MECHANICAL NOCICEPTIVE THRESHOLD TESTING 1 Threshold testing device

Based on the design of the “pressure of palpation device” developed by Slingsby et al (2001), which has been proven to produce reproducible readings of threshold testing both in dogs and cats, a mechanical nociceptive threshold testing device was built with slight

modifications (figure 6). It contained a 2.2mm diameter blunt ended pin with head attached to strain gauge (SG). The SG was connected by a wire to a Wheatstone bridge circuit, which was enclosed in a small fibre box. The box had a digital display, on/off switch and a reset button in the front and the device was battery operated by two nine volt batteries. When switched on an initial reading (- 0.38) appeared each time, which was subtracted from the actual measured value. The device was calibrated using an electronic balance (Model PG 5002-S delta range mercury balance, Watson Victor Ltd. Sydney, Australia). The force applied on the pin head with the thumb, pressed the pin against the plate on the balance and both the weight (g) displayed in the balance and the corresponding reading on the device were recorded. Since there was no means of holding the pressure value, the readings were noted very quickly before a sharp drop, which required help, by another person. Twelve replications were made to keep the standard error as low as possible. 100 g weight was taken as 1Newton (N) and values in Newtons were transformed into their natural log (Ln) for

calibration purpose. A linear graph was built by plotting the log-transformed Newtons against the testing device readings (figure 7). A regression equation obtained from the calibration graph was used to calculate the force from the device reading when dogs were tested.

Figure 6: Mechanical threshold testing device used to assess efficacy of test drugs in dogs

0 2 4 6 8 10 12 0 0.5 1 1.5 2 2.5 3 3.5 Force (Ln N)

Figure 7: Mechanical nociceptive threshold testing device calibration curve. The Y- axis

shows the pressure applied to generate corresponding force in Newtons (y =

2.6658X + 1.0686; r = 0.9927). The X- axis shows the force in natural log of Newtons (Ln N)

3.2.2 Threshold testing

Dogs were allowed 30 minutes after arrival for initial settlement in the new environment (laboratory). They were tested in pairs. Similar background music was played in low volume throughout the testing period with a minimum number of people around. The dogs were not muzzled during the experiment. Ambient temperature in the laboratory was monitored throughout each experiment, and was between 140_{C and 22}0_C.

Thresholds were measured at the ventral abdomen (on the midline, half way between

umbilicus and pubis) of dogs in either standing or recumbent positions. Stimulus was applied at the same site in all dogs. Baseline threshold measurements were taken four times with five minutes between each pair of recordings. The mean±SE of the four baseline values was used for statistical analysis. The behavioural responses indicating the threshold were aggressive teeth bearing, vocalisation, vigorous turning towards the stimulus or standing suddenly from a lying position (Barnhart et al. 2000). This was followed by a baseline ICL test. Following baseline ICL test, the dogs received pre-anaesthetic medication. Thirty to sixty minutes after pre-anaesthetic medication, they were given the analgesic drugs and anaesthesia was then induced and maintained for 30 minutes. Post-treatment nociceptive thresholds were measured using the same procedure as baseline threshold testing, at 20, 40 and 60 minutes after

extubation. A cut-off point of 25 N prevented tissue damage, if any dog did not respond to the stimulus and this value was recorded.

The device was portable, had prompt control over stimulus, easy to operate, and did not require any attachment to the test site. Threshold measurement by single investigator

enhanced the repeatability and therefore, all the four baseline readings remained very close to each other in every dog, which acted as its own control. Dogs tolerated the pressure applied through the probe and produced a clear-cut avoidance (behavioural) response indicating the threshold. Initially, the device was tested with co-researchers in the laboratory and was found to produce no residual pain or tissue damage. Visual examination of the testing site in dogs revealed no signs of tissue injury or persistent skin damage.

The box enclosed the measuring circuit was portable and robust. The joint connecting the SG to the first part of the cable wire was reinforced with strong plastic sleeves, top of which was

sealed with an adhesive tape. This provided sufficient strength to handle the SG and avoided variation in readings due to flexion of the joint (Slingsby 1998).

Conversely, there were few drawbacks to the use of the device:

1. Fast fall in readings immediately after ceasing pressure application on the probe, 2. Amount of charge in the batteries influenced the reading. These findings were

consistent with those of Slingsby (1998) who originally designed the device. Nevertheless, some remedies adept to fix these problems were:

1. Positioning the device close to the testing site and careful observation of the reading during pressure increments, and

2. Changing batteries frequently and calibrating the device each time batteries changed.