Axonal thinning and extensive remyelination without chronic demyelination in spinal injured rats

Abstract

Remyelination following spinal cord injury (SCI) is thought to be incomplete; demyelination is reported to persist chronically and is proposed as a compelling therapeutic target. Yet most reports do not distinguish between the myelin status of intact axons and injury-severed axons whose proximal stumps persist but provide no meaningful function. We previously found full remyelination of spared, intact rubrospinal axons caudal to the lesion in chronic mouse SCI. However, the clinical concept of chronically demyelinated spared axons remains controversial. Since mouse models may have limitations in clinical translation, we asked whether the capacity for full remyelination is conserved in clinically relevant chronic rat SCI. We determined myelin status by examining paranodal protein distribution on anterogradely labeled, intact corticospinal and rubrospinal axons throughout the extent of the lesion. Demyelination was evident on proximal stumps of severed axons, but not on intact axons. For the first time, we demonstrate that a majority of intact axons exhibit remyelination (at least one abnormally short internode, <100 μm). Remarkably, shortened internodes were significantly concentrated at the lesion epicenter and individual axons were thinned by 23% compared with their rostral and caudal zones. Mathematical modeling predicted a 25% decrease in conduction velocity at the lesion epicenter due to short internodes and axonal thinning. In conclusion, we do not find a large chronically demyelinated population to target with remyelination therapies. Interventions may be better focused on correcting structural or molecular abnormalities of regenerated myelin.

Figure 1.

RST and CST anterograde labeling reveal shortened internodes caudal to the injury. A–D, FluoroRuby (A) was injected into the red nucleus to trace RST axons (B) after thoracic contusion. FluoroEmerald (C) was injected into the motor cortex to trace CST axons (D) after cervical hemi-contusion. CASPR-labeled paranodes (B, D, arrowheads) were used to determine internodal length. E, MBP staining demonstrates that FluoroRuby-labeled axons are myelinated, but probable nodes (arrowhead) are difficult to identify using myelin stains. F, Internodes (arrowheads) are easily identified via immunohistochemical staining. G, H, The distribution of internodal lengths is significantly different between injured and control axons in the RST (G) and CST (H). Control RST internodes (G) are never <100 μm long.

Figure 2.

Channel spreading is evident on severed axons; intact axons exhibit thinning and short internodes concentrated at the lesion epicenter. A, Example of isolated FluoroRuby labeled RST axons in stitched confocal z-series (merged) centered on the lesion epicenter. Dystrophic endbulbs of cut axons are visible rostral to the epicenter (white dashed circle). B, A 212 μm internode caudal to the injury, delineated by arrowheads. C, A 68 μm internode at the lesion epicenter. D, Arrowheads delineate a 233 μm internode on an intact axon rostral to injury; white arrows highlight channel spreading on an axon severed after the image edge. E, Severed axons exhibit dystrophic endbulbs with heavy mislocalization of CASPR1 and Kv1.2 protein (arrowheads). Scale bars: A, 500 μm; B, D, 100 μm; C, E, 25 μm. F, All measured internode lengths plotted by the distance of the center of the internode from the lesion epicenter. Short internodes are significantly concentrated within the 2 mm center of the lesion (blue dots). G, The distribution of internodes shorter than 100 μm across the lesion demonstrates a high concentration at the epicenter (blue dots). H, Average RST axon diameter was reduced by injury. I, Axon diameters of individual large-bore axons were thinned at the lesion epicenter compared with rostral and caudal zones. J–L, Conduction velocity of simulated RST axons. J, Velocity (blue) between nodes simulated with data from three examples of real, labeled axons; axon diameter (red) and internode length (axon diagrams shown to scale: black, myelin; green dot, node). Axon 1 exhibited little variation in internode length, axon diameter, and conduction velocity. Axon 2 exhibited short internodes at the lesion epicenter and reduced conduction velocity. Axon 3 exhibited short internodes, axon diameter, and reduced conduction velocity. K, Average velocity between nodes for 100 simulated axons constructed from all measured internode lengths and axon diameters; shortened internodes and axon thinning reduced conduction speed at the lesion epicenter. L, Distributions of conduction velocities through 2 mm regions centered at the lesion (blue) and rostral to the lesion (green); velocities are significantly reduced (p ≪ 0.001) within the lesion.