Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus

The overall goal of this procedure is to evaluate cardiac, electrical function and susceptibility to ventricular arrhythmias in experimental animals following seizures. This is accomplished by first inserting an infusion catheter into a jugular vein of an animal that has undergone seizure activity. Next, electrodes are implanted in thoracic muscles to record cardiac electrocardiograms or ECGs.

ECGs are then recorded under control conditions and during jugular vein infusion of an agent that induces cardiac arrhythmias. Finally, ECG records are evaluated. Ultimately, results can be obtained that show seizures alter electrical cardiac function, and increased risk of lethal ventricular arrhythmias.

Through analysis of ECG recordings, the seizure evoked changes in ECG can serve as a prognostic indicator of the risk of sudden cardiac death. The main advantage of this technique is that these procedures, which are non-invasive in humans, produce recordings of electrical activity to the heart that can be used to predict the risk of lethal cardiac arrhythmias. Furthermore, in animal models, changes in cardiac activity can be directly related to susceptibility of arrhythmias.

This method can be used to address the key question in the field of seizure disorders. That is the relationship between changes in electrical activity of the heart and sudden cardiac death following status epilepticus and an epilepsy. These techniques have implications for the treatment of seizure disorders as individuals identified as being at risk for cardiac dysfunction resulting from seizures.

Using this non-invasive procedure could be placed on cardioprotective therapy to reduce the risk of sudden cardiac death. Though this method can provide insights into cardiac dysfunctions associated with seizure disorders, it can also be applied to any pathological condition, which can be characterized by lethal ventricular arrhythmias and which can be modeled in animals. Generally, individuals familiar with the fundamental and surgical techniques of rodents or other small animals should not have any problems with this method.

We first had the idea for using this method and we were looking for a fast, inexpensive, and easily performed procedure to evaluate cardiac function that was also applicable to diagnosis in humans Before implanting ECG electrodes into the rat for data collection and induction of arrhythmias during IV administration of an arrhythmogenic agent such as aconitine, construct a jugular vein catheter from a 100 millimeter long piece of PE 50 polyethylene tubing by beveling one end, and then blunting the bevel. Then fill the catheter with heparin saline. Next construct ECG recording electrodes from two 100 millimeter lengths of insulated silver wire.

Strip one end of both wires and solder them to a micro connector. Then twist the insulation to form a third wire that is used as a ground After soldering wires are sealed with epoxy. Strip five millimeters of insulation from the distal end of the wires and discard to implant the jugular vein catheter.

First anesthetize the animal by an intraperitoneal injection of urethane. This dose is sufficient to maintain the animal in the appropriate plane of anesthesia for the duration of the procedure. This is a non survival surgery.

Ensure that the animal is at the appropriate plane of anesthesia by performing a toe pinch. Then shave the right side of the neck from the clavicle to the chin and shave the chest. Next, disinfect the shaved portion of the neck with iodine alcohol and make a longitudinal incision in the skin above the carotid artery and open the incision using fine tipped curved dissecting forceps.

Gently separate the skin from the underlying muscle and then the muscles that overlay the jugular vein. Finally, using similar techniques, free the jugular vein from the underlying tissue. Place two pieces of number two, surgical silk under the jugular vein, and position the sutures at the rostral and coddle.

Most portions of the incision tie the rostral ligature to discontinue flow through the vein. Retract the al ligature to lift the vessel off the underlying tissue and using micro scissors or a 23 gauge needle. Make a small cut.

Then insert the blunted beveled end of the previously constructed heparin filled PE 50 polyethylene catheter and advance the tip approximately eight millimeters to the level of the right atrium. Secure the catheter by tying the al ligature around both the vessel and catheter. Finally, close the wound with either wound clips or sutures.

Once the catheter is implanted, place the animal on its back and disinfect the shaved portion of the chest. For placement of the electrodes, make two 10 millimeter longitudinal incisions, one in the upper right and one in the lower left quadrants of the chest and free the overlying skin to expose the thoracic muscles using relatively fine surgical silk suture. One of the exposed five millimeter tips of the silver wire electrodes into the thoracic muscles in each of the exposed areas.

Close the incisions with wound clips and plug the micro connector into the recording device. ECG recordings are made using a power lab data acquisition system and Macintosh computer or similar hardware and software following a 20 to 30 minute equilibration period during which the animal is placed on a grounded metal sheet and loosely wrapped in a towel. To maintain its body temperature record ECG for 20 minutes, attach the jugular vein catheter to a remote syringe placed in a programmable infusion pump.

Initiate the aconitine infusion to deliver a constant dose of five micrograms per kilogram per minute For seven minutes, denote the starting point of the infusion on the computer record following ventricular fibrillation or the end of the infusion period. Euthanize the animal using an approved procedure. The QT interval represents the total duration of ventricular electrical activity.

This figure shows a model ECG recording and the duration of the QT interval. Calculate the mean time between the initiation of the Q wave and termination of the T-wave for all recorded heartbeats over a five minute interval obtained during the 20 minute control recording period to correct the QT interval for differences in heart rate. Use bass's formula shown here where RR is the mean interval between heartbeats.

The RR interval is calculated as the duration between successive Q waves obtained during the analyzed period. The QT dispersion is calculated by subtracting the minimum QT from the maximum QT recorded for each animal. To analyze heart rate variability, use a software program such as lab chart seven that analyzes variability in the RR intervals obtained during the period of analysis and calculates both temporal and or spectral variants in heart rate.

For the analysis of susceptibility to ventricular arrhythmias, calculate the latency from the initiation of aconitine infusion to the first premature ventricular contraction, ventricular tachycardia, and ventricular fibrillation. This figure illustrates one measure of HRV in the spectral domain. The root means squared of the standard deviation of the RR interval or R-M-S-S-D in control rats and in animals that underwent SE one week prior to testing.

This measure of HRV, which represents changes in the parasympathetic control of cardiac function, is significantly decreased in animals following se. This figure shows that both corrected QT interval and QT dispersion are significantly increased. Two weeks following se taken together these data predict that se increases risk of ventricular arrhythmias.

This prediction is confirmed by the findings that the periods of aconitine infusion necessary to induce the experimental arrhythmias observed in these studies were significantly smaller in animals following SE.Generally, this procedure can be done in about an hour and a half when it is performed properly. While attempting this procedure, it's important to remember that the jugular vein catheter must be kept clear and that the ECG electrodes are constructed and implanted in such a way to produce the most noise free signal possible. After watching this video, you should have a good understanding of how to evaluate electrical activity of the heart and relate it to susceptibility to lethal ventricular arrhythmias.

In rodent models of pathologies associated with sudden cardiac death.