Altered fate of subventricular zone progenitor cells and reduced neurogenesis following neonatal stroke

Abstract

Objective: To investigate the effects of neonatal stroke on progenitor cells lining the lateral ventricles.

Methods: Intraventricular injection of replication-incompetent green fluorescent protein (GFP)-expressing lentivirus was performed in postnatal day 1 (P1) rats to specifically label radial glia/type B neural stem cells and ependymal cells of the lateral ventricle. A subset of animals was exposed to transient middle cerebral artery occlusion (MCAO) at P7, with mild or moderate injury confirmed by diffusion-weighted MRI and histology. Newborn cells were identified by GFP expression, location and expression of cell type-specific markers in the striatum, cortex and olfactory bulb using confocal microscopy and systematic random sampling.

Results: Three weeks lentiviral GFP transduction of cells in the lateral ventricle, abundant GFP-expressing neurons and glia were identified in the rostral migratory stream, olfactory bulb and striatum as expected from labeling the subventricular zone (SVZ) type B neural stem cell lineage. Two weeks following mild or severe focal stroke at P7, no GFP-expressing neurons were detected in striatum or cortex although some single-labeled doublecortin+ immature neurons were detected in the penumbra. The densities of GFP+/ glial fibrillary acidic protein (GFAP)+ astrocytes and GFP+/O4+ oligodendrocytes were reduced in the striatum following MCAO (4.8 +/- 1.02 vs. 2.5 +/- 0.4 cells/high-power field, HPF; p = 0.005; 2.8+/- 1 vs. 0.5 +/- 0.2 cells/HPF, p = 0.008). Furthermore, there was a reduction of GFP+ cells in the olfactory bulb following MCAO (58.8 +/- 14.9 vs. 19.6 +/- 5.4 cells/HPF, p = 0.025). Finally, there was an increased percentage of GFP+/GFAP+ cells (70 vs. 50%), with a decreased proportion of GFP+/O4+ cells (14 vs. 30%) in injured animals.

Conclusion: Neurogenesis originating from cells of the lateral ventricle, including SVZ type B cells, is significantly reduced following neonatal stroke. Furthermore, neonatal stroke disrupts gliogenesis in the striatum, decreasing overall numbers of new glia and shifting the population towards astrocytes.

Fig. 1

Intraventricular injection of GFP-lentivirus labels SVZ NSCs. Coronal section of a P3 rat 48 h after GFP-lentiviral intraventricular injection. GFP expression (green) is primarily limited to the SVZ in the ipsilateral hemisphere (A, 4× magnification), with very few cells present in the contralateral hemisphere (A, inset, 4X). Vimentin (Vim) immunohistochemistry (red) shows intense double labeling in the SVZ (B, 10 ×). At higher magnification (C-E, 60×), an example of a double-labeled GFP+ (C)/vimentin+ (D) cell with merge image (E) is seen with typical morphological features of radial glia cells (arrow), with the cell body in the SVZ and a long process moving towards the lateral ventricle. Scale bars: A 250 µm; B 100 µm; C 10 µm. In all figures, colors refer to the online version only.

Fig. 2

Progeny of GFP-lentiviral-labeled NSCs. Analysis of a P21 rat forebrain 3 weeks after intraventricular injection. Sagittal section demonstrates a tangential migration pattern of GFP+ cells (green) labeled in the SVZ at P1 (A, 4× magnification). These cells coexpress DCx (red), a marker of immature neurons (B, 20×), which join the RMS. In the olfactory bulb (OB) cortex, GFP+ cells (C, 60×) with mature neuronal morphology express NeuN (D, red). GFP is stably expressed in the ipsilateral SVZ (E, 10×), with a coronal section showing the fate of cells radially migrating from the SVZ towards the overlying structures. Colabeled GFAP+ astrocytes (red) are seen at the SVZ/striatal boundary (F, 40×), and in the striatum (G). DAPI (blue) is used as a nuclear counterstain. Double labeling of O4+ (H) and NG2+ (I) in the striatum is also seen. Scale bars: A 250 µm; B 50 µm; E 250 µm; F, G 25 µm; H, I 10 µm. HI = Hippocampus; VL = lateral ventricle.

Fig. 3

Histopathology of MCAO leading to mild or severe injury. Anterior to posterior coronal image slices of DW-MRI performed during occlusion demonstrates an ipsilateral subcortical lesion (arrows) in mild injury (A) whereas more diffuse cortical injury is seen in severe injury (D). In coronal sections of P21 rat brain, the ipsilateral hemisphere shows significant reduction (arrow) of neurons (NeuN, red) in the striatum 2 weeks after MCAO that resulted in mild injury (B). GFP+ cells in ipsilateral SVZ and medial striatum in mild injury is also shown (B, inset, 20×). GFAP+ astrocytic expression (green) is localized to the striatum (large arrow) in mild injury, with minimal staining noted in cortical region (small arrow) distal to white matter (asterisks) (C, 10×). In severe injury, more diffuse loss of NeuN+ neurons in ipsilateral striatum (large arrow) and cortex (small arrows) is noted (E, 4×), with fewer GFP+ cells (E, inset, 20×). There is also an increase in GFAP+ staining in cortex (small arrows), indicative of more severe injury (F, 4×). Asterisks indicate region of cortical white matter. Scale bars: 250 µm. WM = White matter; LV = lateral ventricle.

Fig. 4

MCAO alters neurogenesis of GFP-lentiviral-labeled NSCs. Increased expression of DCx+ cells (A, red) in the ipsilateral SVZ (large arrow), white matter (small arrows) and striatum relative to contralateral side at P21 (A, inset, 10×; large arrow = SVZ) and sham-treated animals (not shown). These DCx+ cells did not coexpress GFP (green). At P8 (24 h after MCAO), increased expression of cleaved caspase-3 (red) is seen in the ipsilateral SVZ, some of which coexpress GFP (black arrows) (B, 20×). At P21, GFP expression is shown in ipsilateral SVZ of a control animal (C, 40×), with decreased GFP expression in MCAO animals (D). Detection of BrdU+ cells (red) in SVZ of control (E) and MCAO (F) brain shows marked reduction after injury. GFP+ cells are shown in the olfactory bulb in a control (G) and MCAO animal (H) at P21. Quantification of GFP+ cells in the olfactory bulb (OB) shows a significant (p = 0.025) reduction in MCAO versus control animals (I). Scale bars: C 100 µm; E, G 250 µm.

Fig. 5

MCAO effects on postnatal SVZ-derived glial cells in the striatum. MCAO induces marked astrogliosis (GFAP, red) in ipsilateral striatum (A, 10×). Higher magnification of colabeled cells is also shown (B, 20×). Quantification of GFP+ cells in the striatum showed a significant (p = 0.001) decrease in cells in MCAO brains compared to controls (C). The densities of GFP+/GFAP+ astrocytes and GFP+/O4+ oligodendrocytes were both significantly reduced in injured animals (p = 0.005 and 0.008, respectively) (D). Scale bar: A 250 µm.