Research reported in this publication was supported by National Eye Institute of the National Institutes of Health under award numbers: R01EY022084/S1 (KMC), T32EY007145 (HS) and P30EY014104. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support was provided by the Massachusetts Lions Eye Research Fund (K.M.C.) and a Special Scholar Award from Research to Prevent Blindness (to K.M.C.).

All experiments on animals were handled in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines and regulations set forth by the Massachusetts Eye and Ear Infirmary Animal Care Committee.
The day before the experiment:
1. Preparation of Tubing for Flow Chamber
<ol>
	<li>To prepare the jugular side, connect 1 cm PE10 polyethylene tubing to 6 cm silicon tubing followed by 5 cm polyethylene tubing. For the carotid side, prepare similarly to the jugular side with the addition of a T-tube within the 6 cm silicon tubing about 1.5 cm away from the 1 cm polyethylene tubing (Figure 1A, B). In order to insert the T-tube, widen the silicon tube by using fine forceps if necessary.</li>
	<li>Connect one end of 10 cm of silicone tubing to the pressure transducer then insert the T end of the carotid side T-tube into the other end. Ensure the connection sites are tight and leak free. Apply a small rectangular piece of parafilm over where the tubing fits together, if necessary, to prevent leakage (Figure 1C).</li>
</ol>
2. Coating the Chamber
<ol>
	<li>Prepare the endothelial surface coating protein (e.g., P selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1)) in 0.1% BSA. As a guideline, 1 &#956;g of protein in 200 &#956;l BSA is enough to coat 4-5 chambers. Using a 1 ml syringe connected to a 25 G needle, infuse each chamber (0.04 x 0.4 x 50 mm) with endothelial surface coating protein. Store the coated chambers in a 15 ml tube at 4 &#176;C overnight.</li>
</ol>
The day of the experiment:
3. Preparing the Chamber
<ol>
	<li>Remove the coated chambers from 4 &#176;C and connect the jugular and carotid tubing to each side. Seal the connections by carefully stretching a small rectangular piece of parafilm over where the tubing meets the chamber. Using a syringe, fill the chamber and tubing with 0.1% BSA (check for leakage). Incubate for 45 min.</li>
	<li>Prepare a 35 mm Petri dish to hold the chamber by cutting notches on each side of the dish. Stabilize the chamber in the notches; then use a small drop of silicone to affix the chamber to the dish at the notched site. Make sure the chamber is level by looking under the microscope. Allow the silicone to dry for at least 15 min.</li>
	<li>Fill a 60 ml locking syringe with heparin and connect to the pressure transducer. Connect the pressure transducer to the carotid tubing in the following order: Transducer, silicon tubing, and T-tube.
	<ol>
		<li>Use a 1 cm PE50 between the silicon tubing connected to the transducer and the T-tube to secure the connection. Pass the carotid artery silicon tubing through the pressure clamp (Figure 1D). Run 100USP heparin through the tubing and chamber until there are no air bubbles.</li>
	</ol>
	</li>
</ol>
4. Setting up the Software
<ol>
	<li>To prepare the software, turn on the computer, microscope, and open lab device. Double click on the Leica application suite (LAS) (or equivalent) and prepare a folder to save the video: browse &#8594; acquire &#8594; movie &#8594; define sequence of clips (clips = 15, recording time = 30 sec, and intervals = 15 sec) then click the &#8220;+&#8221; sign to confirm.</li>
	<li>To record the flow pressure open the LabChart (or equivalent) file and coordinate the file name to the recording folder in step 4.1, above.</li>
</ol>
5. Surgical Procedure
<ol>
	<li>Anesthetize an 8-10 week-old mouse using a combination of ketamine (60 mg/kg) and xylazine (6 mg/kg) injected intra-peritoneally (IP). Confirm that deep sedation is achieved within 5-10 min by loss of pedal withdrawal reflex. If necessary, isoflurane delivered via face mask can be given as needed.</li>
	<li>While under anesthesia, place a warming pad under the cage and set to 37 &#176;C to maintain body temperature. Once proper anesthesia is confirmed, place the mouse in a ventral position and coat the eyes with bacitracin ointment to prevent ocular dryness.</li>
	<li>Thereafter, shave the hair at the incision site using a razor blade and thoroughly clean the site with alcohol.</li>
	<li>Once fully anesthetized secure the animal with tape keeping the mouse in a ventral position. Expose the neck area with scissors and remove the thyroid gland to expose the trachea and vessels (Figure 2A).</li>
