Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Summary

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Abstract

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

Introduction

The spatial organization of the lymphatic system provides structural and functional support to efficiently remove extracellular fluid and transport antigens and antigen-presenting cells (APCs) to the draining LNs. The initial lymphatic vessels (also named lymphatic capillaries) are highly permeable due to their discontinuous intercellular junctions, which facilitate the effective collection of fluids, cells, and other materials from surrounding extracellular spaces¹. The initial lymphatic vessels merge into collecting lymphatic vessels, which have tight intercellular junctions, a continuous basement membrane, and lymphatic muscle coverage. Collecting lymphatic vessels are responsible for transporting collected lymph to the draining LNs and eventually returning lymph to the circulation^2,3. The collecting lymphatic vessels that propel lymph into the draining LN are the afferent lymphatic vessels^4,5,6,7. Obstruction of afferent lymphatic vessels can block lymph flow into the LNs, which is a useful technique when studying the function of lymph flow.

Previous studies have shown that lymph flow plays a significant role in transporting antigens and APCs, as well as maintaining LN homeostasis. It is well understood that tissue-derived APCs, typically activated migrating dendritic cells (DCs), travel through the afferent lymphatic vessels to the LN to activate T cells⁸. The idea that free-form antigens, such as microbes or soluble antigens, passively flow with lymph to the LN to activate LN-resident APCs has been gaining acceptance in the past decade^9,10,11,12. Free-form antigens traveling with lymph take minutes after the infection to travel to the LN, and the LN-resident cell activation may occur within 20 min after the stimulation. This is much faster than the activation of migrating DCs, which takes more than 8 h to enter the draining LN⁹. Besides transporting antigens to initiate immune protection, lymph also carries cytokines and DCs to the LN to maintain its microenvironment, and to support immune cell homeostasis^13,14. Previously, blocking lymph flow by suturing the afferent lymphatic vessels demonstrated that lymph is required to maintain the HEV phenotype required for supporting homeostatic T cell and B cell homing to the LN^15,16,17. CCL21 is a critical chemokine that directs DC and T cell positioning in the LN^8,18. Blocking lymph flow interrupts CCL21 expression in the LN and potentially interrupts DC and T cell positioning and/or interaction in the LN¹⁹. Thus, blocking lymph flow can directly or indirectly abrogate antigen/DC access to the draining LN by disrupting the LN microenvironment that regulates immune responses in the LN. To better investigate the function of lymph flow, an experimental protocol is presented (Figure 1) to block lymph flow in mice by suturing the afferent lymphatic vessels from the footpad to the pLN. This method can be an important technique for future studies on lymphatic function in healthy and diseased conditions.

Protocol

All animal work needs to be approved by institutional and governmental ethics and animal handling committee.  This is a non-survival surgery. 

1. Preparation of materials

1. Prepare 100 mL of 70% ethanol by mixing 70 mL of 100% ethanol with 30 mL of sterile water. Autoclave all surgical tools before surgery and keep the tools in 70% ethanol before and during the surgery to maintain sterilization.
2. Prepare an injection apparatus.
   1. Cut ~30 cm of polyethylene tubing (0.28 mm in diameter). Connect the tip of a 30 G x ½ needle (needle A) to one end of polyethylene tubing. Carefully dislodge another 30 G x ½ needle (needle B) and connect the broken side to the other end of polyethylene tubing.
   2. Attach needle A to a 1 mL tuberculin syringe.
      NOTE: For this polyethylene tubing, 1.6 cm of fluid in the tubing corresponds to 1 μL²⁰.
3. Prepare a 10:1 ketamine/xylazine mixture (10 mg/mL ketamine and 1 mg/mL xylazine) in saline (bacteriostatic 0.9% [w/v] sodium chloride). Prepare the solution freshly before use.

2. Preparation of the animal for surgery

NOTE: Use mice aged 6−10 weeks. Both female and male mice can be used. In this study, 6−10-week-old, C57BL/6 female mice were used. This method can be adapted for other strains of mice.

1. Anesthetize the mouse by injecting 250 μL of the ketamine/xylazine mixture intraperitoneally. Wait until the mouse is completely asleep. Ensure mouse does not react to a toe pinch to detect full anesthetization.
2. Shave fur around the legs with hair clippers.
3. Apply the depilatory cream around the leg and wait for 5 min. Wipe off the residual fur and the depilatory cream using a moist tissue and clean the leg with sterile water. Spray 70% ethanol around the leg to sterilize the operating area. The ethanol is restricted to the incision site. 

3. Surgical suturing of afferent lymphatic vessels

NOTE: The right leg is sutured, and the left leg is used as the sham control. The lymphatic suture protocol (steps 3.1−3.8) takes 20−30 min.

