Measurement of botulinum types A, B and E neurotoxicity using the phrenic nerve–hemidiaphragm: Improved precision with in-bred mice C. Rasetti-Escargueil * , R.G.A. Jones, Y. Liu, D. Sesardic Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK article info Article history: Received 29 August 2008 Received in revised form 16 January 2009 Accepted 20 January 2009 Available online 29 January 2009 Keywords: Botulinum neurotoxin Paralysis Mouse Phrenic nerve–hemidiaphragm abstract Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. The current method for assessing the potency of botulinum toxin and antitoxins is the mouse LD 50 assay. The mouse phrenic nerve–diaphragm assay is an in vitro assay that closely mimics in vivo respiratory paralysis. In this study, we have further improved the assay by using gelatin as a non-frothing alternative to albumin and investigated the effects of botulinum toxin serotypes A, B and E on phrenic nerve–hemidiaphragms from out-bred MF1 and in-bred Balb/c mice. Improved reproducibility was found with in-bred mice. Balb/c mice were also found to be much less sensitive to type B toxin perhaps indicating differences in the expression of receptor components. Hemidiaphragm preparations from Balb/c mice were approximately 7 times more sensitive to type A toxin and 7–12 times more sensitive to type E toxin relative to type B toxin. These findings indicate that when fully optimised the mouse nerve–diaphragm preparation can provide a functional in vitro model for accurate and reproducible assessment of toxin activity. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Botulism is a neuromuscular disease characterised by progressive muscle paralysis and possible death from respiratory failure. This disease is caused by seven distinct serotypes of neurotoxins (A–G) inducing prolonged muscle paralysis by blocking the release of neuronal transmitters from peripheral cholinergic nerve endings (Simpson, 1981). Botulinum neurotoxins (BoNTs) act via a complex multi- phasic mechanism: binding to cell surface high affinity receptors of cholinergic nerve endings (Dolly et al., 1984; Simpson, 1986), penetration into the plasma membrane by endocytosis (Black and Dolly, 1986), and pH-dependent translocation of the light chain from the lumen of the vesicles into the cytoplasm. The light chain acts as a zinc- dependent protease that cleaves one or more components of a membrane fusion complex composed of syntaxin, SNAP-25 (synaptosomal associated protein) and synapto- brevin thereby blocking exocytosis (Schiavo et al., 1992, 2000). As a result of this intracellular cleavage event, the neuronal exocytosis docking/fusion machinery becomes impaired, and the release of acetylcholine at the neuro- muscular junction is inhibited, leading to a subsequent flaccid muscular paralysis. Of the seven known BoNTs, serotypes A, B, and E account for almost all cases of human botulism. Botulinum toxin is classified by the Centers for Disease Control and Prevention (CDC) as one of six highest risk ‘‘Category A’’ agents likely to be deployed during a biological assault (Eubanks et al., 2007). However, low doses of the toxin are commonly used therapeutically to locally paralyse specific muscles for clinical or cosmetic benefit (Jankovic, 2004). The most commonly used method for assessing the potency of botulinum toxin is the mouse lethality bioassay where groups of out-bred mice are typically exposed to the toxin by intra-peritoneal injection and monitored over 72 or 96 h. The dose of toxin that kills 50% of the mice is one lethal dose unit (LD 50 ). Due to its lethal endpoint and the * Corresponding author. Tel.: þ44 1707 641382; fax: þ44 1707 641054. E-mail address: cescargueil@nibsc.ac.uk (C. Rasetti-Escargueil). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.01.019 Toxicon 53 (2009) 503–511