IV and IP administration of rhodamine in visualization of WBC‐BBB interactions in cerebral vessels

Abstract

Epi-illuminescence intravital fluorescence microscopy has been employed to study leukocyte-endothelial interactions in a number of brain pathologies. Historically, dyes such as Rhodamine 6G have been injected intravenously. However, intravenous injections can predispose experimental animals to a multitude of complications and requires a high degree of technical skill. Here we study the efficacy of injecting Rhodamine 6G into the peritoneum (IP) for the purpose of analyzing leukocyte-endothelial interactions through a cranial window during real time intravital microscopy. After examining the number of rolling and adherent leukocytes through a cranial window we found no advantage to the intravenous injection (IV). Additionally, we tested blood from both routes of injection by flow cytometry to gain a very precise picture of the two methods. The two routes of administration failed to show any difference in the ability to detect cells. The study supports the notion that IP Rhodamine 6G works as efficaciously as IV and should be considered a viable alternative in experimental design for investigations employing intravital microscopy. Facilitated intravital studies will allow for more exploration into cerebral pathologies and allow for more rapid translation from the laboratory to the patient with less chance of experimental error from failed IV access.

Tau induces blood vessel abnormalities and angiogenesis-related gene expression in P301L transgenic mice and human Alzheimer’s disease

“…Mice received i.v. injections of 200 μL of fluorescein-conjugated dextran (70,000 MW) at a concentration of 12.5 mg/mL (Invitrogen) or, to perform labeling of circulating leukocytes, 100 μL fluorescein–dextran and 100 μL 0.05% rhodamine 6G (Sigma-Aldrich) in PBS ( 13 ).…”

Modeling leukocyte trafficking at the human blood-nerve barrier in vitro and in vivo geared towards targeted molecular therapies for peripheral neuroinflammation

“…Such advances include application of single, and subsequently multibeam two-photon microscopy, dual or multicolor two-photon imaging, and epi-illuminescence fluorescent microscopy facilitated by injecting specific dyes into rodents intravenously or intraperitoneally. These allow for the visualization and tracking of leukocytes in the brain in vivo with improved temporal and spatial resolution [ 68 – 72 ].…”

“…Epi-luminescence fluorescent IVM can be applied to track leukocyte trafficking in vivo by injecting specific fluorophores or fluorescent dyes that facilitate the detection of microvessels. Using Rhodamine 6G to visualize real-time leukocyte-endothelial interactions in the brain, intraperitoneal administration was demonstrated to be as effective as the standard intravenous injection route [ 72 ]. However, the intense excitation of light that may be necessary for visualization can cause phototoxic effects that may manifest as enhanced leukocyte adhesion [ 68 ].…”

Craniula: A cranial window technique for prolonged imaging of brain surface vasculature with simultaneous adjacent intracerebral injection

“…We have followed the approach of the well-known cranial window technique for mice that has been described in detail by Zhang and colleagues [ 9 , 18 ]. We have added several important variations to the procedure that lead to our proposed craniula technique.…”

“…The fluorophore used depends on the equipment capabilities and interests. We used 0.05 % Rhodamine 6G for assessment of leukocyte adhesion, rolling and migration across pial brain vessels [ 18 ]. The dye injection technique does affect the outcome of imaging acquisition, leaving the intravenous injection of any labeling agent as the preferable method [ 21 ].…”