This video demonstrates two pain testing protocols. The first model involves applying heat to the skin to induce pain. In this test, subjects are exposed to gradually increasing temperatures with a ther mode and are asked at which point they experience pain.
The temperature of the ther mode, just as it becomes painful, is the thermal pain threshold. The second model evokes pain using mechanical pressure. Here probes of different weights are applied to the subject who indicates when the probe becomes heavy enough to be painful.
These two pain testing paradigms can independently examine nociceptive processing by the two major peripheral nerve fiber populations that transmit pain. Non myelinated C fibers, which detect burning heat pain, and myelinated a delta fibers, which detect sharp mechanical pain. Hi, I'm Martin Angst from the Department of Anesthesia of Stanford University.
In a previous article published in the Journal of Visualized Experiments, we documented the use of microdialysis techniques for the tissue specific collection of nociceptive and inflammatory biochemicals in human volunteers. Today we will show you pain testing paradigms that we often use in tandem with microdialysis techniques. The combined use of pain test paradigms and microanalysis techniques provides the critical link between behavioral and biochemical data that allows us identifying biochemicals responsible for generating and propagating pain signals.
So let's get started. This procedure uses the TSA two, a commercially available thermos sensory analyzer that delivers heat stimuli to the skin. With a thermo, a central unit generates and transmits the signal to the thermo.
This signal is controlled by proprietary software, typically stored on a laptop connected to the central unit. Also attached to the central unit is a handheld control that allows a study subject to stop the heating of the thermo. Seven sequential heat stimuli are delivered with inter stimulus intervals of 30 seconds.
The basal thermo temperature is set at a comfortable 35 degrees Celsius. The rate at which the thermo heats up is set at one degrees Celsius per second. While the rate at which it cools down is set at 10 degrees Celsius per second, the maximum thermo temperature is set at 52 degrees.
To prepare subjects for the heat pain test seat them comfortably in a reclining chair that provides adequate support for the head, arms, and legs And make yourself comfortable. Would you Like a blanket? Sure.Thanks.Blanket.Okay.The chair should also allow easy access to the examined body sites.
The testing facility should be set at a comfortable room temperature and provide a quiet and neutral environment with no distraction before starting the experiment. All participants should be well-trained in all aspects of the heat. Pain testing training can take place anytime before they experiment.
Interactions between study subjects and investigators should be limited to those necessary for collecting data. Joking and small talk are discouraged because they may compromise data consistency. Study subjects are informed that interactions will be intentionally, somewhat monotonic and neutral.
During the experiments. Keep the instructions clear, simple and consistent. Emphasize that pain is subjective and there are no right or wrong answers.
It is most important to be consistent. We're gonna be doing the heat pain testing today, and in we, one of the things we want is to get very consistent results from you. We want you to be able to clearly identify the threshold, the point at which you first feel pain in order to really, in order for you to be really consistent.
It's important for us to keep the environment very calm and quiet and kind of unexciting.Okay? So during the whole procedure, you and I won't have conversation. We won't be able to joke, we won't be able to talk about the politics of the day.Okay.
We'll just be paying foot, focusing all of our attention on the pain testing itself.Okay? Okay.And again, it's very important for us to get consistent results. And the only way you can give us those consistent results is by keeping the environment very, very calm, very uninteresting, and really rather boring.Okay?
It is critical that a subject knows precisely what to expect during a test session. Any surprises may result in less consistent data because confounders, such as anxiety may be generated, define the heat pain threshold as the point between just about to be painful and just became painful. For example, if seven heat pain stimuli are used to determine the heat pain threshold, the subject should know this.
So how does the testing work? We're gonna be measuring heat pain threshold.Okay. And that's the point at which you first experience pain.Alright.
There will be a repeat of seven exposures.Okay. There will be a 32nd break between each one of the exposures.Okay. The ther mode will be placed on your leg.
It'll start off at a comfortable temperature that will increase gradually in temperatures where you will perceive it to be comfortable, warm, really, really warm, hot, really, really hot.Okay? And at some point it will transition from really, really hot to burning pain. As soon as you experience that burning pain, you press a button, the temperature will immediately drop back down to normal.Okay?
It'll be a 32nd break and it will repeat.Okay? Okay.And it's important for you to know too, that this is the machine is set so that it won't achieve a temperature that is so hot that will actually damage your skin. So while you may feel burning pain, and it's going to feel as if it is burning your skin, it will not actually damage the tissue.
So you don't need to hesitate. You don't need to worry about pressing the button a little too soon, a little before you actually feel that first experience of pain because you will not be Injured. Oh, Okay.Okay.
