Preparing the animal prior to surgery (Time: 20 min)
- Animal preparation
0.1. Anesthetize the rat with an intramuscular injection of a mixture of zolatil (0.3 ml) and xylazine (0.1 ml) by using a 1-ml sterile hypodermic syringe.
0.2. Remove the hair from the abdominal and the flank/hip areas by using a Pet Specialty cordless trimmer shaver. Remove the remaining loose hair by using an alcohol swab and patting the area with tape.
0.3. Fix the rat with its head away from the operator by taping its feet to the operating surgical board. For the protection of rat’s eye from light exposure, cover the rat’s eye with gauze prior to surgery.
0.4. Position the fiber optic illuminators for optimum illumination. Adjust the stereomicroscope and the monitoring system for optimal observation.
Throughout the procedure, the rat is continually monitored for anesthetic state via a toe pinch and examination of the respiratory rate.
Detecting the PVS network (Time: 2 ~3 hours)
- Rat skin incision around the inguinal lymph node (IN)
1.1. During the entire surgical procedure ensure exposed tissue is always kept wet with equilibrated warmed PBS solution.
1.2. Place the rat on a clear surgical board in a supine frog-leg position. Attach the rat by taping each footpad to the board. Check the body temperature of the rat during the procedure.
1.3. Place the rat under a dissecting stereo microscope.
1.4. Make a 2-cm skin midline incision up and down from the navel along the ventral surface of the abdominal cavity and retract the skin towards the rat's spinal column.
Make the incision as small as possible to minimize the damage to the rat.
1.5. After the skin is retracted, pin the skin to the surgery board.
1.6. Place 1 or 2 rolled sheets of clean gauze underneath the skin so that the IN is easier to locate.
- Locating the inguinal lymph node (IN)
2.1. Superficial lymph nodes, such as IN, are bilateral and situated closed to the bifurcation of the superficial epigastric vein. INs are often hidden in the connective tissue and fat that encircles the superficial epigastic vein, so careful observation is required to find the nodes. If you still can’t locate the IN, slightly pull the rat’s lower limb on the finding side towards your body to extend its position.
2.2. Remove the thin layer of connective tissue overlaying the area around the IN. Next, clear the adipose tissue overlaying and surrounding the IN until the micro-vascular bed is exposed. The superficial epigastric vein lays adjacent to the IN and is commonly overlayed by the venules. It is important not to injure the vessels by placing too much force on them when manipulating adipose tissue and the connective tissue around them.
2.3. INs are of variable size in different rats, and usually oval in shape as a small pea or bean. Lymph nodes are normally yellowish brown or tan in color and appear slimy. According to our data, the INs are about 2.5 mm in width and 4.5 mm in length. The lymph nodes of normal rats are small and difficult to distinguish from surrounding adipose and connective tissue, so it is necessary to find lymph vessels connected to INs. You can observe a network of lymph vessels connected to INs along the superior epigastric vein.
- Injecting AB into the IN.
3.1. After exposing the IN, hold the tip of node and slightly lift it up with smooth tip forceps towards your body to ease the injection.
3.2. The prepared 0.2% Alcian blue dye is loaded in a syringe specially designed by our team for reducing the rate of puncturing the blood vessels, which causes leakage of blood inside the node. Inject the loaded dye, about 0.2~0.4 ml, into the node at a slow rate. As we stated, INs are usually grouped with two or three nodes.
3.3. Depending on the injection point in the node, staining dye travels separate ways along different lymph vessels. We recommend choosing the upper most IN among the group for injection to reach to axillary nodes. When staining dye is injected into the middle or the undermost INs, it will travel towards the abdominal area through a network of lymph vessels.
1.Slow and smooth needle injection.
2.Slow injection of AB into the IN: Inject the AB with a max 0.03 mL/4 min into nodes
on each side of the skin. Slower is better.
3.Wait about 3- 5 minutes until the IN is stabilized.
- Slowly and gently pull out the needle after injection.
3.4. Check the rat’s respiratory rate again because without spontaneous pumping, movement of the dye within the lymphatic vessel is limited to a short distance from the injection site.
