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.).

1. Preparation of Surgical Tubing, Vascular Catheters, Chest Wall Window
<ol>
	<li>Intravital microscopy stage. We custom-made a plexiglass stage upon which the anesthetized mouse lies during microscopy. This stage accommodates both a 15 cm by 10 cm flexible plastic cutting board (upon which the mouse lies during induction of anesthesia, tracheostomy placement, and venous catheterization) as well as a similarly-sized heating element (located underneath the cutting board).</li>
	<li>Mouse thorac...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycan+is+a+mechanosensor+on+endothelial+cells.">Heparan sulfate proteoglycan is a mechanosensor on endothelial cells.</a> Circ. Res. 93, e136-e142 (2003).</li><li>Chappell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Glycocalyx+of+the+Human+Umbilical+Vein+Endothelial+Cell:+An+Impressive+Structure+Ex+Vivo+but+Not+in+Culture.">The Glycocalyx of the Human Umbilical Vein Endothelial Cell: An Impressive Structure Ex Vivo but Not in Culture.</a> Circulation Research. 104, 1313-1317 (2009).</li><li>Potter, D. R., Damiano, E. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Luminal+Domains+for+Macromolecules,+Erythrocytes,+and+Leukocytes+Within+Mammalian+Capillaries.">Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries.</a> Circ. Res. 79, 581-589 (1996).</li><li>Marechal, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+glycocalyx+damage+during+endotoxemia+coincides+with+microcirculatory+dysfunction+and+vascular+oxidative+stress.">Endothelial glycocalyx damage during endotoxemia coincides with microcirculatory dysfunction and vascular oxidative stress.</a> Shock. 29, 572-576 (2008).</li><li>Presson, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Imaging+within+the+Murine+Thorax+without+Respiratory+and+Cardiac+Motion+Artifact.">Two-Photon Imaging within the Murine Thorax without Respiratory and Cardiac Motion Artifact.</a> The American Journal of Pathology. 179, 75-82 (2011).</li><li>Looney, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nat. Meth. 8, 91-96 (2011).</li><li>Pearse, D. B., Wagner, E. M., Permutt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ventilation+on+vascular+permeability+and+cyclic+nucleotide+concentrations+in+ischemic+sheep+lungs.">Effect of ventilation on vascular permeability and cyclic nucleotide concentrations in ischemic sheep lungs.</a> J. Appl. Physiol. 86, 123-132 (1999).</li><li>Hossain, M., Qadri, S., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+nitric+oxide+synthesis+enhances+leukocyte+rolling+and+adhesion+in+human+microvasculature.">Inhibition of nitric oxide synthesis enhances leukocyte rolling and adhesion in human microvasculature.</a> Journal of Inflammation. 9, 28 (2012).</li><li>Schmidt, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+guanylyl+cyclase+contributes+to+ventilator-induced+lung+injury+in+mice.">Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice.</a> AJP - Lung Cellular and Molecular Physiology. 295, L1056-L1065 (2008).</li><li>Mead, J., Takishima, T., Leith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+distribution+in+lungs:+a+model+of+pulmonary+elasticity.">Stress distribution in lungs: a model of pulmonary elasticity.</a> J. Appl. Physiol. 28, 596-608 (1970).</li><li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> J. Appl. Physiol. 104, 338-346 (2008).</li><li>Gattinoni, L., Protti, A., Caironi, P., Carlesso, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilator-induced+lung+injury:+the+anatomical+and+physiological+framework.">Ventilator-induced lung injury: the anatomical and physiological framework.</a> Crit. Care Med. 38, 539-548 (2010).</li><li>Tabuchi, A., Kim, M., Semple, J. W., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Lung+Injury+Causes+Pendelluft+Between+Adjacent+Alveoli+In+Vivo.">Acute Lung Injury Causes Pendelluft Between Adjacent Alveoli In Vivo.</a> Am. J. Respir. Crit. Care Med. 183, A2490 (2011).</li><li>Roebuck, K. A., Finnegan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intercellular+adhesion+molecule-1+(CD54)+gene+expression.">Regulation of intercellular adhesion molecule-1 (CD54) gene expression.</a> J. Leukoc. Biol. 66, 876-888 (1999).</li></ol>

No conflicts of interest declared.

