The studies were supported by NIH HL75503 to KP.

All experiments performed on animals were as approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center.
1. Tubing for Perfusing Rat Lung Preparations
<ol>
	<li>Prepare a tubing system with Tygon tubing (#18) for blood perfusion as shown in Figure 1. Connect pressure transducers (P23XL) and place the tubing on the microscope stage.</li>
	<li>Set water bath temperature to 37 &#176;C.</li>
	<li>Connect the lung inlet and outlet temporarily with Tygon tubing.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> 
Figure 1.&#160;Blood perfusion tubing. The schematic shows the tubing setup used for circulating blood through the isolated lung preparation. Also indicated are associated components through which the tubing is routed. A schematic of the heart and lung is included to show sites of connection between the tubing and pulmonary artery (PA), and left atrial (LA) cannula (blue dashed lines).
2. Preparation of Isolated Rat Lungs
<ol>
	<li>Anesthetize rats (male Sprague-Dawley Rats; 250-300 g) with ketamine (80-100 mg/kg) and with xylazine (5-10 mg/kg). After ensuring a surgical plane of anesthesia, place the animal in a supine position.</li>
	<li>Perform tracheostomy, insert a tracheal cannula (PE-90) and secure with surgical suture.</li>
	<li>Infuse heparin (100-200 U) into the heart by cardiac puncture (21 G butterfly needle), wait for 60 sec and exsanguinate blood (~12-15 ml blood).</li>
	<li>Prepare two cannulas (Tygon tubing, 3 mm diameter; 4 cm long; flared at one end) and fill with saline.</li>
	<li>Perform a thoracotomy. Make an incision (3 mm) on the right ventricle and slide the flared end of a cannula into the incision toward the pulmonary artery. Secure cannula to pulmonary artery with surgical sutures.</li>
	<li>Incise (3 mm) the apex of the left ventricle and slide the flared end of the second cannula into the incision. Guide the cannula into the left atrium. Secure cannula to the left ventricle with an umbilical tape (2 mm width).</li>
	<li>Dissect any connective tissue, and remove the lung and heart together with the attached cannulas. Place lung and heart on a petri dish. Reposition the lung so that the diaphragmatic surface is on top. The three cannulas should face the same direction.</li>
	<li>Place the lung preparation on a stage that can be manipulated in the X-Y directions. Any stage that comes with a microscope can be modified to hold the tubing or a custom-made stage built, if needed.</li>
</ol>
3. Blood Perfusion of Isolated Rat Lungs
<ol>
	<li>Mix the exsanguinated blood with an equal volume albumin (5%) solution and add to the reservoir. Start the peristaltic pump and set flow rate to 14 ml/min. Allow blood to fill the tubing.</li>
	<li>With the shunt open, remove the temporary lung inlet-outlet connector. Attach the pulmonary artery and left atrial cannula to the lung inlet and outlet, respectively. Ensure that there are no air bubbles in the tubing.</li>
	<li>Connect the tracheal cannula to a third pressure transducer (P23XL). Inflate lungs with 30% oxygen via the tracheal cannula and maintain pressure at 5 cm H2O.</li>
	<li>Close the shunt with a clamp to begin lung perfusion. Maintain pulmonary artery and left atrial pressures at ~10 and 3 cm H2O respectively.</li>
</ol>
4. Preparing Lung for Microvascular Infusion
<ol>
	<li>Raise the rear end of the extension tubing for the catheter above the level of the lung and secure.</li>
	<li>Prepare an infusion catheter by inserting about 30 cm of a 40 cm long PE10 tubing into a ~30 cm long PE90 tubing. Connect the exposed end of the PE10 tubing to a needle (30 G) attached to a syringe (1 cc).</li>
	<li>Insert the catheter combination into the raised rear end of the extension tubing. Guide the catheter combination through the left atrium and into the lung until it meets resistance. Then, push the PE10 catheter alone further into the lung until it meets resistance. Note that applying excessive force while inserting the catheter will injure the lung.</li>
	<li>Fill the 1 cc syringe with Ringer&#8217;s solution and attach to a syringe pump. Set infusion rate to 10 &#181;l/min.</li>
	<li>Infusion site will become pale compared to rest of the lung.</li>
	<li>Moisten the lung with saline and cover the lung with plastic wrap with a hole large enough to leave the infusion site uncovered.</li>
	<li>Swab some stopcock grease on the bottom of an O-ring (custom-made) and place a glass coverslip (#1.5, 22 mm diameter) on the bottom of the O-ring. Gently position the O-ring with the coverslip on the infusion site. Secure the O-ring with a standard test-tube holder. The coverslip/O-ring combination does not distort the alveolar structures underneath it, as recently reported15.</li>
	<li>Position an objective (20X) of a fluorescence microscope above the O-ring and focus on the lung surface.</li>
</ol>
5. Imaging Fluorescent Dextran Transit Through Microvessels
<ol>
	<li>Prepare the microscope for imaging FITC florescence.</li>
	<li>Select a region to be imaged and acquire images at the rate of 1/minute using image acquisition software.</li>
	<li>Begin infusion of FITC-dextran 20 kD (0.5 mg/ml) using the syringe pump. Fluorescence in blood vessels will gradually increase and reach a maximum. Continue infusion for 60 min.</li>
	<li>Then switch to Ringer&#8217;s infusion to wash off luminal fluorescence. Maintain infusion for over 10 min. Continue image acquisition at 1/min during wash-off.</li>
</ol>
6. Image Analysis
<ol>
	<li>Open the image acquisition file.</li>
	<li>Place regions of interest over microvessels within the image frame.</li>
	<li>Play the image file frame-by-frame and record the fluorescence intensity at each region of interest for all frames.</li>
	<li>Plot the change in fluorescence intensity for each region of interest as a function of time.</li>
	<li>Quantify the maximum fluorescence intensity and the residual fluorescence intensity after 10 min of washoff.</li>
	<li>Calculate the ratio of maximum to residual fluorescence intensity at each region of interest to get the permeability index for microvessels associated with that region of interest.</li>
</ol>

