Conduction failure following spinal cord injury: functional and anatomical changes from acute to chronic stages

Abstract

In the majority of spinal cord injuries (SCIs), some axonal projections remain intact. We examined the functional status of these surviving axons since they represent a prime therapeutic target. Using a novel electrophysiological preparation, adapted from techniques used to study primary demyelination, we quantified conduction failure across a SCI and studied conduction changes over time in adult rats with a moderate severity spinal contusion (150 kdyn; Infinite Horizon impactor). By recording antidromically activated single units from teased dorsal root filaments, we demonstrate complete conduction block in ascending dorsal column axons acutely (1-7 d) after injury, followed by a period of restored conduction over the subacute phase (2-4 weeks), with no further improvements in conduction at chronic stages (3-6 months). By cooling the lesion site, additional conducting fibers could be recruited, thus revealing a population of axons that are viable but unable to conduct under normal physiological conditions. Importantly, this phenomenon is still apparent at the most chronic (6 month) time point. The time course of conduction changes corresponded with changes in behavioral function, and ultrastructural analysis of dorsal column axons revealed extensive demyelination during the period of conduction block, followed by progressive remyelination. A proportion of dorsal column axons remained chronically demyelinated, suggesting that these are the axons recruited with the cooling paradigm. Thus, using a clinically relevant SCI model, we have identified a population of axons present at chronic injury stages that are intact but fail to conduct and are therefore a prime target for therapeutic strategies to restore function.

Figures

Figure 1.

Timeline outlining experimental design. At a number of postinjury time points, spanning acute to chronic stages of spinal cord injury, animals were removed for terminal electrophysiological experiments, for histological assessments, or for EM. Behavioral testing was performed on all remaining animals throughout the study. This allowed a detailed assessment of functional changes as well as pathological and morphological processes that occur over time following contusion injury.

Figure 2.

Schematic diagram illustrating the protocol used for assessing conduction failure in the ascending dorsal column pathway following a spinal contusion injury. Teased dorsal root filaments (arrow) were recorded from both sides of the spinal cord from dorsal roots L3–S1 while first stimulating below the injury site (gray oval), and then using a switch (arrowhead) to stimulate above the injury site, allowing a quantitative measure of the percentage of fibers capable of conducting across a contusion injury and assessment of changes in conduction from acute to chronic injury stages. S, Stimulating electrode; R, recording electrode.

Figure 3.

A 150 kdyn contusion leads to significant and permanent behavioral deficits. A, BBB locomotor rating scores show a severe deficit in locomotor function at acute time points after injury, followed by some spontaneous recovery that plateaus ∼2 weeks after injury. Scores remain stabilized at ∼13 (signifying frequent/consistent stepping, but inconsistent forelimb/hindlimb coordination) into the chronic injury stages (assessed up to 6 months after injury). B, As with BBB locomotor rating scores, the horizontal ladder test shows severe paw placement deficits at acute time points after injury followed by some spontaneous recovery that plateaus ∼4–5 weeks after injury, with animals making ∼20 footslip errors on this task throughout the rest of the testing period. Error bars represent SEM. At every postinjury time point, there was a significant impairment compared with baseline injury scores (p < 0.001, one-way ANOVA, Tukey's post hoc). Correlations of behavioral and electrophysiological data revealed a low correlation between BBB scores and percentage conduction (C) and a high correlation between footslip errors and percentage conduction (D), where animals with improved conduction typically made fewer footslip errors on the horizontal ladder, indicating that this may be a more sensitive test than BBB locomotor rating for revealing subtle changes in behavioral function (R2 value calculated using Pearson's correlation test).

Figure 4.

