A refinement to the formalin test in mice

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.

Machine learning-based automated phenotyping of inflammatory nocifensive behavior in mice

“…The mice in this study were anesthetized which may also influence the timing of the behavior as the early part of the response is clearly reduced. 47 Both the automated system and Bryant 36 showed that C57BL/6N males lick less regardless of the bin choice, but bin size for females heavily influenced the outcome. Experiments using either C57BL/6N or C57BL/6NJ as control mice need to consider the sexes separately, as they show clear differences in timing over Phase II.…”

Formalin Itch Test: Low-Dose Formalin Induces Histamine-Independent, TRPA1-Mediated Itch in Mice

“…Given different pruritogens may induce itch through a distinct mechanism, different acute itch models are still needed to be developed. Since first reported over 40 years ago, the formalin test has been widely used in pain research and evaluation of analgesic drugs in laboratory animals ( 50 ), and formalin test is known to capture some mechanisms that are likely to be relevant to many pain patients in clinic ( 51 ). The nociceptive responses induced by formalin are marked by licking, biting, lifting, and shaking the injected hindpaw in rodents ( 52 ).…”

“…Recently, it was reported that a non-covalent agonist (GNE551) had distinct binding pocket and ligand-interaction mechanism for TRPA1 ( 56 ). Unlike the covalent agonist AITC, GNE551 activated TRPA1 without desensitization and induced persistent pain ( 51 ). Thus, targeting TRPA1 may be a novel strategy for developing new anti-nociception or anti-itch compounds, which is consistent with previous reports ( 7 , 22 , 23 ).…”

Modelos neuropsicobiológicos para estudo da dor e das emoções

“…O teste se baseia na contagem do número de contorções abdominais, caracterizada por contração e rotação do abdômen, seguida pela extensão de uma ou ambas as patas traseiras. O número de contorções abdominais é avaliado durante 20 minutos, iniciando-se dez minutos após a administração de ácido acético (Coolier et al, 1968, Parada et al, 2001Lopes et al, 2019)…”

“…O intervalo de quiescência entre a primeira e a segunda fase é resultado de uma inibição da transmissão nociceptiva através de circuitos supra-espinais e espinais. A resposta comportamental característica é a da lambida da pata (Lopes et al, 2019)…”