Bone marrow stromal cells enhance inter- and intracortical axonal connections after ischemic stroke in adult rats

Abstract

We investigated axonal plasticity in the bilateral motor cortices in rats after unilateral stroke and bone marrow stromal cell (BMSC) treatment. Rats were subjected to permanent right middle cerebral artery occlusion followed by intravenous administration of phosphate-buffered saline or BMSCs 1 day later. Adhesive-removal test and modified neurologic severity score were performed weekly to monitor limb functional deficit and recovery. Anterograde tracing with biotinylated dextran amine injected into the right motor cortex was used to assess axonal sprouting in the contralateral motor cortex and ipsilateral rostral forelimb area. Animals were killed 28 days after stroke. Progressive functional recovery was significantly enhanced by BMSCs. Compared with normal animals, axonal density in both contralateral motor cortex and ipsilateral rostral forelimb area significantly increased after stroke. Bone marrow stromal cells markedly enhanced such interhemispheric and intracortical connections. However, labeled transcallosal axons in the corpus callosum were not altered with either stroke or treatment. Both interhemispheric and intracortical axonal sprouting were significantly and highly correlated with behavioral outcome after stroke. This study suggests that, after stroke, cortical neurons surviving in the peri-infarct motor cortex undergo axonal sprouting to restore connections between different cerebral areas. Bone marrow stromal cells enhance axonal plasticity, which may underlie neurologic functional improvement.

Figure 1

Permanent MCAo induced ischemic lesion in the rat brain. (A) A representative rat coronal brain section shows the ischemic lesion 4 weeks after MCAo. An arrow indicates the location of BDA injection in the right cortex. Rectangle fields in the left cortex (1) and corpus callosum (2) indicate the position of the photomicrograph appearing in Figures 2 and 3, respectively. (B) Quantitative data show no statistical difference in the ischemic lesion size between animal groups treated with PBS or BMSCs. Scale bar=2 mm.

Figure 2

Transcallosal axons in the contralateral cortex labeled with BDA intracortical injection. (A) A representative image shows the BDA-positive labeling in a normal rat brain. The BDA solution was injected into the right cortex 7 days before the kill. The BDA-labeled axons in the boxed area in the contralateral CFA were measured as proportional areas, as shown in B for normal, in C for MCAo, and in D for BMSC-treated groups. The axonal density was significantly increased 28 days after MCAo compared with normal animals (C and E). Bone marrow stromal cell administration further enhanced such stroke-induced axonal reorganization (D and E). Scale bar=1 mm in A and 250 μm in B–D.

Figure 3

Axonal labeling in the corpus callosum. (A) A representative image shows the transcallosal axons in the middle portion of corpus callosum enlarged from the box 2 in Figure 1A. (B) There are no significant differences in axonal quantification among normal animals and ischemic rats with or without BMSC treatment. Scale bar=150 μm.

Figure 4

Coronal sections showing intracortical axonal connections in the right RFA originating from the ipsilateral CFA. In normal rats, few axons were detected in the cortex at the RFA level with BDA injection into the cortex of the CFA (A). Biotinylated dextran amine-positive axons were increased in the ischemic animals treated with PBS (B) and BMSCs (C). Quantitative data showed a significant enhancing effect on both MCAo lesion and BMSC treatment, respectively (D). Insets in A, schematic drawing of the rat brain dorsal view showing the RFA region from which slices were obtained (lines) and BDA-injection position in the CFA (dot). Cross-hatching indicates the ischemic lesion. Scale bar=250 μm.