Musha Jiayinaxi, Zhao Yilin, Zhao Rong, Li Yuan, Sun Rongjuan, Liu Pengfei, Zhan Jianghua
Clinical School of Paediatrics, Tianjin Medical University, Tianjin, China.
Department of General Surgery, Tianjin Children's Hospital, Beichen District, Longyan Road 238, Tianjin, 300134, China.
Appl Biochem Biotechnol. 2025 Apr;197(4):2151-2162. doi: 10.1007/s12010-024-05090-3. Epub 2024 Dec 16.
The purpose of this study was to utilize a two-photon microscope excitation Airy beam to achieve three-dimensional imaging of intrahepatic bile ducts in BDL mice and patients with biliary atresia (BA). Ten male Balb/c mice aged 6-8 weeks underwent extrahepatic bile duct ligation (BDL), and 10 underwent sham operation as control. After the operation, the mice resulted in symptoms such as jaundice, darkened urine, and weight loss. Taken liver tissues from BDL and control mice and trimmed to 553 mm after 10 days. Sixteen patients with BA were included in this study. Liver transplantation was performed in 12 cases of them; liver hilar and liver margin tissues were taken during the operation. Kasai portoenterostomy (KPE) was performed in 4 cases, and liver margin tissues were taken. Intraoperative liver tissue samples were trimmed to a size of 555 mm. The specimens were subjected to tissue fixation, antigen retrieval, antibody incubation, and subsequent tissue hyalinization following the principles of immunofluorescence staining. Subsequently, light-sheet fluorescence microscopy (LSFM) was followed, and intrahepatic bile ducts of the specimen were imaged utilizing Airy beam which was excited with high imaging depth attenuation-compensated two-photon. Deconvolution was applied to image processing to construct a three-dimensional model of intrahepatic bile ducts. Three-dimensional imaging of liver tissue was conducted in both BDL mice and BA patients, and the distribution of intrahepatic bile ducts was visualized. BDL mice exhibited notable widening of intrahepatic bile ducts, accompanied by bile duct hyperplasia. There was no obvious hyperplasia of intrahepatic bile duct in the control group. Significant small bile duct hyperplasia was seen on imaging of the intrahepatic bile ducts in patients with BA. The intrahepatic bile duct was disorganized and hyperplasia especially in patients who performed liver transplantation. The technique of Airy beam three-dimensional reconstruction can effectively image the intrahepatic bile ducts in Balb/c mice and BA patients in three dimensions. This approach contributes to a better understanding of the distribution of intrahepatic bile ducts in BA patients. Moreover, it facilitates the exploration of models that more accurately simulate BA disease by elucidating the distribution of intrahepatic bile ducts in animal models. Understanding the distribution characteristics of intrahepatic bile duct will facilitate the formulation of hilar bile duct microstructure classification, which can guide the operation and evaluate the prognosis better.
本研究的目的是利用双光子显微镜激发艾里光束，对胆管结扎（BDL）小鼠和胆道闭锁（BA）患者的肝内胆管进行三维成像。10只6 - 8周龄的雄性Balb/c小鼠接受肝外胆管结扎（BDL），10只接受假手术作为对照。术后，小鼠出现黄疸、尿色加深和体重减轻等症状。术后10天取BDL小鼠和对照小鼠的肝脏组织，修剪至5×5×3 mm。本研究纳入16例BA患者。其中12例行肝移植手术，术中取肝门和肝边缘组织；4例行Kasai肝门空肠吻合术（KPE），取肝边缘组织。术中肝组织样本修剪至5×5×5 mm大小。按照免疫荧光染色原则，对标本进行组织固定、抗原修复、抗体孵育及后续组织透明化处理。随后，采用光片荧光显微镜（LSFM），利用经高成像深度衰减补偿双光子激发的艾里光束对标本的肝内胆管进行成像。对图像处理应用去卷积，构建肝内胆管三维模型。对BDL小鼠和BA患者的肝组织进行三维成像，观察肝内胆管的分布情况。BDL小鼠肝内胆管明显增宽，伴有胆管增生。对照组肝内胆管无明显增生。BA患者肝内胆管成像可见明显的小胆管增生。肝内胆管排列紊乱且增生，尤其是行肝移植的患者。艾里光束三维重建技术可有效对Balb/c小鼠和BA患者的肝内胆管进行三维成像。该方法有助于更好地了解BA患者肝内胆管的分布情况。此外，通过阐明动物模型中肝内胆管的分布，有助于探索更准确模拟BA疾病的模型。了解肝内胆管的分布特征将有助于制定肝门胆管微观结构分类，从而更好地指导手术和评估预后。
