脑损伤
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Figure 2｜Astrocytic ET-1 overexpression after MCAO promotes NSC proliferation in the DG region of the mouse hippocampus.
结果：Neurogenesis in the SGZ of the hippocampal DG is very important for cognitive function. We used BrdU as a measure of cell proliferation to confirm the influence of ET-1 overexpression on cell proliferation in both GET-1 and WT mice after ischemic stroke (Han et al., 2015; Bowers et al., 2020). We administered an intraperitoneal injection of BrdU to investigate DG cell proliferation at 7 days after ischemic stroke (Figure 2A). As shown in Figure 2B, in both the GET-1 and WT mice, the ipsilateral side (injured) expressed more BrdU+ cells than the contralateral side. Compared with the WT mice, the BrdU+ cells in the ipsilateral DG region of the GET-1 mice were significantly increased 7 days after ischemic injury (P = 0.026 for GET-1 ips vs. WT ips, P < 0.001 for GET-1 ips vs. GET-1 contra, Figure 2B). We conducted double-labeling with BrdU and SOX2 (a known phenotypic marker for NSCs) to identify the proliferating cells (Penisson et al., 2019). We found a conspicuous increase in SOX2+BrdU+ cells in the ipsilateral SGZ of GET-1 mice compared with the contralateral side 7 days after ischemic stroke. Ipsilateral SOX2+BrdU+ expression in the GET-1 mice was also higher than that in the WT mice (P = 0.013 for GET-1 ips vs. WT ips, P < 0.001 for GET-1 ips vs. GET-1 contra; Figure 2C). DCX is often used as a marker of migrating neuroblasts and early differentiated (immature) neurons (Brown et al., 2003). To further investigate whether overexpression of ET-1 in astrocytes was associated with neurogenesis in the DG region, including the regulation of neurogenesis, we performed BrdU and DCX double immunofluorescence labeling. At 7 days post-MCAO, there were more DCX+BrdU+ cells on the ipsilateral side than on the contralateral side in both GET-1 mice and WT mice, and this difference was significant in the GET-1 mice. Additionally, more DCX+BrdU+ cells were observed on the ipsilateral side in GET-1 mice than on the corresponding side in WT mice (P < 0.001 for GET-1 ips vs. WT ips, P < 0.001 for GET-1 ips vs. GET-1 contra; Figure 2D). Consequently, astrocytic ET-1 overexpression reinforced ischemia-induced NSC proliferation in the DG region of the hippocampus, as verified by our results.
Figure 3｜Astrocytic ET-1 overexpression after MCAO impairs neurogenesis in the DG region.
结果：To determine whether NSCs in the WT and GET-1 mice differentiated into astrocytes or neurons after ischemic stroke, we conducted GFAP/BrdU and NeuN/BrdU costaining 28 days after MCAO. We found no significant differences between NeuN+BrdU+ and GFAP+BrdU+ costained cells on the contralateral (normal) side in GET-1 and WT mice (data not shown). However, at 28 days after MCAO, WT mice had more NeuN+BrdU+ cells in the ipsilateral DG region than GET-1 mice (P = 0.034 for GET-1 ips vs. WT ips; Figure 3A and C). In contrast, we observed slightly more GFAP+BrdU+ cells in GET-1 mice than in WT mice, but this difference was not significant (Figure 3B and D). This result suggests that ET-1 overexpression impairs neurogenesis in the DG region after cerebral ischemic injury (Figure 3E). In addition, Ki67 is a protein that labels cells that are actively dividing but not cells that have divided and have exited the cell cycle. We conducted Ki67/NeuN and Ki67/GFAP double immunofluorescence labeling at 3 months after ischemic stroke, and observed similar expression trends in Ki67+NeuN+ and Ki67+GFAP+ cells on the ipsilateral sides in WT and GET-1 mice (Additional Figure 2).
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