Abstract Body: Introduction: Acute cerebral blood flow occlusion by thrombus is a major cause of ischemic stroke. The composition of the resulting thrombus varies depending on the underlying medical condition. Notably, neutrophils, the most abundant white blood cells, play a crucial role in acute ischemic stroke and can form neutrophil extracellular traps (NETs). NETs are web-like structures consisting of DNA, granule proteins, and histones. NETs contribute to thrombo-inflammation and stabilize the thrombus, potentially leading to worse outcomes. While long non-coding RNA (lncRNA) do not encode proteins, they are involved in essential biological processes. The link between lncRNAs and cardiovascular disease is increasingly studied, and their role in ischemic stroke has recently come to attention. We, therefore, hypothesize that lncRNAs within NETs play a critical role in the formation and regulation of thrombi.
Method: Human thrombi obtained from endovascular thrombectomy were categorized based on their underlying medical conditions, such as dyslipidemia, hypertension, or a combination of two or more conditions. Frozen sections of the thrombi were immunostained with markers including Citrullinated H3 (CitH3), CD45, neutrophil elastase (NE), and DAPI, and then analyzed using holotomography, confocal microscopy, and spatial transcriptomics. In spatial transcriptomics analysis, NETs and other leukocytes were selected as distal and proximal ROIs for detailed gene expression profiling. The gene expression of HDAC class 1 genes (HDAC1, HDAC2, HDAC3, and HDAC8) was further examined by qPCR.
Results: Holotomography analysis revealed that cells in the thrombi of patients with underlying conditions exhibited a high mean refractive index. Immunofluorescence imaging also identified a high percentage of NETs in these samples. Spatial transcriptomics analysis confirmed that NETs in patients with hypertension showed upregulated expression of lncRNAs. Additionally, qPCR results demonstrated increased expression of HDAC class 1 genes, which are associated with NET formation, in patients with underlying conditions.
Conclusion: LncRNAs play a crucial role in the formation and regulation of thrombi. Therefore, NETs-related lncRNAs could serve as valuable therapeutic targets and diagnostic markers for acute ischemic stroke patients with underlying diseases.