The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes; however, -the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, a time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that strokeinduced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke, genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is an increase in saccharides, sugar phosphates, ceramides and free fatty acids and a decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery of AAV1-MANF to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.

中文翻译：

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中风后四天大鼠梗塞周围区域的分子谱：MANF研究。

缺血性中风后的梗塞周围区域是许多内源性恢复过程的解剖位置。然而，在梗塞周围区域的分子事件仍然没有很好的表征。在这项研究中，我们检查了卒中后第四天，即修复过程正在进行的时间，梗塞周围区域的分子分布。我们使用了涉及RNA测序的多组学方法，以及基于质谱的蛋白质组学和代谢组学来表征梗塞周围区域的分子变化。我们还利用了我们先前开发的方法在梗塞后区域表达转基因，在卒中后第2天将自互补腺伴随病毒（AAV）载体注射到脑实质中。我们以前已经使用这种方法来显示中脑星形胶质细胞源性神经营养因子（MANF）增强了从中风的功能恢复，并将吞噬细胞募集到梗塞周围区域。在这里，我们首先分析了与假手术动物相比，在中风后第4天中风对梗塞周围区域的影响，发现中风诱发了3345个转录本，341种蛋白质和88种代谢产物的变化。我们发现中风后，与炎症，增殖，凋亡和再生相关的基因被上调，而编码神经活性配体受体和钙结合蛋白的基因被下调。在蛋白质组学中，我们检测到与蛋白质合成相关的蛋白质上调和神经元蛋白质的下调。代谢组学研究表明，中风后组织中的糖，磷酸糖，神经酰胺和游离脂肪酸增加，腺嘌呤，次黄嘌呤，腺苷和鸟苷减少。然后，我们比较了AAV1-MANF向AAV1-eGFP（增强的绿色荧光蛋白）的中风后传递的影响。MANF管理增加了77个基因的表达，其中大多数与免疫反应有关。在蛋白质组学中，MANF给药可降低S100A8和S100A9蛋白质水平。在代谢组学中，未检测到MANF和eGFP治疗之间的显着差异，但相对于假手术组，大多数脂质变化仅在AAV-eGFP组中显着。这项工作描述了缺血性中风恢复过程中梗塞周围区域的分子分布，并为进一步的卒中研究建立了资源。这些结果为实质MANF作为吞噬功能的调节剂提供了进一步的支持。