The rat Digit Abduction Score (DAS) assay: A physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis

Broide, Ron S.; Rubino, John; Nicholson, Gregory S.; Ardila, Maria C.; Brown, Meenakshi S.; Aoki, K. Roger; Francis, Joseph

doi:10.1016/j.toxicon.2013.05.004

Cited by 44 publications

(46 citation statements)

References 35 publications

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“…This encouraged us to test their activity in preventing BoNT toxicity in mice by using as a readout the digit abduction score (DAS) assay, which provides a reliable estimation of the paralysis induced by a local injection of toxin (Broide et al, 2013). Notably, such an experiment avoids the death of the animal and allows one to follow the rate of muscular activity recovery with time.…”

Section: Thioredoxin and Thioredoxin Reductase Inhibitors Inhibit Thementioning

confidence: 99%

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Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

et al. 2014

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Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

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Section: Thioredoxin and Thioredoxin Reductase Inhibitors Inhibit Thementioning

confidence: 99%

“…After 30 min, the muscles were further injected with 15 pg of BoNT/A or 20 pg of BoNT/C (injection volume 25 ml). Hindlimb paralysis was evaluated at least once per day and DAS score provided (Aoki, 2001;Broide et al, 2013).…”

Section: Immunocytochemistry and Immunohistochemistrymentioning

confidence: 99%

Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

et al. 2014

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“…The mouse phrenic nerve hemidiaphragm (PNHD) assay is an ex vivo assay for assessing the neuroparalytic activity of BoNTs, as it directly records the blockade of neuromuscular transmission . In vivo , activity of BoNTs in the digit abduction score (DAS) test in mice and rats is indicative of their muscle‐relaxant properties, whereas reductions in body weight (BW) gain are indicative of toxin spread and migration away from the injected site …”

Section: Introductionmentioning

confidence: 99%

“…16 In vivo, activity of BoNTs in the digit abduction score (DAS) test in mice and rats is indicative of their muscle-relaxant properties, whereas reductions in body weight (BW) gain are indicative of toxin spread and migration away from the injected site. 1,[17][18][19][20] 2 | MATERIALS AND METHODS…”

mentioning

confidence: 99%

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo

Donald

Elliott

Gray

et al. 2018

Pharmacology Res & Perspec

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Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT‐induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well‐tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species‐specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

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“…Interestingly, each inhibitor blocked the different BoNTs tested within a very similar concentration range, suggesting that the molecular environment where reduction takes place and the relevance of the interchain disulfide reduction is closely similar for different BoNTs serotypes. These in vitro results were validated in vivo using the digit abduction score (DAS) assay, a wellestablished model to compare potency and duration of BoNTs (Aoki, 2001;Broide et al, 2013). All tested molecules were very effective in reducing the degree and duration of paralysis induced by the local injection of BoNT/A and BoNT/C.…”

Section: Thioredoxinethioredoxin Reductase Inhibitors As a New Class mentioning

confidence: 92%

The thioredoxin reductase – Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles

Pirazzini

Tehran

Zanetti

et al. 2015

Toxicon

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The rat Digit Abduction Score (DAS) assay: A physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis

Cited by 44 publications

References 35 publications

Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo

The thioredoxin reductase – Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles

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