Fig 2: Effect of hemoglobin-a knockdown on HIF-1a expression in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). (A)/(a) and (B)/(a) Representative photomicrographs of HIF-1a immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-a knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. (A)/(b) and (B)/(b) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in HIF-1a expression in sham after Hb-a knockdown (*: p < 0.05, n = 6). At 7 days after GCI reperfusion (R7d), Hb-a knockdown robustly increased HIF-1a expression in both hippocampal CA1 (A) and CA3 regions (B). (*: p < 0.05, **: p < 0.01, n = 6). Scale bar: 20 µm.

Fig 3: Lenti-CRISPRi efficiently inactivates HBA1/2 (Hb-a) expression in mouse hippocampal CA1 neurons with a resultant increase in hypoxia. (A). Schematic demonstration of the conditional CRISPRi system used to selectively suppress HBA1/2 expression in the hippocampal CA1 and CA3 regions. (B). Representative IHC for Hb-a in hippocampal CA1 region at 2 weeks after infusion with scramble (upper) or HBA1/2 (lower) lenti-CRISPRi. (C). Western Blot analysis showing effectiveness of HBA1/2 lenti-CRISPRi knockdown of Hb-a in mouse hippocampus at 2 weeks after infusion. (D–G). HBA1/2-lenti-CRISPRi effectively knocked down Hb-a in the hippocampal CA1 and CA3 regions, an effect that correlated with significantly increased CA1 and CA3 hypoxia as measured by pimonidazole staining at 2 weeks after lentivirus infusion (*: p < 0.05, n = 6). Scale bar: (B), 10 µm; (D,E), 20 µm.

Fig 4: Effect of hemoglobin-a knockdown on expression of the HIF-1a-regulated pro-apoptotic gene, PUMA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). (A,C) Representative photomicrographs of PUMA immunostaining in 3-month-old male mouse hippocampal CA1 (A) and CA3 (C) regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-a knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. Staining in red represents NeuN, a neuronal marker, and staining in green represents PUMA. (B,D) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in PUMA intensity in sham after Hb-a knockdown. At 7 days after GCI reperfusion (R7d), Hb-a knockdown robustly increased PUMA intensity in the hippocampal CA1 and CA3 regions (**: p < 0.01, n = 6). Scale bar: 20 µm.

Fig 5: Effect of hemoglobin-a knockdown on expression of the HIF-1a-regulated pro-apoptotic gene, NOXA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). (A,C) Representative photomicrographs of NOXA immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-a knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. (B,D). Quantification of mean immuno-fluorescence (IF) demonstrates a mild increase in NOXA expression in sham after Hb-a knockdown (**: p < 0.01, n = 6). At 7 days after GCI reperfusion (R7d), Hb-a knockdown robustly increased NOXA expression in both CA1 (A) and CA3 regions (C). (**: p < 0.01, n = 6). Scale bar: 20 µm.