TITLE: Subcutaneous nerve stimulation in canine model of persistent atrial fibrillation
AUTHORS: Takashi Kusayama, Juyi Wan, Yuan Yuan, Xiao Liu, Xiaochun Li, Changyu Shen, Michael C. Fishbein, Thomas H. Everett IV, Peng-Sheng.Chen  

[DESCRIPTION]
This animal protocol was approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine and the Methodist Research Institute, Indianapolis, IN, and conformed to the Guide for Care and Use of Laboratory Animals. The surgical procedure is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care. 

[STEPS]
SECTION: Surgical Procedure
1. Under isoflurane inhalation general anesthesia, perform left thoracotomy through the 4th intercostal space.
SECTION: Surgical Procedure
2. Implant a radio transmitter (D70-EEE, Data Sciences International), InterStim II neurostimulator (Medtronic Inc.), and a modified Secura implantable cardioverter defibrillator (ICD) (Medtronic Inc.).
SECTION: Surgical Procedure
3. Connect 3 pairs of bipolar electrodes to D70-EEE to record:
Left stellate ganglion nerve activity (SGNA)
Left thoracic vagal nerve activity (VNA)
Subcutaneous nerve activity (ScNA)
SECTION: Surgical Procedure
4. Blindly insert a quadripolar sacral stimulating lead (Medtronic Inc., model 3889) into the subcutaneous fat layer under the cutaneous trunci muscle and connect it to InterStim II in a subcutaneous pocket.
    
SECTION: Surgical Procedure
5. To record ScNA bracket the point of stimulation with a pair of bipolar electrodes from D70-EEE placed in the subcutaneous tissue.
SECTION: Surgical Procedure
6. Screw a 5076 screw-in pacing lead (Medtronic Inc.) into the left atrial appendage and connect it to a modified Secura ICD for rapid atrial pacing to induce sustained AF.
SECTION: Surgical Procedure
7. Turn off the defibrillator function of the ICD.
SECTION: Surgical Procedure
8. 2 weeks after the surgery turn on D70-EEE for baseline recording of ECG and nerve activities (NA) during sinus rhythm (SR).
22. 
SECTION: Atrial Fibrillation (AF) Induction Procedure
9. 2 weeks post surgery turn on D70-EEE. After baseline recording of ECG and NA during sinus rhythm (SR), turn on the ICD.
SECTION: Atrial Fibrillation (AF) Induction Procedure
10. The ICD should deliver high rate continuous atrial pacing (600 bpm, 2X the diastolic threshold output) for at least 3 weeks to induce persistent AF (>48 hours).
SECTION: Atrial Fibrillation (AF) Induction Procedure
11. Halt the pacing protocol every 3 weeks to determine if sustained AF was induced. 
SECTION: Atrial Fibrillation (AF) Induction Procedure
12. Repeat the protocol until sustained AF is induced and study data as the baseline of the sustained AF.
SECTION: Subcutaneous Nerve Stimulation (ScNS)
13. After the baseline recording during SR and sustained AF, turn on the neurostimulator.
SECTION: Subcutaneous Nerve Stimulation (ScNS)
14. Weekly measure the impedance and adjust the voltage output to maintain the desired current output for 4 weeks.
SECTION: Subcutaneous Nerve Stimulation (ScNS)
15. After ScNS for 4 weeks turn off the neurostimulator.
SECTION: Subcutaneous Nerve Stimulation (ScNS)
13.1. In 3.5 mA and 0.25 mA groups:
Program the neurostimulator to 20-s ON and 60-s OFF (10 Hz, 450 μs pulse width).
Program the output current (mA) to deliver ScNS at 3.5 mA group and 0.25 mA group. Progressively increase the output until the desired level is reached.
SECTION: Subcutaneous Nerve Stimulation (ScNS)
13.2. In sham group:
Program the output current to 0 mA (sham stimulation).
SECTION: Subcutaneous Nerve Stimulation (ScNS)
16. Turn on D70-EEE to record final post ScNS state.
SECTION: Subcutaneous Nerve Stimulation (ScNS)
17. Euthanize animals and harvest tissues for additional readouts if needed.