Blocking P2X7 by intracerebroventricular injection of P2X7-specific nanobodies reduces stroke lesions

作者：Maximilian Wilmes, Carolina Pinto Espinoza, Peter Ludewig, Joschi Stabernack, Arthur Liesz, Annette Nicke, Mathias Gelderblom, Christian Gerloff, Simonetta Falzoni, Eva Tolosa, Francesco Di Virgilio, Björn Rissiek, Nikolaus Plesnilla, Friedrich Koch‐Nolte, Tim Magnus · 发表于：Journal of Neuroinflammation · 年份：2022 · DOI：10.1186/s12974-022-02601-z · 被引用次数：49 · 研究领域：Adenosine and Purinergic Signaling、Neuroinflammation and Neurodegeneration Mechanisms、Vagus Nerve Stimulation Research

BACKGROUND: Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. METHODS: Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1β release after incubation with the P2X7-specific nbs. RESULTS: Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found low...