Name: Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice (Video) | JoVE Journal
Uploaded: 2015-08-13T13:00:00.000+00:00
Duration: 5 min 12 s
Description: Learn intravital microscopy to visualize leukocyte-endothelial and platelet-leukocyte interactions in mouse mesenteric veins during inflammation. This method enables real-time analysis of cellular dynamics in vivo.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the Deutsche Forschungsgemeinschaft (DU 1190/3-1).

All animal experiments were performed in compliance with the German animal protection law (TierSchG). The mice were housed and handled in accordance with good animal practice as defined by FELASA (www.felasa.eu/guidelines.php) and the national animal welfare body GV-SOLAS (www.gv-solas.de/index.html). The animal welfare committee of the University of Freiburg as well as the local authorities (Regierungspr&#228;sidium Freiburg) approved all animal experiments.
1. Animal Handling, Preparation and Induction of Inflammation
<ol>
	<li>Use 4 - 6 week old male mice with a weight range of 16 - 20 g. Note: If the mice are older or heavier they present excessive fat surrounding the vessel, limiting the microscopic observation.</li>
	<li>Sterilize all tools and microscope table prior to surgery.</li>
	<li>Inject 20 mg/kg LPS (lipopolysaccharide) intraperitoneally 4 hr&#160;prior to microscopy into the living mouse to induce a bacterial inflammation.</li>
	<li>Prewarm a 0.9% saline solution in a waterbath at 37 &#176;C to humidify the plastic chamber and the mesentery tissue.</li>
	<li>Anesthetize the mouse with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) right before the microscopy procedure. Alternative anesthesia can be used, e.g., 2% isoflurane inhalation.&#160;Confirm proper anesthetization by the loss of response to reflex stimulation (toe or tail pinch with firm pressure).</li>
	<li>Depilate the abdomen using a shaver and remove loose hair with gauze saturated with ethanol 70%.</li>
	<li>Apply vet ointment on the eyes of the mouse to prevent dryness, while under anesthesia.</li>
</ol>
2. Surgery
<ol>
	<li>Sterilize the abdomen using 70% ethanol. This method is not a sterile method and could be lethal at the end of the experiment.</li>
	<li>Perform a median laparotomy: Open the abdominal skin using small, curved forceps and small scissors. Identify the epigastrical vessels and open the peritoneum in the region of the linea alba, to protect the vessels.</li>
	<li>Apply a few drops of prewarmed saline into the abdominal cavity to keep the tissue moist.</li>
	<li>Label leukocytes and platelets fluorescently by injecting 50 &#181;l rhodamine 6G (1 mg/ml) retro-orbitally as described before 9,10.</li>
	<li>Exteriorize a loop of ileumand place it in a petridish with a diameter of 10 cm and make sure to keep the tissue moist by applying the 37 &#176;C prewarmed saline solution (0.9%) every other minute.</li>
</ol>
3. Intravital Microscopy
<ol>
	<li>Place the Mouse underneath the microscope and bring the mesentery vein with a diameter of 200 - 300 &#181;m in the center of view. Choose a vessel with no visible fat surroundings.</li>
	<li>Do not touch the mesentery vessels thereby avoiding stimulation of the endothelium. Handle the ileum loop cautiously.</li>
	<li>Visualize blood cell-endothelial interactions with an inverted or upright microscope and a camera using a microscope software. Record blood cell-endothelial interactions for 1 min&#160;in 4 different veins per mouse.</li>
	<li>Euthanize the mouse by cervical dislocation after the completion of imaging experiments.</li>
</ol>
4. Analysis
<ol>
	<li>Carry out the analysis offline and blinded for all parameters. Carry out analysis using any suitable software program.</li>
	<li>Confirm stable and interindividually comparable blood flow conditions in high time-resolution cine-clips (maximal frame rate) focused on intraluminal blood cell flow.</li>
	<li>Quantify the number of rolling leukocytes. Therefor draw a vertical line through the vein and count all leukocytes crossing this line in 1 min.</li>
	<li>Determine rolling velocity by measuring the time one single leukocyte needs to pass a distance of 50 &#181;m while stably rolling on endothelium. To do this, draw two vertical lines in a distance of 50 &#181;m through the vein.
	<ol>
		<li>Measure the time a representative leukocyte needs to get from one line to the other.Calculate the speed of the leukocytes by dividing 50 &#181;m through the time needed (&#181;m/s).</li>
	</ol>
	</li>
	<li>Measure leukocyte adhesion in a field of 0.04 mm2 . To do this, draw a square with the side length of 200 &#181;m into the vein.
	<ol>
		<li>Count the firm adherent leukocytes, defined as no visible movement for 30 sec, within this square.</li>
	</ol>
	</li>
	<li>Count the number of platelets bound to one leukocyte to quantify platelet-leukocyte interactions.</li>
</ol>

