MouseMet and RatMet: refined mechanical and thermal nociceptive testing

Funding available until: 17 May 2018

Summary | Background | Current state of the art
What could your Solution be used for? | Collaboration | Information on IP | 3Rs impact | References

Summary

Nociceptive threshold testing (NTT) is widely used for testing sensory and nociceptive thresholds in pain research. von Frey filaments and hotplate technologies have been the standard methods for conducting NTT for many years, but have significant scientific and animal welfare limitations. MouseMet and RatMet provide an alternative approach to NTT testing which reduces the number of interventions required, reducing the stress experienced by each subject and improving animal welfare. Mechanical and thermal probes are each mounted on a novel transducer which is not influenced by hand tremor, leading to more reproducible data, therefore allowing fewer animals to be used per study. MouseMet and RatMet testing requires fewer stimuli per data point than filaments, so each animal receives fewer stimuli per study. Both thermal and mechanical testing are applied to the foot in the same specially designed cage to ensure maximum efficiency of the procedure and removing the need to move animals to a new environment for the second modality. We are looking for partners in pain research willing to use and publicise the benefits of this methodology.


Background

Fundamental and pharmacological studies in pain require NTT to detect increases or decreases in sensitivity to a noxious stimulus [1]. Thermal and mechanical sensitivity testing is very widely used in such research, and rodents, primarily mice, are the most common species studied. Testing with von Frey filaments and hot plate technology, principally the Hargreaves system, are currently the accepted methods for mechanical and heat testing respectively. However, these have a number of disadvantages which are discussed below.

In response to the need for better systems Topcat developed MouseMet EvF (electronic von Frey), a novel, rotary force transducer with a range specific for mice (0.1-7gf). This provides a single ramped mechanical stimulus that replaces the succession of “go/no go” tests and post-testing calculations required by filaments. The rotary action transducer is used with species-specific cages designed to make access to the foot and controlled application of the stimulus more efficient; minimising the time taken to obtain results and the time each animal has to spend away from the home cage environment. Threshold force is automatically recorded and the force ramp displayed.

A height adjustable system of one-dimensional cages was developed to ensure that the subject, after first exploring the environment, will sit sideways to the tester. Carefully spaced foot bars ensure that the animal can sit and behave naturally throughout the procedure, while ensuring optimal access to the appropriate test site on the foot. This enhances animal well-being compared with traditional square and mesh-floored cages as testing is quicker and easier. 

   

Subsequently, Topcat Metrology has used its 20 years’ experience of thermal threshold testing in larger species to develop a thermal stimulus for mice (MouseMet Thermal). The miniature probe, (2mm diameter), is mounted on the same MouseMet transducer (thereby minimising operator learning time) and is applied to the plantar surface of the foot in the same way as the mechanical test, with the animal still in the same cage.

A second version of the system, with larger cages, a force range of 1-80gf and a similar thermal probe is available for rats (RatMet).


Current state of the art

Mechanical threshold testing is traditionally carried out using von Frey filaments, where a series of stimuli is applied to the hindpaw. The animal responds by withdrawing its foot, and the threshold value is calculated from the series of stimuli:

  • Filaments are prone to bending damage causing inaccuracies in the applied force as well as errors in measurement. 
  • Each data point requires a sequence of several stimuli and subsequent mathematical derivation of threshold force often using the “up-down” method. This derivation is frequently implemented incorrectly through lack of understanding of the statistical process [2].
  • Each filament has a different tip diameter (depending on its force rating). Mathematical analysis by Topcat Metrology suggests that this artificially expands the true force threshold scale compared to a ramped stimulus with a single probe. Treatment effects are exaggerated and the data are suitable only for non-parametric statistical analysis.
  • Electronic ramped von Frey methodology avoids these problems but current commercially available systems have two drawbacks: (i) the force ranges are too high, particularly for mice, (typically 50gf -500gf), leading to low accuracy, and (ii) they employ “stiff” transducers adversely affected by hand tremor.
  • Testing is usually carried out in square cages with a mesh floor; the animal moves freely in two dimensions and time is lost waiting for measurement opportunities. The grid of floor bars obscures the tester’s view and access to the foot. Such difficulties lead to prolonged testing sessions throughout which the animal is out of its familiar home environment and is increasingly stressed.

Thermal threshold testing is commonly carried out using a Hargreaves or traditional hot plate system.

