This work was supported by the ELSE-Kr&#246;ner-Stiftung and the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center). Special thanks to Hans-Holger G&#228;rtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.

Our study was performed with permission of the state of Saxony-Anhalt, Landesverwaltungsamt Halle, according to section 8 of the German law for animal protection. (&#167; 8, paragraph 1 of the German law for animal protection from 18.05.2016 - BGBI. I S. 1206, 1313, &#167; 31 TierSchVersV from 13.08.2013).

NOTE: For the experiments here, 8 to 12 week old male BALB/c mice were used, and human monocytes from blood donors were used for the visualization of monocytes via intravital microscopy.
1. Cell Preparation
NOTE: For the isolation of monocytes, please see our previous published video on JoVE for instructions: &#34;Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes&#34; by Wagner et al.4
NOTE: When working with the cells all steps must be sterile to avoid contamination.
<ol>
	<li>Cell Staining with 3,3&#39;-Dioctadecyloxacarbocyanine Perchlorate
	<ol>
		<li>Resuspend the cells in serum free culture medium with a density of 1 x 106 cells/mL. 
		NOTE: Only serum free medium enables an efficient staining, since the lipophilic dye would otherwise already be captured by lipophilic components of the serum.</li>
		<li>Add 5 &#181;L of 1 mM 3,3&#39;-Dioctadecyloxacarbocyanine perchlorate in dimethylformamide to 1 mL of the cell suspension and resuspend carefully.</li>
		<li>Incubate the cell solution at 37 &#176;C for 20 min.</li>
		<li>Centrifuge the cells at 37 &#176;C and 500 x g for 5 min.</li>
		<li>Dislodge the supernatant and resuspend the cells with 37 &#176;C warm fetal calf serum supplemented medium.</li>
		<li>Repeat the steps 1.1.4 and 1.1.5 twice.</li>
		<li>Count the cells with the formula: 
		<img alt="Equation 1" src="/files/ftp_upload/56290/56290eq1.jpg" /></li>
		<li>Resuspend the cells with 150 &#181;L sterile 0.9% NaCl solution.</li>
		<li>Inject the cells into the tail vein.</li>
	</ol>
	</li>
</ol>
2. Anesthesia
<ol>
	<li>Inhalation Anesthesia
	<ol>
		<li>Vaporize isoflurane with the help of a vaporizer using a concentration of 5% in a closed bin under a chemical safety hood.</li>
		<li>Handle the mouse carefully and put it into the bin.</li>
		<li>Handle the animal by the skin of the posterior neck after it has stopped moving.</li>
	</ol>
	</li>
	<li>Intraperitoneal Anesthesia 
	NOTE: Use isoflurane anesthesia, described under step 2.1, to perform an intraperitoneal injection. With the fast recovery and short-acting narcotic effect of the isoflurane anesthesia, it is easier to handle the mouse and inject intraperitoneal anesthesia.
	<ol>
		<li>Use a solution of 2.4 mL Ketamine (10%), 0.8 mL Xylazine (2%), and 6.8 mL NaCl (0.9%) for the intraperitoneal injection.</li>
		<li>Weigh the animal before applying anesthetic. 
		NOTE: The formula for the anesthetic is: 
		<img alt="Equation 2" src="/files/ftp_upload/56290/56290eq2.jpg" /></li>
		<li>Use a 1 mL insulin syringe with a 30 G needle to inject the solution into the left underbelly.</li>
		<li>Place the mouse in its cage and wait for narcotic effect. 
		NOTE: The narcotic effect normally appears within 5 min if the injection was successful. The correct depth of the anesthesia can be determined by the absence of either the lid reflex or reaction to toe pinch, as well as a regular, controlled rate of breathing.</li>
	</ol>
	</li>
</ol>
3. Implantation of Basement Membrane-like Matrix
NOTE: This method is used by our group to study tumor angiogenesis after monocyte injection. Depending on the experiments, growth factors can be added to the basement membrane-like matrix. We performed femoral artery ligation before injecting the tumor in the flank of the mouse. The matrix must have a temperature of 4 &#176;C for the injection. At this temperature, the matrix is fluid; the gel hardens to a solid at body temperature (37 &#176;C). For better visibility of the subcutaneous matrix plug, shave the skin of the mouse at the injection site.
Note:Optional: Add 100 ng basic fibroblast growth factor, 300 ng vascular endothelial growth factor, and 26 I.U. heparin under sterile conditions to the basement membrane-like matrix.
<ol>
	<li>Load 1 mL of matrix into a 30 G insulin syringe and store on ice until use.</li>
	<li>Lay the animal on the table and hold the skin of the mouse next to the injection site on the flank.</li>
	<li>Inject 500 &#181;L of the basement membrane-like matrix subcutaneously. 
	NOTE: For practical reasons, it is necessary to inject the basement membrane-like matrix compactly at one location to avoid subcutaneous dispersion. It will be easier to dislodge the artificial tumor from the tissue after sacrificing the mouse at the end of the experiment.</li>
</ol>
4. Tail Vein Injection
