Published: February 22nd, 2013
The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.
The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.
The endothelial glycocalyx is an extracellular layer of proteoglycans and associated glycosaminoglycans lining the vascular intima. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that regulates a variety of endothelial functions including fluid permeability1, neutrophil-endothelial adhesion2, and the mechanotransduction of fluid shear stress3.
Historically, the glycocalyx has been underappreciated due to its aberrance in cultured cell preparations4, 5 and its degradation during standard tissue fixation and processing6.....
1. Preparation of Surgical Tubing, Vascular Catheters, Chest Wall Window
The experimental approach described in steps 1-6 will allow capture of multiple frames of simultaneous DIC (brightfield) and fluorescent images. To determine ESL thickness, recorded images are reviewed by a blinded observer after completion of the experimental protocol. Using an in-focus frame, subpleural microvessels (< 20 μm diameter) are identified; at least 3 microvessels are typically found on a single frame (Figure 10). Using image analysis software (NIS Elements, Nikon), vascular wi.......
Coincident with the expanding use of in vivo microscopy, there is increasing appreciation for both the substantial size of the ESL as well as its numerous contributions to vascular function. These emerging data, however, are primarily derived from studies of the systemic vasculature. Indeed, use of in vivo microscopy in the lung is technically challenging, given significant pulmonary and cardiac motion artifact.
Several recent technical advances have allowed for stabi.......
We thank Drs. Arata Tabuchi and Wolfgang Kuebler (University of Toronto) for instruction regarding intravital microscopy. We thank Andrew Cahill (Nikon Instruments) for assistance in microscopy design and implementation. This work was funded by NIH/NHLBI grants P30 HL101295 and K08 HL105538 (to E.P.S.).....
|Name of Reagent
|FITC-dextran (150 kDa)
|TRITC-dextran (150 kDa)
|Streptavidin-coated fluorescent microspheres
|Dragon Green fluorescence (similar to FITC)
|Anti-ICAM-1 biotinylated antibody
|Isotype biotinylated antibody
|Bipolar cautery forceps
|Temperature control system
|World Precision Instruments
|Pump 11 Elite
|Image processing software
|Circular cover slip
|5 mm, #1 thickness
|Glue (cover slip to membrane)
|For affixing cover slip to membrane
|Glue (cover slip to mouse)
|For attaching membrane to mouse
|Curved surgical forceps
|Straight surgical forceps
|Surgical needle driver
|Kitchen sponges (cut into wedges)
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