This study was supported in part by the Victorian Government's OIS Program, the National Health and Medical Research Council of Australia program and project grants (JPF Chin-Dusting) and postgraduate scholarship (D Michell).

1. Isolating carotid arteries
<ol>
	<li>Euthanase10 week old Sprague Dawley rats via CO2/O2 asphyxiation.</li>
	<li>Excise left and right common carotid arteries with aorta and heart ensuring minimal stretching of the vessels.</li>
	<li>In ice cold Krebs buffer separate the carotid arteries from the aorta and heart and perform close dissection.</li>
	<li>Keep isolated vessels in Krebs on ice before mounting.</li>
</ol>
Approx. time = 45 mins
2. Priming the vessel chamber
<ol>
	<li>At the proximal and distal connectors of the vessel chamber flush Krebs buffer maintained at physiological pH by infusing carbogen gas (95% O2; 5% CO2) through the buffer at 37&deg;C.</li>
	<li>Ensure tubing and cannulas are flushed completely and are aligned.</li>
	<li>Flush Kreb buffer through the P1 (proximal) and P2 (distal) transducers. Close taps to ensure no air bubbles in the transducers.</li>
	<li>Connect the transducers to the corresponding connectors and again flush more Krebs buffer ensuring no air bubbles. Close off taps to the chamber.</li>
</ol>
Approx. time = 15 mins
3. Pressurizing the vessel chamber
<ol>
	<li>Turn on pressure equipment: pressure servo, pressure monitor, peristaltic pump (Figure 1).</li>
	<li>With the peristaltic pump on pressure and the pressure servo on automatic run Krebs buffer through the tubing at 20 mmHg (dial at 1), ensuring no air bubbles.</li>
	<li>Connect tubing to the closed P2 transducer. Pressure will become stable.</li>
	<li>Open P2 tap to the vessel chamber to flush out any bubbles then close off.</li>
	<li>Fill the bath with Krebs buffer (5 - 7 mL).</li>
</ol>
Approx. time = 10 mins
4. Mounting the vessel
<ol>
	<li>Under a dissecting microscope place black polyester ties onto each cannula (Figure 2).</li>
	<li>Move cannulas and cannula holders apart and mount &frac14; vessel (aortic arch end) onto the P1 cannula. Ensure not to tear the vessel.</li>
	<li>Using a syringe filled with Krebs buffer gently flush excess blood out of the vessel via P1 transducer. Close off P1 to chamber.</li>
	<li>Secure vessel to the P1 cannula with the polyester tie.</li>
	<li>Move cannulas/cannula holders closer for mounting onto the P2 cannula.</li>
	<li>Mount distal end of the vessel (~&frac14; of the length) onto distal cannula. Secure with polyester tie.</li>
</ol>
Approx. time = 20 mins
5. Pressurizing the vessel
<ol>
	<li>Adjust the cannula holders to ensure no bend or stretch in the vessel.</li>
	<li>With P1 and P2 closed to the chamber switch the pressure servo from automatic to manual. Check on the pressure servo there is no continual decrease in pressure within 10 seconds. If there is, a leak is occurring and the connections and transducers need to be secured in place.</li>
	<li>Adjust the pressure servo back to automatic then open P2 to the chamber. Under the microscope observe the vessel dilate when the tap is opened to the chamber.</li>
	<li>Switch the pressure servo to manual. Again check for continual decrease in pressure within 10 seconds. If there is a leak then the system is not pressure tight and there is likely to be a hole in the vessel.</li>
	<li>Switch back to automatic open P1 to the chamber. On the manual setting check that the pressure remains stable.</li>
	<li>If no leak, on manual setting increase dial to 2 (40 mmHg).</li>
	<li>Adjust to automatic and observe the vessel under the microscope. If necessary, adjust the cannula holder to ensure no bend in the vessel.Check for leaks on manual setting.</li>
	<li>Repeat steps 5.6 - 5.7 increasing the dial to 3 (60 mmHg), 4 (80 mmHg) and so on until desired pressure is reached.</li>
</ol>
Approx. time = 15 - 30 mins
6. Incubating the pressurized vessel
<ol>
	<li>Connect the temperature controller set to 37&deg;C.</li>
	<li>With a second peristaltic pump perfuse Krebs buffer (1 mL/min) into the vessel chamber bath.</li>
	<li>Connect aspirator to chamber ensuring only the top layer of the bath is removed.</li>
	<li>Incubate pressurized vessel at 37&deg;C for 1 hour with the pressure set to automatic.</li>
	<li>Check the pressure is not leaking (switching to manual) periodically.</li>
	<li>The effects of various pharmacological interventions can be observed by simply adding the compound to the bath during incubation.</li>
</ol>
Approx. time = 1 hour
7. Perfusing the pressurized vessel with whole blood
<ol>
	<li>During incubation obtain at least 7.5 mL human whole blood/vessel collected in 40 U heparin/mL blood. Incubate in a 50 ml falcon at 37&deg;C.</li>
	<li>10 minutes before the end of the incubation label blood with VybrantDil (1:1000) for 10 minutes at 37&deg;C in dark.</li>
	<li>After 10 minutes, collect blood in a syringe clearing any bubbles and attach onto a syringe pump with a heat jacket set at 37&deg;C.</li>
	<li>Close off P1 transducer to the chamber/vessel and connect syringe and waste tubing.</li>
	<li>Purge blood at 1000 &mu;L/min through the waste tubing to remove any bubbles.</li>
	<li>Open P1 to the chamber. The pressure servo will adjust automatically to maintain desired pressure.</li>
	<li>Perfuse blood at 100 &mu;L/min.</li>
	<li>Using a fluorescent microscope coupled to a digital camera record two fields of the perfused vessel for 15 seconds at 1, 3, 5, 7.5 and 10 minutes.</li>
	<li>Post perfusion endothelial integrity and function can be assessed via pharmacological techniques such as myograph and adhesion molecule expression can be determined by immunohistochemistry to further validate the inflammatory response.</li>
</ol>
Approx. time = 15 mins
8. Representative Results:
A schematic diagram of the pressure chamber setup is shown in Figure 1. With a digital camera coupled to a fluorescent microscope results can be visualised instantly via live video recordings. Representative video images are seen in Figure 3 where leukocytes are considered to be adherent to the endothelium if they remained stationary for 10 seconds. With video recordings on continuous loop adhered cells can be counted as an average per field. While both low (Figure 3A) and high (Figure 3B) pressure will cause some amount of adhesion a significant increase in leukocyte adhesion at the higher intraluminal pressure is seen and this is also demonstrated quantitatively (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/3221/3221fig1.jpg" /> 
Figure 1. Pressurised ex vivo vessel chamber schematic. A cannulated vessel connected to a proximal (P1) transducer and a distal (P2) transducers that enables blood pressure to be manipulated within the vessel. Perfusion is through the P1 transducer and pressure is maintained via P2 transducer.
<img alt="Figure 2" src="/files/ftp_upload/3221/3221fig2.jpg" /> 
Figure 2. Cannuala with ties. Black polyester ties are attached to each cannula.
<img alt="Figure 3" src="/files/ftp_upload/3221/3221fig3.jpg" /> 
Figure 3. Representative video images. Dynamic cell adhesion (red arrow) under fluorescence at 80 mmHg (A) and 120 mmHg (B) after 10 minutes of perfusion.
<img alt="Figure 4" src="/files/ftp_upload/3221/3221fig4.jpg" /> 
Figure 4. Leukocyte adhesion in Sprague Dawley carotid arteries after 1 hour incubation at low (80 mmHg) and high pressure (120 mmHg). ***P&lt;0.001, as analysed by 2-way repeated measures ANOVA using Bonferroni post hoc test.

