May 28, 2026
Thermal Nociception Threshold at the Paw in Wistar Rats Protocol
- Ashleigh Abbott1,
- Robert Dalton1,
- Kaitlin Southern1,
- Shane Priester1,
- Folly Patterson1,
- Kyle D. Allen1
- 1University of Florida
Protocol Citation: Ashleigh Abbott, Robert Dalton, Kaitlin Southern, Shane Priester, Folly Patterson, Kyle D. Allen 2026. Thermal Nociception Threshold at the Paw in Wistar Rats Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz4p52lx1/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 28, 2026
Protocol Integer ID: 315902
Keywords: Thermal Sensitivity, Thermal pain, Pain, Pain-like behavior, Thermal nociception, Thermal paw, Paw, Rodent, thermal nociception threshold at the paw, thermal nociception threshold, thermal nociception, thermal threshold recordings at the plantar, thermal threshold recording, thermal probe, response to the thermal probe, wistar rats protocol the aim, wistar rats protocol, surface of the paw, paw, rodent, midplantar paw region
Funders Acknowledgements:
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Grant ID: UC2AR082196
Abstract
The aim for data collection is to collect thermal threshold recordings at the plantar (bottom) surface of the paw with the QST.Lab equipment. This protocol uses a thermal probe that can heat or cool rapidly upon programming with the module. Thermal nociception was determined based on when the rodents physically withdrew and/or vocalized in response to the thermal probe on their midplantar paw region.
Materials
- QST T10 probe
- QST.Lab Computer module (model: TCS II.1.b)
- Acrylic (von Frey) single-animal cages
- Metal mesh platform as flooring for von Frey cages; meshwork must be at least 0.5 inches by 0.5 inches for the T10 probe to pass through
Personal Protective Equipment:
- Disposable gloves
- Face mask (optional)
- Animal handling gown
Before start
Before data is collected, animals must be acclimated to acrylic cages. The acclimation period occurs before baseline data is collected. There is no data collection during acclimation. Each rat should undergo a few days of acclimation to the cages and mesh flooring for 10-20 minutes. It is recommended to do 3 or more days of this acclimation. During acclimation, the conditions in the room should mimic those present during the testing period.
Note: ideally acclimate the rat to the probe touching their paw (while off) during acclimation period.
General Set Up
Lay the 0.5” x 0.5” metal mesh flooring onto a rack shelf in the room.
NOTE: Ensure the height of the rack is adequate enough to be able to place the rat in the cage, but also have access to the underside of the cage.
Standard von Frey cages on a custom testing surface to allow clearance for probe and reaching the paw below the cages
Mesh 0.5"x0.5" to allow clearance of the T10 probe
Line up the mesh and cages so that the QST probe can fit through both sets of metal bars.
Place cages directly on top of the metal mesh flooring.
Plug in the response trigger into the corresponding input (labeled “Response”) on the right side of the QST.Lab module.
Stimulation surface and thermode for the T10 probe ~0.25 inch diameter
QST.Labs T10 Probe (can heat and cool at controlled temperature ranges and rates)
QST.Lab Module (TCS II Model)
Turn on the QST.Lab module using the on/off switch on the right side of the device.
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.
Load either the heat or cool protocol depending on the order of testing. In these experiments, the cool protocol was used first.
Uncap the T10 probe and 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 room but before beginning, acclimate rats to room in its home cages.
Set up the QST.Lab module. 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
Note: test cool for 3 trials before proceeding to the test heat protocol.
Place the rats in the von Frey cage atop the metal wire mesh flooring
Note: needs to be the wire mesh with 0.5x0.5” for the probe to fit
Allow the rats 10-15 minutes to acclimate to the enclosure.
Place the probe on the midplantar region of the paw when ready to stimulate. Ensure as much of the thermode element is flush with the surface of the paw as possible. Keep the probe perpendicular and flush to the paw.
Red circle is the midplantar region of the paw that is stimulated. Image adapted from [Koike, et al., Cell Tissue Res 386, 477–490 (2021). https://doi.org/10.1007/s00441-021-03525-5]
To start the stimulation, tap the Start button. A countdown will begin before the element begins heating/cooling. The experimenter will hold their non-dominant thumb over the response trigger.
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). If no response occurs when the temperature range (~40-60 seconds) has elapsed, proceed to next step.
Remove the probe from the paw once response is noted. The temperature at withdrawal time or once the time is complete with no response will be displayed on the module.
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 and/or a visual withdrawal from the stimulus. Similar to von Frey withdrawals. Further, if the rat licks its paw that is considered positive, however, a lick does not occur during every positive response.
An ambiguous response to the stimulus is if during or after the ten-second count 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 its leg, takes away their leg (an ambiguous response) or licks its paw/withdrawals it’s paw/vocalizes (positive response).
If the type of withdrawal is clear, record the type of response (vocalization, visual withdrawal, reflexive withdrawal).
Repeat steps 15-20 until the number of trials per temperature is achieved (in this case 3 trials total).
Repeat testing of each rat with 30 minutes between each testing session for a total of 3 sessions per paw for one temperature (heat or cool).
Repeat steps 15-21 for the heat protocol for 3 trials per rat after the completion of 3 trials per rat for the cool protocol.
Return rats to the cages and standard housing once complete.