Astrocytic Ca2+ Signaling in the Ischemic Penumbra

缺血半暗带中的星形胶质细胞 Ca2 信号传导

• 批准号: 8208947
• 负责人: Maiken Nedergaard
• 金额: $ 38.61万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8208947
• 关键词: 101 Mouse; Acute; Address; Adenosine; Adenosine A1 Receptor; Animals; Area; Astrocytes; Attention; Bioluminescence; Blood capillaries; Blood flow; Brain; Calcium Signaling; Calculi; Cell Respiration; Cells; Cessation of life; Characteristics; Connexin 43; Cytoprotection; Depressed mood; Detection; Development; Drops; Electrons; Energy Supply; Event; Exhibits; Experimental Models; Extracellular Space; Fluorescence; Glial Fibrillary Acidic Protein; Glucose; Glutamates; Glycolysis; Heterogeneity; Hypoxia; Image; Imaging Techniques; In Situ; Individual; Infarction; Injury; Ischemia; Ischemic Penumbra; Ischemic Stroke; Laser Scanning Microscopy; Life; Light; Link; Mammalian Cell; Maps; Mediating; Membrane Potentials; Metabolic; Microcirculation; Middle Cerebral Artery Occlusion; Modeling; Modification; Monitor; Mus; NADH; Neuronal Injury; Neurons; Neuroprotective Agents; Neurotransmitters; Oxidation-Reduction; P2X-receptor; Pathway interactions; Penetration; Perfusion; Photons; Principal Investigator; Process; Publications; Published Comment; Purinergic P1 Receptors; Purinoceptor; Reporter; Research; Resolution; Resources; Role; Signal Transduction; Specificity; Stroke; Synapses; Synaptic Transmission; Techniques; Testing; Therapeutic Agents; Tissues; Toxic effect; Transgenic Organisms; Validation; Variant; Work; base; capillary; cell type; chelation; expectation; improved; in vivo; innovation; mimetics; molecular imaging; neuronal survival; photolysis; programs; receptor; research study; response; selective expression; sulforhodamine 101; synaptic depression; tool; transmission process; two-photon

The ischemic penumbra was originally defined as peri-infarct tissue with partial reduced blood flow. The blood flow reduction (30-60% of control values) was less severe than in the ischemic core allowing the penumbral tissue to maintain normal transmembrane ionic gradients. The electrical silence of the penumbra was ascribed to the partial reduction in substrate delivery, e.g. the energy supply was sufficient to maintain membrane potential, but not to support synaptic transmission. The fate of astrocytes in the penumbra has received little attention, in part, because the activity of these non-excitable cells only can be analyzed by Ca2+ imaging technique. Combined with recording of local field potential, we can simultaneously monitor local microcirculation, astrocytic Ca2+ signaling, and synaptic activity within the same field. We have observed that astrocytes, as opposed to neurons, are activated and display spontaneous Ca2+ oscillations, as well as propagating Ca2+ waves in the ischemic penumbra. Astrocytic Ca2+ signaling is associated with the release of several neurotransmitters, including glutamate and ATP.