MouseMet EvF
We started with a clean sheet of paper, determined to create exactly what was needed, rather than settling for any compromises.
Thanks are due to Newcastle University, with whom we developed the initial prototype in 2012, and to the University of Queensland who purchased and tested the first batch production units in 2014.
MouseMet EvF is now a mature product, in regular production and usually available from stock.
The first step was to carry out a mouse study ourselves, working at a local CRO, testing with filaments and a borrowed EvF. There's no better way of finding out what does and doesn't work. And we learnt a lot:
Access was poor and it was uncomfortable to test
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The enclosures were typically rectangular with a mesh floor. The mice tended to grip the bars so it was difficult to access the same part of the paw each time (usually the middle of the pad where the "thumb" joins) and easy to hit the grid instead.
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The mice also became aware of the filaments after a while and would avoid it by positioning themselves unhelpfully (with the paw up the side of the enclosure).
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It was difficult to get the cage at the right height, so the tester needed to crouch. The arm motion required to move the filament or probe vertically, ideally without any hand tremor, was difficult and unnatural, especially to do repeatedly. We saw other testers hook their fingers over the edge of the enclosure to steady themselves.
Touch-on responses were a problem with both methods
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The forces were tiny; the threshold for healthy mice is generally 1-4gf but they would sometimes respond to the first touch of a probe. We'd seen this before with larger species: with horses the touch-on response might be 1N when the true threshold was 10N-20N. With filaments, the mice sometimes reacted at 0.4g or 0.16g but then not again until 2g or 4g.
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The number of touch-on responses increased after a lot of filament testing.
The EvF probe was too stiff
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"Touch-on" responses were more likely if there was any sideways movement of the probe, causing it to scratch across the plantar surface. The EVF we used tended to cause this because the probe was stiff, unlike the filaments which flexed from side to side.
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The EvF was also stiff in the axis of measurement which meant that any up-down hand tremor produced a variation in the applied force. So it was difficult to produce a smooth ramp. (The same is true of filaments but once they have buckled, the force produced is pretty constant).
The EvF force range was far too high
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The EvF we used had a force capacity 100 times higher than was required for mice. This meant the signal to noise ratio was poor and it was easy to produce spurious "readings" by just moving the probe in the measuring direction (because of the force required to accelerate the mass of the probe). So we were pressing reset several times before each reading.
EvF and von Frey filaments gave different results
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Because the filaments all have different diameters, the stimulus range is contracted compared to a single probe. We quantified this effect mathematically and concluded we should not expect any EvF to give the same answer as filaments. Both are valid. They just measure slightly different things.
So we went to work:
We designed a transducer that was rotary in action so that the operator could rest their elbows on the bench and apply the force simply by rotating the handles. We found this to be a much more comfortable and controllable action.
We made it battery powered, as cables are a nuisance, with a display to show the force ramp, a readout of the threshold force and a guide plot at a rate of 1g/sec to help testers maintain consistency.
We came up with a "one-dimensional" run so that the mouse, once acclimated, usually sits side on to the operator with the paws easily accessible. The run is wide enough for the mouse to feel comfortable but narrow enough that it has to stand up to turn round.
We also used bars rather than mesh to make the plantar surface more accessible and spaced them carefully for the mouse's feet. And we made the sides of the run completely transparent, without any struts or beading, to allow an un-interrupted view of the mouse's paw
Then we mounted the runs on a height adjustable stand (in 2 cm steps) so that the tester can get comfortable, with their elbows supported on the bench.
And we made the system modular so you can make assemblies of four or six runs,
while making sure everything was suitable for laboratory sterilisation procedures.
We designed the force transducer and its electronics from scratch, with a measurement range aimed specifically at mice:
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Force range of 0.1g to 7g
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Resolution of 0.1g
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Overall accuracy ± 0.1g over entire range
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Over-range warning at 8g
(Rather than make one transducer do two jobs, we designed a second version for rats with exactly the same method of operation but with a force range of 1-100g.)
And we developed a "soft-start" probe:
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0.3mm diameter (the same as a 2g von Frey filament)
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Two stage operation: the first 0.5g of force flattens the probe into the supporting arm, making it very soft (more so than a filament) and substantially reducing the touch-on response rate
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Buckles (like a filament) at about 8 grams making it impossible to over-range
And MouseMet was a success...here's a clip of it in use with MouseCal, our "Mouse foot simulator" and here's the early validation data, taken from a poster presentation at the NC3Rs conference in 2012.
The graphs are a compilation from trials run at three test sites but the message is a common one:
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In all three cases, MouseMet detected a reduction in mechanical nociceptive threshold (MNT) in the same way as filaments. The apparent reduction is less when measured by MouseMet but that's just a matter of scale. As noted above, and explained here, the two methods will always give different numbers because of the varying diameters of the filaments.
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The addition of our soft start probe (Graph D), (which we didn't have at the start of the trials), reduced variability. (It apparently increased the threshold because the data without the soft-start had a greater number of touch-on responses.)
Other organisations then perfomed their own validations:
Thomas A et al, (2013) Validation of a novel electronic von Frey system for use in rodents: MouseMet and RatMet. Proceedings of LAVA, Cambridge 2013
Taylor (2016) Refinements in thermal and mechanical nociceptive threshold testing in mice. Proceedings of the SEB; Improving experimental approaches in animal biology: Implementing the 3Rs. London June 2016, 28
MouseMet EvF, together with MouseMet HOT, are now used worldwide. MouseMet COLD is set to join the suite this year. All in the same ergonomic runs...
