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. 2021 Mar 26;13:612856. doi: 10.3389/fnagi.2021.612856

Figure 5.

Figure 5

Direct reprogramming of astrocytes attenuated microglial activation and astrogliosis in the post-stroke brain. Inflammatory activities including microglia activation and astrogliosis were examined in the peri-infarct region. (A,B) Iba-1 expression was examined 6 weeks post ND1 infection. Iba-1 positive microglia are present in significantly greater numbers in the ipsilateral cortex compared to sham control or contralateral cortex. The iba-1 fluorescence of phagocytic microglia was markedly less in the ND1-treated cortex. (B) The number of microglia in the ischemic cortex was significantly increased after stroke; ND1 injection showed a large reduction in the number of microglia cells in the region. N = 5/group; one-way ANOVA (F(2,12) = 118.4, p < 0.0001), *p < 0.05 vs. sham control, #p < 0.05 vs. empty control vector. (C–F) Astrocyte accumulation or astrogliosis was evaluated in the peri-infarct region using immunohistochemical imaging 6 weeks after stroke after stroke. Images in C show accumulated astrocytes labeled by GFAP (green, arrows). The cell count (D), the thickness of the gliosis (E), and the mean GFAP area (F) were all significantly reduced. N = 6 per group paired t-test, *p < 0.05 vs. stroke controls. (G,H) Gliosis profile analysis by mean gray value across scar transection. The graph plots show median values of stroke control (red lines) and stroke plus ND1 (green lines). The bar graph in (H) quantified the measurement, demonstrating a significant reduction in GFAP positive astrogliosis after ND1 treatment. N = 3 paired t-test, *p < 0.05 vs. stroke controls. (I) Western blotting analysis of the GFAP level. The bar graph shows increased GFAP expression after stroke and the ND1 treatment attenuated this increase. N = 3, one-way ANOVA (F(2,19) = 4.525, p = 0.0247), Holm–Sidak’s Multiple Comparisons Test; *p < 0.05 vs. sham, #p < 0.05 vs. empty vector control. (J) An immunostaining image shows ND1-converted iNeurons (yellow) distributed inside the ischemic core (*). Cells were identified using mCherry (red), YFP (green), and NeuN (blue).