Neural stem/progenitor cells participate in the regenerative response to perinatal hypoxia/ischemia

Abstract

Perinatal hypoxia/ischemia (H/I) is the leading cause of neurologic injury resulting from birth complications. Recent advances in critical care have dramatically improved the survival rate of infants suffering this insult, but approximately 50% of survivors will develop neurologic sequelae such as cerebral palsy, epilepsy or cognitive deficits. Here we demonstrate that tripotential neural stem/progenitor cells (NSPs) participate in the regenerative response to perinatal H/I as their numbers increase 100% by 3 d and that they alter their intrinsic properties to divide using expansive symmetrical cell divisions. We further show that production of new striatal neurons follows the expansion of NSPs. Increased proliferation within the NSP niche occurs at 2 d after perinatal H/I, and the proliferating cells express nestin. Of those stem-cell related genes that change, the membrane receptors Notch1, gp-130, and the epidermal growth factor receptor, as well as the downstream transcription factor Hes5, which stimulate NSP proliferation and regulate stem cellness are induced before NSP expansion. The mechanisms for the reactive expansion of the NSPs reported here reveal potential therapeutic targets that could be exploited to amplify this response, thus enabling endogenous precursors to restore a normal pattern of brain development after perinatal H/I.

Figures

Figure 1.

Diagram of the brain regions analyzed. A, View from the top of the brain. Dashed lines represent the region that was blocked for neurosphere assays and RNA isolation. Dotted line indicates approximate level at which sections were taken for Figures 2–4. B, Coronal section at the level of the dotted line in A. The region bounded by dashed lines delimits the region dissected for neurosphere assays. The smaller region bounded by the dotted line was removed for RNA isolation. C, Representation of the medial SVZDL as shown in Figures 2–4. LV, Lateral ventricle; Ep, ependymal layer; SVZm, 3–4 cell diameters subjacent to Ep; Total, entire medial SVZ, including Ep and SVZm.

Figure 2.

Hypoxia/ischemia increases the number of BrdU+ cells adjacent to or among the ependymal lining in the ipsilateral SVZ. Control and experimental animals received a single intraperitoneal injection of BrdU 1 h before being killed at 48 h recovery from hypoxia/ischemia. Cryostat tissue sections were immunostained for BrdU incorporation (brown) and counterstained with hematoxylin (blue). A, BrdU labeling in a control brain. B, C, BrdU labeling in the ipsilateral hemisphere. Cells with BrdU-labeled nuclei occasionally appeared to have a cytoplasmic extension contacting the lateral ventricle in the ipsilateral SVZ (arrow in C). D, Quantitative results for the number of BrdU-positive cells per section from ipsilateral and contralateral hemispheres. *p < 0.05. Scale bar: A, B, 30 μm; C, 10 μm.

Figure 3.

Hypoxia/ischemia increases the number of proliferating precursors within the NSP niche. A–H, Cryostat tissue sections from control and experimental animals were immunostained for PCNA (red) and nestin (green). DAPI (blue) is the nuclear stain in D–F. Panels represent control (A, D), H/I contralateral (B, E), and H/I ipsilateral (C, F) hemispheres. The number of PCNA+ cells (G) or PCNA/nestin double-positive cells (H) per field of view was quantified for the entire medial SVZ (Total), the ependymal layer (Ep), and those cells within 4 nuclear diameters of the ependymal layer (SVZm). Counts were conducted on three nonadjacent sections per animal. Data represent the mean ± SEM of four independent experiments. *p < 0.002 versus control; **p < 0.0003 versus control by two-tailed t test. I–L, Cryostat sections from ipsilateral (J, L) and contralateral (I, K) SVZs were stained for GFAP, S100, and BrdU (I, J) or nestin, PSA-NCAM, and Ki67 (K, L), and images were obtained on a laser-scanning confocal microscope. In I, J, BrdU administration was the same as in Figure 2.

Figure 4.

