The anesthetized mouse is taped onto a heating board in a supine position. The right jugular vein is separated and the proximal end of the vein is tied off. The 1.1 French OC Apolar catheter is inserted into the vein, and then the catheter is advanced into the right atrium and the right ventricle.
Finally, the distal end of the vein is tied off to secure the catheter position and prevent bleeding. Hi, I'm Naali from the lab of Dr.Zver in the Department of Molecular Physiology and biophysics at Baylor College of Medicine. Today we'll show you a procedure for program the electrical stimulation in mice.
We use this procedure in our lab to study inducible arrhythmias in mice. So let's get started. Prior to the surgery for cannulation and catheter insertion, use hair clippers to shave the fur of the anesthetized mouse from the neck line to mid chest level.
Disinfect the surgical area with 10%povidone iodine. Place the mouse in a supine position and tape its limbs onto ECG electrodes Incorporated. In a heating board, we recommend a system that includes a rectal temperature probe connected to a heating pad controlled by a thermal analyzer system, such that the body temperature is maintained at 37 plus or minus one degree Celsius.
After confirming by a tow pinch that the mouse is fully anesthetized, make a half inch incision to the right of the midline with the codal terminus at the level of the clavicle. Separate the subcutaneous tissue, salivary glands, and lymphatic tissues by blunt dissection. To visualize the right jugular vein, tie the proximal end of the vein with six oh suture.
Place another suture under the vein at the distal end of the visualized segment. Now start computer-based data acquisition to record surface ECG. Connect the catheter to an external stimulator in recording mode.
Next, use micro scissors to make a small incision in the longitudinal direction of the vein while gently pulling on the proximal suture. To help keep the vein straight, advance the 1.1 French Octo puller catheter through the vein into the right atrium and ventricle. Proper catheter position is verified by visualization of the waveforms of the four intracardiac electrograms at the level of the apex of the right ventricle, the base of the right ventricle, the atrial ventricular node and the right atrium finally tie off the distal suture to secure the catheter position and prevent possible bleeding for atrial pacing.
The electrode pair located inside the atrium is switched from recording mode to stimulation mode while the other electrode pairs remain in recording mode. To determine whether a mouse has an increased vulnerability to atrial arrhythmias, programmed electrical stimulation of the right atrium is performed. First, determine the atrial pacing threshold by applying at least 52 millisecond current pulses at different basic cycle lengths or BCL to test for consistency of stimulus capture.
The BCL of the initial pulse train is slightly lower than the intrinsic BCL and is decreased by 10 milliseconds. The typical current amplitude required for stimulus capture is 100 to 200 microamps. After applying a 15 second atrial pacing train at a BC of 100 milliseconds, measure the sinus node recovery time or SNRT, which is defined as the interval between the last stimulus in the pacing train and the onset of the first spontaneous sinus beat.
To determine the atrial effective refractory period or A ERP apply a series of atrial pacing trains at a fixed BCLS one with a couple shorter premature stimulus S two. The S one to S two interval is progressively reduced by two milliseconds in each pacing train from 70 milliseconds to 20 milliseconds with a recovery period of at least 30 seconds between each stimulation protocol. The A ERP is defined as the longest S one to S two coupling interval for atria that failed to generate a propagated beat with S two.
A similar application of atrial pacing trains and premature stimuli is used to determine the effective refractory period of the atrial ventricular node AV NERP. The AV NERP is defined as the longest S one to S two coupling interval at which the premature stimulation delivered to the atrium is followed by a hiss potential, but not by a QRS complex. Inducibility of atrial arrhythmias, including atrial fibrillation, may be tested using the burst pacing protocol where a series of two second bursts is applied.
The first two second burst has a cycle length or CL of 40 milliseconds, and each successive two second burst has a CL of two milliseconds shorter than the previous burst until the final CL of 20 milliseconds. After all pacing protocols are finished, data acquisition is stopped. In the case of terminal EP studies, the knot is gently loosened to release the catheter.
Here we show some representative surface ECG and intracardiac electrograms in a mouse. This surface ECG shows regular sinus rhythm with a frequency of approximately 540 beats per minute. These next four electrograms show bipolar intracardiac electrogram recordings at the level of the right atrium.
The atrial ventricular node, the base of the right ventricle and the apex of the right ventricle. The intracardiac a wave in the electrogram recording at the level of the right atrium corresponds to the P wave on the surface.CCG. The V wave on the ventricular electrogram corresponds to the QRS wave on the surface CCG.
Next we show representative surface CCG and Intracardiac electrograms and a mouse that developed atrial fibrillation after atrial burst pacing on the surface. CCG, the RR interval is irregular and there are no apparent P waves. On the other hand, the atrial electrogram reveals rapid atrial signals that are irregular consistent with atrial fibrillation.
The ventricular electrogram has a slower frequency similar to the surface lead RR intervals. We have just show you how to perform program the electrical stimulation in the mouse by when doing this procedure, it's important to remember to avoid extensive bleeding and to keep the body temperature within the normal range of 36 to 37 degree. In addition, try to minimize the exposure to Asof Florin because exposure to as fuller longer than two hours out to a higher concentration could suppress the cardiac and the respiratory function in the mouse.
Therefore, we recommended that all started be complicated within two hours. So that's it. Thank you for watching and good luck with your experiments.