That's good to know. At any time, the subject can stop the thermo from getting any hotter by pushing the button of the handheld control. Being in control will reduce a subject's anxiety and will make the data more reliable.
And if I have questions about the testing itself, should we talk about those, or should I say close for later? During the training, you can ask questions between the stimulus.Okay. When we're actually conducting the study, you and I won't be able to have any conversations.Okay.
So it's important that the training is conducted properly and that we really get you familiar with what your tasks are.Okay. And then during the data collection piece of the study, you and I will not have any conversation at all.Okay? Okay.I think I can do that.
Okay, great. We'll practice anyway. We'll practice The training session offers an opportunity to judge a study subject's performance.
It may be best to train subjects during two to four test cycles. So their individual pain thresholds vary by no more than 0.8 degrees Celsius, and ideally by no more than 0.6 degrees Celsius between the last two test cycles. Subjects who fail to perform consistently are typically excluded from the study.
Thanks a lot for coming in today. I'm afraid it's not gonna work out for you to participate. It's difficult to be consistent in the way you rate pain, and some people can do it and for whatever reason, some people really have a great time.Okay.
And we just weren't quite consistent enough to be an appropriate candidate for the study. Okay, thank you. To begin the pain threshold testing cycles, mark the placement of the thermo on the subject's skin.
In this experiment, the same inflamed skin lesion used for microdialysis will be tested for pain. To learn about the biochemicals released in response to the pain testing before beginning, give the handheld control to the subject. Initiate each cycle by placing the thermo onto the marked skin area, match the position of the thermo with the markings as closely as possible.
Use constant and gentle pressure to keep the entire surface of the thermo in full contact with the subject's skin. During the testing, allow the temperature between the skin and the thermo to equilibrate to 35 degrees Celsius, a temperature that will cause no pain. This will take about five seconds and will be indicated by a reading of 35 degrees Celsius on the computer screen.
Now start the pre-programmed test algorithm. After 30 seconds, the thermo will start heating up at one degree Celsius per second. As soon as pain is experienced, the subject will press the handheld device.
After the button press, the thermo temperature will automatically be captured and will start decreasing at a rate of 10 degrees Celsius per second down to its baseline temperature of 35 degrees Celsius. The temperature probe will automatically be kept at 35 degrees Celsius for 30 seconds. Then it will start rising again at one degree Celsius per second until the subject again pushes the button of the handheld device.
Remind the subject that the thermo temperature is about to increase. Again, this will keep the subject focused. Repeat the sequence until seven temperature recordings have been registered.
Calculate the average of these temperature readings. This is the heat pain threshold to induce mechanical pain. Punctuated pressure probes, often called von fray hairs are typically used.
These are applied to the skin until the probe bends. Although Von fray hairs are commercially available, custom made, calibrated punctuated pressure probes are recommended because they provide more consistent results. These probes are made of identical cylindrical stainless steel wires 240 microns and diameter.
These are mounted on copper rods of various weights that move freely within a wider handheld aluminum tube. The same general experimental conditions used for heat pain testing will be used for mechanical pain testing. Likewise, the subjects should be prepared and trained the same way.
It is especially important to reassure subjects that the probes are blunt and will not puncture the skin. This will help the subjects to relax and provide consistent data. To determine the mechanical pain threshold.
First, apply the steel wire tip of the lightest probe perpendicularly to the skin. At the center of the experimentally inflamed lesion. The handheld aluminum tube is then lowered toward the skin without touching it.
This way, the entire weight of the copper rod inside rests on the steel wire tip. The study subject then indicates whether the probe causes pain by saying yes or no. If the subject answers no, then the next heavier probe will be used.
This algorithm is called an ascending stimulation sequence. Apply the probes in ascending sequence until the subject answers yes. The average weight of the last two probes in this series is recorded as the first deflection point after the subject.Yes.
Start using a descending stimulation sequence and use the next lighter probe. Apply probes in a descending sequence until the subject answers no. The average weight of the last two probes in this sequence is the second deflection point when the subject answers no switch back to an ascending stimulation sequence, alternate between ascending and descending sequences until seven deflection points are recorded.
The average of these seven points is the mechanical pain threshold. This algorithm can be made more complicated by randomly repeating any probe, which was just used in either ascending or descending sequences. This can help keep the subject blinded to the choice of the next probe.
We have just shown you two paint test paradigms, one using heat, and one using mechanical force. When performing these procedures, it is important for subjects to know that they're always in control of how much pain is inflicted and that it's really up to them of what is painful and what is not. So that's it.
Thanks for watching and good luck with your experiments.
