Comprehensive evaluation and application of tissue clearing techniques for 3-D visualization of splenic neural and immune architecture.

IF 5 2区 生物学 Q2 CELL BIOLOGY
Jianing Li, Letian He, Wenling Wang, Siyu Wang, Dan Zhang, Liyun Liang, Guangping Song, Yijian Zhang, Shaoqing Yu, Lei Wang, Qiuying Han, Shaoyi Huang, Sen Li, Haiqing Tu, Zengqing Song, Huaibin Hu, Huiyan Li, Yang Yang, Min Wu
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引用次数: 0

Abstract

As the largest secondary lymphoid organ, the spleen plays a crucial role in initiating and sustaining immune responses against blood-borne pathogens through antigen capture and delivery. It is innervated by both autonomic and sensory nerves, which allows for neural modulation of its immune responses. The intricate spatial structure and precise coordination between immune and neural components are essential for proper splenic function, necessitating three-dimensional (3-D) imaging to reveal its architecture. However, the dense fibrous capsule and exceptionally rich vasculature of the spleen pose significant challenges for achieving comprehensive 3-D visualization of the entire organ. Here, we systematically evaluated and compared five cutting-edge tissue clearing approaches-ImmuView, fast light-microscopic analysis of antibody-stained whole organs, small-micelle-mediated human organ efficient clearing and labeling (SHANEL), advanced clear, unobstructed brain imaging cocktails and computational analysis (advanced CUBIC), and clearing-enhanced 3-D microscopy-for their effectiveness in rendering the spleen transparent for multiplexed antibody staining and high-resolution 3-D imaging. Our results indicated that SHANEL provided the clearest visualization of essential splenic neural and immune components. Meanwhile, advanced CUBIC achieved the greatest labeling efficacy for immune cells, albeit with slightly reduced transparency. Importantly, our study marked the first application of these optimized protocols to human spleen tissue, successfully revealing the highly organized immune cell zones and neural networks with enhanced clarity. Notably, we identified the nociceptive sensory innervation within human spleen tissue for the first time. Collectively, these findings establish optimal imaging strategies for visualizing splenic immune cells and neural structure in both murine and human tissues, providing profound insights into the intricate neuroimmune interactions and their pivotal roles in the immune functions of the spleen.NEW & NOTEWORTHY This study systematically assessed five tissue-clearing techniques and optimized the conditions of each protocol to overcome the challenges of splenic 3-D imaging posed by its dense structure and high pigmentation. The results demonstrated SHANEL and advanced CUBIC as the optimal methods for 3-D visualization of diverse splenic immune and neural architecture, with which we successfully mapped splenic neuroimmune landscape and identified nociceptive nerves within the human spleen for the first time.

组织清除技术在脾神经和免疫结构三维可视化中的综合评价与应用。
作为最大的次级淋巴器官,脾脏通过抗原捕获和递送,在启动和维持针对血源性病原体的免疫应答中起着至关重要的作用。它是由自主神经和感觉神经支配的,这允许神经调节其免疫反应。脾脏复杂的空间结构以及免疫和神经成分之间的精确协调对于脾脏的正常功能至关重要,因此需要三维成像来揭示其结构。然而,脾脏致密的纤维囊和异常丰富的脉管系统对实现整个器官的全面三维可视化构成了重大挑战。在这里,我们系统地评估和比较了五种尖端的组织清除方法-免疫视图,抗体染色全器官的快速光显微分析,小胶束介导的人体器官有效清除和标记(SHANEL),先进的清晰,无阻碍的脑成像鸡尾酒和计算分析(先进立方),以及清除增强的3d显微镜,因为它们可以有效地使脾脏透明,进行多重抗体染色和高分辨率3d成像。我们的结果表明,SHANEL提供了最清晰的脾神经和免疫成分的可视化。同时,先进的CUBIC对免疫细胞的标记效果最好,尽管透明度略有降低。重要的是,我们的研究标志着这些优化方案首次应用于人体脾脏组织,成功地清晰地揭示了高度组织的免疫细胞区和神经网络。值得注意的是,我们首次在人脾组织中发现了伤害感觉神经支配。总的来说,这些发现为小鼠和人类组织中脾脏免疫细胞和神经结构的可视化建立了最佳成像策略,为复杂的神经免疫相互作用及其在脾脏免疫功能中的关键作用提供了深刻的见解。本研究系统地评估了五种组织清除技术,并优化了每种方案的条件,以克服脾脏致密结构和高色素沉着带来的三维成像挑战。结果表明,SHANEL和先进的CUBIC是脾脏多种免疫和神经结构三维可视化的最佳方法,我们成功地绘制了脾脏神经免疫景观,并首次识别了人体脾脏内的伤害神经。
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
CiteScore
9.10
自引率
1.80%
发文量
252
审稿时长
1 months
期刊介绍: The American Journal of Physiology-Cell Physiology is dedicated to innovative approaches to the study of cell and molecular physiology. Contributions that use cellular and molecular approaches to shed light on mechanisms of physiological control at higher levels of organization also appear regularly. Manuscripts dealing with the structure and function of cell membranes, contractile systems, cellular organelles, and membrane channels, transporters, and pumps are encouraged. Studies dealing with integrated regulation of cellular function, including mechanisms of signal transduction, development, gene expression, cell-to-cell interactions, and the cell physiology of pathophysiological states, are also eagerly sought. Interdisciplinary studies that apply the approaches of biochemistry, biophysics, molecular biology, morphology, and immunology to the determination of new principles in cell physiology are especially welcome.
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