The overall goal of this procedure is to visualize and evaluate the inguinal lymph node feed arterial in vivo. This is accomplished by first creating a skin flap by carefully dissecting the skin on the inguinal region of the mouse, covering the inguinal lymph node. Then the connective tissue overlaying the lymph node area is detached.
Next, the surrounding adipose tissue must be removed to expose the feed arterial vasculature of the lymph node. Finally, changes in the lymph node vasculature are visualized and evaluated by intravital microscopy P, Although the, the preparation you're gonna see today is the inguinal lymph node microcirculation. This, the surgical techniques can be used for other preparations such as ske muscle as well as brain.
Generally, when doing this technique, new individuals struggle for two main reasons. One, being you're dealing with a alive animal, and the second being that because you're doing surgery on a very small area, any kind of disturbances to the vessel itself or the surrounding area could have a negative impact, which would be detrimental to the experiment that you want to conduct. The visual demonstration of this technique is critical for success.
There are a number of intricate steps that are involved that translate not very well from text into actually doing the technique. It's important to see it in order to do it correctly. With much success.
The mouse is first anesthetized and the surgical area shaved. Please see the text manuscript for details. The mouse can then be placed on a clear surgical board In a supine position.
Attach the mouse to the board by taping down each footpad The day before the experiment, mixed thes guard 180 4 according to the manufacturer's instructions. Then using a standard disposable Petri dish as a mold, pour the mixture to an about one quarter inch depth and let it cure overnight. Remove thes guard from the Petri dish and place it on the surgical board.
Place the shaved anesthetized mouse under the dissecting scope. Make a midline incision along the ventral surface of the abdominal cavity using number five stainless steel forceps and straight blades, spring scissors. Then continue the midline incision diagonally towards the limbs and retract the skin towards the massive spinal column.
Once the ln is easily visible, pin the retracted skin using stainless steel insect pins and inox epoxy coated forceps onto the cigar. 180 4 pedestal. Add physiological saline solution or PSS to the area.
Next, remove the thin layer of connective tissue overlaying the area around the ln. Using forceps carefully pick up small pieces of connective tissue and dissect away from the ln. Using spring scissors, then clear the adipose tissue overlaying and surrounding the LN until the microvascular bed is exposed.
For this step, it is important to be careful never to make contact with the vasculature of interest with surgical instruments or to injure the vessels by placing too much force on them when manipulating the tissue surrounding them. The fat is best cleared away from the ln by loosening the connective tissue, holding the adipose together with forceps followed by gently removing the adipose tissue until the field of view is clear. Once the connective and adipose tissues have been removed, the main arterial segment adjacent to the LN and the overlaying accompanying ven can be visualized.
Once the vessel segment of interest has been exposed under the dissecting scope, secure the preparation on the intra vital microscope. Set up the superfusion and suction lines. During this part of the procedure, it is essential that the exposed tissue is always kept super fused with the Equilibrated PSS solution.
Superfusion of PSS occurs by gravity. Feed from an initial reservoir into a water jacket heated reservoir that is perfused by 5%Carbon dioxide balance nitrogen, then down a drip line at a rate of 10 milliliters per minute. A suction line should be used to continue to pull super fused PSS away from the preparation by placing it at the bottom of the skin flap, the temperature of the circulating water bath and the rate of the PSS Superfusion then should be checked and adjusted to achieve an optimal temperature of a about 36 degrees Celsius.
Across the preparation, allow the vasculature to equilibrate with PSS for at least 60 minutes. Ensure the animal does not become hypothermic using a rectal thermometer and supply an external heat lamp. While the PSS is equating, quantify the vessel diameter.
Using the video calipers at multiple time points to ensure the vessel has stabilized stabilization is defined as the diameter size remaining consistent within five micrometers for a period of 20 minutes. Diameter measurements are recorded on a computer using chart lab software. After the equilibration period, assess the health of the vessel using a smooth muscle specific agonist such as phenylephrine as shown here with phenylephrine.
Add the agonist to the suse. Eight agonists should be evaluated at each log, increase in the cumulative concentration of the agonist as it is added to the SUP fate between the administrations of each agonist. A wash phase using PSS should be performed for 30 minutes or until the vessel returns to resting diameter.
Here are representative results of an inguinal ln after the equilibration period. Feeding the arterial with physiological saline solution yields a clear image that allows for identification of both the feed arterial indicated here by the red arrow and the venal indicated by the green arrow, which supply the inguinal lymph node. The graph shows a typical vasoactive response of an inguinal L nfe arterial to the cumulative addition of SUP fused phenylephrine to physiological saline.
From a concentration of 10 to the minus nine molar to 10 to the minus five molar, the presence of a robust vasoconstriction is suggestive of normal smooth muscle function present in the arterial. In this graph, a typical vasoactive response to cumulatively added fused acetylcholine to physiological saline. From a concentration of 10 to the minus nine molar to 10 to the minus five molar is shown.
The presence of a robust vasodilation is suggestive of normal endothelial function present in the arterial. In attempting this preparation, one of the key things to remember is to minimize the disturbance to the vessel and the surrounding area in order to maximize success. Following this procedure, other methods can be introduced such as fluorescence to explore other cell functions such as cell trafficking.
After development of this technique, it has paved the way to bridge the fields of immunology and vascular biology to a new field of vascular immunology.
