This procedure will demonstrate how to prepare an undercut model of post-traumatic epileptogenesis in rats. The first step is to make a simple device that improves the precision and consistency of the undercut surgery. Then the animal is prepared for surgery, and a cranial window is cut on the surface of the skull to provide access to the brain.
Finally, the needle of the undercut device is inserted into the white matter of the cerebral cortex and rotated to produce a fan shape undercut in the target region. Ultimately, results can be obtained that show epileptic form activity in brain slices through field potential recording. The main advantage of this technique is that more precise and consistent cord collision can be made with a simple custom made surgical device.
And this animal model can be used to answer key questions related to epileptogenesis after traumatic brain injury. Though this mice is often used in rice, it can also be applied to mice. Generally, People new to this method, we struggle because making such precise lesion of rodent cortex with free hand is difficult.
The surgical device consists of three parts, a supporting plate, a guiding tube, and a needle bent at 90 degrees close to the tip. To prepare an undercut device for surgery in three to four week old rats, first cut a piece of one to 1.5 millimeter thick, seven to 10 by 30 millimeter rectangular stainless steel or transparent plastic. To make the supporting plate take a 25 gauge and a 22 gauge syringe needle and measure the length of the 25 gauge needle.
Next, cut the plastic end of the 22 gauge needle and measure the total length of the needle mark and cut the 22 gauge needle at a spot so that the final length of the guiding tube is around five to six millimeters shorter than the 25 gauge syringe needle. Sand the end to make it flatten smooth. Use cyanoacrylate glue to fix the guiding tube onto the supporting plate, making sure that the needle is perpendicular and even to the edge of the supporting plate.
Insert the 25 gauge syringe needle into the guiding tube and bend the needle 90 degrees. 2.5 to three millimeters from the tip. Finally, attach a small copper wire or a small piece of tape onto the upper end of the needle in the same direction as the bent tip.
The wire or tape indicates the turning angle of the needle during undercut surgery. Before beginning this procedure, consult with the institutional animal care induced committee to obtain the necessary approval for the following procedures. First, ensure that all surgical instruments are sterilized using an approved method and that a sterile field is available for the surgery.
Anesthetize the animal according to the approved protocol, ensure that the animal is fully anesthetized by performing a tail pinch. Use an electrical trimmer to shave the scalp of the animal around the area where the incision will be made. Then disinfect the scalp using a 10%povidone iodine solution, followed by 70%Ethanol.
Coat the eyes of the animal with ophthalmic ointment to prevent them from drying out. During the surgery, mount the animal onto a stereotaxic apparatus to keep the head in a fixed, stable position. It is best to keep the animal on a heating pad to prevent hypothermia.
Using a scalpel, make a midline anterior posterior incision on the scalp extending from Lambda to between the eyes. Then use hemostats to pull the skin aside and expose the left side of the skull sufficiently. Use the scalpel to make a rectangular cut on the left side of the skull and to scrape off the periosteum.
This step will reduce bleeding and facilitate drilling on the skull. First, clean the skull using sterile saline and several Q-tips to remove blood and dry the area. Then apply a drop of sterile saline on the center of the left side of the skull.
Then begin drilling to produce a four by five millimeter rectangular groove in the center of the left side of the skull. After approximately two thirds of the bone depth is drilled, remove excess saline and clean the drilled area with a Q-tip. Slowly and carefully, continue drilling deeper until the central piece of bone is movable by a gentle touch with forceps.
Carefully remove the central piece of bone by gently grasping the piece of bone with forceps and slowly lifting the bone window to expose the left hemisphere while holding the undercut device. Orient the needle in a para sagittal direction and the supporting plate perpendicular to the midline. Aim the tip of the needle one to two millimeters lateral to the superior sagittal suture in the middle of the cranial window while avoiding large blood vessels.
Next, insert the needle in a horizontal direction through the dura and under the pier. So as desp spare blood vessels, sit the bottom of the undercut device onto both edges of the cranial window so that the needle is positioned normal to the cortical surface. Ensure that the needle is underneath the pier by raising it slightly.
Then slowly lower the needle to create a transcortical cut until the needle cannot penetrate any further. Next, rotate the needle 180 degrees away from the midline to create a half circle white matter deep layer six undercut. Finally, raise the needle again until it is underneath the pier.
Then tilt the undercut device backward and withdraw the needle. Place a piece of plastic film onto the cranial window for protection. Then unclip the hemostats, moisten the scalp with sterile saline if required, and move it back into place.
Close the incision with sutures. Then release the animal from the stereotaxic apparatus and place on a heating pad until the animal recovers from anesthesia. After returning the animal to the home cage, be sure to follow up with analgesia as defined by the relative IA iacuc protocol.
Here, coronal cortical slices have been prepared two weeks after the procedure To assess the success of the undercut surgery. Transcortical and undercut incisions are discernible under the low power objective of a microscope. This field potential recording reveals evoked el lepto formm activity with impartially isolated cortex.
Once master, this technique can be done in about 30 minutes if it is performed properly. Well, attempting this procedure, it's important to remember to practice the use of the surgical device before the surgery. After watching this video, you should have a good understanding of how to prepare uncut model of post-traumatic epileptogenesis in the rodents.
