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. 2009 May 13;29(19):6285-95.
doi: 10.1523/JNEUROSCI.5885-08.2009.

Blockade of Nogo receptor ligands promotes functional regeneration of sensory axons after dorsal root crush

Pamela A Harvey  1 , Daniel H S Lee, Fang Qian, Paul H Weinreb, Eric Frank
Affiliations

Blockade of Nogo receptor ligands promotes functional regeneration of sensory axons after dorsal root crush

Pamela A Harvey et al. J Neurosci. .

Abstract

A major impediment for regeneration of axons within the CNS is the presence of multiple inhibitory factors associated with myelin. Three of these factors bind to the Nogo receptor, NgR, which is expressed on axons. Administration of exogenous blockers of NgR or NgR ligands promotes the regeneration of descending axonal projections after spinal cord hemisection. A more detailed analysis of CNS regeneration can be made by examining the growth of specific classes of sensory axons into the spinal cord after dorsal root crush injury. In this study, we assessed whether administration of a soluble peptide fragment of the NgR (sNgR) that binds to and blocks all three NgR ligands can promote regeneration after brachial dorsal root crush in adult rats. Intraventricular infusion of sNgR for 1 month results in extensive regrowth of myelinated sensory axons into the white and gray matter of the dorsal spinal cord, but unmyelinated sensory afferents do not regenerate. In concert with the anatomical growth of sensory axons into the cord, there is a gradual restoration of synaptic function in the denervated region, as revealed by extracellular microelectrode recordings from the spinal gray matter in response to stimulation of peripheral nerves. These positive synaptic responses are correlated with substantial improvements in use of the forelimb, as assessed by paw preference, paw withdrawal to tactile stimuli and the ability to grasp. These results suggest that sNgR may be a potential therapy for restoring sensory function after injuries to sensory roots.

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Figures

Figure 1.
Figure 1.
A, Schematic diagram of experimental preparation. All cervical dorsal roots that supply the brachial plexus, from C5–T1, were crushed, as described in Materials and Methods. B, Experimental time course. Dorsal roots were crushed in an initial surgery, and infusion of sNgR was begun immediately into the lateral ventricle via osmotic minipump and was continued for 28 d. Behavioral testing was performed once or twice each week for the duration of the recovery period. One week before electrophysiological recording, peripheral nerves were injected with CTB. After the recording session, rats were perfused and the spinal cords processed for histology.
Figure 2.
Figure 2.
Spinal projections of myelinated sensory axons in untreated rats. Projections of axons crushed within the dorsal roots 4 weeks previously are shown in A–C, E, and G; projections of uncrushed axons are shown in B, D, F, and H. A, B, Motoneurons (arrows) and sensory cell bodies (large arrow in A) are labeled on crushed and uncrushed sides. Projections of sensory axons (double arrow) within the spinal gray matter are only present on the uncrushed side, and these projections avoid the most dorsal spinal laminae in the dorsal horn (arrowheads in B and D). C, D, Labeled axons (shown at higher power in G and H) are present in dorsal roots on both sides, but after an untreated crush, they end in terminal endbulbs (arrow in G) and do not cross into the DREZ. Uncrushed axons project into the dorsal white matter, and many turn to run longitudinally within the dorsal columns (small dots in F). No labeled axons are seen in the white matter after root crush (E). Scale bar in C applies to all panels: A, B, 500 μm; C, D, 200 μm; E, F, 100 μm; G, H, 50 μm.
Figure 3.
Figure 3.
Recovery of spinal projections of lesioned sensory axons in rats treated with sNgR. A, B, By 2 weeks, axons labeled with CTB by injection into brachial nerves project into the DREZ (A), and occasional axons are seen within the dorsal white matter (B). Individual axons are shown at higher magnification in inset panels. C, D, By 4 weeks, labeled axons have grown extensively into the dorsal white matter, although they project in a disorganized manner (arrowheads in D, which is a higher-magnification view of the white matter shown in C). Many project ventrally into the upper laminae of the dorsal horn (C, arrows). E, Six weeks after root crush, labeled axons project throughout the dorsal horn (arrows) but still have a disorganized growth pattern in the dorsal white matter (arrowheads). F, Uncrushed CTB-labeled axons in a treated rat maintain their normal projection pattern to the dorsal horn, including an absence of projections to laminae I and IIo (F, arrows). G, H, Regenerated axons supplying a small patch of skin in the arm project diffusely throughout the dorsal horn (G, arrows), unlike the topically restricted projection of uncrushed cutaneous sensory axons on the contralateral side of the same rat (arrow in H). Scale bars: (in H) A–C, E–H, 200 μm; D, 100 μm.
Figure 4.
Figure 4.
Distribution and density of CGRP expression in treated and untreated rats 4 weeks after unilateral root crush. Each pair of panels is from the two sides of the same transverse section. Pixel density of fluorescently labeled sections was thresholded as described in Materials and Methods and reversed so pixels exceeding the threshold are black. The images were superimposed on schematic drawings of the section. The dorsal horn is shown in green. There is little recovery of CGRP expression after root crush with or without sNgR treatment.
Figure 5.
Figure 5.
Functional recovery of synaptic activity evoked by peripheral nerve stimulation in treated spinal cords. A, Examples of extracellular field potentials recorded in the spinal cord after stimulation of the median and ulnar nerves. The first peak after the stimulus (arrowheads) represents synchronized action potentials in the DRG and dorsal roots. The second peak (arrows) represents postsynaptic responses in spinal neurons. B, Time course of recovery of synaptic function after root crush. Representative traces at 2, 4, and 6 weeks are shown in the top; red traces are evoked by sensory axons that were previously crushed, green traces from the contralateral, control side of the same rat. Bars indicate fractional recovery of synaptic potential amplitudes. Error bars indicate ±1 SEM. The numbers above each column indicate the number of rats with measurable synaptic responses versus the total number of rats tested at each time point.
Figure 6.
Figure 6.
Behavioral recovery of sNgR-treated and untreated rats after root crush. The number of rats tested at each time point is indicated above each bar. The legend applies to all panels. Asterisks indicate significant differences from treated rats at 1 week: *p < 0.01; **p < 0.001. Error bars indicate ±1 SEM. A, Paw preference test. B, Ability to grasp. C, Withdrawal of forelimb to tactile stimulation of paw. The dotted line in C represents the minimum sensitivity this test could measure (see Materials and Methods). Paw withdrawal was only measured at 4 weeks for untreated rats.
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