NOTE: Practice the tail vein injection with NaCl solution on test animals before experimentation. If the monocytes cannot be adequately injected in the tail vein, there will be no systemic effect on the collateralization. In this protocol, we injected 2.5 million monocytes. Try to inject no more than 5 &#181;L/g of bodyweight.
<ol>
	<li>Use the monocyte solution prepared under step 1.1.8 containing 2.5 million monocytes.</li>
	<li>Use a 30 G needle and a 1 mL insulin syringe for the injection.</li>
	<li>Carefully handle the mouse, restrain the animal in the restrainer, and make sure the mouse is not harmed and has adequate space for breathing.</li>
	<li>Put the restrainer on the heating pad so that the tail can contact the plate.</li>
	<li>Identify the tail veins, which are located on the lateral side of the tail.</li>
	<li>Turn the tail 90 &#176;, so the tail vein appears on the upper side of the tail.</li>
	<li>Disinfect the injection side before injecting the monocytes.</li>
	<li>Try to inject the monocyte solution in a flat angle with the bevel of the needle facing up. 
	NOTE: Stop the injection if a blister appears, as this is a sign of a failed injection. Attempt the procedure again more proximally.</li>
	<li>Stop bleeding at the injection site by applying gentle pressure on the tail for about 60s.</li>
	<li>Observe the animal for 30 min to monitor for systemic side effects and place the mouse in its cage after the animal has fully recovered.</li>
</ol>
5. Intravital Microscopy
<ol>
	<li>Preparation
	<ol>
		<li>Place the anesthetized mouse on the heating pad (37 &#176;C) to keep a constant temperature and fix the paws in place with adhesive tape.</li>
		<li>Disinfect the skin at the site of the leg or flank that is used for microscopy.</li>
		<li>Shave the region of interest for better handling and to avoid interference with hair.</li>
		<li>Excise the skin with a sterile scalpel and fine forceps in a square area of 0.5 x 0.5 cm. 
		NOTE: It is important to keep the region of interest moist; otherwise, the quality of images and tissue will be compromised. NaCl solution can be used to moisten the area.</li>
		<li>Place the leg between two adjustable stamps and position a cover glass on top of the stamps.</li>
		<li>Ensure the tissue is wet and is in contact with the glass.</li>
		<li>Inject 50 &#181;L rhodamine dextran retrobulbar into the venous system for better visibility of the vessels.</li>
		<li>Start microscopy and adjust the position of the leg if needed.</li>
	</ol>
	</li>
	<li>Intravital microscopy settings
	<ol>
		<li>Turn on the microscope, computer, electronic interfaces, and lasers.</li>
		<li>Turn on the heating unit of incubation chamber and/or heating stage (plate/pad), and set the temperature to 37 &#176;C.</li>
		<li>Start the acquisition software. If the microscope is equipped with a resonant scanner, select this fast scanning mode.</li>
		<li>Wait until the temperature reaches a constant level (35 - 37 &#176;C) 
		NOTE: A stable temperature is important for: (a) the mouse, which under anesthesia cannot control its body temperature, and (b) avoiding or at least minimizing focal drift. This could take 1 h to several hours, depending on the surrounding conditions (e.g., air conditioning system and the number of heat sources in the room, including people). If immersion objectives are used, the contact zone between the lens and cover glass (or tissue) is a typical source of instability. Heating the lens with an objective heater may help; alternatively, a microscope with an autofocus system is highly recommended.</li>
		<li>Select a magnification lens with a high numeral aperture that fulfils the resolution requirements of the experiment (see note at the end of this section).</li>
		<li>Optimize the microscope settings with respect to gain, channel settings, scan speed, pixel resolution, depth volume, step size, and averaging before placing the experimental animal on the stage.</li>
		<li>Scan bidirectionally to reduce acquisition time.</li>
		<li>Place the anesthetized mouse on the prewarmed microscope stage after the microscope has reached stable conditions and the settings have been tested with the help of a dummy.</li>
		<li>Bring the region of interest within the basement membrane-like matrix into focus by using bright light illumination and inspect briefly the capillaries by ultraviolet light with adequate filter settings for the applied fluorophores.</li>
		<li>Switch to scanning mode; start scanning at low resolution 256 x 256 mode and enhance gain and laser power until a signal is observed on the monitor.</li>
		<li>Focus on the structure of interest. Zoom in until all details are visible.</li>
		<li>Define &#34;start&#34; and &#34;end&#34; positions in the axial direction.</li>
		<li>Stop scanning.</li>
		<li>Define stepping size (e.g., 0.5 &#181;m) and number of focal planes (e.g., 10 - 20).</li>
		<li>Switch to the final pixel resolution (512 x 512 or 1,024 x 1,024) depending on the speed of the moving subcellular structures or cells and select the scan speed (scan frequency) that fits best to the movements. Bidirectional scanning is recommended to fasten the acquisition of stacks. 
