The Nogo-Nogo receptor pathway limits a spectrum of adult CNS axonal growth

Abstract

The hypothesis that Nogo-A (Reticulon 4A) and Nogo-66 receptor (NgR1) limit adult CNS axonal growth after injury is supported by both in vitro experiments and in vivo pharmacological studies. However, genetic assessment of the role of Nogo-A in corticospinal tract (CST) axons after spinal cord dorsal hemisection has yielded conflicting results. CST regeneration is detected in homozygous nogo-ab(trap/trap) mice, but not in nogo-ab(atg/atg) mice. CST regeneration is also present after pharmacological NgR blockade, but not in ngr1(-/-) mice. To assess the nogo-ab(atg) and ngr1-null alleles for other axon growth phenotypes, we created unilateral pyramidotomies and monitored the uninjured CST. There is robust pyramidotomy-induced growth of nogo-ab(atg/atg) and ngr1(-/-) CST axons into denervated cervical gray matter. This fiber growth correlates with recovery of fine motor skill in the affected forelimb. Thus nogo-ab and ngr1 play a modulated role in limiting CNS axonal growth across a spectrum of different tracts in various lesion models.

Figure 1.

PyX ablates corticospinal input along one side of the spinal cord. Schematic of brain and spinal cord illustrating mature termination pattern of the adult CST (A) and location of lesion and predicted sprouting response from intact CST (B) into deafferented side (stippled line). Photomicrographs C, E, and G show brainstem, cervical, and lumbar spinal cord, respectively, from intact wild-type animals and D, F, and H from pyramidotomized wild-type animals. Anti-GFAP-reactive astrocytes demarcate the lesion (D), but no astrocytic response is evident in sham-lesioned animals (C). Scale bar, 200 μm. Sections from C7 spinal cord of naive (E) and PyX wild-type (F) animals illustrate an identical termination pattern of BDA⁺ CST axons (red) and myelin compaction (green). Scale bar, 200 μm. Inspection of L4 spinal cord shows bilateral PKCγ immunoreactivity in the ventrodorsal columns of intact mice (G) and unilateral PKCγ immunoreactivity in PyX mice (H). Scale bar, 200 μm.

Figure 2.

PyX-induced sprouting of intact CST fibers in ngr1 ^−/− mice. Photomicrographs A–C illustrate C7 transverse sections of spinal cord from ngr1 ^−/− sham, ngr1 ^+/+ PyX, and ngr1 ^−/− PyX-lesioned animals. Fasciculated BDA⁺ axons can be seen in the left ventrodorsal column (A) from which CST collaterals project unilaterally into both dorsal and ventral horns in sham-lesioned ngr1 ^−/− (A) and PyX ngr1 ^+/+ mice (B). PyX ngr1 ^−/− mice illustrate robust sprouting of intact BDA⁺ CST axons into the deafferented side of the spinal cord (C). Scale bar, 100 μm. The optical density of BDA⁺ axons from the pial surface into gray matter was assessed in zones I–VI (A). Quantification of zones IV–VI is shown in D–F. The integrated CST density (area under the curve) of ngr1 ^−/− animals is significantly greater in zones IV and V than in sham-lesioned or PyX ngr1 ^+/+ mice (*p < 0.05, ANOVA). G, H, As another measure of CST innervation, fiber length per cross-sectional area was measured throughout the spinal cord gray matter. Fiber length was not significantly different on the intact side between ngr1 ^+/+ and ngr1 ^−/− mice after sham or PyX lesion (G); however, CST fiber length was significantly greater on the injured side of ngr1 ^−/− mice (H) compared with ngr1 ^+/+ PyX, ngr1 ^−/− sham, and ngr1 ^+/+ sham-lesioned mice (*p < 0.001, ANOVA). For G and H, data are the mean ± SEM for n = 7–10 mice.

Figure 3.

CST fibers crossing the midline of the spinal cord. A–C, Photomicrographs illustrate significant numbers of BDA-immunoreactive CST axons sprouting across the midline in ngr1 ^−/− mice after PyX compared with ngr1 ^−/− sham and ngr1 ^+/+ PyX-lesioned mice. Scale bar, 100 μm. D, The average number of CST fibers crossing the midline of the cervical spinal per transverse section is significantly greater in ngr1 ^−/− mice compared with ngr1 ^+/+ PyX, ngr1 ^−/− sham, and ngr1 ^+/+ sham-lesioned mice (*p < 0.001, ANOVA). Data are the mean ± SEM for n = 7–10 mice.

Figure 4.