  • The Hargreaves system provides an infra-red stimulus to the foot applied through a solid glass floor, so animals must be moved to a new environment. This is stressful and requires considerable acclimation time. Furthermore, urine, inevitably produced throughout testing, prevents reproducible heat transfer through the glass floor to the animal’s foot.
  • Traditional hot-plate systems suffer a number of disadvantages compared to our technology: animals cannot act as their own controls (increasing animal use in studies) [3], the system is not suitable for all studies, is susceptible to learning phenomenon [1] and is more stressful than plantar tests.

MouseMet and RatMet provide significant improvements to traditional thermal and mechanical nociceptive threshold testing: fewer mechanical stimuli are required, parametric data are produced without mathematical derivation and handling stress is reduced as both tests are applied in the same environment.


What could your Solution be used for?

Testing for thermal and mechanical hyperalgesia, allodynia and hypoalgesia is a fundamental technique used in pain research ranging from understanding of gross and molecular structure and function of the nervous system to broader understanding of the concept of pain in the whole animal. NTT is used across these studies in fundamental pain research, pharmaceutical and chemical screening, drug efficacy testing and disease modelling [1, 4-6].

MouseMet and RatMet are suitable for use in all circumstances where NTT is used and provide refinement over the current methodologies used for mechanical and thermal stimuli.  

MouseMet technology was developed out of a need for robust systems for NTT in mice and has clear welfare and methodological advantages over current approaches. The method’s additional applicability to rats using the RatMet system makes it appropriate for all rodent studies. Both the EvF and Thermal systems have been validated [3, 7-13] and provide significant refinement [14, 15] due both to reduced animal stress, as well as production of more robust data allowing smaller group sizes and reducing the need for repeat experiments. Widespread employment of MouseMet and RatMet technologies in laboratories conducting rodent based research in the pain field would benefit both animal welfare and the quality of the science.


Need for collaboration

MouseMet technology is validated and now requires widespread acceptance as a refinement over traditional von Frey filaments and Hargreaves methodology to realise the animal welfare benefits.

Increased employment of MouseMet technology and consequent publication of results would publicise the benefits of this method in providing both better welfare and better science for all rodent-based research in the pain field. 

Topcat Metrology is seeking partners using rodent NTT studies for drug development or basic research to assist with this in exchange for a discount on either one or both of the MouseMet and RatMet systems. Topcat would collaborate in further refining testing protocols (for example establishing appropriate time intervals between tests) and in providing any training required in how to use the system and interpret data.  


Information on IP

MouseMet is covered by GB patents 2489793 and 2489933, US patent 8,944,008 and by European application 12161418.4. Any further IP resulting will be the property of Topcat Metrology Ltd.


3Rs impact assessment

Pain is a complex whole animal phenomenon requiring conscious perception. Hence, although some mechanisms can be studied in vitro, conscious animals must be studied at some stage. NTT is used in the majority of rodent pain studies, from initial molecule screening, through fundamental pain physiology to preclinical testing of analgesics. Thousands of mice and rats undergo such testing per annum: for example, every issue of the monthly journal Pain, the key publication for research in pain, contains at least one, and usually two or three articles including data collected from nociceptive testing in rodents, utilising between 50 and 250 animals each. Replacing traditional methods with our technology would make a significant contribution to refinement and reduction where there is no alternative to in vivo studies.

Refinement

A single ramped stimulus replaces the series of stimuli (usually at least five) required for testing with filaments. Combined with one-dimensional MouseMet and RatMet cages, this offers a quicker, and therefore less stressful approach, as the animal has to be handled less and remains in an optimal position for measurement. Furthermore, since MouseMet/RatMet EvF and Thermal are used for dual testing without the need to transfer the animals to a second environment, acclimation time is halved and stress further reduced. Additionally, the use of our thermal devices avoids the need for animals to be placed on hotplates up to 50oC where nocifensive reactions (licking/shaking of the hind paws, licking of the genitals, jumping) are assessed over two minute periods [6], presenting a significant welfare improvement.

Reduction

Mechanical NTT data from an electronic transducer with an appropriate force range are less variable [16] and less prone to errors than current approaches. Additionally, thermal NTT data obtained using our system exhibits less variability than the Hargreaves approach [3, 17]: these characteristics may permit smaller animal group sizes of five or six animals [9, 16].


