Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state

Abstract

Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well-established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.

Figure 1

Stimulus-response maps created from dorsal column electrical stimulation. Maps in uninjured and untreated animals show position of conducting sensory axons in the dorsal columns of the spinal cord at T9 (A) and T7 (B). Maps display axons below the injury site in animals that received a peripheral nerve conditioning-lesion and control, non-neutralizing anti-NG2 antibodies (C) or neutralizing anti-NG2 antibodies (E). Above the lesion, spatial distribution of regenerated sensory axons differs depending on treatment. In animals with conditioning-lesion and control antibodies (D), regenerated sensory axons are distributed more superficially and bilaterally. Sensory axons in animals with conditioning-lesion and neutralizing anti-NG2 antibodies (F) regenerated beyond the injury within deeper regions of the ipsilateral dorsal columns. Dashed lines on maps delineate the midline and the surface of the spinal cord. Response amplitude is expressed as % of the maximum compound action potential elicited at that site and is presented as gray-scale intensity. Drawings of coronal sections are adapted from Paxinos and Watson, 2004.

Figure 2

Sensory axons induced to regenerate rostral to the injury site are electrically functional. A) Nissl-stained sagittal section (18μm thick) of a bilaterally dorsal-transected spinal cord at T8. Dense cell labeling demarcates the lesion core (*). A pin (black arrow) was placed in the spinal cord tissue at an arbitrary location within T9—caudal to the injury. The rostral-caudal distances of all stimulus-response maps were calibrated to this pin. B) In this animal, four matrices created from stimulus-evoked compound action potentials and mapped with three coordinates, relative to the pin, the midline and surface of the spinal cord. The rostral-caudal distance bar is not to scale. C, and D) To assess the regeneration of sensory axons induced to grow, four-days prior to electrophysiological recordings, the transganglionic tracer, CTB, was injected into the left sciatic nerve. CTB-labeled sensory axons can be found rostral to the injury site. Scale bar A = 500μm; C, D = 50μm

Figure 3

Histological and physiological representations of spared and regenerated fibers. Partial or no dorsal column injury results in presence of CTB-filled profiles in the gracilis nucleus (arrowheads). Sample raw traces from above (T7) or below (T9) injury stimulation indicate differences in temporal dispersion and amplitude of CAP in these preparations. Scale bar = 500μm.

Figure 4

Regenerating axon populations stimulated above the injury exhibited lower mean conduction velocity. (A) Schematic of the electrophysiological preparation. Stim = stimulating electrode above (black) and below (faded) the injury. a and b are pairs of recording electrodes on the dorsal root. CV_dr was determined from the distance and conduction time between the electrode pairs a and b. CV_sc was determined from distance and time between the stimulating electrode and the proximal-most recording electrode on the dorsal root. CV_sc is the average of CV_r (regenerated fiber segments) and CV_i (intact segment). Dashed and solid black lines represent superficial and deep axon populations, respectively. (B) Conduction velocity of the fastest component of the CAP. CV_sc incorporates the CV of both the regenerated and spared segments of the axon, whereas CV_dr is from the intact dorsal root. Stimulation of superficial and deep sub-populations of the dorsal columns above the lesion (CV_sc) elicited volleys with much lower conduction velocity than stimulation of the dorsal root in the same experiments (CV_dr) (* = p<0.001; one-way ANOVA on ranks with Dunn's test). Stimulation of the dorsal columns below the lesion (CV_i) elicits volleys with conduction velocity similar to that of dorsal root. (C) Data from single units recorded in dorsal root filaments in response to stimulation of the same “deep” fiber above and below the lesion indicate that the regenerated segment had a much lower CV than the spared segment. (* = p<0.001; Student's t-test). Graphs are mean ± s.e.m and the number of axons included in analysis is in parentheses

Figure 5

Regenerated sensory axons have pathologic conduction properties. (A) Fidelity of conduction of single axons stimulated above and below the injury decreased with increasing stimulus frequency, but the decrease was greater for axons stimulated above the injury. (B) CV decreased (expressed here as latency increase) with increasing stimulation frequency. The latency delay for above-injury stimulation was greater than below-injury stimulation. (C-D) Confocal z-stack images of spinal cord tissue above (C) and below (D) the injury site immunostained against myelin-basic protein (green) and transganglionically transported cholera toxin-B (red). White arrow indicates an exposed CTB-labeled axon at a node of Ranvier. (*=p<0.01; ** = p<0.001; Mann-Whitney Rank Sum Test). Graphs are mean ± s.e.m. All scale bars = 50μm.