Fig. 1: Optic nerve glial cell survival under glucose deprivation requires FA utilization.

a, Schematic representation of the experimental pipeline. Below, myelinated optic nerves from Cnp-mEOS reporter mice, maintained ex vivo (n = 5). Left, longitudinal section showing mEOS⁺ oligodendrocytes (black on white). Right, all DAPI⁺ cell nuclei (black on white). b, Higher magnification of DAPI⁺ (blue) optic nerve glia and PI⁺ (white) dying cells (arrows). Left, cells surviving in 10 mM glucose (Glc). Right, without glucose, cells surviving up to 16 h, but many dying after 24 h (quantified in c). c, Cell survival quantified by subtracting (PI⁺DAPI⁺) dying cells from total (DAPI⁺) cells including data from d–f (16 h: N = n = 8; 24 h: N = n = 12; mean ± s.e.m., Kruskal–Wallis test, Dunn’s multiple comparison). d, Different vulnerabilities of glial subtypes to 24-h glucose withdrawal. Optic nerve longitudinal sections are labeled by DAPI (all cells), PI (dying cells) and genetically expressed cell-specific markers (oligodendrocytes: Cnp-PTS1-mEOS; OPCs: Ng2-YFP; microglia: Cxcr1-GFP; astrocytes: Aldh1l1-GFP). Note the shrunken cell nuclei in glucose-free medium and lack of overlap between oligodendrocytes and dying cells. e, Frequency of glial subtypes after 24-h incubation in 10 mM or 0 mM glucose (oligodendrocytes: 10 mM, N = n = 5; 0 mM, N = n = 4; microglia: 10 mM, N = n = 3; 0 mM, N = n = 5; OPCs and astrocytes: N = n = 3, both) with data from d. f, Survival rate of glial subtypes after 24 h in 10 mM or 0 mM glucose, normalized to cells in glucose-containing aCSF (100%) with data from e (mean ± s.e.m., one-way ANOVA, Tukey’s multiple comparison). g, Glial cells that survive 16-h glucose deprivation dying under hypoxia (bottom), demonstrating oxidation of an endogenous energy reserve other than glucose (N = n = 3, each). h, 4-Br (a mitochondrial β-oxidation inhibitor) treatment of glucose-deprived optic nerves causing widespread cell death, demonstrating FAs as an energy reserve (N = n = 3). Note that 4-Br is not cytotoxic by itself (mean ± s.e.m., unpaired, two-tailed Student’s t-test, heteroscedastic). i, Thio (a peroxisomal β-oxidation inhibitor) treatment not increasing glial death which indicates mitochondrial β-oxidation sufficient for glial survival (N = n = 3). Animals, both sexes, are aged 2 months. Percentages (in g–i) were calculated relative to overall survival for 16 h with glucose under normoxia (in c). N, individual optic nerves; n, independent experiments. Error bars in e, g and i: mean ± s.e.m., unpaired, two-tailed Student’s t-test.

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