Optimisation of CUBIC tissue clearing technology based on perfusion methods

GONG Chuan-Hui; QIU Jia-Yi; YIN Ke-Xin; ZHANG Ji-Ru; HE Cheng; YUAN Ye; YUAN Ye Guang-Ming

doi:10.16098/j.issn.0529-1356.2024.03.016

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Acta Anatomica Sinica ›› 2024, Vol. 55 ›› Issue (3) : 363-370. DOI: 10.16098/j.issn.0529-1356.2024.03.016

Technology and Methodology

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-ming^1,3*

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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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GONG Chuan-hui QIU Jia-yi YIN Ke-xin ZHANG Ji-ru HE Cheng YUAN Ye LÜ Guang-ming. Optimisation of CUBIC tissue clearing technology based on perfusion methods[J]. Acta Anatomica Sinica. 2024, 55(3): 363-370 https://doi.org/10.16098/j.issn.0529-1356.2024.03.016

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