to limb withdrawal and skin twitches, although over a longer period of time (15–24 hours after onset of testing) an increase in latencies to these responses was observed, possibly either due to diurnal variation or release of endogenous opioids with repeated testing (Kamerling et al. 1985a). A diurnal variation in beta-endorphin concentrations and skin twitch and hoof withdrawal latencies have been reported (Hamra et al. 1993). Thermode based systems Equipment for measuring thermal nociceptive thresholds, initially designed and validated for use in cats (Dixon et al. 2002), has been used in studies examining the antinociceptive effects of analgesics in horses (Robertson et al. 2005; Sanchez et al. 2007, 2008; Love et al. 2008; Elfenbein et al. 2009). The equipment consists of a probe containing a heating element and temperature sensor held against a clipped area of skin over the withers using an elastic surcingle. The pressure at which the probe contacts the skin can be modified by inflation or deflation of a blood pressure cuff, therefore it is possible to standardise the skin contact pressure of the probe. Before each test, skin temperature was recorded and then the probe heated at a constant rate of 0.6 °C second)1 until a behavioural response (skin twitch or turn of the head) was noted, at which point heating was stopped and the threshold temperature recorded. In order to prevent skin burns, should an animal not respond, for example after administration of an analgesic, a thermal cutout is built into the design. This was set at 55 °C in the earliest study (Robertson et al. 2005) and was then reduced to 45 °C in the three subsequent studies performed at the same institution (Sanchez et al. 2007, 2008; Elfenbein et al. 2009) due to concerns over the development of skin lesions following testing. Pre- and post-treatment threshold temperatures were not reported in one study (Elfenbein et al. 2009) and it is not possible to determine if the absence of a treatment effect was a 6

genuine result or if it was simply that the low cutout temperature employed limited the scope to detect increases in thermal thresholds after analgesic administration. The effects of varying the heating rate using the equipment described above have been investigated (Love et al. 2008). A clearer end-point (skin twitch) and more consistent threshold temperatures were obtained when the rate of heating was reduced from 0.85 °C second)1 to 0.5 °C second)1 and 0.2 °C second)1 although some horses became restless during probe heating at the lowest heating rate. The probe was removed from the skin as soon as possible after the end of each test and allowed to cool before being replaced on a new skin site. Occasional small wheals were observed on the test site when the cut-out temperature (53 °C) was reached, although these resolved within a few hours and no lasting skin damage occurred. A wireless thermal threshold testing system with a heating rate of 0.5 °C second)1 and cut-out of 53 °C has been developed to enable thermal threshold testing to be performed on unrestrained horses within their stable, due to the difficulties encountered working with horses restrained in stocks for prolonged periods (Love 2009). In addition, new probes which cool more rapidly and are less likely to result in skin lesions following testing have been developed (Dixon & Taylor 2009). Skin and threshold temperatures can easily be measured using a thermode based system although the temperatures measured on the skin surface may not accurately reflect the temperature at the level of the nociceptors. The depth and density of Ad and C fibres in the epidermis in horses has not been established, although it is likely that the nociceptors are located at a greater depth than in cats or rodents since the epidermis in horses is at least twice as thick compared to cats and rats (Monteiro-Riviere et al. 1990). This could lead to a greater temperature difference between the skin surface and nociceptors in horses compared to cats and rats. It is also possible that the hairy skin, despite clipping, acts to insulate the deeper layers of the epidermis from the heat source. A slow heating rate may result in more time for equilibration between the skin surface temperature and the temperature at the level of the nociceptor and this may account for the reduced numbers of skin lesions observed when the rates of heating were decreased (Love et al. 2008). One advantage of a thermode based system is that it can be adjusted so that the increase in

