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 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.
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.
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. (§ 8, paragraph 1 of the German law for animal protection from 18.05.2016 – BGBI. I S. 1206, 1313, § 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: "Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes" by Wagner et al.4
NOTE: When working with the cells all steps must be sterile to avoid contamination.
2. Anesthesia
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 °C for the injection. At this temperature, the matrix is fluid; the gel hardens to a solid at body temperature (37 °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.
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 µL/g of bodyweight.
5. Intravital Microscopy
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 can occur if the injection is conducted in the arterial system.
If basement membrane-like matrix is used, injecting slowly to avoid dispersion will help with plug explantation for further histological examination (Figure 2, Figure 3). After sacrificing the mouse, the basement membrane-like matrix plug will be explanted from the flank of the mouse. Within the basement membrane-like matrix plug, we can measure vascularization by counting capillaries within different experimental settings (Figure 4, Figure 5).
Another condition for successful experiments is the microscope setting, which depends on software, hardware, and animal preparation (Figure 6). If subcellular structures (<2 µm) need to be identified, an upright microscope with 2-Photon-excitation and water immersion objectives (20X or 25X, numeral aperture 1.0) with long working distance (>2 mm) are advisable. Since water immersion objectives with high numeral aperture are extremely sensitive to refractive index variations, the optimal image resolution and brightness must be adjusted by moving a correction collar at the objective. Unfortunately, adjustment by hand is quite difficult due to limited space. Therefore, expensive objectives with a motorized collar correction are offered by microscope manufacturers.
If cellular resolution (5 – 10 µm) is sufficient, a 10x dry lens with a numeral aperture 0.4 or higher and a long working distance (>2 mm) is recommended. In this case, an upright or an inverted microscope stand can be selected. Live cell imaging with a 10X dry lens (numeral aperture 0.4) at higher zoom factors (>3) is much easier and cheaper because classical confocal laser scanning microscopy (i.e., 1 Photon excitation) can be used to obtain image stacks with sufficient resolution. If much time is required for the acquisition of images, the amount of rhodamine dextran within the vessel decreases. For a better visibility of the vessels, the injection of the fluorophore must be repeated (Figure 7).
It is most effective to sample different adjustments and decide the best image quality possible. The use of positive probes for cells or basement membrane-like matrix (without transplantation) can help obtain optimal settings. We could detect labeled monocytes via intravital microcopy within the blood flow and muscle tissue (Figure 8, Figure 9). Histological examination of tissue sections verifies our findings (Figure 10).
Figure 1: Injection of 2.5 million monocytes into the tail vein. Veins are located on the lateral side of the tail, with arteries on the dorsal and ventral side. Please click here to view a larger version of this figure.
Figure 2: Injection of basement membrane-like matrix. Handle the skin of the mouse and inject the basement membrane-like matrix in the flank. Please click here to view a larger version of this figure.
Figure 3: Explanted matrix plug from the flank of the mouse (see point 3.5). Incorporated vessels five days after monocyte transplantation via the tail vein. Please click here to view a larger version of this figure.
Figure 4: Comparison of vascularization within the basement membrane-like matrix ( + SD, n = 3 in each group). Vessels within the basement membrane-like matrix plug, with no growth factor added (red bar), compared to vessels of basement membrane-like matrix plug with growth factor added. The supplementation of basement membrane-like matrix with growth factor leads to increased vessel growth. Please click here to view a larger version of this figure.
Figure 5: Vessels within the basement membrane-like matrix plug. Visualization of blood flow (red) within vessels (arrows) and monocyte homing (green) within the basement membrane-like matrix plug. Please click here to view a larger version of this figure.
Figure 6: Mouse prepared for image acquisition. The paws are fixed with adhesive tape and a cover glass is positioned on the top of two adjustable stamps. The region of interest was excised with a scalpel. NaCl is used to moisten the area so the quality of images and tissue will not be compromised. Please click here to view a larger version of this figure.
Figure 7: Decreased visibility of vessels. 3,3'-Dioctadecyloxacarbocyanine perchlorate stained monocytes (arrows) within basement membrane-like matrix next to a vessel (longitudinal section, *). Please click here to view a larger version of this figure.
Figure 8: Monocyte flushed into vessel. In vivo visualization of 3,3'-Dioctadecyloxacarbocyanine perchlorate stained and transplanted monocyte (green, arrow) within the collateral artery (*). Please click here to view a larger version of this figure.
Figure 9: Intravital microscopy. 3,3'-Dioctadecyloxacarbocyanine perchlorate stained monocytes (arrows) within collateral vessels with typical corkscrew formation (*) stained with rhodamine dextran. Please click here to view a larger version of this figure.
Figure 10: Immunohistological staining of thigh musculature. Vessels (red: alpha smooth muscle actin, *), macrophages (green: 3,3'-Dioctadecyloxacarbocyanine perchlorate, arrows), cell nucleus (blue). Please click here to view a larger version of this figure.
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 the devices.
There are other difficulties connected with this protocol that can be avoided with practice. In the beginning, there can be problems with positioning the mouse under the microscope, and image quality can suffer under these circumstances. Another critical point is the tail vein injection. Monocytes can only be seen in the veins if injected properly. Therefore, it is advisable to practice the injection before positioning the mouse.
Monocyte isolation is also critical. Monocytes can be isolated from different species using multiple protocols, which often lead to various results and cell yields12,13,14. It is necessary to work in sterile conditions to avoid contamination. Cell damage should be prevented by pipetting carefully and maintaining constant temperatures.
Despite these disadvantages, this method is practical and easy to perform, enabling users to shed light on tumor angiogenesis and the basic mechanisms behind peripheral arterial disease.
The authors have nothing to disclose.
