Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

Summary

This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode.

Abstract

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical and thermal stimuli. QST is used to determine normal dogs' sensory thresholds and evaluate alterations in peripheral and central sensory pathways caused by various disease states, including osteoarthritis, spinal cord injury, and cranial cruciate ligament rupture. Mechanical sensory thresholds are measured by electronic von Frey anesthesiometers and pressure algometers. They are determined as the force at which the dog exhibits a response indicating conscious stimulus perception. Hot thermal sensory thresholds are the latency to respond to a fixed or ramped temperature stimulus applied by a contact thermode.

Following a consistent protocol for performing QST and paying attention to details of the testing environment, procedure, and individual study subjects are critical for obtaining accurate QST results for dogs. Protocols for the standardized collection of QST data in dogs have not been described in detail. QST should be performed in a quiet, distraction-free environment that is comfortable for the dog, the QST operator, and the handler. Ensuring that the dog is calm, relaxed, and properly positioned for each measurement helps produce reliable, consistent responses to the stimuli and makes the testing process more manageable. The QST operator and handler should be familiar and comfortable with handling dogs and interpreting dogs' behavioral responses to potentially painful stimuli to determine the endpoint of testing, reduce stress, and maintain safety during the testing process.

Introduction

Quantitative sensory testing (QST) assesses the responses elicited by externally applied stimuli; it is used to evaluate the function of the somatosensory system in humans and animals¹. Mechanical stimuli in the form of punctate pressure or deep pressure are applied as a ramped stimulus. The sensory threshold is determined as the force that evokes a psychophysical response¹. Hot or cold thermal stimuli can be used as a ramped stimulus or as a fixed intensity stimulus. The sensory threshold is determined as the temperature at which there is a response or the latency to respond to the stimulus. Punctate pressure sensory thresholds are measured using electronic von Frey anesthesiometers or von Frey hair filaments, deep pressure is measured using handheld pressure algometers, and thermal sensory thresholds are determined using a variety of contact thermode systems.

QST provides information about the functioning of both peripheral and central sensory pathways and can be used to evaluate alterations in these sensory pathways (algoplasticity) in various disease processes, particularly those that cause chronic pain¹. Meissner's corpuscles detect punctate pressure, and the sensation is transmitted by Aβ afferent fibers at non-noxious levels and Aδ afferent fibers when the stimulus is of a noxious intensity^1,2. Deep pressure is detected by Pacinian corpuscles and transmitted by C afferent fibers, noxious heat is detected by Ruffini corpuscles and transmitted by Aδ and C afferent fibers, and noxious cold is detected by Krause corpuscles and transmitted by C afferent fibers^1,2. QST can be used to detect both inhibition (decreased sensitivity, hypoesthesia) and facilitation (increased sensitivity, hyperesthesia) of these receptors and pathways. In dogs, QST has been used to evaluate alterations in sensory thresholds secondary to acute spinal cord injury^3,4,5, Chiari-like malformation and syringomyelia⁶, cranial cruciate ligament rupture^5,7, and osteoarthritis (OA)^8,9,10. Additionally, some studies have used QST to assess pain alleviation provided by certain analgesics^6,11,12,13 and surgical procedures¹⁴. These studies have provided important insights into the mechanisms of pain sensation in dogs, such as evidence for peripheral and central sensitization after surgery and diseases causing chronic pain states such as cranial cruciate ligament rupture and OA. This information can help improve the detection and treatment of pain in dogs.

Validation studies of mechanical and hot thermal QST in dogs have shown good feasibility, repeatability, and reliability of QST results over time in normal dogs and dogs with chronic pain from OA^8,9,15,16. However, several studies have found poor repeatability and reliability of cold thermal and occasionally von Frey QST^1,15,17. These studies used different equipment and methodology but provided evidence that mechanical and hot thermal QST is an accurate, semi-quantitative method of measuring sensory thresholds in dogs. However, attention to precise details, including the setting of the measurements, is critical to optimizing QST in dogs, necessitating a standardized protocol for QST. Sanchis-Mora et al. detailed a sensory threshold examination protocol (STEP) for mechanical and hot and cold thermal QST but encountered difficulty with dogs not responding to the cold thermal QST or the highest gram force von Frey filament used in the study¹⁷. The following protocol provides a standard method for mechanical and hot thermal QST in dogs; this protocol can assess sensory thresholds in normal dogs or dogs with various disease processes affecting the somatosensory system. The development of standardized protocols may allow for comparing results across studies and meta-analyses of data to improve the utility of QST in veterinary medicine.

