This work was supported by the German Ministry for Education and Research (BMBF 01EO1003 and BMBF 01EO1503).

Experiments were conducted on male mice age 8 to 12 weeks old under the approval of the ethics committee on animal experimentation from Rhineland-Palatinate (authorization number 23 177-07/G12-1-002 and 23 177-07/G15-1-051).
1. Mouse Anesthesia and Surgery Preparation
<ol>
	<li>Prior to surgery, verify the good health and condition of the animals. Before surgery, monitor animals once per day for 2-3 days for their general condition (appearance, posture, spontaneous behavior) as well as body weight, food, and water consumption.</li>
	<li>Prepare the master mix for anesthesia with midazolam (5 mg/kg body weight), medetomidine (0.5 mg/kg body weight), and fentanyl (0.05 mg/kg body weight) to intraperitoneally (i.p.) inject 200 &#181;L/30 g mouse in 1 mL syringe with a 26 G needle.</li>
	<li>Osmotic pump implantation and animal preparation for IVM experiments are performed on an operation field with a 39 &#176;C warming platform. Sterilize all tools in a disinfection bath and disinfect the warming platform.</li>
	<li>During the procedures, protect the animal eyes with eye ointment. Remove the fur with razors before osmotic pump implantation. Use a hair removal cream and cotton swabs for isolation of carotids and jugular catheter implantation in IVM experiments. Disinfect the animal skin using two skin disinfectants: one antiseptic and disinfectant containing povidone iodine and one skin antiseptic containing octenidine in alcohol-based solution.&#160;</li>
	<li>Prepare the master mix to antagonize anesthesia (&#34;antisedan mix&#34;) with atipamezol (0.05 mg/kg body weight) and flumazenil (0.01 mg/kgbody weight), and prepare 200 &#181;L/mouse in 1 mL syringe with a 26 G needle to be subcutaneously (s.c.) injected.</li>
</ol>
2. Osmotic Pump Preparation and Implantation
NOTE: The subcutaneous AngII infusion using osmotic pumps was described in detail by Lu et al.17
<ol>
	<li>Prepare the AngII by reconstituting the lyophilized powder with sterile saline and adjust the concentration with the animal weight and the delivery rate of the pump. Keep the reconstituted AngII in plastic tubes (do not use glass tubes).</li>
	<li>Fill the pumps with the AngII solution to deliver 1 mg/(kg&#183;d) for 7 days. After the preparation, keep the pumps in sterile saline for 4-6 h minimum at 37 &#176;C.</li>
	<li>Test the complete anesthesia of the mouse after injection of the anesthesia mix with rear foot reflexes and place it on the warm operation field. Anesthesia induction takes 10-15 min and works better if animals are placed in a dark environment.</li>
	<li>Shave the lower back of the mouse and disinfect it with an alcoholic skin antiseptic.</li>
	<li>Make a 1 cm incision perpendicular to the spine using scissors. Create a pocket for the pump using a strait hemostat and insert the pump (flow moderator first). The pocket must allow free movement of the pump with no pressure on the wound.</li>
	<li>Close the wound with sutures, and not clips, in order to limit inflammation; then disinfect with skin antiseptic.</li>
	<li>Antagonize the anesthesia by s.c. injection of 200 &#181;L of the antisedan mix and return the mouse to its cage.</li>
	<li>Carry out postoperative analgesia with buprenorphine (0.075 mg/kg s.c.) one time after the surgery and on demand depending on the animal behavior.</li>
</ol>
3. Jugular Catheter Implantation and Carotids Preparation
<ol>
	<li>Test the complete anesthesia of the mouse after injection of the anesthesia mix with rear food reflexes and place the anesthetized animal in dorsal recumbence on the 39 &#176;C surgical plate&#160;with a rectal probe in order to maintain the temperature and put the ointment over the eyes to protect them during the procedure.</li>
	<li>Remove neck hairs using the hair removal cream. Use a cotton swab to spread and rub the cream for 2 min until the hairs start to fall out. After an additional 2 min, remove the cream and hairs with a spatula and disinfect the skin with an alcoholic skin antiseptic.</li>
	<li>Perform the surgery under a stereomicroscope. Make a 1-1.5 cm long incision in the skin longitudinally in the neck, 1 cm next to the trachea. Then make two other incisions perpendicularly to both extremities of the first incisions.</li>