	<li>Clean the carotid artery until it is fully exposed taking care not to damage the vagus nerve (Figure 2B). Fold a piece of suture and pass the bend underneath the carotid artery then cut at the bend so there are now 2 pieces of suture underneath the carotid. Loop each suture into knots (do not tighten the knots).</li>
	<li>Repeat the same procedure to expose the left jugular vein (Figure 2C), preparing the sutures in the same manner (Figure 2D).</li>
	<li>Connect the tubing to the mouse vasculature.
	<ol>
		<li>Heparinize the mouse by injecting 1 ml of heparin IP.</li>
		<li>Tighten the upper suture on the carotid artery. Place the vessel clamp as low as it can go on the carotid artery (Figure 3A). Use forceps to hold the artery by the upper suture and make a small incision, about 1/8 of the circumference of the artery, using the microscissors (Figure 3B).</li>
		<li>Pass the carotid artery tubing through the second knot in the upper suture and into the incision made in the artery. Tie off the lower suture so that the artery is sealed around the tubing (several knots should be made to ensure a tight seal; Figure 3C).</li>
		<li>Repeat the same procedure for inserting the jugular vein tubing (the vessel clamp is not needed for the jugular side; Figure 3D).</li>
		<li>Test the flow by releasing the vessel clamp. If there are no leaks move the mouse to the microscope and connect the carotid tubing and jugular vein to the chamber tubing (see attached video for demonstration of surgical procedure).</li>
	</ol>
	</li>
</ol>
6. Recording Rolling Velocity
<ol>
	<li>Open the carotid clamp to fill the chamber, making a circuit with the mouse circulatory system. Allow the blood to circulate for 3-5 min so that the flow conditions stabilize. During the waiting period of 3-5 min, adjust the microscope stage so that the chamber is level from one end to the other with the microscope objective and its edge is parallel with the video recording window on the screen.</li>
	<li>Fill the modified Petri dish containing the glass chamber with water then lower the 10X water immersion objective into the dish so that the cells flowing through the chamber are visible. The light intensity may have to be adjusted for adequate viewing of the cells. Generally a lower intensity is better.</li>
	<li>Adjust the clamp on the tubing so that the transducer reads a steady 30 mm Hg, correlating to a medium physiological flow pressure, or as desired.</li>
	<li>Press &#8220;start&#8221; to initiate recording both the video and the pressure with the first time lapse close to the jugular end and moving against the flow toward the carotid end until completed. 
	NOTE: Several coating conditions using different adhesion molecules can be studied in the same mouse by using multiple chambers. Only one chamber is recorded at a time.
	<ol>
		<li>When recording is finished, close the valve at the input site, stopping the blood flow. Remove the chamber and fit a new chamber (coated with a different adhesion molecule) to the circuit using fine forceps in one hand and grooved forceps in the other hand. Resume recording for the new chamber as outlined in the steps 6.1-6.4.</li>
	</ol>
	</li>
	<li>Euthanize all mice by CO2 asphyxiation followed by a spinal cord dislocation at the end of the experiment. The procedure is terminal, therefore, the mice are euthanized by a spinal cord dislocation at the end of the experiment.</li>
</ol>
7. Interpreting the Results
<ol>
	<li>To analyze the recorded data use the &#8220;M TrackJ&#8221; plugin for ImageJ software23.</li>
	<li>Convert the file to grayscale by Select file &#8594; import &#8594; AVI &#8594; convert to grayscale.</li>
	<li>Standardize the frames by Select &#8594; image &#8594; property &#8594; enter the frame intervals (i) &#8594; global. Calculate the frame intervals by dividing the total video duration (usually 30 sec) by the number of frames in one video (shown in the upper corner of the Image J video window).</li>
	<li>Measure the flow of each leukocyte by Select plugin &#8594; M TrackJ &#8594; Add tracks &#8594; select a leukocyte &#8594; Track it with each frame for 15 - 20 sec &#8594; Merge.</li>
	<li>Repeat step 7.4 for all the leukocytes in view.</li>
	<li>Determine the mean rolling velocity of the leukocytes by Select measure &#8594; two columns appear &#8594; Copy all of the M TrakJ:Tracks into a spreadsheet. The column labeled &#8220;mean V&#8221; is the leukocyte rolling measured in pixels/second. Convert into &#956;m/sec by dividing by 2/3.</li>
	<li>To analyze the pressure data highlight and export into a spreadsheet and perform the following calculation: &#916;P (mm Hg) and t (dyne/cm2) for a comparison between each experimental group. Once calculated, copy the data into prism software for graphing.</li>
</ol>

