Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.
In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.
The purpose of the present article is to describe the technique of intravital microscopy (IVM) applied on the mouse cremaster microcirculation for direct observation and analysis of peripheral bone marrow stem cell migration.
The current concept of stem cell based tissue and organ regeneration involves the homing of bone marrow derived stem cells to the injured tissue1. A crucial step for successful stem cell migration includes stem cell interaction with the local endothelium within the injured organ followed by transendothelial migration and eventually organ engraftment2. Intravital analysis of these stem cell – endothelial cell interactions forms an ideal parameter for the quantification of a stem cell based regenerative response in different pathophysiological settings in vivo. Furthermore, it seems conceivable, that, in the future, intravital analysis of the migratory capacity of specific stem cell populations within standardized microcirculatory environments, might be applied prior advancing these populations to further clinical testing.
Intravital microscopy has been initially developed for the observation and quantification of leucocyte-endothelial cell interaction in vivo in the field of inflammatory research3. First successful recordings of intravital microscopy studies were reported by Cohnheim already in the 19th century, studying frogs' tongues and mesenteries under a light microscope4. Since its first use IVM has undergone a tremendous technical development and today certain essential components form prerequisites in order to perform quantitative IVM: (i) tissue preparations that permit optical access, (ii) molecular probes that can be detected by a microscope, (iii) a microscope connected to a detection system and (iv) computer based analysis systems that can extract parameters of interest from the image data set4.
A variety of tissue preparations has been introduced for IVM studies including the mesenteries and liver of the mouse and rat5, the dorsal skinfold chambers of mouse 6 and hamster, the rabbit ear and the hamster cheek pouch to name a few.
However, in the following we will focus on the mouse cremaster muscle, representing an ideal tissue for intravital observations, as preparation and visualization are possible by a well-standardized surgical procedure, and in general no problems of movement artifacts occur. The open cremaster muscle preparation was carried out for the first time in the 1970s by Baez and colleagues 7. Originally described for rats, it has been adopted successfully also to the mouse8. After previous studies had mainly focused on leucocyte interactions with the vessel wall, our own group recently introduced the mouse cremaster muscle preparation as a valuable tool for direct visualization and quantitative analysis of stem cell-endothelium interactions within a defined microenvironment9. Various stem cell subpopulations have been studied utilizing this model, including murine c-kit+ bone marrow stem cell and mesenchymal stem cells, as well as human CD 133+ bone marrow stem cells10-12. Following cell isolation from donor bone marrow and fluorescent labeling for visualization, the stem cells are selectively applied into the cremaster microcirculation utilizing an arterial injection via the femoral artery, thereby avoiding any cell entrapment within remote organs. Furthermore, the cremaster muscle model is particularly useful since various chemokines potentially mediating local stem cell migration within the respective settings, can be topically applied to the target tissue by simple superfusion technique.
The entire protocol after cell isolation takes approximately 2 hr.
1. Microsurgical Preparation
2. Intravital Microscopy
In general, interaction of directly injected stem or progenitor cells with the vascular endothelium within the cremaster muscle microcirculation is a rare event and occurs exclusively in postcapillary venules (diameter: 30-80 μm). Due to fluorescence labeling rolling and firmly adherent stem cells can be clearly quantified in separation from circulating endogenous leucocytes in the observed venules (Figure 2).
The microcirculatory conditions represented by the blood flow velocity and wall shear rate usually do not differ significantly between the respective experimental groups with different chemokine treatment (Table 1).
Local treatment of the cremaster muscle tissue with different chemokines or mediators of inflammatory response, e.g. SDF-1α (stromal cell-derived factor-1 alpha) or TNF-α (tumor necrosis factor-alpha) is capable to mimic specific microenvironmental conditions. Such conditions enhance stem cell-endothelial cell interactions in a different amount, depending on the specific chemokine/mediator applied and the stem cell population injected (Figure 3)12. The possibility to clearly quantify these differing patterns of stem cell-endothelial cell interactions enables for evaluation of the migratory potential of specific stem cell populations within defined microenvironments in vivo prior advancing these populations to further clinical testing.
Furthermore, the impact of certain pathophysiological conditions affecting the endothelial function, such as nitric-oxide deficiency, has been shown by the use of knockout animals9.
Figure 1. Illustration of the key microsurgical steps (a-f) in mouse cremaster muscle preparation for subsequent intravital microscopy.
