中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (34): 8939-8945.doi: 10.12307/2026.888

• 脊柱组织构建 spinal tissue construction • 上一篇    下一篇

组织透明化技术在大鼠慢性脊髓损伤模型中的应用

王志壮1，许  博1，马国梁1，张  丹2，秦晓宽1，冯敏山1，陈  忻1，杨克新1，杨博文1，银  河1   

  1. 1中国中医科学院望京医院，北京市  100102；2清华大学生物医学测试中心共享仪器平台，北京市  100804
  • 收稿日期:2025-10-14 修回日期:2026-02-08 出版日期:2026-12-08 发布日期:2026-04-13
  • 通讯作者: 银河，博士，副主任医师，中国中医科学院望京医院，北京市 100102 通讯作者：杨博文，博士，主治医师，中国中医科学院望京医院，北京市 100102
  • 作者简介:王志壮，男，2000年生，河南省洛阳市人，汉族，硕士，主要从事脊柱疾病的临床与基础研究。
  • 基金资助:
    国家自然科学基金青年科学基金项目(82405442)，项目负责人：杨博文；中国中医科学院基本科研业务费优秀青年科技人才培养专项项目(ZZ18-YQ-031)，项目负责人：杨博文；中国中医科学院基本科研业务费优秀青年科技人才培养专项项目(ZZ14-YQ-022)，项目负责人：银河

Application of tissue clearing technology in a rat model of chronic spinal cord injury

Wang Zhizhuang1, Xu Bo1, Ma Guoliang1, Zhang Dan2, Qin Xiaokuan1, Feng Minshan1, Chen Xin1, Yang Kexin1, Yang Bowen1, Yin He1   

  1. 1Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China; 2Shared Instrument Platform of Biomedical Testing Center, Tsinghua University, Beijing 100804, China
  • Received:2025-10-14 Revised:2026-02-08 Online:2026-12-08 Published:2026-04-13
  • Contact: Yin He, PhD, Associate chief physician, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China Co-corresponding author: Yang Bowen, PhD, Attending physician, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China
  • About author:Wang Zhizhuang, MS, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China
  • Supported by:
    The National Natural Science Foundation of China (Youth Science Fund Project), No. 82405442 (to YBW); The Excellent Young Scientific and Technological Talents Training Program of the Fundamental Research Funds for the China Academy of Chinese Medical Sciences, Nos. ZZ18-YQ-031 (to YBW) and ZZ14-YQ-022 (to YH) 

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摘要：



文题释义：
组织透明化技术：是一种通过化学或物理方法处理生物组织，使其从浑浊不透明状态转变为光学透明的技术。组织透明化技术的核心目标是消除光在组织中传播时因折射率不匹配(如脂质、蛋白质等成分)导致的散射，从而实现对完整器官甚至生物体的高分辨率三维成像。
脊髓损伤：指由创伤性或非创伤性因素导致脊髓结构/功能的破坏，引起损伤平面以下运动、感觉及自主神经功能的障碍。脊髓损伤的核心特点是神经信号传导中断和不可逆性神经元损伤，具有高致残率、年轻化趋势和终身性影响。

