Research

Journal of Neuroinflammation

, 10:23

Open Access This content is freely available online to anyone, anywhere at any time.

Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia

  • Linli YaoAffiliated withKey Laboratory of the Ministry of Education for Experimental Teratology, Shandong Provincial Key Laboratory of Mental Disorders, Department of Histology and Embryology, Shandong University School of MedicineDepartment of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore
  • , Enci Mary KanAffiliated withDefense Medical and Environmental Research Institute, DSO National Laboratories
  • , Jia LuAffiliated withDefense Medical and Environmental Research Institute, DSO National Laboratories
  • , Aijun HaoAffiliated withKey Laboratory of the Ministry of Education for Experimental Teratology, Shandong Provincial Key Laboratory of Mental Disorders, Department of Histology and Embryology, Shandong University School of Medicine
  • , S Thameem DheenAffiliated withDepartment of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore
  • , Charanjit KaurAffiliated withDepartment of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore Email author 
  • , Eng-Ang LingAffiliated withDepartment of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore Email author 

Abstract

Background

Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury.

Methods

One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-κB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1α) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining.

Results

TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-α, IL-1β and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-α, IL-1β, iNOS, ROS and NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-κB activation. Furthermore, HIF-1α antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-α, IL-1β and iNOS on microglia post-hypoxia.

Conclusion

Activated microglia TLR4 expression mediated neuroinflammation via a NF-κB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.

Keywords

Toll-like receptor 4 Microglia NF-κB Hypoxia-inducible factor-1α Hypoxia Inflammation