title: Real-time differential labeling of blood, interstitium, and lymphatic and single-field analysis of vasculature dynamics in vivo.
abstract: Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd. (MEM-L55), La Jolla, CA 92037, USA.
authors.forename: Gor
authors.initials: G
authors.lastname: Sarkisyan
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Cahalan
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Gonzalez-Cabrera
authors.forename: Nora B
authors.initials: NB
authors.lastname: Leaf
authors.forename: Hugh
authors.initials: H
authors.lastname: Rosen
mesh_terms: D000818:Animals; D003199:Computer Systems; D042783:Endothelial Cells / Q000737:chemistry / Q000502:physiology*; D045604:Extracellular Fluid / Q000737:chemistry* / Q000502:physiology; D008198:Lymph Nodes / Q000098:blood supply / Q000737:chemistry / Q000502:physiology; D042601:Lymphatic Vessels / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D049349:Receptors, Lysosphingolipid / Q000096:biosynthesis / Q000097:blood; D012039:Regional Blood Flow / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D049349:Receptors, Lysosphingolipid