We’ve got some experiments coming up, and I’m thinking about testing all the different modalities in a single day. One concern is that performing all these assays will stress the mice out. The other question is the order of assays.
Assays I’d like to perform:
- Von Frey
- Cotton Brush
- Pin Prick
- Cold Plantar
What order would you do these in? And would you separate these across days?
As far as order, generally one moves from less to more noxious. And my rationale for this order is that thermal assays I think can modify/sensitize the tissue more so than the punctate mechanical assays.
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We normaly do mechanical tests in one day and cold (eg acetone) in the next day.
Definitely separate these tests over multiple days if it’s possible. Stress will play a huge role here. In the Mogil lab, we usually separate tests over many days i.e. max two tests in a day, on two different modalities (one thermal, one mechanical). Even then, I’ve always only ever done one test per day. Some people go as far to give them a day off in between, but that might be overkill.
With rats during my PhD, I kept it to one test per day, and didn’t test consecutive days. Now working with mice, I have done at most 3 assays per day, depending on how stressful they are. Most recently, I’ve done von Frey -> acetone -> 1 hour break -> rotarod. While rotarod isn’t very painful, it is stressful for the mice, so we do it at the end of testing.
This seems like a lot for one cohort. I’ve done Von Frey followed by rotarod on the same day with a hour break in between, but haven’t tried anything more than that in one day.
Also, it depends on the genotype of your mouse. Suppose, if you are working on touch sensitivity by playing with the LTMRs, then avoid trying to mingle VFH, dynamic brush and pin-prick on the same day. Also, as per my experience, the light in optogenetic experiments should be used with a significant gap with other behavior experiments to avoid the sensitization of the other modalities on the same day (Or it would be best to use a separate group for this).
Apology for the late answer. I would separate these tests in 2 or even 3 days. Usually we perform 3 assays per day, with 1 hour break between tests. I suggest: von Frey -> cotton brush -> Hargreaves in the first day and then pin prick -> cold plantar -> rotarod.
We decided to stick with just one test, the von Frey. We are studying the effect of stress of nociception, which is huge. One way we stress our rats is called the novel environment stress, which basically consists in moving the rats or mice to a new cage near a light. So, you would need to habituate your animals to ALL your testing equipment, for a minimum of 30 min per day, for 3 days. And this will not guarantee that your animals are not stressed. Maybe you should measure cortisol levels? Otherwise, I would assume that all the animals subjected to multi-tests, even on different days, are stressed animals.
Here are some papers showing the effect of stress on nociception in chronic pain models:
- Rivat, C., Laboureyras, E., Laulin, J.P., Le Roy, C., Richebe, P. & Simonnet, G. (2007) Non-nociceptive environmental stress induces hyperalgesia, not analgesia, in pain and opioid-experienced rats. Neuropsychopharmacology, 32, 2217-2228.
- Le Roy, C., Laboureyras, E., Gavello-Baudy, S., Chateauraynaud, J., Laulin, J.P. & Simonnet, G. (2011) Endogenous opioids released during non-nociceptive environmental stress induce latent pain sensitization via a NMDA-dependent process. Journal of Pain, 12, 1069-1079.
- Laboureyras, E., Aubrun, F., Monsaingeon, M., Corcuff, J.B., Laulin, J.P. & Simonnet, G. (2014) Exogenous and endogenous opioid-induced pain hypersensitivity in different rat strains. Pain Res Manag, 19, 191-197.
- Chen, W., Taché, Y. & Marvizon, J.C.G. (2018) Corticotropin Releasing Factor in the brain and blocking spinal descending signals induce hyperalgesia in the latent sensitization model of chronic pain. Neuroscience, 381, 149-158.
- Jennings, E.M., Okine, B.N., Roche, M. & Finn, D.P. (2014) Stress-induced hyperalgesia. Prog. Neurobiol., 121, 1-18.