<ol>
	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. 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=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Bhattacharya, J., Matthay, M. 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+and+repair+of+the+alveolar-capillary+barrier+in+acute+lung+injury.">Regulation and repair of the alveolar-capillary barrier in acute lung injury.</a> Annu Rev Physiol. 75, 593-615 (2013).</li><li>Parthasarathi, K., 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=Connexin+43+mediates+spread+of+Ca2+-dependent+proinflammatory+responses+in+lung+capillaries.">Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries.</a> J Clin Invest. 116, 2193-2200 (2006).</li><li>Parthasarathi, K., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Acid+instillation+by+a+wedged-catheter+method+reveals+a+role+for+vascular+gap+junctions+in+spatial+expansion+of+Acid+injury.">Localized Acid instillation by a wedged-catheter method reveals a role for vascular gap junctions in spatial expansion of Acid injury.</a> Anat Rec (Hoboken). 294, 1585-1591 (2011).</li><li>Gorin, A. B., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+permeability+of+endothelial+and+epithelial+barriers+to+albumin+flux.">Differential permeability of endothelial and epithelial barriers to albumin flux.</a> J Appl Physiol Respir Environ Exerc Physiol. 47, 1315-1324 (1979).</li><li>Boutoille, 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=FITC-albumin+as+a+marker+for+assessment+of+endothelial+permeability+in+mice:+comparison+with+125I-albumin.">FITC-albumin as a marker for assessment of endothelial permeability in mice: comparison with 125I-albumin.</a> Exp Lung Res. 35, 263-271 (2009).</li><li>Thorball, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-dextran+tracers+in+microcirculatory+and+permeability+studies+using+combined+fluorescence+stereo+microscopy,+fluorescence+light+microscopy+and+electron+microscopy.">FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.</a> Histochemistry. 71, 209-233 (1981).</li><li>Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+heterogeneity+of+pulmonary+endothelial+cells.">Functional and molecular heterogeneity of pulmonary endothelial cells.</a> Proc Am Thorac Soc. 8, 453-457 (2011).</li><li>Ofori-Acquah, S. F., 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=Heterogeneity+of+barrier+function+in+the+lung+reflects+diversity+in+endothelial+cell+junctions.">Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions.</a> Microvasc Res. 75, 391-402 (2008).</li><li>Kandasamy, K., 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=Real-time+imaging+reveals+endothelium-mediated+leukocyte+retention+in+LPS-treated+lung+microvessels.">Real-time imaging reveals endothelium-mediated leukocyte retention in LPS-treated lung microvessels.</a> Microvasc Res. 83, 323-331 (2012).</li><li>Kandasamy, K., 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=Lipopolysaccharide+induces+endoplasmic+store+Ca2+-dependent+inflammatory+responses+in+lung+microvessels.">Lipopolysaccharide induces endoplasmic store Ca2+-dependent inflammatory responses in lung microvessels.</a> PloS One. 8, (2013).</li><li>Qiao, R. L., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+barrier+properties+of+the+pulmonary+microvascular+bed.">Segmental barrier properties of the pulmonary microvascular bed.</a> J Appl Physiol. 71, 2152-2159 (1991).</li><li>Parthasarathi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+connexin43+mediates+acid-induced+increases+in+pulmonary+microvascular+permeability.">Endothelial connexin43 mediates acid-induced increases in pulmonary microvascular permeability.</a> Am J Physiol Lung Cell Mol Physiol. 303, (2012).</li><li>Wu, Y., Perlman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+methods+for+assessing+alveolar+mechanics.">In situ methods for assessing alveolar mechanics.</a> J Appl Physiol 1985. 112, 519-526 (2012).</li><li>Kuebler, W. M., 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=A+novel+signaling+mechanism+between+gas+and+blood+compartments+of+the+lung.">A novel signaling mechanism between gas and blood compartments of the lung.</a> Journal Clin Invest. 105, 905-913 (2000).</li></ol>

The authors have nothing to disclose.

