Lymph node mapping in the mouse

doi:10.1016/j.jim.2007.11.012

Journal of Immunological Methods

Volume 332, Issues 1–2, 20 March 2008, Pages 170-174

https://doi.org/10.1016/j.jim.2007.11.012 Get rights and content

Abstract

Accurate identification of lymph nodes in the mouse is critical for studies of tumor metastasis, and of regional immune responses following immunization. However, these small lymphatic organs are often difficult to identify in mice using standard dissection techniques, so that larger rats have been used to characterize rodent lymphatic drainage. We developed techniques injecting dye into the mouse footpad or tail, to label the lymphatic drainage of the hind leg and flank, pelvic viscera, prostate and mammary glands. While lymphatic drainage patterns were similar in mice and rats, the inguinal lymph nodes showed distinct differences in afferent and efferent drainage. These techniques allow accurate and rapid identification of lymph nodes and lymphatic drainage in normal as well as diseased mice.

Introduction

The lymphatic system plays a crucial role in immune responses to foreign antigens and tumors, and in tumor metastasis in humans and rodent models (Saharinen et al., 2004). For example, foot-draining popliteal LNs were used to characterize the LN response to an acute inflammatory stimulus in the foot (Angeli et al., 2006). In addition, sentinel or tumor-draining LNs are used for the diagnosis of metastasis of a variety of cancers (Morton et al., 2003, Turner et al., 1997). Mice are widely used as the experimental model of choice for immunology and tumor biology experiments. However, the lymphatic drainage of different body regions has been extensively characterized only in rats (Hebel and Stromberg, 1976, Tilney, 1971), even though there could be species-specific differences in lymphatic drainage in mice and rats. While enlarged LNs can be readily identified in mice with acute inflammation or advanced cancer, the LNs of normal mice are small and difficult to distinguish from surrounding adipose and connective tissue. Historically, dyes such as colloidal India ink, or Evans Blue, a low molecular weight dye with high affinity for serum albumen, have been used to map lymphatic drainage in rodents. These dyes are of the correct diameter (less than 100 nm) to be preferentially drained via the lymphatics (Swartz, 2001). Here we report the development of two dye injection methods using Evans Blue dye to identify commonly studied LNs, and to characterize lymphatic drainage in the mouse.

Section snippets

Materials and methods

Male or female C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME) were used at 5 weeks to 1 year of age. Experimental methods involving animals were approved by the Fred Hutchinson Cancer Research Center Animal Care and Use Committee. Dye injections were performed with 1% or 5% Evans Blue dye (Sigma, St. Louis, MO) in 25 μL Hank's Buffered Salt Solution, delivered using an 0.5 ml syringe with a 27^1/2 g needle (Becton-Dickinson, Franklin Lakes, NJ).

Mice were anesthetized with 2.5%

Lymphatic drainage of the hind leg

We developed methods to identify the hind leg lymphatic drainage of mice, to facilitate our studies of lymph node responses to cancer. The popliteal LN drains the hind leg, as illustrated in Fig. 1A. Injection of 1% Evans Blue dye into the foot readily labeled this LN after subcutaneous foot injection, so that the LN was easily distinguished from the adipose and connective tissue of the popliteal fossa (Fig. 2A). Injection time was not critical, as euthanasia at any time after injection gave

Discussion

Murine lymph nodes are normally small and indistinct, which makes them difficult to reliably identify by gross dissection. The dye injection strategies we describe in this study allow confident identification of a number of major LNs in normal healthy mice, or in diseased mice. This study also characterized two major lymphatic drainage basins in the mouse (hindfoot and tail), which should be useful for local immune response or tumor metastasis studies.

The first dye injection approach of

Acknowledgments

We are most grateful to Katrina Elsaesser for drawing the illustrations, and Deborah Kwok, Momoko Furuya, and Karen Kelly-Spratt for the advice and assistance on surgical procedures. This work was supported by NIH grant NCI R01 CA 68328 to A. Ruddell.

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Technical noteLymph node mapping in the mouse

Abstract

Introduction

Section snippets

Materials and methods

Lymphatic drainage of the hind leg

Discussion

Acknowledgments

Immunity

American Journal of Pathology

American Journal of Pathology

Trends in Immunology

Advanced Drug Delivery Reviews

Technical note
Lymph node mapping in the mouse