Anatomical Plasticity of Rostrally Terminating Axons as a Possible Bridging Substrate across a Spinal Injury

Abstract

Transfer of information across a spinal lesion is required for many aspects of recovery across diverse motor systems. Our understanding of axonal plasticity and which subpopulations of neurons may contribute to bridging substrates following injury, however, remains relatively incomplete. Most recently, attention has been directed to propriospinal neurons (PSNs), with research suggesting that they are capable of bridging a spinal lesion in rodents. In the current study, subpopulations of both long (C5) and short (T6, T8) PSNs-as well as a supraspinal system, the rubrospinal tract (RST)-were assessed following low thoracic (T9) hemisection in the cat using the retrograde tracer Fluoro-Gold. Acutely, within 2 weeks post-hemisection, the numbers of short and long PSNs, as well as contralateral RST neurons, with axons crossing the lesion were significantly decreased relative to uninjured controls. This decrease persisted bilaterally and was permanent in the long PSNs and the contralateral red nucleus (RN). However, by 16 weeks post-hemisection, the numbers of ipsilesional and contralesional short PSNs bridging the lesion were significantly increased. Further, the number of contralesional contributing short PSNs was significantly greater in injured animals than in uninjured animals. A significant increase over uninjured numbers also was seen in the ipsilateral (non-axotomized) RN. These findings suggest that a novel substrate of undamaged axons, which normally terminates rostral to the lesion, grows past a thoracic lesion after injury. This rostral population represents a major component of the bridging substrate seen and may represent an important anatomical target for evolving rehabilitation approaches as a substrate capable of contributing to functional recovery.

Keywords: collateral sprouting; feline; hemisection; propriospinal; rubrospinal; spinal cord injury.

FIG. 1.

Spinal injection site and retrograde labeling. A three-dimensional rendering of a typical injection site shows the areal extent of tracer spread and four injection locations from a dorsal view (*; A). Most cats showed tracer coverage throughout the cross-sectional extent of the spinal cord with the exception of varying degrees of labeling in the dorsal columns as shown in this example (B; dorsal orientation is up). This composite image (B1) is simplified to a single coronal slice (B2) showing that tracer is present across the entire spinal cord cross-section (black) with the exception of an area in the right dorsal column (white). Propriospinal neurons (C) and red nuclei neurons (D) were counted only if Fluoro-Gold was seen in the somas (scale bars = 100 μm). The inset in C is a higher magnification (scale bar = 20 μm) of a labeled neuron identified by the arrow (white) at lower magnification. CC, central canal.

FIG. 2.

Cross-sectional representations of lesions. Tissue sparing and loss can be readily identified using cresyl violet and myelin stained sections. Comparison of histological sections from T9 in a normal control (A) and an animal with a lateral spinal hemisection (B) illustrate the typical asymmetrical loss seen. Using a drawing of the normal cord as a template, the extent of tissue loss and sparing for each acute (C-I) and chronic (J-O) injury was determined for each animal in the study. The representative drawing composite for the histological cross-section shown (B) lies directly below it (E) and shows the modest sparing of the medial part of the ipsilesional dorsal columns. Although there is some variation at the ventral and dorsal midlines across animals, only one lesion (J) showed notable contralesional damage. This animal was included as the damage did not appear to extend into the area of the rubrospinal tract.

FIG. 3.

Ipsilateral and contralateral projections in the normal spinal cord. Unilateral injections (n = 4) labeled more long propriospinal neurons on the contralateral side than ipsilateral, whereas bilateral injections (n = 5) yielded similar numbers on both sides (A). Short propriospinal neurons at both T6 (B) and T8 (C) have similar numbers of neurons that project contralaterally and ipsilaterally. In both cases, bilateral injections labeled more neurons. Following a unilateral injection, labeled neurons were seen almost exclusively in the contralateral red nucleus with <5 neurons ipsilaterally. Bilateral injections labeled neurons in both red nuclei and numbers in each nuclei were similar to that seen in the contralateral nucleus after a unilateral injection (D). Similarity of groups determined by Wilcoxon Signed Test (all p values >0.68). Note: To allow comparison, the use of ipsilateral and contralateral labels for the bilateral injection group are matched to the corresponding unilateral injection side.

FIG. 4.

Changes in the long and short propriospinal neurons (PSN) numbers following injury. Neurons were counted at C5 (long PSNs), T6 and T8 (short PSNs). Tracing of a T6 section is shown in (A) to indicate that counts were made in the grey matter. Neurons typically were present in the intermediate and ventral lamina (scale bar = 500 μM). High magnification (20 × ) photomicrographs in the normal (control, B) and injured (C) T6 spinal cords show examples of Fluoro-Gold–labeled PSNs that were counted (scale bar = 100 μM). The median number of retrogradely labeled ipsilateral and contralateral long propriospinal neurons at C5 (D) shows that following injury, the number of the long propriospinal neurons significantly decreases (*) bilaterally at both acute (n = 7) and chronic (n = 4) time-points compared with normal controls. As illustrated in (E) the number of T6 PSNs decreased significantly on the ipsilateral side of the spinal cord (*) acutely compared with normal controls, but significant increases (‡) were seen bilaterally at the chronic, compared with the acute, time-point. T8 PSNs (F) also showed a significant ipsilateral decrease (*) acutely with significant bilateral increases chronically (‡) from the acute time-point. The number of T8 neurons on the contralateral side at the chronic time-point were significantly greater than in normal controls (†). See the Results section for specific p values.

FIG. 5.

Changes in rubrospinal tract (RST) neurons following injury. A line drawing of the midbrain (A) depicts the areas (red nuclei) where Fluoro-Gold–labeled RST neurons were counted (hatched area, scale bar = 500μM). Photomicrographs show examples of RST neurons counted in the normal control (B) and injured (C) animals. As illustrated in (D), the ipsilateral (non-axotomized) red nucleus had significantly more (†) neurons at both the acute and chronic time-points compared with normal controls. The contralateral (axotomized) red nucleus had significantly fewer (*) neurons at both time-points compared with normal controls, but there was a significant increase from the acute to chronic time-point (‡). See the Results section for specific p values.

FIG. 6.

Retrograde labeling and mechanisms of growth. In the intact spinal cord, neuronal cell bodies with axons extending into the area injected with Fluoro-Gold will be labeled (A). The immediate effect of a lateral hemisection (Hx) is to cut all axons on one side at the lesion level and spare those on the opposite side (B). The neurons with spared axons extending into the area of Fluoro-Gold, caudal to the Hx level, will be labeled. There are several potential mechanisms of growth that may occur after Hx to re-innervate areas below the lesion level. The first is that spared axons of passage, left intact contralateral to the Hx, may develop axonal collaterals (C). This growth is not captured in the neuronal counts as the tracer also is transported by the originally spared axon. Increases in the number of contributing neurons compared with normal controls seen in this study can occur by a combination of at least two potential mechanisms. The first is regeneration of neurons that were axotomized (D). The second mechanism is growth of a non-axotomized subset of neurons with axons that normally terminate rostral to the lesion site (E). The cell bodies of these neurons were not labeled in the normal (intact spinal cord) control group because their axonal termination sites are normally rostral to the injection site. Following injury however, they extended axons into more caudal spinal segments, thus bridging the lesion site and placing them at the level of the injection site.