Protocol

All procedures were approved by the Institutional Animal Care and Use Committee of North Carolina State University.

1. Room set-up and study subject acclimatization

1. Perform QST in a dedicated space where there is ample room for a QST operator, handler, and dog of any size to move about comfortably. Minimize potential auditory and visual distractions and use a white noise machine to block out ambient sound.
2. Place a large yoga mat or similar padding on the floor to ensure that the dogs are comfortable in lateral recumbency during testing.
3. Allow the dog at least 10 min to freely explore and acclimate to the room and become comfortable with the QST operator and handler. Offer fresh water ad libitum in the room, and give occasional food rewards.
4. Randomize the testing site (left or right side) by a coin flip. Clip an approximately 2 x 4 cm section of fur centered around the space between the dorsal surface of the third and fourth metatarsals halfway between the tarsometatarsal joint and the metatarsophalangeal joint. Clip an approximately 1 x 2 cm fur section on the lateral antebrachium just proximal to the antebrachiocarpal joint over the ulna.

2. Electronic von Frey anesthesiometer

1. Instrument set-up
   1. Gently apply a rigid 0.9 mm von Frey tip to the load cell and ensure that the load cell is securely screwed into the handpiece. Connect the cord from the handpiece to the recording device through the M0 channel (Figure 1A,B).
   2. Turn on the recording device and press the MAX button so that the device will record and display the maximum force achieved when the dog responds to the applied stimulus (Figure 1C).
   3. Zero the instrument by pressing the CLR button.
2. Data collection
   1. Place the dog in lateral recumbency for measuring thresholds.
      NOTE: Dogs are placed in right lateral recumbency for measuring thresholds on the left limbs or placed in left lateral recumbency for measuring thresholds on the right limbs. If the dog will not willingly lay in lateral recumbency when given verbal cues, the QST operator and handler can manually lay the dog in lateral recumbency.
   2. Apply minimal to moderate restraint as needed to maintain the dog in lateral recumbency and relatively still.
      NOTE: The handler performs this step.
   3. Apply the stimulus once the dog is calm and relaxed and the limb being tested is in at least 70% extension. Provide gentle manual support to the limb being tested to keep the limb off the floor and provide stable backing to apply force against while not preventing the dog from withdrawing the limb.
      NOTE: The QST operator performs this step.
   4. Apply the von Frey tip perpendicular to the skin of the area being tested. If the dog exhibits reflex movements (e.g., twitching of the paw or withdrawal of the limb before force is being applied) from the sensation of the von Frey tip on the skin, allow the dog to relax the limb again before reapplying the von Frey tip. Take a measurement when the skin does not cause reflex movements by applying the von Frey tip.
   5. Apply steadily increasing force with the von Frey tip (~20 g/sec) until the dog withdraws the limb, vocalizes, turns to look at the stimulus, or exhibits other movements or behavioral responses that indicate the conscious perception of the stimulus. Remove the stimulus when the dog withdraws the limb, or the maximum force is reached.
      NOTE: Do not exceed 1,000 g of force.
   6. Record the maximum force applied that is displayed on the recording device.
      NOTE: If the safety cut-off of 1,000 g of force is reached, 1,000 g is recorded as the sensory threshold, and it is noted that there was no response before the safety cut-off.
   7. Repeat the measurements for a total of five trials, allowing 1 min between each measurement (inter-trial interval). Zero the instrument between each step by pressing the CLR button.
      1. Allow the dog to remain in lateral recumbency during the inter-trial intervals if they remain relatively calm and relaxed with no or minimal restraint. Otherwise, allow the dog to sit, stand, or move about the QST room to maintain their comfort. Place the dog back in lateral recumbency before the subsequent measurement.
   8. Record a feasibility score of 0-5 to indicate the ease with which the data was collected.
      NOTE: Feasibility scores are as follows: 0 = no problem, 1 = mild difficulty, 2 = moderate difficulty, 3 = significant difficulty, 4 = extremely difficult, 5 = impossible. The rubric used for assigning feasibility scores is provided in Table 1.
   9. Give the dog a 5 min break before starting measurements with the blunt probed pressure algometer.