		NOTE: Select the highest scan rate of scanning that gives sufficient image quality. Typical point scanners provide speeds between 400 Hz and 1.4 kHz. Resonant scanners provide an additional 8 kHz or 12 kHz. By reducing the number of lines in the y-direction, the scan rate can be further increased (typical values are 512 x 512, 512 x 256, or 512 x 200 pixel resolution). Depending on the signal-to-noise ratio, one can try to improve the image quality by adding line averaging between 2 and 4. Typically, a setting of 512 x 200 with no averaging results in an acquisition time/frame of 18 ms in the bidirectional mode with an 8 KHz resonant scanner; with 512 x 200 and 4x averaging, it slows down to 56 ms per frame.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Volz, K. S., Miljan, E., Khoo, A., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pluripotent+stem+cells+for+vascular+therapy.">Development of pluripotent stem cells for vascular therapy.</a> Vascular pharmacology. 56 (5-6), 288-296 (2012).</li><li>Henry, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VIVA+trial:+Vascular+endothelial+growth+factor+in+Ischemia+for+Vascular+Angiogenesis.">The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis.</a> Circulation. 107 (10), 1359-1365 (2003).</li><li>Wagner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+intravenous+injection+of+murine+bone+marrow+derived+monocytes.">Isolation and intravenous injection of murine bone marrow derived monocytes.</a> Journal of visualized experiments JoVE. (94), (2014).</li><li>Herold, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+monocytes:+a+novel+strategy+for+in+vivo+augmentation+of+collateral+vessel+growth.">Transplantation of monocytes: a novel strategy for in vivo augmentation of collateral vessel growth.</a> Human gene therapy. 15 (1), 1-12 (2004).</li><li>Ito, W. D., Arras, M., Scholz, D., Winkler, B., Htun, P., Schaper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+but+not+collateral+growth+is+associated+with+ischemia+after+femoral+artery+occlusion.">Angiogenesis but not collateral growth is associated with ischemia after femoral artery occlusion.</a> The American journal of physiology. 273 (3 Pt 2), H1255-H1265 (1997).</li><li>Herold, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetanus+toxoid-pulsed+monocyte+vaccination+for+augmentation+of+collateral+vessel+growth.">Tetanus toxoid-pulsed monocyte vaccination for augmentation of collateral vessel growth.</a> Journal of the American Heart Association. 3 (2), e000611 (2014).</li><li>. Matrigel Matrix Available from: <a target="_blank" href="https://www.corning.com/au/en/products/life-sciences/products/surfaces/matrigel-matrix.html">https://www.corning.com/au/en/products/life-sciences/products/surfaces/matrigel-matrix.html</a> (2017)</li><li>Eubank, T. D., Galloway, M., Montague, C. M., Waldman, W. J., Marsh, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF+induces+vascular+endothelial+growth+factor+production+and+angiogenic+activity+from+human+monocytes.">M-CSF induces vascular endothelial growth factor production and angiogenic activity from human monocytes.</a> Journal of immunology (Baltimore, Md. 1950). 171 (5), 2637-2643 (2003).</li><li>Fridman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+tumor+growth+of+both+primary+and+established+human+and+murine+tumor+cells+in+athymic+mice+after+coinjection+with+Matrigel.">Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel.</a> Journal of the National Cancer Institute. 83 (11), 769-774 (1991).</li><li>Woodman, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caveolin-1+knockout+mice+show+an+impaired+angiogenic+response+to+exogenous+stimuli.">Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli.</a> The American journal of pathology. 162 (6), 2059-2068 (2003).</li><li>Francke, A., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C., Herold, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+bone+marrow+derived+monocytes:+a+novel+approach+for+augmentation+of+arteriogenesis+in+a+murine+model+of+femoral+artery+ligation.">Transplantation of bone marrow derived monocytes: a novel approach for augmentation of arteriogenesis in a murine model of femoral artery ligation.</a> American journal of translational research. 5 (2), 155-169 (2013).</li><li>Houthuys, E., Movahedi, K., Baetselier, P., de Van Ginderachter, J. o. A., Brouckaert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+and+purification+of+mouse+peripheral+blood+monocytes.">A method for the isolation and purification of mouse peripheral blood monocytes.</a> J. Immunol. Methods. 359 (1-2), 1-10 (2010).</li><li>Berthold, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+monocytes+by+Ficoll+density+gradient+centrifugation.">Isolation of human monocytes by Ficoll density gradient centrifugation.</a> Blut. 43 (6), 367-371 (1981).</li></ol>

