Ability of Local Clearance of Senescent Cells in Ipsilateral Hemisphere to Mitigate Acute Ischemic Brain Injury in Mice

Abstract

Senolytic treatment has potential therapeutic efficacy for acute ischemic stroke (AIS). However, the systemic treatment of senolytics may produce off-target side effects and a toxic profile, which affect analysis of the role of acute senescence of neuronal cells in pathogenesis of AIS. We constructed a novel lenti-INK-ATTAC viral vector to introduce INK-ATTAC genes to the ipsilateral brain and locally eliminate senescent brain cells by administering AP20187 to activate caspase-8 apoptotic cascade. In this study, we have found that acute senescence is triggered by middle cerebral artery occlusion (MCAO) surgery, particularly in astrocytes and cerebral endothelial cells (CECs). The upregulation of p16^INK4a and senescence-associated secretory phenotype (SASP) factors including matrix metalloproteinase-3, interleukin-1 alpha and -6 were observed in oxygen-glucose deprivation-treated astrocytes and CECs. The systemic administration of a senolytic, ABT-263, prevented the impairment of brain activity from hypoxic brain injury in mice, and significantly improved the neurological severity score, rotarod performance, locomotor activity, and weight loss. The treatment of ABT-263 reduced senescence of astrocytes and CECs in MCAO mice. Furthermore, the localized removal of senescent cells in the injured brain through the stereotaxical injection of lenti-INK-ATTAC viruses generates neuroprotective effects, protecting against acute ischemic brain injury in mice. The content of SASP factors and mRNA level of p16^INK4a in the brain tissue of MCAO mice were significantly reduced by the infection of lenti-INK-ATTAC viruses. These results indicate that local clearance of senescent brain cells is a potential therapy on AIS, and demonstrate the correlation between neuronal senescence and pathogenesis of AIS.

Keywords: acute ischemic stroke; lenti-INK-ATTAC viral vector; senolytic treatment.

Figure 1

Expression of p16^INK4a, a typical marker of senescence, and SASP factors in the ipsilateral brain of C57BL/6 mice after MCAO. (A) A representative photo of the immunohistochemistry staining of p16^INK4a in the ipsilateral (Ipsi) brain of mice subjected to MCAO taken using the EVOS FL Cell Imaging System. The photograph represents three similar experiments. Blue depicts the nucleus, red depicts p16^INK4a, and the white bar represents 1 mm. (B) The mRNA levels of cellular senescence markers, namely p16^INK4a, IL-6, CCL8, and CXCL2, were determined in the cortex, hippocampus, and striatum of the mice 6, 12, and 24 h after MCAO. All data are represented as means ± SEMs (n = 8); ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001, compared with sham control group. The percentage change in senescent neuronal cells, namely cerebral endothelial cells, astrocytes, microglia, and neurons (C) in the brain of 6 weeks, 18 weeks and 24 months old mice or (E) in the ipsilateral brain 24 h and 48 h after MCAO was analyzed using flow cytometry. (D&F) Quantification of the flow cytometry analysis. All data are represented as means ± SEMs (n = 4 (C); n = 6 (E)). ^**P < 0.01, and ^***P < 0.001, compared with the 6-weeks group (C) or contralateral (Con) group (E).

Figure 2

Acute cellular senescence was provoked in astrocytes and CECs of ipsilateral cortex through MCAO and in OGD-stimulated isolated primary astrocytes and CECs. The expression of senescence marker p16^INK4a in (A) astrocytes and (B) CECs was observed using immunohistochemistry staining and confocal microscopy in the ipsilateral cortex of mice 24 h after MCAO. Arrows indicate senescent cells, and white bars represent 125 μm. (C) The content of SASP factors, namely MMP-3, IL-6, and IL-1α, in the brain tissue of mice without or with MCAO surgery was evaluated using ELISA kits. Data are presented as means ± SEMs (n = 4-6); ^***P < 0.001, compared with the sham control group. The expression of p16^INK4a in the isolated primary astrocytes (D) and CECs (E) was triggered by 4.5-h OGD and determined after 24-h by using confocal microscopy. The white bars represent 50 μm. (F) The concentration of MMP-3, IL-1α, and IL-6 in the conditioned medium of primary astrocytes and CECs stimulated by OGD was determined using ELISA kits. Data are presented as means ± SEMs (n = 3); ^*P < 0.05 and ^**P < 0.01, compared with the resting group.

