Sandoval Ruben M, Molitoris Bruce A, Palygin Oleg
Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN, USA.
Indiana Center for Biological Microscopy, Indianapolis, IN, USA.
Methods Mol Biol. 2019;2018:151-175. doi: 10.1007/978-1-4939-9581-3_7.
The rat is a favored model organism to study physiological function in vivo. This is largely due to the fact that it has been used for decades and is often more comparable to corresponding human conditions (both normal and pathologic) than mice. Although the development of genetic manipulations in rats has been slower than in mice, recent advances of new genomic editing tools allow for the generation of targeted global and specific cell type mutations in different rat strains. The rat is an ideal model for advancing imaging techniques like intravital multi-photon microscopy or IVMPM. Multi-photon excitation microscopy can be applied to visualize real-time physiologic events in multiple organs including the kidney. This imaging modality can generate four-dimensional high resolution images that are inherently confocal due to the fact that the photon density needed to excite fluorescence only occurs at the objective focal plane, not above or below. Additionally, longer excitation wavelengths allow for deeper penetration into tissue, improved excitation, and are inherently less phototoxic than shorter excitation wavelengths. Applying imaging tools to study physiology in rats has become a valuable scientific technique due to the relatively simple surgical procedures, improved quality of reagents, and reproducibility of established assays. In this chapter, the authors provide an example of the application of fluorescent techniques to study cardio-renal functions in rat models. Use of experimental procedures described here, together with multiple available genetically modified animal models, provide new prospective for the further application of multi-photon microscopy in basic and translational research.
大鼠是研究体内生理功能的常用模式生物。这主要是因为它已经被使用了几十年，并且在正常和病理情况下，与相应的人类状况相比，通常比小鼠更具可比性。尽管大鼠基因操作的发展比小鼠慢，但新的基因组编辑工具的最新进展使得在不同大鼠品系中产生靶向的全局和特定细胞类型突变成为可能。大鼠是推进诸如活体多光子显微镜（IVMPM）等成像技术的理想模型。多光子激发显微镜可用于可视化包括肾脏在内的多个器官中的实时生理事件。这种成像方式可以生成四维高分辨率图像，由于激发荧光所需的光子密度仅发生在物镜焦平面，而不是在其上方或下方，因此本质上是共聚焦的。此外，较长的激发波长允许更深地穿透组织，改善激发效果，并且与较短的激发波长相比，本质上光毒性更小。由于手术过程相对简单、试剂质量提高以及既定检测方法的可重复性，应用成像工具研究大鼠生理学已成为一种有价值的科学技术。在本章中，作者提供了一个在大鼠模型中应用荧光技术研究心肾功能的例子。使用这里描述的实验程序，以及多种可用的转基因动物模型，为多光子显微镜在基础研究和转化研究中的进一步应用提供了新的前景。