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<li>Muller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukocyte-endothelial-cell+interactions+in+leukocyte+transmigration+and+the+inflammatory+response%5BTitle%5D)+AND+%22Trends+in+Immunology%22%5BJournal%5D)">Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response.</a> Trends in Immunology. 24, 327-334 (2003).</li>
<li>Alon, R., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cells+on+the+run%3A+shear-regulated+integrin+activation+in+leukocyte+rolling+and+arrest+on+endothelial+cells%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells.</a> Current Opinion in Cell Biology. 20, 525-532 (2008).</li>
<li>Mehta, P., Cummings, R. D., McEver, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Affinity+and+kinetic+analysis+of+P-selectin+binding+to+P-selectin+glycoprotein+ligand-1%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1.</a> The Journal of Biological Chemistry. 273, 32506-32513 (1998).</li>
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<li>Ding, X., Patel, M., Chan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+pathology+of+age-related+macular+degeneration%5BTitle%5D)+AND+%22Progress+in+Retinal+and+Eye+Research%22%5BJournal%5D)">Molecular pathology of age-related macular degeneration.</a> Progress in Retinal and Eye Research. 28, 1-18 (2009).</li>
<li>Xu, H., Chen, M., Forrester, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Para-inflammation+in+the+aging+retina%5BTitle%5D)+AND+%22Progress+in+Retinal+and+Eye+Research%22%5BJournal%5D)">Para-inflammation in the aging retina.</a> Progress in Retinal and Eye Research. 28, 348-368 (2009).</li>
<li>Peterson, K., et al. Drug Class Review: Nonsteroidal Antiinflammatory Drugs (NSAIDs): Final Update 4 Report. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPeterson%2C+K+%22Drug+Class+Reviews%22">Drug Class Reviews</a>. , Oregon Health & Science University. Portland, OR. (2010).</li>
<li>Ulbrich, H., Eriksson, E. E., Lindbom, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukocyte+and+endothelial+cell+adhesion+molecules+as+targets+for+therapeutic+interventions+in+inflammatory+disease%5BTitle%5D)+AND+%22Trends+in+Pharmacological+Sciences%22%5BJournal%5D)">Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease.</a> Trends in Pharmacological Sciences. 24, 640-647 (2003).</li>
<li>Stamper, H. B., Woodruff, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphocyte+homing+into+lymph+nodes%3A+in+vitro+demonstration+of+the+selective+affinity+of+recirculating+lymphocytes+for+high-endothelial+venules%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Lymphocyte homing into lymph nodes: in vitro demonstration of the selective affinity of recirculating lymphocytes for high-endothelial venules.</a> The Journal of Experimental Medicine. 144, 828-833 (1976).</li>
<li>Lawrence, M. B., McIntire, L. V., Eskin, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+flow+on+polymorphonuclear+leukocyte%2Fendothelial+cell+adhesion%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion.</a> Blood. 70, 1284-1290 (1987).</li>
<li>Abbitt, K. B., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characteristics+of+leucocyte+adhesion+directly+observed+in+flowing+whole+blood+in+vitro%5BTitle%5D)+AND+%22British+Journal+of+Haematology%22%5BJournal%5D)">Characteristics of leucocyte adhesion directly observed in flowing whole blood in vitro.</a> British Journal of Haematology. 112, 55-63 (2001).</li>
<li>Smith, M. L., Sperandio, M., Galkina, E. V., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoperfused+mouse+flow+chamber+reveals+synergistic+neutrophil+accumulation+through+P-selectin+and+E-selectin%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">Autoperfused mouse flow chamber reveals synergistic neutrophil accumulation through P-selectin and E-selectin.</a> Journal of Leukocyte Biology. 76, 985-993 (2004).</li>
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The authors (L.M., H.S., and K.M.C.) have no competing financial interests to disclose.