PyX-induced sprouting of intact CST fibers in nogo-ab^atg/atg mice. Photomicrographs A–C illustrate C7 transverse section of spinal cord from nogo-ab^atg/atg sham, nogo-ab ^+/+ PyX, and nogo-ab^atg/atg PyX-lesioned animals, respectively. Fasciculated BDA⁺ axons can be seen in the left ventrodorsal column (A) from which CST collaterals project unilaterally into both dorsal and ventral horns in sham-lesioned nogo-ab^atg/atg (A) and PyX nogo-ab ^+/+ mice (B). PyX nogo-ab^atg/atg mice illustrated increased sprouting of intact BDA⁺ CST axons into the deafferented side of the spinal cord (C). Scale bar, 200 μm. The optical density of BDA reactivity from the pial surface into gray matter was assessed in zones I–VI (A). Quantification of zones IV–VI is shown in D–F. Assessment of the integrated axonal density (area under the curve) revealed that nogo-ab^atg/atg animals have significantly more BDA reactivity in zones IV and V in comparison to sham-lesioned and PyX nogo-ab ^+/+ mice (*p < 0.05, ANOVA). G, H, The absolute length of CST axon per cross-sectional area was measured throughout the spinal cord gray matter for each condition. Fiber length was not significantly different on the intact side between nogo-ab ^+/+ sham and nogo-ab ^+/+ PyX-lesioned mice (G). However, PyX-lesioned ngr1 ^−/− mice had significantly greater CST length compared with nogo-ab^atg/atg sham-lesioned mice (*p < 0.05, ANOVA). CST fiber length was also significantly greater on the injured side of nogo-ab^atg/atg mice (H) compared with nogo-ab ^+/+ PyX, nogo-ab^atg/atg sham, and nogo-ab ^+/+ sham-lesioned mice (*p < 0.001, ANOVA). For G, H, data are the mean ± SEM for n = 7–10 mice.

Figure 5.

Increased numbers of nogo-ab^atg/atg CST fibers crossing the midline of the spinal cord after PyX. A–C, Photomicrographs illustrate significant numbers of BDA-immunoreactive CST axons sprouting across the midline in nogo-ab^atg/atg mice after PyX compared with nogo-ab^atg/atg sham and nogo-ab ^+/+ PyX-lesioned mice. Scale bar, 100 μm. CC, Central canal. D, The average number of CST fibers crossing the midline of the cervical spinal per transverse section was significantly greater in nogo-ab^atg/atg mice compared with nogo-ab ^+/+ PyX, nogo-ab^atg/atg sham, and nogo-ab ^+/+ sham-lesioned mice (*p < 0.001, ANOVA). Data are the mean ± SEM for n = 7–10 mice.

Figure 6.

Pyridotomized ngr1 ^−/− and nogo-ab^atg/atg mice recover fine forelimb function. A, ngr1 ^+/+ and ngr1 ^−/− mice were trained to retrieve food pellets through an aperture displaced to one side of a transparent plastic box. Ipsilateral paw usage was identical between genotypes during presurgical training. At 4 weeks after PyX, the ngr1 ^+/+ illustrated a significant inability to retrieve food with their ipsilateral paws (*p < 0.001, ANOVA). ngr1 ^−/− mice recovered the ability to use their ipsilateral paws and were not significantly different from sham-lesioned controls. B, Ipsilateral paw usage was identical between nogo-ab ^+/+ and nogo-ab^atg/atg mice during presurgical training. At 28 d after PyX, the nogo-ab ^+/+ showed a significant inability to retrieve food with the ipsilateral forepaw (*p < 0.001, ANOVA). In contrast, nogo-ab^atg/atg mice fully recovered the ability to use their ipsilateral paws and were not significantly different from sham-lesioned controls.

Figure 7.

PyX fails to influence wiring of primary afferent terminals or descending raphe axons. Photomicrographs show transverse sections of C7 spinal cord (dorsal horn) immunostained for CGRP from ngr1 ^+/+ (A) and ngr1 ^−/− (B) mice after PyX (left side intact). Scale bar, 200 μm. Assessment of CGRP axon density illustrates normal termination pattern (lamina II) in ngr1 ^+/+ (C) and ngr1 ^−/− (D) mice. PyX does not result in primary afferent reorganization in ngr1 ^+/+ or ngr1 ^−/− mice. Also shown are photomicrographs of C7 spinal cord (ventral horn) immunostained for 5-HT from ngr1 ^+/+ (E) and ngr1 ^−/− (F) mice after PyX (left side intact). No significant difference is found in 5-HT-immunoreactive spatial axon density in dorsal or ventral horn of ngr1 ^+/+ or ngr1 ^−/− mice after PyX (G). Photomicrographs show transverse sections of C7 spinal cord (dorsal horn) immunostained for CGRP from nogo-ab ^+/+ (H) and nogo-ab^atg/atg (I) mice after PyX (left side intact). Assessment of CGRP axon density illustrates normal termination pattern (lamina II) in nogo-ab ^+/+ (J) and nogo-ab^atg/atg (K) mice. PyX does not result in primary afferent reorganization in nogo-ab ^+/+ or nogo-ab^atg/atg. Also shown are photomicrographs of C7 spinal cord (ventral horn) immunostained for 5-HT from nogo-ab ^+/+ (L) and nogo-ab^atg/atg (M) mice after PyX (left side intact). No significant difference is found in 5-HT-immunoreactive axon density in dorsal or ventral horn of nogo-ab ^+/+ or nogo-ab^atg/atg mice after PyX (N). Scale bar, 200 μm.