The overall goal of this procedure is to determine the thermal nociceptive threshold of control and diabetic rats. The first step is to place an animal on the incremental hot plate and analgesia meter. Next, the temperature of the hot plate is gradually increased while the surface is heating.
The animal is observed for no offensive behaviors. When this behavior is observed, the heating is stopped and the plate's temperature at that moment is recorded. Ultimately, results can be obtained that show changes in the thermal nociceptive threshold in animals treated with analgesics or suffering from painful diabetic neuropathy.
The main advantage of this technique over existing methods like the hot plate, which is normally set as super threshold temperature, is that our methods gradually increases the temperature of the plate, allowing for the accurate determination of the thermal nociceptive threshold. The thermal nociceptive threshold or TNT is determined using the incremental hot plate analgesia, meter, or IH pam. The components of this system include a plexiglass observation chamber, a temperature controlled aluminum plate, a keypad used for programming, and a foot switch for remote control of the unit.
Data are collected using customized software on a nearby computer. Alternatively, hard copies can be printed using a serial port located on the unit. Place a mirror behind the plexiglass chamber to increase the visibility of the animal's behavior.
Next, set the desired heating rates as well as the initial standby and final cutoff temperatures. Press the foot switch to start and stop the system. Confirm that the initial standby and final cutoff temperatures are recorded as well as the time it takes to reach the final temperature.
If desired, the experimenter can test the system on themselves. Withdrawing the hand from the metal plate at the moment of pain. Perception must neither cause harm to the skin, nor produce any later discomfort.
Begin acclimation the night before by placing cages on the bench where the testing will take place. Two hours before testing. Turn on the IH pan to familiarize the animals with the background noise of the unit.
At this point, the system can be set up and tested as seen earlier. After the animals have acclimated to the environment, gently place a rat into the plexiglass chamber on top of the warm surface. Allow the animal to explore the chamber for a few minutes.
Begin testing. Once the animal appears comfortable to start the trial, press the foot switch to begin heating the plate. Observe the animal's behavior as the plate heats.
Generally, individuals new to this method will struggle because identification of mass offensive behavior is not always straightforward and requires practice. No offensive behaviors are characterized by the licking, shaking, or lifting of either hind pore. It is useful to note that the first behavior observed may be the licking of the four pores, followed by a reaction by the hind pores.
When a most offensive behavior is observed, press the foot switch to terminate the trial and remove the animal back to its home cage. The temperature that evokes this behavior is regarded as the thermal nociceptive threshold or TNT of the control animal. The unit is cleaned and the test is repeated for the next animal seen.
Here are the thermal nociceptive thresholds of normal young adult rats and mice. The data here show that the TNT of rats did not vary when they were tested over a four day period. Similarly, the TNT of mice was consistent between different ages.
In this example, TN TS of young adult STZ diabetic rats were repeatedly measured. A steady decrease in the thermal nociceptive thresholds can be seen starting two weeks after the induction of diabetes. Here, the IH pam was used to test the acute antinociceptive properties of the analgesic compound.
A, the compound produced a mild analgesic effect in control animals compared to those receiving vehicles. However, analgesia was more pronounced in diabetic animals receiving the compound compared to vehicle controls. While attempting this procedure, it is very important to remember to monitor continuously the animal's behavior, particularly those that occur near the cept T of the animal.