The process of leukocyte recruitment is a crucial step in the inflammatory response; it involves the migration of leukocytes from the circulatory system towards target tissues, where they are able to exert their effector function. Leukocyte recruitment is integral in a variety of inflammatory conditions such as atherosclerotic plaques, myocardial infarction, ischemia/reperfusion, and transplant surgery1, as well as multiple CNS related neuro-inflammatory conditions10-12,20,24,25. Considering the diversity of the disease conditions that leukocyte recruitment spans, the autoperfused microflow chamber provides an indispensible tool allowing the investigator the ability to study leukocyte migration dynamics.
Over the past several decades, a variety of in vitro assays have been developed to study the dynamics of leukocyte cell adhesion26. Unfortunately, all of these assays require the extraction of the leukocytes from the blood, introducing mechanical activation. To get closer to the in vivo environment, adaptations were made to collect whole blood samples and control the hemodynamic flow conditions16,17. Here, we extend the previous advances in the field by linking a coated chamber to the mouse circulatory system. We are able to regulate the flow of the blood to a physiologic range and study the dynamics of leukocyte rolling. The coated chamber gives us the ability to study leukocyte interactions with specific adhesion molecules. Since the system is functioning as a unit operated by a live mouse, it more closely mimics the natural environment, allowing us to study leukocyte-endothelial interactions in a variety of immunologic mouse models. Additionally, this system allows us to take advantage of the multiple genetic mouse models available. While the system does not completely replicate the in vivo environment, it provides a platform to study specific elements of the leukocytes under physiologic conditions, a feat that has not previously been possible. Even though this takes us a step closer to a more physiologic environment there are limitations to the system. It does not completely replicate the complex 3D matrix of the vasculature so we are only able to assess certain elements of leukocyte interactions that are restricted to the chamber coating. In addition, great care should be taken to ensure a closed system to the mouse circulation by following all the steps mentioned in the protocol. The introduction of air bubbles will greatly affect the accuracy and reproducibility of the experiments.
While we describe the use of the Autoperfused Microflow chamber for assessing leukocyte rolling dynamics the procedure has the potential to be personalized by investigators to study a variety of diseases. For example, cancer cells metastasize by expressing many of the common integrins shared by leukocytes. Studying their rolling dynamics in a setting that more closely mimics an in vivo environment could help in our knowledge, and possibly the prediction, of the invasive nature of certain cancer cells. One possible approach could be to combine a labeling technique for the cancer cells, such as GFP27, along with the flow chamber assay to track rolling dynamics of the cancer cells expressing GFP. Given the flexibility of coating the chamber with a variety of substances and connecting multiple chambers to the same mouse it will be interesting to see how this procedure is modified for use in other labs in combination with genetically modified mice and disease models. The technique we describe here just touches on a much broader application potential that is only limited by the creativity of the investigator.

Inflammation is the body&#8217;s universal response to injury and is a crucial step in both innate and adaptive immune system function. In response to injury and/or inflammatory stimuli, endothelial cells upregulate specific adhesion molecules; this leads to leukocyte extravasation through the microvascular endothelium, primarily in post capillary venules. This process starts with tethering of the free-flowing leukocytes in the bloodstream on the endothelium. Stable rolling and firm adhesion of leukocytes, which in turn leads to transmigration and the secretion of cytotoxic agents, follow this tethering1,2. Selectins are known to mediate the early steps of the cascade3-5; integrins are responsible for the later steps of firm adhesion and transmigration1,6-8.
A growing body of evidence suggests leukocytes and endothelial adhesion molecules have vital roles in animal models of ischemia reperfusion injury, asthma, psoriasis, multiple sclerosis, and age-related macular degeneration9-12. Under these conditions, the inflammatory response is misdirected to attack one&#8217;s own body, resulting in the breakdown of healthy tissue. Existing anti-inflammatory agents (such as non-steroidal anti-inflammatory drugs, corticosteroids, or other chemotherapeutic agents) carry the risk of severe side effects with long-term use13. Therefore, it is of great interest to have the appropriate tools with the ability to identify disease-specific molecules, which can ultimately be targeted to have the desired anti-inflammatory effect while remaining non-toxic14.
Existing in vitro methods such as the static leukocyte adhesion assay were used as early as 197615. The parallel flow chamber was first used in vitro in 1987 to study leukocyte-endothelial interactions under flow conditions. In these experiments, stimulated human polymorphonuclear leukocytes (PMNs) from venous blood were perfused over a monolayer of primary human umbilical vein endothelial cells (HUVECs). To control for the hemodynamic flow conditions, the Harvard Apparatus syringe pump was employed16. Alternatively, to avoid artificial leukocytes isolation, whole blood was used in combination with the glass chamber coated with immobilized adhesion molecules17.
To avoid leukocyte stimulation and to mechanistically study their interaction with the adhesion molecules under approximate physiologic conditions, an ex vivo autoperfused, extracorporal, arteriovenous circuit was developed16. In this circuit, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing interaction of native PBLs within a glass microflow chamber coated with single or co-immobilized adhesion molecules. A major advantage to this system is the ability to employ genetically engineered mice, in which inflammatory pathways are directly or indirectly manipulated. Additionally, there is the ability to pinpoint the isolated contribution of leukocyte adhesion molecules to inflammation, free of external activation, under flow conditions. The application of a flow regulator at the input point of the flow chamber provides a wide range of experimental variations of shear forces to mimic either arterial or venous systems18-22. Here we describe in great detail a protocol concerning the preparation and performance of the ex vivo autoperfused microflow chamber assay.

