This method interrupts lymph flow with minimal damage to lymphatic endothelial cells, and with precise control of blockade timing, it can be used to study how lymph flow impacts homeostasis and immune responses in the lymph node. Helping to demonstrate the procedure will be Jingna Xue, a graduate student from my laboratory. To prepare an injection apparatus, cut approximately 30 centimeters of polyethylene tubing.
Connect the tip of needle A to one end of the tubing. Carefully dislodge another needle, and connect the broken side to the other end of polyethylene tubing. Then attach needle A to a one-milliliter tuberculin syringe.
Immediately before use, prepare a 10:1 ketamine-xylazine mixture in saline. After anesthetizing the mouse by injecting 250 microliters of the ketamine/xylazine mixture intraperitoneally, ensure full anesthetization with a toe pinch. Shave the fur around the legs with hair clippers, then apply depilatory cream around the leg.
After five minutes, wipe off the residual fur and the depilatory cream using a moist tissue, then clean the leg with sterile water. Spray 70%ethanol around the leg to sterilize the operating area. Place the mouse in a prone position, and use surgical tape to expose the operation area on the right leg.
Intradermally inject five microliters of 1%Evans blue dye, or nine centimeters of the fluid from the injection apparatus tubing into the right foot pad of the mouse. Gently massage the footpad to help the fluid enter the lymphatic vessels. Under a dissecting microscope, locate an incision site five millimeters from the bottom edge of the popliteal fossa.
Using a pair of scissors, make a small incision approximately five millimeters in length. Then use fine operation forceps to stretch the incision and expose the collecting lymphatic vessels. Identify both of the afferent lymphatic vessels leading to the popliteal lymph nodes.
Using a needle holder, cautiously insert the suture needle between the afferent lymphatic vessels and the saphenous artery. Gently pull the needle out around the afferent lymphatic vessels. Carefully pull the suture string, leaving about two centimeters of the suture string behind.
Use the needle holder to help tie a surgeon's knot. Gently massage the foot pad to ensure no Evans blue dye passes the suture site, then cut the excess string with scissors. Suture the other afferent lymphatic vessels, then close the skin incision with the same suture that was used for the lymphatic vessels.
For the sham control, intradermally inject five microliters of 1%Evans blue dye in the left foot pad, and massage the foot pad. Open the skin with an incision, and then close the wound without suturing the vessel. When monitoring the mouse post-surgery, the right leg should show edema, with Evans blue dye spreading to the thigh, while the control leg will show Evans blue dye restricted to the foot pad.
Immediately after the surgery, intradermally inject 10 microliters of 2%FITC in both the right foot pad and the left. Two, six, and 12 hours after FITC injection, collect the popliteal lymph nodes from the fossa of euthanized mice. Carefully remove the perinodal adipose tissue around the pLNs.
Embed the pLNs in optimal cutting temperature compound with the medullary sinus area facing to the side of the cryo mold. Use a cryotome to prepare 20-micron frozen sections. To determine the FITC distribution in the pLNs, image the cryo-sections under a confocal microscope.
Confocal images captured two, six, and 12 hours after FITC injection show substantially reduced FITC accumulation in the popliteal lymph nodes after suturing the afferent lymphatic vessels. The residual FITC in the pLNs was preferentially accumulated in the lymph node sinuses. Confocal images of the perinodal adipose tissue show when lymphatic vessels are blocked by suturing, FITC enters the tissue when the lymph node sinuses, but it's not effectively distributed throughout the lymph nodes.
When attempting this method, it's important to remember that inserting the suture needle between the blood vessel and the lymphatic vessel is critical. Failure of this step may break the vessels. After performing this method, it's possible to immunize mice with infection or other immune stimulation to study how lymph flow regulates immune protection.
The function of lymph flow is not well understood. This method can be used to study cell migrations in the lymph node in the absence of lymph flow.
