CHANGES IN THE MICROENVIRONMENT AFTER BRAIN INJURY OF VARYING SEVERITY AND RELATION TO FUNCTIONAL OUTCOME

Ph.DDOCTOR OF PHILOSOPH

Scutellarin regulates the Notch pathway and affects the migration and morphological transformation of activated microglia in experimentally induced cerebral ischemia in rats and in activated BV-2 microglia

10.1186/s12974-014-0226-zJournal of Neuroinflammation12

Notch-1 signaling regulates microglia activation via NF-κB pathway after hypoxic exposure in vivo and in vitro.

Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain

Up-regulation of Notch-1 and Delta-1 expression in primary cultured microglia following hypoxia.

<p>(<b>A</b>) 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>B and C</b>) 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>B</b>) and 40 µm (<b>C</b>).</p

DAPT pretreatment inhibited the increase in NF-κB immunoexpression in microglia of neonatal rats after hypoxic treatment.

<p>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.</p

Antibodies used for western Blotting.

<p>Antibodies used for western Blotting.</p

Notch signaling was activated in primary cultured microglia exposed to hypoxia.

<p>(<b>A</b>) 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>B</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 *<i>p</i><0.05 and ** <i>p</i><0.01. Scale bars  = 50 µm (<b>A</b>).</p