- Visualization of the PVS
4.1. Put the skin back to the original position to keep the body warm.
4.2. Stop the lymph flow from the IN to the axillary node to induce the proper staining of the PVS by tying with a string in the upper chest for 30 minutes.
4.3 Release the tie after 30 minutes for proper lymph fluid flow.
4.4. Wash the residual AB staining in the lymph ducts by natural flow. This takes about one and half hours.
1.Finding the optimal time and speed for the AB to be absorbed by the PVS in the lymph the flow-blocking period is critical. It is about 30 minutes. This process is critical because the PVS will absorb the AB during this period. Various techniques can be implemented to slow down the lymph flow: Put a pillow under the shoulder to keep a slanted position. Put a weight on the skin near the entrance to the axillary node. Tie the upper chest to keep pressure on the lymph ducts. Other methods can be tried.
Another optimal condition is to make the lymph flow fast during the washing period to clean the lymph ducts. Otherwise, the lymph ducts will be stained blue. This is critical because the remnant AB should be completely cleared from the lymph flow.
Observation of the lymph ducts
5.1. Second incision of the skin.
Recall steps 1.4 through 1.6. This second incision starts from the endpoint of the first cutting near the navel about 4 cm towards the cranium. Overturn the skin towards the spinal column and pin it to a surgical board for clear observation of the lymph ducts.
5.2. Tracing lymph ducts along the epigastric blood vessel.
The main lymph duct to observe is located along the superior epigastric vein. It connects the IN to the axillary node (AN), and most of the time, it branches out to the sides on the skin, so extra care is required, especially when connective tissue is removed for the observation.
5.3. Locating the AN.
The AN is easily identifiable due to the blue color of the AB which flowed in the lymph ducts from the IN. ANs are regularly situated internal to the deep fascia of the upper limb; more specifically, they are present in the axillary fossa. Brachial and retoscapular lymph nodes, in proximity to the angle of the scapula, are found in groups of more than one large lymph node.
- Observation of the PVS
6.1. When the conditions are satisfied (such as washing time, concentration of AB, temperature, etc.), a large network of the PVS appears floating inside the clear lymph vessels between the IN and the AN. The primo vessel is more prominently stained with AB than the lymphatic duct. From a stereomicroscope examination along the lymph ducts in the skin, freely-floating, thin, blue PVS lines will be seen inside the washed lymph vessels.
6.2. Try to find the primo nodes (PNs). A PN is irregularly located along the PV, and it looks like a thicker oval-shaped blob (Fig. 3A).
6.3. The PV forms a network structure with branches. Try to observe as many PVS specimens as possible (Fig. 3B).
6.4. A PV often passes lymph valves, so this phenomenon should be observed as carefully as possible (Fig. 3C).
6.5. PVs in efferent ducts flowing from the IN are to be observed (Fig. 3D).
6.6. PVs in afferent ducts flowing to the AN are to be observed (Fig. 3E).
- The PV is sometimes only partially stained, and a few blue dots appear sporadically. The PV is transparent and hard to notice between these blue dots. However, the thread is continuous, and the uncolored parts are better for an analysis of the genuine PVS (Fig. 3F).
- When the washed lymph duct is shaken gently but briskly by touching the outside with a smooth tip forcep, the PVS lines floating inside a lymph duct move like a wave in lymph fluid and shine in a moment, so they are best detected through straightforward (but careful) real-time observation.
On-site criteria to discern the PVS candidate from artifacts.
- Shake the lymph ducts gently, and the blue threadlike structure should remain unbroken. The aggregates of dye are easily broken. The PVS undulates in this process and remain intact as shown in the movie (Supplementary Information).
- The PV’s thickness is uniform and is about 20 -30 μm.
- When a PV is cut by accident inside the lymph duct, it will roll-up due to its elasticity and can be easily pulled out from the duct without breaking when the sample is extracted.
Harvesting the PVS (Time: 1~ 2 hour)
- Isolation of the lymph ducts and PVS specimens.
7.1. After the PVS has been detected in the lymph ducts, the lymph ducts with the PVS in them can be isolated in two alternative methods.
