Glial Reaction to Ischemic Brain Injury

描述(申请人提供)：大脑通过活动变得对损伤更有弹性，包括扩散性抑郁(SD)。虽然这一内在过程的机制仍不清楚，但它们涉及到先天的细胞因子。重要的是，天生的细胞因子是多余的和多效性的，所以多种细胞因子可以有相似的作用，而单一的细胞因子可以有不同的作用。这是由于在配体、受体和磷蛋白水平上的复杂的相互作用信号。更复杂的是脑细胞类型的多样性，每种细胞都能产生独特的表达变化。到目前为止，这种复杂性使他们无法进行系统的研究。我们将通过两种策略的结合来解决这一空白，以实现定义SD诱导的神经保护的固有细胞因子信号的总体目标。第一种方法是同时、多路测量先天细胞因子途径变量(即细胞因子、受体、磷蛋白)。第二种是应用经典的生化途径分离策略，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。研究将在海马体器官培养(HOTC)中进行，它类似于体内的对应细胞；缺乏混淆的炎症变化的证据，并且是孤立的，因此不受全身免疫变化的影响。我们的数据显示，HOTCs经历了NMDA介导的损伤，SD和SD诱导的神经保护，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，通过代偿性、适应性反应。与新的工具相结合，HOTCs非常适合我们定义固有细胞因子的特定目标：1)配体；2)受体；以及3)与SD诱导的NMDA损伤相比，与NMDA损伤相关的相关磷蛋白变化-作为一种手段，建立引起SD诱导的神经保护的细胞和组织固有细胞因子途径变化的包罗万象的特征。这些目标将使用：新开发的基于微球的流式细胞术分析，以同时检测固有的细胞因子(IL-1α、IL-1β、TNF-α、干扰素-γ、IL-6、IL-10)及其受体(IL-1R1、TNFR1、IL-6Rpha/gp130、IFNGammaR、IL-10R)以及相关的下游磷蛋白(激酶(ERK1/2、p38MAPK、JNK)和转录因子(ATF-2、NFkappaB、STAT3))。变化将通过激光解剖显微镜获得的特定细胞增强样本(细胞因子)和双标记免疫组织化学(受体和磷蛋白)获得的mRNA多重实时RT-PCR定位于细胞。先天的细胞因子通过基因激活来触发改变的功能。我们认为，识别先天细胞因子系统的代偿性变化将预测该系统内(以及其他信号系统内)哪些基因和相关的蛋白质通路将成为模仿内在神经保护保护能力的最佳治疗靶点。

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.