Yu-Song Yuan1,2, Fei Yu1,2, Su-Ping Niu3, Hao Lu1,4, Yu-Hui Kou5,6, Hai-Lin Xu7,8. 1. Department of Trauma and Orthopaedics, Peking University People's Hospital, Peking University, Beijing, 100044, China. 2. National and Local Joint Engineering Research Center of Orthopaedic Biomaterials, Department of Bone & Joint Surgery, Peking University Shenzhen Hospital, Shenzhen, 518036, China. 3. Office of Academic Research, Peking University People's Hospital, Peking University, Beijing, 100044, China. 4. Diabetic Foot Treatment Center, Peking University People's Hospital, Peking University, Beijing, 100044, China. 5. Department of Trauma and Orthopaedics, Peking University People's Hospital, Peking University, Beijing, 100044, China. yuhuikou@bjmu.edu.cn. 6. Key Laboratory of Trauma and Neural Regeneration (Peking University), Ministry of Education, Beijing, 100044, China. yuhuikou@bjmu.edu.cn. 7. Department of Trauma and Orthopaedics, Peking University People's Hospital, Peking University, Beijing, 100044, China. xuhailinfa@163.com. 8. Diabetic Foot Treatment Center, Peking University People's Hospital, Peking University, Beijing, 100044, China. xuhailinfa@163.com.
Abstract
OBJECTIVE: Wallerian degeneration is a pathological process closely related to peripheral nerve regeneration following injury, and includes the disintegration and phagocytosis of peripheral nervous system cells. Traditionally, morphological changes are observed by performing immunofluorescence staining after sectioning, which results in the loss of some histological information. The purpose of this study was to explore a new, nondestructive, and systematic method for observing axonal histological changes during Wallerian degeneration. METHODS: Thirty male Thy1-YFP-16 mice (SPF grade, 6 weeks old, 20±5 g) were randomly selected and divided into clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) optical clearing (n=15) and traditional method groups (n=15). Five mice in each group were sacrificed at 1st, 3rd, and 5th day following a crush operation. The histological axon changes were observed by CUBIC light optical clearing treatment, direct tissue section imaging, and HE staining. RESULTS: The results revealed that, compared with traditional imaging methods, there was no physical damage to the samples, which allowed for three-dimensional and deep-seated tissue imaging through CUBIC. Local image information could be nicely obtained by direct fluorescence imaging and HE staining, but it was difficult to obtain image information of the entire sample. At the same time, the image information obtained by fluorescence imaging and HE staining was partially lost. CONCLUSION: The combining of CUBIC and Thy1-YFP transgenic mice allowed for a clear and comprehensive observation of histological changes of axons in Wallerian degeneration.
OBJECTIVE: Wallerian degeneration is a pathological process closely related to peripheral nerve regeneration following injury, and includes the disintegration and phagocytosis of peripheral nervous system cells. Traditionally, morphological changes are observed by performing immunofluorescence staining after sectioning, which results in the loss of some histological information. The purpose of this study was to explore a new, nondestructive, and systematic method for observing axonal histological changes during Wallerian degeneration. METHODS: Thirty male Thy1-YFP-16 mice (SPF grade, 6 weeks old, 20±5 g) were randomly selected and divided into clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) optical clearing (n=15) and traditional method groups (n=15). Five mice in each group were sacrificed at 1st, 3rd, and 5th day following a crush operation. The histological axon changes were observed by CUBIC light optical clearing treatment, direct tissue section imaging, and HE staining. RESULTS: The results revealed that, compared with traditional imaging methods, there was no physical damage to the samples, which allowed for three-dimensional and deep-seated tissue imaging through CUBIC. Local image information could be nicely obtained by direct fluorescence imaging and HE staining, but it was difficult to obtain image information of the entire sample. At the same time, the image information obtained by fluorescence imaging and HE staining was partially lost. CONCLUSION: The combining of CUBIC and Thy1-YFP transgenic mice allowed for a clear and comprehensive observation of histological changes of axons in Wallerian degeneration.