Rapid isolation of intact retinal astrocytes: a novel approach.

IF 6.2 2区 医学 Q1 NEUROSCIENCES
Paul F Cullen, Arpan G Mazumder, Daniel Sun, John G Flanagan
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Abstract

Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA.

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快速分离完整的视网膜星形胶质细胞:一种新方法。
星形胶质细胞是中枢神经系统中一类主要的神经胶质支持细胞,在健康和疾病中发挥着各种重要作用。随着我们对这些细胞不同功能的理解提高,星形胶质细胞群体之间的异质性程度已成为一个关键的研究领域。视网膜星形胶质细胞形成视网膜神经节细胞胞体和轴突的直接细胞环境,在人类青光眼和该疾病的动物模型中都会发生反应性反应,但它们对其病理学和进展的贡献仍然相对未知。这种知识差距很大程度上是由于分离技术不足,部分原因是这些细胞的稀疏性及其与更丰富的视网膜Müller细胞的相似性。在这里,我们提出了一种分离视网膜星形胶质细胞并富集其RNA的新方法,在正常和高眼压小鼠(一种常见的实验性青光眼模型)中进行了测试。我们的方法结合了一种新的酶辅助视网膜星形胶质细胞显微切割和使用Ribotag方法的选择性核糖体免疫沉淀。我们的显微切割方法快速,保留了星形胶质细胞的形态,导致短暂的死后间隔,并最大限度地减少了这些细胞远端区域的RNA损失。显微切割和Ribotag免疫沉淀都需要最少的专业设备或试剂,通过将它们结合使用,我们能够实现 > 星形胶质细胞RNA富集100倍。
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来源期刊
Acta Neuropathologica Communications
Acta Neuropathologica Communications Medicine-Pathology and Forensic Medicine
CiteScore
11.20
自引率
2.80%
发文量
162
审稿时长
8 weeks
期刊介绍: "Acta Neuropathologica Communications (ANC)" is a peer-reviewed journal that specializes in the rapid publication of research articles focused on the mechanisms underlying neurological diseases. The journal emphasizes the use of molecular, cellular, and morphological techniques applied to experimental or human tissues to investigate the pathogenesis of neurological disorders. ANC is committed to a fast-track publication process, aiming to publish accepted manuscripts within two months of submission. This expedited timeline is designed to ensure that the latest findings in neuroscience and pathology are disseminated quickly to the scientific community, fostering rapid advancements in the field of neurology and neuroscience. The journal's focus on cutting-edge research and its swift publication schedule make it a valuable resource for researchers, clinicians, and other professionals interested in the study and treatment of neurological conditions.
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