Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

The overall goal of this surgical intervention is to observe the effect of the vagus nerve stimulation in inflammatory conditions. To analyze and understand the different mechanisms and cells involved in the so-called cholinergic anti-inflammatory pathway. This method was developed in the Kevin Tracey lab in the Feinstein Institute.

The method can help answer key questions in the neuroimmunology field. As recent advances establish a close link between the nervous and immune systems. The main advantage with this technique is that it can be easily set up in a laboratory.

But important rules have to be followed regarding animal care and the surgery protocol. The wandering course of the vagus nerve throughout the whole body, and its ability to down regulate information in various animal models implies that its stimulation could be useful in the treatment of many inflammatory diseases. Usually individuals new to this technique will struggle to identify and isolate the vagus nerve from the carotid artery under microscopic observation.

However, although it is tricky at the beginning it becomes easier with time and practice. Begin by turning on the computer and the data acquisition system linked to the stimulating electrode. Launch the data acquisition and analysis software.

Then follow an aliquot of 5 milligrams per milliliter lipopolysaccharide or LPS and dilute it to zero point five milligrams per milliliter with saline. Anesthetize a 25 gram mouse in an induction chamber. When the desired level of anesthesia is reached, move the animal from the chamber to the mask.

Turn the three-way connector to feed the mask, and adjust the flow regulator. Check for a surgical plane of anesthesia by loss of the righting reflex and a regular respiratory rate of around 100 breaths per minute before starting the surgical procedure. Once the mouse is properly anesthetized, fix the legs of the mouse to the workbench with adhesive tape.

Make sure that the nose of the animal is still carefully positioned in the mask. Properly disinfect the surgical area according to the approved procedures. Then, after making a one to one point five centimeter incision in the skin at the level of the neck, position a stereo microscope with a 12 point five x objective to view the surgical area.

Locate the sternocleidomastoid muscle by removing the layers of skin and fat with forceps. Retract the sternocleidomastoid muscle and move the forceps behind both the left vagus nerve and the carotid artery. Isolate the nerve from the artery by placing the forceps very carefully between the nerve and artery.

This is a critical step in the procedure, as the nerve and the artery are close to each other, it's very easy to cut the vessels and kill the animals. However by placing very carefully the forceps between the nerve and the artery, the eventually separate and it's possible to isolate the vagus nerve. Now, place the electrode under the vagus nerve.

As the needle electrode is quite long, the nerve will always be in contact with the electrode even if it moves slightly. Next perform an intraperitoneal injection of LPS and wait five minutes before starting the stimulation. After five minutes, stimulate the vagus nerve for five minutes at five volts and one hertz by pushing the start button in the acquisition software.

When the stimulation period is over, remove the electrode and then stitch the wound of the animal with surgical suture thread. Spray a no-sting barrier film on the wound in order to improve healing and to protect from infections. Finally, move the animal into a clean cage and monitor as it regains full consciousness.

The following images demonstrate the effect of reduced LPS concentration and increased latency to sacrifice on levels of TNF alpha and IL one beta following vagus nerve stimulation. Here, it can be seen that the original paradigm significantly decreases both TNF alpha and IL one beta compared to sham operated controls. The protocol demonstrated in this video significantly reduces the levels of TNF alpha but not IL one beta.

As seen here, a six-hour recovery without LPS injection does not affect levels of TNF alpha or IL one beta. Once mastered, this technique can be done in approximately 20 minutes if it is performed properly. After its development, this technique has paved the way for researchers in the field of neuroimmunology.

To study the connection between the central nervous and the immune system. And also study the vagus nerve innervation of new organs and the effect of the cholinergic anti-inflammatory pathway in chronic inflammatory diseases. After watching this video, you should have a good understanding of how to locate, isolate and stimulate the vagus nerve.

And also keep in mind that the level of induced inflammation and the time for recovery of the animal might be important issues to take into account.