3. Blunt probed pressure algometer

1. Instrument set-up
   1. Ensure that the small blunt probe is securely screwed into the device (Figure 2A).
   2. Turn on the recording device and press the MAX button to continue when prompted on the screen. Press the UNIT button until the unit is displayed as grams (g) at the top of the screen (Figure 2B).
   3. Zero the instrument by pressing the ZERO button.
2. Data collection
   1. Place the dog in lateral recumbency for measuring thresholds.
      NOTE: Dogs are placed in right lateral recumbency for measuring thresholds on the left limbs or placed in left lateral recumbency for measuring thresholds on the right limbs. If the dog will not willingly lay in lateral recumbency when given verbal cues, the QST operator and handler can manually lay the dog in lateral recumbency.
   2. Apply minimal to moderate restraint as needed to maintain the dog in lateral recumbency and relatively still.
      NOTE: The handler performs this step.
   3. Apply the stimulus once the dog is calm and relaxed and the limb being tested is at approximately 70% extension. Provide gentle manual support to the limb being tested to keep the limb off the floor and provide stable backing to apply force against, while not preventing the dog from withdrawing the limb.
      NOTE: The QST operator performs this step.
   4. Apply the blunt probe perpendicular to the skin of the area being tested (Figure 2C). If the dog exhibits reflex movements (e.g., twitching of the paw or withdrawal of the limb before force is being applied) from the sensation of the blunt probe on the skin, allow the dog to relax the limb again before reapplying the blunt probe. Take a measurement when the application of the blunt probe to the skin does not cause reflex movements.
   5. Apply steadily increasing force with the probe (~20 g/s) until the dog withdraws the limb, vocalizes, turns to look at the stimulus, or exhibits other movements or behavioral responses that indicate the conscious perception of the stimulus. Remove the stimulus when the dog withdraws the limb or the maximum force is reached.
      NOTE: Do not exceed 2,500 g of force.
   6. Record the maximum force applied that is displayed on the recording device.
      NOTE: If the safety cut-off of 2,500 g of force is reached, 2,500 g is recorded as the sensory threshold, and it is noted that there was no response before the safety cut-off.
   7. Repeat the measurements for a total of five trials, allowing 1 min between each measure (inter-trial interval). Zero the instrument between each step by pressing the ZERO button.
      1. Allow the dog to remain in lateral recumbency during the inter-trial interval if they remain relatively calm and relaxed with no or minimal restraint. Otherwise, allow the dog to sit, stand, or move about the QST room to maintain their comfort. Place the dog back in lateral recumbency before the subsequent measurement.
   8. Record a feasibility score of 0-5 to indicate the ease with which the data was collected.
      NOTE: Feasibility scores are as follows: 0 = no problem, 1 = mild difficulty, 2 = moderate difficulty, 3 = significant difficulty, 4 = extremely difficult, 5 = impossible.
   9. Give the dog a 5 min break before starting measurements with the hot thermal probe.

