Mechanical threshold testing on mice is usually carried out using von Frey filaments. They are simple to use and, as long as they are in good condition, will apply the force reliably. But the process is time consuming, requiring several readings to produce one result and some analysis to extract the threshold force from the data. The data are also non-parametric: the threshold forces that result from the popular up-down” method are a discrete range of values and this should be taken into account when analysing the results statistically.
Not surprisingly, several companies have produced “electronic von frey systems” in an attempt to replace the use of several filaments with a single, analogue test consisting of a force ramp until threshold and a peak-hold of the value reached. These electronic force transducers, while useful for larger animals, have not yet gained wide acceptance for threshold testing in mice.
Here’s why it’s difficult:
The test is awkward to apply
The mouse is typically housed in a rectangular enclosure with a mesh floor, with bars of about 1mm diameter and spaced about 5 mm apart. The mouse is free to move in all directions while the tester pokes the filament, or transducer probe, up between the bars and onto the plantar surface of the hind paw. As mice tend to grip the bars it can be awkward to access the same part of the paw each time (usually the middle of the pad where the “thumb” joins) and it is easy to accidentally hit the grid with the filament or probe. Also, if the cage is not at the right height, the operator must crouch or stretch to see and perform the test. There is often no support for the arms, and we’ve noticed that many people tend to hook their fingers over the edge of the mouse enclosure to steady themselves.
None of this is ideal for delicate, highly repetitive testing!
The “touch-on” force
The forces are tiny; the threshold for healthy mice is generally 1-4gf but they will also sometimes respond to the very first touch of a probe or filament. We call this the “touch-on” force and we see it in mechanical testing of many species; data falls into two groups, those where the subject responded to the first touch and those where it ignored (or did not feel) it and allowed the force to rise to a true threshold before reacting.
We have found that, with von Frey filaments, the mouse might react sometimes at 0.4gf or even 0.16gf but then not again until 2g or 4g.
A reaction to “touch-on” is more likely if the force rises quickly, or if there is any sideways movement of the probe, leading it to slide or scratch across the plantar surface. Filaments avoid both these problems, being unable to apply more than their buckling force and being inherently “bendy” from side to side, thus adsorbing any hand tremor.
The effect of hand tremor
Unlike a filament, a probe mounted on an electronic force transducer tends to be stiff, or non-bendy, from side-to-side. This means that, with the slightest amount of hand tremor, it’s more likely to slide across the plantar surface and to elicit a touch-on response than a filament is.
A force transducer also tends to be stiff in the vertical direction. This means that any up-down hand tremor produces an instant and relatively large variation in the applied force. This doesn’t happen with filaments; once they have buckled, the force produced is pretty constant and so if your hand tremors during the 2-3 seconds that the filament is held in contact, the force remains the same.
Finally, it appears that the electronic von frey systems currently available are based around force transducers with a much higher force capacity than is required for mice. Force ranges of 500gf are not uncommon, but perhaps not ideally suited for a measurement that rarely exceeds 5gf.
So why not stick to filaments?
They are simple, relatively cheap and certainly well proven. Their main drawback is the number of measurements required to achieve a threshold reading. The up-down method requires 6 readings “around the threshold force” and, if starting with filaments that are not expected to produce a reaction, it is common to start counting at the first positive response (going down one filament and then taking four more measurements after that). If the first response is a spurious one (perhaps a touch-on) then the subsequent row of Os while you wait for the next (higher) positive can reduce the validity of the data.
There is also no doubt that mice become aware of the filaments and, if tested too much or too often, will avoid the filament by positioning themselves unhelpfully (perhaps with the relevant paw up the side of the enclosure) or just by reacting continually to touch-on. A test method which reduces the number of measurements is, therefore, desirable.
Finally, the stimulus applied by a filament is actually a rather complex one; although a flat, circular end is presented during the application of the force, this becomes an edge contact at the point at which the filament buckles. The effect of this is not really understood at present (although readers might be interested in the poster abstract presented recently by Dr Michael Dixon at the WCVA conference in Capetown, concerning the effect of probe area on threshold force in conventional algometry).