The authors declare that they have no competing financial interests.

The method described here sheds light on the development of collateral arteries, the behavior of monocytes in these vessels, and the process of arteriogenesis. The steps for applying this protocol are easy to learn and can be used in other fields of science. Despite these advantages, there are some disadvantages. For instance, microscopic equipment is required to execute the described techniques. Obtaining equipment for one experiment is unsustainable, so it is important to collaborate with other institutions to share th...

Cardiovascular diseases, including coronary heart disease or peripheral arterial disease, are the most common causes of death globally1. Cell therapy is a promising approach to treat cardiovascular disease, particularly for people who are not able to undergo surgical interventions. There are several approaches to use cells or their secreted substances as a therapeutic tool2,3, with the overall goal to improve the perfusion and maintain function of ischemic and underperfused tissue. One attempt to achieve this goal is to improve arteriogenesis, which enhances the development of collateral arteries. Monocytes are an important cell type associated with collateralization. Our group has focused on researching the effects of monocytes in areas of inflammation4,5, in particular using the hindlimb ischemia model to induce ischemia and subsequent inflammation6. Monocytes home to areas of inflammation and cause complex systemic responses that lead to the development of collateralization7.
With the use of intravital microscopy, we can study the behavior of these cells in vivo and observe the homing of injected monocytes to areas of inflammation. Most former studies only describe post mortem analyses, which hold disadvantages including an introduction of histological artifacts and large numbers of animal required for preparations. With our approach, we can investigate immunological processes and collateral formation via live imaging at multiple time points.
In addition to the development of collateral arteries in ischemic areas, monocytes also influence tumor growth. To investigate these processes, we inject a basement membrane-like matrix extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins8, and analyze using intravital microscopy. This matrix is used to screen test molecules for either endothelial cell network formation or anti-cancer therapies through angiogenic inhibition; in this case, we will assess the tumor angiogenic potential of monocytes for cell therapy9,10,11.
The aim of this protocol is to demonstrate an easy and efficient way to study immunological processes caused by ischemia in an in vivo model. We can generate a more realistic test environment compared to histological workup of post mortem muscle tissue.