-ATP is, in the extracellular space, rapidly degraded to adenosine - anendogenous neuroprotective agent. On the basis of these observations, this proposal will test the proposition that astrocytic Ca2+ signaled- release of ATP, and the latter's rapid degradation to adenosine, mediates the electrical silence of the ischemic penumbra. In Aim 1, we will characterize ATP release in the setting of focal ischemia. Aim 2 intends to define astrocytic Ca2+ signaling in the penumbra, using 2-photon laser scanning microscopy to establish which transmitters (glutamate, ATP) trigger the abnormal Ca2+ signaling by local application of receptor antagonists. Aim 3 will directly image NADH to assess the cellular metabolic responses to reduced blood flow in the ischemic penumbra, and will define the respective contributions of astrocytes and neurons in this regard. Combined imaging of capillary flow and Ca2+ or NADH imaging will allow a correlation between local perfusion and astrocytic Ca2+ signaling or NADH on a single cell level in live animals. For these experiments, we will use of transgenic Thy1-YFP loaded with the astrocyte-specific indicator, sulforhodamine 101 mice. Aim 4 then asks if astrocytic Ca2+ signaling by release of ATP/adenosine reduce synaptic transmission, lower metabolic demands, and thereby increase neuronal survival in the penumbra. In specific, we will define the role of adenosine A1 receptors in synaptic depression in the penumbra. Preliminary observations show that the adenosine A1 receptor antagonist, DPCPX, triggered a robust increase in synaptic activity in the penumbra without a concomitant increase in blood flow. Our expectation is that a more precise mechanistic understanding of the role of astrocytes in this process will ultimately justify the development and assessment of adenosine mimetics and modulators as therapeutic agents in ischemic stroke. PHS 398 (Rev. 09/04) Page 71. Form Page 2 PO1 NS050315 Project 1 Principal Investigator/Program Director (Last, First, Middle): Nedergaard, Maiken Introduction to revised application This is a second revision of our PPG application entitled The role of astrocytes in ischemic stroke, and of my section therein, Astrocytic calcium signaling in the ischemic penumbra. The past submission of this proposal was praised for being highly innovative, and for addressing several fundamentally new areas in stroke research. However, the referees raised concerns regarding: 1) our technical ability to causally link acute ischemic events, e.g. ATP release and Ca2+ signaling, to neuronal death; 2) the concept of ATP as an excitatory transmitter, and its potential to trigger neuronal death in ischemia; and, 3) the specificity of P2X receptor antagonists. Based on the reviewers' comments, we have chosen to focus this revised application on acute cellular responses to ischemia, thereby taking maximal advantage of our 2-photon imaging approaches to assessing both intracellular and intercellular signaling events in situ. I have also dropped Aim 3, which correlated ATP release with delayed neuronal injury, and which was viewed as too preliminary by the referees. We have since last application refined the technique of in vivo NADH imaging in the ischemic penumbra. NADH is the principal electron carrier in glycolytic and oxidative metabolism, and is an intrinsic indicator of cellular redox state. Recent developments in 2-photon imaging have improved its spatial resolution substantially, so much so as to resolve subcellular changes in NADH within the ischemic cortex. Another