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Szczech, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+kidney+injury+and+cardiovascular+outcomes+in+acute+severe+hypertension.">Acute kidney injury and cardiovascular outcomes in acute severe hypertension.</a> Circulation. 121, 2183-2191 .</li><li>Rodriguez-Garcia, J. L., Botia, E., de La Sierra, A., Villanueva, M. A., Gonzalez-Spinola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+elevated+blood+pressure+and+its+management+on+the+short-term+outcome+of+patients+with+acute+ischemic+stroke.">Significance of elevated blood pressure and its management on the short-term outcome of patients with acute ischemic stroke.</a> Am J Hypertens. 18, 379-384 (2005).</li><li>Levy, D., Larson, M. G., Vasan, R. S., Kannel, W. B., Ho, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progression+from+hypertension+to+congestive+heart+failure.">The progression from hypertension to congestive heart failure.</a> JAMA. 275, 1557-1562 (1996).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat Rev Immunol. 7, 678-689 (2007).</li><li>Riou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+pressure+promotes+monocyte+adhesion+to+the+vascular+wall.">High pressure promotes monocyte adhesion to the vascular wall.</a> Circ Res. 100, 1226-1233 (2007).</li><li>Ley, K., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial,+not+hemodynamic,+differences+are+responsible+for+preferential+leukocyte+rolling+in+rat+mesenteric+venules.+Circ+Res.">Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules. Circ Res.</a> 69, 1034-1041 (1991).</li><li>Bernhagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIF+is+a+noncognate+ligand+of+CXC+chemokine+receptors+in+inflammatory+and+atherogenic+cell+recruitment.">MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.</a> Nat Med. 13, 587-596 (2007).</li><li>Woollard, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+levels+of+soluble+P-selectin+mediate+adhesion+of+leukocytes+to+the+endothelium+through+Mac-1+activation.">Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation.</a> Circ Res. 103, 1128-1138 (2008).</li><li>Eberth, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+pulsatility+in+hypertensive+carotid+artery+growth+and+remodeling.">Importance of pulsatility in hypertensive carotid artery growth and remodeling.</a> J Hypertens. 27, 2010-2021 (2009).</li><li>Cooke, J. P., Rossitch, E., Andon, N. A., Loscalzo, J., Dzau, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+activates+an+endothelial+potassium+channel+to+release+an+endogenous+nitrovasodilator.">Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.</a> J Clin Invest. 88, 1663-1671 (1991).</li></ol>

