Less disruption of the blood-brain barrier (BBB) after severe ischemic stroke is one of the beneficial outcomes of ischemic preconditioning (IP). However, the effect of IP on tight junctions (TJs), which regulate paracellular permeability of the BBB, is not well understood. In the present study, we examined IP-induced changes in TJs before and after middle cerebral artery occlusion (MCAO) in mice, and the association between changes in TJs and tolerance to a subsequent insult. After IP, we found decreased levels of transmembrane TJ proteins occludin and claudin-5, and widened gaps of TJs with perivascular swelling at the ultrastructural level in the brain. An inflammatory response was also observed. These changes were reversed by inhibition of extracellular signal-regulated kinase1/2 (ERK1/2) via the specific ERK1/2 inhibitor U0126. After MCAO, reduced brain edema and inflammatory responses were associated with altered levels of angiogenic factors and cytokines in preconditioned brains. Pretreatment with U0126 reversed the neuroprotective effects of IP against MCAO. These findings suggest that ERK1/2 activation has a pivotal role in IP-induced changes in TJs and inflammatory response, which serve to protect against BBB breakdown and inflammation after ischemic stroke.
Toll-like receptors (TLRs), which have been implicated in various neuroinflammatory responses, are thought to act in defense mechanisms by inhibiting neuronal cell death in Alzheimer's disease. In this study, we evaluated the effects of TLR2 on amyloid beta peptide 25-35 (Aβ25-35)-induced neuronal cell death, synaptic dysfunction, and microglial activation in organotypic hippocampal slice cultures (OHSCs) from wild-type (WT) C57BL/6 mice and TLR2-knockout (KO) mice. In WT mice, Aβ25-35 induced β-amyloid aggregation and surrounding TLR2 expression. And, propidium iodide (PI) uptake, which is a measure of cell death, increased in a dose-dependent manner in slices with Aβ25-35 treatment. In the Aβ25-35-treated TLR2-KO OHSCs, the PI uptake was significantly attenuated to the control level, indicating that the cells were less susceptible to Aβ25-35-induced neuronal toxicity. In the ultrastructural analysis, nuclear shrinkage, slightly swollen mitochondria, and degraded organelles were detected in the Aβ25-35-treated slices from WT mice but not in the Aβ25-35-treated slices from TLR2-KO, suggesting the resistance of TLR2-KO to Aβ25-35-induced neurotoxicity. In Aβ25-35-treated OHSCs of WT mice, the levels of phosphorylated tau were increased and the levels of synaptophysin were decreased in a dose-dependent manner, but they were not changed in OHSCs of TLR2-KO mice. In WT mice, Aβ25-35 increased total protein level and immunoreactivity of Iba-1, which was colocalized with TLR2. However, there were no significant changes in the slices of Aβ25-35-treated TLR2-KO mice. These results suggested that TLR2 may play a role in Aβ25-35-induced neuronal cell loss and synaptic dysfunction through the activation of microglia in OHSCs.
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