advantage of 2-photon NADH imaging is that we can identify and isolate changes in redox state, in both individual cells and their processes. Use of Thy1-YFP mice, that selectively express YFP in central neurons, combined with loading of the astrocyte specific indicator, sulforhodamine 101, has enabled us to separately assess the respective metabolic responses of neurons and astrocytes within the same field of view. Our preliminary observations have indicated a strong compartmentalization of metabolic responses in the ischemic penumbra. Combined imaging of NADH and capillary flow should therefore enable us to precisely define changes in neuronal and astrocytic redox state, in response to experimentally-defined reductions in blood flow. By imaging NADH and Ca2+, we intend to assess the interdependence of hypoxia-associated NADH and Ca2+ increases. Our collaborator, Frank Kirchhoff, Gottingen, has established a set of spectrally distinct GFAP reporter mice that include GFAP-EGFP, GFAP-AMCyan, and GFAP-mRFP1, which will permit us to directly visualize astrocytes concurrently with NADH and Ca2+ imaging. In light of the upgrade of our 2-photon imaging setup to 3 channel detection, these mice will greatly facilitate the combined analysis of Ca2+, capillary perfusion, and NADH in astrocytes and neurons. By this means, we intend to assess the interaction between Ca2+ signaling and NADH levels among defined cell types within the ischemic penumbra. In our revised aim 3, 1intend to test the hypothesis that astrocytic ATP release comprises a conserved mechanism of cell protection. Our studies so far have suggested that adenosine's neuroprotective effects outweigh P2XR-mediated excitotoxic injury in the cortex. We found that P2X receptor antagonists have little effect upon neuronal injury in the penumbra, whereas adenosine receptor antagonists potently increased ischemic injury after MCA occlusion. ATP is released by astrocytes in response to reduced perfusion in the ischemic penumbra. Besides its activation of local purinergic receptors, it is also rapidly converted to adenosine, which potently inhibits excitatory transmission while lowering cellular energy demands. One of the defining characteristics of the ischemic penumbra is its electrical silence. On that basis, we suspect that neuronal activity is depressed by adenosine; as a corollary to this postulate, we have found that adenosine receptor antagonists increase electrical activity in the ischemic penumbra (Fig. 15). We believe these additions and modifications to our proposal allow us to take better advantage of our available imaging and molecular resources, while expanding both the breadth and rigor of our analysis of the role of astrocytes in stroke. Specific points: Reviewer 2 was unconvinced by the rationale for correlating rates of glycolysis with ATPrelease. We have removed this aim. The validity of the proposed experiments with BAPTA-AM and uncaging was questioned by reviewer2. Chelation of cytosolic Ca2+ with BAPTA is widely used approach to study the functional significance of Ca2+ signaling. BAPTA does efficiently block cytosolic Ca2+ increases, and has low toxicity. We agree with the reviewer that BAPTA-AM and uncaging are unphysiological approaches, but also would like to point out that they are excellent tools to define the impact of Ca2+ signaling. Photolysis of caged Ca2+ is considered state-of- the-art in study of astrocytes and we used this approach in a recent publication (Takano et al., 2006). Few approaches exist that selectively target astrocytes. Use of BAPTA and caged Ca2+ can add important PHS 398/2590 (Rev. 09/04) Page 72~ Continuation Format Page