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...

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. The increasing use7 of intravital microscopy (in vivo microscopy, IVM) has coincided with heightened scientific interest in the importance of the ESL to vascular function during health and disease. The ESL is invisible to light microscopy and cannot be easily labeled in vivo, given the propensity of fluorescent glycocalyx-binding lectins to cause RBC agglutination8 and fatal pulmonary emboli (unpublished observations). Several indirect approaches have therefore been developed to deduce ESL thickness (and, by extension, glycocalyx integrity) in non-moving vascular beds such as the cremasteric and mesenteric microcirculations. These techniques include the measurement of differences in circulating microparticle velocity as a function of distance from the endothelial membrane (microparticle image velocimetry9) as well as the measurement of the exclusion of bulky, fluorescently-labeled vascular markers (e.g. dextrans) from the endothelial surface (dextran exclusion technique10, 11). Of these techniques, only dextran exclusion is capable of estimating ESL thickness from measurements made at a single point in time. By simultaneously measuring vascular widths using brightfield microscopy (a width inclusive of the "invisible" ESL) and fluorescent microscopy of a vascular tracer excluded from the ESL, ESL thickness can be calculated as one-half the difference between vascular widths2.
	The use of an instantaneous measure of ESL thickness is well-suited for study of the pulmonary glycocalyx. Intravital microscopy of the lung is challenging, given significant pulmonary and cardiac motion artifact. While recent advances allow for immobilization of mouse lungs in vivo12, 13, concerns exist regarding the physiologic impact of lung stasis. Lung immobility is associated with decreased endothelial nitric oxide signaling14, a signaling pathway that impacts both neutrophil adhesion15 and lung injury16. Furthermore, immobilization of an area of lung exposes surrounding mobile alveoli to injurious shear forces (so-called "atelectrauma"), in accordance with the classic physiologic concepts of alveolar interdependence17.
	In 2008, Arata Tabuchi, Wolfgang Kuebler and colleagues developed a surgical technique allowing for intravital microscopy of a freely-moving mouse lung18. Respiratory artifact arising from this technique can be negated by use of high-speed imaging, including simultaneous measurement of brightfield and fluorescent microscopy. In this report, we detail how instantaneous dextran exclusion imaging can be employed to measure ESL thickness in the subpleural microcirculation of a freely-moving, in vivo mouse lung. This technique can be easily modified to determine glycocalyx function-specifically, the ability of an intact ESL to exclude circulating elements from the endothelial surface. We have recently used these techniques to determine the importance of pulmonary ESL integrity to the development of acute lung injury during systemic inflammatory diseases such as sepsis2.

