Repetition of the behavioral tests in rats (Eg., Mechanical allodynia) doesn't produce the same result!

Today on POD 6, I did nociceptive testing (Mechanical allodynia followed by thermal hyperalgesia) in my experimental rat which had undergone a hind-paw incision (model of postoperative pain). When I measured mechanical allodynia using von-Frey filaments via Up-Down method (Chaplan et al., 1994), it was around 6.7 g then after a gap of 20 minutes, I measured thermal hyperalgesia using Hargreaves apparatus in the same rat.
Later, my supervisor tried to re-confirm/validate my results so, after 30 minutes of the first phase of the nociceptive testing which was done by me, he himself repeated testing for mechanical allodynia. Surprisingly, he got the different readings (~2 g) from that of mine and rat was showing a brisk flick after administration of the filaments to the medial site of incision.
I would be highly thankful to the forum members if they could torch some light on the enigma of validation/re-confirmation of the nociceptive assays’ results in this situation.

Thanks and regards,
Mayank Gautam
AIIMS, New Delhi

@neurotemple Thanks for joining the forum.

Things to consider:

  • The thermal testing may affect the sensitivity of subsequent mechanical testing. Thus, the order of your testing matters.
  • Your PI is a different tester and may consider different responses as ‘positive’ and negative. Also, the animals may be responding to the smell or presence of a different tester.
  • There may be subtle differences in the manner of testing. For example, the duration of application of the VF filament. Longer applications (5 sec) have been shown to result in decreased thresholds compared to shorter applications (1-2 s).

I would not put too much stock into the differences between testers with respect to magnitudes. Directions of change should be similar, but the actual numerical values will likely differ, unless both testers do things exactly the same and the animals have been habituated to the presence of both testers. It’s hard to say what a one-off test from a previously uninvolved tester means.

Good luck.

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