缺血半暗带最初被定义为血流量部分减少的梗塞周围组织。 血流量减少（对照值的 30-60%）不如缺血核心严重，从而允许 半影组织维持正常的跨膜离子梯度。半影的电寂静 归因于底物输送的部分减少，例如能源供应足以维持 膜电位，但不支持突触传递。半影中星形胶质细胞的命运 很少受到关注，部分原因是这些非兴奋细胞的活性只能通过 Ca2+ 来分析 成像技术。结合局部场电位的记录，我们可以同时监测局部 同一区域内的微循环、星形细胞 Ca2+ 信号传导和突触活动。我们观察到 与神经元相反，星形胶质细胞被激活并表现出自发的 Ca2+ 振荡，以及 在缺血半暗带中传播 Ca2+ 波。星形胶质细胞 Ca2+ 信号传导与释放 多种神经递质，包括谷氨酸和 ATP。-ATP 在细胞外空间中迅速降解 腺苷 - 一种内源性神经保护剂。 根据这些观察结果，该提案将检验星形胶质细胞 Ca2+ 发出信号的命题—— ATP 的释放以及后者快速降解为腺苷，介导了 ATP 的电沉默 缺血半暗带。在目标 1 中，我们将描述局灶性缺血情况下 ATP 释放的特征。目标 2 打算 为了定义半影中的星形细胞 Ca2+ 信号传导，使用 2 光子激光扫描显微镜建立 哪些递质（谷氨酸、ATP）通过受体的局部应用触发异常的 Ca2+ 信号传导 对手。目标 3 将直接对 NADH 进行成像，以评估细胞对血流量减少的代谢反应 在缺血半暗带中，并将定义星形胶质细胞和神经元在该区域中各自的贡献 看待。毛细血管流和 Ca2+ 或 NADH 成像的组合成像将允许局部之间的相关性 活体动物单细胞水平上的灌注和星形细胞 Ca2+ 信号传导或 NADH。对于这些实验， 我们将使用装载有星形胶质细胞特异性指示剂的转基因 Thy1-YFP，磺胺罗丹明 101 小鼠。 目标 4 然后询问星形细胞 Ca2+ 信号通过释放 ATP/腺苷是否会减少突触传递，降低 代谢需求，从而增加半影中神经元的存活率。具体来说，我们将定义 腺苷 A1 受体在半暗带突触抑制中的作用。初步观察表明 腺苷 A1 受体拮抗剂 DPCPX 引发了突触活性的强劲增加 半暗带不伴随血流量增加。我们的期望是更精确的机械 了解星形胶质细胞在此过程中的作用将最终证明开发和评估的合理性 腺苷模拟物和调节剂作为缺血性中风治疗剂的研究。 PHS 398（修订版 09/04）第 71 页。表格第 2 页 PO1 NS050315 项目 1 首席研究员/项目总监（姓、名、中）：Nedergaard, Maiken 修订申请简介 这是我们的 PPG 申请的第二次修订，题为星形胶质细胞在缺血性中风中的作用，以及 我的部分是缺血半暗带中的星形胶质细胞钙信号传导。过去提交的这个 该提案因其高度创新性和针对中风的几个全新领域而受到赞扬 研究。然而，裁判员对以下方面提出了担忧：1）我们将急性因果关系联系起来的技术能力 缺血事件，例如ATP 释放和 Ca2+ 信号传导导致神经元死亡； 2）ATP的概念 兴奋性递质及其在缺血时引发神经元死亡的潜力；以及，3) P2X 的特异性 受体拮抗剂。根据审稿人的意见，我们选择将此修订后的应用程序重点关注 细胞对缺血的急性反应，从而最大限度地利用我们的 2 光子成像方法 原位评估细胞内和细胞间信号传导事件。我还放弃了目标 3， 将 ATP 释放与迟发性神经元损伤联系起来，裁判认为这过于初步。 自上次应用以来，我们改进了缺血性脑损伤体内 NADH 成像技术 半影。 NADH是糖酵解和氧化代谢中的主要电子载体，是一种内在的电子载体。 细胞氧化还原状态的指标。 2 光子成像的最新进展提高了其空间分辨率 基本上，足以解决缺血皮质内 NADH 的亚细胞变化。其他 双光子 NADH 成像的优点是我们可以识别和隔离氧化还原态的变化，无论是 单个细胞及其过程。使用在中枢神经元中选择性表达 YFP 的 Thy1-YFP 小鼠， 与星形胶质细胞特异性指示剂磺基罗丹明 101 的负载相结合，使我们能够分别 评估同一视野内神经元和星形胶质细胞各自的代谢反应。我们的 初步观察表明，缺血区的代谢反应存在强烈的区隔性。 半影。因此，NADH 和毛细血管流的联合成像应该使我们能够精确定义 神经元和星形细胞氧化还原状态的变化，响应于实验定义的血流量减少。 通过对 NADH 和 Ca2+ 进行成像，我们打算评估缺氧相关的 NADH 和 Ca2+ 的相互依赖性 增加。我们的合作者，来自哥廷根的 Frank Kirchhoff，建立了一套光谱上不同的 GFAP 包括 GFAP-EGFP、GFAP-AMCyan 和 GFAP-mRFP1 的报告小鼠，这将使我们能够直接 与 NADH 和 Ca2+ 成像同时可视化星形胶质细胞。鉴于我们的2光子成像的升级 设置为3通道检测，这些小鼠将极大地促进Ca2+、毛细血管的联合分析 灌注，以及星形胶质细胞和神经元中的 NADH。通过这种方式，我们打算评估之间的相互作用 缺血半暗带内特定细胞类型的 Ca2+ 信号传导和 NADH 水平。 在我们修订后的目标 3 中，1 打算检验以下假设：星形胶质细胞 ATP 释放包含保守的 细胞保护机制。迄今为止我们的研究表明腺苷的神经保护作用 超过 P2XR 介导的皮质兴奋性毒性损伤。我们发现 P2X 受体拮抗剂几乎没有 对半暗带神经元损伤的影响，而腺苷受体拮抗剂则有效增加 MCA 闭塞后缺血性损伤。星形胶质细胞响应减少的灌注而释放 ATP 缺血半暗带。除了激活局部嘌呤能受体外，它还能迅速转化为 腺苷，可有效抑制兴奋性传递，同时降低细胞能量需求。中的一个 缺血半暗带的定义特征是其电静默。在此基础上，我们怀疑 腺苷抑制神经元活动；作为这个假设的推论，我们发现腺苷 受体拮抗剂增加缺血半暗带的电活动（图15）。我们相信这些补充 对我们提案的修改使我们能够更好地利用我们现有的成像和分子技术 资源，同时扩大我们对星形胶质细胞在中风中作用的分析的广度和严格性。 具体要点： 审稿人 2 不相信糖酵解速率与 ATP 释放相关的基本原理。 我们已经取消了这个目标。 所提出的 BAPTA-AM 和解笼实验的有效性受到审稿人 2 的质疑。 用 BAPTA 螯合胞质 Ca2+ 是广泛用于研究功能意义的方法 Ca2+ 信号传导。 BAPTA 能有效阻止胞浆 Ca2+ 增加，且毒性低。我们同意 审稿人认为 BAPTA-AM 和解笼是非生理方法，但也想指出 它们是定义 Ca2+ 信号传导影响的优秀工具。笼中 Ca2+ 的光解被认为是状态- 这是星形胶质细胞研究的最新技术，我们在最近的出版物中使用了这种方法（Takano 等，2006）。很少 存在选择性靶向星形胶质细胞的方法。使用 BAPTA 和笼式 Ca2+ 可以增加重要的 PHS 398/2590 (Rev. 09/04) 第 72 页~ 续格式页