	<li>Carefully remove tissues next to the skin and remove the piece of skin covering the left jugular vein and the trachea.</li>
	<li>Carefully isolate the parotid gland; sublingual and submaxillary glands from the zone of interest with 2 curved forceps. Do not cut or damage the glands, place them to allow the best access to the jugular vein and to the carotids.</li>
	<li>To isolate the jugular vein, use thin forceps and slowly open and close it to gently free the vessel from the surrounding tissue. Once the vein is completely clean, position the forceps under the vessel and place two 7-0 sutures of 10 cm each under it.</li>
	<li>Close the suture proximal to the head with two knots. On the other suture, prepare one knot but do not close it.</li>
	<li>Hold the catheter (0.28 mm inside diameter, 0.61 mm outer diameter) filled with 37 &#176;C sterile saline with forceps with one hand; while with the other hand, make a small incision into the vessel with a thin scissors or a curved 26 G needle. Then insert the catheter into the jugular vein and close the knot two times. Close the suture proximal to the head over the catheter in order to ensure complete immobilization.</li>
	<li>To isolate and prepare the carotid arteries, dissect the area covering the trachea using thin curved forceps.</li>
	<li>Once the carotids are free from surrounding tissues, remove the vagus nerve (it looks like a white line adjacent to the carotid) from the vessel (but do not cut it). The thin forceps can be closed and slowly opened to gently mobilize the nerve.</li>
	<li>Once both arteries are completely clean, place the forceps under the first carotid artery and place a small black 3 mm wide x 2.5 cm length plastic piece under the vessel. It is very important not to overstretch the vessel and to check that the blood flow is present once the vessel holder is placed. Place the second artery over the vessel holder. If the mouse is not placed directly under the microscope, put back the glands over the trachea and apply 37 &#176;C sterile saline to avoid drying of the zone.</li>
</ol>
4. Assessment of Rolling and Adhering Leukocytes/LysM+ Cells with IVM
<ol>
	<li>Prepare acridine orange from a 2 mg/mL stock solution stored at -20 &#176;C and dilute it 1:4 in sterile saline (0.5 mg/mL final concentration) in a 1 mL syringe. Protect the syringe from light and use 200 &#181;L per one mouse.
	<ol>
		<li>Test different conditions: (1) Injection of acridine orange with the jugular catheter; (2) Injection of acridine orange directly into the tail lateral vein (with this condition no jugular catheter was implanted); (3) Measurement without acridine orange using the fluorescence LysMCre+IRG+ mice (here again no jugular catheter was implanted).</li>
	</ol>
	</li>
	<li>Once the catheter is in place and the two carotids are isolated, position the mouse under the microscope. Perform measurements with a high-speed wide-field fluorescence microscope using a long-distance condenser and a 10X (NA 0.3) water immersion objective with a monochromator, a beam splitter, and a charge-coupled device camera. Perform image acquisition and analysis with real-time imaging system.</li>
	<li>Focus the microscope objective in the middle of the carotid artery at the endothelium surface. Slowly inject 50 &#181;L of acridine orange (0.5 mg/mL) (take into account the dead volume of the catheter for the first injection). Set the software to record 100 images with an exposure time of 120 milliseconds per image.</li>
	<li>Make four videos per carotid artery (left and right) at different locations on the artery for each video. If the fluorescence signal decreases, inject another 50 &#181;L of acridine orange.</li>
	<li>After recording all the videos euthanize the animal by cervical dislocation.</li>
	<li>Set the video speed at 10 images per second and quantify the rolling and adherent cells in a 200 &#181;m x 250 &#181;m rectangle view placed in the middle of the vessel for each video. Count rolling and adherent cells; adherent cells are defined as cells that do not move or detach from the endothelium within the 10 s video. To simplify quantification, remove the channel with the red fluorescence and use only the green fluorescence to count rolling and adhering cells.</li>
	<li>For experiments using acridine orange, make a manual threshold of the fluorescence in order to remove background made by endothelial cells.</li>
</ol>

