Figure 1.

Cytoplasmic Calcium Levels Predict Axonal Fate in Neuroinflammatory Lesions

(A) In vivo multiphoton maximum intensity projection of spinal cord axons of healthy (left) and EAE (peak of disease, 2 days after onset; right) Thy1-CerTN-L15 mice. Top: grayscale images of YFP channel; bottom: ratiometric (yellow fluorescent protein/cyan fluorescent protein; YFP/CFP) images color coded for axonal cytoplasmic calcium ([Ca²⁺]_cyt).

(B) Representative images of control axons in healthy spinal cord and normal-appearing (stage 0), swollen (stage 1), and fragmented (stage 2) axons in acute EAE (2–3 days after onset), color coded for [Ca²⁺]_cyt.

(C) [Ca²⁺]_cyt of single axons in healthy and acute EAE mice, plotted as YFP/CFP channel ratios and normalized to the mean of control axons. Top: percentage of axons with [Ca²⁺]_cyt ≥3 SD above control mean, shown as mean ± SEM (tested per animal in n = 6 control and n = 11 EAE mice, Mann-Whitney U test).

(D) Multiphoton ratiometric time-lapse images of EAE lesions showing a normal-appearing axon rising in [Ca²⁺]_cyt (top), a high [Ca²⁺]_cyt axon undergoing focal axonal degeneration (middle), and the morphological recovery of a low [Ca²⁺]_cyt axon (bottom).

(E) Transition probabilities of axons between morphological stages per observed axon hour in high [Ca²⁺]_cyt (≥control mean + 3 SD, yellow) and low [Ca²⁺]_cyt (magenta) states (n = 307, 8 mice, 1,201 axon hours).

Scale bars in (A), 25 μm, and in (B) and (D), 10 μm. ^∗p < 0.05; ^∗∗∗p < 0.001. See also Figure S1.