<table><tbody><tr><td>Micro glass chamber 0.4 x 0.04 x 50 mm</td><td>VitroCom</td><td>2540-050</td><td></td></tr><tr><td>Polyethylene tubing PE 10</td><td>Fisher Scientific</td><td>427400</td><td></td></tr><tr><td>Polyethylene tubing PE 60</td><td>Fisher Scientific</td><td>427416</td><td></td></tr><tr><td>Silicone tubing 002</td><td>Fisher Scientific</td><td>11-189-15A</td><td></td></tr><tr><td>Y tube</td><td>Value Plastics</td><td>Y210-6</td><td></td></tr><tr><td>T tube</td><td>value plastics</td><td>T410-6</td><td></td></tr><tr><td>Silicone gel</td><td>Hardware store - Home Depo</td><td></td><td></td></tr><tr><td>35 mm Petri dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>Parafilm</td><td>Pechiney Plastic Packaging</td><td>PM996</td><td></td></tr><tr><td>Fine forceps</td><td>FST</td><td>11253-25</td><td></td></tr><tr><td>Fine scissors</td><td>FST</td><td>15000-08</td><td></td></tr><tr><td>Tube holder</td><td>FST</td><td>00608-11</td><td></td></tr><tr><td>Clamp applicator</td><td>FST</td><td>18057-14</td><td></td></tr><tr><td>Vascular clamp</td><td>FST</td><td>18055-04</td><td></td></tr><tr><td>6-0 silk sutures</td><td>George Tiemann &amp;amp; Co</td><td>160-1215-6/0</td><td></td></tr><tr><td>25 x 1 G needles</td><td>BD</td><td>305125</td><td></td></tr><tr><td>30 x 1/2 G needles</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Heparin 100 USP units/ml</td><td>Hospital pharmacy</td><td></td><td></td></tr></tbody></table>

assessing leukocyte-endothelial interactions under flow conditions in an ex vivo autoperfused microflow chamber assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

During each recording only interacting leukocytes are evident on the chamber. Results from a representative experiment can be seen in Figure 4. The white arrows point to individual leukocytes that were captured by the adhesion molecule of interest and are rolling along the chamber amongst other free-flowing ones within the blood flow. A common artifact outside the chamber can be seen with the large dark spheres in all the images. The dark spheres help the reader appreciate the leukocyte movement in relation to a fixed point. Leukocytes advancement over a period of 22 sec can be seen in relation to the spheres. The graph in the same figure shows a final plot of the data where Tx1 decreases the rolling velocity (a shift to the left) and Tx2 increases the velocity (a shift to the right) compared to the control.
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig1.jpg" width="700" /> 
Figure 1: Preparation of tubing for the flow chamber. (A) Tools required for tubing and chamber assembly (micro glass chamber 0.4 x 0.04 x 50 mm, polyethylene tubing PE10, polyethylene tubing PE60, silicon tubing 002, Y tube, T tube, 35 mm Petri dish, fine forceps, fine scissors, tube holder, vascular clamp, 7-0 silk suture. (B) Preparation of the carotid artery and jugular vein tubing for re-directing the mouse blood flow through the coated chamber. (C) Connecting the carotid artery tubing to the pressure transducer. (D) Pass the tubing from the transducer through the clamp to adjust the rate of the blood flow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52130/52130fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig2.jpg" width="700" /> 
Figure 2: Exposing the carotid artery and jugular vein. (A) Carefully cut the neck area to expose the trachea. (B) Clean the right carotid artery so that a piece of suture can be passed underneath the vessel (dotted yellow line outlines the carotid artery). (C) Clean the left jugular vein so that a piece of suture can be passed under the vessel (dotted yellow line outlines the jugular vein). (D) Arrange loosely tied sutures at the upper and lower regions of the carotid artery and jugular vein (arrows indicated the upper and lower regions of the vessels). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52130/52130fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig3.jpg" width="700" /> 
Figure 3: Connecting the tubing to the carotid artery and jugular vein. (A) Tighten the upper knot on the carotid and place the clamp below the lower knot. (B) Using the microscissors, make a small incision in the carotid, about 1/8 the circumference (dotted yellow circle indicates the incision). (C) Insert the tubing into the carotid and secure with at least two sutures (green dotted line shows the tubing and the yellow dotted line the carotid artery). (D) Similarly insert tubing in the jugular (green dotted line shows the tubing and the yellow dotted line the jugular vein). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52130/52130fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig4.jpg" width="700" /> 
Figure 4: Representative time course of leukocytes rolling through the flow chamber (Scale bar = 100 &#956;m) (white arrowheads point out rolling leukocytes). Graph depicts representative results after exporting into a graphing program (Tx1 and Tx2 are experimental treatment groups and CTR is the control group). Tx1 represents a treatment in which leukocytes slow down leading to a decrease in leukocytes rolling velocity and a left shift of the graph compared to the untreated group (CTR), while Tx2 leads to a right shift reflecting an increase in leukocytes rolling velocity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52130/52130fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay at JoVE.com