<table border="1">
	 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Name of Reagent</td>
 </tr>
 <tr>
 <td>FITC-dextran (150 kDa)</td>
 <td>Sigma</td>
 <td>FD150S</td>
 <td></td>
 </tr>
 <tr>
 <td>TRITC-dextran (150 kDa)</td>
 <td>Sigma</td>
 <td>T1287</td>
 <td></td>
 </tr>
 <tr>
 <td>Streptavidin-coated fluorescent microspheres</td>
 <td>Bangs Laboratories</td>
 <td>CP01F/10428</td>
 <td>Dragon Green fluorescence (similar to FITC)</td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Moore Medical</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Moore Medical</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-ICAM-1 biotinylated antibody</td>
 <td>eBioscience</td>
 <td>Clone YN1/1.7.4</td>
 <td>1:50 dilution</td>
 </tr>
 <tr>
 <td>Isotype biotinylated antibody</td>
 <td>eBioscience</td>
 <td>IgG2b eB149/10H5</td>
 <td>1:50 dilution</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Mechanical ventilator</td>
 <td>Harvard Apparatus</td>
 <td>Inspira</td>
 <td></td>
 </tr>
 <tr>
 <td>Tracheostomy catheter</td>
 <td>Harvard Apparatus</td>
 <td>730028</td>
 <td></td>
 </tr>
 <tr>
 <td>Electrocautery apparatus</td>
 <td>DRE Medical</td>
 <td>Valleylab SSE-2L</td>
 <td></td>
 </tr>
 <tr>
 <td>Bipolar cautery forceps</td>
 <td>Olsen Medical</td>
 <td>10-1200I</td>
 <td>9.9cm McPherson </td>
 </tr>
 <tr>
 <td>Temperature control system</td>
 <td>World Precision Instruments</td>
 <td>ATC1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe pump</td>
 <td>Harvard Apparatus</td>
 <td>Pump 11 Elite</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope (widefield)</td>
 <td>Nikon</td>
 <td>LV-150</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope (confocal)</td>
 <td>Nikon</td>
 <td>A1R</td>
 <td></td>
 </tr>
 <tr>
 <td>Image splitter</td>
 <td>Photometrics</td>
 <td>DV2</td>
 <td></td>
 </tr>
 <tr>
 <td>CCD camera</td>
 <td>Photometrics</td>
 <td>CoolSNAP HQ2</td>
 <td></td>
 </tr>
 <tr>
 <td>Image processing software</td>
 <td>Nikon</td>
 <td>NIS Elements</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyvinylidene membrane</td>
 <td>Kure Wrap</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Circular cover slip</td>
 <td>Bellco</td>
 <td>5CIR-1-BEL</td>
 <td>5 mm, #1 thickness</td>
 </tr>
 <tr>
 <td>Glue (cover slip to membrane)</td>
 <td>Pattex</td>
 <td>Flussig (liquid)</td>
 <td>For affixing cover slip to membrane</td>
 </tr>
 <tr>
 <td>Glue (cover slip to mouse)</td>
 <td>Pattex</td>
 <td>Gel</td>
 <td>For attaching membrane to mouse</td>
 </tr>
 <tr>
 <td>Surgical tubing</td>
 <td>Intramedic</td>
 <td>PE50, PE10</td>
 <td></td>
 </tr>
 <tr>
 <td>Suture</td>
 <td>Fisher</td>
 <td></td>
 <td>4:0 silk</td>
 </tr>
 <tr>
 <td>Electric razor</td>
 <td>Oster</td>
 <td>78997</td>
 <td></td>
 </tr>
 <tr>
 <td>Curved surgical forceps</td>
 <td>Roboz</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Straight surgical forceps</td>
 <td>Roboz</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical scissors</td>
 <td>Roboz</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical microscissors</td>
 <td>Roboz</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical needle driver</td>
 <td>Roboz</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical tape</td>
 <td>Fisher</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Kitchen sponges (cut into wedges)</td>
 <td>various</td>
 <td></td>
 <td></td>
 </tr>
	 </tbody>
 </table>

in vivo measurement of the mouse pulmonary endothelial surface layer

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 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 (&lt; 20 &mu;m 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...

Watch this Scientific Journal Video about In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer at JoVE.com

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

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.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

in-vivo-measurement-of-the-mouse-pulmonary-endothelial-surface-layer

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14.5K visualizações.  University of Colorado School of Medicine. A camada de superfície endotelial glicocálice / endotelial está idealmente estudada por microscopia intravital. Microscopia intravital é tecnicamente desafiante em um órgão em movimento, tais como o pulmão. Demonstramos como brightfield simultânea e microscopia de fluorescência pode ser utilizado para estimar a espessura da camada endotelial da superfície em um movimento livremente In vivo Pulmão do rato.