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 ...

Hello, my name is Christian Deffge. I'm a medical doctor at university hospital in Magdeburg and I'm working in the cardiovascular research group of Dr.Herold and Professor Braun-Dullaeus. We investigate the augmentation of collateral growth.Therefore, we use imaging. In this video, we want to explain our methods. Resuspend the cells in serum-free culture medium with a density of one times 10 to the sixth cells per milliliter.Add five microliters of one millimolar DIO and dimethylformamide to one milliliter of the cell suspension and resuspend carefully. Incubate the cell solution at 37 degrees Centigrade for 20 minutes. Centrifugate the cells at 500 g for five minutes.Dislodge the supernatant and resuspend the cells with 37 degrees Centigrade warm FCS supplemented medium. Repeat this step twice. Count the cells with the help of a Neubauer counting chamber and light microscopy.Resuspend the cells with sodium chloride solution. Inject the cells into the tail vein. Matrigel matrix is a reconstituted basement membrane preparation extracted from the squamous sarcoma.It screens test molecules for either endothelial cell network formation or anti-cancer therapies through angiogenic inhibition. Reassess the tumor angiogenic potential of monocytes we strive to use for cell therapy. Add 100 nanogram BFGF, 300 nanogram VEGFA and 26 international units heparin under sterile conditions to the Matrigel.After vortexing the solution, fill it up into one milliliter insulin syringe and store on ice until usage. For better visibility of the subcutaneous Matrigel plaque, shave the skin of the mouse at the injection site. You can use a sharp scalpel or depilatory cream.Lay the animal on the table and hold the skin of the mouse next to the injection site on the flank. Inject 500 microliters of Matrigel subcutaneously. For practical reasons, it is necessary to inject the Matrigel compactly at one location to avoid subcutaneous dispersion.It will be easier to dislodge the Matrigel matrix from the tissue after sacrificing the mouse at the end of the experiment. Practice the monocyte injection with sodium chloride solution before experimentation. If the monocytes cannot be applied systemically, there will be no systemic effect.Within this protocol, we injected 2.5 million monocytes. Try to inject no more than five microliters per gram. Make sure the mouse is warmed with the help of a heating pad while the whole procedure.For systemic application of the cells, you will need a restrainer and one milliliter insulin syringe, a 30 g needle and the monocytes resuspended and sodium chloride solution. Carefully handle the mouse and restrain the animal in the restrainer. Make sure the mouse is not harmed and has adequate space for breathing.Put the restrainer on the heating pad so that the tail can contact the plate. Make sure you disinfect the injection site to avoid an infection. Identify the tail veins which are located on the lateral side of the tail.Turn the tail 90 degrees so the tail vein appears on the upper side of the tail. Try to inject the monocyte solution in a flat angle. Observe the animal for 30 minutes to look for systemic side effects.Place the mouse in its cage after the animal has fully recovered. Place the anesthetized mouse on the prewarmed microscope stage after the microscope has reached stable conditions and the settings have been tested by a dummy. Fix the paws with adhesive tape.Disinfect the skin at the site of the leg or flank that is used for microscopy. Shave the region of interest for better handling and to avoid interference with hair. Keep the region of interest moist.Otherwise, the quality of images and tissue will be compromised. Place the leg between two adjustable stems. Position a cover glass on top of the stems.Start the microscopy and adjust the position of the leg if needed. Microscope settings depend on the software that is used. Please see the protocol for further information.Our data demonstrates that intravital microscopy represents a potent tool for visualization of transplanted monocytes within the blood flow in small animal models. The monocytes must be injected into the venous system to maintain systemic effects and to avoid emboli which can occur if the injection is conducted in the arterial system. If Matrigel is used, injecting slowly to avoid dispersion will help with plaque explantation for further histological examination.Within the Matrigel plaque, we are able to measure vascularization by counting capillaries within different experimental settings. Intravital microscopy findings can be verified by histological examination. We are able to find monocytes detected by intravital microscopy within all tissue sections.If much time is required for the acquisition of images, the amount of rhodamine dextran within the vessel decreases. For better visibility of the vessels, the application of the rhodamine dextran must be repeated. The use of positive probes for cells on Matrigel can help obtain optimal settings.The goal for peripheral arterial disease or ischemic heart disease therapeutics is to increase blood flow to other perfused areas caused by hemodynamic stenosis. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is a noninvasive option for patients without indications for surgery. In addition to the development of collateral arteries in ischemic areas, monocytes can also influence the growth of tumors.To investigate these processes, we use Matrigel injection in connection with intravital microscopy. Our aim with this protocol is to demonstrate an easy and efficient way to study immunological processes caused by ischemia in an in vivo model. With this new method, we are able to generate a more realistic test environment compared to histological workup of postmortem muscle tissue.

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Research

JoVE Journal

Medicine

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...