We have been using 45°C on our Columbus Instruments Hot/Cold Plate Analgesiometer with 3-6 month old Sprague-Dawley rats. Previously, this gave us a reliable baseline latency of 100-200 seconds before paw-licking. Recently, all the animals have started to respond below 100s, some of them dramatically below. Has anyone experienced this effect? Any ideas as to the cause?
I assume you have made sure the hot plate is actually at 45 degrees and that there isnt an issue with the kit.
There an numerous factors what can affect pain behaviour:
Firstly, have you changed supplier or have the supplier started sending you rats from a different colony? Are you certian they are sending SD rats? Has the constituents of the diet changed? (Seltzer et al saw a significant reduction in neuropathic pain behaviour when soy protein was added to their rat chow without them knowing). Have you changed experimenters, if not have you changed after shave or shampoo? Have there been any changed in animal care staff? Are there any works in the facility or in the surrounding buildings? Stress induced hyperalgesia is as real a problem as stress induced analgesia. The list could go on!
Another point is that over the years I've seen dramatic alteration of pain behaviour over the course of a year with a marked reduction over winter months. I've never had the resource to look into this effect methodically but I'm certain its not an artifact.
All of Ian Robinson's suggestions are valid reasons for the problem you are experiencing. I would definitely look into facilities issues such as construction, changes in personnel or methods in room procedures. Any disruption in normal routines, personnel, scents, procedures, and environmental factors such as cage enrichment, vibrations, temperature, humidity, food, etc. can cause changes in any nociceptive assay. You should try to decrease traffic in your housing and testing rooms as well as limit the number of experimenters that are in contact with the animals.
I also have a few things to add. First, are these naive or injured animals? I am assuming naive as you mention baseline latencies. Second, Are you doing any training for the procedure? It is well known that there is learning in the hot plate test and experiments that do not use a short training procedure may produce much more variable results. Successive days of hotplate testing will show a gradual decrease in hotplate latencies. These differences will level out after just a couple short training sessions. Please refer to the following publication:
Jacqueline Crawley. (2010). What’s wrong with my mouse: Behavioral phenotyping of transgenic and knockout mice. New Jersey: John Wiley & Sons Publishing.
Third, what other testing procedures are you performing on these rats and are any of these procedures done on the same day or in close proximity to the hotplate testing? Are you also performing testing of cold thresholds? Testing both hot and cold thresholds in proximity of each other (even 24 hours) can cause reductions in baseline thresholds in both tests.
Did you change testing rooms? apparatus? experimenters? sex of experimenter? feed? room change schedules? were cages changed on testing day? Fire alarms? Room sanitization?
My last suggestion would be to verify that the temperature of your hotplate is actually what the apparatus is displaying as the temperature. Sometimes these can be off and need to be re-calibrated to accurately reflect the temperature of the plate.
Thanks, Ian and Renee. Clearly, there are a lot of variables to consider.
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