PMC

Increased neurogenesis from fate mapped astroglial lineage cells following hypoxia. GCE;R26R and GCE;eGFP mice were tamoxifen-injected at P12–P14 and analyzed at P35. Hypoxic reared mice had increased numbers of reporter+/DCX+ immature neurons (D–F) and reporter+/NeuN+ mature neurons (J–L) in the dentate gyrus as compared to normoxic mice (A–C, G–I). Scale bar, 40 μm.

Environmental enrichment increases stem cell proliferation and survival. GCE;R26R and GCE;eGFP normoxic-reared mice were tamoxifen injected from P12–14, subsequently injected with CldU from P15–16 (to examine cell survival) (see schematic outline in A), reared in enriched or standard environment from P21–P35, and then injected with IdU 1 h before sacrifice (to examine cell proliferation) at P35. Both Sox2+ cell proliferation (A–C) and Sox2+ cell survival (D–F) were significantly increased in enriched mice. * denote significant increases from NSE.

Effect of hypoxia and environmental enrichment on stem cell proliferation. GCE;R26R mice reared under normoxic or hypoxic conditions followed by environmental enrichment as indicated were analyzed at P35. A–H, Representative double staining of Sox2 and Ki67 at P35. Stereological quantification showed an increase in the total number (G) and proportion (H) of proliferating Sox2+ cells in response to environmental enrichment. No increase in stem cell proliferation was observed in response to hypoxic rearing (G–H). * denote significance from all other groups.

Reporter expression is initiated and maintained in dentate gyrus astrocytes and neural progenitors/stem cells. A, B, GCE;R26R and GCE;CAG-EGFP hypoxic mice were analyzed at P15 after tamoxifen (A) or vehicle (B) injections at P12–P14. Mice that received tamoxifen injections exhibited lacZ-positive cells in the hippocampus (A, indicated with arrows). No cells were observed in response to vehicle only injections (B). C–H, Over 90% of reporter-positive cells (shown in green) are colocalized with GFAP (red) (C–E) and no reporter+ cells were colocalized with NeuN (shown in red) (F–H). Scale bar, 10 μm.

Environmental enrichment reverses hypoxia-induced cognitive deficits. Mice were reared in normoxia or hypoxia from P3 to P11 and subsequently in standard or enriched environment from P21 to P35. Behavioral testing was conducted on P35–36. A–C, Results of the Morris water maze. A, The proportion of mice that successfully found the platform in under 60 s, averaged across six trials, during the acquisition phase on the maze. B, The latency to find the platform across trials. C, Probe trial. D, E, Results from the open field test. D, Proportion of distance traveled in the center/total distance traveled. E, Time spent in the center. *, Significance from all other groups; **, significance from NSE and NEn groups, #, HSE and NEn are different from all other groups.

Schematic representation depicting hypoxia- and enrichment-induced changes in astroglial lineage. There is an increase in the number of neuronal cells in hypoxic-reared as well as normoxic enriched mice, shown in purple (DCX+) and blue (NeuN+). HEn mice show the greatest increase in total number of reporter+ neurons, suggesting an additive effect of hypoxia and enrichment on neurogenesis arising from GFAP+ cells. HSE mice show a greater proportion of the stem cell pool that is committed to the neuronal lineage (Sox2+DCX+, shown in red); this effect remains apparent in hypoxic mice that were subsequently enriched. In NEn mice there is no change in fate potential of NSC but rather a robust increase in the total stem/progenitor cell pool (shown in green, yellow, red) that is still present in Hen mice.

Hypoxia and subsequent environmental enrichment increase the number of neurons arising from GFAP+ cells in an additive way. A, Schematic of the time course of hypoxia and enrichment experiment. B–I, Representative images showing the increase in reporter+/DCX+ neurons in hypoxic, enriched mice (F–I) as compared to hypoxic, standard reared mice (B–E) at P35; stereological quantification of the reporter+/DCX+ and reporter+/NeuN+ cell numbers is shown in N and O, respectively. J–M, Long-term increase in mature reporter+/NeuN+ neurons in response to hypoxia and enrichment at P90, with stereological quantification of reporter+/NeuN+ cell numbers at P90 shown in P. Q, The total number of eGFP+ cells at P35 increases in hypoxic and enriched mice. R, Proportion of reporter+ cells that attain neuronal (NeuN+), astroglial (GFAP+) and oligodendroglial (Olig2) fate are shown across groups. * denote significant increases from NSE; # denotes significant difference from all other groups.

Hypoxia pushes stem cells to a neuronal fate, whereas enrichment increases the total stem cell pool. A–T, Representative Sox2/GFAP/eGFP immunostaining for all four groups; note the increase in number of Sox2+ cells expressing GFAP with environmental enrichment. U, Environmental enrichment also increased the number of reporter+/Sox2+ cells in both normoxic and hypoxic reared mice. V, Proportion of reporter+ cells that attain an immature neuronal (DCX+) or stem cell (Sox2+) fate is shown across groups. W, The percentage of Sox2+ stem cells that were committed to a neuronal lineage (colocalized with DCX) increased with hypoxic rearing, and this effect was attenuated with enrichment. X, The percentage of Sox2+ stem cells that remained in the NSC lineage (colocalized with GFAP) increased with enrichment. Y, Schematic representation of cell lineages arising from fate mapped GFAP+ astroglia. * denote significant increases from NSE; ** denotes significant increase from both NSE and HSE groups; — denotes significance from all other groups.