Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

The overall goal of this procedure is to perform a recording guided implantation of micro electrodes into the subthalamic nucleus of rats, and thereafter a long-term stimulation of this nucleus. This is accomplished by first drilling an electrode hole through the exposed skull. The second step is to advance the electrode through the electrode hole into the brain, and simultaneously record the electrical activity in different regions of the brain until the specific activity of the STN is detectable.

Next, the electrode is fixed by applying dental cement around the electrode shank, and the head plug is attached to the electrode pin. The final step is to fix the plug by dental cement and to connect it to the wire leading to the stimulator. Ultimately, the harvested brain is cut in eight micron thick slices and stained with hemat, toin, and eoin to show that the tip of the electrode is placed in the subthalamic nucleus.

The main advantage of this technique over existing methods like a blind electrode implantation, is that recording guided implantation of micro electrodes improves targeting the subthalamic nucleus with high accuracy Animal experiments were approved by the University of Burg and the legal state authorities and performed according to the recommendations for research in experimental stroke studies and the current animal research reporting of in vivo experiments. Guidelines begin by anesthetizing the rat in an induction chamber with air delivered at two liters per minute and isof fluorine at 3.5%Remove the rat from the induction chamber and shave the area between the ears and the eyes. Use a cotton swab soaked with antiseptic solution to swab the shaved area.

To remove any loose hair, switch the airflow to the nose cone and adjust the anesthesia to 2.5%Isof fluorine delivered at a flow rate of one liter per minute. Position the rat on the nose cone and place it in the Stereotaxic frame. Check the level of anesthesia by pinching the interdigital area.

If the rat is anesthetized adequately, the defensive reflexes are abolished. Apply vet ointment on eyes to prevent dryness while under anesthesia. Monitor and maintain the rat's body temperature at 37 degrees Celsius.

Inject 0.2 milliliters of mepivacaine subcutaneously into the center of the shaved area to anesthetize the surgical area, ensuring sterility from this point on. Use a scalpel to make a midline incision beginning between the ears and extending forwards for about two centimeters. Ensure that the periosteum is also incised, and then expose the skull with four clamps.

Use a cotton bud to gently remove the periosteum until the coronal and sagittal sutures are exposed. Stach any blood with cotton wool. Fix a needle onto a probe holder and then mark the tip of the needle with a black felt tip pen.

Using the anterior posterior midline lateral and dorso ventral drive screws. Position the tip of the needle directly over bgma. Take the AP and ML veneer scale readings.

Subtract 3.6 millimeters from the AP reading and 2.5 millimeters from the ML reading for electrode implantation into the right STN. Lower the tip of the needle to the surface of the skull to mark the injection site with the dye from the felt tip pen, clamp the dental drill onto the large probe holder of the stereotaxic instrument. Move the dental drill to the marked point on the skull, and while looking through the microscope, drill a hole of about one millimeter diameter through the skull until the dura is visible.

This is the hole for the electrode. Retract the dura using microdissection forceps or a sterile needle as the dura is tough enough to destroy the tip of the electrode. Drill a hole with the dental drill in each frontal squama and in the parietal squama opposite to the hole for the electrode.

Thereafter, drill a hole in each inter parietal squama. Screw a bone screw into each of the five holes. Avoid threading the screws in too deep, which will put pressure on the brain.

The number of turns will depend on the pitch of the screw. Two to three turns of each screw is used here. Disconnect the probe holder from the stereotaxic instrument and clamp the probe holder with the electrode in the micro manipulator.

Using the AP ML and DV drive screws, move the probe holder with the electrode until the tip is almost touching Breg ma. Note the AP ML and DV veneer scale readings at breg ma. Then raise the electrode a few millimeters to prevent the electrode from scraping the skull during movement.

To determine the coordinates of the position where the electrode has to be inserted into the hole, add 3.6 millimeters to the AP reading and add 2.5 millimeters to the ML reading. Using the AP and ML drive screws, move the electrode to the calculated position. At this point, the electrode tip should be situated directly over the drilled electrode hole.

Next, while looking through the microscope, lower the electrode to the level of the dura. Note this as zero in the DV direction. Then gently insert the tip of the electrode into the brain.

While looking through the microscope, connect the electrode pin to the connector of the recording system. Ground the stereotaxic instrument with the counter poise of the room. Then place a faraday cage over the rat in the stereotaxic instrument in place of a faraday cage.

You can also use aluminum foil as shown here since its effect is the same as that of a Faraday cage. Start the recording system if available. Use a loudspeaker to obtain an acoustic signal of discharges of single units while advancing the electrode.

Slowly insert the electrode into the brain while recording the electric activity during the advancement of the electrode. During the recording, reduce anesthesia as much as possible to 0.8 to 1%as low anesthetized animals show a clearer electric brain activity. The specific electrical activity of the STN is usually detectable at a depth of between 7.5 and 8.1 millimeters from the dura.

The typical activity of neurons in the STN is characterized by an irregular firing pattern and a high firing rate. Now, swab away any blood or cerebrospinal fluid that was displaced at the surface of the skull when lowering the electrode, then mix up a small amount of dental cement and then use a small spatula to apply it around the electrode and around four of the five screws. When the dental cement has hardened, disconnect the electrode pin from the electrode holder and connector of the recording system.

Then unscrew the fifth screw that was not fixed by dental cement. Put the plug on the electrode pin and fix the ground wire of the plug with the fourth screw. Apply dental cement around the plug as the cement thickens.

Mold it around the plug to form a cap. Avoid sharp edges of the dental cement that may harm the animal and remove them during hardening. Debride the wound edges and close them with a suture behind the cap and at the front disinfect the wound edges.

Connect the head plug to the wire that is fixed on a swivel. Then remove the rat from the stereotaxic instrument, administer appropriate postoperative analgesia. 12.5 milligrams per kilogram.

Tramadol is used here. Place the rat in a clean cage with thermal support and fix the swivel on this cage. Observe the recovery of the animal.

This image shows an eight micron thick section stained with hemat, toin, and eoin from an animal sacrificed 14 days after implantation and continuous stimulation to visualize the position where the tip of the electrode was located. A solid line surrounds the STN. A small lesion is visible where the electrode tip was located during a 14 day period of stimulation.

It is of note that there is no penetration canal of the electrode visible, indicating that the electrode does not disrupt the surrounding tissue. This image shows the same region at higher magnification. A small number of inflammatory cells were detected around the lesion due to the reaction of the brain tissue to the electrode tip.

Once master, this technique can be done in one and a quarter hours if it's performed properly.