Nogo-A expression after focal ischemic stroke in the adult rat

Abstract

Background and purpose: The Nogo-A protein is an important inhibitor of axonal remodeling after central nervous system injuries, including ischemic stroke. Interfering with the function of Nogo-A via infusion of a therapeutic anti-Nogo-A antibody after stroke increases neuronal remodeling and enhances functional recovery in rats. In this study, we describe the regional distribution of cortical neurons expressing Nogo-A in normal rats and following middle cerebral artery occlusion (MCAO).

Methods: Normal and post-MCAO neuronal Nogo-A expression were described via immunohistochemical analyses. All brains were processed for Nogo-A and parvalbumin expression. The level of Nogo-A expression was scored for each cortical area or white matter structure of interest. The number and fluorescent intensity of layer V neurons in contralesional sensorimotor forelimb cortex were also assessed at each time point.

Results: Nogo-A expression was observed in both cortical pyramidal neurons and parvalbumin-positive interneurons. Neuronal expression of Nogo-A changed over time in ipsilesional and contralesional cortical areas after MCAO, becoming globally elevated at 28 days after stroke. Nogo-A expression was not observed to fluctuate greatly in the white matter after stroke, with the exception of a transient increase in Nogo-A expression in the external capsule near the stroke lesion.

Conclusions: Neuronal Nogo-A expression is significantly increased at 28 days post-MCAO in all examined brain regions. Because of their robust expression of Nogo-A after stroke lesion, both excitatory and inhibitory neurons represent potential targets for anti-Nogo-A therapies in the poststroke cerebral cortex.

Figure 1

Scope of lesion and brain regions assessed. The typical cortical lesion observed at 28 days poststroke is depicted (gray shading). The cortical areas assessed for Nogo-A and PV staining included cingulate cortex (Cg), medial agranular cortex (AGm), lateral agranular cortex (AGl), retrosplenial cortices (RSA/RSG), sensorimotor forelimb cortex (FL), sensorimotor hindlimb cortex (HL), primary and secondary somatosensory cortices (S1 and S2), Insular cortex (Insular), and Piriform cortex (Piri). We also examined white matter structures including the corpus callosum (cc), cingulum bundle (cingulum), the external capsule (ec), and the internal capsule (ic). Figure adapted with permission from Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 5th Ed., New York: Academic Press; 2005.

Figure 2

Neuronal Nogo-A expression. Many neurons in the cerebral cortex express Nogo-A. Immunohistochemical staining for Neurofilament-200 (A, scale bar 20 μm) and Nogo-A (B) confirm the identity of Nogo-A positive cells as neurons via their colocalization (C). Parvalbumin-positive (PV⁺) interneurons (D, scale bar 20 μm) in the cerebral cortex also express Nogo-A (E). Colocalization of Nogo-A and PV occurs in many PV⁺ cells, extending into the axons (F). PV⁺ neurons were located in cortical layers II–VI (G, red, scale bar 600 μm), and neurons expressing Nogo-A were most dense in cortical layer V (area between lines; H). PV⁺/Nogo-A⁺ neurons (seen in higher magnification in F) were visible in layers II–VI in normal adult rats, but were also most prominent in layer V (I).

Figure 3

Neuronal Nogo-A and PV expression are diminished in perilesional cortex at early time points. At day 7 after stroke, expression of Nogo-A and PV were reduced in the perilesional cortex (P), located immediately adjacent to the stroke lesion (A, green is Nogo-A, red is PV, scale bar 600 μm; higher magnification inset scale bar 50 μm; dashed line shows edge of perilesional cortex). At day 14, Nogo-A and PV expression returned to the spared perilesional cortical tissue (B; higher magnification inset).

Figure 4

Neuronal Nogo-A expression in contralesional sensorimotor forelimb cortex. A significant increase in the number of anti-Nogo-A immunolabeled layer V neurons was detected in contralesional sensorimotor forelimb cortex at 28 days after stroke compared to earlier time points (A; *P<0.05; **P<0.001). The elevated number of Nogo-A immunopositive neurons at 28 days after stroke corresponded with a significant increase in the number of neurons with a midlevel fluorescence intensity over time and a reduction in the number of cells in the high and low immunofluorescence groups (B).