The Journal of Neuroscience, December 13, 2006, ():
A Neurovascular Niche for Neurogenesis after Stroke
J. Neurosci. Ohab et al.
26: 13007
Supplemental Data
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Supplementary Figure 1. Cell death and injury after focal cortical stroke. The nissl stain indicates the location of infarction in frontal and parietal cortex. The box on the right illustrates the region in panels a,c,e,g. The box on the left illustrates the region in panels b,d,f,h. Scale bar for nissl stain, 200µm. All panels represent the point of maximal staining. a,b, fluorojade staining (green) at 12 hours after stroke. Fluorojade+ cells are located in peri-infarct cortex with a sharp border at the junction with subcortical white matter. Staining was present at all time points examined, but maximal at 12 hours and one day after stroke. Fluorojade+ cells were localized to the infarct core and approximately 200µm of peri-infarct cortex. There were no immunoreactive cells in the SVZ. c,d, HNE staining (red) of oxidized lipids at 30 minutes after stroke. HNE staining was greatest at 30 minutes and declined progressively from 12 hours to one day after stroke. HNE staining was localized to blood vessels in the infarct core and peri-infarct cortex at 30 minutes and then in peri-infarct cortex at 12 hours and one day after stroke. e,f, 8OHdOG (red) and fractin (green) staining at one day after stroke. Both staining patterns label cells in the infarct core and in peri-infarct cortex, but not the white matter, striatum or SVZ. 8OHdOG staining was increased from 30 minutes to a maximum at one day after stroke, with a decline at three days after stroke. 8OHdOG was localized to peri-infarct cells with large cell bodies and apical processes suggestive of neurons. Fractin staining was present in the infarct core at 12 hours, one and three days after stroke, and in closely adjacent peri-infarct cortex. Fractin+ cells appeared fragmented, with beading of local processes g,h, Activated caspase 3 at one day after stroke. Staining is present in infarct core and peri-infarct cortex, but not white matter, striatum or SVZ. Activated caspase 3 immunoreactive cells were present in the infarct core at 12 hours, and in peri-infarct cortex at 12 hours, one and three days after stroke. These cells occupied a rim of cortical tissue adjacent to the infarct. Scale bar, 100µm. cc, corpus callosum; lv, lateral ventricle; svz, subventricular zone.
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Supplementary Figure 2. Population histograms for vector analysis of DCX+ cells in white matter after stroke. The position of DCX+ cells in white matter in each treatment condition was digitized and rendered as a polar plot with a vector angle (θ) that represents the angle of migration of neuroblasts from the SVZ. The solid line represents the mean angle for all DCX+ cells in vehicle-treated (a) and SDF1β (b) and Ang1 (c) gain- and loss-of-function animals at day 7 after stroke. Blue triangles represent 5 cells. Error bars indicate S.D. Because the vectors for each high and low group in Ang1 and SDF1β are co-extensive, the data from these conditions were grouped into common Ang1 and SDF1β groups for statistical analysis.
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Supplementary Figure 3. Migrating neuroblasts do not express markers of microglia/macrophages or pericytes after stroke. a,b, DCX+ cells (red) do not co-localize with OX-42+ (a) (green) or desmin+ (b) (green) cells in peri-infarct cortex after stroke. Scale bars, 25µm. c, stereological quantification of BrdU+ cells in the olfactory bulb at day 7 after stroke. #p=0.004. Error bars indicate S.D.
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Supplementary Figure 4. Lentivirus and BrdU microinjections. a,b, shows the location of the pipette track (arrows) and infected cells with GFP expression in the SVZ (box). The pipette track is located through cingulate cortex, 2-4 millimeters from the infarct. Scale bar in a, 100µm. Scale bar in b, 200µm. c-e, DCX+ cells (red) do not co-localize with BrdU+ cells (green) following microinjection of BrdU into cortex at the time of stroke, indicating that DCX+ after stroke are not derived from cortex. Scale bar, 25µm. cc, corpus callosum; lv, lateral ventricle; svz, subventricular zone.
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Supplementary Figure 5. Neuroblasts associate with blood vessels. a, DCX+ cells (red) form close physical associations with peri-infarct blood vessels immunoreactive for von Willebrand factor (green). Scale bar, 25µm. b-e, fluoroscein-conjugated Lycopersicon esculentum (green), when administered intravascularly in non-stroke animals at the time of sacrifice, binds all PECAM-1+ endothelial cells (purple) that receive vascular perfusion. Note the absence of DCX+ cells (red) in the non-stroke cortex. Compare this complete overlap of vascular perfusion and endothelial cell markers in the normal adult to that seen in the area of vascular remodeling in peri-infarct cortex after stroke (Fig. 5a-e). Scale bar, 50µm.
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Supplementary Figure 6. Vascular remodeling and angiogenesis are causally linked to neurogenesis after stroke. All blood vessels receive vascular flow in the healthy adult brain. a-c, individual confocal images from Fig. 5f show DCX+ cells (b) and PECAM-1+ blood vessels (a) are co-extensive in peri-infarct cortex at day 7 after stroke. The co-labeling of BrdU (c) with DCX+ and PECAM-1+ cells indicates that cells are newly born. d, additional confocal image demonstrating that DCX+ cells (red) are located in close physical proximity to endothelial cells (green) in peri-infarct cortex after stroke. Arrows indicate DCX+ cells that are newly born (co-label with BrdU, green) and arrowheads indicate endothelial cells that are newly born (co-labeled with BrdU, green). e, stereological quantification of DCX+ cells in peri-infarct cortex in endostatin- and vehicle-treated animals at day 7 after stroke. *p<0.0001. Error bars indicate S.D.
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Supplementary Figure 7. Ang1 and SDF1 expression after stroke. a, Ang1 expression (red) in PECAM-1+ endothelial cells (green) remains unchanged at 3 days after stroke and increases at 7 days after stroke compared to non-stroke animals. b, SDF1 expression (red) in PECAM-1+ endothelial cells (green) increases at day 3 after stroke and persists in blood vessels through day 7 after stroke compared to non-stroke animals. Asterisks indicate the region of infarction. Scale bar, 50µm.
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Supplementary Figure 8. Systemic delivery of vascular growth factors and chemokines. a,b, SDF1β, Ang1, and a function-blocking anti-Tie2 antibody were conjugated to biotin and delivered systemically via osmotic minipump for five days after stroke. Panels show streptavidin-Alexa488 fluorescence in cortex contralateral to stroke (a) and peri-infarct cortex (b). Representative confocal images were taken from the biotin-conjugated anti-Tie2 treatment condition. Scale bar, 25µm. c,d, Evans blue fluorescence in cortex contralateral to stroke (c) and peri-infarct cortex (d) at day 7 after stroke. e,f, Evans blue fluorescence in cortex contralateral to stroke (e) and peri-infarct cortex (f) at day 14 after stroke. g, quantification of infarct volume in Ang1 (left) and SDF1β (right) gain- and loss-of-function animals at day 7 after stroke. There is no statistically significant difference across conditions (p>0.34). h, quantification of DCX+ cells in subcortical white matter of vehicle-treated and Ang1 (left) and SDF1β (right) gain- and loss-of-function animals at day 7 after stroke. There is no statistically significant difference across conditions (p> 0.08). H and L indicate high and low doses. Error bars indicate S.D.
