Effects of primary microglia and astrocytes on neural stem cells in in vitro and in vivo models of ischemic stroke

Abstract

Transplantation of neural stem cells (NSCs) can protect neurons in animal stroke models; however, their low rates of survival and neuronal differentiation limit their clinical application. Glial niches, an important location of neural stem cells, regulate survival, proliferation and differentiation of neural stem cells. However, the effects of activated glial cells on neural stem cells remain unclear. In the present study, we explored the effects of activated astrocytes and microglia on neural stem cells in vitro stroke models. We also investigated the effects of combined transplantation of neural stem cells and glial cells after stroke in rats. In a Transwell co-culture system, primary cultured astrocytes, microglia or mixed glial cells were exposed to glutamate or H₂O₂ and then seeded in the upper inserts, while primary neural stem cells were seeded in the lower uncoated wells and cultured for 7 days. Our results showed that microglia were conducive to neurosphere formation and had no effects on apoptosis within neurospheres, while astrocytes and mixed glial cells were conducive to neurosphere differentiation and reduced apoptosis within neurospheres, regardless of their pretreatment. In contrast, microglia and astrocytes induced neuronal differentiation of neural stem cells in differentiation medium, regardless of their pretreatment, with an exception of astrocytes pretreated with H₂O₂. Rat models of ischemic stroke were established by occlusion of the middle cerebral artery. Three days later, 5 × 10⁵ neural stem cells with microglia or astrocytes were injected into the right lateral ventricle. Neural stem cell/astrocyte-treated rats displayed better improvement of neurological deficits than neural stem cell only-treated rats at 4 days after cell transplantation. Moreover, neural stem cell/microglia-, and neural stem cell/astrocyte-treated rats showed a significant decrease in ischemic volume compared with neural stem cell-treated rats. These findings indicate that microglia and astrocytes exert different effects on neural stem cells, and that co-transplantation of neural stem cells and astrocytes is more conducive to the recovery of neurological impairment in rats with ischemic stroke. The study was approved by the Animal Ethics Committee of Tongji University School of Medicine, China (approval No. 2010-TJAA08220401) in 2010.

Keywords: astrocytes; glutamate; microglia; neural stem cells; neurogenesis; neurons; peroxide; repair; stroke.

Figure 1

Schematic diagram of the experimental procedure. Glu: Glutamate; MCAO: middle cerebral artery occlusion; NSC: neural stem cell.

Figure 2

Characterization of NSCs from newborn rat hippocampus. (A, B) Phase contrast microscopy images of a neurosphere in NSC culture for 8 days (A) and a nestin-positive (green, Cy3) neurosphere (B). Nuclei were labeled by Hoechst 33258 (blue). (C, D) NSCs differentiated into MAP2-positive neurons (C; red, Cy3), GFAP-positive cells (C, D; green, FITC), and Galc-positive oligodendrocytes (D; red, Cy3) in differentiation medium for 8 days. Nuclei were labeled by Hoechst 33258 (blue). Scale bars: 50 μm in A and B, 100 μm in C and D. Galc: Galactose cerebroside; GFAP: glial fibrillary acidic protein; MAP2: microtubule associated protein 2; NSC: neural stem cell.

Figure 3

Identification of primary cultured mixed glia, astrocytes and microglia. (A) Phase-contrast micrographs showing mixed glial cells (left), microglia (middle), and astrocytes (right). Solid arrows indicate astrocytes; dotted arrows indicate microglia. (B) Microglia in microglia culture stained with GSI-B4-FITC (B, green). (C) Astrocytes in astrocyte culture stained with GFAP antibody (C, red, Cy3). Nuclei were labeled by Hoechst 33258 (blue). Scale bars: 50 μm in A, 100 μm in B and C. (D) Quantitation of the purity of microglia and astrocytes. Data are expressed as the mean ± SEM from three independent experiments. GFAP: Glial fibrillary acidic protein.

Figure 4

Effects of glial cells pretreated with glutamate or H₂O₂ on NSC neurospheres in a Transwell co-culture system. (A) Phase-bright photomicrographs showing adherent neurospheres after NSCs were plated on uncoated wells in growth medium for 7 days with astrocytes, microglia or mixed glia with or without glutamate or H₂O₂ pretreatment. In NSC cultures, adherent neurospheres displayed a spherical shape. In NSC/microglia co-cultures, most adherent neurospheres displayed a spherical shape. In NSC/astrocyte and NSC/mixed glia co-cultures, adherent neurospheres showed irregular shapes and had a few of cells migrating away. Scale bar: 50 μm. (B–D) Quantification of the ratio of differentiated neurospheres in NSC co-cultures with microglia (B), astrocytes (C), and mixed glial cells (D). Data are expressed as the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by a least significant difference test). Glu: Glutamate; NSC: neural stem cell.

