Chondroitinase ABC-mediated plasticity of spinal sensory function

Abstract

Experimental therapeutics designed to enhance recovery from spinal cord injury (SCI) primarily focus on augmenting the growth of damaged axons by elevating their intrinsic growth potential and/or by nullifying the influence of inhibitory proteins present in the mature CNS. However, these strategies may also influence the wiring of intact pathways. The direct contribution of such effects to functional restoration after injury has been mooted, but as yet not been described. Here, we provide evidence to support the hypothesis that reorganization of intact spinal circuitry enhances function after SCI. Adult rats that underwent unilateral cervical spared-root lesion (rhizotomy of C5, C6, C8, and T1, sparing C7) exhibited profound sensory deficits for 4 weeks after injury. Delivery of a focal intraspinal injection of the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC (ChABC) was sufficient to restore sensory function after lesion. In vivo electrophysiological recordings confirm that behavioral recovery observed in ChABC-treated rats was consequent on reorganization of intact C7 primary afferent terminals and not regeneration of rhizotomized afferents back into the spinal cord within adjacent segments. These data confirm that intact spinal circuits have a profound influence on functional restoration after SCI. Furthermore, comprehensive understanding of these targets may lead to therapeutic interventions that can be spatially tailored to specific circuitry, thereby reducing unwanted maladaptive axon growth of distal pathways.

Figure 1.

In vivo digestion of CSPGs with chondroitinase ABC. A, Schematic diagram illustrating the site of a single microinjection of ChABC or saline (shaded area in C7 spinal segment) and relative location of rhizotomized dorsal roots (C5, C6, C8, and T1). Ipsilateral and contralateral spinal cord segments from animals microinjected with saline or ChABC were harvested for protein analysis 14 d after lesion and treatment. B, Immunoblot analysis of spinal cord lysates probed with anti-chondroitin-4-sulfate antibody (C4S) revealed digestion of all endogenous 4-sulfated CSPGs 14 d after ChABC delivery [N indicates naive spinal cord lysate from a control animal incubated in vitro in the presence (+) or absence (−) of ChABC]. C, Analysis of lysates from experimental animals treated with saline (red bars) or ChABC (green bars) revealed that ChABC injected unilaterally at C7 diffused bilaterally and significantly (*p < 0.005, ANOVA) digested 4-sulfated CSPGs from C7 to T1. Photomicrograph of a sagittal (D) and a transverse (E) section of spinal cord 14 d after ChABC microinjection (asterisk, injection site) immunostained with C4S. Digestion of intrinsic 4-sulfated CSPGs can be seen spreading both rostrocaudally and mediolaterally from the injection site. Scale bars: D, E, 500 μm. Photomicrographs of transverse sections from C7 [intact segment (F–H)] and C8 [rhizotomized segment (I–K)] after spared-root lesion. Spared-root lesion does not digest intrinsic CSPGs within intact (F) and rhizotomized (I) segments, indicated by the absence of C4S immunoreactivity. Neurocan (red) is highly expressed in spinal gray matter (Gm) within intact segment C7 (G) and does not increase after rhizotomy (Rhx) (J). Phosphacan (red) is also highly enriched in spinal gray matter within intact C7 (H) and is unaltered after rhizotomy (K). Scale bar, 200 μm. Quantification of CSPG expression within dorsal horn gray matter (G, boxed area) confirms that both neurocan and phosphacan levels are constitutively high in spinal gray matter and remain unchanged after RhX (bar graphs in J and K, respectively). Error bars indicate SEM.

Figure 2.

Chondroitinase ABC-mediated restitution of sensory function after spared-root lesion. A, During spontaneous exploration of a Plexiglas cylinder (assessed over 21 d), intact animals use both forelimbs simultaneously to vertically explore the cylinder ∼50–60% of the time; the reminder of the time individual paws are used. The experimental forelimb (left) score was ∼85% (injured limb plus both limbs) presurgery. Spared-root lesion and saline treatment resulted in a significant deficit in the ability of the animals to explore laterally with their injured forelimb throughout the experimental period. In contrast, spared-root lesion animals treated with ChABC illustrated a deficit at only at 2 d after lesion, but recovered and were indistinguishable from sham-lesioned control animals within 7 d (*p < 0.01, multivariate ANOVA). B, C, In an adhesive tape removal task, spared-root-lesioned animals treated with saline displayed a significant deficit in sense scores up to 14 d after injury and motor scores at 2 d after lesion, compared with sham-lesioned control animals. In contrast, spared-root-lesioned animals treated with ChABC failed to show a deficit at any time point after injury compared with sham animals (*p < 0.01, multivariate ANOVA). Error bars indicate SEM.

