解剖学报

解剖学报 ›› 2024, Vol. 55 ›› Issue (3): 363-370.doi: 10.16098/j.issn.0529-1356.2024.03.016

• 技术方法 • 上一篇    下一篇

基于灌注系统对CUBIC组织透明化技术的优化

 宫川惠1   邱家怡2   印可馨2   张继茹2   何铖1   袁野1   吕广明1,3*
  

  1. 1.南通大学医学院人体解剖学系，江苏 南通 226001; 2.南通大学医学院2019级临床医学五年制，江苏 南通 226001;3.南通大学神经生物学研究所，江苏省神经再生重点实验室，江苏 南通  226001
  • 收稿日期:2023-02-23 修回日期:2023-05-16 出版日期:2024-06-06 发布日期:2024-06-11
  • 通讯作者: 吕广明 E-mail:gmlu@ntu.edu.cn
  • 基金资助:
    江苏省研究生科研与实践创新计划项目

Optimisation of CUBIC tissue clearing technology based on perfusion methods

GONG  Chuan-hui1  QIU  Jia-yi2  YIN  Ke-xin2  ZHANG  Ji-ru2  HE  Cheng1  YUAN  Ye1  LÜ Guang-ming1,3*   

  1. 1.Department of Anatomy, Medical School of Nantong University, Jiangsu Nantong 226001, China; 2.Grade 2019, Clinical Medicine of Five-year, Medical School of Nantong University, Jiangsu Nantong 226001, China; 3.Institute of Neurobiology, Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Jiangsu Nantong  226001, China
  • Received:2023-02-23 Revised:2023-05-16 Online:2024-06-06 Published:2024-06-11
  • Contact: LÜ Guang-ming E-mail:gmlu@ntu.edu.cn

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摘要：

目的 为了缩短清晰无障碍脑成像鸡尾酒和计算分析（CUBIC）技术的透明时间，提高透明效率，探索亲水性组织透明技术应用的可能性，本研究对CUBIC技术进行灌注优化后，与4种亲水性透明化方法在组织透明效果、透明时间、面积变化、体积变化及腺相关病毒(AAV)荧光保留情况等方面进行了比较。 方法 取6只成年美国癌症研究所（ICR）小鼠的脑、肝、脾和肾分别采用SeeDB、FRUIT、ScaleS、CUBIC的方法进行透明化处理，使用Image J 1.8.0 测算样本的面积和灰度值，排水法测量透明前后体积，比较各组的透明效果、时间以及大小变形。通过改进灌注速率与最佳灌注剂量对CUBIC技术进行灌注优化，每组实验样本量为6。另在16只成年小鼠的大脑运动皮层定位注射AAV，4周后取其颈髓节段透明化处理，荧光照片经过ImageJ1.8.0测量平均荧光强度，评估不同技术的荧光保存情况。 结果 灌注CUBIC的最佳灌注速率和灌注剂量分别是15 ml/min和200 ml。对于透明能力和速度，灌注CUBIC技术的平均灰度值最低且用时最短，而CUBIC消耗的时间最长，SeeDB、FRUIT、ScaleS并没有显示出良好的透明能力。在面积和体积变化方面，几种技术对组织或器官透明后均有不同程度的膨胀。在荧光保留方面，灌注CUBIC对绿色荧光蛋白(GFP)荧光信号的保留效果最好，其次是 CUBIC、ScaleS、FRUIT和 SeeDB。 结论 灌注CUBIC技术与其他技术相比，组织透明效果最好，透明时间最短，AAV荧光保留最多。

关键词: 组织透明化, 光学透明, 清晰无障碍脑成像鸡尾酒和计算分析, 亲水性组织透明化技术, 小鼠

Abstract:

Objective  In order to shorten the transparency time of clear, unobstructed brain imaging cocktails and computational analysis(CUBIC), improve the transparency efficiency, and explore the possibility of applying hydrophilic tissue transparency technique, this study was conducted to optimize the perfusion of CUBIC technique and compare it with four hydrophilic tissue clearing method  in terms of tissue transparency effect, transparency time, area change, volume change and adeno-associated virus (AAV) fluorescence retention. Methods  Brain, liver, spleen and kidney of 6 adult Institute of Cancer Research(ICR) mice were subjected to clearing treatment by SeeDB, FRUIT, ScaleS and CUBIC method, respectively. The area and gray value of the samples were measured by Image J 1.8.0, and the volume before and after transparency was measured by drainage method  to compare the transparency effect, time and size deformation of each group. Perfusion optimization of the CUBIC was performed by improving the perfusion rate with the optimal perfusion dose, each group of the experimental sample size was 6. Fluorescence preservation by different techniques was evaluated by injecting AAV in the motor cortex of 16 adult mice and taking the cervical spinal segments for transparency treatment after four weeks, and the fluorescence photographs were measured by Image J 1.8.0 to measure the mean fluorescent intensity.  Results The optimal perfusion rate and dose of CUBIC was 15 ml/min and 200 ml respectively. For transparency ability and speed, the perfusion CUBIC had the lowest mean gray value and took the shortest time, while CUBIC consumed the longest time, and SeeDB, FRUIT, and ScaleS did not show good transparency ability. In terms of area and volume changes, several techniques showed different degrees of expansion after transparency of tissues or organs. In terms of fluorescence retention, perfusion CUBIC showed the best retention of green fluorescent protein (GFP) fluorescence signal, followed by CUBIC, ScaleS, FRUIT, and SeeDB. Conclusion Perfusion CUBIC technique shows the best tissue transparency, the shortest transparency time, and the most AAV fluorescence retention compared with other techniques.

Key words: Tissue clearing, Optical clearing, Clear, unobstructed brain imaging cocktails and computational analysis, Hydrophilic reagent-based tissue clearing technology, Mouse

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