Figure 1: NAD⁺ prevents demyelination and axon loss.

(a) Behavioural scores of EAE in C57BL/6 mice treated at the day of MOG immunization daily with intraperitoneal injection of 60 mg of NAD⁺ or a placebo solution (PBS). An additional group of mice was treated with intraperitoneal injection of 60 mg of NAD⁺ 15 days after MOG immunization (n=8–11 per group). (b–i) Myelin and neurofilament (NF) assessment in spinal cord of: (b) mice that were treated daily with a placebo solution (PBS), (c) mice that were treated daily with NAD⁺ and (d) mice that were treated with NAD⁺ 15 days after MOG immunization (when mice developed hindlimb paralysis). (b–d) Paraffin sections were stained with luxol fast blue (LFB) to assess demyelination, H&E for lymphocyte infiltration, myelin basic protein (MBP) to assess demyelination, NF 200 for axon loss and DAPI to label nuclei. Merged images of MBP, NF and DAPI are also shown. The boxed regions in a–c for LFB and H&E staining have been magnified in d–f, respectively. The white stars in a–c for MBP, NF, DAPI and merged images depict regions that are shown in higher magnification in d–f. Scale bars: a, 100 μm (applies to b–d), d, 50 μm (applies to e–g). (h,i) Quantification of myelination and NF-positive areas showing a significant protection and recovery of (h) myelin and (i) NFs following daily NAD⁺ treatment and administration 15 days after MOG immunization. (j,k) Vimentin/Ki67 (j) and Vimentin/Musashi (k) co-labelling revealed increased ependymal cell proliferation in NAD⁺-treated spinal cord when compared with PBS-treated spinal cord. Scale bars: (b), 100 μm (applies to c,d); (e), 50 μm (applies to f,g); (j), 20 μm (applies to k); n=8–11; data derived from three independent experiments. Data represent mean±s.d. *P<0.05; **P<0.01; ***P<0.001, NS, not significant. Student’s t-test and Mann–Whitney U-test were used accordingly to compare between groups.