Erythropoietin increases neurogenesis and oligodendrogliosis of subventricular zone precursor cells after neonatal stroke

Abstract

Background and purpose: Stroke is a common cause of neonatal brain injury. The subventricular zone is a lifelong source of newly generated cells in rodents, and erythropoietin (EPO) treatment has shown benefit in different animal models of brain injury. The purpose of this study is to investigate the specific role of exogenous EPO on subventricular zone progenitor cell populations in response to neonatal stroke.

Methods: Intraventricular injections of green fluorescent protein (GFP)-expressing lentivirus to label subventricular zone precursor cells were made in postnatal day 1 (P1) Long-Evans rats, which then underwent transient middle cerebral artery occlusion on P7. Middle cerebral artery occlusion and sham rats were treated with either vehicle or EPO (1000 U/kg) at reperfusion, 24 hours, and 7 days later. The density of double-labeled DCx+/GFP+, NeuN+/GFP+, O4+/GFP+, GFAP+/GFP+, as well as single-labeled GFP+ and Ki67+ cells, was calculated to determine cell fate outcome in the striatum at 72 hours and 2 weeks after stroke.

Results: There was a significant increase in DCx+/GFP+ and NeuN+/GFP+ neurons and O4+/GFP+ oligodendrocyte precursors, with decreased GFAP+/GFP+ astrocytes at both time points in EPO-middle cerebral artery occlusion animals. There was also a significant increase in GFP+ cells and Ki67+ proliferating cells in EPO compared with vehicle-middle cerebral artery occlusion animals.

Conclusions: These data suggest that subventricular zone neural progenitor cells proliferate and migrate to the site of injury after neonatal stroke and multiple doses of EPO, with a shift in cell fate toward neurogenesis and oligodendrogliosis at both early and late time points. The contribution of local cell proliferation and neurogenesis remains to be determined.

Figure 1

Experimental protocol (A). (B) Anterior to posterior coronal image slices of DW-MRI during occlusion demonstrate lesion (arrows) involving ipsilateral striatum and parieto-temporal cortex.

Figure 2

Intraventricular injection of GFP-lentivirus labels SVZ NSCs. Coronal sections of P21 rat forebrain (2 weeks after MCAO) in EPO-MCAO (A) and vehicle-MCAO (B) animal. LV: lateral ventricle, STR: striatum, CTX: cortex (tiled, 10x images). GFP+ cell density in striatum was increased in EPO-MCAO versus vehicle-MCAO animals at P10 (*p<0.05) and P21 (*p<0.05,**p<0.04) (C). Striatal density of Ki67+ cells also increased in EPO-MCAO versus vehicle-MCAO animals (*p<0.05**p<0.04). VS=vehicle-sham, ES=EPO-sham, VO=vehicle-MCAO, EO=EPO-MCAO. Scale bars: 250-μm.

Figure 3

Neuronal cell fate of GFP-labeled NSCs and progeny. Analysis of ipsilateral striatum in EPO-MCAO animal demonstrates co-expression of DCx (A, red, 20x) and NeuN (B, red, 20x) at P21. DAPI (blue) is used as a nuclear counterstain, double-labeled cells marked with arrows. There was an increased density of DCx+/GFP+ co-labeled cells (*p<0.02,**p<0.05,***p<0.01 at P10; *p<0.04,**p<0.05 at P21), and NeuN+/GFP+ cells (*p<0.05) in EPO-MCAO versus vehicle-MCAO animals. Y-axis shows proportion of GFP+ cells that co-labeled with specified marker. Scale bars: 100-μm.

Figure 4

Glial cell fate of GFP-labeled cells. Analysis of ipsilateral striatum at P21 in EPO-MCAO animal demonstrates co-expression of O4 (A, red, 20x) and GFAP (B, red, 20x). Increased proportion of O4+/GFP+ co-labeled cells at both time points in EPO-treated animals (*p<0.05). Following MCAO, there were more GFAP+/GFP+ cells, which was reduced in both EPO-MCAO and EPO-sham animals (*p<0.02,**p<0.0005,***p<0.0001 at P10; *p<0.05,**p<0.005,***p<0.0005 at P21). Scale bars: 100-μm.