Anesth Analg 2005;100:1220-1221
© 2005 International Anesthesia Research Society
LETTER TO THE EDITOR
Leukocytes with Bright Fluorescence in Rats
John K. Hayes, PhD,
Dmytro M. Havaleshko, MD,
Roman V. Plachinta, MD, and
George F. Rich, MD, PhD
Department of Anesthesiology; University of Virginia Health System; Charlottesville, VA; jkh2n{at}virginia.edu
In Response:
We thank Sato et al. for their interest in our article (1) and for inquiring into the method we used to visualize leukocytes in our study. Our experimental preparation used the mesentery of the rat. This established model has been used for years by other researchers (2,3) and is well suited for intravital microscopy because it is readily accessible, easily surgically isolated, thin (<200 µm thick), and transparent, thus allowing for direct transillumination of vessels and cellular morphotic elements.
In the present study (1), we used an upright epifluorescence microscope (Olympus BX51) with water immersion lens (magnification 20x or 40x; numerical aperture = 0.5 or 0.8, respectively) operating in bright field mode. Due to the exceptional clarity of the morphotic elements within the mesenteric microcirculation, no fluorescent dye was needed in our study to observe and quantitate leukocyte-endothelial interactions (adhesion and leukocyte rolling velocities).
Leukocytes, as mentioned in the first paragraph of our study (1), is a general term that we and others (4) use to describe polymorphoneutrophils in in vivo flow conditions. Most leukocytes that we observed in the microvessels of our rats were probably granulocytes (5,6). However, the distinction between granulocytes and mononuclear cells could not readily be made during real-time flow conditions when employing intravital microscopy; hence we use the general term leukocyte. The off-line analysis system we used to analyze the number of adherent leukocytes, and the velocity of rolling leukocytes as they adhered to or traveled along the endothelium of a postcapillary venule from videotape, generally allowed us to differentiate granulocytes from the smaller lymphocytes (Fig. 1).
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Figure 1. Bright-field transillumination of rat mesentery venule showing granulocyte and smaller lymphocyte.
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However, on occasions when rolling velocities of a specific cell type is required, or when tracking the movement of leukocytes in tissues or organs is not amiable to transillumination, for example, in parenchymatous or opaque organs of the body such as in the liver, brain and lung, then other techniques, such as using fluorescent markers, may be required to visualize leukocyte dynamics. As pointed out by Sato et al., some fluorescent dyes may induce phototoxic effects especially in leukocytes (7). To avoid these potential problems, many researchers, including ourselves (Fig. 2), use transgenic animal models that express green fluorescent protein (GFP) in certain cell lines, such as the murine lysozyme M GFP mouse (8). These mice express GFP in their mature neutrophils, while other cell lineages are fluorescent negative.
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Figure 2. Epifluorescence illumination of lys-M GFP mouse showing GFP labeled neutrophils in a cremasteric venule.
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References
- Hayes JK, Havaleshko DM, Plachinta RV, Rich GF. Isoflurane pretreatment supports hemodynamics and leukocyte rolling velocities in rat mesentery during lipopolysaccharide-induced inflammation. Anesth Analg 2004;98:999–1006.[Abstract/Free Full Text]
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- Woodman RC, Teoh D, Payne D, Kubes P. Thrombin and leukocyte recruitment in endotoxemia. Am J Physiol Heart Circ Physiol 2000;279:H1338–45.[Abstract/Free Full Text]
- Atherton A, Born GVR. Quantitative investigations of the adhesiveness of circulating polymorphonuclear leukocytes to blood vessels. J Physiol (Lond) 1972;222:447–74.[Abstract/Free Full Text]
- Fiebig EK, Ley K, Arfors KE. Rapid leukocyte accumulation by "spontaneous" rolling and adhesion in the exteriorized rabbit mesentery. Int J Microcirc Clin Exp 1991;10:127–44.[ISI][Medline]
- Saetzler RK, Jallo J, Lehr HA, et al. Intravital fluorescence microscopy impact of light-induced phototoxicity on adhesion of fluorescently labeled leukocytes. J Histochem Cytochem 1997;45:505–13.[Abstract/Free Full Text]
- Faust N, Varas F, Kelly LM, Heck S, Graf T. Insertion of enhanced green fluorescent protein into the lysozome gene creates mice with green fluorescent granulocytes and macrophages. Blood 2000;96:719–26.[Abstract/Free Full Text]
