Elsevier

Neuroscience

Volume 171, Issue 1, 24 November 2010, Pages 258-267
Neuroscience

Neurodegeneration, Neuroprotection, and Disease-Oriented Neuroscience
Research Paper
Toll-like receptor 4 (TLR4), but not TLR3 or TLR9, knock-out mice have neuroprotective effects against focal cerebral ischemia

https://doi.org/10.1016/j.neuroscience.2010.08.054Get rights and content

Abstract

Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is a specific immunologic response to systemic bacterial infection. We investigated whether cerebral ischemia induced by the middle cerebral artery occlusion (MCAO) for 2 h differed in mice that lack a functional TLR3, TLR4, or TLR9 signaling pathway. TLR4, but not TLR3 or TLR9, knock-out (KO) mice had significantly smaller infarct area and volume at 24 h after ischemia-reperfusion (I/R) compared with wild-type mice. In addition, TLR4 KO mice improved in neurological deficits after I/R compared with wild-type mice. Moreover, we investigated the expression of TLR4 in the ischemic brain with immunohistochemistry. The number of TLR4-positive cells gradually increased from 1 h after MCAO to 22 h after I/R. We also examined the localization of TLR4 in the ischemic area. TLR4 was localized with CD11b-positive microglial cells in the ischemic striatum and the number of CD11b-positive microglial cells was smaller in TLR4 KO mice than in wild-type mice. In addition, we investigated the translocation of NF-κB among TLR3, 4, and 9 KO mice after I/R injury using western blotting. NF-κB's p65 subunit was decreased in TLR4 KO mice compared to wild-type mice, but not TLR3 or 9 KO mice. These data suggest that TLR4 knockout, but not TLR3 or TLR9 knockout, may play a neuroprotective role in ischemic brain injury induced by MCAO in mice.

Section snippets

Experimental materials

The experiments were conducted in accordance with the Animal Care Guidelines issued by the Animal Experiments Committee of Gifu Pharmaceutical University. All efforts were made to minimize both suffering and the number of animals used. The TLR3 KO and TLR9 KO mice were backcrossed nine generation onto a BALB/c background. TLR4 KO mice were backcrossed nine generation onto a C57BL/6 background or a BALB/c background. The animals were housed at 24±2 °C under a 12 h light-dark cycle (lights on

Physiological parameters

There were no significant differences in mean arterial blood pressure (MABP) or heart rate (HR), in arterial pH, pCO2, pO2, or in rCBF between the wild-type and TLR4 KO mice (data not shown). Surface rCBF was reduced to approximately 20% of the baseline value immediately after MCAO in all mice (data not shown).

Infarction and neurological deficits after ischemia

Twenty-four hours after I/R, mice developed infarcts affecting the cortex and striatum (Fig. 1, Fig. 2). TLR4 KO mice had significantly smaller infarct area and volume at 24 h after MCAO

Discussion

The main aim of this study was to investigate the stroke outcomes of TLR3, TLR4, or TLR9 KO mice. The present study showed that TLR4 KO mice have smaller cerebral infarctions compared with wild-type mice. On the other hand, in TLR3 KO and TLR9 KO mice, there were no significant differences in stroke outcomes (infarct area, infarct volume, or neurological deficits), suggesting that TLR3 or TLR9 signaling pathway is not directly related to ischemic brain injury induced by I/R. Activation of TLRs

Conclusion

In conclusion, TLR4, but not TLR3 or TLR9, KO mice showed a neuroprotective effect on the neuronal damage and neurological deficits after ischemia, and TLR4 was up-regulated in response to ischemia-reperfusion in the brain, suggesting that TLR4 may play a pivotal role in the pathogenesis of the ischemic brain damage induced by MCAO.

Acknowledgments

The authors thank Dr. Tsuyoshi Sugiyama (Microbiology, Gifu Pharmaceutical University) for technical support.

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