I am currently using the Hargreaves test for mice after CCI. I train the mice for around 6 days before measuring baseline, putting them in the boxes for 15 minutes twice a day. I normally try to measure the baseline (after the training period) as soon as I put them in the box, however they are still relatively frantic (even if I wait 10-15mins), and I really think the baseline measurements are inaccurate as a result. Does anyone have any advice or tips? After CCI they are much more subdued, and since they sit still, the paw withdrawal latency for the uninjured paw is lower than the baseline measurement.
An interesting Question asked... if I was to give my input. I agree with Ryan about the exploring grooming resting phase!! in our lab (and over the past few years) I have been working quite a bit of Hargreaves. I have devised the following protocol:
1. day 1 BL- pop the animals into the boxes - let them acclimatise for 45min-1 hour and then start recordings- the recordings are very variable but this is simply a training exercise. (it is important that you remain in the room during the habituation/acclimatisation period)
2. Day 3 BL 2 - repeat the above- but this set of data is the first real recordings that you will keep.
3. Day 5 BL 3 - repeat step no. 1 but you will notice the mice begin to acclimatise quicker- ie. within 30-40 mins. once resting- you can start testing!
4. Day 7 BL4 - repeat step no. 3 and this is your last baseline. use baselines 2,3,4 to get your average BL values. by this point the animals are well trained with consistent left and right paw values.
notice that i test these animals and then leave a gap of at least one day before testing again for the next BL. (you don't want to exhaust the animals- and you will notice this by drop in weight!)
it is quite important to note that the more BL's you take- the lower the latency to withdraw from the radiant heat source- this happens as a result of training and learning. you will notice the threshold values will begin to shift to lower latencies through the entire course of your study.
another point- the sex of the experimenter seems quite important- a little far fetched i know but recent nature paper by Jeff Mogil looks into this further (male mice show more analgesic behaviour when it is a male experimenter vs when it is a female experimenter). we saw this effect with our hargreaves data! a male post doc in the lab and then I testing the same cohort of mice and we found a difference in the well trained BL values.
GOOD LUCK!!
http://www.nature.com/news/male-researchers-stress-out-rodents-1.15106
http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2935.html
We always let rats habituate to testing boxes for ~30min prior to testing, and sometimes will discount the first session. Do you let them acclimatise to the testing room before placing them in the apparatus?
Yes, all the training in the boxes is done in the same room as the final testing (every day for 6 days) so they should have acclimatised. Also, I am using mice, who are significanly stupidier than rats.
haha true ;)
The room acclimatization is more to let them calm down after being moved from another room, but we do still let rats acclimatize to the box for a further ~30 in (15-30 usually) until they are more relaxed, although too long and they just sleep, which isn't ideal either.
I'll speak to my colleagues who use mice and see if they have any thoughts :)
I have NEVER met an infuriating C57Bl/6 mouse...I have NEVER met an infuriating animal come to think of it..(except for the homo sapien variety)...
And as for 'significantly stupider than rats' ...well we won't be asking to see the stats on that !
It is not the caged animal who is stupid
Your mice need to understand what is going on in their tiny furry lives !!!
I suggest you spend some time with your animals on a regular basis and let them get to know you...once they trust you they are less fearful of whatever procedure you are about to embark on. It will also mean the result will be less hampered by anxiety.
But before you do so can I suggest you modify your attitude towards your animals ? The hostility you clearly show on this issue won't help things. Mice like all creatures are highly sensitive. They sense a vibe from you and it makes them nervous. They can't learn when they are frightened...
OK so saying 'mice are significantly stupider than rats' probably doesn't come across as very scientific or humane...
but if you were to spend ~ 15 minutes a day watching and talking quietly to them so they get to know your voice...preferably on your own if this is how the experiments will be run...Then occasionally take out a cage and let them see you don't intend any trauma to them...they worry !
it probably sounds daft but believe me it will make a VAST difference to how they behave. Also mice are obligate talkers (alas we cannot hear in their frequency) but they communicate to each other what is going on...so if 1 cage has a traumatic time they tell the others when they return. This then sets the standard for how the rest may respond.
Finally ask yourself why it is that mice have migrated across continents with us from as far back as records began? They must like us !!! (or our food), so we should at least like them back - none of us are going anywhere without our research subjects so it makes sense to afford them the credit they deserve.
An interesting Question asked... if I was to give my input. I agree with Ryan about the exploring grooming resting phase!! in our lab (and over the past few years) I have been working quite a bit of Hargreaves. I have devised the following protocol:
1. day 1 BL- pop the animals into the boxes - let them acclimatise for 45min-1 hour and then start recordings- the recordings are very variable but this is simply a training exercise. (it is important that you remain in the room during the habituation/acclimatisation period)
2. Day 3 BL 2 - repeat the above- but this set of data is the first real recordings that you will keep.
3. Day 5 BL 3 - repeat step no. 1 but you will notice the mice begin to acclimatise quicker- ie. within 30-40 mins. once resting- you can start testing!
4. Day 7 BL4 - repeat step no. 3 and this is your last baseline. use baselines 2,3,4 to get your average BL values. by this point the animals are well trained with consistent left and right paw values.
notice that i test these animals and then leave a gap of at least one day before testing again for the next BL. (you don't want to exhaust the animals- and you will notice this by drop in weight!)
it is quite important to note that the more BL's you take- the lower the latency to withdraw from the radiant heat source- this happens as a result of training and learning. you will notice the threshold values will begin to shift to lower latencies through the entire course of your study.
another point- the sex of the experimenter seems quite important- a little far fetched i know but recent nature paper by Jeff Mogil looks into this further (male mice show more analgesic behaviour when it is a male experimenter vs when it is a female experimenter). we saw this effect with our hargreaves data! a male post doc in the lab and then I testing the same cohort of mice and we found a difference in the well trained BL values.
GOOD LUCK!!
http://www.nature.com/news/male-researchers-stress-out-rodents-1.15106
http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2935.html
Thanks a lot for all your answers! Sanam, your protocol looks good and I will definitely give it a try :) Thanks for taking the time to write such a helpful response. Have a great weekend!