Murine Bilateral Renal Lymphadenectomy

Summary

This protocol describes a survival procedure for safe and well-tolerated murine bilateral renal lymphadenectomy.

Abstract

Autoimmune diseases are the result of immune cells inappropriately targeting self-tissue, leading to pathology and damage to vital organs. Secondary lymphoid organs serve as a site for B and T cell activation and peripheral immune tolerance, consequent to antigen drainage and antigen-presenting cell (APC) trafficking through local lymphatics. The role of local draining lymph nodes as the initial site of lymphocyte activation has been described in settings of tumors, infection, and organ-specific autoimmune diseases, but not in systemic autoimmunity. Systemic lupus erythematosus, one such disease, is a widespread autoimmune disease with common kidney involvement leading to kidney injury and eventual failure. Further understanding of the role of renal lymph nodes, which are the primary site of antigen draining and APC trafficking from the kidneys, is limited by an inability to selectively perturb this tissue. Described here is a survival procedure for safe and well-tolerated murine bilateral renal lymphadenectomy. Included are detailed instructions, visual aids, helpful tips for improved success, and troubleshooting procedural pitfalls. Long-term survival is excellent and has been observed for at least 4 months postoperatively. Resected renal lymph nodes are intact and immediately ready for downstream processing. This procedure is best suited for studies of the local effect of renal lymph nodes.

Introduction

Autoimmune diseases span every organ in the human body from relatively organ-restricted diseases like autoimmune hepatitis, uveitis, and type 1 diabetes to widespread diseases like systemic sclerosis and sjögrens^1,2,3,4,5. Immunologic tolerance is a critical stage in B and T cell development to prevent self-reactivity⁶. While central tolerance via clonal deletion in the thymus is necessary and effective, this alone leaves gaps in the defense against autoimmunity

Protocol

All procedures are performed in compliance with Yale Institutional Animal Care and Use Committee protocols. To develop this procedure, 8-12-week-old Balb/c strain, Lupus-prone mice (MRL/MpJ-Fas^lpr/J /J) and age/sex/genotype matched sham controls were utilized¹⁵. In addition, 8-12-week-old wild type C57BL/6 strain mice were also tested, and nodes were safely resected. Both male and female mice were included. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of instruments, animals, and the operating room

1. Sterilize all surgi....

Results

This procedure can be performed in 15-20 min per mouse. To confirm the accuracy of this procedure, bilateral renal lymphadenectomies were performed on five 8-12 week-old, age, sex, and genotype-matched littermates. All mice tolerated the procedure well with 100% perioperative survival. Excised samples were placed in PBS on ice. All samples immediately sunk, confirming low adipose content. Excised samples (and four separate visceral adipose controls) were then crushed through a 100 µm cell strainer and washed with PBS. Ce.......

Discussion

Described here is a survival procedure for safe and well-tolerated murine bilateral renal lymphadenectomy. The major limitation of this procedure is the relatively small size of renal lymph nodes, necessitating an operating microscope. Without an operating microscope, confident resection can be difficult to achieve. Some genetic mouse models have enlarged lymph nodes, making identification and technique learning easier¹⁷. In otherwise healthy, wild-type mice, lymph nodes can be small and difficult.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthesia circuit	Any		Or injectable anesthetic
BD LSRII flow cytometer (or equivalent)	BD Biosciences		Equipped with FACS Diva Software
Buprenorphine XR	Any		For analgesia
Cell culture media	Any		(for live cell techniques; ie. DMEM or RPMI)
DAPI	Biolegend	422801	Working concentration: 15 ug/mL
Dual/twin gooseneck microscope LED lights	Any
Ethanol (70% in water)	Any		For cleaning
FITC-conjugated anti-mouse CD45.2 (104)	Biolegend	109806	Working concentration: 5 ug/mL
Forceps: Angled fine-tipped forceps (x2)	Roboz	RS-5058	Material: Dumoxel; Pattern #5, 45 degree angle; tip size 0.1 X 0.06mm
Forceps: Fine-tipped forceps	Roboz	RS-4960	Material: Dumostar; Pattern #1; Tip size: 0.20 X 0.12mm
Forceps: Fine-tipped forceps	Roboz	RS-4966	Material: Dumostar, Biologie tip; Pattern #3; Tip size: 0.08 x0.04mm
Lidocaine	Any		For analgesia
Mice	Jax		For C57BL/6 strain, wild type mice: The jackson laboratories #000664
Needle drivers	Roboz	RS-6410	Castroviejo needle holder; Straight
Operating microscope	Leica	MZ9.5	Leica Mz9.5 stereo head operating microscope
OPTIONAL: Evans Blue dye	Sigma	E2129-10G	For lymph tracing.
Phosphate-buffered saline (PBS)	Any
Povidone/Iodine solution	Any		For skin preparation/sterilization
Scissors: Extra fine-tipped scissors	Roboz	RS-5605	McPherson-Vannas; 3mm cutting edge; curved
Scissors: Medium scissors	Roboz	RS-5676	Noyes; 13mm cutting edge; straight
Surgical glue	Any		e.g. 3M Vetbond tissue adhesive
Suture: 6-0 blue monofilament polypropylene	Any
Tissue fixative	Any		(for planned histology; ie. 10% neutral buffered formalin or 4% paraformaldehyde)
Warm-water circulating heating pad	Any
Mice	The Jackson laboratories		For Balb/c strain, Lupus prone mice (MRL/MpJ-Fas-lpr/J): #000485