Fig. 3. Folate-induced transgenerational enhancement of CNS axon regeneration is independent of route of administration, breeding protocol, and genus.

(A) Oral folate supplementation of progenitors enhances spinal axon regeneration in F1-F6 progeny on an unsupplemented diet. Graph shows the percentage of DRG neurons that regenerated into sciatic nerve grafts in S0 animals and the untreated offspring (F1-F6) of F0 progenitors with a methyl-supplemented diet, compared to an un-supplemented ‘normal’ diet (F1-F4). (n (high methyl-purple, normal-blue) = S0: 13, 6; F1: 7, 9; F2: 11, 9; F3: 14, 11; F4: 9, 10; F5: 10, - ; F6: 9, - ; the “normal diet” lineage was not bred beyond F4; *p<0.05; The high methyl diet includes supplemental folic acid and other methyl donor compounds comprising betaine, choline, and vitamin B12). (B) Folate supplementation of inbred Fisher rat progenitors enhances in vivo regeneration of injured spinal axons in untreated F1-F3 progeny. Percentage of DRG neurons of the S0 animals and the untreated offspring (F1-F3) of folate supplemented F0 progenitors that have regenerated spinal axons into the sciatic nerve grafts (n (FA-purple, DDI-blue) = S0: 12, 11; F1: 5, 11; F2: 8, 9; F3: 11, 11; *p<0.05). (C) Folate supplementation of mouse progenitors enhances in vivo regeneration of injured spinal axons in untreated F1-F3 progeny. Percentage of DRG neurons of S0 animals and the untreated offspring (F1-F3) of folate supplemented progenitors that have regenerated spinal axons into the sciatic nerve grafts (n (FA-purple, DDI-blue) = S0: 6, 4; F1: 7, 10; F2: 7, 9; F3: 7, 7; *p<0.05). Boxes extend from the 25^th to 75th percentiles, and whiskers from the minimum to the maximum, except for outliers indicated by a circle. Comparisons of differences within generations were made using the Wilcoxon rank-sum test, *p<0.05 (S0: unbred single generation control).