Method 1: (Fresh Sample)
7.2. Cut out the whole lymph nodes and ducts (that is IN, AN, and the ducts between them) containing the PVS from the skin with micro-scissors under a stereomicroscope.
7.3. Carefully incise the lymph vessel along the vessel’s wall by using microscissors.
7.4. Use sharp-ended curved forceps to hold both side splits of a lymph vessel and tear the vessel apart towards the direction of the AN. Be careful not to damage the PVS specimen floating inside the vessel during this procedure.
7.5. When an appropriate length of PVS specimen has been exposed, gently pull it out toward your body. A PV is elastic, so it can be pulled out with a constant force. This specimen is ready for identification by using a histological analysis.
Method 2: (Fixed Sample)
- 1’. Cut out the whole lymph nodes and ducts containing the PVS from the skin.
7.2’. Fix the whole isolated samples with either 4% PFA solution or 10% NBF solution for a day or two for further analysis. Fixed samples are to be stored at 4ºC in a refrigerator until use.
7.3’. Wash the fixed sample with 1x PBS solution three times and prepare a glass slide with 1 drop of 1x PBS solution to protect the sample from drying during the procedure.
7.4’. Put the fixed whole sample on a slide. Use two 31G insulin syringe needle tips to cut and tear the lymph duct to extract the PVS specimen. A useful tip for isolating the PVS from the duct is to cut one end of the lymph vessel obliquely at 45˚from the surface of skin, which will give space for separation.
- Tear the lower end of a harvested lymph vessel with sharp-end forceps and expose the end part of the PVS in it. Pull the PV gently out.
- The following is an on-site check list for identification of the PVS from artifacts such as the coagulation of AB with lymph fluids in a string like form: ① The PVS is a floating threadlike structure not attached to the lymph walls. ② The PVS thread is elastic, but the AB coagulation is not and is, therefore, easily broken. ③ The diameter of a PV is around 20 - 30 μm. ④ The PNs are thicker parts of the PV, and their numbers, thicknesses and lengths vary. ⑤ The PVs pass through lymph valves. ⑥. The PVs branch when the lymph vessels branch. ⑦ If the thickness of a PV is larger than 50 μm, the PV most likely has adhered blood cells or lymphocytes.
Identifying the PVS (Time: 40 min)
8.Identification of the PVS with DAPI and Phalloidin.
8.1. Gently wash the PVS specimen in a drop of 1x PBS solution, place it on a clean glass slide then flatten the tissue slightly. Do this step under a stereomicroscope.
8.2. Phase contrast microscope: Check the bundle structure of the PV.
8.3. DAPI: Check the alignment of rod-shaped nuclei of the endothelial cells lining the primo sub-vessel. Stain the specimen with Prolong Gold Antifade reagent with DAPI again for 10 min to examine the nuclei in the endothelial cells of the PV. When applying the DAPI, mix thoroughly, but take care not to create too many bubbles. Drain excess solution from the slide. Apply two separate drops of Gel Mount – DAPI on the sections, and lower the cover slip on the sections. Let the slide be stay in darkness for a few minutes, and seal the cover slip with a transparent manicure.
8.4. Phalloidin: See the f-actin distribution in cells. The f-actin molecules should be along the PV. In order to show the f-actins in the endothelial cells of the PV, stain the specimen with 6.6-μM Phalloidin for 20 min, followed by three PBS washes.
Avoid light on the sample during the Phalloidin and DAPI staining procedures.
8.5. Examine the specimens with a fluorescence phase contrast microscope (Olympus BX51, Olympus) and a confocal laser scanning microscope (CLSM; C1 plus, Nikon, Japan) to observe f-actins and the rod-shaped nuclei (Figs. 4A, B, C = phase contrast, DAPI, Phalloidin).
- Morphological characteristics of the PV are thickness (20 - 30 μm), bundle structure of several sub-vessels, rod-shaped nuclei (length: 15 - 20 μm) of endothelial cells aligned in broken parallel curves, and sometimes the presence of DNA-containing granules inside sub-vessels. F-actins are also aligned along the PV.
- Non-pure, thick samples have coagulated lymphocytes surrounding the PV.