The identification of endogenous signals that lead to microglial activation is a key step in understanding neuroinflammatory cascades. As ATP release accompanies mechanical strain to neural tissue, and as the P2X7 receptor for ATP is expressed on microglial cells, we examined the morphological and molecular consequences of P2X7 receptor stimulation in vivo and in vitro and investigated the contribution of the P2X7 receptor in a model of increased intraocular pressure (IOP). In vivo experiments involved intravitreal injections and both transient and sustained elevation of IOP. In vitro experiments were performed on isolated mouse retinal and brain microglial cells. Morphological changes were quantified in vivo using Sholl analysis. Expression of mRNA for M1- and M2-like genes was determined with qPCR. The luciferin/luciferase assay quantified retinal ATP release while fura-2 indicated cytoplasmic calcium. Microglial migration was monitored with a Boyden chamber. Sholl analysis of Iba1-stained cells showed retraction of microglial ramifications 1 day after injection of P2X7 receptor agonist BzATP into mouse retinae. Mean branch length of ramifications also decreased, while cell body size and expression of Nos2, Tnfa, Arg1, and Chil3 mRNA increased. BzATP induced similar morphological changes in ex vivo tissue isolated from Cx3CR1+/GFP mice, suggesting recruitment of external cells was unnecessary. Immunohistochemistry suggested primary microglial cultures expressed the P2X7 receptor, while functional expression was demonstrated with Ca2+ elevation by BzATP and block by specific antagonist A839977. BzATP induced process retraction and cell body enlargement within minutes in isolated microglial cells and increased Nos2 and Arg1. While ATP increased microglial migration, this required the P2Y12 receptor and not P2X7 receptor. Transient elevation of IOP led to microglial process retraction, cell body enlargement, and gene upregulation paralleling changes observed with BzATP injection, in addition to retinal ATP release. Pressure-dependent changes were reduced in P2X7−/− mice. Death of retinal ganglion cells accompanied increased IOP in C57Bl/6J, but not P2X7−/− mice, and neuronal loss showed some association with microglial activation. P2X7 receptor stimulation induced rapid morphological activation of microglial cells, including process retraction and cell body enlargement, and upregulation of markers linked to both M1- and M2-type activation. Parallel responses accompanied IOP elevation, suggesting ATP release and P2X7 receptor stimulation influence the early microglial response to increased pressure.

中文翻译：

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P2X7 受体刺激和眼压升高诱导小胶质细胞的快速形态变化以及混合小胶质细胞激活状态标记物的上调

识别导致小胶质细胞激活的内源信号是理解神经炎症级联的关键步骤。由于 ATP 释放伴随着神经组织的机械应变，并且由于 ATP 的 P2X7 受体在小胶质细胞上表达，我们检查了体内和体外 P2X7 受体刺激的形态和分子后果，并研究了 P2X7 受体在模型中的贡献眼压（IOP）升高。体内实验涉及玻璃体内注射以及短暂和持续的眼压升高。对分离的小鼠视网膜和脑小胶质细胞进行了体外实验。使用 Sholl 分析对体内形态变化进行量化。通过 qPCR 测定 M1 和 M2 样基因的 mRNA 表达。荧光素/荧光素酶测定定量视网膜 ATP 释放，而 fura-2 指示细胞质钙。用博伊登室监测小胶质细胞的迁移。对 Iba1 染色细胞的 Sholl 分析显示，将 P2X7 受体激动剂 BzATP 注射到小鼠视网膜 1 天后，小胶质细胞分支收缩。分枝的平均分支长度也减少，而细胞体大小和 Nos2、Tnfa、Arg1 和 Chil3 mRNA 的表达增加。BzATP 在从 Cx3CR1+/GFP 小鼠分离的离体组织中诱导了类似的形态变化，表明不需要招募外部细胞。免疫组织化学表明原代小胶质细胞培养物表达 P2X7 受体，而功能性表达通过 BzATP 升高 Ca2+ 并被特定拮抗剂 A839977 阻断来证明。在分离的小胶质细胞中，BzATP 在几分钟内诱导突起收缩和细胞体增大，并增加 Nos2 和 Arg1。虽然 ATP 增加了小胶质细胞的迁移，但这需要 P2Y12 受体而不是 P2X7 受体。除了视网膜 ATP 释放外，眼压短暂升高还导致小胶质细胞突起收缩、细胞体增大和基因上调，与 BzATP 注射观察到的变化平行。P2X7−/− 小鼠中压力依赖性变化减少。在 C57Bl/6J 小鼠中，视网膜神经节细胞的死亡伴随着眼压的增加，但 P2X7−/− 小鼠则不然，并且神经元损失显示出与小胶质细胞活化存在一定的相关性。P2X7 受体刺激诱导小胶质细胞快速形态激活，包括突起收缩和细胞体增大，以及与 M1 和 M2 型激活相关的标记物上调。平行反应伴随着 IOP 升高，表明 ATP 释放和 P2X7 受体刺激影响早期小胶质细胞对压力升高的反应。