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. 2001 Apr 10;98(8):4710-5.
doi: 10.1073/pnas.081011098.

Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat

K Jin  1 , M Minami, J Q Lan, X O Mao, S Batteur, R P Simon, D A Greenberg
Affiliations

Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat

K Jin et al. Proc Natl Acad Sci U S A. .

Abstract

Because neurogenesis persists in the adult mammalian brain and can be regulated by physiological and pathological events, we investigated its possible involvement in the brain's response to focal cerebral ischemia. Ischemia was induced by occlusion of the middle cerebral artery in the rat for 90 min, and proliferating cells were labeled with 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUrd) over 2-day periods before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia increased the incorporation of BrdUrd into cells in two neuroproliferative regions-the subgranular zone of the dentate gyrus and the rostral subventricular zone. Both effects were bilateral, but that in the subgranular zone was more prominent on the ischemic side. Cells labeled with BrdUrd coexpressed the immature neuronal markers doublecortin and proliferating cell nuclear antigen but did not express the more mature cell markers NeuN and Hu, suggesting that they were nascent neurons. These results support a role for ischemia-induced neurogenesis in what may be adaptive processes that contribute to recovery after stroke.

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Figures

Figure 1
Figure 1
BrdUrd labeling of ischemic brain. One MCA was occluded for 90 min, followed by reperfusion. (Upper) BrdUrd was given as four injections over one of the 2-day spans indicated by shading (days 5 and 6, 12 and 13, or 19 and 20 after ischemia), and animals were killed on day 7, 14, or 21 after ischemia. (Lower) Brain regions examined for BrdUrd incorporation in SVZ and DG (long arrows) are shown with the corresponding TTC-stained brain slices 1 week after ischemia; red is viable and white is nonviable (short arrows) tissue. White matter tracts including the corpus callosum also appear white with this method. Neither SVZ nor DG is within the infarct area. TTC staining was repeated three times with similar results. BrdU, BrdUrd.
Figure 2
Figure 2
Distribution of injury and DNA damage in ischemic brain. Sections were obtained from the contralateral control (Left) or ipsilateral ischemic (Center and Right) hemisphere of brains 1 week after ischemia. DG, SVZ, cerebral cortex (Ctx), and caudate-putamen (CPu) were stained with cresyl violet to detect ischemic neuronal loss (Left and Center) or with the Klenow fragment of DNA polymerase I to label DNA strand breaks (Right). Neither DG nor SVZ, which are outside the ischemic territory, showed neuronal loss or DNA damage, whereas the ipsilateral cortex and caudate-putamen, which are within this territory, showed both. The experiment was repeated three times with similar results.
Figure 3
Figure 3
BrdUrd incorporation into DG. Sections through DG were stained for BrdUrd and visualized with DAB. BrdUrd labeling was increased on the ipsilateral ischemic side relative to nonischemic control brains and the contralateral side at 1 week but not at 2 or 3 weeks. The experiment was repeated three times with similar results.
Figure 4
Figure 4
BrdUrd incorporation into SVZ. Sections through SVZ were stained for BrdUrd and visualized with DAB. BrdUrd labeling was increased both ipsilateral and contralateral to ischemia at 1 and 2 weeks, relative to nonischemic control brains and 3 weeks. The experiment was repeated three times with similar results.
Figure 5
Figure 5
BrdUrd-immunoreactive cell counts in DG and SVZ. BrdUrd-reactive cells were counted in nonischemic brains (time = 0) and in ischemic brains on the ischemic side (ipsilateral, ●) and nonischemic side (contralateral, ○) at 1–3 weeks. Data are the mean ± SEM (n = 8–15). In DG, cell counts were increased at 1 week relative to time = 0 on both the ischemic (P < 0.001, Student's t test) and the nonischemic (P < 0.05, Student's t test) side, and at 1 week on the ischemic relative to the nonischemic side (P < 0.001, paired Student's t test). In SVZ, cell counts were increased relative to time = 0 at 1 week on the ischemic (P < 0.05, Student's t test) and nonischemic (P < 0.005, Student's t test) sides and at 2 weeks on the ischemic (P < 0.005, Student's t test) and nonischemic (P < 0.001, Student's t test) sides. BrdU, BrdUrd.
Figure 6
Figure 6
Relationship between BrdUrd incorporation and NeuN expression. Double immunolabeling for BrdUrd (Left, red) and NeuN (Center, green) was performed in DG and SVZ from control brains (con) and the ischemic side of brains 1 week after MCA occlusion (isch). Merged images show lack of coexpression of BrdUrd and NeuN and localize BrdUrd labeling to the SGZ of DG and the SVZ. The experiment was repeated three times with similar results. BrdU, BrdUrd.
Figure 7
Figure 7
Relationship between BrdUrd incorporation and expression of Hu, DCX, and PCNA in DG. Double labeling for BrdUrd (Left, red) and Hu, DCX, and PCNA (Center; top to bottom, green) was performed in sections through DG ipsilateral to MCA occlusion, taken 1 week after ischemia. Merged images show no coexpression of BrdUrd with Hu, but extensive coexpression of BrdUrd with DCX and PCNA. The experiment was repeated three times with similar results. BrdU, BrdUrd.
Figure 8
Figure 8
Relationship between BrdUrd incorporation and expression of Hu, DCX, and PCNA in SVZ. Double labeling for BrdUrd (Left, red) and Hu, DCX, and PCNA (Center; top to bottom, green) was performed in sections through SVZ ipsilateral to MCA occlusion, taken 1 week after ischemia. Merged images show no coexpression of BrdUrd with Hu, extensive coexpression of BrdUrd with DCX, and expression of PCNA in only a small percentage of BrdUrd-labeled cells. The experiment was repeated three times with similar results. BrdU, BrdUrd.
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