A subset of newly generated cells differentiate along the neuronal lineage after perinatal H/I. Sections from the contralateral (A) and ipsilateral (B) hemisphere of animals killed at 3 weeks of recovery were processed for Dcx immunostaining. Whereas Dcx staining was primarily restricted to the SVZ in the contralateral hemisphere (as well as in controls), there were numerous cells with migratory profiles streaming out of the SVZ into the striatum of the ipsilateral hemisphere. When BrdU was injected between 3 and 5 d recovery after H/I and animals killed at 3 weeks after H/I, numerous BrdU+/Dcx+ cells could be found in the ipsilateral striatum (C). Another cohort of animals was perfused 5 weeks after H/I, and BrdU+/NeuN+ cells could be found in the ipsilateral striatum. All images were captured using a Zeiss LSM 410 confocal microscope. Scale bars: A, B, 100 μm; C, 50 μm; D, 10 μm.

Figure 5.

Hypoxia/ischemia increases the number of colony-forming NSPs and the self-renewal of NSPs in the ipsilateral SVZ. Control and experimental animals were sterilely decapitated at the designated time points, and the SVZs were dissociated into single-cell suspensions. These suspensions were cultured in the presence of FGF-2 (10 ng/ml) and EGF (20 ng/ml). Each replication consisted of pools of two to three animals per group. The number of NSPs per hemisphere for controls was compared with H/I ipsilateral (A) and H/I contralateral (B) hemispheres. Representative neurospheres from ipsilateral H/I (C), contralateral H/I (D), and control (E) SVZs demonstrate the larger size of spheres from the ipsilateral SVZ. Scale bar, 20 μm. The diameter of 10 neurospheres from each of four animals per condition was measured (F; error bars represent a 95% confidence interval). Primary neurospheres generated at 3 d of recovery were dissociated and subcultured to determine the relative self-renewal rates of each group (G). Values represent the mean ± SEM of four independent experiments. *p < 0.05 versus control by two-tailed t test.

Figure 6.

Hypoxia/ischemia increases the proportion of multipotential SVZ-derived neurospheres. NSPs were harvested from animals at 48 h of recovery and cultured in vitro as neurospheres in the presence of both FGF-2 and EGF. Spheres were plated onto coverslips and differentiated in growth factor free medium for 5 d. Neurospheres from control (A), contralateral (B), and ipsilateral (C) SVZs were immunostained for neuronal, astrocytic, and oligodendrocytic markers, and the percentage of glial or neuron/glial producing spheres was quantified. D depicts a representative multipotential clone from the ipsilateral SVZ stained for a neuronal marker (Tuj1, green), an astrocytic marker (GFAP, blue), and an oligodendrocyte marker (O4, red). Values embedded in the chart are the mean numbers of colonies counted from six independent experiments with three to four animals per group per experiment. *p < 0.005 for the percentage of ipsilateral multipotential spheres versus control multipotential spheres using ANOVA, followed by Bonferroni’s/Dunn’s post hoc test.

Figure 7.

Signaling pathways involved in control of NSP fate are induced by perinatal H/I. Ipsilateral H/I and control hemispheres were dissected out after 48 h of recovery. A, Total RNA was isolated and amplified by qRT-PCR using primers specific for EGFR and Notch1 and normalized to expression of 18S. Values in parentheses indicate CV for the target gene in the sample groups; n = 10 for ipsilateral and contralateral conditions, and n = 4 for sham condition. *p < 0.05 versus sham; †p < 0.05 versus contralateral by pairwise fixed reallocation randomization. Immunostaining for Notch1 was performed on cryostat sections from H/I (B) and control (C) animals. In situ hybridization was performed on cryostat sections of contralateral (D, G), ipsilateral (E, H), and control (F, I) hemispheres using a digoxigenin-labeled RNA probe for Hes5 (D–F) and Hes1 (G–I). Arrows in E delineate the region of increased Hes5 expression. Inset in E shows high-magnification 100× Nomarski image of the Hes5+ region showing a subependymal Hes5+ cell body (arrowhead) with processes (arrow) projecting through the ependyma. There was no evident change in Hes1 expression. Scale bars: inset in E, 4 μm; F, 10 μm. cp, Choroid plexus; V, ventricle.