Cardiac and respiratory dysfunction contributes to sudden death after a seizure. Therefore, to study this phenomenon, a multi-system approach was applied to a rabbit model. This technique enables simultaneous monitoring and later analysis of the neurologic, cardiac, and respiratory function of multiple rabbits surrounding seizures, arrhythmias, and preceding death.
While a disease may be primarily neuronal or cardiac, it may include electrical disturbances in both the brain and the heart, such as seizures and arrhythmias, as well as respiratory dysfunction. It can be difficult to work with rabbits because of their hind leg strength. Take care to keep the rabbits calm when moving them, and when attaching the monitors.
Begin by connecting the computer to an amplifier with a 64-pin headbox. To make every eighth pin on the headbox a reference, set the reference electrode to independent. For implantation of the ECG electrodes, secure a rabbit in the supine position with the head at the investigator's knees and lower than the rest of its body.
With a second investigator spreading the fur to expose the underlying skin, subdermally insert 35-degree angle bent electrodes into each axilla. Place leads on the chest, posterior to the right and left forelimbs. And on the abdomen, anterior to the left hind limb.
Then place a ground pin electrode anterior to the right hind limb on the abdomen. When all of the ECG leads have been properly placed, secure the rabbit in an appropriately sized restrainer with the hind limbs under the animal. Using a 45-degree angle approach, insert subdermal straight-pin electrodes into the scalp.
Place one EEG lead on the right anterior, left anterior, right occipital, and left occipital regions of the head. And place a central reference lead at the central point between the other four leads. Run the wires between the ears, and loosely tether them to the restrainer behind the head.
To monitor respiration during the experiment, attach a pulse oximeter to one ear over the marginal ear vein, and gently secure a face mask with capnography tubing over the mouth and nose of the animal. Then attach the other end of the tubing to the vital signs monitor. To record video EEG-ECG, open an appropriate commercially-available EEG software program and adjust the video such that all of the rabbits can be observed within the field of view.
Perform a baseline recording for the animal for a minimum of 10 to 20 minutes, or until the heart rate stabilizes to 200 to 250 beats per minute for at least five minutes. Set the low frequency filter to one hertz and the high frequency filter to 59 hertz. Add time-locked notes in real-time to indicate the timing of interventions, neuro-cardiac events, and motor or investigator artifacts.
For photic stimulation, place a light source with a circular reflector 30 centimeters in front of the rabbit at eye level with the flash intensity set to the maximum. And place two mirrors on each side of the head, and one mirror behind the rabbit, so that the light enters the rabbit's eyes. Attach the light to a controller with an adjustable rate, intensity, and duration.
With the photic stimulator set to one hertz and the flash on, record the response for 30 seconds. Cover the rabbit's eyes with a mask to simulate or cause eye closure and stimulate for another 30 seconds. After recording the eyes open and eyes closed response at each frequency, turn off the photic stimulator for 30 seconds and set the controller to the next frequency setting.
After recording for 60 seconds at each frequency from one to 25 hertz in two hertz intervals, decrease the frequency from 60 to 25 hertz in five hertz increments, and record for 30 seconds with the eyes open and 30 seconds with the eyes closed. At the end of the experiment, remove the EEG and ECG leads from the rabbit and return the animals to their home cages for routine care by the husbandry staff. Before administering the medication, collect 10 to 20 minutes of baseline EEG-ECG video from the untreated rabbit as demonstrated.
For the oral administration of a medication of interest, mix 0.3 milligrams per kilogram of the drug of interest in three milliliters of food-grade applesauce, and load the mixture into a three milliliter oral syringe. Gently lifting the upper lip of the rabbit, slide the tip of the syringe into the side of the mouth that is unobstructed by the rabbit's teeth, and inject the entire volume of drug-supplemented applesauce into the rabbit's mouth. Then collect two hours of EEG-ECG video data before returning the rabbit to its home cage for routine care.
Before recording the EEG-ECG response to intravenous medication administration, shave the posterior surface of the rabbit's ear and use 70%ethanol to disinfect the site, and to dilate, the marginal ear vein. Carefully, cannulate the marginal ear vein with a 25-gauge angiocatheter, and place an injection plug at the end of the catheter. Tape a splint created from three rolled pieces of gauze to the ear to secure the catheter in place and to hold the ear upright.
Inject one milliliter of 10 USP units per milliliter of heparinized saline to keep the catheter patent. When baseline recording has finished, inject saline supplemented with one to 10 milligrams per kilogram of the drug of interest into the catheter once every 10 minutes. After each dose, carefully monitor the video EEG-ECG, oximetry capnography, for any neuro, cardiac, electrical and respiratory abnormalities or visual evidence of epileptiform activity.
Note these changes in real-time, as well as during post analysis. To analyze the ECG video, use an appropriate commercially-available software to identify periods of tachycardia, brachycardia, ectopic beats, or other arrhythmias within the ECG data. To reduce the amount of data to review, create a tachogram to increase the ease with which periods of tachycardia, bradycardia, or irregularities of the RR interval can be identified.
For video EEG analysis after a drug administration experiment, visually scroll through the EEG tracing to distinguish epileptic versus non-epileptic movements for at least one minute after each dose of drug. For video EEG analysis after a photic stimulation experiment, create a spectral analysis plot in an appropriate EEG analysis software program. Then analyze the occipital leads of the EEG for the presence and absence of the occipital driving rhythm.
The occipital driving rhythm will create a peak in the spectral analysis that corresponds to the frequency of the photic stimulator. Assessment of the ECG morphology allows the detection of abnormal heart rates, conduction, and ECG wave forms. The traces can also be used to quantify the heart rate, RR interval, PR interval, P duration, QRS interval, QT interval, QTc, JT interval, and T-peak to T-end interval.
Recordings from the occipital EEG leads typically exhibit a higher amplitude than that observed for frontal lead data. And the dominant frequency in all of the leads are commonly measured in the Delta range. Here, sleep spindle waves from a representative rabbit experiment are shown.
Multiple EEG montages of one period of sleep demonstrate that these waves arise from the center of the head, which is consistent with human findings. In addition to normal EEG changes, various conscious, non-epileptic rabbit movements can also be observed during baseline recordings that can be used to distinguish these data from epileptiform discharges. It is important to place the ECG lead securely into the skin to keep the wires away from the legs and to take care that the electrodes remain attached when transferring rabbits into the restrainer.
This multi-system recording apparatus will enable future drug safety and efficacy studies, and facilitate a comprehensive understanding of various acquired and inherited diseases. This technique captures the course of potentially fatal, multiorgan dysfunction after a seizure, which will lead to a better understanding of the mechanism of SUDEP.