Figure 3

Oral administration of senolytic ABT-263 alleviates the impairment of brain activity and neurological deficits through MCAO in C57BL/6 mice. (A) 18F-FDG PET of the ipsilateral cortex, hippocampus, and striatum in mice subjected to MCAO was performed on Days 1, 3, and 7 after surgery. Mice were treated with or without ABT-263. (B) The relative 18F-FDG binding ratio of the ipsilateral cortex, hippocampus, and striatum to the cerebrum in the mice treated with or without ABT-263 on Days 1, 3, and 7 post-MCAO. (C) The NSSs (a), rotarod test (b), locomotor activity (c), and body weight change (d) of the mice treated with or without ABT-263 were examined 0, 1, 3, 7, 14, 21, 28 days after MCAO. (D) Astrocyte (cell marker: GFAP) and CECs (cell marker: CD31) of mice treated with or without ABT-263 7 days after MCAO were sorted by FACS. (E) The mRNA levels of astrocytes and CECs in the mice treated with or without ABT-263. The mRNA expression of each gene was normalized using GAPDH mRNA expression. Data are presented as means ± SEMs (n = 8) ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001, compared with the vehicle group.

Figure 4

Elimination of senescent cells through infection of lenti-INK-ATTAC viruses in H₂O₂-treated HEK293 cells and C57BL/6 mice subjected to MCAO. (A) The third-generation lenti-viral vector was used to label p16^INK4a-expressing cells. LTR: long terminal repeats, CMV: cytomegalovirus. (B) Full sequence of the novel lent-INK-ATTAC viral vector. (C) In vitro experiment. HEK293 cells were transfected with lenti-INK-ATTAC viruses for 48 h and then stimulated with or without H₂O₂ (150 μM) for 4 h. H₂O₂ -treated cells were then treated with or without AP20187 for 24 h. (D) The GFP-positive cells were observed using confocal microscopy. The white bar represents 75 μm. (E) The infection of lenti-INK-ATTAC viruses was performed 5 days before MCAO and AP20187 was treated 1 day before MCAO for 5 consecutive days. (F) Immunohistochemistry staining was used to determine the expression of GFPs in lenti-INK-ATTAC-infected C57BL/6 mice with MCAO and with or without AP20187 treatment. Blue depicts the nucleus, green depicts GFPs, and the white bar represents 50 μm. Data are presented as means ± SEMs (n = 5); ^***P < 0.001, compared with the H₂O₂ control group.

Figure 5

Neuroprotective effects of local elimination of senescent cells through stereotaxical injection of lenti-INK-ATTAC viruses in the ipsilateral brain of C57BL/6 mice after acute hypoxic brain injury. (A) Lenti-GFP or Lenti-INK-ATTAC virus injection, AP20187 administration, and data collection. (B) Body weight change (C) The NSSs (a), rotarod test (b), locomotor activity (c and d) of mice infected with or without lenti-GFP or lenti-INK-ATTAC and treated with or without AP20187 (AP) were examined 0, 1, 3, 7, 14, 21 days after MCAO. Data are presented as means ± SEMs (n = 12); ^###P < 0.001, compared with the sham group. ^*P < 0.05 and ^***P < 0.001, compared with the INK-ATTAC+MCAO group. (C and D) The concentration of MMP-3, IL-1α, and IL-6, and the mRNA expression of p16^INK4a in the brain tissue of MCAO mice infected with or without lenti-GFP or lenti-INK-ATTAC and treated with or without AP20187 were evaluated using ELISA kits and quantitative reverse transcription PCR, respectively. Data are presented as means ± SEMs (n = 8); ^###P < 0.001, compared with the sham group. ^*P < 0.05 and ^**P < 0.01, compared with the INK-ATTAC+MCAO group.

Figure 6

Local Elimination of Senescent Cells in Ipsilateral Hemisphere to Reduce Ischemic Brain Injury in a Mouse Model of Acute Ischemic Stroke. MCA: Middle Cerebral Artery; SASP: Senescence Associated Secretory Phenotype