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temperature is linear. Another advantage in horses is that it is possible to maintain a relatively constant skin contact pressure using a thermode, therefore standardising the rate of thermal transfer (Yarnitsky & Ochoa 1990). Difficulty in standardising skin contact pressure in small laboratory animals has been a limiting factor in the use of thermodes in thermal threshold assays in rodents. An alteration in the stimulus–response relationship was observed when a thermode device was compared to application of heat by a laser beam in people (Svensson et al. 1997). A linear relationship between stimulus intensity and perception of pain was described for heat produced using a laser which was not in direct contact with skin, whereas a non-linear, positively accelerating relationship was described when a thermode in direct contact with the skin was used (Svensson et al. 1997). The authors hypothesised that mechanical contact with the skin by the thermode activates cutaneous mechanosensitive afferents (Svensson et al. 1997). From the data presented (positive accelerating stimulus response curve) this could be interpreted as a facilitatory effect at higher temperatures or an inhibitory effect at lower temperatures. However, it has been argued that once contact between the thermode and skin has been established for a relatively short period of time, firing of the low threshold mechanical afferents is reduced due to fatigue and adaptation which will reduce the confounding effects on thermal thresholds (McMullan et al. 2004). Deep pain has also been evaluated in ponies following implantation of a heating element over the periosteal surface of the radius, though the results were highly variable with long reaction times, and tissue damage was not evaluated (Pippi et al. 1979). The requirement for surgical implantation of the heating element means that this technique is relatively invasive and the surgical procedure could potentially alter the distribution and function of the nerve fibres surrounding the site. Cold pressor testing Cold pressor testing by immersion of a limb in cold water (Che´ry-Croze 1983), or by use of a peltiertype stimulator (Yarnitsky & Ochoa 1991), is performed in people and has been used to investigate the antinociceptive effects of opioids (Koltzenburg et al. 2006). To date, the technique has not been reported in horses, possibly due the practical difficulties associated with immersion of a limb in cold

water, although the use of peltier-type stimulators are likely to be more practical and merit investigation. Mechanical stimulation Mechanical stimuli are used daily in clinical practice, for example hoof testers are used to localise areas of discomfort in horses with hoof pain. However, most devices used clinically rely on subjective evaluation of the degree of force required to produce a response. Devices have been developed for use under laboratory conditions to measure mechanical nociceptive thresholds on the surface of the skin by application of force over a given area and by distending hollow viscera by the application of pressure. Force usually is generated using a pneumatic cylinder (actuator). The gas pressure within the actuator is increased until a response is elicited from the animal. The surface pressure applied to the animals’ tissues will be directly proportional to (but not the same as) the gas pressure within the actuator, provided that the area of tissue in contact with the device remains constant with even pressure distribution, conditions almost impossible to achieve in practice. Practically, in order to apply a force at a constant rate the actuator should be perpendicular to the site selected for testing, and there should be minimal amounts of distensible tissue to reduce the spread of pressure across a larger area as the pressure within the actuator is increased. The advantage of measuring thresholds on the dorsal aspect of the cannon bone is that there are minimal anatomical variations between horses, with very little soft tissue between the skin and periosteum. Applying a force at a constant rate is much more difficult in techniques designed to measure visceral thresholds since the area of stimulation will increase with visceral distension. Mechanical threshold testing devices for use on the limbs of large animals have been developed and validated for use in horses (Chambers et al. 1990, 1994), sheep (Nolan et al. 1987) and cattle (Whay et al. 1998). These devices are attached to a limb using a cuff so that a blunt ended pin is in contact with the skin on the dorsal aspect of the cannon bone. The cuff is attached securely to the limb and pressure of increasing intensity is then applied to the leg by the pin until a withdrawal reflex (lifting of the limb) is observed at which point the stimulus is terminated.

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The rate of application of force must remain constant between tests to ensure reproducible results are obtained and, while thresholds remain fairly constant in the short term (15 minutes), after this time an increase in the variability of the results was seen as the horses became restless (Chambers et al. 1994). With repeated testing over several weeks thresholds may decrease as horses learn to respond when the stimulus is applied, though this effect was prevented when the horses were sedated with acepromazine (Chambers et al. 1990). Mechanical threshold testing using this method was able to differentiate between sedation and analgesia. No increases in thresholds were measured following administration of acepromazine, a sedative with no analgesic properties (Chambers et al. 1990; Love 2009). However, increases in thresholds were recorded after administration of detomidine, a sedative with analgesic properties (Chambers et al. 1990). Chambers (1992) studied the effects of butorphanol (0.1 mg kg)1 IV) in this model but found that although it was possible to measure thresholds, the end-point was not as well defined as normal, and the marked excitatory effects following butorphanol administration made it difficult to handle the horses to perform mechanical threshold testing safely and the study was discontinued. More recently, a sharp-ended pin has been used as part of a device for measuring nociceptive thresholds in horses though the repeatability of the results and the effect of using a pointed pin on the tissues was not described (Moens et al. 2003). A hand-held pressure algometer with a broader (1-cm diameter) flat-ended probe has been used to measure mechanical nociceptive thresholds in painfree horses with good repeatability (Haussler & Erb 2006a). In horses with induced back pain three measurements at each testing site were taken during a single testing session and some measurements were repeated after 1 year (Haussler & Erb 2006b). Marked decreases in mechanical nociceptive thresholds were measured following surgery to implant (and subsequently remove) pins in the dorsal spinous processes of two vertebrae suggesting that this is a sensitive method for detection of musculoskeletal pain. The stability of the thresholds over shorter periods (e.g. 1 week) and the potential confounding effect of learning by the horse remain to be established. One potential disadvantage of this technique is that operator bias and variations in the rates of application could influence the manual 8

