Fluorescent Imaging and Microscopy for Dynamic Processes in Rats

Abstract

The rat is a favored model organism to study physiological function in vivo. This is largely due to the fact 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 have been slower than in mice, recent advances of new genomic editing tools allows 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 4-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 photo-toxic 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 genetic modified animal models, provide new prospective for the further application of multi-photon microscopy in basic and translational research.

Intravital Multiphoton Microscopy as a Tool for Studying Renal Physiology, Pathophysiology and Therapeutics

“…Large molecular weight fluorescent molecules create shadows of blood cells in the vasculature and the velocity of these cells can be inferred from the angles of these streaks in 2D images, or more accurately from the angle of the streaks in kymographs derived from line scans. This allows for assessment of red blood cell (RBC) flow rates and the degree of white blood cell rolling and attachment following ischemic injury and during sepsis ( Dunn et al, 2002 , 2021 ; Molitoris and Sandoval, 2005 , 2011 ; Sharfuddin et al, 2009 ; Sandoval and Molitoris, 2017 ; Sandoval et al, 2019 ). In disease states red blood cells can stack together to form rouleaux reducing their oxygen delivery capacity and resulting in partial or complete peritubular capillary obstruction.…”

“…In MWF rats surface glomeruli are easily identified allowing the dynamic aspects of glomerular capillary vessel diameter, RBC flow rates, single nephron GFR, and permeability to be quantified ( Dunn et al, 2002 , 2021 ; Sandoval et al, 2019 ). Of these different parameters the measurement of glomerular permeability of macromolecules is likely the most clinically important and has created controversy in the literature.…”

“…They also function to “sense” the internal environment and have immunologic surveillance capabilities ( Hato et al, 2013 ). Intravital MPM has played an important role in understanding the processes involved, intracellular trafficking and catabolism of the reabsorbed material ( Molitoris and Sandoval, 2005 ; Horbelt et al, 2007 ; Sandoval and Molitoris, 2008 , 2017 ; Sandoval et al, 2019 ). This has been particularly important for macromolecules including therapeutic agents ( Sandoval et al, 2004 ; Molitoris and Sandoval, 2006 ; Molitoris et al, 2009 ; Kalakeche et al, 2011 ).…”

“…Careful consideration to the background fluorescence must also be accounted for when quantifying uptake of any compound into the lysosomal/endosomal pool. This value must be subtracted from the raw images to determine true and meaningful intensity values ( Sandoval et al, 2019 ). We typically take three background 3D volumes of proximal tubules at different laser transmissivities prior to imaging, and calculate average intensity values at each laser power to mathematically compensate for saturating intensities.…”

Lysine metabolism conveys kidney protection in hypertension

“…All surgical, imaging, rat uorescent albumin, and image analysis procedures were performed as described previously 53,54 . Imaging was conducted using an Olympus FV1000 microscope adapted for an intravital two-photon microscopy with high-sensitivity gallium arsenide nondescanned 12-bit detectors.…”