Murine Superficial Lymph Node Surgery

Summary

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

Abstract

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells^1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x10⁶ cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others^5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

Protocol

1. Preparation Before The Surgery

Mice were treated in accordance to the Canadian Council on Animal Care guidelines.

1. Anesthetise mice by injection of ketamine/xylazine (150/300 mg/kg, i.p.) or by inhalation of isoflurane (2%, 1L oxygen). If available, isoflurane should be used as it allows for a better control of anaesthesia time and depth. Also, oxygen is administered at the same time which supports the physiological functions of the animal. Moreover, isoflurane is directly eliminated by the lungs and thus does not require hepatic or kidney metabolism.
2. Inject buprenorphine (0.05 - 0.1 mg/kg, s.c.) or another analgesic....

Discussion

We have described a protocol of LN surgery which can apply to many experimental systems. Although very useful to follow immune responses or immune cell homeostasis, this technique has a few limitations. First, only four LNs can be harvested. Second, the immune response studied must be systemic or occur in the superficial inguinal or brachial (skin-draining) LNs. Finally, the number of cells harvested is influenced by the quality of the LN removal, a technical limitation which may contribute to experimental variability wh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Graefe Fine Pattern Premium Forceps	Harvard Apparatus	52-2144	Strongly Curved, 10 cm (4 in), 0.8 mm tip
Michel Clip Applying and Removing Forceps	Harvard Apparatus	52-3779	12.5 cm (5 in)
Michel Clip 100	Harvard Apparatus	52-3746	7.5-1.75 mm
Temgesic (Buprenorphine hydrochloride)	Merck & Co.
Vetalar (Ketamine hydrochloride)	Bioniche Animal Health		DIN: 01989529
Rompun (Xylazine)	Bayer AG		DIN: 02169592
Isoflurane	Abbott Laboratories		DIN: 02032384
Hypotears eye ointment	Novartis AG		DIN: 02133288
Baxedin (Clorhexidine gluconate 2% in isopropyl alcohol 70%)	Omega Engineering, Inc.	L0000017	DIN: 02251477