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The authors have nothing to disclose

LysM+ monocytes were previously shown to be implicated in the development of hypertension5. Here we show that LysM+ immune cells roll and adhere to the endothelial layer in response to AngII infusion. This finding was obtained using LysMCre+IRG+ mice from our previous studies exploring the role of immune cells in hypertension in vivo by visualizing leukocytes with injection of acridine orange6,10. Thus, discrimination of cell types adhering to the endothelium was not possible.
The IVM is a very useful tool for vascular studies and in vivo observations of cells directly in the vasculature, but the impact of injecting dyes with the help of a surgically inserted catheter in the inflammatory context of hypertension remains unknown. To evaluate the potential effect of catheter and acridine orange injection we took advantage of the LysMCre+IRG+ mice. AngII infusion increased the number of rolling LysM+ cells to the endothelium. Insertion of a catheter into the jugular vein amplified the effect and more rolling and adhering leukocytes were detected in AngII-infused mice instrumented with a catheter compared to mice without catheter implants. This indicates that implantation of a carotid catheter causes a systemic inflammatory reaction in the context of wound healing that overlays with the immune reaction seen in hypertension18. In addition, due to the proximity of the jugular vein to the carotid artery an additional immune activation might have occurred by affecting the jugular vein. Since this effect was not present when injections were made directly into the tail vein, we can assume that the effect was due not to the dye but to the procedure of inserting the catheter. We demonstrated here the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.
One critical step of the procedure is the AngII infusion. Tail cuff assessment of the blood pressure can be made in order to control the effectiveness of AngII delivery by the pump. Blood pressure should increase after 2&#8722;3 days of infusion. To limit inflammation that could influence LysM+ cells activation, closure of the incision where the pump is implanted should be made with sutures instead of clips. One limitation must be noted about tail vein injection: to make the best possible data acquisition, 4 videos are usually taken for each carotid. If the fluorescent signal decreases, 50 &#181;L of acridine orange are injected but with tail injection it is more difficult to make several injections and to keep the carotids stable under the microscope objective. The duration of the presence of a catheter in the jugular vein might also modulate the immune cells activation since it is affecting the coagulation activation in platelet-rich plasma prepared 4 h after the catheter implantation19. This aspect of catheter implantation needs further investigation.
Finally, even if we appreciate the impact of jugular catheter implantation on LysM+ cell activation after AngII infusion, we cannot fully estimate the role of the preparation, skin removal, and carotid isolation on a potential LysM+ cell activation. We conclude that the use of fluorescence reporter mice like the LysMCre+IRG+ mouse is recommended to avoid disruption of vessel integrity during the animal preparation for in vivo imaging.

Arterial hypertension increases the risk of cardiovascular disease and death and promotes the development of atherosclerosis, coronary heart disease, and arterial or venous thromboembolisms1. The development of hypertension depends on the interaction of environmental, genetic, endocrine, and hemodynamic factors. Currently, immunity and related inflammation are appreciated to play an important role in the etiology of hypertension2.
Among immune cells, T lymphocytes as well as monocytes and macrophages were found to be causally involved in AngII-induced vascular inflammation and hypertension, in part related to their ability to trigger reactive oxygen species3. Macrophage colony-stimulating factor deficient mice showed reduced response to AngII regarding blood pressure increase and vascular inflammation4. In a previous work, we could show that LysM+ monocytes drive vascular dysfunction and inflammation in AngII-induced hypertension5. More recently, we described a novel pathway in which coagulation factor XI cooperates with platelets and the vessel wall to induce thrombin-dependent vascular inflammation6. The current knowledge about the role of the immune system in hypertension was recently summarized and reviewed by Rodriguez-Iturbe et al.7
Since the involvement of immune cells in the development of hypertension became evident, models and techniques to study the interaction between the vessel and immune cells became necessary. Epifluorescence IVM of blood vessels is a useful tool to observe in vivo interactions between circulating blood cells and the endothelium8,9,10. With this technique, injection of dyes intercalating with DNA (such as acridine orange) can visualize nucleated cells (circulating as well as from the endothelium). Isolated platelets stained ex vivo with rhodamin-6G or dichlorofluorescein (DCF) can be injected to visualize platelet-rich thrombus in arterial or venous injury models.
Typically, a jugular vein catheter is used to inject tracers or marked platelets. Alteration of the endothelium and subsequent activation of the coagulation cascade are both known to have effects on monocyte activation. Endothelial injury immediately leads to platelet activation via subendothelial matrix molecules to seal the tissue, with ensuing monocyte attraction and activation11. On the contrary, an intact endothelium is known to have anticoagulant properties (e.g., via tissue factor pathway inhibitor or thrombomodulin)12 and direct inhibitory effects on the monocyte, e.g., through the secretion of extracellular vesicles containing microRNAs13. Monocytes are known to produce a tissue factor, the extrinsic activator of the coagulation cascade and express protease-activated receptors (PARs) that can be activated by thrombin and participate in monocyte activation14,15. Therefore, any activation of platelets or of the coagulation cascade due to vascular injury may have unexpected effects on monocyte activation and interfere with the observed phenomenon. With the help of IRG transgenic LysM Cre transgenic mice, a double-fluorescent Cre reporter mouse (LysMCre+IRG+), we propose to study in detail the effect of injections with a catheter and alternative methods on LysM+ myelomonocytic cells in a mouse model of arterial hypertension16.