This work was supported by the ELSE-Kröner-Stiftung and the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center). Special thanks to Hans-Holger Gärtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.
10% fetal calf serum (FCS) | Sigma Aldrich, Hamburg, Germany | ||
1% penicillin/streptomycin | Sigma Aldrich, Hamburg, Germany | ||
1mL Omnifix -F insuline syringe | B. Braun, Melsungen AG, Melsungen, Germany | ||
50 ml syringe | Fresenius Kabi AG, Bad Homburg, Germany | Injectomat- syringe 50 ml with canule | |
6-well-ultra-low-attachement-plates | Corning Incorporated, NY, USA | ||
8- 12 week old, male, C57BL/6, BalbC mice | Charles River, Sulzfeld, Germany | ||
Adhesive tape | TESA SE, Hamburg, Germany | ||
Acquisition Software | Leica, Wetzlar, Deutschland | Leica Application Suite Advanced Fluorescence (LAS AF); Version: 2.7.3.9723 | |
Canules | B. Braun, Melsungen AG, Melsungen, Germany | 29G, 30G | |
Cell culture dish | Greiner Bio-One GmbH, Frickenhausen, Germany | ||
Cell culture medium | Manufactured by our group with single components | Medium199, 10% Fetal calf serum, 1% Antibiotic (penicillin/streptomycin) | |
Centrifuge | Beckman Coulter GmbH, Krefeld, Germany | Allegra X-15R centrifuge | |
Depilatory cream | Veet, Mannheim, Germany | ||
DiO | Invitrogen Eugene, Oregon, USA | ||
Disinfection agent | Schülke&Mayr GmbH, Norderstedt, Germany | ||
Disposable scalpel No.10 | Feather safety razor Co.Ltd, Osaka, Japan | ||
EDTA | Sigma Aldrich, Hamburg, Germany | ||
Erlenmeyer flask | GVB, Herzogenrath, Germany | ||
Ethanol 70% | Otto Fischar GmbH und Co KG, Saarbrücken, Germany | ||
Fetal Calf Serum | Sigma Aldrich, Hamburg, Germany | ||
Fine Forceps | Rubis, Stabio, Switzerland | ||
Flurophor/Rhodamindextran | Thermo Fischer Scientific, Waltham, MA USA | Katalognummer: D-1819 | |
Gloves | Rösner-Matby Meditrade GmbH, Kiefersfelden, Germany | ||
Heating pad | Labotect GmbH, Göttingen, Germany | Hot Plate 062 | |
Human macrophage-colony stimulating factor | Sigma Aldrich, Hamburg, Germany | SRP3110 | |
Humane leucocyte filters | Blood preservation | ||
Incubator | Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany | ||
Isoflurane | Baxter Deutschland GmbH, Unterschleißheim, Germany | ||
Ketamine (10%) | Ketavet, Pfizer Deutschland GmbH, Berlin , Germany | ||
Leukocyte separation tubes (tubes with filter) | Bio one GmbH, Frickenhausen, Germany | ||
Light microscope | Carl Zeiss SMT GmbH, Oberkochen, Germany | Axiovert 40 C | |
Lymphocyte separation medium LSM1077 | GE Healthcare, Pasching, Austria | ||
Matrigel | Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA | ||
Medium M199 | PAA Laboratories GmbH, Pasching, Austria | ||
Microbiological work bench | Thermo Electron, LED GmbH, Langenselbold, Germany | Hera safe | |
Microscope slide | Carl Roth GmbH + Co. KG, Karlsruhe | Art. Nr. 1879 | |
Microscope stand with incubator and heating unit | Leica DMI 6000, Pecon, Germany | ||
Monocyte wash buffer | Manufactured by our group with single components | PBS, 0,5% BSA, 2mM EDTA | |
Mouse restrainer | Various | ||
Multi-photon microscope | Leica, Wetzlar, Deutschland | Leica SP5 Confocal microscope, Cameleon, Coherent | |
NaCl (0,9%) | Berlin Chemie AG, Berlin, Germany | ||
Neubauer counting chamber | Paul Marienfeld GmbH und Co.KG, Lauda-Königshofen, Germany | ||
Objective | Leica, Wetzlar, Deutschland | Leica HC PL APO 10x/0.4 CS | |
PBS | Life technologies GmbH, Darmstadt, Germany | ph 7,4 sterile | |
Penicillin/Streptomycin | Sigma Aldrich, Hamburg, Germany | ||
Percoll | Manufactured by our group with single components | 90 % Percoll, 10% 1,5M NaCl, ρ= 1,064 g cm-3 | |
Percoll solution | GE Healthcare, Bio-Science AB, Uppsala, Sweden | ||
Pipettes | Eppendorf AG, Hamburg, Germany | 10µL/100µL/200µL/1000µL | |
Pipettes serological | Greiner Bio-One GmbH, Frickenhausen, Germany | Cellstar2ml, 5ml, 10ml | |
Pipetting heads | Eppendorf AG, Hamburg, Germany | ||
Pipetus | Eppendorf AG, Hamburg, Germany | ||
Polystyrol tube | Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany | ||
Scissor | Word Precision Instruments, Inc., Sarasota, USA | ||
Scale | Mettler PM4800 Delta Range, Mettler-Toledo GmbH, Gießen, Germany | ||
Suction unit | Integra bioscience, Fernwald, Germany | Vacusafe comfort | |
Surgical scissors | Word Precision Instruments, Inc., Sarasota, USA | ||
Trypan blue solution 0,4 % | Sigma Aldrich, Hamburg, Germany | ||
Tubes with cap | Greiner Bio-One GmbH, Frickenhausen, Germany | 15ml, 50ml Cellstar | |
Xylazine (2 %) | Ceva Tiergesundheit GmbH, Düsseldorf, Germany |