application of the stimulus although the simplicity of the equipment means that it does have a potential application in clinical practice to detect and monitor the treatment response in horses with musculoskeletal injuries. Monofilaments – von Frey, or Semmes Weinstein filaments – are commonly used to measure mechanical sensory thresholds in people, and laboratory animals (Bove 2006). The original von Frey filaments were made of horse hair but the more modern Semmes Weinstein testing kits comprise a set of standard lengths of monofilament nylon, of varying thickness, attached to plastic handles. The tip of the filament is placed in contact with the skin and pressure applied until the filament bends. In theory, the force produced by the filament is independent of the degree of bending, and is determined by the thickness of the filament. The mechanical threshold is determined by the size of filament at which a behavioural (animal), or verbal (human) response is obtained. Monofilaments have been used in horses to quantify skin sensitivity following banding and microchip insertion (Lindegaard et al. 2009), and to investigate the effect of epidural ketamine on a skin wound in an experimental setting (Redua et al. 2002). Despite the ease of application and portability of the equipment the use of monofilaments does have a number of limitations. These have been reviewed recently by Bove (2006). A number of mechanical testing methods have been used to evaluate visceral pain in horses by reproducing the intestinal distension that may occur in horses with colic. These visceral pain models have particular clinical relevance since colic is a relatively common condition in horses. However, the severity of the experimentally induced pain is unlikely to be as marked as may occur in clinical conditions which result in ischaemic sections of intestine. This may explain why the responses to analgesic administration in clinical cases are not as consistently profound as the results of laboratory experiments suggest. Several balloon models of visceral pain have been developed: caecal distension with a balloon inserted via a surgically created caecal fistula (Lowe et al. 1970; Pippi et al. 1979); duodenal distension with a balloon inserted via a permanent implanted gastric cannula (Merritt et al. 2002); and colorectal distension with a balloon inserted into the rectum (Skarda & Muir 2003; Sanchez & Merritt 2005). In each case, pressures inside the balloon are

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measured and increased until a response is reached. Different techniques have been used for balloon inflation. Sanchez & Merritt (2005) and Merritt et al. (2002) used a computer-driven inflation procedure where air was pumped into a Mylar balloon so that the pressure within the balloon was increased by 1 mmHg and maintained for 20 seconds before a further increase of 1 mmHg. The Mylar balloon has infinite compliance therefore, changes in compliance should not influence the internal pressure of the balloon. In contrast, the technique developed by Skarda & Muir (2003) used a latex balloon which was inflated by a constant flow of compressed nitrogen, with continuous monitoring of the balloon volume, and pressure, by use of a sphygmomanometer and respirometer. The elastic recoil of the latex may have contributed to the pressure recorded in the balloon and partially account for both the considerably higher pressures (mean baseline threshold pressures 130– 152 mmHg) reported in comparison to the results reported by Sanchez & Merritt (2005) (mean baseline threshold pressure 14.7 mmHg), and for the absence of tissue damage, despite the recorded pressures being close to the reported bursting strength of the equine rectum (Hanson et al. 1988). Regardless of the method used for balloon inflation these techniques suffer from the inherent problem that the horses’ behavioural responses to abdominal pain (kicking at the abdomen, flank watching, shifting pelvic limbs) are relatively nonspecific and interpretation is subjective. This makes determination of an end-point challenging, and delays in interpretation of the end-point may mean that the stimulus is not terminated until a pressure beyond the threshold is achieved. A clearer endpoint was used in one of the colorectal distension models where the balloon was expelled from the rectum in 44 out of 48 experiments. However, the authors suggested that this may be more representative of an ‘urge to defecate’ or perception of the balloon rather than a true nociceptive threshold (Sanchez & Merritt 2005). Consistency in the rate of application of pressure is extremely important since both low and high threshold mechanoreceptors innervate the viscera (Gebhart 2000) and different patterns of inflation of viscera preferentially activate different nociceptors (Sabate et al. 2000). The variability in methods used for balloon inflation and differences in the elastic properties of the materials used in the construction of balloons employed in different studies for distension of