Figure 5

Effect of microglia on proliferation of NSC co-cultures. (A) Phase-bright photomicrographs showing floating neurospheres in NSC/microglia co-cultures after NSCs were seeded in lower uncoated wells and microglia with or without glutamate or H₂O₂ pretreatment were plated in the upper inserts and cultured for 7 days in the growth medium. In NSC cultures and NSC/microglia co-cultures, neurospheres were mainly floating without apparent difference in size. NSC cultures were a control. Scale bar: 100 μm. (B) Quantification of the perimeter of the floating neurospheres in NSC cultures and co-cultures with microglia. Data are expressed as the mean ± SEM from three independent experiments and were analyzed by one-way analysis of variance followed by the least significant difference test. Glu: Glutamate; NSC: neural stem cell.

Figure 6

Effects of astrocytes, microglia and mixed glial cells with or without glutamate or H₂O₂ pretreatment on apoptosis of adherent neurospheres in NSC co-cultures. (A) Representative images showing neurospheres stained by Hoechst 33258 after 7 days in growth medium. The viable cells (solid arrows) are characterized by regular and round nuclei with a pallid blue fluorescence; apoptotic cells (dotted arrow) are condensed and fragmented. The right image is an enlargement of the rectangle. Scale bars: 100 μm (left) and 25 μm (right). (B) Quantitative analyses showed no difference in apoptosis of adherent neurospheres in NSC/microglia co-cultures compared with NSC cultures. (C, D) Quantitative analyses showed a lower apoptotic rate in the NSC/astrocytes (C) and NSC/mixed glial cell (D) co-cultures compared with NSC cultures. Data are expressed as the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by the least significant difference test). Glu: Glutamate; NSC: neural stem cell.

Figure 7

Effects of astrocytes and microglia exposed to glutamate or H₂O₂ on neuronal differentiation of NSC co-cultures. (A–C) Fluorescence confocal images showing MAP2-positive cells (red, Cy3) and GFAP-positive cells (green, FITC) in NSC cultures (A), co-cultures of NSCs and microglia (B) or astrocytes (C) with or without glutamate or H₂O₂ pretreatment for 7 days. All nuclei were labeled by Hoechst 33258 (blue). In NSC cultures, there was no difference in MAP2 differentiation. In NSC/microglia and NSC/astrocyte cultures, there was a significant increase in MAP2 differentiation. Scale bars: 100 μm. (D, E) Quantification of MAP2-positive cells in NSC cultures and NSC co-cultures. Data are expressed as the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by the least significant difference test). Glu: Glutamate; GFAP: glial fibrillary acidic protein; MAP2: microtubule associated protein-2; NSC: neural stem cell.

Figure 8

Effect of transplantation of NSCs with astrocytes or microglia on the functional recovery of rats after ischemic stroke. Neurological function of rats after stroke was determined by modified neurological severity scores. Data are expressed as the mean ± SEM (n = 5). **P < 0.01, ***P < 0.001 (two-way analysis of variance followed by a least significant difference test). Ctl: ischemic rats treated with vehicle; NSCs: ischemic rats treated with NSCs; NSCs-microglia: ischemic rats treated with NSCs and microglia; NSCs-astrocyte: ischemic rats treated with NSCs and astrocytes.

Figure 9

Co-transplantation of NSCs with astrocytes or microglia decreases ischemic volume in rats after stroke. (A) Representative brain slices stained with 2,3,5-triphenyltetrazolium show lesion regions at 7 days after stroke. The normal area is red and the ischemic region is white. Compared with the control group, NSC-, NSC/microglia-, and NSC/astrocyte-treated rats had significantly smaller ischemic volumes. Notably, NSC/microglia-, and NSC/astrocyte-treated rats were less affected than NSC-treated rats. (B) Quantitative analysis of ischemic volume evaluated by 2,3,5-triphenyltetrazolium staining. Data are expressed as the mean ± SEM (n = 5). **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by a least significant difference test). Ctl: Ischemic rats treated with vehicle; NSCs: ischemic rats treated with NSCs; NSCs-astrocyte: ischemic rats treated with NSCs and astrocytes; NSCs-microglia: ischemic rats treated with NSCs and microglia.