Sensory nociceptive measurements

Pain is a multifactorial entity which cannot be measured by a single method (STEAGALL et al., 2007). Nociceptive threshold testing involves the administration of a quantifiable stimulus to a body part until a behavioral or physiological reply is observed, at which point’s application of the stimulus is terminated. The stimuli should have characteristics such as reliability, repeatability and to be easy to apply with a clear end-point.

Moreover, an increase in nociceptive thresholds after application of an analgesic is accepted as an indication of an anti-nociceptive impact (LOVE et al., 2011).

Electrical, thermal and mechanical nociceptive threshold tests are the most often used tests (Luna et al., 2014).

2.6.1.1. Mechanical threshold

Mechanical nociceptive threshold tests are well accepted strategies which can be applied to investigate of pain and analgesia as well as post-operation hyperalgesia (DIXON et al., 2007; BRENNAN et al., 1996; LASCELLES et al., 1998; DAHL et al., 1990 ).

Veterinarians always use different mechanical devices to measure pain in

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animals. For example, a reproducible device includes of a pneumatically operated piston with a pressed blunt ended pin on the animal’s leg under the control of computer has been designed to use on sheep, horse as well as human’s leg (CHAMBERS et al., 1994). Another example of those devices is a small, silent, low friction and linear actuator for mechanical nociceptive testing in cats and dogs which is light and easy to administer to the limb of small animals.

Another benefit to use such those devices is that, using the small and manually driven syringe to generate the driving pressure can make the system completely silent and therefore does not disturb the animals at all. However, a mechanical stimulus particularly could be commonly generated by using a pneumatic actuator to drive a pin into the tissue in larger animals (DIXON et al., 2010). To measure the nociceptive threshold in dairy cows, a pneumatically actuated blunt pin with 2 mm in diameter can be applied. This pin could be pressed against the dorsal aspect of the metatarsus and thereafter the required pressure to generate a cow’s response would be identified. One point to use this device could be that, the type of foot’s injuries or lesions does not have any significant effect on the response of the nociceptive threshold (LAVEN et al., 2008).

Using mechanical threshold device to examine the analgesia effect of drugs has been applied in several studies. Being simple to use and the capacity of producing reproducible responses are some criteria of the mechanical equipment which would be applied for that aim. Furthermore, to apply the mechanical device, not only the stimulus must be the same but also its rate of administration must be the same from trial to trial (CHAMBERS et al., 1990).

Pressure nociceptive threshold testing device could be applied in different species. For instance, in domestic cats, the pressure stimulation is produced via a plastic bracelet weighing 5 gm which is taped around of forearm of each cat with non-elastic masking tape (DIXON et al., 2007). In addition, we can improve the nociceptive threshold responses under different pins’ application to test the nociceptive threshold responses. As an example of behavioral reactions to mechanical stimulation, cats’ behavioral reactions to the pressure stimulus is picking up the leg and shaking it, turning the head towards the bracelet, licking or biting the bracelet as well as vocalization (DIXON et al., 2007).

Analgesia under drug’s administration has some impact on the nociceptive threshold responses. For instance, the use of non-steroidal anti-inflammatory drugs (NSAIDs) has a potential to decrease the impact of the hyperalgesia resulting from lameness. As an example, the application of ketoprofen could increase considerably the mean nociceptive thresholds, even though ketoprofen does not suppress the hyperalgesia completely, the

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modulation is a significant step in the control of long-term or chronic pain associated with lameness (WHAY et al., 2005). In other words, it is the contribution of hyperalgesia to chronic pain (REEH and SAUER, 1997). Another example could be detomidine which can produce a significant increase in mechanical thresholds (CHAMBERS et al., 1994).

Dosage of analgesic could be another point which has an impact on mechanical threshold responses in animal models. For instance, low dose ketamine infusions minimally can affect mechanical as well as thermal anti-nociception in conscious cats (AMBROS and DUKE, 2013). Instead of dosage, type of drugs has an effect on mechanical threshold responses. For example, pressure thresholds after buprenorphine administration is remarkably higher than after carprofen application at 2 hours. The mean pressure thresholds after buprenorphine is above the upper 95 % CI from 2 - 3 hours and from 6 - 8 hours after treatment while mean pressure threshold after carprofen application remains within the 95 % CI (STEAGALL et al., 2007). Additionally, the timing of the test as well as the weight could be effective in mechanical threshold responses (JANCZAK et al., 2012).