Figure 3.

Chondroitinase ABC enhances anatomical reorganization of intact CTB-IR fibers. Photomicrographs illustrate CTB-IR primary afferent terminals in contralateral (A–D) and ipsilateral (F–I) spinal cord in saline-treated animals and contralateral (K–N) and ipsilateral (P–S) spinal cord in ChABC-treated animals after spared-root lesion. Horizontal dorsal horn mappings (E, J, O, T) illustrate the mediolateral distribution of CTB-IR terminals at the level of the lamina III–IV border after peripheral C7 dermatome CTB injections in spared-root-lesioned animals after vehicle (E, J) or ChABC treatment (O, T). In each graph, the midline is set at 0 μm, and the lateral extent of the dorsal columns is marked by the first dashed line; the gray matter extends between the first and second dashed lines, and the dorsolateral columns extend between second and third dashed lines. On this map, the area occupied by CTB-IR terminals is marked by the horizontal lines at different rostrocaudal levels. The rostrocaudal extent of CTB-IR terminal distribution is significantly reduced at all spinal levels on the ipsilateral side in saline treatment compared with ChABC treatment (U) (*p < 0.001, one-way ANOVA), as shown by the histogram below, in which data are expressed as average ± SEM mediolateral occupation of CTB-IR terminals.

Figure 4.

Restoration of postsynaptic activity after spared root lesion and chondroitinase ABC treatment. A, Shown are examples of CDPs (arrow indicates N-wave) recorded at the midline at C7 after electrical stimulation of the C7 dorsal root from sham-lesioned intact animals (black line) and spared-root-lesioned animals that received saline treatment (red line) or ChABC treatment (green line); schematic illustrates placement of stimulating and recording electrodes. B, Stimulus–response curves were created by increasing the stimulus intensity in 10 μA steps. A maximum response of −2.0 mV recorded in sham-lesioned animals plateaued at 60 μA (black circles); acute rhizotomy of the C7 dorsal root abolished activity (open circles). Spared-root lesion and saline treatment significantly reduced the maximum CDP recorded at C7 (red circles), with a maximum recorded CDP of −0.6 mV (*p < 0.05, ANOVA). Spared-root lesion and ChABC-treated animals (green circles) exhibited CDPs significantly higher than saline-treated animals (*p < 0.05), but were statistically indistinguishable from sham-lesioned animals. C, CDPs were recorded from adjacent spinal segments after spared-root lesion; shading distinguishes intact segments from areas of deafferentation. Intact animals showed a peak in the N-wave of CDP at C7 (black circles); CDPs diminished when the recording electrode was moved either rostrally or caudally. Lesion- and saline-treated animals showed a significant decrease in CDP recorded at all spinal segments compared with sham-lesioned intact animals (red circles; *p < 0.05, ANOVA). Lesion- and ChABC-treated animals displayed CDPs that were not significantly different from sham-lesioned controls (green circles). Error bars indicate SEM.

Figure 5.

Restoration of spatially defined activity in the dorsal horn after spared-root lesion and chondroitinase ABC treatment. A–I, Field potential recordings were made from 12 sites (surface of gray matter and descending in 100 μm steps) along a track 400 μm lateral to the midline, 7, 14, and 28 d after sham, spared-root, and saline or ChABC treatment. Three-dimensional reconstructions of field potential recordings (A, B, D, E, G, H) illustrate activity changes (color coded; 0.5 mV, red, to −4.0 mV, indigo) recorded at different depths (x-axis) in spinal gray matter after electrical stimulation of the C7 dorsal root (time, z-axis). Contralateral spinal cords from sham-lesioned (A), lesion- and vehicle-treated (D), and lesion- and ChABC-treated (G) animals illustrate equivalent activity profiles. The peak amplitude of the N-wave and latency of N-wave recorded from contralateral spinal cords were not significantly different between groups at any time point after lesion. Lesioned animals that received saline treatment illustrated significantly reduced activity ipsilateral to the lesion (E; original trace shown in F, J) (*p < 0.05, ANOVA) compared with sham-lesioned animals (B; original trace, C) at all time points after lesion (J). The latency of the N-wave was significantly elevated in saline-treated animals compared with sham-lesioned animals at all postlesion time points (K) (*p < 0.05, ANOVA). In contrast, lesioned animals that received ChABC treatment illustrated robust activity in the dorsal horn ipsilateral to the lesion (H; original trace, I). Assessment of the peak amplitude of N-wave showed that it was not significantly reduced compared with sham-lesioned animals at 7 and 14 d after lesion (J) and was significantly higher than saline-treated animals at all postinjury time points (J) (^#p < 0.01, ANOVA). The latency of the N-wave from ChABC-treated animals was significantly reduced compared with saline-treated animals (K) (^#p ≤ 0.05, ANOVA), but not significantly different from sham-lesioned animals (K). Error bars indicate SEM.

