Name: in vivo measurement of the mouse pulmonary endothelial surface layer
Uploaded: 2013-02-22T13:00:00
Description: Watch this Scientific Journal Video about In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer at JoVE.com

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>
	<li>Negrini, D., Tenstad, O., Passi, A., Wiig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+degradation+of+matrix+proteoglycans+and+edema+development+in+rabbit+lung.">Differential degradation of matrix proteoglycans and edema development in rabbit lung.</a> AJP - Lung Cellular and Molecular Physiology. 290, L470-L477 (2006).</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=The+pulmonary+endothelial+glycocalyx+regulates+neutrophil+adhesion+and+lung+injury+during+experimental+sepsis.">The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.</a> Nat. Med. 18, 1217-1223 (2012).</li><li>Florian, J. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamically+relevant+endothelial+cell+glycocalyx+observed+in+vivo+is+absent+in+vitro.">The hydrodynamically relevant endothelial cell glycocalyx observed in vivo is absent in vitro.</a> Circ. Res. 102, 770-776 (2008).</li><li>Weinbaum, S., Tarbell, J. M., 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>

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

Research

JoVE Journal

Medicine

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...