A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

The overall goal of this procedure is to perform local injections of neurochemicals directly into the noose ganglia to study the effect of neurochemicals on physiological behavior relating to the vagus nerve. This is accomplished by first conducting neck surgery to visualize the noose ganglia and vagus nerves. The second step is to isolate and clean the noose ganglia of connective tissues.

Next, inject the neurochemical of interest into the no dose ganglia. The final step is to measure the physiological and behavioral effects of modulating vagal nerve activity by local injections of neurochemicals into the no dose ganglia. This method can help answer key questions in the neuroscience field, such as whether or naer locally administered neurochemicals modulate vagus nerve activity.

Generally, individuals new to this method may struggle because it takes very fine motor skills to inject into the no dose ganglia. Practice is key. First, prepare a stock solution of 0.05 molar serotonin hydrochloride in PBS.

Then dilute the stock solution with PBS to a final concentration of point 203 millimolar serotonin next dilute dronabinol in sesame oil to a concentration of 20 micrograms per microliter. Now use scissors to cut a 20 centimeter length of polyethylene PE 50 tubing. Then cut a bevel tip at one end and insert a 23 gauge needle at the other square end.

Connect the needle to a one milliliter syringe and fill it with 0.3 milliliters of 50 units per milliliter heparin. Finally, cut four pieces of four zero braided silk thread and sterilize the instruments for the procedure using 70%ethanol or a bead sterilizer. After anesthetizing, a spro dolly rat with ketamine xylazine, pinch the toe of the rat and observe any movement To confirm a proper level of anesthesia, shave the ventral aspect of the left thigh and the ventral aspect of the neck.

Then secure the rat in a supine position on a surgical board, and use the surgical scissors to cut away skin on the left hind thigh. Next, use forceps to blunt, dissect the superficial muscles to expose the femoral vein and separate the vein from the femoral artery. Place two threads around the femoral vein.

Now hold the curved grief forceps in one hand and pull up the vein to stop the blood flow. Pick up a 22 gauge needle in the other hand and puncture the femoral vein while still holding the vein with the grief forceps. Insert the bevel den of the PE 50 tubing into the vein.

Then gently retract the plunger of the syringe. If the PE 50 tubing is inserted correctly, blood will enter the tubing. Finally, tie each of the two threads around the vein and PE 50 tubing and secure each with a knot.

Place a pizo electric strain gauge around the rat to measure respiration. Set the electronic amplifier to amplify and ban pass. Filter the respiratory signals obtained from the strain gauge via the amplifier software on the computer Set high pass filter at AC at one hertz and low pass filter at 10 hertz.

Then set the amplification by inputting 10 for initial gain and 100 for total gain. Change the settings for sampling rate by opening analog input to digitize using an analog to digital converter. Set sample at 1000 hertz and skip every other recording point by one, and then record the signal using the software.

Once the parameters are set. Remove the one milliliter syringe from the catheter and insert a 500 microliter precision glass syringe filled with point 203 millimolar serotonin into the catheter. Next, place the glass syringe into an infusion pop set to infuse 12.5 micrograms per kilogram of serotonin at a rate of 63 milliliters per hour.

Perform multiple infusions and observe apnea in the rat, which can be seen in the recorded respiratory signals on the computer. Monitor as a pause in breathing that lasts longer than 2.5 seconds. Before proceeding with neck surgery, monitor the breathing pattern and check for a pain reflex with a toe pinch in the rat if breathing is irregular or if there is a pain reflex from the toe.

Pinch administer ketamine xylazine, and then reconfirm a proper level of anesthesia. Begin the next surgery by securing the anesthetized rat in a supine position on a surgical board. Then use surgical scissors to make a midline longitudinal cut at the neck blunt.

Dissect the platysma muscle and clamp using two micro clamps to keep the muscle clear of the surgical site exposed. The Sterno high Aus and Omo Hi Aus muscles. Separate these muscles to expose the internal carotid artery and one of its branches.

The tego palatine artery Observe the vagus nerve as it runs along the internal carotid artery, and then along the tego palatine artery. Notice that the noose ganglion is displayed as a swelling of the vagus nerve right before it enters the posterior lacerated foramen. Using the grief forceps, separate the vagus nerve from the arteries.

Then place a piece of thread around the vagus nerve and place a clamp on the thread to apply slight tension on the vagus nerve. Finally, clean the noose ganglion of any connective tissue to provide less resistance. When injecting to confirm that the noose ganglion is undamaged, repeat the infusion of serotonin to induce apnea as previously demonstrated.

Bill a 10 microliter gas type precision glass pipette affixed with a custom made 28 gauge half inch syringe needle with a 35 degree bevel tip with five microliters of ol in sesame oil. Puncture the no dose ganglion. Taking care not to pierce through it, depress the syringe slowly to inject the entire contents of the syringe.

Note that some of the syringe contents will leak outta the ganglion. Repeat serotonin induced apnea as previously demonstrated. This image shows sample recordings from acute serotonin induced apnea experiments before and after injections of 100 micrograms dronabinol in five microliters of sesame oil into the no dose ganglia of the vagus nerves.

Respiratory recordings were taken before surgery after surgery, and after no dose ganglia injections. The red line signifies intravenous serotonin infusion to induce apnea. After watching this video, you should have a good understanding of how to locally inject neurochemicals directly into the noose ganglia.

To study the effects of neurochemicals have on physiological behavior relating to the vagus nerve once mastered, this technique can be done in less than an hour if it is performed properly.