The overall goal of this procedure is to measure no susceptive responses to mechanical pressure in the hind paw of mice. This is accomplished by first placing the animals on a wire mesh floor and allowing them to habituate when ready. A polypropylene tip is applied to the underside of the hind paw, and the pressure is gradually increased.
When the appropriate behavioral response is noted, the stimulus is withdrawn. Once baseline values are collected, the animals receive an experimental intervention before further testing. Ultimately, changes in hind paw withdrawal thresholds are compared across treatment groups to determine the effect of a particular treatment on changes in tactile sensitivity to pressure.
Pain is a cardinal component of inflammation. It is important to measure and characterize pain in different contexts. This relatively simple method of pain assessment can be applied to many different models of inflammation.
Demonstrating the procedure will be Tiana ov a research technician in my laboratory, Madison Mack, who was involved in this work as an undergraduate and Akila Sykes. Currently an undergraduate student in my laboratory. Be begin by setting up the test apparatus for easy access to the hind paws.
Place individual chambers on top of a mesh floor. The mesh floor should be at a comfortable height and on a sturdy surface. Next place the electronic Von Frey apparatus onto the benchtop.
Ensure that the probe and cable are appropriately connected to the display unit and turn the instrument on in the setup mode. Set the readout to zero and gently place the provided test weight onto the cone of the Von Frey probe. Record the readout and repeat this procedure again at the end of testing, if there is a discrepancy between the two values, the apparatus may need to be recalibrated.
Select a polypropylene tip of the appropriate size for the tissue to be tested. Rigid tips work well for the less sensitive, tougher hind paw tissue. Gently mount the tip onto the cone of the probe.
Next switch from setup mode to operation mode. This will display the maximum applied pressure when the probe is retracted. Two investigators are needed to measure hind paw withdrawal thresholds.
One, to operate the apparatus and carry out the measurements, and another to record those measurements In order to reduce bias. The investigator operating the anesthesi TER should be blinded to the treatments as well as to recorded withdrawal threshold values. Furthermore, to ensure consistency between measurements, the same individual should record all baseline and withdrawal threshold values.
48 hours before treatment, the first set of baseline measurements are recorded. Place absorbent material below the stand to collect excreted waste. After mounting the tip and zeroing the probe, the experimenter recording the measurements should place the mice into individual chambers.
Cover the chambers with perforated lids, and place a heavy object on top. To prevent the mice from escaping, allow the mice to acclimate to the chamber for 15 minutes before proceeding. Generally, individuals new to this technique will struggle with the gradual application of increased pressure, and with the establishing internal consistency with their own measurements, practice is essential to generating reproducible results.
When ready, use two hands to raise the probe slowly and stimulate the footpad of either the left or right hind paw. Increase the pressure gradually until a nociceptive behavior is observed. These are identified by hind paw retraction, hind paw licking, or for paw jumping.
Instruct the other investigator to record the pressure that elicited the observed behavior. While measuring it is useful to encourage the mouse to hold still by placing a food palate just outside the chamber or making a slight noise such as a gentle tap before the measurement is taken. Continue to the next mouse until each has been tested once and repeat this procedure four more times.
After collecting five baseline values, obtain an average for each mouse. Repeat the baseline measurements 24 hours later and use these values to calculate another average. To ensure that all mice have consistent baseline responses, exclude animals with an average baseline withdrawal threshold below 3.5 grams or with baseline thresholds differ by two grams or more.
Lastly, assign mice into separate treatment groups so that the average withdrawal baseline of each is as similar as possible Here, withdrawal thresholds were measured after hind paw, administration of the venom, ROS raca. Saline treated control. Mice showed little change compared to baseline at all time.
Points tested nociceptive responses in treated mice were clearly maintained up to six hours. In addition, tissue edema, characteristic of inflammation was evident in venom, but not saline treated hind paws. Venom induced changes in tactile sensitivity were accompanied by a neutrophil influx of almost 1000 fold.
In comparison to saline treated hind paws in saline treated paws, plantar mast cells in the hind paw tissue were largely intact or showed mild degranulation. In contrast, in mated hind showed 15 to 20%moderate and extensive degranulation of mast cells. This technique can be adapted to assess mechanical or tactile sensitivity in a wide array of tissues other than the hind paw.
Therefore, this technique can be incorporated into many different rodent models of inflammation and pain.
