TITLE: Vagus Nerve Selective Stimulation and EIT recording
AUTHORS: Enrico  Ravagli, Svetlana  Mastitskaya, Nicole  Thompson, Kirill  Aristovich, David  Holder

[DESCRIPTION]
This electrophysiology protocol describes the electrical stimulation and measurement steps needed to localize branches and image functional activity in the cervical; vagus nerve of pigs.

[STEPS]
SECTION: Preparation
1. Animal (pig) is anaesthetized (Isofluorane) and cervical vagus is exposed through surgery.
SECTION: Preparation
2. Clean electrodes of the nerve cuffs by dipping into ethanol and then physiological solution (or saline solution).
SECTION: Preparation
3. Apply nerve cuffs to exposed cervical left vagus and connect to the ScouseTom system.
SECTION: Selective Stimulation - Pulmonary Branch
5. Set initial parameters for selective stimulation of the pulmonary branch: suggested 50µs pulse width, 0.4mA pulse amplitude, 20Hz repetition frequency, 30s on / 30s off stimulation. Set animal ventilation setup to spontaneous breathing.
SECTION: Selective Stimulation - Pulmonary Branch
6. Run selective stimulation with above parameters and observe ETCO2 response to stimulation (breathing slowdown or complete block) on different electrode pairs.
SECTION: Selective Stimulation - Pulmonary Branch
7.  Repeat stimulation rounds with increased or decreased pulse amplitude until ETCO2 alteration is localised to one or a few stimulation pairs. Restore mechanical ventilation after completion.
SECTION: Selective Stimulation - Recurrent Laryngeal Branch
8. Set initial parameters for selective stimulation of the recurrent laryngeal branch: suggested 50µs pulse width, 50-100µA pulse amplitude, 20Hz repetition frequency, 5s on / 5s off stimulation. Animal can stay in mechanical ventilation during this phase.
SECTION: Selective Stimulation - Recurrent Laryngeal Branch
9. Run selective stimulation with above parameters and observe EMG needle response to stimulation (activation of larynx shows bursts of activity on EMG recordings) on different electrode pairs.
SECTION: Laryngeal EIT
11. Connect stimulator output of ScouseTom to bipolar cuff placed on laryngeal branch. Connect ScouseTom in EIT mode to vagus nerve EIT cuff.
SECTION: Selective Stimulation - Cardiac Branch
13. Switch anaesthetic from isofluorane to α-chloralose and wait 30-40 minutes for settling.
SECTION: Selective Stimulation - Cardiac Branch
14. Set initial parameters for selective stimulation of the recurrent laryngeal branch: suggested 1ms pulse width, 1mA pulse amplitude, 20Hz repetition frequency, 30s on / 30s off stimulation. Animal can stay in mechanical ventilation during this phase.
SECTION: Selective Stimulation - Cardiac Branch
15. Run selective stimulation with above parameters and observe heart rate response to stimulation, computed in real time from peaks in blood pressure or ECG on different electrode pairs.
SECTION: Selective Stimulation - Cardiac Branch
16. Repeat stimulation rounds with increased or decreased pulse amplitude until heart rate change in response to stimulation is localised to just one or a few stimulation pairs.
SECTION: Spontaneous EIT
17. Perform spontaneous fast neural EIT with following parameters:
- No stimulation
- EIT: skip-5 pattern, 200 µA, 6kHz, 10 minutes each injection pair
- Recording ECG, blood pressure, End-Tidal CO2 on auxiliary channels of the recording system
SECTION: Preparation
4. Apply other required electrical interfaces: EMG needles on larynx, bipolar stimulation cuff on recurrent laryngeal branch (approx. 40cm from cervical vagus cuffs).
SECTION: Selective Stimulation - Recurrent Laryngeal Branch
10. Repeat stimulation rounds with increased or decreased pulse amplitude until EMG bursts in response to stimulation are localised to just one or a few stimulation pairs.
SECTION: Laryngeal EIT
12. Perform evoked fast neural EIT according to published work with following parameters:
- EIT: skip-5 pattern, 200 µA, 6kHz, 15s each injection pair
- Stimulation: 50 µs pulse width, 1.2 mA amplitude, 20 Hz repetition during each injection