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1Department of Pharmacology, University of Saskatchewan
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We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.
Xu, N., Lei, X., Liu, L. Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy. J. Vis. Exp. (55), e3296, doi:10.3791/3296 (2011).
The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.
1. Preparation of Chemoattractant in Agarose Gel
2. Preparation of Cremaster Muscle for Intravital Microscopy (Figure 1)
Note: All procedures from 2.6 to 2.14 must be performed very gently5.
3. Cell Tracking Using ImageJ
4. Analysis of Neutrophil Recruitment Parameters
5. Representative Results:
Although bightfield intravital microscopy is used for the study of leukocyte-endothelial cell interactions and may not be necessarily for neutrophils, we confirmed that, by our histology studies, more than 95% of the recruited cells in neutrophil chemoattractant-treated cremaster muscles were indeed neutrophils. In this report, using neutrophil-selective chemoattractants, we present procedures of tracking the recruitment of neutrophils in vivo. Specifically, we describe a protocol of tracking neutrophil intraluminal crawling, transendothelial migration, and chemotaxis in cremaster muscle tissue in anesthetized mice using time-lapse intravital video microscopy and ImageJ. The chemoattractant-containing agarose gel on the cremaster muscle slowly releases chemoattractant and allows a chemoattractant gradient to be established in tissue. Neutrophil chemoattractant induces neutrophil-endothelial cell interactions in cremasteric postcapillary venules in mice. The whole experiment is visualized under an upright brightfield intravital microscope with video images projected by a color video camera to a TV monitor and recorded by a video recorder. We determined the neutrophil intraluminal crawling, transendothelial migration and migration and chemotaxis in muscle tissue in response to neutrophil chemoattractant MIP-2 and WKYMVm prepared in agarose gel (Figure 2). We found that MIP-2 (at 0.5 µM) and WKYMVm (at 0.1 mM) elicited neutrophil intraluminal crawling at similar velocity, neutrophil transendothelial migration and detachment from the venule for comparable length of time, neutrophil migration and chemotaxis in muscle tissue at nearly the same velocity and with similar neutrophil chemotaxis indexes (P > 0.05, Student t test).
Figure 1. The schematic illustration of an intravital microscope system. The mouse cremaster muscle is exteriorized on the clear viewing pedestal of cremaster muscle board on microscope stage and superfused with 37°C-warmed bicarbonate-buffered saline. The upright microscope is connected with a CCD color video camera for brightfield intravital microscopy. A monochrome deep-cooled CCD digital camera is also connected to microscope port for fluorescence intravital microscopy, the images from which are directly processed by a computer.
Figure 2. Neutrophil recruitment parameters of brightfield intravital microscopy. Neutrophil recruitment was induced by the gradual release of neutrophil chemoattractant MIP-2 or WKYMVm in the agarose gel preparation placed 350 µm adjacent to the postcapillary venule. Time-lapsed video data were analyzed by ImageJ after processing the real time video recording of the experiment. Neutrophil intraluminal crawling (A), transmigration time and detachment time (B), migration velocity and chemotaxis velocity in tissue (C), and chemotaxis index in cremaster muscle (D) were determined after the administration of MIP-2 or WKYMVm agarose gel on cremaster muscle in C57BL/6 mice (n = 3, # of tracked cells = 22 (in A and B) and 27 (in C and D) respectively for MIP-2, and = 26 (in A and B) and 44 (in C and D) respectively for WKYMVm).
Intravital microscopy is the essential tool for revealing the cellular and molecular mechanisms of leukocyte recruitment during inflammation. Quantitative visualization for determination of leukocyte-endothelial cell interactions in microvasculature of transluscent tissues such as cremaster muscle and mesentery remains the gold standard for the application of the technique1,5. The conventional brightfield intravital microscopy has many unique technical features and the recruitment mechanisms revealed in these tissues are applicable to most tissues in vivo1,2,6. However, the mechanisms of leukocyte recruitment in some other tissues such as the lung, liver and the brain have been found quite different from those revealed in cremaster muscle and mesentery1. In addition, fluorescence microscopy has to be used in some less transparent tissues.
Compared with the fluorescence-based microscopy which is known for photodamage to the functions of live cells, brightfield intravital microscopy is more physiological and less harmful to the cells and tissues (therefore with less artifacts) when long-time imaging for observing dynamic cellular behaviors in live animals is necessary7. It is also more convenient, less costly and no fluorescent labeling is needed. On the other hand, with transillumination imaging in intravital microscopy, automatic tracking cellular movement is impossible with currently available commercial imaging software on the market. However, it is very easy to set up a separate fluorescence intravital imaging system (e.g., by projecting the images to a fluorescence CCD camera and computer) on the same brightfield intravital microscope (Figure 1). This provides the convenience of switching between brightfield microscopy and fluorescence imaging on the same sample preparation in one single experiment. This also makes it possible to automatically track the movement of fluorescently labeled cells under fluorescence intravital microscopy when the contrast between the fluorescence intensity of labeled cells and the background is large enough and suitable imaging software is installed on the computer.
As depicted here in our presentation, we demonstrate the value of this in vivo technique for direct observation of the whole process of neutrophil recruitment and for the determination of the functions of cells and molecules in each recruitment step. With chemoattractant contained in agarose gel preparation and held on the tissue, a unidirectional chemotactic gradient can be established in the tissue that induces leukocyte recruitment responses resembling those naturally occurring during local inflammation8,9. The directional leukocyte movement from postcapillary venule toward the source of chemoattractant can be clearly visualized by brightfield intravital microscopy and video photography. With time-lapse video processing, cell movement can be tracked by ImageJ and a series of highly reproducible parameters can be measured. With specific transgenic mice, inhibitors and selective chemoattractants, the assay system helps us to reveal the functions of specific proteins in leukocyte recruitment. For example, this technique assisted us to identify the role for LSP1 in endothelial cells as the gatekeeper in the regulation of neutrophil transendothelial migration10, the role for Mac-1 (αMβ2 integrin) in neutrophil intraluminal crawling, an essential step for optimal transendothelial migration3, and the role for PI3Kγ in neutrophil chemotaxis in tissue11.
No conflicts of interest declared.
This work was supported by a research grant from Canadian Institutes of Health Research (CIHR, MOP-86749). L. Liu is a recipient of CIHR New Investigator Award (MSH-95374).
|Polyethylene tubing, PE10||Becton Dickinson||427401||I.D. 0.28mm × O.D. 0.61mm|
|India ink||Speedball Art||Super Black||100% carbon black pigment, no dyes|
|Xylazine||Bayer HealthCare, Bayer Inc.||DIN 02169592|
|Ketamine hydrochloride||Bioniche Animal Health Canada, Inc.||DIN 01989529|
|Murine recombinant MIP-2||R&D Systems||452-M2|
|WKYMVm||Phoenix Pharmaceuticals, Inc.||072-12|
|3CCD color video camera||SONY||DXC-990|
|HD-DVD video recorder||LG Electronics Inc.||RH398H-M|
|TV monitor||LG Electronics Inc.||22LG30|
|Water circulator||Thermo Scientific||HAAKE DC10|
|Peristaltic pump||Gilson; Pharmacia||Gilson MINIPULS 3; Pharmacia P-3|
|Cremaster muscle board||University of Saskatchewan||Home-made|
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