1. Keep mouse at a prone position and fix it with surgical tape to expose the operation area on the right leg.
2. Intradermally inject 5 µL of 1% Evans blue dye or 9 cm of the fluid of the injection apparatus tubing into the footpad. Gently massage the footpad to help Evans blue enter the lymphatic vessels.
   NOTE: The insulin syringe is not easy to control for small volume injection. The volume can be controlled more accurately using the injection apparatus. Lymphatic vessels are visualized by blue dye under the skin. With extensive training, both afferent lymphatic vessels can be seen with the naked eye as transparent vessels in the adipose tissue, parallel to the Saphenous artery. With extensive training, it is possible to suture the vessels without injecting Evans Blue dye in cases where there are concerns of potential disturbances from the dye.
3. Under a dissecting microscope, choose an incision site 5 mm from the bottom edge of the popliteal fossa. Make a small incision (~5 mm) in the skin with scissors. Using fine operation forceps, stretch the incision, and expose the collecting lymphatic vessels (Figure 1A).
   NOTE: If necessary, a small skin fragment can be removed to expose the lymphatic vessels.
4. Identify both afferent lymphatic vessels leading to the pLNs under the dissecting microscope (Figure 1B).
   NOTE: There are two afferent lymphatic vessels from the footpad to the pLN. Both need to be sutured to block lymph flow completely.
5. Using a needle holder, cautiously insert the suture needle (0.7 metric or smaller) between the afferent lymphatic vessel and the Saphenous artery and pull the needle gently out around the afferent lymphatic vessel. Gently pull the suture string and leave about 2 cm of the suture string behind. Use the needle holder to help tie the string tightly to suture one lymphatic vessel with a surgeon’s knot (Figure 1C).
   NOTE: The tissue underneath the incision may dry out with prolonged exposure to air. Making the incision as small as possible and performing the suture quickly (i.e., within 5 min) will prevent the tissue from drying out. Maintain the tissue moisture by applying a small volume of saline with a cotton swab.
6. Gently massage the footpad to ensure no Evans Blue dye passes the suture site and then cut the excess string with scissors.
7. Perform the same suture steps (i.e., steps 3.5 and 3.6) on the other afferent lymphatic vessel (Figure 1D). Close the skin incision with the same suture that was used to suture the vessels in step 3.5 (Figure 1E).
8. For the sham control, intradermally inject 5 µL of 1% Evans blue dye at the left footpad and massage the footpad to visualize the lymphatic vessels. Open the skin with an excision and then close the wound without suturing the vessel (Figure 1F).
9. Optionally, monitor the operated mice for 2−4 h. The suture side of the leg should show edema with Evans Blue spread to the thigh, while the control leg will show restricted Evans blue dye in the footpad. If mice awaken, an additional ketamine/xylazine mix will be injected to keep anesthetized until euthanasia.

4. Tracking of the lymph flow

1. Immediately after the surgery, intradermally inject 10 µL of 2% fluorescein isothiocyanate (FITC) in the footpad of both the control and the lymphatic sutured leg.
2. Euthanize the mice with 400 μL of ketamine/xylazine mixture and perform cervical dislocation when the mice are fully anesthetized.
3. Collect pLNs from the popliteal fossa and carefully remove the perinodal adipose tissue around the pLNs under the dissection microscope at 2, 6, and 12 h after FITC injection.
4. Embed the pLNs with the medullary sinus area facing to the side of the cryomold in optimal cutting temperature (OCT) compound (Figure 1G,H).
5. Prepare 20 µm frozen sections using a cryotome.
6. Image the cryosections under a confocal microscope to determine FITC distribution.

Results

Lymphatic vessel suture has been used in previous studies^15,16,17,19, where it served as an important tool to study the function of lymph flow before the molecular biology of lymphatic vessels was better understood. Blocking lymph flow interrupts LN homeostasis, which leads to HEVs losing the critical gene expression needed for optimal lymphocyte homing to the LN^15,...

Discussion

Blocking lymph flow will have broad applications in manipulating antigen delivery to the LN in healthy and diseased conditions. It is possible to use this method to control the timing of antigen delivery in order to study how continuous lymph flow regulates immune response in draining LNs. This method of lymph flow interruption can also be used to study how lymph impacts cell compartmentalization, cell activation, cell migration, and cell-cell interactions in the LN.

Mice specifically expressi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Saline	Baxter	JB1323
100% ethanol	Greenfield Global		University of Calgary distribution services UN1170.
Depilatory cream	Nair		Nair Sensitive Formula Hair Removal Crème with Sweet Almond Oil and Baby Oil, 200-ml. Or similar product.
Evans Blue dye	Sigma Life Science	E2129-10G	For 1 ml of Evans blue dye, add 0.1g Evans blue to 10 ml PBS. The Evens Blue solution will be filtered through 0.22 mm filters and kept sterile in 1ml aliquots.
Fluorescein isothiocyanate isomer I (FITC)	Sigma Life Science	F7250-1G
Forceps Dumont #3	WPI	500337
Forceps Dumont #5	WPI	500233
Injection apparatus			Connect one end of polyethylene tubing to 30G × ½ needle. Attach a 1ml TB syringe to the needle. Dislodge needle shaft from another 30G × ½ needle. Insert the blunt end of the 30G × ½ needle shaft into the other end of the tubing. The inside diameter of this tubing is 0.28mm. Thus, 1.6 cm of fluid in the tubing is 1 μl.
Insulin syringe	Becton Dickinson and Company (BD)	329461
IRIS Forcep straight	WPI	15914
IRIS scissors	WPI	14218-G
Ketamine	Narketan	DIN 02374994	The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.
Needles (26Gx3/8)	Becton Dickinson and Company (BD)	305110
Needles (30Gx1/2)	Becton Dickinson and Company (BD)	305106
Paton Needle Holder	ROBOZ	RS6403	Straight, Without Lock; Serrated
Phosphate-Buffered Saline (PBS)	Sigma Life Science	P4417-100TAB
Polyethylene tubing	Becton Dickinson and Company (BD)	427401
Surgical tape (1.25cmx9.1m )	Transpore	1527-0
Surgical tape (2.5cmx9.1m )	Transpore	1527-1
Suture	Davis and Geck CYANAMID Canada	11/04	0.7 metric monofilament polypropylene
Syringe (1ml)	Becton Dickinson and Company (BD)	309659
VANNAS scissors	World Precision Instruments (WPI)	14122-G
Xylazine	Rompun	DIN02169606	The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.
Equipment
Dissecting microscope	Olympus		Olympus S261 (522-STS OH141791) with light source: Olympus Highlight 3100
Confocal microscope	Leica		SP8