A 150 kDa contusion leads to a substantial decrease in the percentage of fibers capable of conducting across a contusion injury and a slowing of their conduction velocity. A, Functionality of the ascending sensory pathway is initially abolished postinjury, with a complete lack of conduction acutely (1 d), followed by a partial recovery of conduction over the subacute phase (with ∼14% of sampled fibers capable of conducting across the contusion injury at 4 weeks); no further improvements in conduction properties of ascending dorsal column axons were observed, with the functionality of the dorsal column pathway remaining severely impaired at the chronic postinjury time points (∼16% of sampled fibers capable of conducting across the contusion injury at chronic stages of spinal cord injury). B, Conduction velocity measurements of individual fibers capable of conducting through the lesion site (above injury recordings) revealed a significant slowing of conduction velocity at every postinjury time point, compared with the conduction velocity of fibers activated below the injury (below injury recordings), where this tract would be relatively intact (although, as with the percentage conduction measurements, there was some recovery in conduction velocity over the subacute stages of injury). Representative traces recorded while stimulating below (C) and then above (D) the injury site highlight the differences in conduction properties between the “intact” and injured pathway. Three single units are present while stimulating below the injury, but only one survives to conduct through the lesion. The delayed latency of the unit conducting through the lesion indicates impaired conduction velocity. Single units of activity (arrows) represent the activity of single nerve fibers. E and F show representative traces from above the injury where the cooling technique was applied. By cooling the lesion site with cold mineral oil, additional conducting fibers could be recruited at every postinjury time point (the asterisk in F highlights a single unit added following cooling), thus revealing an important population of axons that are viable but unable to conduct under normal physiological conditions. Error bars represent SEM. The asterisks in A denote significantly impaired conduction of dorsal column axons following contusion, compared with conduction properties in the uninjured (naive) spinal cord (p < 0.001, one-way ANOVA, Tukey's post hoc); the asterisks in B denote significantly impaired conduction velocity of dorsal column axons recorded above, compared with below, the injury (p < 0.01, two-way RM ANOVA, Bonferroni's post hoc).

Figure 5.

Time course of demyelination and subsequent remyelination of dorsal column axons at the contusion lesion epicenter. A, Electron micrograph of dorsal column axons from an uninjured (naive) spinal cord illustrating healthy myelin sheaths. B, At 1 week after injury, extensive demyelination is apparent (asterisks) and remaining myelin appears unhealthy as it becomes less compact and often appears to be unraveling (white arrowhead). C, By 4 weeks after injury, some remyelination is apparent and both nonmyelinated and thinly remyelinated axons are found throughout the dorsal columns. Some axons associated with Schwann cells are also apparent (black arrows). D, Remyelination progresses and at 12 weeks after injury many axons have myelin sheaths that are thick and compact in appearance and are often associated with Schwann cells (black arrows). E, G ratio frequency distributions show a significant shift toward high proportions of thinly myelinated axons at 1 and 4 weeks after injury compared with naive (uninjured) dorsal column axons (p < 0.0001, Kolmogorov–Smirnov test), with this shift reversed by 12 weeks after injury. F, Graphic plot displaying numbers and diameter of nonmyelinated axons measured at each time point shows that significant populations of large-diameter axons (>1 μm) that lacked myelin sheaths were present following contusion injury at all postinjury time points, compared with uninjured spinal cords where only axons with a diameter of ≤1 μm were nonmyelinated (p < 0.001, one-way ANOVA, Tukey's post hoc test). Scale bar, 2 μm.

Figure 6.

Transverse semithin spinal cord sections, stained for toluidine blue, taken from an uninjured (naive) spinal cord (A) or from the lesion epicenter at 1 week (B), 4 weeks (C), and 12 weeks (D) after contusion injury. The boxed areas indicate the regions processed for electron microscopy, containing the ascending dorsal column axons, and correspond to the electron micrographs in Figure 5A–D. Shown are a scatter plot (E) and frequency distribution (F) of diameters of myelinated axons. No significant changes in axon calibre were found at any of the postinjury time points assessed.

Figure 7.

