Profiling lamina specific pyramidal neurons using postmortem human formalin fixed paraffin embedded frontal cortex tissue in combination with digital spatial profiling

IF 2.7 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-06-03 DOI:10.1016/j.jneumeth.2025.110512

Aleksandra Stanisavljevic , Kyrillos W. Ibrahim , Philip H. Stavrides , Christopher Bare , Melissa J. Alldred , Adriana Heguy , Ralph A. Nixon , Stephen D. Ginsberg

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引用次数: 0

Abstract

Background

Digital spatial profiling (DSP) is an innovative approach to perform RNA sequencing (RNA-seq), including in neuronal populations. DSP enables expression profiling linking RNA-seq data to spatially characterized samples utilizing tissue bound probes. We employ the GeoMx DSP system for spatial characterization of transcriptomic data from lamina specific pyramidal neurons and cortical ribbons containing admixed cell types using human postmortem brain tissue.

New method

We established a protocol using human postmortem formalin fixed paraffin embedded (FFPE) frontal cortex tissue from nondemented human control brains. Layer III (L3) and Layer V (L5) pyramidal neurons from Brodmann area 9 were identified with the neuronal marker Ca^2 + /calmodulin-dependent protein kinase II and selected for probe collection.

Results

This approach significantly reduced the amount of FFPE tissue needed for robust single population RNA-seq. We demonstrate ∼20 identified L3 or L5 pyramidal neurons or one lamina-specific cortical ribbon from a single 5 µm thick section is sufficient to generate robust RNA-seq reads. Bioinformatic analysis of neurons and ribbons showed notable similarities and differences reflective of the single neuron and multiple admixed cell types within the former and latter, respectively.

Comparison with existing methods

Protocols exist for DSP of postmortem human FFPE brain tissue. However, this new approach enables profiling small groups of ∼14–21 pyramidal neurons using the GeoMx DSP platform.

Conclusions

This optimized DSP assay provides high resolution RNA-seq data demonstrating utility and versatility of the GeoMx platform for individually characterized neurons and isolated cortical ribbons within postmortem FFPE human brain tissue for downstream analyses.

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利用人死后福尔马林固定石蜡包埋额叶皮层组织，结合数字空间剖面，对层特异性锥体神经元进行剖面分析。

背景：数字空间分析（DSP）是一种执行RNA测序（RNA-seq）的创新方法，包括在神经元群体中。DSP支持表达谱分析，利用组织结合探针将RNA-seq数据与空间特征样本联系起来。我们使用GeoMx DSP系统对人类死后脑组织中含有混合细胞类型的层特异性锥体神经元和皮质带的转录组数据进行空间表征。我们建立了一种使用人类死后福尔马林固定石蜡包埋（FFPE）额叶皮层组织的方法，该组织来自非痴呆的人类对照大脑。用神经元标志物Ca2+/钙调素依赖性蛋白激酶II对Brodmann区9的第III层（L3）和第V层（L5）锥体神经元进行鉴定，并选择探针收集。结果：这种方法显著减少了强大的单群体rna测序所需的FFPE组织的数量。我们证明，从单个5 μ m厚的切片中，约20个已鉴定的L3或L5锥体神经元或一个层状特异性皮质带足以产生稳健的RNA-seq读取。对神经元和条带的生物信息学分析显示，前者和后者分别反映了单个神经元和多个混合细胞类型的相似性和差异性。与现有方法的比较，对人死后FFPE脑组织的DSP有不同的处理方案。然而，这种新方法可以使用GeoMx DSP平台分析约14-21个锥体神经元的小群体。结论：该优化的DSP分析提供了高分辨率RNA-seq数据，证明了GeoMx平台在死后FFPE人脑组织中单个表征神经元和分离皮质带的实用性和多功能性，可用于下游分析。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.