Activation of Toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion

Abstract

Toll-like receptors (TLRs) play a critical role in the induction of innate immune responses which have been implicated in neuronal death induced by global cerebral ischemia/reperfusion (GCI/R). The present study investigated the role and mechanisms-of-action of TLR4 signaling in ischemiainduced hippocampal neuronal death. Neuronal damage, activation of the TLR4 signaling pathway, expression of pro-inflammatory cytokines and activation of the PI3K/Akt signaling pathway in the hippocampal formation (HF) were assessed in wild type (WT) mice and TLR4 knockout mice (TLR4 -/-) mice after GCI/R. GCI/R increased expression of TLR4 protein in the hippocampal formation (HF) and other brain structures in WT mice. Phosphorylation of the inhibitor of kappa B (p-IκB) as well as activation of nuclear factor kappa B (NFκB) increased in the HF of WT mice. In contrast, there were lower levels of p-IκB and NFκB binding activity in TLR4 -/-mice subjected to GCI/R. Pro-inflammatory cytokine expression was also decreased, while phosphorylation of Akt and GSK3β were increased in the HF of TLR4 -/-mice after GCI/R. These changes correlated with decreased neuronal death/apoptosis in TLR4 -/-mice following GCI/R. These data suggest that activation of TLR4 signaling contributes to ischemia-induced hippocampal neuronal death. In addition, these data suggest that modulation of TLR4 signaling may attenuate ischemic injury in hippocampal neurons.

Protective role of isoflurane pretreatment in rats with focal cerebral ischemia and the underlying molecular mechanism

“…TLR4-mediated NF-κB signaling contributes to myocardial ischemia-reperfusion injury (23). Quantitative studies using western blot analysis were performed to further observe whether ISO pretreatment affected the expression levels of TLR4, MyD88 and NF-κB.…”

Preconditioning with a TLR2 specific ligand increases resistance to cerebral ischemia/reperfusion injury

“…To evaluate infarct size, brains were harvested and stained with thiphenyltetrazolium chloride (TTC). To examine the levels of tight junction proteins, brain tissues were harvested and cellular proteins isolated (Hua et al 2007b). For immunohistochemistry staining, mice were anesthetized with Ketamine and transcardially perfused with normal saline followed by 30 ml of 4% buffered paraformaldehyde, pH 7.4.…”

“…For immunohistochemistry staining, mice were anesthetized with Ketamine and transcardially perfused with normal saline followed by 30 ml of 4% buffered paraformaldehyde, pH 7.4. The brain was removed, postfixed, embedded in paraffin and cut into sections (7μm) as described previously (Hua et al 2007b). …”

“…Cellular proteins were prepared from ischemic cerebral hemispheres (Hua et al 2007b), electrophoresed with SDS-polyacrylamide gel and transferred onto Hybond ECL membranes (Amersham Pharmacia, Piscataway, NJ) (Hua et al 2007a;Hua et al 2005;Hua et al 2007b). The ECL membranes were incubated with the appropriate primary antibody followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc.).…”

“…The IHC staining for tight junction protein ZO-1 were performed as described previously (Hua et al 2005;Hua et al 2007b). The primary antibody employed was rabbit anti-ZO-1 (40-6100, Invitrogen, South San Francisco, CA).…”