Abstract
The constant refinement of tests used in animal research is crucial for the scientific community. This is particularly true for the field of pain research, where ethical standards are notably sensitive. The formalin test is widely used in pain research and some of its mechanisms resemble those underlying clinical pain in humans. Immediately upon injection, formalin triggers two waves (an early and a late phase) of strong, nociceptive behaviour, characterised by licking, biting, lifting and shaking the injected paw of the animal. Although well characterised at the behaviour level, since its proposal over four decades ago, there has not been any significant refinement to the formalin test, especially those combining minimisation of animal distress and preservation of behavioural outcomes of the test. Here, we propose a modified and improved method for the formalin test. We show that anaesthetising the animal with the inhalable anaesthetic sevoflurane at the time of the injection can produce reliable, robust and reproducible results whilst animal distress during the initial phase is reduced. Importantly, our results were validated by pharmacological suppression of the behaviour during the late phase of the test with gabapentin, the anaesthetic showing no interference with the drug. In addition, we demonstrate that this is also a useful method to screen for changes in pain behaviour in response to formalin in transgenic lines.
Highlights
Since first reported over 40 years ago, the formalin test[1] has been widely used in pain research and is known to capture mechanisms that are likely to be relevant to many pain patients in clinic[2,3], including the poorly localized, burning and throbbing pain sensation[4]
Our newly proposed method showed that whilst the pain behaviour in the first phase was suppressed by the anaesthetic, the response in the late phase remained comparable to the control, non-anaesthetised group
We demonstrate that this is a useful method to use while screening transgenic lines for changes in pain behaviour in response to formalin
Summary
Since first reported over 40 years ago, the formalin test[1] has been widely used in pain research and is known to capture mechanisms that are likely to be relevant to many pain patients in clinic[2,3], including the poorly localized, burning and throbbing pain sensation[4]. The 3Rs concept (reduce, refine and replace) ensures that for every experiment, the use of animals is absolutely necessary[13,15,16,17] Further to these ethical considerations, and of particular relevance to the formalin test, other evidence suggests that restraining induces stress, behavioural and other physiological changes in the animals[18,19,20,21,22,23,24,25], including hyperalgesia[26,27,28], which can impact the outcome of the test. We posed the question of whether anaesthetising the animal at the time of formalin injection could result in more consistent injections and reduce the stress experienced by the animals, without losing the behavioural effects that formalin triggers
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