viscera could account for the large differences in reported thresholds (Sanchez & Merritt 2005) and could confound comparison of results obtained from different laboratories. Factors influencing the results of nociceptive threshold testing Temperature A wide range of environmental conditions may be encountered when working with large animals in their home environment. Seasonal and daily fluctuations in temperature may potentially influence nociceptive threshold testing via both animal factors (skin temperature, perfusion and moisture content) and interference with the equipment itself. The effects of environmental temperature on nociceptive threshold temperature are complex, with conflicting results reported in the literature. Variations in skin temperature have been reported to influence the tail flick latency (a thermal nociceptive test) in rats (Lascelles et al. 1995) though, using the same method, another investigator found no effect of skin or core temperature on the tail flick response (Lichtman et al. 1993). Variations in environmental temperature would be expected to result in cutaneous vasodilation or vasoconstriction that could influence the dissipation of heat and, therefore, temperature in the tissues surrounding nociceptors during a thermal nociceptive test. No effects attributable to environmental temperature were detected on either the function of the thermal nociceptive testing equipment or skin temperatures recorded from the ears of calves when recordings were taken at five different ambient temperatures between 5 and 27 °C (Whay 1998). However, when the initial recorded skin temperatures were low, the temperature at which the nociceptive response occurred was higher than when the initial skin temperatures were higher (Whay 1998). In the same study, the performance of mechanical threshold testing equipment varied with changes in environmental temperature although the calves’ mechanical nociceptive thresholds did not change with skin temperature. In contrast to these results, ambient temperature was found to have a significant effect on mechanical nociceptive thresholds in sheep, with markedly increased thresholds recorded at environmental temperatures below 8 °C (Chambers et al. 1994). The authors suggested that the increase in

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mechanical threshold was due to cold-induced vasoconstriction and ischaemia of small nerve fibres. The effect of environmental temperature on nociceptive thresholds measured in horses has not been comprehensively evaluated, but limited unpublished data suggest that at environmental temperatures below 10 °C thermal thresholds are increased compared to those measured at temperatures between 10 and 20 °C, but there did not appear to be significant effects on mechanical thresholds. Sweating may occur in high environmental temperatures, following drug administration (e.g. alpha-2-adrenoceptor agonists), following exercise or excitement. This could alter probe or actuator contact with the skin and heat transfer from a thermode. Drug induced alterations in cutaneous blood flow could potentially influence skin temperature and dissipation of heat during thermal threshold testing. Acepromazine (0.05 mg kg)1 IV) did not influence skin or threshold temperatures measured using a thermode based thermal threshold testing system (Love 2009). In contrast, when a similar testing system was used, administration of detomidine (10 and 20 lg kg)1 IV) resulted in a significant decrease in skin temperatures compared to baseline values (Elfenbein et al. 2009). The influence of alpha-2-adrenoceptor agonists on skin temperature and thermal thresholds in horses requires further investigation. Environmental stimuli and learning More consistent responses to mechanical threshold testing with less variability in thresholds were reported in sheep kept for experimental use compared to naı¨ve hill sheep introduced to the laboratory environment (Welsh & Nolan 1995). Training and acclimatisation to the testing stimulus was thought to be responsible for this (Welsh & Nolan 1995). However, the variability of the responses of heifers to a similar mechanical stimulus was not influenced by a period of training suggesting that adaptation to a new environment is also an important factor influencing nociceptive thresholds (Whay 1998). Cattle also appear to be receptive to environmental stimuli during the ‘wakeful’ state and these stimuli may act as distractions from the testing stimulus (Whay 1998). Distractions are used clinically in people to relieve pain (Wismeijer & Vingerhoets 2005) and distraction induced analgesia, as well as alterations in pain behaviour are produced in 10

response to fearful or stressful stimuli in rodents (Kavaliers et al. 1991). Exposure to novel environments and objects also reduces formalin evoked nociceptive behaviour in rats (Ford et al. 2008). It has been suggested that mice housed in pairs show empathy in response to observation of pain-related distress in their cage mate and this can facilitate nociceptive mechanisms (Langford et al. 2006). Empathy in horses has not yet been investigated but housing the horses in close proximity to each other could potentially influence the results of nociceptive threshold testing. Conversely, interaction with other horses is important to maintain social bonds, and separation of horses, as may occur during experimental procedures, may lead to abnormal behaviour (VanDierendonck et al. 2009). This could potentially also influence the results of testing as well as making the procedures hazardous for researchers. Chambers et al. (1994) reported that the variability of mechanical thresholds increased after fifteen minutes when the horses became ‘bored and easily distracted’. In horses, the majority of analgesiometry studies have been performed with the animals restrained in stocks. This introduces confounding variables, such as the stress of moving them to a different environment, disruption of social bonds, as well as the potential for boredom during prolonged testing schedules. Wireless thermal and mechanical threshold testing equipment have been developed to enable testing to be performed on horses in their stable with minimal restraint (Dixon et al. 2008; Love 2009). This has facilitated testing with consistent baseline thresholds obtained. After prolonged testing, horses may learn to respond to a stimulus as soon as it is perceived rather than when it becomes aversive (Kamerling et al. 1985b; Chambers et al. 1994). This may be eliminated by regular ‘sham testing’ (Kamerling et al. 1985b) or sedation (Chambers et al. 1990). The experience of the operator may also influence the results of nociceptive threshold testing through familiarity with the testing system, animal handling and an ‘as yet unexplained factor’ (Chesler et al. 2002). There is undoubtedly an operator learning curve associated with each technique. This may influence detection of end-points, and reaction times, as well as application of the stimulus. Gender differences Gender differences in nociception and pain have been reported in rodents (Mogil et al. 1993) and

