The overall goal of this presentation is to show the general procedures involved in the stereotactic surgery for Kenly implantation into specific brain regions With special attention to procedural refinements to this longstanding procedure, these refinements are achieved mainly by monitoring and supplementing blood oxygen levels, while also monitoring heart rate and body temperature throughout the surgery. These procedural refinements results in an increased survival rate and overall increased animal welfare. The main advantage of this technique over a standard methods for a stereotactic surgery is that due to our modification, animal survival and welfare are significantly improved.
Generally, individuals unfamiliar to this method would struggle putting the rat in the stereotactic frame and determining the coordinates After thoroughly sterilizing the stereotaxic apparatus and autoclaving, the surgical tools begin by setting up the workspace and prewarm the heating pad. Once the stereotaxic apparatus is set up, place the cannula in its support and check to make sure it is straight. Turn on the gas system to begin the flow of ambient air and 30 to 35%oxygen.
Once the rat has been fully anesthetized, shave the head area going from the ears to just in between the eyes. With an electric razor, place the rat on the heating pad with its nose in front of the air tubing. Use an oximeter according to the manufacturer's instructions to ensure that the rat has a blood oxygenation level between 90 and 99%Note the decrease in the blood oxygenation level if the oxygen tubing is removed.
And be sure to keep an eye on the position of the tubing. Apply eye cream on both corneas to avoid dehydration and check the rat's reflexes to ensure that it is adequately anesthetized if no toe pinch reflexes shown. Place the rat in the stereotaxic apparatus.
Adjust the ear bars so that it shows equal reading on both sides. And place the air tubing in front of the animal by fixing it with the nose bar. Check again.
If the rat shows a blood oxygenation level between 90 and 99%if not, adjust either the tubing bringing it closer to the nose or increase the flow of oxygen. Monitor the blood oxygenation level and heart rate throughout the surgery. Continuously Monitor the rat's temperature with the rectal thermometer and adjust the heating pad or use a blanket to maintain a body temperature of 37.5 to 38.5 degrees Celsius.
After a subcutaneous injection of analgesic, clean the shaved area of the head with disinfectant.Locally. Inject a mixture of lidocaine and adrenaline for local anesthesia and vasoconstriction to prevent excessive bleeding. Make an anterior posterior incision of about 2.5 centimeters on the midline of the scalp, going from between the eyes until the back of the ears.
Use four to six bulldog clamps to pinch off the skin and keep the incision open. Remove any conjunctive tissue with a spatula and or cotton swabs and clean the area to expose the skull surface. Check if the head is level by first finding Lambda and placing the guide cannula exactly over this location, touching the skull.
Record the dorsal ventral coordinate, and then place the guide cannula exactly over bgma and record. Its dorsal ventral.Coordinate. These two coordinates should be identical.
If the difference is greater than 0.3 millimeters, adjust the nose bar to correct it Using a sterilized hand drill, make a small hole approximately five millimeters anterior to the cannula location in one of the hemispheres, and then make another five millimeters posterior to the cannula location. In the contralateral hemisphere, place two sterile screws into the holes until they're tightly anchored without being inserted completely into the skull. With the guide cannula placed exactly at BMA record the anterior, posterior, and lateral coordinates the correct location of the guide.
Cannula placement for each brain region can be calculated by adding or subtracting from bgma. After all the cannula coordinates have been recorded and marked, make the bur holes using a hand drill. Checking the size and the correct location with the aid of the guide cannula.
Once the holes are made, use the sterile needle to gently punch through the meninges to allow for unobstructed insertion of the cannula. Place the cannula into the first hole and lower it carefully until it reaches the final ventral. Coordinate and generously apply dental cement to fix the cannula.
Once the cement is dried completely, remove the cannula support by turning the dorsal ventral bar upwards. Repeat with the second cannula and then cover the screws and a large surface of the cannulas with the cement. Before the cement is dry, remove any surplus from the skin subcutaneously.
Inject warm sterile saline to ensure rehydration. After the cement is completely dried, remove the cannula support and place a sterile pin into each cannula. To prevent obstruction, clean the wound area with sterile saline and suture the front and the back of the wound.
Remove the animal from the stereotaxic apparatus replacing the gas tubing in front of its nose. Continue to monitor the oxygen saturation level and body temperature. If an injectable anesthetic with dexa eide is used, inject its antagonist a tip, asle, and wait approximately five minutes until the animal wakes up.
Place the rat in a recovery cage to avoid hypothermia. Place the cage in an incubator at 28 degrees Celsius or on a heating pad in a place where you can observe the animal for at least one hour before returning it to the vivarium room.Here. The non survival rate of 20 cohorts of 20 rats, each that have undergone stereotaxic surgery, that includes oxygen supplementation and the continuous monitoring of blood oxygenation levels.
Heart rate and body temperature is significantly reduced when compared to rats that have undergone a standard protocol. Moreover, post-surgical weight loss of rats operated with a modified protocol was also significantly reduced at postoperative days one and two as compared to that of rats operated with the standard procedure. After watching this video, you should have a good understanding of how to perform the different steps involved in a stereotactic surgery and how to increase animal welfare during and after this procedure.
Once masta, this technique can be done in approximately 45 minutes if it is performed properly.
