Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

The overall goal of this procedure is to effectively implant the epic transmitter to record high quality EEG in rodents, such as rats and mice. This is accomplished by first exposing and drying the surface of the skull. The second step is to drill holes in the skull for insertion of the transmitter, electrodes and any bone screws needed.

Next, the bone screws are inserted and the transmitter electrodes are aligned. The final step is to insert the electrodes and secure the transmitter to the skull with acrylic adhesives. Ultimately, data acquisition video and review software are used to monitor EEG activity 24 7 to capture all EEG events such as seizures and interictal spikes.

Visual demonstration of this method is critical as the implantation steps are difficult to learn. The size of the rodent skull results in a complex but trainable surgical procedure. The main goal of this presentation is to simplify this implantation procedure and make it easily reproducible.

We first had the idea for this method when we were having difficulty making long-term electrographic recordings from animal models of epilepsy. During convulsive seizures, wired recordings are prone to electrical artifacts and untoward stress on the electrode skull interface. Wired recording systems can fail due to the animal chewing on the tether and scratching at the implant.

This wireless system addresses all of these problems while providing up to six months of continuous uninterrupted EEG recordings. In preparation for the surgery, be certain to fully sterilize the transmitter, soak it in 70%ethanol, rinse it in sterile saline, and submerge it in sterile saline. Leave it there until it is needed.

The last preparation is to make certain everything at the surgical site is sterilized. Spray and wipe down the surrounding area and equipment with 70%ethanol. After anesthetizing the animal, be sure to confirm a lack of response to a toe pinch before commencing with the surgery while the animal is anesthetized.

Do this every 15 minutes. The anesthetized animal should be first fixed in position in a stereotaxic frame. Use ear bars, but do not tighten them too much as the skin of young pups is very soft.

After secured, apply a nose cone to deliver 3%iso fluorine. To keep the animal warm, keep a heating pad underneath it at 37 degrees Celsius. For adult animals Also lubricate the eyes.

Now sterilize the incision site First, clean off the scalp with 70%ethanol, and then with Betadine, rub the skin in a circular motion. Then drape the animal. Begin by very cautiously making an incision in the scalp, slightly behind the ears, along the midline.

Extend the incision about two centimeters. Young pups are very soft skull. Also, make this incision in just one cut so it heals quicker.

Thus expose the skull. Next, using aneurysm clips, grasp the skull and expose and dry an area where the transmitter will be attached. Keep in mind, in young pups, the skull bones are not fused.

Then dry the skull where the transmitter will attach For a strong adhesive bond, use an electrocautery pen to stop the bleeding on the skull. Now using anatomical landmarks and a brain atlas, identify the skull location to place the bur hole for recording. Then using a Dremel and bur type bit, create two holes at the recording position that are no more than 300 microns in diameter.

Next, over the cerebellum and behind the Lambda, place a bur hole. For the reference electrode, check that the transmitter's wires are aligned to the bur holes. If not, check the fit of the transmitter and gently bend the electrodes to line up with the holes using fine tipped forceps.

Now trim them using surgical scissors to the desired length. They should extend to above the dura for EEG recordings or penetrate down to defined brain structures. Now without coating the electrodes apply enough cyanoacrylate to the transmitter to completely cover it.

Just do not get glue on the electrodes. If the transmitter is going to take surface recordings, lower the transmitter into the holes and just hold it in place for about a minute using slight pressure. If the recording is going to be taken from deep brain structures, mount the transmitter on a cannula holder and place it on a stereotaxic arm.

It is critical to position the transmitter electrodes into the bur holes without glue contamination. Contamination will result in degraded signal. Next, add more cyanoacrylate gel to sturdy the attachment of the transmitter.

Apply the glue in circles covering the transmitter's walls. Make sure not to cover the antenna. Then apply 100 microliters of a chemical accelerant around the transmitter's base from a syringe.

Prevent it from contacting the surrounding tissues. To remove the accelerant thoroughly wash the area with half a milliliter of sterile saline. Now suture the skin around the transmitter without covering it.

The top of the transmitter should be externalized. For good signal transmission, use Vicryl or silk suture. When working with delicate pups, the skin should be reasonably tight around the transmitter and the surrounding glue.

Now transfer the animal from the frame to a warm blanket For recovery, postoperative care details are provided in the text protocol. Transfer a single implanted animal to a recording chamber. Only one implanted animal can be monitored at a time, but other non implanted animals can be with it.

To set up the recorder, connect the power supply to the receiver base. Next, connect the base to a data acquisition system via BNCs. Now place the cage on the signal receiver base.

The signal light should illuminate. Data can now be recorded. First, connect the receiver base to an ad converter and connect the converter to a computer.

Using the software settings, set the sample rate of the recording to 500 per second. The transmitter sends EEG signals between 0.1 to 100 hertz with an eight decibel per VE roll-off at either end Setting an appropriate sample rate that is above nyquist is critical. We recommend 500 samples per second While the animals are being recorded from watch for signs of stress and dehydration.

Pups should not be in the chamber for more than two hours. The transmitter was designed with a minimally damaging form factor and long battery life recording could be performed continuously for six months depending on the battery capacity. Mice at P 12 and older tolerated the transmitter very well as did rats as young as P six.

The cyanoacrylate attachment to the skull did not hinder development permitting continuous data collection throughout the animal's development into adulthood Once mastered, this technique can be done in just minutes if it is performed properly. Remember that it's important to keep electrodes clear of glue contamination Following this procedure. Other methods like quantitative seizure detection analysis can be performed in order to answer additional questions like the time course and severity of status epilepticus.

This technique will pave the way for epilepsy researchers to study neonatal seizures and rodent models of perinatal stroke.