背景：研究表明，组织透明化技术能够对脊髓损伤区域的神经元进行三维可视化，清晰地呈现神经元的形态变化，包括细胞体萎缩、树突断裂以及轴突退行性改变等。
目的：系统评估组织透明化技术在大鼠慢性脊髓损伤模型中的应用潜力。
方法：将36只雌性SD大鼠随机均分为正常组(n=12)、假手术组(n=12)和手术组(n=12)，正常组不进行任何处理，假手术组C5-C7椎管内植入聚乙烯醇丙烯酰胺互穿网络水凝胶后立刻取出，手术组C5-C7椎管内植入聚乙烯醇丙烯酰胺互穿网络水凝胶压迫脊髓建立慢性脊髓损伤模型。术后第1，3，7，14天，采用 BBB 评分和改良 Rivlin 斜板实验评估大鼠的运动功能；术后第14天取材，苏木精-伊红染色观察脊髓组织形态，组织透明化结合神经元特异性核蛋白免疫荧光标记对脊髓组织进行三维重建分析和横断面视图分析。
结果与结论：①手术组术后第1，3，7，14天的BBB 评分与斜板实验角度测量值均低于正常组、假手术组(P < 0.001)；②苏木精-伊红染色显示，手术组大鼠脊髓损伤显著，灰质中的神经细胞出现肿胀和破坏，白质结构失去均一稳定性，部分细胞核消失，细胞数量减少，压迫区域大量胶质细胞增殖聚集，白质结构混乱，大量空洞形成；③脊髓组织三维重建与横断面分析显示，正常组和假手术组脊髓外观连续饱满，神经元特异性核蛋白红色荧光信号均匀分布，灰质前角神经元呈层状密集排列，白质纤维束完整；手术组脊髓外观凹陷甚至中断，压迫节段神经元特异性核蛋白荧光强度明显减弱，灰质神经元层结构破坏，出现区域性荧光中断。结果表明组织透明化技术能够有效展示脊髓损伤后的组织结构变化，为脊髓损伤的病理机制研究提供了有力支持。
https://orcid.org/0009-0009-1072-8971(王志壮)


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 组织透明化技术, 脊髓损伤, 大鼠模型, 三维成像, 神经细胞核免疫荧光标记, 组织构建

Abstract: BACKGROUND: Studies have shown that tissue clearing technology enables the three-dimensional (3D) visualization of neurons in the spinal cord injury area, clearly presenting morphological changes of neurons, including soma atrophy, dendrite fragmentation, and axonal degeneration.
OBJECTIVE: To systematically evaluate the application potential of tissue clearing technology in a rat model of chronic spinal cord injury.
METHODS: Thirty-six female Sprague-Dawley rats were randomly and equally divided into a normal group (n=12), a sham surgery group (n=12), and a surgery group (n=12). The normal group received no treatment. The sham group underwent implantation and immediate removal of a poly(vinyl alcohol)/polyacrylamide interpenetrating network hydrogel into the C5-C7 spinal canal. The surgery group received implantation of the hydrogel to compress the spinal cord at C5-C7 to establish a chronic spinal cord injury model. Motor function was assessed using the Basso-Beattie-Bresnahan score and the modified Rivlin inclined plane test on postoperative days 1, 3, 7, and 14. On day 14, spinal cord tissue was harvested. Hematoxylin-eosin staining was used to observe tissue morphology. Tissue clearing combined with immunofluorescence labeling for neuron-specific nuclear protein (NeuN) was performed for 3D reconstruction analysis and cross-sectional view analysis of the spinal cord.
RESULTS AND CONCLUSION: (1) The Basso-Beattie-Bresnahan scores and inclined plane angles in the surgery group on postoperative days 1, 3, 7, and 14 were significantly lower than those in the normal and sham groups (P < 0.001). (2) Hematoxylin-eosin staining revealed significant spinal cord injury in the surgery group: neurons in the gray matter showed swelling and disruption, the white matter structure lost uniformity and stability, some nuclei disappeared, cell numbers decreased, extensive glial cell proliferation and aggregation occurred in the compression area, white matter structure was disordered, and numerous cavities formed. (3) 3D reconstruction and cross-sectional analyses of the spinal cord tissue showed that the spinal cord appeared continuous and full with uniformly distributed NeuN red fluorescence signal in the normal and sham groups. Neurons in the ventral horn of the gray matter were densely arranged in layers, and white matter fiber tracts were intact. In the surgery group, the spinal cord appeared sunken or even interrupted. NeuN fluorescence intensity was markedly weakened in the compressed segments, the layered structure of gray matter neurons was disrupted, and regional fluorescence interruption was observed. These findings indicate that tissue clearing technology can effectively reveal the structural changes of spinal cord tissue after injury, providing strong support for research into the pathological mechanisms of spinal cord injury.

Key words: tissue clearing technology, spinal cord injury, rat model, three-dimensional imaging, neuron-specific nuclear protein immunofluorescence labeling, tissue construction

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