Comparison of the state of myelination of dorsal column axons located either furthest away from the cavity edge (dorsal from cavity) or adjacent to the cavity (closest to cavity). A–D, G ratio frequency distributions reveal no differences in myelin sheath thickness in the two regions in uninjured (naive) spinal cords (A) or 12 weeks after injury (D). However, at 1 week (B) and 4 weeks (C) after injury, many more thinly myelinated axons are located close to the cavity border while axons with thicker myelin sheaths at these postinjury time points are predominantly found away from the cavity edge (*p < 0.05; **p < 0.001; two-way ANOVA Bonferroni's test). E, Measurements of the proportion of nonmyelinated axons with diameters >1 μm relative to their location from the cavity border reveals that at 1 week and 4 weeks after injury significantly more nonmyelinated axons are found close to the cavity border (*p < 0.05; **p < 0.001; two-way ANOVA Bonferroni's test). F, Illustration of compared locations of axons relative to the cavity border. Axons furthest away from the cavity border (red) were compared with axons located close to the cavity (green).

Figure 8.

Remyelination of dorsal column axons at the lesion epicenter is frequently associated with Schwann cells. Costaining of axons (NF 200; red) and Schwann cell-associated myelin (P0; green) illustrates the presence of remyelinating Schwann cells in the dorsal column of the spinal cord at the lesion epicenter at 12 weeks after injury. The bottom panels (D–F) depict the boxed area in the top panels (A–C) at higher magnification (63× oil). Scale bars: A–C, 50 μm; D–F, 20 μm.

Figure 9.

Costaining of axons (NF 200; red) and Schwann cell-associated myelin (P0; green) illustrates the presence of remyelinating Schwann cells in the dorsal columns of the spinal cord at the lesion epicenter at 4 weeks (B) but not at 1 week (A) after injury.

Figure 10.

A 150 kdyn contusion leads to progressive cell loss, reactive gliosis, and cavitation. GFAP and NeuN immunohistochemistry (A–F) and eriochrome cyanine staining (G–L) in transverse sections of the spinal cord through epicenter of the injury demonstrate the pathological changes taking place from acute to chronic time points after injury (1 d to 12 weeks) and can be compared with uninjured spinal cord (A, G). GFAP (astrocytes; red) and NeuN (neuronal cell bodies; green) costaining illustrates the progressive destruction of the gray matter and neuronal cell loss that occurs following injury as well as the pronounced increase in reactive gliosis acutely following injury (B), which at later stages becomes more localized to the borders of the cavity, forming a dense glial scar (C–F). Staining with eriochrome cyanine (to demarcate gray and white matter areas) highlights the initial phase of mass necrosis in the spinal parenchyma at early stages (H–J) followed by clearance of the debris, resulting in large central cavities surrounded by a spared rim of white matter at chronic postinjury time points (K, L). Scale bar, 500 μm.

Figure 11.

Cavity formation and rostrocaudal degeneration gradually increases over time following spinal contusion. A, Eriochrome cyanine staining of serial tissue sections from 3 mm rostral to 3 mm caudal to the lesion site (0 mm indicates the lesion epicenter) at a number of postinjury time points shows stereotypical tissue degeneration and progressive cavity formation; mass destruction of both white and gray matter is apparent at the lesion epicenter at all postinjury time points; rostrocaudal degeneration was mainly restricted to the dorsal columns at early postinjury time points, with increasing rostrocaudal cavitation observed at later stages. B, Quantification of cavity area (expressed as percentage of spinal cord area) at 800 μm intervals through the extent of the injury confirms significantly increased cavity size and rostrocaudal degeneration in the later stages of injury (from 4 weeks). C, Similarly, quantification of total lesion volume, expressed as percentage of total spinal cord volume through the same section of spinal cord shows a significantly increased lesion volume at all chronic injury time points (from 4 weeks onward), compared with the earlier postinjury time points (up to 2 weeks). Error bars represent SEM. The asterisks denote significantly increased cavity area (<0.05, two-way RM ANOVA, Bonferroni's post hoc) and cavity volume (p < 0.01, one-way ANOVA, Tukey's post hoc) at all chronic injury time points (4 weeks, 12 weeks, and 6 months), compared with acute and subacute injury time points (1 d, 1 week, 2 weeks). Scale bar, 500 μm.