组织透明化技术在器官可视化应用中的初步探讨

doi:10.13418/j.issn.1001-165x.2024.4.06

摘要/Abstract

摘要： 目的　对CUBIC（clear, unobstructed brain imaging cocktails and computational analysis ）组织透明化技术在小鼠不同器官中的适应性进行探讨，并探究该技术与细胞核染色及病毒示踪神经元标记技术的兼容性。方法　（1）摘取并解剖新鲜灌注小鼠的脑、心以及肝，分别取1.0 cm×1.5 cm×0.5 cm的组织块，使用clear, unobstructed brain imaging cocktails and computational analysis-L (CUBIC-L)和clear,unobstructed brain imaging cocktails and computational analysis-R (CUBIC-R)组织清除液进行透明化处理；（2）对小鼠整脑进行透明化处理，应用碘化丙啶（PI）对鼠脑神经元的细胞核染色，激光片层显微镜（LSFM）获取小鼠左、右脑半球及小脑脑干的细胞核体积三维图像；（3）分别在鼠脑海马和黑质区显微注射腺相关病毒(AAV)-U6-增强型绿色荧光蛋白（EGFP），标记特定解剖部位的神经元，对完成病毒转染后的鼠脑进行透明化处理，LSFM进行三维立体成像。结果　经过透明化处理的脑组织、肝组织、心脏组织都呈现透明凝胶样状态，但不同组织块的清除效率不同，脑组织清除效率最高，其次为肝脏组织，最后为心脏组织。LSFM可以对透明化并核染的脑组织进行整体积三维立体成像。AAV可以清楚的标记不同部位的神经元，配合透明化处理后可以观察到这些神经元的空间分布情况，同时还可以观察到不同部位神经元的物理投射。结论　CUBIC组织透明化技术在小鼠脑组织、心组织、肝组织中具有普遍应用性，其中对脑组织的透明效率最高。CUBIC组织透明化方法与细胞核染色技术及病毒示踪神经元标记技术有很好的适配性。

关键词: CUBIC, 　透明化, 　组织, 　器官, 　可视化

Abstract: Objective To explore the adaptability of clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue transparency techniques in different organs of mice, and explore the compatibility of tissue transparency technology with nuclear staining and virus tracer neuron labeling technology. Methods （1） The brains, hearts and livers of freshly injected mice were extracted and dissected. Tissue blocks of 1.0cm×1.5cm×0.5cm were taken respectively, clear, unobstructed brain imaging cocktails and computational analysis-L (CUBIC-L) and clear, unobstructed brain imaging cocktails and computational analysis-R (CUBIC-R) tissue cleaning solution were used for transparency treatment. （2） The whole brain of mice was treated with transparency, and the nucleus of mouse brain neurons was stained with propidium iodide (PI). Three-dimensional nuclear volume images of the left and right hemispheres and cerebellar brainstem were obtained by light sheet fluorescence microscopy (LSFM). （3） Adeno-associated virus (AAV) -U6-enhanced green fluorescent protein (EGFP) was microinjected into the hippocampus and substania nigra of rat brain, respectively, to label neurons at specific anatomical sites. The rat brain after virus transfection was treated transparently. LSFM performs 3D stereoscopic imaging. Results The brain tissue, liver tissue and heart tissue all showed a transparent gel-like state after transparent treatment. However, the removal efficiency of different tissue blocks was different, and the removal efficiency of brain tissue was the highest, followed by liver tissue and heart tissue. LSFM could perform whole-volume three-dimensional imaging of transparent and nucleated brain tissue. AAV can clearly mark neurons in different parts, after transparent processing, the spatial distribution of these neurons could be observed, and the physical projection of neurons in different parts could also be observed. Conclusions CUBIC tissue transparency technology has universal application in brain tissue, heart tissue and liver tissue of mice, and the transparency efficiency of brain tissue is the highest. CUBIC tissue transparency method has good compatibility with nuclear staining and virus tracer neuron labeling technology.

Key words: CUBIC, , , , Transparency, , , Tissue, Organ, , , Visualization

中图分类号:

R445.2

唐倩, 侯兰伟, 刘宇宁, 娄云霞, 滕野, 周文娟, 刘树伟. 组织透明化技术在器官可视化应用中的初步探讨[J]. 中国临床解剖学杂志, 2024, 42(4): 393-398.

Tang Qian, Hou Lanwei, Liu Yuning, Lou Yunxia, Teng Ye, Zhou Wenjuan, Liu Shuwei. Preliminary study on the application of tissue transparency technology in organ visualization[J]. Chinese Journal of Clinical Anatomy, 2024, 42(4): 393-398.

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