<ol><li>Lehr, H. A., Vollmar, B., Vajkoczy, P., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+fluorescence+microscopy+for+the+study+of+leukocyte+interaction+with+platelets+and+endothelial+cells%5BTitle%5D)+AND+%22Methods+in+enzymology%22%5BJournal%5D)">Intravital fluorescence microscopy for the study of leukocyte interaction with platelets and endothelial cells.</a> Methods in enzymology. 300, 462-481 (1999).</li>
<li>Atherton, A., Born, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+investigations+of+the+adhesiveness+of+circulating+polymorphonuclear+leucocytes+to+blood+vessel+walls%5BTitle%5D)+AND+%22The+Journal+of+physiology%22%5BJournal%5D)">Quantitative investigations of the adhesiveness of circulating polymorphonuclear leucocytes to blood vessel walls.</a> The Journal of physiology. 222, 447-474 (1972).</li>
<li>Duerschmied, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serotonin+stimulates+platelet+receptor+shedding+by+tumor+necrosis+factor-alpha-converting+enzyme+%28ADAM17%29%5BTitle%5D)+AND+%22Journal+of+thrombosis+and+haemostasis+%3A+JTH%22%5BJournal%5D)">Serotonin stimulates platelet receptor shedding by tumor necrosis factor-alpha-converting enzyme (ADAM17).</a> Journal of thrombosis and haemostasis : JTH. 7, 1163-1171 (2009).</li>
<li>Chauhan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ADAMTS13%3A+a+new+link+between+thrombosis+and+inflammation%5BTitle%5D)+AND+%22The+Journal+of+experimental+medicine%22%5BJournal%5D)">ADAMTS13: a new link between thrombosis and inflammation.</a> The Journal of experimental medicine. 205, 2065-2074 (1084).</li>
<li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy%5BTitle%5D)+AND+%22Microvascular+research%22%5BJournal%5D)">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvascular research. 5, 384-394 (1973).</li>
<li>Leister, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+peritoneal+cavity+chamber+for+intravital+microscopy+of+the+liver+under+conditions+of+pneumoperitoneum%5BTitle%5D)+AND+%22Surgical+endoscopy%22%5BJournal%5D)">A peritoneal cavity chamber for intravital microscopy of the liver under conditions of pneumoperitoneum.</a> Surgical endoscopy. 17, 939-942 (2003).</li>
<li>Duerschmied, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+serotonin+promotes+the+recruitment+of+neutrophils+to+sites+of+acute+inflammation+in+mice%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice.</a> Blood. 121, 1008-1015 (2013).</li>
<li>Herr, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+fluoxetine+treatment+induces+slow+rolling+of+leukocytes+on+endothelium+in+mice%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Acute fluoxetine treatment induces slow rolling of leukocytes on endothelium in mice.</a> PloS one. 9, e88316(2014).</li>
<li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retro-orbital+injections+in+mice%5BTitle%5D)+AND+%22Lab+animal%22%5BJournal%5D)">Retro-orbital injections in mice.</a> Lab animal. 40, 155-160 (2011).</li>
<li>Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+as+routes+of+intravenous+drug+delivery+in+a+transgenic+mouse+model%5BTitle%5D)+AND+%22Lab+animal%22%5BJournal%5D)">Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model.</a> Lab animal. 37, 26-32 (2008).</li>
<li>Duerschmied, D., Bode, C., Ahrens, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+functions+of+platelets%5BTitle%5D)+AND+%22Thrombosis+and+haemostasis%22%5BJournal%5D)">Immune functions of platelets.</a> Thrombosis and haemostasis. 112, 678-691 (2014).</li>
<li>Wagner, D. D., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+vessel+wall+and+its+interactions%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">The vessel wall and its interactions.</a> Blood. 111, 5271-5281 (2008).</li>
<li>Gawaz, M., Fateh-Moghadam, S., Pilz, G., Gurland, H. J., Werdan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+activation+and+interaction+with+leucocytes+in+patients+with+sepsis+or+multiple+organ+failure%5BTitle%5D)+AND+%22European+journal+of+clinical+investigation%22%5BJournal%5D)">Platelet activation and interaction with leucocytes in patients with sepsis or multiple organ failure.</a> European journal of clinical investigation. 25, 843-851 (1995).</li>
<li>Sarma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+platelet+binding+to+circulating+monocytes+in+acute+coronary+syndromes%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Increased platelet binding to circulating monocytes in acute coronary syndromes.</a> Circulation. 105, 2166-2171 (2002).</li>
<li>Sumagin, R., Sarelius, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TNF-alpha+activation+of+arterioles+and+venules+alters+distribution+and+levels+of+ICAM-1+and+affects+leukocyte-endothelial+cell+interactions%5BTitle%5D)+AND+%22American+journal+of+physiology.+Heart+and+circulatory+physiology%22%5BJournal%5D)">TNF-alpha activation of arterioles and venules alters distribution and levels of ICAM-1 and affects leukocyte-endothelial cell interactions.</a> American journal of physiology. Heart and circulatory physiology. 291, H2116-H2125 (2006).</li>
</ol>