<table><tbody><tr><td>10% fetal calf serum (FCS)</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>1% penicillin/streptomycin</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>1mL Omnifix -F insuline syringe</td><td>B. Braun, Melsungen AG, Melsungen, Germany</td><td></td><td></td></tr><tr><td>50 ml syringe&amp;nbsp;</td><td>Fresenius Kabi AG, Bad Homburg, Germany</td><td></td><td>Injectomat- syringe 50 ml with canule</td></tr><tr><td>6-well-ultra-low-attachement-plates</td><td>Corning Incorporated, NY, USA</td><td></td><td></td></tr><tr><td>8- 12 week old, male, C57BL/6, BalbC mice&amp;nbsp;</td><td>Charles River, Sulzfeld, Germany</td><td></td><td></td></tr><tr><td>Adhesive tape</td><td>TESA SE, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Acquisition Software</td><td>Leica, Wetzlar, Deutschland&amp;nbsp;</td><td></td><td>Leica Application Suite Advanced Fluorescence (LAS AF); Version: 2.7.3.9723</td></tr><tr><td>Canules</td><td>B. Braun, Melsungen AG, Melsungen, Germany</td><td></td><td>29G, 30G</td></tr><tr><td>Cell culture dish</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Cell culture medium</td><td>Manufactured by our group with single components</td><td></td><td>Medium199, 10% Fetal calf serum, 1% Antibiotic (penicillin/streptomycin)</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter GmbH, Krefeld, Germany</td><td></td><td>Allegra X-15R centrifuge</td></tr><tr><td>Depilatory cream</td><td>Veet, Mannheim, Germany</td><td></td><td></td></tr><tr><td>DiO</td><td>Invitrogen Eugene, Oregon, USA</td><td></td><td></td></tr><tr><td>Disinfection agent</td><td>Sch&amp;uuml;lke&amp;amp;Mayr GmbH, Norderstedt, Germany</td><td></td><td></td></tr><tr><td>Disposable scalpel No.10&amp;nbsp;</td><td>Feather safety razor Co.Ltd, Osaka, Japan&amp;nbsp;</td><td></td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Erlenmeyer flask</td><td>GVB, Herzogenrath, Germany</td><td></td><td></td></tr><tr><td>Ethanol 70%</td><td>Otto Fischar GmbH und Co KG, Saarbr&amp;uuml;cken, Germany</td><td></td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Fine Forceps</td><td>Rubis, Stabio, Switzerland</td><td></td><td></td></tr><tr><td>Flurophor/Rhodamindextran</td><td>Thermo Fischer Scientific, Waltham, MA USA</td><td>Katalognummer: D-1819</td><td></td></tr><tr><td>Gloves</td><td>R&amp;ouml;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td><td></td><td></td></tr><tr><td>Heating pad&amp;nbsp;</td><td>Labotect GmbH, G&amp;ouml;ttingen, Germany&amp;nbsp;</td><td></td><td>Hot Plate 062</td></tr><tr><td>Human macrophage-colony stimulating factor</td><td>Sigma Aldrich, Hamburg, Germany</td><td>SRP3110&amp;nbsp;</td><td></td></tr><tr><td>Humane leucocyte filters</td><td>Blood preservation</td><td></td><td></td></tr><tr><td>Incubator</td><td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Baxter Deutschland GmbH, Unterschlei&amp;szlig;heim, Germany</td><td></td><td></td></tr><tr><td>Ketamine (10%)</td><td>Ketavet, Pfizer Deutschland GmbH, Berlin , Germany</td><td></td><td></td></tr><tr><td>Leukocyte separation tubes (tubes with filter)&amp;nbsp;</td><td>Bio one GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Light microscope&amp;nbsp;</td><td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td><td></td><td>Axiovert 40 C</td></tr><tr><td>Lymphocyte separation medium LSM1077</td><td>GE Healthcare, Pasching, Austria</td><td></td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td><td></td><td></td></tr><tr><td>Medium M199&amp;nbsp;</td><td>PAA Laboratories GmbH, Pasching, Austria</td><td></td><td></td></tr><tr><td>Microbiological work bench</td><td>Thermo Electron, LED GmbH, Langenselbold, Germany</td><td></td><td>Hera safe</td></tr><tr><td>Microscope slide</td><td>Carl Roth GmbH + Co. KG, Karlsruhe</td><td>Art. Nr. 1879</td><td></td></tr><tr><td>Microscope stand with incubator and heating unit</td><td>&amp;nbsp;Leica DMI 6000, Pecon, Germany</td><td></td><td></td></tr><tr><td>Monocyte wash buffer</td><td>Manufactured by our group with single components</td><td></td><td>PBS, 0,5% BSA, 2mM EDTA</td></tr><tr><td>Mouse restrainer</td><td>Various</td><td></td><td></td></tr><tr><td>Multi-photon microscope&amp;nbsp;</td><td>Leica, Wetzlar, Deutschland&amp;nbsp;</td><td></td><td>Leica SP5 Confocal