Abstract
Background: Accumulating evidence suggests that neuroinflammation plays a critical role in early brain injury after subarachnoid hemorrhage (SAH). Pannexin-1 channels, as a member of gap junction proteins located on the plasma membrane, releases ATP, ions, second messengers, neurotransmitters, and molecules up to 1 kD into the extracellular space, when activated. Previous studies identified that the opening of Pannexin-1 channels is essential for cellular migration, apoptosis and especially inflammation, but its effects on inflammatory response in SAH model have not been explored yet.Methods: Adult male Sprague-Dawley rats were divided into six groups: sham group (n = 20), SAH group (n = 20), SAH + LV-Scramble-ShRNA group (n = 20), SAH + LV-ShRNA-Panx1 group (n = 20), SAH + LV-NC group (n = 20), and SAH + LV-Panx1-EGFP group (n = 20). The rat SAH model was induced by injection of 0.3 ml fresh arterial, non-heparinized blood into the prechiasmatic cistern in 20 s. In SAH + LV-ShRNA-Panx1 group and SAH + LV-Panx1-EGFP group, lentivirus was administered via intracerebroventricular injection (i.c.v.) at 72 h before the induction of SAH. The Quantitative real-time polymerase chain reaction, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting were performed to explore the potential interactive mechanism between Pannexin-1 channels and TLR2/TLR4/NF-κB-mediated signaling pathway. Cognitive and memory changes were investigated by the Morris water maze test.Results: Administration with LV-ShRNA-Panx1 markedly decreased the expression levels of TLR2/4/NF-κB pathway-related agents in the brain cortex and significantly ameliorated neurological cognitive and memory deficits in this SAH model. On the contrary, administration of LV-Panx1-EGFP elevated the expressions of TLR2/4/NF-κB pathway-related agents, which correlated with augmented neuronal apoptosis.Conclusion: Pannexin-1 channels may contribute to inflammatory response and neurobehavioral dysfunction through the TLR2/TLR4/NF-κB-mediated pathway signaling after SAH, suggesting a potential role of Pannexin-1 channels could be a potential therapeutic target for the treatment of SAH.
Highlights
Subarachnoid hemorrhage (SAH), especially aneurysmal SAH, accounting for 5% of all stroke types, is a catastrophic cerebrovascular disease with high morbidity and mortality throughout the world
To determine the time course of Pannexin-1 channels expression after SAH, rat brain tissues were obtained at different time points after SAH and assayed by Western blot
For the first time, we demonstrated that Pannexin-1 channels were involved in TLR2/TLR4/NF-κBmediated inflammatory responses following SAH
Summary
Subarachnoid hemorrhage (SAH), especially aneurysmal SAH, accounting for 5% of all stroke types, is a catastrophic cerebrovascular disease with high morbidity and mortality throughout the world. During the inflammatory response process after SAH, activation of TLRs-mediated pathway is reported to initiate the inflammatory cascades and damage neurons and white matter (Ascenzi et al, 2005; Pradilla et al, 2010). The signaling cascades initiated by TLR2 and TLR4 show much overlap and cooperativity Activation of these receptors results in the activation of NF-κB via the MyD88-/TRIF-dependent pathways, which induces the upregulation of leukocyte adhesion molecules and immunomodulatory cytokines, and increases the production of pro-inflammatory cytokines. Previous studies identified that the opening of Pannexin-1 channels is essential for cellular migration, apoptosis and especially inflammation, but its effects on inflammatory response in SAH model have not been explored yet. Results: Administration with LV-ShRNA-Panx markedly decreased the expression levels of TLR2/4/NF-κB pathway-related agents in the brain cortex and significantly ameliorated neurological cognitive and memory deficits in this SAH model. Administration of LV-Panx1-EGFP elevated the expressions of TLR2/4/NF-κB pathway-related agents, which correlated with augmented neuronal apoptosis
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