<table><tbody><tr><td>Midazolam</td><td>Ratiopharm GmbH</td><td></td><td>Anesthesia mix</td></tr><tr><td>Medetomidine</td><td>Pfizer Deutschland GmbH</td><td></td><td>Anesthesia mix</td></tr><tr><td>Fentanyl</td><td>Janssen-Cilag GmbH</td><td></td><td>Anesthesia mix</td></tr><tr><td>Alzane</td><td>Zoetis</td><td></td><td>Antisedan mix</td></tr><tr><td>Flumazenil</td><td>Hikma pharma</td><td></td><td>Antisedan mix</td></tr><tr><td>Braunol</td><td>B Braun</td><td></td><td></td></tr><tr><td>Octeniderm</td><td>Sch&amp;uuml;lke</td><td></td><td></td></tr><tr><td>Osmotic pumps</td><td>Alzet</td><td>1007D</td><td>Osmotic pump implantation</td></tr><tr><td>Angiotensin II</td><td>Sigma-Aldrich</td><td></td><td>Osmotic pump implantation</td></tr><tr><td>7-0 prolene suture</td><td>Ethicon</td><td></td><td>Osmotic pump implantation</td></tr><tr><td>Acridine orange</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Catheter</td><td>Smiths Medical Deutschland GmbH</td><td></td><td>Inside diameter, 0.28 mm; outer diameter, 0.61 mm</td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX51WI fluorescence microscope</td><td></td></tr><tr><td>Beam Splitter</td><td>Photometrics</td><td>CMR-DV2-SYS - DualView2 MicroImager</td><td></td></tr><tr><td>Objective</td><td>Olympus</td><td>UMPLFLN10X</td><td>Water immersion objective with a monochromator</td></tr><tr><td>Camera</td><td>Hamamatsu Photonics</td><td>ORCA-R2</td><td></td></tr><tr><td>Image acquisition and analysis software</td><td>Olympus</td><td>Realtime Imaging System eXcellence RT</td><td></td></tr><tr><td>Mice</td><td>The Jackson Laboratory</td><td>Jax 008705</td><td>B6.Cg-Tg(CAG-DsRed,-EGFP)5Gae/J</td></tr><tr><td>Mice</td><td>The Jackson Laboratory</td><td>Jax 004781</td><td>LysM Cre (B6.129P2-&lt;em&gt;Lyz2&lt;sup&gt;tm1(cre)Ifo&lt;/sup&gt;&lt;/em&gt;/J )</td></tr></tbody></table>

visualizing leukocyte rolling and adhesion in angiotensin ii-infused mice: techniques and pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

