Name: intravital microscopy of monocyte homing and tumor-related angiogenesis in a murine model of peripheral arterial disease
Uploaded: 2017-08-26T13:00:00
Description: Watch this Scientific Journal Video about Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease at JoVE.com

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 microsc...

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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>. 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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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">10% fetal calf serum (FCS)</td>
			<td style="width:495px;">Sigma Aldrich, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% penicillin/streptomycin</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1mL Omnifix -F insuline syringe</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml syringe&#160;</td>
			<td>Fresenius Kabi AG, Bad Homburg, Germany</td>
			<td style="width:179px;"></td>
			<td>Injectomat- syringe 50 ml with canule</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well-ultra-low-attachement-plates</td>
			<td>Corning Incorporated, NY, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8- 12 week old, male, C57BL/6, BalbC mice&#160;</td>
			<td>Charles River, Sulzfeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive tape</td>
			<td>TESA SE, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition Software</td>
			<td>Leica, Wetzlar, Deutschland&#160;</td>
			<td></td>
			<td>Leica Application Suite Advanced Fluorescence (LAS AF); Version: 2.7.3.9723</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Canules</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td style="width:179px;"></td>
			<td>29G, 30G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dish</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture medium</td>
			<td>Manufactured by our group with single components</td>
			<td style="width:179px;"></td>
			<td>Medium199, 10% Fetal calf serum, 1% Antibiotic (penicillin/streptomycin)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Beckman Coulter GmbH, Krefeld, Germany</td>
			<td style="width:179px;"></td>
			<td>Allegra X-15R centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Depilatory cream</td>
			<td>Veet, Mannheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiO</td>
			<td>Invitrogen Eugene, Oregon, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disinfection agent</td>
			<td>Sch&#252;lke&#38;Mayr GmbH, Norderstedt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable scalpel No.10&#160;</td>
			<td>Feather safety razor Co.Ltd, Osaka, Japan&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erlenmeyer flask</td>
			<td>GVB, Herzogenrath, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 70%</td>
			<td>Otto Fischar GmbH und Co KG, Saarbr&#252;cken, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Forceps</td>
			<td>Rubis, Stabio, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flurophor/Rhodamindextran</td>
			<td>Thermo Fischer Scientific, Waltham, MA USA</td>
			<td>Katalognummer: D-1819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gloves</td>
			<td>R&#246;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad&#160;</td>
			<td>Labotect GmbH, G&#246;ttingen, Germany&#160;</td>
			<td style="width:179px;"></td>
			<td>Hot Plate 062</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human macrophage-colony stimulating factor</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td>SRP3110&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Humane leucocyte filters</td>
			<td>Blood preservation</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Baxter Deutschland GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine (10%)</td>
			<td>Ketavet, Pfizer Deutschland GmbH, Berlin , Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leukocyte separation tubes (tubes with filter)&#160;</td>
			<td>Bio one GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope&#160;</td>
			<td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td>
			<td style="width:179px;"></td>
			<td>Axiovert 40 C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lymphocyte separation medium LSM1077</td>
			<td>GE Healthcare, Pasching, Austria</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel&#160;</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium M199&#160;</td>
			<td>PAA Laboratories GmbH, Pasching, Austria</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microbiological work bench</td>
			<td>Thermo Electron, LED GmbH, Langenselbold, Germany</td>
			<td style="width:179px;"></td>
			<td>Hera safe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope slide</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe</td>
			<td>Art. Nr. 1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope stand with incubator and heating unit</td>
			<td>&#160;Leica DMI 6000, Pecon, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monocyte wash buffer</td>
			<td>Manufactured by our group with single components</td>
			<td style="width:179px;"></td>
			<td>PBS, 0,5% BSA, 2mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse restrainer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-photon microscope&#160;</td>
			<td>Leica, Wetzlar, Deutschland&#160;</td>
			<td></td>
			<td>Leica SP5 Confocal microscope, Cameleon, Coherent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (0,9%)</td>
			<td>Berlin Chemie AG, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauer counting chamber&#160;</td>
			<td>Paul Marienfeld GmbH und Co.KG, Lauda-K&#246;nigshofen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective</td>
			<td>Leica, Wetzlar, Deutschland&#160;</td>
			<td></td>
			<td>Leica HC PL APO 10x/0.4 CS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td style="width:179px;"></td>
			<td>ph 7,4 sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll</td>
			<td>Manufactured by our group with single components</td>
			<td style="width:179px;"></td>
			<td>90 % Percoll, 10% 1,5M NaCl, &#961;= 1,064 g cm-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll solution</td>
			<td>GE Healthcare, Bio-Science AB, Uppsala, Sweden</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td>10&#181;L/100&#181;L/200&#181;L/1000&#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes serological</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td>&#160;Cellstar2ml, 5ml, 10ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetting heads</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetus</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polystyrol tube</td>
			<td>Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissor</td>
			<td>Word Precision Instruments, Inc., Sarasota, USA</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scale</td>
			<td>Mettler PM4800 Delta Range, Mettler-Toledo GmbH, Gie&#223;en, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction unit</td>
			<td>Integra bioscience, Fernwald, Germany</td>
			<td style="width:179px;"></td>
			<td>Vacusafe comfort</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Word Precision Instruments, Inc., Sarasota, USA</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue solution 0,4 %</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubes with cap</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td>15ml, 50ml Cellstar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (2 %)</td>
			<td>Ceva Tiergesundheit GmbH, D&#252;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 ...

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