Vascular Pharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Monash University
Michell, D. L., Andrews, K. L., Woollard, K. J., Chin-Dusting, J. P. Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure. J. Vis. Exp. (54), e3221, doi:10.3791/3221 (2011).
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.
1. Isolating carotid arteries
Approx. time = 45 mins
2. Priming the vessel chamber
Approx. time = 15 mins
3. Pressurizing the vessel chamber
Approx. time = 10 mins
4. Mounting the vessel
Approx. time = 20 mins
5. Pressurizing the vessel
Approx. time = 15 - 30 mins
6. Incubating the pressurized vessel
Approx. time = 1 hour
7. Perfusing the pressurized vessel with whole blood
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).
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.
Figure 2. Cannuala with ties. Black polyester ties are attached to each cannula.
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.
Figure 4. Leukocyte adhesion in Sprague Dawley carotid arteries after 1 hour incubation at low (80 mmHg) and high pressure (120 mmHg). ***P<0.001, as analysed by 2-way repeated measures ANOVA using Bonferroni post hoc test.
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.
The study protocol was approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee and the Alfred Hospital Ethics Committee.
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).
|Microscope||Carl Zeiss, Inc.||SteREO Discovery V.20|
|PHD 2000 Syringe Pump||Harvard Apparatus||70-2016|
|Digital Camera and Controller||Hamamatsu Corp.||C4742-95|
|Fluorescence Illumination System||Lumen Dynamics||X-Cite 120|
|Vessel Chamber||Living Systems Instrumentation||CH/1/SH|
|Pressure Servo Controller and Peristaltic Pump||Living Systems Instrumentation||PS - 200|
|Perfusion Pressure Monitor||Living Systems Instrumentation||PM - 4|
|2 x Pressure Transducer||Living Systems Instrumentation||PT - F|
|Temperature Controller||Living Systems Instrumentation||TC-01|
|Peristaltic Pump||Instech Laboratories, Inc||P720|
|VybrantDil cell-labelling solution||Invitrogen||V-22885||Use 1:1000|