The authors have nothing to disclose.

Herein we describe the preparation and performance of intravital microscopy of mesentery veins of mice in vivo. This method gives us the opportunity to observe leukocyte-endothelial and platelet-endothelial interactions11 directly in the living organism.
As an early response to inflammation the endothelium gets activated and interacts with leukocytes and platelets resulting in rolling, adhesion and transmigration12. But leukocytes also interact with platelets for...

The purpose of this protocol is to describe a simplified technique of intravital microscopy of mesentery veins in living mice for direct observation of leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions.
Intravital microscopy was developed to study leukocyte-endothelial interactions in vivo under inflammatory conditions1,2, which is not trivial but important for understanding inflammatory leukocyte recruitment and function. The method we describe in this protocol was developed based on previously published publications. Similar to visualizing platelet incorporation in a growing thrombus3 and in parallel to what has been published earlier4, an exteriorized mesentery vein is examined by transmitted light microscopy. There are various other models of intra-vital microscopy such as the cremaster muscle of rats5 or liver of mouse and rats6.
Intravital microscopy of mesentery vein has been developed and applied in previous studies by several groups. We have used this technique to observe differences in leukocyte recruitment between wild type mice and tryptophane hydroxylase1 (Tph1) -/- mice, which are deficient of non-neuronal serotonin and compared the findings to mice whose platelet serotonin was pharmacologically depleted by long-term application the a selective serotonin reuptake inhibitor fluoxetine7. We have also examined leukocyte-endothelial interactions after acute fluoxetine treatment8.
In this protocol we focus on intravital microscopy of mesentery veins, because this model can be performed quickly, and allows valid measurements of leukocyte-endothelial and platelet-leukocyte interactions. This is much more challenging and time-consuming in intravital microscopy of other organs, such as bone, liver, skin, or cremaster muscle. The model described here is ideal for reproducible evaluation of inflammatory cell-cell interaction after challengeing it with an inflammatory stimulus, such as intraperitoneal injection of lipopolysaccharide.

<table><tbody><tr><td>Rhodamine 6G</td><td>Sigma-Aldrich</td><td>&amp;nbsp;989-38-8</td><td></td></tr><tr><td>Lipopolysaccharides from Escherichia coli 055:B5</td><td>Sigma-Aldrich</td><td>L2637&amp;nbsp;</td><td></td></tr></tbody></table>

intravital microscopy of leukocyte-endothelial and platelet-leukocyte interactions in mesenterial veins in mice

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.

An overview of the experimental setting described in this protocol is shown in Figure 1. It shows a mouse with an exteriorized ileum-loop and its vessels, which can be observed in intravital microscopy. Figure 2 shows certain degree of activation in untreated animals due to the procedure itself. But there is almost no slow rolling or firm adhesion of leukocytes compared to LPS-treated mice. Figure 2 also shows the different results of intravital microscopy in wildtype mi...

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 ...