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.6K Views.  Harvard Medical School, Massachusetts Eye & Ear Infirmary.  Hier presenteren we een protocol waarmee de onderzoeker leukocytaantrekking dynamiek ex vivo evaluatie door er een kamer bekleed met endotheliaalverkregen adhesiemoleculen aan het circulatiesysteem van een muis. Deze methode biedt aanzienlijke voordelen, omdat het zorgt voor leukocyten beoordeling op grond van de relatieve biologische omstandigheden. 

Video: Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

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.

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. ...

Immunology and Infection

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone and inflamed tissues.  E-selectin expression is constitutive on dermal and bone microvessels and is inducible by pro-inflammatory cytokines, such as IL-1&#945;/  and TNF-&#945;, on microvessels in inflamed tissues.  This lectin receptor mediates weak binding interactions with carbohydrate counter-receptor ligands on circulating leukocytes, which results in a characteristic  rolling  behavior.  Because these interactions precede more stable adhesive events and diapedesis activity, characterization of leukocyte rolling activity and identification of leukocyte E-selectin ligands have been major goals in studies of leukocyte trafficking and inflammation and in the development of anti-inflammatory therapeutics (1-5).  The intent of this report is to provide a visual, comprehensive description of the most widely-used technology for studying E-selectin   E-selectin ligand interactions under physiologic blood flow conditions.  Our laboratory in conjunction with the Harvard Skin Disease Research Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied by digital visualization and new recording software, NIS-Elements.  This technology allows us to analyze adhesion events in real time for onscreen visualization as well as record rolling activity in a video format.  Cell adhesion parameters, such as rolling frequency, shear resistance and binding/tethering efficiency, are calculated with NIS-Elements software, exported to an Excel spreadsheet and subjected to statistical analysis.  In the demonstration presented here, we employed the parallel-plate flow chamber to investigate E-selectin-dependent leukocyte rolling activity on live human bone marrow endothelial cells (hBMEC).  Human hematopoietic progenitor KG1a cells, which express a high level of E-selectin ligand, were used as our leukocyte model, while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our endothelial cell model (6).  To induce and simulate native E-selectin expression in the flow chamber, HBMEC-60 cells were first activated with IL-1 .  Our video presentation showed that parallel-plate flow analysis is a suitable method for studying physiologic E-selectin-mediated leukocyte rolling activities and that functional characterization of leukocyte E-selectin ligand(s) in the flow chamber can be ascertained by implementing protease or glycosidase digestions.

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

Biology

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 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Developmental Biology

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Intravital Microscopy to Analyze Blood Cell-Endothelial Interactions in an Inflammation Mouse Model

Source: Herr, N. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53077">Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the real-time visualization of blood cell-endothelial interactions in the mesenterial veins of a mouse model injected intraperitoneally with lipopolysaccharide.

Source: Herr, N. et al., Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

An In Vitro Flow Investigation of the Effect of Stromal Cells on Leukocyte Recruitment

Source: Munir, H. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52480">Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow.</a> J. Vis. Exp.&#160;(2015)
The video demonstrates a method for investigating the impact of stromal cells on leukocyte recruitment. In a parallel plate flow chamber model, pro-inflammatory cytokines induce the expression of adhesion receptors by endothelial cells, facilitating neutrophil recruitment and migration. The cross-talk between stromal cells and endothelial cells diminishes neutrophil recruitment to the endothelial cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation and Culture of Primary ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

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