4. Hot thermal probe

1. Instrument set-up
   1. Connect the thermosensory analyzer to the computer via the USB cable and ensure that the 16 x 16 mm thermode is connected to the analyzer. Turn on the analyzer.
   2. Open the thermosensory analyzer software on the computer and select the TSA II analyzer from the startup menu. Click on OK on the pop-up warning for the analyzer self-test. Ensure that the thermode is not connected to the study subject during the self-test.
   3. In the TEST tab (upper right-hand corner), under the Select Patient prompt (left-hand side of the screen), select the appropriate patient by double-clicking on the name in the list.
      1. To create a new patient, click on the PATIENTS tab to the right of the TEST tab. Click on the New Patient icon in the lower left-hand corner and fill in the patient details (department, patient first and last name, ID, gender, and date of birth).
   4. Under the Select Program prompt in the TEST tab, select the appropriate program by double-clicking on the program in the list.
      1. To create a new program, single-click on the PROGRAMS tab to the right of the PATIENTS tab. Click on the New Program icon in the lower left-hand corner and fill in the program details.
         NOTE: For this protocol, the program details are given in Table 2. A body site does not need to be selected under the Select Body Site prompt in the TEST tab.
   5. Once the appropriate patient and program have been selected, click on the Go to Test prompt under the TEST tab. Single click on the Pre-Test button in the lower left-hand corner to calibrate the analyzer to the specified program.
   6. Once the Pre-Test is complete, the Pre-Test button is replaced by the Start button, and the test window displays: Press Start button to start the test (Figure 3A).
   7. Unwind the thermode cable and ensure that the thermode is easily accessible.
2. Data collection
   1. Place the dog in lateral recumbency for measuring thermal latency.
      NOTE: Dogs are placed in right lateral recumbency for measuring thresholds on the left limbs or placed in left lateral recumbency for measuring thresholds on the right limbs. If the dog will not willingly lay in lateral recumbency when given verbal cues, the QST operator and handler can manually lay the dog in lateral recumbency.
   2. Apply minimal to moderate restraint as needed to maintain the dog in lateral recumbency and relatively still.
      NOTE: The handler performs this step.
   3. Apply the stimulus once the dog is calm and relaxed and the limb being tested is in approximately 70% extension. Provide gentle manual support to the limb being tested to keep the limb off the floor while not preventing the dog from withdrawing the limb. Also, hold and operate a stopwatch with the hand supporting the limb.
      NOTE: The QST operator performs this step.
   4. Apply the thermode to the skin of the area being tested (Figure 3B). If the dog exhibits reflex movements (e.g., twitching of the paw or withdrawal of the limb before heat is being applied) from the sensation of the thermode on the skin, allow the dog to relax the limb again before reapplying the thermode. Take a measurement when the application of the thermode to the skin does not cause reflex movements.
   5. Click on the Start button in the lower left-hand corner of the TEST tab to start the test.
      NOTE: The QST operator signals to the handler to start the test (e.g., by nodding their head), and the QST operator simultaneously starts the stopwatch.
   6. Remove the thermode when the dog withdraws the limb, vocalizes, turns to look at the stimulus, or exhibits other movements or behavioral responses that indicate the conscious perception of the stimulus or when the maximum latency is reached while simultaneously stopping the stopwatch.
      NOTE: The QST operator performs this step. Do not exceed 20 s of thermode application or 49 °C of maximum thermode temperature.
   7. Record the latency to withdrawal. If the safety cut-off of 20 s of thermode application is reached, record 20 s as the sensory latency, and note that there was no response before the safety cut-off.
   8. Repeat the measurements for a total of five trials, allowing 1 min between each measure (inter-trial interval). Click on the End Test button, and then the Pre-Test button between each measurement to stop heating the thermode and recalibrate the thermode to prepare for the next application.
      NOTE: The handler performs this step.
      1. Allow the dog to remain in lateral recumbency during the inter-trial interval if they remain relatively calm and relaxed with no or minimal restraint. Otherwise, allow the dog to sit, stand, or move about the QST room to maintain their comfort. Place the dog back in lateral recumbency before the subsequent measurement.
   9. Record a feasibility score of 0-5 to indicate the ease with which the data was collected.
      NOTE: Feasibility scores are as follows: 0 = no problem, 1 = mild difficulty, 2 = moderate difficulty, 3 = significant difficulty, 4 = extremely difficult, 5 = impossible.

Results

Mechanical and thermal QST has been performed to detect sensory thresholds in both research and client-owned dogs under various clinical conditions, including normal, healthy dogs, dogs with chronically painful conditions such as OA, dogs with acute spinal cord injury, and to assess post-operative pain and effectiveness of analgesics. Though there is a growing body of work on QST in dogs, no normal range of values has been established for any testing modalities. However, several studies have assessed the feasibility and ...

Discussion

It is crucial to the acquisition of accurate data - that reflects the dog's sensory thresholds - that the dog is as calm, relaxed, and positioned adequately as possible for each measurement. A previous study noted that agitation from restraint or distraction from factors within or outside the testing environment affected dogs' responses to the QST stimuli¹⁶. If the dog becomes agitated from recumbency or restraint or is distracted, the dog should be given time to settle before a measuremen...

Materials

Name	Company	Catalog Number	Comments
Electronic von Frey anesthesiometer	IITC Life Science Inc.	Item # 23931	Custom made with a 1000g max force load cell
Medoc Main Station software	Medoc	(supplied with TSA-II)
SMALGO: SMall Animal ALGOmeter	Bioseb	Model VETALGO
TSA-II NeuroSensory Analyzer	Medoc	DC 00072 TSA-II	No longer manufactured - new model is TSA-2 with same probes and same function