A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

The overall goal of this method is to induce consistent secondary lymphedema in the hindlimb of mice without causing severe morbidity. This method can be used to induce secondary lymphedema in the hindlimb of mice. The main advantage of this method is that it resolves insignificant lymphedema lasting at least eight weeks without causing severe morbidity.

Someone new to the procedure may struggle with the microsurgical techniques. We therefore recommend microsurgical training and several practice procedures to ensure a successful result, but also to prepare the researcher so that he or she can perform the procedure more efficiently. Set up the source of radiation and anesthetize the mouse as described in the protocol.

Place a 1.5 millimeter thick lead pad to ensure that only the area that undergoes surgery gets irradiated. The shown example is of a post-surgery mouse to clarify the area. Administer a dose of 10 gray irradiation.

The researchers must take precautions when working with radiation. During this experiment all irradiation was performed in a radiation insulated room. And the source of radiation was only turned on when all personnel had left the room.

Weigh the mouse pre-surgery. Examine anesthetic depth by paw or tail pinch test. Shave the leg chosen for the procedure.

Set the flow of oxygen to 0.8 Liters per minute. Connect it with a nose cone. Apply ophthalmic ointment.

Inject 0.5 milliliters of saline subcutaneously, preferably in the scruff of the mouse to prevent hypovolemia during surgery. Place the mouse on the surgical cloth in supine position. Place the nose cone over the snout.

Fixate the end of the hindlimbs gently with tape, to prevent the mouse from shifting during surgery. Lift the skin with smooth forceps and clip a small opening approximately five millimeters proximal to the popliteal fossa. Slide sharp scissors into the opening and clip towards the knee, so that the incision ends just above the knee.

Make sure not to puncture the underlying vessels, by lifting the skin with forceps while clipping. Move the mouse to prone position and complete the circumferential incision. Dissect the skin to a couple of millimeters above the angle.

Remember to keep the tissue damp with sterile saline during the whole procedure. Retract the skin of the proximal woundage with an elastic retractor. And make sure the sciatic vein is visible as shown.

Inject 0.01 milliliters Patent Blue V subcutaneously between the second and third toe. Gently press the paw a couple of times to distribute the Patent Blue V.The distribution of the Patent Blue V can be seen through the microscope. The structures needed to be visualized are, the proximal lymph vessel, PLV, the popliteal lymph node, PLN, the first distal lymph vessel, DLV1, and the second distal lymph vessel, DLV2.

Magnify to visualize the PLV and ligate it using a 10-0 nylon suture. Press the paw to ensure no Patent Blue passes proximal to the ligature. Ligate the two distal lymph vessels as shown.

Press the paw several times again to make sure that no Patent Blue V passes proximal to the ligature. In this example, it can be seen that one of the lymph vessels bursts due to the ligature hindering the lymph flow. Remove the popliteal lymph node, the lymph node has a smooth pearl-like surface and is colored by the Patent Blue V in contrast to the surrounding fat tissue.

Before removing the inguinal fat pad, cauterize this vessel as shown. Then remove the fat pad in one piece. Make sure that there is no active bleeding.

This is an example of a located inguinal lymph node. The lymph node located in the inguinal fat pad is rarely colored by the Patent Blue V and can be hard to differentiate from the fat. Removing the fat pad in one piece is the best way to ensure that the lymph node has been removed.

Suture the skin edges down to the muscle fascia, with a 6-0 nylon suture using forceps and needle holder, leaving a gap of two to three millimeters, to constrain the superficial lymph flow. This is an example of the finished sutures, in the popliteal fossa, the lateral side of the hindlimb, above the knee, and the medial side of the hindlimb. Administer analgesia and weigh the mouse post-surgery, then place it in a cabinet heated for recovery.

Repeat the procedure for pre-surgery irradiation. This graph shows the combined mean hindlimb volume of three separate studies in the eight weeks after surgery. 31 mice were included in total.

The x-axis represents weeks after surgery. The y-axis represents hindlimb volume in cubic millimeters. The arrow bars represent the standard deviation.

It is to be expected that the swelling from the surgery and the induced lymphedema will decrease during the first four weeks. After the first four weeks we see a relative stable state of lymphedema. The volume of the control hindlimb increases during the eight weeks, thus strictly saying the difference in volume between the lymphedema hindlimb and the control hindlimb, this was unexpected.

The cause is speculated to be that the mice used their non-operated hindlimb more than the operated hindlimb in the weeks following surgery. This could lead to hypertrophy of the control hindlimb and maybe explain the increase in volume. For individual figures on the three studies included please read the protocol.

This table shows Sidak’s multiple comparison test for the combined volume measurements of the three included studies. The calculated P-value shows that there is statistically significant difference between the hindlimbs with induced lymphedema and the control hindlimbs, during all eight weeks of follow up. For individual statistical analysis on the three included projects please read the protocol.

This method induces statistically significant lymphedema, lasting at least eight weeks. The procedure can be used to study the petrificiality of lymphedema and to research novel treatment options. After watching this video, you should have gained an understanding of how to induce secondary lymphedema in the hindlimb of mice, using a combination of microsurgery and pre and post-surgery irradiation.

Once mastered, the surgical procedure can be performed in 45 minutes.