Figure 6.

Chondroitinase ABC restores peripheral drive to the deafferented dorsal horn. A, Summary of single-unit activity after sham or spared-root-lesioned and saline or ChABC treatment. The table illustrates data collected from animals that had undergone mass wave and field potential recordings (n = 5 per treatment at each time point). Units were identified by lightly brushing the rat forelimb area while driving a sharp tungsten electrode into the dorsal horn. On identifying a single unit, its receptive field was mapped and electrically stimulated with pin electrodes placed in the skin within the center of the receptive field. Receptive fields mapped in sham-lesioned animals revealed that single units isolated in the C7 spinal segment were driven by peripheral stimulation of the medial digits and medial forearm (B). Lesion and saline treatment resulted in significant reduction in the size of the receptive fields; the few units that were identified had receptive fields restricted to the medial digits (C). In contrast, lesioned animals that received ChABC treatment illustrated expanded receptive fields that in many cases encompassed fields within deafferented spinal segments (D). E, Single unit isolated from a sham-lesioned animal, responding to light brushing, light touch, and pinch. PSTH (G) generated from pin electrodes stimulating the receptive field (2 mA at 0.5 ms). F, Single unit identified in a ChABC-treated animal 14 d after lesion. Uniform spikes were recorded after brush, touch, and pinch stimulus; PSTH recorded from this unit (H) illustrates that this cell receives both A- and C-fiber inputs.

Figure 7.

Peripheral noxious stimulus phosphorylates MAP kinase in deafferented spinal segments. A–F, Photomicrographs show capsaicin-induced phosphorylation of MAP kinase as detected by phosphospecific antibodies to ERK1/2 (pERK). Phospho-ERK is detected in the medial aspect of lamina I and II contralateral to the spared-root lesion in saline (A)- and ChABC (D)-treated animals. Significantly reduced numbers of dual pERK-IR/NeuN+ cells are seen ipsilateral after saline treatment (B, C). ChABC-treated animals maintain robust numbers of pERK-IR/NeuN+ cells ipsilateral to the lesion (E, F). G, Quantification of the number and rostrocaudal distribution of dual pERK/NeuN-immunoreactive cells from C5 to T1 after bilateral noxious thermal stimulus. The number of pERK-IR cells on the contralateral side was the same between treatment groups at all segments. Lesion- and saline-treated animals illustrated a significant reduction (*p < 0.005, ANOVA) in the number of pERK-IR cells on the ipsilateral side compared with the contralateral intact side at all segments. Lesion- and ChABC-treated animals showed a significant increase in the number of ipsilateral pERK-IR neurons compared with saline-treated animals (^#p < 0.005, ANOVA). There was no significant difference in the number of pERK/NeuN+ cells between ipsilateral and contralateral sides after ChABC treatment at any spinal segment assessed. Error bars indicate SEM.

Figure 8.

Effect of chondroitinase ABC on the deafferented spinal cord. Shown is a schematic representation of the influence of ChABC after spared-root lesion. Within the “intact” spinal cord, small diameter primary afferents enter through the dorsal root entry zone and synapse on local second-order neurons in the dorsal horn. Three color-coded spinal levels are shown, axons from each segment synapse on dorsal horn neurons (of the same color) that mediate adult sensory function. The inset shows that axons from one spinal segment (shown in blue) project terminals into the vicinity of second-order neurons in adjacent segments (into the green axon territory). However, these terminals remain nonfunctional or silent under normal physiological conditions. Second-order dorsal horn neurons and primary afferent terminals are surrounded by extracellular matrix rich in chondroitin sulfate proteoglycans (shown as reticulum of yellow sugar chains, attached to a red core protein). Primary afferent terminals retract in “lesioned” animals (pink and green axons). Spinal deafferentation removes peripheral input to (pink and green) second-order neurons. Local chondroitinase ABC infusion (blue cloud) removes sugar side chains from CSPG core protein (inset), thus creating a more permissive environment for terminal reorganization. We propose that at least two mechanisms mediate ChABC-associated restoration of spinal sensory function after spared-root lesion. First, axons from the intact (blue) spinal segment sprout into deafferented territories and make de novo connections on second-order neurons (blue/green cells) and hence maintain a measure of appropriate sensory input. Second, previously silent connections are unmasked and begin to relay cutaneous information. Both of these mechanisms are made possible by ChABC-induced reduction in the inhibitory environment associated with the extracellular matrix in the dorsal horn.