Figure 4. Aging induces accumulation of NfL in SJL/J A/T EAE mice and loss of brain volume at the postacute disease stage.

(A) Serum neurofilament light chain (NfL) levels in young (n = 4–12) and old (n = 4–7) SJL/J A/T EAE mice at the acute (day 11) and postacute (day 25) disease stages. Age-matched, unimmunized controls included for comparison at the postacute time point (young, n = 4; old, n = 4). (B) Correlation analysis of clinical severity represented by AUC with levels of serum NfL. (C) Representative images in the somatosensory cortex (SSC) of old versus young SJL/J A/T EAE mice. (D) Quantification of IHC staining for NfL using %NfL⁺ area. (E) Correlation analysis of NfL immunohistochemistry and serum levels. (F) Clinical course of old (n = 8) versus young (n = 8) SJL/J A/T EAE mice followed for 40 days post-A/T. Statistical analysis by 2-way ANOVA with Bonferroni’s correction for multiple comparisons; error bars indicate mean ± SD. (G) Ex vivo MRI image of an SJL/J A/T EAE mouse showing the axial length of the skull from the nasal cavity (green arrow) to the base of the skull (red arrow). (H) Brain volume of young (n = 3–5) versus old (n = 5–6) SJL/J A/T EAE mice expressed as a ratio to axial length. (I) Percentage change in SJL/J A/T EAE mice from age-matched controls (young n = 3, old n = 3) at acute and postacute disease stages. Statistical analysis by Mann-Whitney U test; error bars indicate mean ± SD. (J) Correlation analysis of clinical severity with brain volume. (K) SSC volume of each mouse expressed as a ratio to axial length. (A, H, and K) Statistical analysis by 1-way ANOVA with Bonferroni’s correction for multiple comparisons; error bars indicate mean ± SD. (D) Statistical analysis by Kruskal-Wallis with correction for multiple comparisons; error bars indicate mean ± SD. (B and E) Statistical analysis by Spearman’s correlation test. (J) Statistical analysis by Pearson’s correlation test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.