Figure 2. Intravital microscopy of c-kit+ cells in a murine cremaster muscle preparation. Intravascular background is provided by rhodamin-dextran. (a-f) A series of six consecutive images with rolling (black arrows) and firmly adherent (white arrow) carboxy-fluorescein d-acetate succinimidylester- labeled c-kit+ cells in the venular vasculature. Lab Invest, 2008, Vol 88. Kaminski, A., Ma, N., Donndorf, P. et al.
Figure 3. Quantitative analysis of interactions between c-kit+ cell and endothelial cells based on intravital fluorescence microscopic images. (a) The number of rolling c-kit+ cells on venular endothelium (expressed in percentage of all passing cells) in murine cremaster muscles exposed to SDF-1α, TNF-α or a combination of both compared to c-kit+ cell injection without chemokine pretreatment (control). Treatment with SDF-1α alone and the combination of SDF-1α+TNF-α increased percentage of rolling c-kit+ cells. (b) The number of firmly adherent c-kit+ cells per mm2 of venular endothelial lining is significantly increased only after treatment with the combination of SDF-1α+TNF-α. Data are expressed as mean ± s.e.m. (*P<0.05 vs control). Lab Invest, 2008, Vol 88. Kaminski, A., Ma, N., Donndorf, P. et al.
Mouse Type | Experimental Group | red blood cell velocity (mm/sec) | wall shear rate (sec-1) |
Wild type | Control | 0.7±0.2 | 43±32 |
Wild type | SDF-1α | 0.5±0.2 | 51±31 |
Wild type | TNF | 0.4±0.2 | 45±24 |
Wild type | SDF-1 α +TNF α | 1.0±0.5 | 117±26 |
Table 1. Red blood cell velocities and wall shear rates in venules of murine cremaster muscles at baseline. Analysis was performed using CapImage Software (Zeintl, Heidelberg, Germany) in muscle preparations of wild-type mice after treatment with SDF-1α, TNF-α. Data are expressed as mean ± s.d. Differences between the individual groups were not found being significant. Lab Invest, 2008, Vol 88. Kaminski, A., Ma, N., Donndorf, P. et al.
Intravital fluorescence microscopy allows for direct visualization and quantitative analysis of stem cell-endothelium interactions. The cremaster muscle model is particularly useful, since microsurgical exposure and techniques intravital visualization have been well established during the research on leucocyte-endothelium interactions and various chemical components can be selectively applied to the target tissue by simple superfusion technique. Due to the absence of movement artifacts and severe image noise, this particular model, in comparison to other intravital-microscopy settings, provides optimal conditions for visualization of cell-cell interactions.
The model allows revealing essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism. If required, endogenous leucocytes can serve as positive controls for validation and prove of principle of stem cell behavior.
Representing an acute animal model and utilizing direct arterial injection instead of venous application, the potential limitations of chronic cell migration models and/or systemic cell delivery – e.g. cell loss due to entrapment within remote organs – can be avoided. On the other hand, acute surgical tissue trauma is likely to induce a certain degree of tissue activation itself. Therefore, an atraumatic surgical preparation is mandatory and stable microcirculatory conditions following cremaster muscle preparation need to be achieved in a reproducible fashion prior starting with experimental groups.
The presence of extensive leucocyte accumulation within the postcapillary venules and significant leakage of the fluorescent dye to the surrounding tissue at the beginning of the intravital microscopy recordings in control animals indicate major surgical tissue trauma.
However, even when applying meticulous preparation techniques, results derived from different investigators are not necessarily completely comparable due to slightly differing baseline tissue activation. This is why subsequent experiments should be performed by the same investigator whenever possible.
Designed for the analysis and quantification of the initial steps of stem cell recruitment to target tissues, the current model does not allow statements on definite stem cell organ-engraftment.
The authors have nothing to disclose.
Name of Reagent | Company | Catalog Number |
Carboxy-fluorescein diacetate succinimidylester (CFDA) | Invitrogen, Carlsbad, CA, USA | C1157 |
Rhodamine 6 G (1%) | Sigma-Aldrich, Munich, Germany | 83697 |
Ketamin (Ketanest) | Pfizer, Berlin, Germany | not available |
Xylacin (Rompun) | Bayer, Leverkusen, Germany | not available |
Dulbecco's Phosphate – buffered saline (PBS) | PAN Biotech, Aidenbach, Germany | P04-36500 |