References

  1. Le Bars D, Gozariu M, Cadden SW (2001). Animal models of nociception. Pharmacological Reviews 53:597-652.
  2. Dixon MJ, Roughan JV, Chamessian AG,Taylor PM (2014). Mechanical nociceptive threshold testing in mice: evaluation of the errors incurred using the up-down method to analyse von Frey filament data. Presented to the Association of Veterinary Anaesthetists, April 2014, Nottingham.
  3. Deuis JR, Vetter I (2016). The thermal probe test: A novel behavioural assay to quantify thermal paw withdrawal thresholds in mice. Temperature 3:1-9.
  4. Koga K, Honda K, Ando S, Harasawa I, Kamiya HO, Takano Y (2004). Intrathecal clonidine inhibits mechanical allodynia via activation of the spinal muscarinic M1 receptor in streptozotocin-induced diabetic miceEuropean Journal of Pharmacology 505:75-82.
  5. Huang W, Calvo M, Pheby T, Bennett DLH, Rice ASC (2017). A rodent model of HIV protease inhibitor indinavir induced peripheral neuropathyPain 158(1):75-85.
  6. Tzschentke TM, Linz K, Frosch S, Christoph T (2017). Antihyperalgesic, Antiallodynic, and Antinociceptive Effects of Cebranopadol, a Novel Potent Nociceptin/Orphanin FQ and Opioid Receptor Agonist, after Peripheral and Central Administration in Rodent Models of Neuropathic Pain. Pain Practice 17 [Epub ahead of print].
  7. Thomas A, Flecknell P, Roughan J (2014). Validation of the RatMet electronic von Frey for measurement of mechanical nociceptive thresholds in rats. Presented to the Association of Veterinary Anaesthetists, October 2013, Moscow. Veterinary Anaesthesia & Analgesia 41, A38 doi:10.1111/vaa.12114a (poster: RatMet).
  8. Thomas A, Taylor PM, Dixon MJ (2013). Validation of a novel electronic von Frey system for use in rodents: MouseMet and RatMet. Proceedings of the Laboratory Animal Veterinary Association, November 2013, Cambridge.
  9. Deuis JR, Zimmermann K, Romanovsky AA, Possani LD, Cabot PJ, Lewis RJ, Vetter I (2013). An animal model of oxaliplatin-induced cold allodynia reveals a crucial role for Nav1.6 in peripheral pain pathways. Pain 154:1749-57 doi: 10.1016/j.pain.2013.05.032.
  10. Deuis JR, Whately E, Brust A, Inserra MC, Asvadi NH, Lewis RJ, Alewood PF, Cabot PJ, Vetter I (2014). Analgesic effects of clinically used compounds in novel mouse models of polyneuropathy induced by oxaliplatin and cisplatin. Neuro-Oncology Epub 2014 April 8 doi:10.1093/neuro/nou048.
  11. Deuis JR, Whately E, Brust A, Inserra MC, Asvadi NH, Lewis RJ, Alewood PF, Cabot PJ, Vetter I (2015). Activation of κ Opioid Receptors in Cutaneous Nerve Endings by Conorphin-1, a Novel Subtype-Selective Conopeptide, Does Not Mediate Peripheral AnalgesiaACS Chemical Neuroscience. Epub Aug 12 doi:10.1021/acschemneuro.5b00113.
  12. Schuster CJ, Pang DSJ (2015). Assessment of a novel mechanical sensory threshold testing device. Proceedings of the 66th meeting of the American Association of Laboratory Animal Scientists. Phoenix, November 2015. (Abstract 224), 71-72 (poster: RatMet).
  13. Taylor PM, Dixon MD, Holmes F, Davletov B (2012). Comparison of mechanical threshold testing in mice using von Frey filaments and the MouseMet electronic system. Proceedings of the NC3Rs conference, London, October 2012.
  14. Taylor PM (2016). Refinements in mechanical and thermal nociceptive threshold testing in mice. Proceedings of the Laboratory Animal Science Association, November 2016, London, 21-22.
  15. Taylor PM, Dixon MJ, Deuis JR, Vetter I (2016). Refinements in mechanical and thermal nociceptive threshold testing in mice. Proceedings of the Society for Experimental Biology Annual Conference, June 2016, London (REF15), 28-29 (poster).
  16. Miller AL, Leach MC, Dixon MJ, Scotto di Perrotolo M, Roughan NJV, Flecknell PA, Taylor PM (2011). Evaluation of a new electronic von Frey system for pre and post surgical measurement of mechanical thresholds in mice. Proceedings of the Association of Veterinary Anaesthetists Conference, Liverpool, September 2011, 33.
  17. Dixon MJ, Roughan NJV, Chamessian AG, Taylor PM (2014). Mechanical nociceptive threshold testing in mice: evaluation of the errors incurred using the up-down method to analyse von Frey filament data. Presented to the Association of Veterinary Anaesthetists, April 2014, Nottingham. Veterinary Anaesthesia & Analgesia 41:A63 (poster).

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