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Original article

Toll-like receptor 4/nuclear factor-kappa B signaling detected in brain after early subarachnoid hemorrhage

MA Chun-xiao, YIN Wei-ning, CAI Bo-wen, WU Jian, WANG Jun-yi, HE Min, SUN Hong, DING Jun-li

and YOU Chao

Keywords: subarachnoid hemorrhage; toll-like receptor 4; nuclear factor-kappa B; inflammatory cytokine

Background Inflammation and immunity play a vital role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). Nuclear factor-kappa B (NF-κB) regulates many genes essential for inflammation and immunity and is activated by toll-like receptor (TLR). This study aimed to detect the expression of the toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling in the rat brain after early SAH.

Methods The rats were decapitated and their brains were removed at 0, 2, 4, 6, 12, 24 and 48 hours after a single injection of blood into the prechiasmatic cistern. mRNA expression of TLR4 was measured by Taqman real-time RT-PCR, and protein expression by immunohistochemistry and Western blotting. NF-κB activity and concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA).

Results TaqMan real-time RT-PCR and Western blotting identified a biphasic change in TLR4 expression in both mRNA and protein: an initial peak (2–6 hours) and a sustained elevation (12–48 hours). Immunohistochemical staining showed the inducible expression of TLR4-like immunoreactions predominantly in glial cells and vascular endothelium. A similar biphasic change in the activation of NF-κB subunit p65 as well as the production of NF-κB-regulated proinflammatory cytokines (TNF-α, IL-1β and IL-6) were detected by ELISA.

Conclusions These data suggest that experimental SAH induces significant up-regulation of TLR4 expression and the NF-κB signaling in early brain injury. Activation of the TLR4/NF-κB signaling may regulate the inflammatory responses after SAH.

Chin Med J 2009;122(13):1575-1581

ubarachnoid hemorrhage (SAH) is associated with high mortality: 12.4% result in sudden death before receiving medical attention1 and up to 40%–60% of such patients die within the first 48 hours because of initial bleeding.2 Brain injury secondary to the initial bleeding largely contributes to unfavorable outcomes after SAH.3 However, the mechanisms leading to secondary brain injury, in particular during the early period after SAH, remain poorly understood.

The inflammatory response after SAH is likely to contribute to brain injury.4 Proinflammatory mediators including cytokines, chemokines, adhesive molecules, etc, have been shown to be associated with SAH.5 In addition, inflammatory and immune responses are positively correlated with the poor outcomes of SAH.6,7 However, the sources or mechanisms for the inflammatory responses are not well understood. The nuclear factor kappa B (NF-κB) is a predominant regulator for inflammatory cytokines such as interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8). Thus, the signaling of NF-κB has been shown to be important in SAH.8-10

Many stimulators, including the above-mentioned cytokines, can stimulate the signaling of NF-κB activation. Toll-like receptors (TLRs) and IL-1 receptors share a common signaling pathway leading to NF-κB activation.11 TLR4 is the first identified member of the TLR family and has been widely investigated in various central nervous system (CNS) injury models, in particular stroke12,13 and neurodegenerative diseases.14,15 However, few data are available regarding the role of TLR4 in SAH-induced brain injury. A recent study demonstrates that the expression of TLR4 is significantly increased in the brain and basil artery on days 3, 5 and 7 after experimental SAH in rabbits.13-16 We hypothesized that elevation of proinflammatory cytokines after SAH may result from the activation of TLR4/NF-κB signaling in early brain injury.

In the present study, we detected the expression of TLR4/NF-κB signaling in the process of inflammation in

DOI: 10.3760/cma.j.issn.0366-6999.2009.13.019

Department of Neurosurgery, West China Hospital, Sichuan

University, Chengdu, Sichuan 610041, China (Ma CX, Yin WN,

Cai BW, Wu J, Wang JY, He M, Sun H and You C)

Institute of Digestive Surgery, Key Laboratory of Transplant

Engineering and Immunology, Ministry of Health, West China

Hospital, Sichuan University, Chengdu, Sichuan 610041, China

(Ding JL)

Correspondence to: Dr. YOU Chao, Department of Neurosurgery,

West China Hospital, Sichuan University, Chengdu, Sichuan

610041, China (Tel: 86-28-85422490. Fax: 86-28-85164009.