The overall goal of this procedure is to investigate leukocyte, endothelial cell and platelet leukocyte interactions during inflammation in vivo. This is accomplished by first pretreating, a mouse with the substances of interest and labeling the blood cells an ileum loop, and the accompanying mesentary vessels are then exteriorized and the STO cell interactions that take place within a selected mesentary vein are imaged by intra vital microscopy. Ultimately, the interactions between the leukocytes and the platelets with the endothelial or each other can be analyzed.This method can help answer key questions in the field of inflammation, such as what kinds of leukocyte endothelial, or platelet leukocyte interactions occur in vivo during different models of inflammation or after different pre-treatments of interest. Four hours before the intra vital imaging inject 20 milligrams per kilogram of LPS IP into a 16 to 20 gram, four to six week old male mouse to induce the mock bacterial inflammatory response. Prewarm a 0.9%saline solution in a 37 degree Celsius water bath for humidifying the plastic chamber and mesentary tissue at this time as well.Next, confirm the appropriate depth of anesthesia by a lack of response to toe pinch in the LPS treated mouse, and then apply ointment to the animal size, dete the abdomen with a shaver, removing any loose hair with 70%ethanol soaked gauze. Then use a small curved forceps and scissors to open the abdomen, identify the epigastric vessels and open the peritoneum in the linear alba region to protect the vessels. Apply a few drops of prewarm saline into the abdominal cavity to keep the tissue moist, followed by a retroorbital injection of 50 microliters of rod Domine six G to label the circulating blood cells.Then place the mouse in a 10 centimeter Petri dish, exteriorize a loop of illium and administrate the prewarm saline every other minute to keep the tissue moist. To visualize the inflammatory response by intravital microscopy, place the mouse underneath the microscope and bring a mesentary vein with the diameter of 200 to 300 micrometers and as little as possible visible surrounding fat into the field of view. Using the appropriate microscope software.Record the blood cell endothelial interactions for one minute in four different veins per mouse. Then to analyze the cell to cell responses to confirm the stable and enter individually blood flow conditions. To quantify the number of rolling leukocytes, draw a vertical line through the vein and manually count all of the leukocytes that cross this line in one minute.To determine the rolling velocity, draw two vertical lines through the vein 50 micrometers apart, and measure the time it takes a single leukocyte to cross between the lines while stably rolling on the endothelium. To measure the leukocyte adhesion, draw a 200 by 200 micrometer square in the vein. Then manually count the firmly adherent leukocytes that exhibit, no visible movement within the square for 30 seconds.Finally, to quantify the platelet leukocyte interactions manually count the number of platelets bound to one leukocyte. Although there is a certain degree of activation in untreated animals due to the procedure itself, there is almost no slow rolling or firm adhesion of untreated leukocytes. Indeed, higher numbers of rolling and adherent leukocytes are observed with a decrease in the rolling velocity of the circulating blood cells in LPS challenged mice.In this experiment, intravital microscopy was performed without further LPS challenge immediately after acute intraperitoneal treatment with fluoxetine. Note, the higher numbers of firmly adherent leukocytes and the lower rolling velocities in the fluoxetine treated animals indicating an influence of the acute fluoxetine treatment on leukocyte endothelial interactions. Further, as observed in this representative image of platelet leukocyte interactions with an e mesenteric vein during LPS induced peritonitis, a ting of the platelets around the leukocytes can be observed as well as the interactions of these complexes with the vessel wall.After watching this video, you should have a good understanding of how to perform inal microscopy to investigate leukocyte endothelial and platelet leukocyte interactions.

intravital-microscopy-leukocyte-endothelial-platelet-leukocyte

Research

JoVE Journal

Immunology and Infection

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

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.

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

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

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

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

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

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.

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

This protocol presents a method for inducing mesenteric ischemia-reperfusion injury and visualizing neutrophil extracellular traps (NETs) in mesenteric venules using intravital microscopy. Blood flow in the superior mesenteric artery was restricted for 1 h, followed by reperfusion, allowing direct quantification of leukocyte adhesion and NETosis.

Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Intravital Microscopy of the Inguinal Lymph Node

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

Learn intravital microscopy to visualize leukocyte-endothelial and platelet-leukocyte interactions in mouse mesenteric veins during inflammation. This method enables real-time analysis of cellular dynamics in vivo.

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

Explore More Videos