Elsevier

Brain Research

Volume 1388, 4 May 2011, Pages 148-156
Brain Research

Research Report
Disruption of downstream MyD88 or TRIF Toll-like receptor signaling does not protect against cerebral ischemia

https://doi.org/10.1016/j.brainres.2011.02.074Get rights and content

Abstract

Toll-like receptor (TLR) signaling plays an important role in cerebral ischemia, but downstream signaling events, which can be organ-specific, are incompletely understood. We thereby investigated involvement of the MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling pathways in 2 in vitro and 2 in vivo models of cerebral ischemia. For in vitro studies, we used a model of oxygen-glucose deprivation (OGD) followed by flow cytometric analysis to determine:1) viability of PC12 cells following knock-down with MyD88 siRNA compared to negative control siRNA and 2) viability, apoptosis and necrosis of cortical neurons from MyD88 null (−/−) , TRIF mutant, and wild type (WT) mice. In addition, in vivo, 1) We examined CA1 neuronal survival 7 days after global forebrain ischemia and 2) infarct volumes 24 h after Middle Cerebral Artery Occlusion (MCAO) in all 3 types of mice. OGD: 1) There were no differences in either percent viability of PC12 cells transfected with MyD88 compared to negative control siRNA or 2) in percent viability, apoptosis and necrosis of cortical neurons from MyD88−/−,TRIF mutant and WT mice. Global ischemia: neuronal survival was similar in all 3 groups of mice. Finally, MCAO: infarct volumes were not statistically different among all 3 groups of mice: MyD88−/−, 23.9 ± 9.9 mm3, TRIF mutant, 26.7 ± 5.8 mm3 and WT, 17.9 ± 8.4 mm3. These findings show that disruption of MyD88 or TRIF signaling does not confer protection in brain ischemia and suggests the possibility of additional or alternate downstream adaptors during TLR signaling in cerebral ischemia.

Research highlights

► MyD88 knock-down does not affect PC12 cell viability in vitro. ► Disruption of MyD88 or TRIF has no effect on cortical neuron viability in vitro. ► CA1 neuronal survival is unaffected by MyD88 or TRIF disruption in global ischemia. ► No differences in infarct sizes in WT, MyD88 and TRIF mutant mice after pMCAO.

Introduction

Stroke remains the third leading cause of death and the leading cause of long-term serious disability in the United States and new approaches for treatment are desperately needed. One such approach involves understanding the role of pathways leading to inflammation such as the Toll-like receptor (TLR) signaling pathway in controlling the brain's response to ischemia.

Toll-like receptors are evolutionarily conserved pattern-recognition receptors important for initiation of innate immune responses to pathogen-associated molecular patterns (PAMPs). However, the “danger model” of immunity suggests that “danger signals” that activate the innate immune response to infection are not restricted to PAMPs such as lipopolysaccharide (LPS;TLR4 ligand) (Matzinger, 2002a, Matzinger, 2002b), but can include endogenous danger-associated molecular patterns (DAMPs) released during ischemic stress or during the subsequent restoration of blood flow (reperfusion) (Medzhitov, 2001).

Evidence supporting the involvement of the TLR signaling pathway in the innate immune response to brain ischemia includes elevated TLR4 message levels in blood cells of patients with acute cerebral ischemia compared to controls (Yang et al., 2008) and protection against cerebral ischemia in mice with Toll-like receptor deletions/mutations (Caso et al., 2007, Ziegler et al., 2007, Tang et al., 2007). While these studies focus on upstream TLR signaling events, very little is known about the molecules that propagate TLR signals downstream during cerebral ischemia. Understanding these downstream signaling events is important as it may lead to the identification of specific points along the pathway that can be modulated during cerebral ischemia. Also, there is evidence that downstream TLR signaling pathways may be organ-specific. For instance in cardiac ischemia, disruption of myeloid differentiation factor 88 (MyD88) signaling is protective (Hua et al., 2005). In hepatic ischemia–reperfusion, however, downstream TLR signaling seems to occur via the Toll/IL-1 Receptor-containing adapter inducing IFN-β (TRIF) as opposed to the MyD88 pathway (Tsung et al., 2006). In addition, in renal ischemia, downstream TLR 2 signaling seems to be mediated via a MyD88-independent pathway, as mice with deletions of TLR2 are better protected from renal ischemia compared to mice with disruptions of the currently known TLR2 downstream adaptor, MyD88 (Shigeoka et al., 2007).

Currently, downstream TLR signaling is known to occur via two major pathways, the MyD88-dependent pathway and the MyD88-independent pathway or TRIF pathway (Kumar et al., 2009).

In light of the fact that the Toll-like receptors previously shown to be important in cerebral ischemia both use the MyD88 and TRIF adaptors for downstream signaling, we sought to systematically determine whether one, both, or neither, of these two known TLR adaptors, MyD88 and TRIF, were important or preferentially used in downstream TLR signaling during cerebral ischemia. We used two in vitro and two in vivo models of cerebral ischemia in our study.

Section snippets

Confirmation of MyD88−/− and TRIF mutant genotypes

Polymerase Chain Reaction (PCR): PCR of tail DNA, from all 3 types of mice, using MyD88 primers, to confirm the genotype of MyD88−/− mice compared to TRIF mutant and WT mice, yielded the expected DNA size fragments (data not shown).

MyD88 knock-down does not affect PC12 cell viability in vitro

PC12 cells transfected

Discussion

Our results showed that neither deletion of MyD88 nor disruption of TRIF had any clear effect on 1) cell viability in in vitro ischemia, 2) CA1 neuron survival in global forebrain ischemia or 3) on infarct size in a model of permanent focal ischemia in contrast to the previously reported roles of MyD88 and TRIF in downstream TLR signaling in cardiac and hepatic ischemia respectively (Hua et al., 2005, Tsung et al., 2006). However, there was an unexpected trend toward larger infarct volumes in

Animals

We used 12- to 16-week-old, male, WT C57BL6/J and TRIF mutant mice (Jackson Laboratories) and MyD88 knock-out mice (breeder pairs from Institute for Systems Biology, Seattle, WA). Genotypes of both MyD88−/− and TRIF mutant mice were confirmed by Polymerase Chain Reaction (PCR) and DNA sequencing. WT C57BL6/J mice served as controls since both TRIF mutant and MyD88 knock-out mice were on a C57BL6/J background. All mice were housed and bred together in the institutional animal facility. Mice had

Disclosures

We have no conflict of interest and nothing to disclose.

Acknowledgments

Dr. Maria Spatz for editorial and scientific suggestions and Dr. Sungyoung Auh for help with the statistical analysis. This research was supported by the Intramural Research Program of the National Institutes of Health, National institute of Neurological Disorders and Stroke.

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