microscope, Cameleon, Coherent</td></tr><tr><td>NaCl (0,9%)</td><td>Berlin Chemie AG, Berlin, Germany</td><td></td><td></td></tr><tr><td>Neubauer counting chamber&amp;nbsp;</td><td>Paul Marienfeld GmbH und Co.KG, Lauda-K&amp;ouml;nigshofen, Germany</td><td></td><td></td></tr><tr><td>Objective</td><td>Leica, Wetzlar, Deutschland&amp;nbsp;</td><td></td><td>Leica HC PL APO 10x/0.4 CS</td></tr><tr><td>PBS</td><td>Life technologies GmbH, Darmstadt, Germany</td><td></td><td>ph 7,4 sterile</td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Percoll</td><td>Manufactured by our group with single components</td><td></td><td>90 % Percoll, 10% 1,5M NaCl, &amp;rho;= 1,064 g cm-3</td></tr><tr><td>Percoll solution</td><td>GE Healthcare, Bio-Science AB, Uppsala, Sweden</td><td></td><td></td></tr><tr><td>Pipettes</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td>10&amp;micro;L/100&amp;micro;L/200&amp;micro;L/1000&amp;micro;L</td></tr><tr><td>Pipettes serological</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td>&amp;nbsp;Cellstar2ml, 5ml, 10ml</td></tr><tr><td>Pipetting heads</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Pipetus</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Polystyrol tube</td><td>Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Scissor</td><td>Word Precision Instruments, Inc., Sarasota, USA</td><td></td><td></td></tr><tr><td>Scale</td><td>Mettler PM4800 Delta Range, Mettler-Toledo GmbH, Gie&amp;szlig;en, Germany</td><td></td><td></td></tr><tr><td>Suction unit</td><td>Integra bioscience, Fernwald, Germany</td><td></td><td>Vacusafe comfort</td></tr><tr><td>Surgical scissors</td><td>Word Precision Instruments, Inc., Sarasota, USA</td><td></td><td></td></tr><tr><td>Trypan blue solution 0,4 %</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Tubes with cap</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td>15ml, 50ml Cellstar</td></tr><tr><td>Xylazine (2 %)</td><td>Ceva Tiergesundheit GmbH, D&amp;uuml;sseldorf, Germany</td><td></td><td></td></tr></tbody></table>

intravital microscopy of monocyte homing and tumor-related angiogenesis in a murine model of peripheral arterial disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Intravital microscopy for the examination of tumor and collateral vessel growth triggered by monocytes can help reveal new aspects in the molecular mechanisms of tumor angiogenesis and arteriogenesis. Cells must be prepared and injected carefully using the steps of the protocol. Differences can lead to variations between single experiments. The monocytes must be injected into the venous system (Figure 1) to maintain systemic effects and avoid emboli, which ca...

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

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.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

intravital-microscopy-monocyte-homing-tumor-related-angiogenesis

Research

JoVE Journal

Medicine

7.1K Views.  University of Magdeburg. Monocyten zijn belangrijke bemiddelaars/mediators van arteriogenesis in het kader van perifere arteriële ziekte. Met behulp van een kelder membraan-achtige matrix en intravital microscopie, onderzoekt dit protocol monocyt homing en tumor-gerelateerde angiogenese na monocyt injectie in de femorale slagader afbinding lymfkliertest model.

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

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Cancer Research

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

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

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

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

Immunology and Infection

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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 ...

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.

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 ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

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

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

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.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

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

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