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people (Fillingim et al. 2009). Results of somatic nociceptive testing in people have indicated that women have lower thresholds, less tolerance and give higher pain ratings than men (Berkley 1997). It is likely that the gender differences in pain tolerance as well as perception and response to analgesic administration in people are due to multiple factors including social, cultural, and environmental variables as well as genetic and hormonal influences (Craft et al. 2004). The effect on nociceptive thresholds of gender, the oestrus cycle in mares and castration of male animals have not been established in horses. The high cost associated with experimental procedures in horses limits the numbers of animals included in studies and this seriously restricts the power to detect relatively small differences attributable to gender. It is possible that the inclusion of both geldings and mares in studies has contributed to the variability in nociceptive thresholds reported with some techniques (Pippi et al. 1979; Hamra et al. 1993). Some investigators have limited nociceptive threshold testing on mares to the behavioural dioestrus phase of the oestrus cycle (Sanchez et al. 2007) since the behavioural effects of oestrus could potentially confound interpretation of behavioural end-points. Tissue damage Injuries such as tendon damage in horses and claw lesions in cattle have been reported to result in hyperalgesia and a decrease in mechanical nociceptive thresholds (Chambers et al. 1993; Whay et al. 1998). In contrast, in cows with mastitis, increases in mechanical nociceptive thresholds in the contralateral pelvic limbs, indicating hypoalgesia, were detected (Kemp et al. 2008). The authors’ hypothesis was that the increases in thresholds were due to the cows’ reluctance to bear weight on the pelvic limb on the side of the mastitic quarter (Kemp et al. 2008). A complete and thorough physical examination should be performed during animal selection for studies investigating antinociceptive effects of analgesics since the presence of a painful lesion could alter thresholds and influence results. Tissue damage due to repeated application of a noxious stimulus or the use of a stimulus that results in injury may also lead to altered thresholds, and this should be considered when designing equipment for nociceptive threshold testing. Prolonged heat stimulation of the skin may occur during the cooling phase if a thermode is fixed in

position. This may be partially overcome by removing the thermode from the skin as soon as possible after the threshold temperature has been achieved, moving the thermode to different sites between tests and with the development of more rapidly cooling heating elements. Data on the distribution and density of nociceptors in different areas of horse skin would also help to determine optimum regions of the body for nociceptive threshold testing and also the optimum skin surface area that should be stimulated to obtain a response. This has the potential to reduce skin damage while ensuring that a response is obtained and may reduce the ‘trial and error’ associated with the development of nociceptive threshold testing devices. In an attempt to prevent tissue damage the duration and/or magnitude of each type of stimulus are limited. This may limit evaluation of effective analgesics and mean that it is not possible to fully investigate dose-response relationships. However, it is often still possible to establish the duration of antinociceptive effects (Love 2009). Conclusions Thermal, mechanical and electrical nociceptive threshold testing have valuable roles in the identification of altered nociceptive function and the preclinical evaluation of analgesics in horses. When setting up an experiment a number of factors should be considered. Ideally the environmental temperature should be regulated, although if this is not possible it should at least be recorded. Minimising disruption and distractions to the horse can be achieved by testing within the animal’s normal environment and allowing it time to express normal behaviour between tests. Administration of a sedative without analgesic properties may reduce excitement following opioid administration and facilitate testing (Love 2009). Careful selection of animals is essential and will be determined by the study objectives; healthy horses without any painful conditions are essential for pre-clinical assessment of analgesic drugs, but deliberate selection of animals with a painful condition may be appropriate if this relates to the aims of the study. The concurrent use of different threshold testing methods has the potential to advance pre-clinical understanding of the differences between peripheral and central hyperalgesia and to determine the most appropriate analgesics for clinical evaluation. This may also be used to gain an insight into

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