Glial Reaction to Ischemic Brain Injury

描述（由申请人提供）：大脑变得更有弹性，通过活动，包括从扩散性抑郁症（SD）的损伤。虽然，这一内在过程的机制仍不明确，它们涉及先天性细胞因子。重要的是，先天性细胞因子是多余的和多效性的，因此多种细胞因子可以具有相似的作用，并且单一细胞因子可以具有可变的作用。这是由于配体、受体和磷蛋白水平上复杂的相互作用信号。进一步的复杂性源于脑细胞类型的多样性，每种细胞都有独特的表达变化。迄今为止，这种复杂性阻碍了对其进行系统研究。我们将通过两种策略的组合来解决这一空白，以实现定义SD诱导的神经保护的先天细胞因子特征的总体目标。第一种方法使用先天性细胞因子途径变量的同时多重测量（即，细胞因子、受体、磷蛋白）。第二个应用经典的生物化学途径解剖策略来模拟（通过应用重组细胞因子配体）和调节（通过应用细胞因子途径抑制剂）对NMDA损伤或SD改变的生物反应。研究将在海马器官型培养物（HOTC）中进行，其类似于其体内对应物;缺乏混淆炎症变化的证据，并且是孤立的，因此不受全身免疫变化的影响。我们的数据表明，HOTC经历NMDA介导的损伤，SD和SD诱导的神经保护作用，就像在体内看到的那样。此外，这种神经保护依赖于细胞因子，通过补偿性，适应性反应。与新的工具结合，HOTC非常适合我们定义先天性细胞因子的特定目标：1）配体; 2）受体;和3）与单独的NMDA损伤相比，与SD诱导的NMDA损伤的神经保护相关的相关磷蛋白变化-作为建立引起SD诱导的神经保护的细胞和组织先天性细胞因子途径变化的涵盖特征的手段。这些目标将通过以下方式实现：新开发的基于微球的流式细胞术测定，以同时测量先天性细胞因子（IL-1 α、IL-1 β、TNF-α、IFN-γ、IL-6、IL-10），其受体（IL-1 R1、TNFR 1、IL-6 R α/gp 130、IFN γ R、IL-10 R）和相关下游磷蛋白（激酶（ERK 1/2、P38 MAPK、JNK）和转录因子（ATF-2、NF κ B、STAT 3））。使用来自激光解剖显微镜获得的特异性细胞增强样品（细胞因子）和双标记免疫组织化学（受体和磷蛋白）的mRNA的多重实时RT-PCR，将变化定位于细胞。先天性细胞因子通过基因激活触发改变的功能。我们认为，先天性细胞因子系统的代偿性变化的识别将预测哪些基因和相关的蛋白质通路，在系统内（和其他信号系统内）将是最佳的治疗靶点，以模仿内在神经保护的保护能力。

DESCRIPTION (provided by applicant): Brain becomes more resilient to injury through activity, including that from spreading depression (SD). While, mechanisms for this intrinsic process remain undefined, they involve innate cytokines. Importantly, innate cytokines are redundant and pleiotropic so multiple cytokines can have similar effects and a single cytokine can have variable effects. This is due to complex interactive signaling at the level of ligands, receptors, and phosphoproteins. Further complexity stems from the diversity of brain cell types, with each capable of unique expression changes. To date such complexity has precluded their systematic study. We will resolve this void via combination of two strategies to accomplish the general goal of defining the innate cytokine signature of SD-induced neuroprotection. The first uses simultaneous, multiplexed measurement of innate cytokine pathway variables (i.e., cytokines, receptors, phosphoproteins). The second applies classical biochemical pathway dissection strategies to mimic (by application of recombinant cytokine ligands) and modulate (by application of cytokine pathway inhibitors) the biological response to NMDA injury or its alteration by SD. Studies will be performed in hippocampal organotypic cultures (HOTCs), which resemble their in vivo counterparts; lack evidence of confounding inflammatory changes and are isolated, and thus, are not influenced by systemic immune changes. Our data show that HOTCs undergo NMDA-mediated injury, SD, and SD-induced neuroprotection like that seen in vivo. Furthermore, this neuroprotection depends on cytokines, via compensatory, adaptive responses. Coupled with novel tools, HOTCs are well suited to our specific aims of defining innate cytokine: 1) ligand; 2) receptor; and 3) related phosphoprotein changes associated with SD-induced neuroprotection from NMDA injury compared to NMDA injury alone - as a means to establish an encompassing signature of the cellular and tissue innate cytokine pathway changes that evoke SD-induced neuroprotection. These aims will be accomplished using: newly developed microsphere-based flow cytometric assays to simultaneously measure innate cytokines (IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-10), their receptors (IL-1R1, TNFR1, IL-6Ralpha/gp130, IFNgammaR, IL-10R), and related downstream phosphoprotein (kinases (ERK1/2, P38MAPK, JNK) and transcription factors (ATF-2, NFkappaB, STAT3)). Changes will be localized to cells using multiplexed real-time RT-PCR of mRNA derived from laser dissection microscopy acquired specific cell enhanced samples (cytokines) and double label immuno- histochemistry (receptors and phosphoproteins). Innate cytokines trigger altered function via gene activation. We suggest that identification of compensatory changes of the innate cytokine system will predict which genes, and related protein pathways, within the system (and within other signaling systems) will be optimal therapeutic targets to emulate protective capacities of intrinsic neuroprotection.