The study protocol was approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee and the Alfred Hospital Ethics Committee.

This is a modified method to study leukocyte adhesion to the endothelium in intact isolated blood vessels under pressurised conditions in real time. Perfusion of the vessel chamber alone enables a quick validation of pro-inflammatory strains of large mice and rat vessels. Enabling pressure manipulation allows dynamic cell interactions to be observed from low to very high intraluminal pressures, thus better mimic-ing physiological and pathophysiological conditions. Diameter of vessels can also be measured using a sufficient cell-imaging program and therefore shear flow and rate can be determined and therefore manipulated. 
With its myograph capabilities, pharmacological interventions placed in the bath add another dimension to the experimental conditions possible with this model enabling studies of mechanistic and signalling pathways. While endothelial preservation cannot be confirmed during pressure manipulation, responses to ACh and PE can be conducted post perfusion9. 
It should be noted that this setup demonstrates the effects of intraluminal pressure on cell-to-cell interactions not the effects of pulsatile blood flow nor systolic or diastolic pressures. Furthermore, while acute pressure changes on leukocyte adhesion were observed, this setup can be also utilised to look at chronic pressure effects (ie increasing incubation times and using a chronic pressure animal model). Sprague Dawley common carotid arteries are demonstrated in this set up but other strains and species may be used with appropriate adjustments to the cannula size. Indeed, it is important to note that the age and weight of animals affect vessel size and thus that the set up needs to be individualised for each vessel. Close and careful dissection of the connective tissue can improve visualisation of leukocytes immensely.

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent/equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Microscope </td>
 <td>Carl Zeiss, Inc</td>
 <td>SteREO Discovery V.20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PHD 2000 Syringe Pump</td>
 <td>Harvard Apparatus</td>
 <td> 70-2016</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Camera and Controller</td>
 <td>Hamamatsu ORCA-ER</td>
 <td> C4742-95</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluorescence Illumination System</td>
 <td>Lumen Dynamics</td>
 <td> X-Cite 120</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vessel Chamber</td>
 <td>Living Systems Instrumentation</td>
 <td>CH/1/SH</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pressure Servo Controller and Peristaltic Pump</td>
 <td>Living Systems Instrumentation</td>
 <td>PS - 200</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Perfusion Pressure Monitor </td>
 <td>Living Systems Instrumentation</td>
 <td>PM - 4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2 x Pressure Transducer</td>
 <td>Living Systems Instrumentation</td>
 <td>PT - F</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Temperature Controller</td>
 <td>Living Systems Instrumentation</td>
 <td> TC-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Instech Laboratories, Inc</td>
 <td>P720</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>VybrantDil cell-labelling solution</td>
 <td>Invitrogen</td>
 <td>V-22885 </td>
 <td>&nbsp;Use 1:1000</td>
 </tr>
 </tbody>
</table>

imaging leukocyte adhesion to the vascular endothelium at high intraluminal pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Watch this Scientific Journal Video about Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure at JoVE.com

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

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Immunology and Infection

15.7K Aufrufe.  Monash University. Dies ist eine Methode zur Leukozyten-Adhäsion an das Endothel in geerntet Druckbehälter zu visualisieren. Die Technik ermöglicht das Studium vaskulären Haftung unter Scherströmung mit unterschiedlichen intraluminale Drücke bis 200 mmHg damit nachahmen-Ing den pathophysiologischen Bedingungen des hohen Blutdrucks.

Serie: Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...