2.6.1.2. Thermal threshold

Heat stimulation of the skin is applied to simulate superficial or cutaneous pain.

Different techniques of heat stimulation are used which includes the measurement of the latency to a response after a constant temperature exposure or measurement of the temperature at which a response occurs when there is a ramped increase in temperature (LOVE et al., 2011). Another differentiation can be made by the type of heat applied, either radiant heat or contact heat.

Radiant heat by laser light stimulation was used on the bovine hind leg with a tail flick as end-point. Tail flicks with an overall average of 5.5 ± 2.5 as well as an average of 0.5 ± 1 tail presses per 25 sec can be the dairy cows’ responses during laser stimulation (RASMUSSEN et al., 2011). Application of a laser at a range of power settings (2.0, 3.0, 4.0, 4.5, 5.0 and 5.5 W) showed that, response latencies decrease as power increase up to 4.5 W, after which no further change occur (VEISSIER et al., 2000).

Contact heat is nowadays often applied via wireless systems, to allow free movement of the animal, as restraint can interfere with the response.With this technique, the heat stimulus is provided by a small probe, which includes a thermal element as well as a temperature sensor. The probe temperature is increased by 0.5 to 0.6˚C per second with a safety cut-off between 55˚C to 60°C to prevent skin damage (AMBROS and DUKE, 2013;

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DIXON et al., 2002; TAYLOR et al., 2007; BARTER and KWIATKOWSKI, 2013).

The endpoint of stimulation depends on the site of stimulation, it can be a reflex movement or a more complex behavioural response. Different behaviors in response to different sites of stimulus application can be seen. For example, stimulation at the withers followed by a skin flick while stimulation at the nostrils results in head shaking or rubbing the face against an object in horses (POLLER et al., 2013a; POLLER et al., 2013b).

2.6.1.3. Electrical nociceptive threshold

The application of electrical stimuli has the advantages of being quantifiable, reproducible, and noninvasive and of producing synchronized afferent signals. However, it also has disadvantages. Electrical stimuli are not a natural type of stimulus like those encountered by an animal in its normal environment. More importantly, intense electrical stimuli excite in a non-differential fashion all peripheral fibers, including large diameter fibers, which are not directly implicated in nociception, as well as fine Aδ and C fibers, which mediate sensations of cold and hot as well as nociceptive information. Furthermore, this type of stimulation completely short-circuits peripheral receptors (Le Bars et al., 2001). There are difficulties introduced by variations in the impedance of tissue being stimulated, although these can be minimized by the use of a constant current stimulator and the monitoring of the voltage as well as the current of the applied stimulus. Electricity can be applied in a very brief and sudden fashion. This results in the signals in the afferent nerve fibers being synchronized.

Thus, electrical stimuli can release a vast repertoire of behavioral responses that are graded as a function of intensity from spinal reflexes, through complex vocalizations, and up to very organized types of behavior (escape, aggression and so on). The electrical thresholds of individual fibers are related to their diameters; therefore, when the applied intensity of an electrical stimulus to a cutaneous nerve is increased progressively, it is first the Aβ, then the Aδ, and finally the C fibers that are activated (Le Bars et al. 2001).

There is no standardization of current and electrode position in electrical nociceptive threshold testing. Needle electrodes or surface electrodes can be located in the tooth pulp as well as the coronary band or even to the digital palmar nerve (LUNA et al., 2014). Stimulation protocols also can vary, like constant current stimulation in humans with delivering sine-wave stimuli at 5 Hz via two gold electrodes and by increasing its intensity from 0 to 20 mA by 0.2 mA s^-1 (NEDDERMEYER et al., 2008), or like in horses with two adhesive electrodes by administration of an alternating current square wave of 50 Hz and 10

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ms duration by starting at 1 V and increasing that each 5 second until observation of the withdrawal’s responses or reaching the maximum of 15 V (LUNA et al., 2014).