Let’s try this again. How do we measure pain in rodents???
In humans, we can just ask! How severe is it on a scale of 1 to 10, how would you describe it, where does it hurt?
This is too confusing for the rats, though.
(This is a real slide from a talk I’ve given on this subject!)
This is too confusing for the rats, though.
(This is a real slide from a talk I’ve given on this subject!)
So to measure pain in rodents, we often use behavioral assays. With these assays, we can measure either spontaneous pain or evoked pain.
Spontaneous pain isn’t dependent on a stimulus, and because it is less affected by the experimenter, it is considered more “natural.” It involves observing the animals in their home environment.
Measuring spontaneous pain can be tricky in prey animals like rodents, because they tend to hide their pain, so that they are less likely to be targeted by predators when injured.
One of the most popular measures of spontaneous pain is the rat (or mouse) grimace scale. This scale grades rodent facial expressions on things like squinting their eyes and flattening their ears to quantify how severe their pain is.
Think about the faces you make when you’re in pain. Do you squeeze your eyes 😣 or puff your cheeks 😬? A human grimace scale would probably look pretty similar to the rodent ones! Hopefully it would be more detailed than the classic pain scale faces...
Evoked pain is dependent on a painful stimulus that is applied by the experimenter. Evoked pain can be measured across different sensory modalities (like heat, cold, mechanical stimuli), which is useful because these different stimuli activate different receptors and neurons
To measure heat-evoked pain, two typical assays are the hot plate test and the Hargreaves’ test (named for the author of the paper first describing it)
The hot plate test has been around for a long time, and is pretty straightforward. An animal is placed on a hot plate, and the time until it exhibits a pain behavior (also called a nocifensive behavior) is timed. The faster it responds, the lower its pain threshold.
(In this test, animals are removed once they respond or reach the cut off time. They are typically on the plate for only a few seconds. We don’t want them to suffer)
The Hargreaves’ test is similar to the hot plate test, but instead of a direct application of heat, it uses radiant heat from an infrared beam. This is useful because it can be localized to one paw if needed, allowing us to compare left vs right responses
Interestingly, even though heat and cold are both thermal types of pain, they activate distinct receptor types and neuron populations, so they may be affected differently in the same pain pathology. In humans with chronic pain, ❄️ sensitivity is more common than 🔥 sensitivity!
Cold-evoked pain is typically measured with either the cold plate test (similar approach as the hot plate test) or the acetone test.
In the acetone test, a drop of liquid acetone is touched to the paw, and its evaporation causes a cold sensation. Either time to a nocifensive behavior or number of nocifensive behaviors in a set time are measured to determine the cold pain threahold
Acetone is sometimes in nail polish remover, so you may have felt this sensation yourself when changing the color of your fingernails! 💅
Lastly, to measure pain from mechanical stimuli, the von Frey assay is commonly used. This is also one of the most widely used behavior pain assays for rodents.
In the von Frey assay, a series a filaments are touched to the paw of a rodent. The first filaments exert less force, an feel similar to pressing a hair from your head into your hand. The force gradually increases as filaments go up
Once the animal exhibits nocifensive behavior (flinching, licking the paw, shaking the paw), we know we have reached the threshold. When the rodents are in pain, this threshold will be lower
Okay! I think I covered all the things lost in the original thread. Let me know what questions you have about these assays!