The isolated blood-perfused lung preparation coupled with real time imaging provides a simple tool for determination of permeability changes in single lung microvessels. We applied this method to define permeability changes in response to infusions of LPS. Our data clearly suggest that LPS infusion caused an increase in microvascular permeability. Further, the data also indicate that permeability changes induced by LPS were similar in both venules and capillaries. Thus, a major advantage of this technique is the ability ...

Increased microvascular permeability in lungs leads to development of alveolar edema and compromised gas exchange and is a major characteristic of acute lung injury (ALI)1-3. Thus, estimates of vascular permeability are important in defining the extent of lung injury and efficacy of proposed therapeutic interventions. Gravimetric analysis such as blood free lung wet-to-dry ratio and microvascular filtration coefficient are widely used methods to estimate permeability4,5. Other methods include quantifying the retention of radioactive or fluorescent probes in lung tissue6-8. However, the above methods require postexperiment processing of lung tissue samples toward elucidating the permeability data. Moreover, since one animal can be only used for a single treatment protocol, large animal numbers may be needed for a complete study. A common characteristic of the above methods is that they determine the mean vascular permeability for all blood vessels within the tissue sample. However, it is well established that pulmonary micro- and macro-vessels are phenotypically different9. Hence, permeability responses may be heterogeneous among the various vessel segments as well9,10. Thus, quantifying mean permeability of all pulmonary vessels in a tissue sample may not adequately reflect this heterogeneity.
In the isolated blood-perfused lung preparation, blood vessels on the lung surface can be visualized by an upright microscope4,11,12. This enables characterizing responses in single vessels and thus, addressing any heterogeneity in the responses13. In addition, by utilizing fluorescence imaging of microvessels, fluorescence based assays can be incorporated. Further, a left atrial microcatheter can be used to deliver agents and fluorescence probes into blood vessels11,14. The microcatheter limits the delivery to a small lung region, thus exposing only the blood vessels within the region to the infused agents and fluorophores. This allows multiple small regions within the same lung to be used for separate experiments, leading to an overall reduction in animals needed for a study.
Real time imaging enables capture of dynamic changes in vascular and extravascular fluorescence of single microvessels of the isolated lung preparation. Thus, for each microvessel within an image field, changes in fluorescence during infusion of fluorophores and washoff can be recorded, and quantified offline14. Using values of maximum and residual vascular fluorescence, a permeability index for each microvessel within the imaging field can be determined. To determine permeability changes in response to inflammatory or injurious agents, the desired agent can be administered first and then the permeability index determined. In addition, the image field can be set anywhere within the lung region infused by the microcatheter, thus enabling a high degree of flexibility in selecting the desired vascular network. Thus, the isolated blood-perfused lung preparation in tandem with real time imaging provides an attractive experimental model to quantify permeability in single lung microvessels.

<table><tbody><tr><td>Tygon Tubing</td><td>Fisher Scientific</td><td></td><td>#18</td></tr><tr><td>Pressure Transducer</td><td>Data Sciences International</td><td>P23XL</td><td>Need quantity 3</td></tr><tr><td>Butterfly Needle</td><td>Greiner Bio-One</td><td>450081</td><td>21 G</td></tr><tr><td>Peristaltic pump</td><td>Cole Parmer</td><td>Masterflex L/S</td><td></td></tr><tr><td>PE-90 tubing</td><td>Becton Dickinson</td><td>427421</td><td>30 cm needed</td></tr><tr><td>PE-10 tubing</td><td>Becton Dickinson</td><td>427401</td><td>40 cm needed</td></tr><tr><td>Syringe Pump</td><td>Braintree Scientific</td><td>BS8000</td><td></td></tr><tr><td>O-ring</td><td></td><td></td><td>Custom made with a 20 mm diamter hole and a handle to secure O-ring to holder</td></tr><tr><td>Upright fluorescence microscope</td><td>Olympus America</td><td>BX61WI</td><td></td></tr><tr><td>Image Acquisition Software</td><td>Molecular Devices</td><td>Metamorph</td><td></td></tr><tr><td>FITC Dextran 20KD</td><td>Sigma Aldrich</td><td></td><td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&amp;nbsp;</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma Aldrich</td><td></td><td>Serotype 0111:B4</td></tr></tbody></table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

An isolated blood-perfused lung preparation connected to the perfusion tubing and related equipment is shown in Figure 2. For demonstration purposes, we used a Sprague Dawley rat, though the procedures described herein can be used with any rat species. Infusions through a left atrial microcatheter reach only a small region of the lung. The infused region can be identified by the infusion-induced discoloration (Figure 3). The lung preparation as positioned for real-time imaging is shown i...

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Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

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9.4K Views.  The University of Tennessee Health Science Center. The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

Video: Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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 ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

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. ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Summary

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Chapters in this video

An in vivo Assay to Test Blood Vessel Permeability

Angiogenesis in the Ischemic Rat Lung

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

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

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion