Isoflurane preconditioning provides neuroprotection against stroke by regulating the expression of the TLR4 signalling pathway to alleviate microglial activation

Scientific Reports, Jun 2015

Excessive microglial activation often contributes to inflammation-mediated neurotoxicity in the ischemic penumbra during the acute stage of ischemic stroke. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation via the NF-κB pathway. Isoflurane preconditioning (IP) can provide neuroprotection and inhibit microglial activation. In this study, we investigated the roles of the TLR4 signalling pathway in IP to exert neuroprotection following ischemic stroke in vivo and in vitro. The results showed that 2% IP alleviated neurological deficits, reduced the infarct volume, attenuated apoptosis and weakened microglial activation in the ischemic penumbra. Furthermore, IP down-regulated the expression of HSP 60, TLR4 and MyD88 and up-regulated inhibitor of IκB-α expression compared with I/R group in vivo. In vitro, 2% IP and a specific inhibitor of TLR4, CLI-095, down-regulated the expression of TLR4, MyD88, IL-1β, TNF-α and Bax, and up-regulated IκB-α and Bcl-2 expression compared with OGD group. Moreover, IP and CLI-095 attenuated microglial activation-induced neuronal apoptosis, and overexpression of the TLR4 gene reversed the neuroprotective effects of IP. In conclusion, IP provided neuroprotection by regulating TLR4 expression directly, alleviating microglial activation and neuroinflammation. Thus, inhibiting the activation of microglial activation via TLR4 may be a new avenue for stroke treatment.

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Isoflurane preconditioning provides neuroprotection against stroke by regulating the expression of the TLR4 signalling pathway to alleviate microglial activation

Abstract Excessive microglial activation often contributes to inflammation-mediated neurotoxicity in the ischemic penumbra during the acute stage of ischemic stroke. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation via the NF-κB pathway. Isoflurane preconditioning (IP) can provide neuroprotection and inhibit microglial activation. In this study, we investigated the roles of the TLR4 signalling pathway in IP to exert neuroprotection following ischemic stroke in vivo and in vitro. The results showed that 2% IP alleviated neurological deficits, reduced the infarct volume, attenuated apoptosis and weakened microglial activation in the ischemic penumbra. Furthermore, IP down-regulated the expression of HSP 60, TLR4 and MyD88 and up-regulated inhibitor of IκB-α expression compared with I/R group in vivo. In vitro, 2% IP and a specific inhibitor of TLR4, CLI-095, down-regulated the expression of TLR4, MyD88, IL-1β, TNF-α and Bax, and up-regulated IκB-α and Bcl-2 expression compared with OGD group. Moreover, IP and CLI-095 attenuated microglial activation-induced neuronal apoptosis, and overexpression of the TLR4 gene reversed the neuroprotective effects of IP. In conclusion, IP provided neuroprotection by regulating TLR4 expression directly, alleviating microglial activation and neuroinflammation. Thus, inhibiting the activation of microglial activation via TLR4 may be a new avenue for stroke treatment. Introduction Ischemia/reperfusion (I/R)-induced brain injury is implicated in the pathophysiology of stroke and results in an initial area of neuronal death known as the core, surrounded by an area vulnerable to further damage known as the penumbra1. Furthermore, neuronal apoptosis and necrosis in the penumbra can be viewed as primary causes of aggravated cerebral injury and functional impairment. Additionally, neuronal apoptosis can be exacerbated by the excessive formation of inflammatory reactions after an ischemic stroke, which has been demonstrated to be the one cause contributing to cerebral I/R injury2. Although several studies have been devoted to developing methods to reduce inflammatory reactions during ischemic cerebral injury, effective methods have not yet been established. Inflammatory processes are involved in a broad range of pathologies and diseases of the central nervous system (CNS) including stroke, brain trauma and brain infection3. Previous studies have found that acute inflammatory cascade reactions that cause overactivation of microglia and the release of inflammatory substances can aggravate neuronal injury in the cerebral penumbra4. Microglia are macrophage-like cells that are considered the major immune cells in the brain4,5. During the response to various endogenous or exogenous stimuli, microglia can be activated to produce inflammatory cytokines such as IL-1, TNF and inducible nitric oxide (iNOS), which can induce neuronal apoptosis and aggravate brain injury6,7. Therefore, reducing microglial overactivation in the penumbra could be beneficial and might provide neuroprotection against cerebral ischemia. Isoflurane is commonly used in the clinic and is a relatively safe volatile anesthetic. Many studies have shown that isoflurane preconditioning (IP) can induce ischemic tolerance and exert neuroprotection against cerebral I/R injury8,9,10,11. Zuo found that rats that were pretreated with 2% isoflurane could reduce neuronal apoptosis and regulate the expression of anti-apoptotic protein Bcl-2 after stroke in vivo12 and attenuate microglial activation in vitro13. However, despite the important role of IP in inhibiting microglial activation, little information is available regarding how IP reduces microglial activation and provides neuroprotection during the process of the ischemic stroke. TLRs are key mediators of innate immunity that respond to injury-induced endogenous ligands from necrotic cells such as HSPs and diverse microbial products14. In particular, Toll-like receptor 4 (TLR4) is expressed primarily in microglia15,16,17. We and other studies have reported that TLR4 can induce microglial activation and cytokine production in brain injury, trauma and neurodegenerative diseases18,19. Moreover, Hara et al. reported that TLR4 knockout (KO) mice had significantly smaller infarct volumes and better neurobehavioural recovery at 24 h after I/R injury compared with wild-type mice. Furthermore, these authors found that attenuated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in TLR4 KO mice can provide neuroprotection during acute focal cerebral I/R injury20. Another study also showed that TLR4 expression in activated microglia mediated neuroinflammation via an NF-κB signalling pathway in response to hypoxia and that TLR4 inhibition can attenuate TNF-α and IL-1 expression against hypoxia21. Hence, microglial TLR4 may be as a potential therapeutic target to inhibit microglial activation for ischemic stroke treatm (...truncated)


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Meiyan Sun, Bin Deng, Xiaoyong Zhao, Changjun Gao, Lu Yang, Hui Zhao, Daihua Yu, Feng Zhang, Lixian Xu, Lei Chen, Xude Sun. Isoflurane preconditioning provides neuroprotection against stroke by regulating the expression of the TLR4 signalling pathway to alleviate microglial activation, Scientific Reports, 2015, Issue: 5, DOI: 10.1038/srep11445