Fig. 2. Vitamin B₃/NAM supplementation protects against glaucoma development in mice.

(A) NAD(t) levels were increased in Vitamin B₃/NAM treated D2 retinas as measured by colorimetric assay (n = 22/group). (B and E) NAM intervention protected from optic nerve degeneration as assessed by PPD staining (a sensitive stain for damaged axons). Green = no or early damage (<5% axon loss; NOE), yellow = moderate damage (~30% axon loss; MOD), red = severe (>50% axon loss; SEV) damage. Early start indicates mice that started treatment at 6 mo (prior to IOP elevation in most eyes in our colony, thus prophylactic). Late start indicates mice that started treatment at 9 mo (when the majority of eyes have had continuing IOP elevation, thus interventional). Fisher’s exact test: ** = P < 0.01, *** = P < 0.001. (C and E) NAM protected from RGC soma loss (number of RBPMS+ cells, n = 8/group, the density drop between D2 and D2-Gpnmb⁺ is due to pressure induced stretching). (D) NAM protected from early loss in PERG amplitude (n > 20/group). (E) NAM protected from RGC soma loss (n = 8/group), retinal NFL and IPL thinning (n = 8/group), optic nerve degeneration (n > 50/group), and loss of anterograde axoplasmic transport (n = 20/group). Corresponding markers and color keys are beneath each column. Scale bars; RBPMS (a specific marker of RGCs; immunofluorescence) = 20 μm, Nissl (a pan-neuronal stain; light microscopy) = 20 μm, PPD (light microscopy) = 20 μm, CT-β = 100 μm (retina; immunofluorescence), 200 μm (LGN, Sup. Col.). ONH = optic nerve head, LGN = lateral geniculate nucleus, Sup. Col. = superior colliculus. White asterisk denotes loss of axonal transport at the site of the ONH. (F) Heatmap of gene expression (all expressed genes) shows that NAM treated RCGs were molecularly similar to controls. (G) Individual gene expression plots show metabolic and DNA damage pathways were returned to normal in NAM treated RGCs. Dots represent individual genes, grey = not differentially expressed, red = differentially expressed at q < 0.05 compared to D2-Gpnmb⁺ 9 mo control. (B, D, and E) * = P < 0.05, ** = P < 0.01, *** = P < 0.001 (Student’s t test). For boxplots, center hinge represents the mean, and the upper and lower hinges represent the first and third quartiles, whiskers represent 1.5 * interquartile range, values beyond the whiskers are plotted as outliers. See also Fig. S4, Tables 1–3.