MouseMet:Topcat Metrology Ltd is a small, highly innovative company run by Dr Polly Taylor, an internationally known veterinary anaethestist and Dr Michael Dixon, an engineer with 20 years experience of difficult and unusual measurement problems. We designed “Mousemet” from first principles, attempting to retain the many positive aspects of traditional von Frey filaments while removing the need for repetitive measurements and post test analysis. The result is a unique “soft” force transducer of an appropriate range for mice, software which allows quick assessment of the ramp rate and profile and a cage system that is ergonomically sound and maximises throughput.
The initial evaluation was performed at Newcastle University and presented at the autumn meeting of the AVA in Liverpool, 2011. Please click here for a PDF of the poster. Following further improvements to the system, evaluations have now also been carried out at the Department of Medical Science, Bristol University and by workers at the Medical Research Council. Preliminary results were presented as a poster abstract at the recent NC3Rs meeting in London. Finally, a paper describing validation by an unrelated university has been accepted for publication in “Pain”.
The mouse is contained in a “one-dimensional run”. This enclosure is wide enough for the mouse to feel comfortable in, thereby ensuring natural behaviour, but not wide enough to turn round in without standing up. So, for the vast majority of the test session (after the initial exploratory behaviour), the mouse will be pointing left to right (or right to left). The run has bars which are sized and spaced specifically for the mouse’s paws, allowing him to grip the bars comfortably with his toes but allowing the maximum plantar area for testing and a lid making it easy to decant the mouse safely back into his cage.
MouseMet is supplied with four runs and a four run support system. Additional runs may be purchased to save more time; the next four mice can be exploring while you test the current four. All the runs and supports are suitable for laboratory sterilisation procedures.
The run supports are adjustable, in 2cm click steps, so that, when you are sitting at the bench with your elbows on the table the bars of the run will be at eye level. The sides of the run are transparent, so you have a clear view of the mouse’s paws and the probe. It also means that your hands will be at the right height to operate MouseMet.
MouseMet is a highly sensitive rotary force transducer with a measurement range of 0.1-5g. The innovative rotary design makes it insensitive to slight hand tremor in the vertical direction; forces down to 0.1g can be applied without any difficulty.To apply a force with MouseMet, hold the instrument with the probe arm horizontal, touch the probe tip onto the plantar surface, and then rotate the black handles. This is intrinsically easy and comfortable with your forearms properly supported and gives you full control over the placement of the probe tip.
Hand tremor in the horizontal plane (front to back or side to side) can also affect a reading because it can lead to the probe tip sliding sideways or scratching across the plantar surface, especially at touch-on. This is not problem with von Frey filaments because they are flexible in this direction, even before they reach their buckling force but it can be an issue with a rigid force transducer. MouseMet’s probe tip, however, is similar to a filament but operated always below its bucking force, so it’s flexible and absorbs slight side to side movement. In addition, it has a 90 degree bend which means that, over the first few milimetres (or 0.5 grams force) of operation, it’s even softer, increasing the chance of placing the filament successfully on the plantar surface without generating a touch-on response.
The force ramp is displayed immediately on the notebook computer and the peak force calculated for you. Reject tests are inevitable, because the mouse moved, the probe slipped or just because the scientist wasn’t sure if it was a valid response.
MouseMet allows you to select immediately via a thumbswitch. Press the switch within 5 seconds of the test, and that ramp is stored. Otherwise it’s ignored. Similarly, we’ve watched people trying to achieve a pre-determined force ramp rate, by watching guide lights, or symbols on a computer screen. This really isn’t practical; you should be watching the mouse. So MouseMet allows you to preset the force ramp rate that you’re aiming for, and overlays it onto the test result displayed. The two ramps on the left below are good, at a rate of 1gf/sec. The right hand ramp is rather too fast and ended up over-range. A response like this is more likely to be as a result of the leg being simply lifted by the probe, rather than the mouse withdrawing it and hence should not, we suggest, be included in the data. After the test series, MouseMet’s software allows you to select the ramps you want to keep and then exports the results to Excel.
Purchase or Lease
MouseMet is available for outright purchase, for short term lease or for lease-to-buy. For details of terms please call Dr Michael Dixon on +44 (0)7739913696 or email email@example.com.