May 25, 2026
Thermal Nociception Threshold at the Knee in Wistar Rats Protocol
- Ashleigh Abbott1,
- Robert Dalton1,
- Kaitlin Southern1,
- Folly Patterson1,
- Kyle D. Allen1
- 1University of Florida
Protocol Citation: Ashleigh Abbott, Robert Dalton, Kaitlin Southern, Folly Patterson, Kyle D. Allen 2026. Thermal Nociception Threshold at the Knee in Wistar Rats Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl847b8g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 28, 2026
Last Modified: May 25, 2026
Protocol Integer ID: 315904
Keywords: Thermal Knee, Knee sensitivity, Thermal pain, Thermal nociception, Rodents, Thermal sensitivity, Withdrawal latency, Physical withdrawal, Behavior, Pain-like behavior, Nociceptive assay, thermal nociception threshold at the knee, thermal nociception threshold, thermal threshold recording, wistar rats protocol description, cold measurement, locations on the knee, location on the knee, nociception, knee, temperature
Funders Acknowledgements:
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Grant ID: UC2AR082196
Abstract
Description: The aim for data collection is to collect thermal threshold recordings at three locations on the knee (medial, frontal, and lateral) with the QST.lab device. A warm and cold measurement will be recorded at each location on the knee, resulting in 6 trials collected/knee/rodent/temperature (12 total trials). The estimated time can vary pending animals behavior but will take roughly 6-8 hours for 12 rats.
Image Attribution
Figure 1: Anatomical regions of the knee used for mechanical sensitivity testing. Medial testing was performed at the medial tibiofemoral joint line, corresponding anatomically to the level of the BB landmark (fibular head)on the lateral aspect of the knee where the joint space is palpable (left image). Anterior testing was conducted inferior to the patella near the anterior joint line (middle image). Lateral testing was performed at the lateral tibiofemoral joint line directly superior to the BB landmark (right image). Red star = BB landmark; red circle = testing region.
Materials
- QST T10 probe
- QST.Lab Computer module (Model: TCS II.1.b)
Personal Protective Equipment (PPE):
- Disposable gloves
- Face mask (optional)
-Disposable PPE gown
Safety warnings
Protocols and behavioral collection requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Ethics statement
Protocols and behavioral collection described here were approved by the local Institutional Animal Care and Use Committee (IACUC) and university ethics committee.
Before start
Acclimation (1-2 weeks)
Before data is collected, animals must be acclimated to experimenter handling and the tuning forks. The acclimation period occurs before baseline data is collected. There is no data collection during acclimation. On the first week, each rat should undergo acclimation to handling for periods of 5-10 minutes (routine handling acclimation). The second week of acclimation will consist of handling in the c-hold and acclimation to palpation of the knee. It is important the animals are comfortable through testing to not skew results, so increase acclimation time as needed. During acclimation, the conditions in the behavior room should mimic those present during the testing period.
General Set Up
Plug in the response trigger into the corresponding input (labeled “Response”) on the right side of the QST.Lab module.
Turn on the QST.Lab module using the on/off switch on the right side of the device.
Thermode of stimulation on the T10 probe ~0.25" diameter
QST.Labs T10 Probe (can heat and cool at controlled temperature ranges)
QST.Lab Module (TCS II Model)
If you have not already done so, create an experimental protocol to set the temperature range, the ramp rate of temperature, the series of the test, and the start/stop settings.
To open an experiment protocol, tap on the following menu options:
Thresholds and Standard Protocols
Select your correct protocol either cool or heat
Uncap the T10 probe, Hold the T10 probe in the experimenter’s dominant hand, Hold the response trigger in the experimenter’s non-dominant hand.
Data Collection
After bringing the rat cages to the testing but before handling, let the rats acclimate to the room for 30 minutes.
Set up the QST.Lab module (see above section, “Set Up of QST.lab equipment”). Use the program which consists of either heating or cooling until response or time limit is reached:
Heating parameters: baseline of 30°C with temperature ramp increase of 0.5°C/second to 50°C
Cooling parameters: baseline of 30°C with temperature ramp decrease of -0.5°C/second to 0 °C
To begin collection, the first experimenter (handler) should be in a seated position holding the first rodent. A second experimenter is necessary to apply the thermal probe. The handler will hold the rodent in the C-hold against their chest with the dorsal side of the rat facing the handlers chest and the knee facing out.
Once the rat is settled and not squirming, the second experimenter will palpate the knee lightly to locate the knee joint space.
Note: To locate the knee joint space, feel the knee to find where there is a space or where you can feel the BB-like structure (fibular head) on the lateral side of the knee. The knee may be higher than expected, so feel for this joint space. It should be with relatively light pressure that you can feel the joint space between the femur and tibia.
Figure 1: Anatomical regions of the knee used for mechanical sensitivity testing. Medial testing was performed at the medial tibiofemoral joint line, corresponding anatomically to the level of the BB landmark (fibular head)on the lateral aspect of the knee where the joint space is palpable (left image). Anterior testing was performed on the patellar tendon inferior to the patella (middle image). Lateral testing was performed at the lateral tibiofemoral joint line directly superior to the BB landmark (right image). Red star = BB landmark; red circle = testing region.
Apply the thermode flush with the skin overlying the joint space, maintaining a perpendicular angle of the probe to the knee.
To start the stimulation, tap the Start button. A countdown will begin before the element begins heating/cooling. Press the response trigger immediately when/if a response is noted. Pressing the response button will stop the stimulation and return the element to baseline temperature (30°C).
Remove the probe from the knee and record the temperature at withdrawal. Return the rodent to their cage.
Record the type of response as either positive (X) or negative (O) for each trial, along with the data displayed on the QST.lab device (response temperature, delta of temp., etc.) at the end of each trial.
A positive response is an audible vocalization to the ear, a visual withdrawal from the stimulus, or a reflexive withdrawal felt by the rat handler.
An ambiguous response to the stimulus is if during or after the temperature range the rat moves/slightly raises or shakes its leg from the thermode without vocalizing.
If a rat has an ambiguous response, repeat the measurement or application of the probe again; record as positive (X) or a negative (O) response along with QST.lab data.
A negative or non-response to the stimulus is, if after the ten-second count, the rat neither moves/takes away their leg (an ambiguous response) or vocalizes (positive response).
If the type of withdrawal is clear, record the type of response (vocalization, visual withdrawal, reflexive withdrawal).
Once you have completed the trials at one aspect of the knee (1 application/trial), proceed to the next rat (i.e. do all medial trials once for all rats). The rats are returned to their home cages between testing.
Typically, all rats are tested in the medial aspect, then all rats for the anterior aspect, and all rats for the lateral aspect of the knee joint space. This approach spaces out the testing of each testing region.
Repeat Steps 8-14 for the next two aspects of the knees. Leave 30 minutes between trials on the same rat.
Depending on the number of rodents you are testing at once, there may have already been 30 minutes elapsed between testing the first and last rats. In this case, you can continue onto the next location.
Repeat all previous steps for heat after doing cool. This should result in one trial/aspect of the knee/temperature (heat or cool).