Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy
Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy
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DOI：
10.1007/s00535-009-0187-7
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发表时间：
2010-05-01
影响因子：
6.3
通讯作者：
Kusunoki, Masato
中科院分区：
文献类型：
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作者：
Toiyama, Yuji;Mizoguchi, Akira;Kusunoki, Masato
双光子激光扫描显微镜（TPLSM）是一种强大的诊断工具，用于实时，高分辨率的结构成像。然而，获得高质量的体内腹腔内器官的TPLSM图像仍然是技术上的挑战。利用TPLSM对转绿色荧光蛋白（GFP）基因小鼠的肝脏、脾脏、肾脏和肠道等内脏器官进行了体内实时成像。还在简单手术后在制备的GFP小鼠中研究了使用葡聚糖硫酸钠（DSS）诱导的结肠炎的细菌易位模型。这允许使用体内TPLSM捕获形态学真实的图像。ZO-1的免疫组化分析进行支持TPLSM的形态学研究结果。我们建立了一个器官稳定系统，以评估实时成像的肌动蛋白-绿色荧光蛋白转基因小鼠的内脏器官在体内TPLSM。DSS诱导的结肠炎表现为隐窝结构不规则、隐窝消失、炎性细胞浸润和沿着脉管系统的白色血细胞滚动增加。此外，在DSS结肠炎期间，肠粘膜中隐窝中的粘膜细胞和肠壁中的血管内皮细胞的细胞间距离增加。在DSS结肠炎中，粘膜和血管内皮ZO-1表达显著丧失，这可以通过ZO-1染色的减少看出。总之，我们的观察结果表明，我们的TPLSM成像系统可以用于澄清各种疾病的病理生理变化，在同一动物在很长一段时间内的微观变化的纵向研究。
Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging.An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM.We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin-GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.