Carotids of LysMCre+IRG+ mice infused with AngII were observed using IVM. Acridine orange was injected using a jugular catheter. We aimed to look at the proportion of LysM+ cells in contact with the vascular wall compared to all nucleated circulating cells (which could also interact with the vessel since discrimination of the cell type interacting with the vessel remained an open question from our previous work). At baseline, the presence of a jugular catheter causes adhesion of LysM+ cells (Figure 2A, D). After acridine orange injection, the same cells were fluorescent, but also the endothelial cells were fluorescent (Figure 2B, E). After the reduction of the background to limit endothelial related fluorescence, the data indicate that all nucleated adhering cells are LysM+ (Figure 2C, F); these results hold true after AngII infusion confirming our previous results and demonstrating that the LysMCre+IRG+ is a good model to observe the effect of AngII on LysM+ cell activation.
We evaluated the role of administration routes of acridine orange on LysM+ cell activation after AngII infusion in LysMCre+IRG+. After one week of AngII infusion, leukocyte endothelium interactions in carotid arteries of LysMCre+IRG+ mice were imaged and visualized by IVM with or without acridine orange injection via a jugular catheter or through the tail vein. Without any catheter or injection, rolling of LysM+ cells was significantly increased after AngII infusion compared to untreated mice, and adhesion showed an increase (Figure 3). Injection of acridine orange with a jugular catheter increased adhesion and rolling in AngII treated mice to a greater extent compared with mice without a catheter (Figure 3). Injection of acridine orange in the tail vein leads to similar adhesion and rolling compared to the mice that did not receive injection of acridine orange (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56948/56948fig1.jpg" /> 
Figure 1. Scheme for angiotensin II infusion and assessment of LysM+ cells rolling and adhesion in mice. LysMCre+IRG+ mice were infused 7 days with AngII and adhering as well as rolling LysM+ cells over the carotids were quantified with or without injection of acridine orange through a jugular catheter or by tail vein injection. <a href="https://cloudflare.jove.com/files/ftp_upload/56948/56948fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56948/56948fig2.jpg" /> 
Figure 2. Assessment of LysM+ cell adhesion and rolling in carotid arteries of LysMCre+IRG+ mice. Adhesion of LysM+ cells in LysMCre+IRG+ mice before (A, D) and after (B, E) acridine injection via a jugular catheter. Red and green fluorescence were recorded, LysM+ cells (in green) and smooth muscle cells (in red) were visible. After injection of acridine orange, endothelial cells are also visible in green but removing background fluorescence allows only circulating nucleated cell visualization (C, F). <a href="https://cloudflare.jove.com/files/ftp_upload/56948/56948fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56948/56948fig3.jpg" /> 
Figure 3. Evaluation of administration routes of fluorescent dyes in angiotensin II infused LysMCre+IRG+ mice. Quantification of rolling (A) and adhering (B) LysM+ cells in sham operated or AngII-infused animals and with or without injection of acridine orange using a jugular catheter or by tail vein injection. Representative pictures of the different conditions (C). Results are mean &#177; standard error of the mean. 2-way ANOVA was performed and Bonferroni&#39;s post hoc test, n = 3-12/groups; *p &#60; 0.05. <a href="https://cloudflare.jove.com/files/ftp_upload/56948/56948fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls at JoVE.com

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

visualizing-leukocyte-rolling-adhesion-angiotensin-ii-infused-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.6K Views.  University Medical Center Mainz. This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium. 

Video: Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

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 ...

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. ...

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.

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.

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

Bioengineering

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 ...

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 ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

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 ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Biology

Quantification of Diabetes-induced Adherent Leukocytes in Retinal Vasculature

Leukostasis refers to the attachment of leukocytes to the luminal wall of the vasculature. This interaction of leukocytes with the wall of blood vessels is characteristic of inflammation and has been causally linked to capillary occlusion in a variety of tissues and diseases, including diabetic retinopathy. 
Leukostasis has been reported for years as a life-threatening complication of hyperleukocytosis and can only be diagnosed clinically. Given the importance of the phenomenon, intensive research has been done to understand the potential mechanism(s) that lead to its manifestation; however, there is no gold-standard technique in laboratory settings to visualize and quantify the severity of the event.
In the method summarized below, the vasculature is initially perfused with a buffer to remove blood, and then, concanavalin A is perfused into the vasculature where it binds to all exposed cell walls and causes especially bright staining of leukocytes. If the perfusion to remove all unbound blood cells was successful, the remaining fluorescently labeled leukocytes are bound to the vasculature, and they can be manually quantified using any available fluorescence microscope.

We demonstrate a method to label the walls of the retinal vasculature and adherent leukocytes. These adherent leukocytes can then be counted under a fluorescence microscope as a parameter of inflammation or the response of that inflammation to therapies.

Leukostasis refers to the attachment of leukocytes to the luminal wall of the vasculature. This interaction of leukocytes with the wall of blood vessels ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Summary

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Chapters in this video

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

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

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

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

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

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

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Quantification of Diabetes-induced Adherent Leukocytes in Retinal Vasculature