Table 1.
Gene sequence used for RT-PCR.
Table 2.
Antibodies used for western Blotting.
Figure 1.
Up-regulation of Notch-1 and Delta-1 expression in primary cultured microglia following hypoxia.
(A) Reverse transcription (RT)-PCR analysis of Notch-1 and Delta-1 mRNA expression in primary microglia exposed to hypoxia for 2, 4, 6, 12 and 24 h and control (c). Note the significant increase in Notch-1 and Delta-1 mRNA expression after hypoxia. (B and C) Confocal images showing Notch-1 expression (Bb, Bf; red) in primary cultured microglia labeled with lectin (Ba, Be; green) and Delta-1 expression (Cb, Ce; green) colocalized with OX-42 (Ca, Cd; red)) in both control and hypoxia for 12 h. Nuclei are stained with DAPI (blue). Note Notch-1and Delta-1 immunoflurosence intensity is markedly enhanced after hypoxia exposure (Bg, Cf) in comparison with the control (Bb, Cb). The values represent the mean ±SD in triplicate. Scale bars = 50 µm (B) and 40 µm (C).
Figure 2.
Notch signaling was activated in primary cultured microglia exposed to hypoxia.
(A) Immunofluorescence images showing NICD expression in primary microglia labeled with lectin (a, e; green). The expression is intensely augmented both in the cytoplasm and nucleus after hypoxic treatment for 12 h (f, g) compared with the control (b, c). (B) Reverse transcription (RT)-PCR analysis of RBP-Jκ and Hes-1 mRNA expression in primary microglia exposed to hypoxia for 2, 4, 6, 12 and 24 h and control (c). Note the significant increase in RBP-Jκ and Hes-1 mRNA expression after hypoxia. The values represent the mean ±SD in triplicate. Significant differences between control and hypoxic BV-2 cells are expressed as *p<0.05 and ** p<0.01. Scale bars = 50 µm (A).
Figure 3.
Notch signaling was expressed and activated in BV-2 cells following hypoxia.
(A) RT-PCR analysis showing the mRNA expression of Notch-1, Delta-1 and Hes-1 mRNA in BV-2 cells exposed to hypoxia was significantly increased compared with the control. (B) Western blotting of Notch-1, NICD, RBP-Jκ and Hes-1 protein expression in BV-2 cells exposed to hypoxia for 4, 6, 8 and 12 h and control (c). The left panel shows specific bands of Notch-1 (120 kDa), NICD (80 kDa), RBP-Jκ (56 kDa), Hes-1 (37 kDa) and β-actin (43 kDa). The right panel is bar graphs showing significant changes in the optical density following hypoxic exposure. Note significant increase in Notch-1, NICD, RBP-Jκ and Hes-1 expression after hypoxic treatment of varying durations in BV-2 cells. Significant differences between control and hypoxic BV-2 cells are expressed as *p<0.05 and ** p<0.01. The values represent the mean ±SD in triplicate.
Figure 4.
Notch signaling blockade in primary microglia and BV-2 cells by DAPT.
(A) No obvious morphological difference was observed in Hypoxia and Hypoxia+DAPT groups compared with the control primary microglia under the phase-contrast microscope. (B) The mRNA expression of RBP-Jκ and Hes-1 in primary microglia was significantly decreased in Hypoxia+DAPT group compared with Hypoxia group shown by RT-PCR analysis. (C) Confocal images showing NICD expression in BV-2 cells of different groups. NICD immunofluorescence intensity was reduced both in cytoplasm and nucleus in Hypoxia +DAPT BV-2 cells (Cc) compared with hypoxic BV-2 cells (Cb). (D) Western blotting of Notch-1 and Hes-1 protein expression in BV-2 cells after DAPT pretreatment. The left panel shows specific bands of Notch-1 (120 kDa), Hes-1 (37 kDa) and β-actin (43 kDa). The right panel is bar graphs showing Notch-1 protein expression was increased in Hypoxia+DAPT group compared with hypoxic BV-2 cells; while increase in Hes-1 protein expression after hypoxia was significantly inhibited in DAPT pretreated hypoxic BV-2 cells. Significant difference between control vs hypoxia groups is shown as *p<0.05 and **p<0.01; Significant difference between hypoxia vs hypoxia+DAPT groups is shown as #p<0.05 and ##p<0.01. The values represent the mean ±SD in triplicate. Scale bar in C = 40 µm.
Figure 5.
Notch blockade altered the mRNA expression of inflammatory cytokines and iNOS induced by hypoxic stress in primary microglia.
Reverse transcriptase (RT)-PCR analysis of TNF-α, IL-1β, iNOS, TGF-β1, M-CSF, IL-10 and IL-6 gene expression in primary microglia exposed to different duration of hypoxia with or without DAPT pretreatment. Note that mRNA expression of all the above mentioned genes is increased significantly to varying extents after hypoxic exposure for different duration. Significant difference between control vs hypoxia groups is shown as *p<0.05 and **p<0.01; significant difference between hypoxia vs hypoxia+DAPT groups is shown as #p<0.05 and ##p<0.01. The values represent the mean ±SD in triplicate.
Figure 6.
Notch blockade altered protein expression of inflammatory cytokines, iNOS and nitric oxide (NO) secretion in hypoxic BV-2 cells.
