Swiss mice

Human fetuses were obtained after a legal abortion with the written informed consent of the patient. The procedure for the procurement and use of human fetal CNS tissue was approved and monitored by the “Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale” of Henri Mondor Hospital, France. The cells are declared at the “Centre des Ressources Biologiques” of the University Hospital in Angers with reference numbers at the Research Ministry: declaration No DC-2011-1467; authorization No AC-2012-1507.
All animals used in this study were housed under standard conditions within a specific-pathogen-free facility at GIGA, C.H.U, Sart Tilman, Belgium, with ad libitum access to food and water. All animal procedures were approved by the Animal Ethics Committee of the Liege University (#16-1829) and are compliant with the Belgian Ministry of Agriculture, in agreement with the European Community Laboratory Animal Care and Use Regulations (86/609/CEE, Journal Officiel des Communautés Européennes L358, 18 December 1986). Time-mated immunocompetent adult SWISS mice (2–3 months of age, purchased from Janvier Laboratories, France), were allowed to acclimate for at least 24 h (hrs) prior to any procedure and were used when noon of the day after mating was considered embryonic day 0.5 (E0.5).

The MPN assay was performed employing 20–30 g Swiss mice (Janvier SA, France) as described previously^{57 (link)}. Mice were euthanized by CO₂ anesthesia and subsequently exsanguinated. The phrenic nerve hemidiaphragm tissue was explanted, placed into an organ bath and continuously stimulated at 5–25 mA with a frequency of 1 Hz and a 0.1 ms pulse duration. Isometric contractions were transformed using a force transducer and recorded with VitroDat Online software (FMI GmbH, Germany). The time required to decrease the amplitude to 50% of the starting value (paralytic half-time) was determined. To allow comparison of the altered neurotoxicity of mutants with BoNT/E1 wild-type (displaying a specific activity of 0.41 × 10⁸ LD₅₀/mg), its MPN assay dose-response-curve logarithmic function (y(BoNT/E1 wild-type; 2.0, 4.0, 8.0 pM) = −23.61 ln(x) + 104.11, R² = 0.999) consisting of three concentrations determined in 5–8 technical replicates as described previously was employed^{67 (link)}. Mean (n = 3–5) of resulting paralytic half-times of the BoNT/E1 mutants were converted to concentrations of the wild-type employing the above function and finally expressed as relative neurotoxicity.

Unilateral hindlimb ischemia (HLI) was induced on 9-week-old female Swiss mice (Janvier Labs, n = 8) after femoral artery excision under 2% isoflurane anesthesia as previously described [40 (link)]. LASER Doppler perfusion imaging (Perimed, Craponne, France) was used to assess revascularization from day 0 to day 21 after surgery. Perfusion measurements were expressed as an ischemic-to-contralateral ratio of hind limb blood flow normalized to the day of surgery.