Figure 1. Irradiation decreases neural precursor proliferation and growth potential. a, Inset shows the caudal half of a rat brain coronally bisected to expose the dorsal-medial poles of the hippocampal formations (red box outlines the right hippocampal formation shown in panel). The hippocampal formations are banana-shaped structures that extend caudally and ventrally from this plane. The regions within the hippocampus scored for this study include the hilus, the granule cell layer (GCL, blue) and the subgranule zone (SGZ, green), which is a thin lamina at the boundary of the GCL and hilus. Cell counts were made within the indicated GCL including a 50-µm hilar margin of the SGZ (blue and green areas combined) or within the hilus, b and c, Confocal micrograph (x10 magnification, zoom of 1) of the dentate gyrus in a control animal (b) and an irradiated animal (c). NeuN (mature neurons) are shown in blue; BrdU (proliferative cells) are green. Scale bar, 200µm d, Radiation-induced suppression of proliferation is documented by scoring the total number of BrdU⁺ cells per GCL/SGZ and hilus of the entire hippocampal dentate gyrus in control animals () and at two months post-irradiation (). Data are means +/- s.e.m.; n = 4 animals per group. e - f, Precursor cell growth in culture. Adult rats were sacrificed one month after exposure to 0 Gy (), 2 Gy () or 10 Gy () and hippocampi (e) or whole brain (f) were used to establish cultures highly enriched for precursor cells (n = 3 animals per group).

Figure 2. Irradiation alters the cell fate profile of the hippocampus. a and b, Relative proportion (a) and absolute number (b) of precursors adopting a recognized cell fate. BrdU-labeled cell phenotype was determined by confocal analysis (full z-stack resolution). Only those nuclei clearly associated with the indicated marker were scored. Phenotype was scored only for BrdU-labeled cells in the GCL+SGZ of controls () or animals that had received 10 Gy cranial irradiation (). Data are means +/- s.e.m.; n = 4 animals per group. The relative proportions of cells adopting specific fates were corrected for total newborn cells per SGZ+DG to give an estimate of total cells of each type produced in irradiated and non-irradiated hippocampi (b). While the relative proportion of immature oligodendrocytes is increased (a), correction of percent-positive values into total endogenous newborn oligodendrocytes shows that the number produced in control and irradiated brains is not significantly different (b). c-h, Confocal micrographs (x40 magnification, zoom of 4) demonstrating examples of each cell type. In all images, BrdU-labeled nuclei are shown in red. Cell markers were: c, NeuN (a nuclear antigen in mature neurons, green); d, ß-tubulin recognized by the Tuj-1 antibody (tubule-associated protein specifically expressed by immature neurons, green); e, GFAP (an intermediate filament protein in astrocytes, blue); f, NG2 (a chondroitin sulfate proteoglycan expressed by immature oligodendrocytes and their precursors, green); g, Rat endothelial cell antigen-1 (RECA, luminal surface antigen expressed by endothelial cells, blue). Nuclei of capillary endothelium are flattened and wrap tightly around the vessel wall. The morphology is diagnostic and this example is imaged in cross-section. h, IB4/CD18 (a cell-surface marker expressed by all cells of the monocyte/microglial lineage, green) and ED1 (a lysosomal antigen expressed specifically by activated monocytes/microglia, blue). The BrdU-labeled cells shown here are within the subgranule zone and the granule cell layer is positioned in the upper left quadrant of each panel. The hilus is in the lower right. Scale bar, 10µm

Figure 3. Precursor cells from irradiated brains can differentiate into neurons in vitro. a, Differentiation of precursor cells isolated from animals exposed to 0 Gy (), 2 Gy () or 10 Gy () cranial radiation. Data are means +/- s.e.m.; n = 3 animals per group. b-d, Confocal micrographs of precursor cells isolated from brains exposed to either 0 Gy (b), 2 Gy (c) or 10 Gy (d) that were cultured for 5 d under differentiation conditions. Cells were immunostained for type III ß-tubulin (immature neurons, green), GFAP (astrocytes, red) and NG2 (immature oligodendrocytes, blue). Scale bar, 50 µm

Figure 4. Irradiation disrupts the neurogenic microenvironment. a and b, Nonirradiated neural stem/precursor cells were transplanted into the dentate gyrus of non-irradiated (a) and irradiated (b) animals. Transplanted cells were pre-labeled with BrdU. Low-power confocal micrographs show an overview of the surviving cells within the hippocampus. a, Non-irradiated hippocampus stained for NeuN (mature neurons, blue) and BrdU (transplanted cells, red). b, 10 Gy-irradiated hippocampus stained for NG2 (immature oligodendrocytes, blue) and BrdU (transplanted cells, red). Distributions of transplanted cells are roughly equivalent in control and irradiated hippocampi. c, Cell-fate profile of neural stem/precursors transplanted to the normal () or 10 Gy () irradiated hippocampus (n = 3 animals per group). d-g, Confocal micrographs of transplanted cells. d, mature neuron (NeuN, blue; BrdU, red); e, immature neuron (Tuj-1, green; BrdU, red); f, astrocyte (GFAP, blue; BrdU, red); g, immature oligodendrocyte (NG2, green; BrdU, red). Scale bars, 200µm (a and b) and 10 µm (d-g). The tissues surrounding the transplanted cells shown in d-g were also reconstructed in three dimensions (see Supplementary information).

Figure 5. Irradiation induces a chronic inflammatory response. a-b Confocal micrographs demonstrate the increase in activated microglia in the dentate gyrus at two months post-irradiation (b) compared with control (a). ED1 (activated microglia, green); IB4 (microglia, blue); BrdU (proliferative cells, red). c, Detail of activated microglia in the subgranule zone at two months post-irradiation. ED1, red; IB4, blue; BrdU, green. d, Quantification of activated microglia (dividing + non-dividing ED1⁺ cells) in the GCL+SGZ in controls () and at two months after 10 Gy irradiation (). Data are means +/- s.e.m., n = 4 animals per group. Scale bars, 50 µm (a and b) and 10 µm (c).

Figure 6. Irradiation disrupts the neuro-vascular relationship. a and b, Confocal images of non-irradiated (a) and irradiated (b) hippocampal formations after 6 daily BrdU doses given one month post-radiation exposure. Sections are stained for BrdU (green), ED1⁺ microglia (red) and tomato lectin-binding endothelial cells (blue). c and d, Higher magnification confocal micrographs illustrate that the vessel-associated proliferative clusters seen in the control dentate gyrus (c) are lost in the irradiated dentate gyrus (d). e, Distance from BrdU-labeled cells to nearest vessel at 1 mo post-irradiation , 0 Gy; , 10 Gy (n = 8 per group, 40-µm sections per animal). f, In normal animals, precursors divide in clusters within the SGZ. The number of cells per cluster and the proximity to the nearest vessel is plotted for individual clusters in controls () and irradiated animals (). The mean values are also plotted for controls ( and irradiated (animals. Error bars for s.e.m. are smaller than the symbol itself. The distance value for each cluster is the average of distances for all cells within a cluster. Scale bars, 200 µm (a and b) and 10 µm (c and d).