Hi, I am Brenda Geiger from the Laboratory of Emmanuel POTOs in the Department of Pharmacology and Experimental Therapeutics at Tufts University School of Medicine. And I'm Lauren Frank, also from the lab. Today we will demonstrate survivable stereotactic surgery in rodents.
This procedure involves setting up the instruments and preparing the animal for surgery and then performing the stereotaxic surgery. So let's get started. Before starting the procedure, set up the stereotaxic instrument and all the materials needed.
Make sure the area and instruments are cleaned and sterilized. Next, prepare the animal for surgery. I will demonstrate today's surgery using a 10 week old Sprague dolly rat.
We also use three month old spray dolly rats and two month old C 57 black six J mice, which require a specialized platform adapter. Anesthetize the rat with an IP injection of a cocktail containing 60 milligrams per kilogram, ketamine, and 10 milligrams per kilogram.Xylazine. Keep the animal warm by using a thermostat regulated heating pad.
After ensuring that the animal is sedated and is stabilized at a proper body temperature, proceed with the surgery. To begin the surgery, shave off the animal's fur with an electric razor shaved from the ears to just in between the eyes. Apply iodine to the shaved area, but make sure to protect the eyes from it.
Now mount the animal onto the stereotaxic apparatus. To do this first mount, one ear bar in the ear canal and then hold it in place and slide in the other ear bar so that the head cannot be moved side to side, but the snout can be moved up and down. Secure the mouth with the anterior mount of the Stereotaxic instrument user ruler to make sure that the head is level.
There should be a 90 degree angle between the ruler and the middle of the animal scalp. Once the animal is in place, make an anterior posterior incision on the scalp with a sterile scalpel. Extend the incision from the lambda to just in between the eyes of the animal.
Use hemostats to pinch off the skin and keep the incision open. Dry off the exposed skull surface with several cotton swabs. Next, put the guide cannula onto its mount.
Then find bgma on the skull and position the guide cannula directly over this location. Write down the anterior, posterior and lateral coordinates from bgma. Find the correct coordinates needed for the placement of your probe.
With the aid of the stereotactic atlas position the guide cannula to the correct coordinates by adding or subtracting from bgma. Lower the guide cannula until it is touching the skull, and then record this ventral coordinate. Make a pencil mark at this location on the skull.
This is where you will be drilling. To begin drilling. Remove the guide cannula and sterilize your drill bit.
Then drill a 0.5 millimeter hole at the pencil mark until you get through the width of the skull. Check with the guy cannula to see if it would clear the hole without touching the sides. Keep drilling and checking until the cannula can clear the hole in a straight path.
Be careful not to penetrate through the meningeal membranes or blood vessels. Once the hole is made, use a sterile needle to gently punch the meninges in order to allow unobstructed insertion of the guide cannula. Next, using a hand drill makes six holes for skull screws.
Two anterior, two lateral, and two posterior to the cannula hole. Sterilize six stainless steel bone screws and place them onto the skull until they are tightly anchored in. After inserting the screws, clean the guide cannula with ethanol and allow it to dry then mount and slowly lower the guide to the proper ventral coordinate.
The cannula is lowered up and down to prevent damaging the brain. Make sure that the sides are not touching and that it is going in perfectly vertical. Then remove the hemostats.
Mix a thin match of liquid dental cement and cover the guide cannula, screws and the rest of the skull with a spatula in a thin layer. Next, place the anchor screw medially and behind the posterior skull screws and hold it in place with tweezers. Make another batch of cement thicker this time and completely cover the cannula and anchor screws with enough cement to secure them when the cement becomes thicker.
But before it solidifies, separate the skin from the cement cap and mold the cement cap with the spatula to make sure it is smooth all around and does not irritate the skin. Later, more cement is added to reinforce the guide cannula and bridge it with the rest of the head mount. After the dental cement is completely dry, remove the animal from the apparatus.
Then apply the local antibiotic bacitracin all the way around the cement cap. Next, administer an intramuscular injection of penicillin followed by a oneill subcutaneous injection of saline. Place the animal in its cage and monitor the animal until it becomes conscious.
Then return the animal to its room to recover. After recovery. Monitor animal daily until the end of the experiment for signs of infection and evaluation of pain and distress.
Administer 0.1 to 0.5 milligrams per kilogram subcutaneous buprenorphine twice daily on an as needed basis if animal appears to be in pain. In addition, local bacitracin treatment and systemic penicillin treatment are administered. If postoperative infections occur, if any of the symptoms persist longer than 12 hours after surgery, the animal is euthanized.
We, we've just shown you how to perform survivable stereotactic surgery in rodents. When doing this procedure. It is important to remember to maintain a sterile environment during surgery and monitor the animal closely following surgery to avoid infection and that place the animal on the air bars correctly so that the location of the guide cannula is correct.
So that's it. Thanks for watching and good luck with your experiments.
