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. 2008 Dec;56(16):1747-54.
doi: 10.1002/glia.20722.

Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brain

Li Hong Shen  1 , Yi Li, Qi Gao, Smita Savant-Bhonsale, Michael Chopp
Affiliations

Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brain

Li Hong Shen et al. Glia. 2008 Dec.

Abstract

The glial scar, a primarily astrocytic structure bordering the infarct tissue inhibits axonal regeneration after stroke. Neurocan, an axonal extension inhibitory molecule, is up-regulated in the scar region after stroke. Bone marrow stromal cells (BMSCs) reduce the thickness of glial scar wall and facilitate axonal remodeling in the ischemic boundary zone. To further clarify the role of BMSCs in axonal regeneration and its underlying mechanism, the current study focused on the effect of BMSCs on neurocan expression in the ischemic brain. Thirty-one adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by an injection of 3 x 10(6) rat BMSCs (n = 16) or phosphate-buffered saline (n = 15) into the tail vein 24 h later. Animals were sacrificed at 8 days after stroke. Immunostaining analysis showed that reactive astrocytes were the primary source of neurocan, and BMSC-treated animals had significantly lower neurocan and higher growth associated protein 43 expression in the penumbral region compared with control rats, which was confirmed by Western blot analysis of the brain tissue. To further investigate the effects of BMSCs on astrocyte neurocan expression, single reactive astrocytes were collected from the ischemic boundary zone using laser capture microdissection. Neurocan gene expression was significantly down-regulated in rats receiving BMSC transplantation (n = 4/group). Primary cultured astrocytes showed similar alterations; BMSC coculture during reoxygenation abolished the up-regulation of neurocan gene in astrocytes undergoing oxygen-glucose deprivation (n = 3/group). Our data suggest that BMSCs promote axonal regeneration by reducing neurocan expression in peri-infarct astrocytes.

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Figures

Fig 1
Fig 1
A-D: BMSC administration significantly increases GAP43 expression in the ischemic boundary zone. *P<0.05 vs. MCAo, #P<0.01 vs. normal control. Scale bars: A, B = 25 μm.
Fig 2
Fig 2
A-D: Neurocan protein is barely detectable in the contralateral hemisphere (A), is increased in the peri-infarct region after MCAo (B), and is significantly reduced after BMSC treatment (C); D: Quantitative analysis of immunostaining and Western blot assay show the similar changes in neurocan expression in the IBZ, neurocan is upregulated after ischemic attack, and BMSC treatment significantly decreases neurocan expression; E-H: Double immunostaining shows that neurocan is concentrated around reactive GFAP positive astrocytes, and confocal analysis reveals the colocalization of the two proteins. *P<0.05 vs. MCAo, #P<0.01 vs. normal control, A-C = 50 μm, E-G = 25 μm, H = 20 μm.
Fig 3
Fig 3
A: Hematoxylin and eosin-stained coronal section illustrates the ischemic boundary regions from which reactive astrocytes are cut by LCM (area 1-8); B,C: GFAP immunofluorescent staining before and after cell capture; D: Neurocan gene expression in reactive astrocytes significantly decreases in BMSC treated animals, compared to that of the MCAo alone rats. E: 5hrs of OGD up-regulates neurocan gene expression in primary astrocytes, and coculture with BMSC during reoxygenation markedly suppresses neurocan gene expression; *P<0.05, **P<0.01 vs. ischemia alone; ##P < 0.01 vs. normoxia. Scale bar: B, C = 25 μm.
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