Dear community,
I felt in trouble with the Von Frey test that I could not overcome. I have a classic before-and-after experiment with alogene injection into the hindpaw. In about 5 days, I am trying to measure the baseline level of mechanical sensitivity. Before VF, I acclimated mice to VF cages for 2 days, and then I was planning to measure baseline. But about half of the mice do not settle. I have already tried 5 days, but still, some of them fail to calm down even after spending 4 hours in the VF cage. They just run around the cage and bite the mesh floor. Sometimes they groom, but grooming behavior seems a bit abnorman, nervous, with interruptions. I noticed that some of them tried to put their hind paws on a wall while biting the grid, sometimes even tumbling over. It looks as if they were afraid of the floor, which is strange since no stimuli are being applied.
Experimental conditions.
We use C57BL/6 mice. VF cagese are transparrent plastic boxes (small lunch boxes) that are about 400 mL in volume (Length Width Height - 12 x 8 x 4 cm). I have also covered the long sides and the top with tape to prevent observation by neighbors and make the environment inside a little darker. When we used taller cages from a company, mice exhibited a lot of rearing and climbing behavior. Thus, we changed to smaller ones to prevent such kinds of behavior.
The floor is a metal grid with 0.5-cm-square cells. Every time after an experiment, the metal grid is thoroughly washed with tap water (no soap), followed by 70% ethanol. Mice acclimate in a room for at least 1 hour before being placed in a VF cage, where they habituated for at least 30–40 minutes until they settled down. Usually I start at 8 a.m. I transfer all the mice (2 groups) to the behavioral room. After I finished the first group, I cleaned the cages and aparatus, rinsed them carefully, completely dried them, and then put on the second group. Despite the second group, in fact, acclimates for hours in a room, they still fail to settle down in aparatus. The white noise is playing at 60 dB from acclimation until the end to mask any noises from gloves, experimenter movement, crying, and suffering. The aparatus height is 26 cm. I do not use any perfumes or scent-based deodorants.
I have heard that Bl6 mice are quite special in terms of anxiety, but I didn't expect them to be so special! I am completely frustrated because it is just a baseline PWT with no stimulation. What will they behave like after stimulation? All of the articles describe how they measured a PWT 30 minutes after stimulation. Some of them even show the results 30 minutes, 1 hour, 2 hours, etc. after intervention. I just do not understand how it is possible or what I am doing wrong. Please, if somebody can shed some light, I will be relieved and alive again.
Amen
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