Nogo receptor antagonism promotes stroke recovery by enhancing axonal plasticity

Abstract

After ischemic stroke, partial recovery of function frequently occurs and may depend on the plasticity of axonal connections. Here, we examine whether blockade of the Nogo-NogoReceptor (NgR) pathway might enhance axonal sprouting and thereby recovery after focal brain infarction. Mutant mice lacking NgR or Nogo-AB recover complex motor function after stroke more completely than do control animals. After a stroke, greater numbers of axons emanating from the undamaged cortex cross the midline to innervate the contralateral red nucleus and the ipsilateral cervical spinal cord; this axonal plasticity is enhanced in ngr -/- or nogo-ab -/- mice. In rats with middle cerebral artery occlusion, both the recovery of motor skills and corticofugal axonal plasticity are promoted by intracerebroventricular administration of a function-blocking NgR fragment. Behavioral improvement occurs when therapy is initiated 1 week after arterial occlusion. Thus, delayed pharmacological blockade of the NgR promotes subacute stroke recovery by facilitating axonal plasticity.

Figure 1.

Behavioral recovery after stroke in ngr -/- mice. A, B, Cresyl violet-stained coronal brain sections 28 d after photothrombosis. There is no difference in total infarct volume between ngr +/- and ngr -/- mice. C, Staircase test of skilled forelimb reaching ability. The photothrombotic lesion reduces the number of pellets retrieved with the contralateral forepaw. Significantly greater recovery during the postoperative period is observed in the ngr -/- mice. ^**p < 0.01 (two-way parametric ANOVA). D, Rotarod performance at baseline is presented as the time spent on an accelerating rotarod at final pre-stroke training session. ^*Significantly different from ngr +/-; p < 0.05 (Student's t test). E, Rotoarod performance is expressed as a ratio to baseline (pre-lesion) performance at various times after stroke in mice of the indicated genotypes. Performance was significantly better in ngr -/- mice after stroke. ^*p < 0.05 (two-way parametric ANOVA). Data are mean ± SEM from different animals. n = 10 mice for ngr +/- and n = 11 mice for ngr -/- in B-E.

Figure 2.

Corticofugal axonal plasticity after stroke in ngr -/- mice. A, B, BDA-labeled axons traced from the uninjured cerebral cortex are illustrated in coronal sections at the level of the red nucleus (A) and transverse sections of cervical spinal cord (B) from different mice. Both ngr +/- and ngr -/- mice without stroke possess very few BDA-labeled fibers in the red nucleus contralateral to the tracer injection (arrow) or in the spinal gray matter ipsilateral to the injection (arrows). After stroke, the number of such fibers increases to a greater extent in the ngr -/- mice. Dorsal is up and the lesioned side is to the right in this and all micrographs. C, The total BDA-positive axon length per area of a coronal section centered on the red nucleus is plotted as a function of the side, genotype, and stroke. D, The total number of BDA-positive axons in the gray matter ipsilateral to the injection site per transverse section of the cervical spinal cord is plotted for the indicated conditions. Data are mean ± SEM from different animals. n = 10 mice for ngr +/- and n = 11 mice for ngr -/- in C, D. ^*p < 0.05; ^**p < 0.01 (Student's t test).

Figure 3.

Recovery from cortical photothrombosis in mice lacking Nogo-AB. A, B, Cresyl violet-stained coronal brain sections 28 d after photothrombosis. Total infarct volume in nogo-ab -/- mice is smaller than nogo-ab +/- mice, although the difference is not significant at the p < 0.05 level. C, D, Staircase test and rotarod test performance after stroke. Performance across the postoperative period is significantly improved in nogo-ab -/- mice relative to the nogo-ab +/- mice. ^***p < 0.001 (two-way parametric ANOVA). Data are mean ± SEM from different animals. n = 10 mice for nogo-ab +/- and n = 9 nogo-ab -/- mice in B-D.

Figure 4.

Corticofugal axonal plasticity after stroke in mice lacking Nogo-AB. A, B, BDA-labeled axons traced from the uninjured cerebral cortex are illustrated at the level of the red nucleus (A) and cervical spinal cord (B) from different mice. In non-stroke mice there are few BDA-labeled fibers in the red nucleus contralateral to the tracer injection (arrow) or in the spinal gray matter ipsilateral to the injection (arrows), regardless of genotype. After stroke, the number of fibers sprouting into these areas increases to a higher level in the nogo-ab -/- mice. C, The total BDA-positive axon length per area of a coronal section centered on the red nucleus is plotted as a function of the side, genotype, and stroke. D, The total number of BDA-positive axons in the gray matter ipsilateral to the injection site per transverse section of the cervical spinal cord is plotted for the indicated conditions. Data are mean ± SEM from different animals. n = 10 mice for nogo-ab +/- and n = 9 nogo-ab -/- mice in C, D. ^*p < 0.05; ^**p < 0.01 (Student's t test).

Figure 5.

NgR(310)Ecto-Fc treatment of rats with MCAO stroke. A, Cresyl violet-stained sections of rat brain 77 d after MCAO and after intracerebroventricular treatment from day 7 to 63 with either IgG or NgR(310)Ecto-Fc. B, Stroke volume. There is no significant effect of NgR(310)Ecto-Fc on infarct volume. C, Staircase test. The number of pellets retrieved with the contralateral forepaw is significantly increased in the NgR(310)Ecto-Fc group compared with the control group. ^***p < 0.001 (two-way parametric ANOVA). D, Rotarod test. Postoperative performance ratio to baseline was significantly better in rats treated with NgR(310)Ecto-Fc. ^**p < 0.01 (two-way parametric ANOVA). Data are mean ± SEM from different animals. n = 16 rats for IgG and n = 17 rats for NgR(310)Ecto-Fc in B-D.

Figure 6.

Corticofugal axonal plasticity after MCAO in rats treated with NgR(310)Ecto-Fc. A, B, BDA-labeled axons traced from the uninjured cerebral cortex are illustrated at the level of the red nucleus (A) and cervical spinal cord (B) from different rats. Control rats with no stroke and no treatment exhibit very few BDA-labeled fibers in the red nucleus contralateral to the tracer injection (arrow) or in the spinal gray matter ipsilateral to the injection (arrows). After stroke, many BDA-labeled fibers sprouting into the territory normally innervated by the infracted cortex are present. This is more prominent in rats treated with NgR(310)Ecto-Fc than in control IgG rats. C, The total BDA-positive axon length per area of a coronal section centered on the red nucleus is plotted as a function of the side, protein infusion, and stroke. D, The total number of BDA-positive axons in the gray matter ipsilateral to the injection site per transverse section of the cervical spinal cord is plotted for the indicated conditions. Data are mean ± SEM from different animals. n = 16 rats for IgG and n = 17 rats for NgR(310)Ecto-Fc in C, D. ^*p < 0.05; ^**p < 0.01 (Student's t test).