(A and B) Western blotting of TNF-α, IL-1β and iNOS (A); TGF-β1, M-CSF and IL-10 (B) protein expression in BV-2cells following 8 h of hypoxic exposure and DAPT pretreatment. The upper panel shows specific bands of TNF-α (25.6 k Da), IL-1β (17 kDa), iNOS (130 kDa) and β-actin (43 kDa) (A); TGF-β1 (25 kDa), M-CSF (18.5 kDa), IL-10 (17 kDa) and β-actin (43 kDa) (B). The lower panel in A and B are bar graphs showing significant changes in the optical density in protein expression of different groups. Note the decrease in TNF-α, IL-1β and iNOS expression (A) as well as TGF-β1 and M-CSF expression (B) in hypoxia+DAPT group compared with hypoxic BV-2 cells. A noteworthy feature was the increase in IL-10 protein expression after DAPT pretreatment in hypoxic BV-2 cells (B). (C) NO production in supernatant of different groups of cells. Note the NO production, which is increased after hypoxic exposure for 8 h is decreased nearly to basal level after hypoxic exposure in the DAPT treated BV-2 cells. Significant difference between control vs hypoxia groups is shown as *p<0.05 and **p<0.01; significant difference between control vs hypoxia and hypoxia vs hypoxia+DAPT groups is shown as #p<0.05 and ##p<0.01. The values represent the mean ±SD in triplicate.
Figure 7.
DAPT treatment inhibited NF-κB activation and translocation induced by hypoxic stress in BV-2 cells.
(A). Western blot analysis of NF-κB/p65 protein expression in BV-2 cells of different groups. The upper panel shows specific bands of NF-κB/p65 (65 kDa) and β-actin (43 kDa) and the lower panel bar graph showing significant changes in the optical density of different groups. Note the NF-κB/p65 protein expression, which is increased after hypoxic exposure in control BV-2 cells, is significantly decreased after hypoxic exposure in DAPT treated BV-2 cells. (B) ELISA analysis of phospho-NF-kB/p65 in nucleus of different groups of BV-2 cells showing the content of phospho-NF-kB/p65 in nucleus is increased in BV-2 cells after hypoxic stress; however, phospho-NF-kB/p65 content is drastically reduced in hypoxic BV-2 cells pretreated with DAPT compared with the hypoxic BV-2 cells. Significant difference between control vs hypoxia groups is shown as *p<0.05 and **p<0.01; significant difference between hypoxia vs hypoxia+DAPT groups is shown as #p<0.05 and ##p<0.01. The values represent the mean ±SD in triplicate.
Figure 8.
TLR4/MyD88/TRAF6 pathway was inhibited in hypoxic microglia with Notch signaling blockade.
(A) Notch blockade suppressed TLR4 mRNA expression. RT-PCR analysis showing the hypoxia induced increase in mRNA expression of TLR4 in primary microglia was significantly suppressed when pretreated with DAPT. (B) Western blotting of MyD88 and TRAF6 protein expression in BV-2 cells exposed to hypoxia for 2, 4, 6, 8, 12 and 24 h and control (c). The upper panel shows specific bands of MyD88 (38 k Da), TRAF6 (60 k Da) and β-actin (43 kDa). The lower panel is bar graphs showing significant changes in the optical density following hypoxic exposure. Note the MyD88 and TRAF6 protein expression after hypoxia is significantly increased. (C) RT-PCR analysis showing the hypoxia induced increase in mRNA expression of MyD88 and TRAF6 in primary microglia is significantly suppressed when pretreated with DAPT. (D) Western blotting of MyD88 and TRAF6 protein expression in BV-2 cells exposed to hypoxia for 8 h, hypoxic BV-2 cells pretreated with DAPT and corresponding control (c). The bar graphs showing increase in MyD88 and TRAF6 protein expression induced by hypoxia is significantly suppressed in DAPT pretreated group. Significant difference between control vs hypoxia groups is shown as *p<0.05 and **p<0.01; significant difference between hypoxia vs hypoxia+DAPT groups is shown as #p<0.05 and ##p<0.01. The values represent the mean ±SD in triplicate.
Figure 9.
Delta-1 expression was increased in the microglial cells in subventricular zone and corpus callosum of neonatal rats following hypoxic exposure.
Confocal images showing the distribution of lectin (green) and Delta-1 (red) immunoreactive microglial cells in the subventricular zone (a–f) and corpus callosum (g–l) of neonatal rats at 3 days after hypoxic exposure and the corresponding control. Very weak Delta-1 expression (arrows) is detected in the SVZ of control rats, but the immunoflurorescence intensity is enhanced and more Delta-1 positive microglial cells are observed after hypoxia. In the corpus callosum, Delta-1 expression is barely detected in microglia of control rats (h and i) and some Delta-1 positive cells colocalized with lectin (arrowheads) are seen after hypoxia (k and l). Scale bar = 40 µm.
Figure 10.
NICD expression was increased in the corpus callosum of neonatal rats following hypoxic exposure.
Confocal images showing the expression of NICD (red) in the corpus callosum of neonatal rats 3 and 7 days after hypoxia and the corresponding control. Microglial cells were labeled with lectin (green). Very week NICD immunofluorescence intensity was observed in lectin-positive microglia in the control rats of both 3 (b–c) and 7 (g–i) days. NICD immunofluorescence intensity in microglia is enhanced after hypoxic exposure at 3 (d–f) and 7 (j–l) days after hypoxia, especially at 3 days (d–f) in comparison with the control (j–l)). Nuclei are stained with DAPI (blue). Scale bars = 20 µm.
Figure 11.
DAPT pretreatment inhibited the increase in NF-κB immunoexpression in microglia of neonatal rats after hypoxic treatment.
Confocal images showing the expression of NF-κB in lectin-labeled (green) microglia (arrows) in the corpus callosum of control (a–c), hypoxia (d–f) and hypoxia +DAPT (g–i) rats at 24 h after hypoxic exposure. Increase in NF-κB expression in microglia of the corpus callosum was evident in hypoxic rats (e,f). In hypoxia +DAPT rats, increase in NF-κB was inhibited when compared with that in the hypoxic rats (h,i). Note lack of NF-κB expression in lectin positive blood vessels (arrowhead). Scale bar = 20 µm.