Anesth Analg 2005;100:1219-1220
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000149018.17929.9D
LETTER TO THE EDITOR
Leukocytes with Bright Fluorescence in Rats
Aki Sato, MD*
,
Yuki Sato, MD*,
Yukio Sato, MD, PhD
,
Yasunori Sohara, MD, PhD,
Norimasa Seo, MD, PhD, and
Eiji Kobayashi, MD, PhD
*The first two authors contributed equally to this work. Department of Anesthesiology; Division of Thoracic Surgery, Department of Surgery; Division of Organ Replacement Research; Animal Resource Project; Center for Molecular Medicine; Jichi Medical School; Tochigi, Japan; eijikoba{at}jichi.ac.jp
To the Editor:
We have a great interest in the recent article by Hayes et al. (1). Because leukocytes act as a major role for the tissue/organ damages, there has been much focus on in vivo visualization of these circulatory leukocytes. Intravital fluorescent microscopy has been established as a versatile technique for the study of cell-cell interaction and blood flow at the level of the microcirculatory unit. While Hayes et al. (1) did not mention how they labeled and detected circulating leukocytes, fluorescent markers such as acridine orange and rhodamine 6G are usually used for direct visualization of leukocytes during these processes (2). However, there are some pitfalls of these labeling methodologies. It has been mentioned that these fluorescent dyes often induce phototoxic effects and arteriolar vasospasm at high concentration levels (2). These dyes are taken up predominantly by leukocytes, but their fluorescent intensity is transient and quickly taken up by hepatocytes 10–20 minutes after IV injection (data not shown). Therefore, this labeling technique does not permit stable identification of individual cell types during observation.
To address this issue, we have established a new method for in vivo visualization of leukocyte trafficking using our ubiquitously expressed GFP transgenic (Tg) rats (3). Characteristically, this Tg rat has two peaks of strong GFP intensity in the mononuclear cells and GFP positive granulocytes. Almost all of the granulocytes are detected separately from lymphocytes according to their size and fluorescent intensities (4). Thus, using fluorescent microscopy and charge-coupled-device (CCD) camera, we could visualize the trafficking of GFP-positive leukocytes in the lung and liver in vivo (Fig. 1) (see supplemental Figure 1 as a video file available at anesthesia-analgesia.org).
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Figure 1. Successful visualization of GFP positive leukocytes in the liver of a GFP Tg rat using fluorescent microscopy with CCD camera. Arrowhead = leukocytes with bright GFP.
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Considering the results of our study, we would like to ask how the authors elegantly visualized the activated leukocytes to monitor their dynamics in postcapillary venules using the epifluorescence microscope (1).
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]
- 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]
- Hakamata Y, Tahara K, Uchida H, et al. Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophys Res Commun 2001;286:779–85.[Web of Science][Medline]
- Sakuma Y, Sato Y, Inoue S, et al. Lympho-myeloid chimerism achieved by spleen graft of green fluorescent protein transgenic rat in a combined pancreas transplantation model (TI03–029). Transpl Immunol 2004;12:115–22.[Medline]
