Focal cerebral ischemia induces a multilineage cytogenic response from adult subventricular zone that is predominantly gliogenic

Abstract

The purpose of this study was to ascertain the relative contribution of neural stem/progenitor cells (NSPCs) of the subventricular zone (SVZ) to lineages that repopulate the injured striatum following focal ischemia. We utilized a tamoxifen-inducible Cre/loxP system under control of the nestin promoter, which provides permanent YFP labeling of multipotent nestin(+) SVZ-NSPCs prior to ischemic injury and continued YFP expression in all subsequent progeny following stroke. YFP reporter expression was induced in adult male nestin-CreER(T2):R26R-YFP mice by tamoxifen administration (180 mg kg(-1), daily for 5 days). Fourteen days later, mice were subjected to 60-min transient middle cerebral artery occlusion (MCAO) and sacrificed at 2 days, 2 weeks, or 6 weeks post-MCAO for phenotypic fate mapping of YFP(+) cells using lineage-specific markers. Migration of YFP(+) cells from SVZ into the injured striatal parenchyma was apparent at 2 and 6 weeks, but not 2 days, post-MCAO. At 2 weeks post-MCAO, the average percent distribution of YFP(+) cells within the injured striatal parenchyma was as follows: 10% Dcx(+) neuroblasts, 15-20% oligodendrocyte progenitors, 59% GFAP(+) astrocytes, and only rare NeuN(+) postmitotic neurons. A similar phenotypic distribution was observed at 6 weeks, except for an increased average percentage of YFP(+) cells that expressed Dcx(+) (20%) or NeuN (5%). YFP(+) cells did not express endothelial markers, but displayed unique anatomical relationships with striatal vasculature. These results indicate that nestin(+) NSPCs within the SVZ mount a multilineage response to stroke that includes a gliogenic component more predominant than previously appreciated.

Figure 1

A) Experimental design. B) Fluoro-Jade at day 2 post-MCAO. C) YFP immunofluorescence 2 days post-MCAO. D) YFP immunofluorescence at 2 weeks post-MCAO. LV (lateral ventricle), ST (Striatum). Scale Bars = 100 µm.

Figure 2

A) Dcx (red) and YFP (green) immunofluorescence in vehicle-injected control Nestin-CreER^T2:R26R-YFP mice. B) Nestin (red), GFAP (blue) and YFP (green) immunofluorescence in tamoxifen-treated Nestin-CreER^T2:R26R-YFP mice at d14 post-MCAO. LV (lateral ventricle). Scale bar = 100 µm.

Figure 3

A) Doublecortin (red) and YFP (green) immunofluorescence at 2 weeks post-MCAO. B) Higher power images with orthogonal view of confocal z-stack. DCX⁺/YFP⁺ cells (arrows) and DCX⁻/YFP⁺ cells (arrowheads). C, E) Percentage of YFP reporter⁺ cells that co-express Dcx (C) or NeuN (E). D) NeuN+ (red) and YFP (green) immunofluorescence at 6 weeks post-MCAO. ND = <1 cell per section. *p<0.05. Scale bars = 100 µm (A) or 10 µm (B,D).

Figure 4

Dual immunofluorescence for YFP (green) with A) NG2 (red) B) Olig2 (red) and C) Iba-1 (red) at 2 weeks post-MCAO. D) Percentage of YFP⁺ cells that express Olig2 and NG2 at 2 and 6 weeks post-MCAO. Scale bars = 20 µm (A,C) or 10 µm (B).

Figure 5

Dual immunofluorescence for YFP (green) with GFAP (red or blue) at 2 weeks post-MCAO. Graph depicts the percentage of YFP⁺ cells that express GFAP at 2 and 6 weeks post-MCAO. Scale bars = 20 µm.

Figure 6

A) Boxed images on histological section are shown in higher power in B–D. Solid area represents region of ischemic damage. B) Dual immunofluorescence for YFP (green) and GLUT-1 (red) demonstrating YFP⁺ processes from radial glial like cells in the SVZ with endothelial end-feet. C) YFP reporter⁺ cells (green) that have migrated into the ischemic border zone and are associated with laminin⁺ (red) cerebral blood vessels. D) YFP reporter⁺ cells (green) within the ischemic striatum associated with processes of laminin+ (red) angiogenic blood vessels. Scale bars = 10 µm (B–D).