Abstract
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.
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References
Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ et al (2002) Functional studies of the kidney of living animals using multicolor two-photon microscopy. Am J Phys Cell Phys 283(3):C905–C916. https://doi.org/10.1152/ajpcell.00159.2002
Svoboda K, Denk W, Kleinfeld D, Tank DW (1997) In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385(6612):161–165. https://doi.org/10.1038/385161a0
Peti-Peterdi J, Morishima S, Bell PD, Okada Y (2002) Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus. Am J Physiol Ren Physiol 283(1):F197–F201. https://doi.org/10.1152/ajprenal.00356.2001
Ferrell N, Sandoval RM, Bian A, Campos-Bilderback SB, Molitoris BA, Fissell WH (2015) Shear stress is normalized in glomerular capillaries following (5/6) nephrectomy. Am J Physiol Ren Physiol 308(6):F588–F593. https://doi.org/10.1152/ajprenal.00290.2014
Kleinfeld D, Mitra PP, Helmchen F, Denk W (1998) Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc Natl Acad Sci U S A 95(26):15741–15746
Schiessl IM, Bardehle S, Castrop H (2013) Superficial nephrons in BALB/c and C57BL/6 mice facilitate in vivo multiphoton microscopy of the kidney. PLoS One 8(1):e52499. https://doi.org/10.1371/journal.pone.0052499
Chobanian AV (2009) Shattuck Lecture. The hypertension paradox--more uncontrolled disease despite improved therapy. N Engl J Med 361(9):878–887. https://doi.org/10.1056/NEJMsa0903829
Cowley AW Jr (1997) Genetic and nongenetic determinants of salt sensitivity and blood pressure. Am J Clin Nutr 65(2 Suppl):587S–593S
Mattson DL, Dwinell MR, Greene AS, Kwitek AE, Roman RJ, Jacob HJ et al (2008) Chromosome substitution reveals the genetic basis of Dahl salt-sensitive hypertension and renal disease. Am J Physiol Ren Physiol 295(3):F837–F842. https://doi.org/10.1152/ajprenal.90341.2008
Russo LM, Sandoval RM, Campos SB, Molitoris BA, Comper WD, Brown D (2009) Impaired tubular uptake explains albuminuria in early diabetic nephropathy. J Am Soc Nephrol 20(3):489–494. https://doi.org/10.1681/ASN.2008050503
Ilatovskaya DV, Levchenko V, Lowing A, Shuyskiy LS, Palygin O, Staruschenko A (2015) Podocyte injury in diabetic nephropathy: implications of angiotensin II-dependent activation of TRPC channels. Sci Rep 5:17637. https://doi.org/10.1038/srep17637
Slaughter TN, Paige A, Spires D, Kojima N, Kyle PB, Garrett MR et al (2013) Characterization of the development of renal injury in Type-1 diabetic Dahl salt-sensitive rats. Am J Phys Regul Integr Comp Phys 305(7):R727–R734. https://doi.org/10.1152/ajpregu.00382.2012
Endres BT, Sandoval RM, Rhodes GJ, Campos-Bilderback SB, Kamocka MM, McDermott-Roe C et al (2017) Intravital imaging of the kidney in a rat model of salt-sensitive hypertension. Am J Physiol Ren Physiol 313(2):F163–F173. https://doi.org/10.1152/ajprenal.00466.2016
Rhodes GJ (2017) Surgical preparation of rats and mice for intravital microscopic imaging of abdominal organs. Methods 128:129–138. https://doi.org/10.1016/j.ymeth.2017.07.003
Sandoval RM, Molitoris BA (2013) Quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in Munich Wistar rats. J Vis Exp (74). https://doi.org/10.3791/50052
Russo LM, Sandoval RM, McKee M, Osicka TM, Collins AB, Brown D et al (2007) The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states. Kidney Int 71(6):504–513. https://doi.org/10.1038/sj.ki.5002041
Wagner MC, Campos-Bilderback SB, Chowdhury M, Flores B, Lai X, Myslinski J et al (2016) Proximal tubules have the capacity to regulate uptake of albumin. J Am Soc Nephrol 27(2):482–494. https://doi.org/10.1681/ASN.2014111107
Asgeirsson D, Venturoli D, Rippe B, Rippe C (2006) Increased glomerular permeability to negatively charged Ficoll relative to neutral Ficoll in rats. Am J Physiol Ren Physiol 291(5):F1083–F1089. https://doi.org/10.1152/ajprenal.00488.2005
Tojo A, Endou H (1992) Intrarenal handling of proteins in rats using fractional micropuncture technique. Am J Phys 263(4 Pt 2):F601–F606. https://doi.org/10.1152/ajprenal.1992.263.4.F601
Sandoval RM, Wagner MC, Patel M, Campos-Bilderback SB, Rhodes GJ, Wang E et al (2012) Multiple factors influence glomerular albumin permeability in rats. J Am Soc Nephrol 23(3):447–457. https://doi.org/10.1681/ASN.2011070666
Yamamoto T, Tada T, Brodsky SV, Tanaka H, Noiri E, Kajiya F et al (2002) Intravital videomicroscopy of peritubular capillaries in renal ischemia. Am J Physiol Ren Physiol 282(6):F1150–F1155. https://doi.org/10.1152/ajprenal.00310.2001
Sharfuddin AA, Sandoval RM, Berg DT, McDougal GE, Campos SB, Phillips CL et al (2009) Soluble thrombomodulin protects ischemic kidneys. J Am Soc Nephrol 20(3):524–534. https://doi.org/10.1681/ASN.2008060593
McCurley A, Alimperti S, Campos-Bilderback SB, Sandoval RM, Calvino JE, Reynolds TL et al (2017) Inhibition of alphavbeta5 integrin attenuates vascular permeability and protects against renal ischemia-reperfusion injury. J Am Soc Nephrol 28(6):1741–1752. https://doi.org/10.1681/ASN.2016020200
Sandoval RM, Wang E, Molitoris BA (2014) Finding the bottom and using it: offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy. Dermatol Int 2(1):e23674. https://doi.org/10.4161/intv.23674
Acknowledgments
The authors acknowledge grant support from the National Institutes of Health (NIH) (DK091623 and DK079312), the Veterans Administration through a Merit Review award (to B.A.M.), and the American Heart Association 17SDG33660149 (to OP).
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Sandoval, R.M., Molitoris, B.A., Palygin, O. (2019). Fluorescent Imaging and Microscopy for Dynamic Processes in Rats. In: Hayman, G., Smith, J., Dwinell, M., Shimoyama, M. (eds) Rat Genomics. Methods in Molecular Biology, vol 2018. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9581-3_7
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DOI: https://doi.org/10.1007/978-1-4939-9581-3_7
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