Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain

Abstract

Reactive gliosis is the universal reaction to brain injury, but the precise origin and subsequent fate of the glial cells reacting to injury are unknown. Astrocytes react to injury by hypertrophy and up-regulation of the glial-fibrillary acidic protein (GFAP). Whereas mature astrocytes do not normally divide, a subpopulation of the reactive GFAP(+) cells does so, prompting the question of whether the proliferating GFAP(+) cells arise from endogenous glial progenitors or from mature astrocytes that start to proliferate in response to brain injury. Here we show by genetic fate mapping and cell type-specific viral targeting that quiescent astrocytes start to proliferate after stab wound injury and contribute to the reactive gliosis and proliferating GFAP(+) cells. These proliferating astrocytes remain within their lineage in vivo, while a more favorable environment in vitro revealed their multipotency and capacity for self-renewal. Conversely, progenitors present in the adult mouse cerebral cortex labeled by NG2 or the receptor for the platelet-derived growth factor (PDGFRalpha) did not form neurospheres after (or before) brain injury. Taken together, the first fate-mapping analysis of astrocytes in the adult mouse cerebral cortex shows that some astrocytes acquire stem cell properties after injury and hence may provide a promising cell type to initiate repair after brain injury.

Fig. 1.

Proliferation and fate of genetically labeled astrocytes in the intact or injured mouse neocortex. (A and A′) β-gal⁺ cells exhibit an astrocytic morphology and coexpress S100β in the intact cortex (DAPI is blue in A, causing the white color in A′). (B) Identity of β-gal⁺ cells in the intact cortex and hours postlesion (hpl) or days postlesion (dpl). (C) Percentage and phenotype analysis of β-gal⁺ cells that incorporated BrdU provided in the drinking water (colors as indicated in B). (D–E′) A large number of genetically labeled β-gal⁺ astrocytes incorporated BrdU (arrows) 3 dpl (D) or 30 dpl (E) after BrdU in drinking water (dashed line, lesion track). Arrows in D point to BrdU⁺/β-gal⁺ cells displaying GFAP up-regulation. (F–G′) β-gal⁺ astrocytes incorporating BrdU (arrows) 2 h after a single BrdU injection. (Scale bars: 100 μm in A, E, and G and 40 μm in A′, D, E′, F, F′, and G′.)

Fig. 2.

LCMV-targeted cells in the intact cortex and their progeny after lesion. (A–F) Most LCMV-infected GFP⁺ cells in the intact cortex have the morphology and antigen profile (S100β or GLT1, red in A–C′, arrows in B–C′) of protoplasmic astrocytes and do not contain Sox10 (red, arrows in E), whereas some are GSA⁺ microglia (red, arrows in D′). (F) Histogram depicting the proportion of cell types infected by the LCMV virus. (G–K) Progeny of GFP⁺ cells after injury with examples of GFP⁺ and GFAP⁺ (G and G′), GLT1⁺ (H and H′), and S100β⁺ (I–I″) astrocytes that incorporated BrdU (arrows point to triple-labeled cells) 7–30 dpl. (J and K) Histograms depict the identity of all GFP⁺ (J) or BrdU⁺/GFP⁺ (K) cells. Arrowheads in C′ and G′ indicate processes positive for GLT1 (C′) or GFAP (G′). (Scale bars: 100 μm in A and 30 μm in B-I″.)

Fig. 3.

Neurosphere-forming cells upon injury. (A–D and F–N) Micrographs depicting examples of neurospheres formed by cells isolated from the lesioned cortex (10 days in vitro) (A, F, and I–L) and their progeny after 10 days in differentiating conditions (B–D, G, H, M, and N). (E) The sorting profile of cortical cells isolated 3 dpl from the injury site of GLAST::CreERT2;Z/EG mice (E, sortings for F–H) with the GFP⁺ signal in yellow. (Scale bars: 50 μm in A, 20 μm in B–D and F–H, and 10 μm in I.)