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Actions of Botulinum Toxin A in nerve-muscle-tissue-cultures: A proof-of-principle-study
Project
Project code: BfR-ZEBET-08-1328-456
Contract period: 01.10.2010
- 30.04.2013
Purpose of research: Project preparation activity
This project aims to develop a cell-culture based in vitro-system to be used for reducing the number of animal experiments performed for testing the efficacy of medical products containing botulinum neurotoxins A. In organotypic co-cultures composed of spinal cord and muscle tissue, spontaneous contractions of muscle fibers are observed. This muscle activity is evoked by spinal motor neurons which form synapses directly onto muscle cells. Specifically, the hypothesis that these muscle contractions are depressed by botulinum toxin A is tested.
Actions of botulinum toxin A were studied in 35 cell-cultures, derived from the spinal cord and surrounding muscle tissue of embryonic mice. 32 cultures underwent sham-treatment and served as internal controls. Muscle activity in sham- and botulinum toxin treated tissue cultures was quantified 24, 48 and 72 hours after starting these treatments. Muscle activity was significantly different between the sham and botulinum toxin treated group (Kruskal-Vallis test, p-values for 24, 48 and 72 hours: 1.8x10-5, 1.9x10-2 und 6.0x10-4, respectively). In more than half of the cultures exposed to botulinum toxin, muscle activity declined to less than 0.15 Hz. Under control conditions the corresponding value was 1.58 Hz. However, a small subgroup of cultures was almost unaffected by botulinum toxin, even 72 hours after starting the treatment. The reason for this lack of response is unknown. Muscle activity monitored in sham-treated but not in botulinum toxin treated cultures was normally distributed. Recovery of muscle activity was not observed within 72 hours in cultures exposed to the neurotoxin. In summary we conclude that the efficacy of botulinum toxin A can be estimated by conducting studies in nerve muscle tissue cultures. Furthermore, Xeomin®, a botulinum toxin A-containing medical product was tested in different concentrations to assess the sensitivity of the test system. The concentrations ranged between 1 and 50 mouse-LD50-units. It was observed that Xeomin depressed muscle activity up to 90%. The limits of detection of the test system were between 1 and 2 mouse-LD50-units. The results suggest that testing of 30 cell cultures should be statistically sufficient to show whether or not a test solution contains botulinum toxin A at a concentration corresponding to 2 mouse-LD50-units or higher. The number of cell cultures that can be prepared from a single mouse ranged between 30 and 50.
Section overview
Subjects
- Biotechnology
Framework programme
Funding programme
Excutive institution
BfR - Centre for Documentation and Evaluation of Alternatives to Animal Experiments (BfR - ZEBET)
