Figure 5.

FK866 is effective ex vivo and preserves functional axons and neuromuscular synapses. (A) FK866 delays Wallerian degeneration ex vivo. (a) NAD (left panel) and NMN (right panel) levels were determined in mouse sciatic nerve explants cultured for 30 h in the presence of 10 μM FK866 or vehicle (DMSO) and normalized against the total adenylate pool (n=10, mean±S.E.M., unpaired Student's t-test, **P<0.01, ***P<0.001, ****P<0.0001). (b) Rate of axon degeneration in YFP-H nerve explants cultured for 135 h in the presence of 10 μM FK866 or DMSO control. The upper panel shows the time until the first axon break appeared in each nerve. The lower panel shows the percentage of surviving axons in each nerve after 48 h (n=5–10 nerves; mean±S.E.M., one-way ANOVA with Bonferroni's post-hoc test, *P<0.05, ****P<0.0001). (B) FK866 applied to WT muscles mimics synaptic protection conferred by Wld^S ex vivo. The number of responsive fibres, that is, those showing EPPs upon stimulation and/or spontaneous miniature EPPs, was analysed in isolated tibial nerve/FDB muscle preparations that had been incubated in MPS containing FK866 in panels (a and b) for 16–20 h (n=4–5 muscles in each group) and in panel (c) for 42–48 h (n=8 muscles in both groups). FK866 dose-dependently delayed synaptic degeneration in WT muscles at 16–20 h (P<0.05; ANOVA with post-hoc Dunnett's test) and potentiated the protective effect of Wld^S at 48 h (P<0.05; t-test). Note that synaptic degeneration in FDB muscles from Wld^S mice was markedly more rapid ex vivo than in vivo,⁶⁴ probably reflecting the shorter nerve stump length and altered environment. However, the phenotype is readily distinguished from WT ex vivo, with incubation in MPS at 32 °C. het=heterozygous